Nusantara Bioscience vol. 2, no. 3, November 2010

Membrane damage of Spodoptera litura photo by Y Sanjaya

| Nus Biosci | vol. 2 | no. 3 | pp. 109‐156| November 2010 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

| Nus Biosci | vol. 2 | no. 3 | pp. 109‐156 | November 2010 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 109-115 November 2010

Serum lipid profile and retinol in rats fed micronutrient rich edible vegetable oil blend HAMID NAWAZ KHAN1,♥, HUMAIRA FAROOQI2, SHAKIR ALI2, JAFAR SALAMAT KHAN1 ¹Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India. Tel.: +91-1126059688 Ext. 5616, Fax.: +91-1126059663, ♥email: hamidramzan@rediffmail.com 2 Departments of Biotechnology, Faculty of Science, Jamia Hamdard (Hamdard University) Hamdard Nagar, New Delhi-110062, India.

Manuscript received: 20 October 2010. Revision accepted: 26 November 2010.

Abstract. Khan HN, Farooqi H, Ali S, Khan JS. 2010. Serum lipid profile and retinol in rats fed micronutrient rich edible vegetable oil blend. Nusantara Bioscience 2: 109-116. The animal rats were given 10% oil mixed in fat free diet for one month or six months. In the experiment, the groups of rats were fed with the micronutrient (MN) rich blends mixed previously with 1% cholesterol, and their effects were tested on serum lipid profile. Most significant changes in the High Dencity Lipoprotein (HDL) cholesterol were observed in onemonth study where HDL increased from 24 mg/dl in group to 64 mg/dl in the Mustard palm olein oil blend (MP); in mustard oil (MO) alone fed rats, the HDL was 36 mg/dl. Serum retinol was analyzed as one of the important MN in rats receiving the diet mixed with the blend for various duration of time. The results assume great significance as MO or palm olein oil (PO) alone could not bring the maximum beneficial effects, and the blends appear to have more merit as health oils in alleviating adverse health condition such as coronary heart disease (CHD), diabetes, obesity and hypertension.

Key words: mustard oil, palm olein oil, oil blend, lipid profile, micronutrient, retinol, diabetes, hypertension, coronary heart disease.

Abstrak. Khan HN, Farooqi H, Ali S, Khan JS. 2010. Profil serum lipid dan retinol pada tikus yang diberi makanan yang dicampur minyak sayur yang kaya mikronutrien. Nusantara Bioscience 2: 109-116. Tikus percobaan diberi minyak 10% dicampur dalam diet bebas lemak selama satu bulan atau enam bulan. Dalam percobaan, kelompok tikus diberi makan yang kaya mikronutrien (MN) dicampur dengan kolesterol 1% sebelumnya, dan efeknya diuji pada profil serum lipid. Sebagian besar perubahan signifikan dalam High Dencity Lipoprotein (HDL) kolesterol teramati dalam penelitian satu bulan, dimana HDL meningkat dari 24 mg/dl menjadi 64 mg/dl pada campuran minyak mustard-kelapa sawit olein (MP); pada tikus yang hanya diberi minyak mustard (MO) saja, nilai HDL adalah 36 mg/dl. Serum retinol dianalisis sebagai salah satu mikronutrien penting pada tikus yang menerima diet dengan campuran minyak pada berbagai durasi waktu. Hasil menunjuknya secara signifikan bahwa pemberian mustard MO atau kelapa sawit olein (PO) saja tidak membawa efek yang menguntungkan secara maksimum, adapun campuran keduanya tampaknya memberikan manfaat lebih tinggi untuk mengurangi kondisi yang merugikan kesehatan seperti penyakit jantung koroner (PJK), diabetes, obesitas dan hipertensi.

Kata kunci: minyak mustard, minyak kelapa sawit olein, campuran minyak, profil lipid, mikronutrien, retinol, diabetes, hipertensi, penyakit jantung koroner.

INTRODUCTION Fats and oils are integral to our diets, and are important sources of calorie density in the diet. Besides, they provide essential fatty acids and increase the absorption of fatsoluble vitamins. Oils such as palm olein oil and rice bran oil are considered as good sources of micronutrients (MN) that include β-carotene, tocopherols and tocotrienols that are known for their beneficial actions in human.

Tocotrienols, for example, have been reported for lowering the serum cholesterol level in hypercholesterolemic subjects (Qureshi et al. 1991a) and have been recommended for metabolic disorders such as coronary heart disease (CHD), diabetes, obesity and hypertension.

The MN present in oils are also recognized for a number of other beneficial effects on body, and have been suggested effective in conditions as wide as cancer and kidney stones, to name a few. However, in spite of a high MN content, the

use of unconventional but MN rich oils is limited mainly because of the regional preferences of specific individual oils. In the following section, the effect of three MN rich blends of edible vegetable oils on serum lipid composition and the retinol level is presented. The study is important, as limited data is available on the effects of oil blends on the physiological systems, although extensive research has been conducted on many conventional and unconventional vegetable oils. The oils rich in PUFA, for example, cause a decrease in ‘bad’ cholesterol and triglycerides. Rice bran oil and palm oil are particularly rich in many MN that have been reported for health benefits (Tomeo et al. 1995). The impact of palm oil on cardiovascular disease and cancer has been investigated (Elson and Qureshi 1995).

Tocotrienols from the palm olefin oil inhibit protein oxidation and lipid peroxidation in rat liver microsomes (Kamat et al. 1997). The ω-3 fatty acids (linolenic acid) in oils can increase the level of circulating good cholesterol.

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These and other such findings encouraged us to use the unconventional MN rich oils for preparing various blends that are being tested in this study on experimental animals such as MP vegetable oil blends.

MATERIALS AND METHODS Materials Refined vegetable oils such as palm olein oil and expeller-pressed, unrefined mustard oil (MO) were purchased from the local market of Alaknanda market, New Delhi, India; obtained from the manufacturers, and used before the ‘best before date’.

Preparation of oil blends and analysis A binary blend of the MP represents mustard oil - palm olein oil blend (35:65) in proportions. The blends marked with a superscript ‘C’ denote the oil blend in which 1% cholesterol was mixed. The blending was based on the fatty acid composition of the oil, which were mixed in such a way that an ideal fatty acid profile was obtained in the blends. Animals were fed the experimental diet and boiled and purified water on an ad libitum basis; the composition of fat free diet provided to the animals during the study period is provided in the Table 1.

Measurement of serum lipid profile Male rats weighing 160-190 g were divided into groups with each group consisting of 6 rats. Three groups were fed 10% oil/blend in diet for one month or six months before analyzing the serum lipid profile. In yet another group, the rats were fed similarly but a high cholesterol diet (HCD).

Animals were maintained in individual cages and supplied water and diet adlib (ad hbidn). Daily food intake and weekly body weights were recorded. Following the completion of the tenure of feeding, the animals were sacrificed and 4 mL of blood collected from each animal by cardiac puncture. The liver was dissected, weighed and the liver weight of each animal was calculated as percentage of body weight of each animal calculated. 1 gm of liver from each animal was homogenized in a buffer and total cholesterol (TC), triglycerides (TG), HDL-C LDL + VLDL-C were measured from the serum. The level of triglycerides, cholesterol, and other lipid moieties in rat serum were estimated using the diagnostic kits. Serum triglycerides were hydrolyzed to glycerol and free fatty acids by lipase. The intensity of the color developed was proportional to the triglycerides concentration and was measured photo metrically at 546nm (530 to 570nm) or with Green filter. Normal cholesterol levels are affected by stress, age, hormonal balance and pregnancy. The concentration of cholesterol in the sample is directly proportional to the intensity of the red complex (red quinone), which is measured at 500 nm.

Table-1: The composition of fat free diet

Casein

Fat free diet (g/100 g diet) 15.0

Starch

66.3

Salt mixture

04.0

Vitamin mixture

01.0

Cellulose

02.0

Cholesterol

01.0

Ingredients

Cholic acid

00.5

Choline chloride

00.2

Note: Salt mixture contained: 4.6% NaCl; 9.3% Na2HPO4.H2O; 25.6% K2HPO4; 14.5% CaH4(PO4)2.H2O; 3.3% Fe(C6H5O7).

5H2O; 34.9% Ca(C3H5O3)2.5 H2O; 7% MgSO4; 0.05% ZnSO4.7H2O; 0.9% KI; 0.02% Cr(C2H3O2)3. Vitamin mixture contained: 150 mg riboflavin; 100 mg thiamine; 1000 mg nicotinic acid; 100 mg pyridoxine; 1 mg cyanocobalamine; 500 mg pantothenic acid; 50 mg folic acid; 3750 mg ascorbic acid; 100 mg vitamin K; 100 mg vitamin E; 2,50,000 IU vitamin A; 20,000 IU vitamin D, and starch to make up to 100.

Chemicals Authentic standards of tocopherols (α, β, γ, δ) tocotrienols (α, β, γ, δ) and β- carotene were purchased from E. Merck (Germany) and fatty acid methyl esters from Sigma Chemical Company, USA. HPLC grade methanol, acetonitrile, water, n-hexane, alcohol, BF3 and isopropanol were procured from Merck (India). Other chemicals and reagents were of analytical grade and were obtained from standard commercial sources in India.

Determination of serum retinol by HPLC The technique has several advantages over other available techniques for retinol estimation, and is a rapid micro procedure (Bieri et al. 1979). We have used this method for assessing the status of retinol in animals given the oil blend on a Shimadzu (model LC-10ATvp) HPLC equipped with a binary gradient and a multiple wavelength detector (SPD-10Avp) and C-18 column (alpha Bond C18 125A 10μm300 x 4.60mm) protected by a guard column was used. The system was operated using SPINCHROM software. Methanol/HPLC water (95/5) was used as a mobile phase, and the flow rate was set to 1.2 mL/minute.

Retention time of retinol was 6.80 minutes at this flow rate in the C-18 column used in this study. In the injector of the machine, 20 µl aliquot (from serum) was injected and the results were calculated with the help of an internal standard added to the sample before injection. Detection wavelength was 326 nm.

Statistical analysis Data were analyzed by ANOVA to ascertain if the dietary treatments were a source of variance related to various lipid parameters measured. Significance was accepted at the p<0.05 levels (Snedecor and Cochran 1990).

RESULTS AND DISCUSSION The effect of dietary fats on serum lipids and MN profile has long been recognized (Rukmini and Raghuram

KHAN et al. – Serum lipid profile and retinol after vegetable oil treatment

1991; Manorama et al. 1996). Public health programs for the prevention of coronary heart diseases (CHD) in developed nations recommend changes in the dietary habits, especially in the quality and the quantity of fats. The American Heart Association recommends (AHA Medical/Scientific Statement 1990) that total fat in diet should not exceed 30% of energy calories, out of which the energy percent from saturated fatty acids (SFA) should not exceed 8-10%, PUFA (poly unsaturated fatty acid) 10 energy per cent, and monounsaturated fatty acids (MUFA) by difference (that is 10 - 12%). The recommended dietary allowances (RDA) for a balanced diet as per the ICMR Nutrient Requirements and RDA guidelines for Indians (ICMR Expert Group Report: 1989; ICMR 1990) suggest that fat in diet should not exceed 20 energy% with 3 energy% derived from PUFA. In 1985, Lasserre et al reported that linolenic acid (18:3), an essential polyunsaturated fatty acid, should provide 0.5-1% of total calories. These and various other recommendations were made to ensure optimum serum lipid composition in human for nutritional and health benefits. Studies have revealed that the type of fatty acid in diet is critical. Replacement of dietary saturated by unsaturated fatty acids, for example, is a very effective way of lowering serum cholesterol. A desirable serum lipid composition in human is the one, which has low cholesterol (<180 mg/dl), low triglycerides (<100 mg/dl), low LDL-cholesterol and high HDLcholesterol (Ausman et al. 2005).

Unconventional oils such as rice bran oil, soyabean oil, cotton seed oil and palm olein oil have been used in suitable proportions for nutritional, health and other benefits as per the consumer’s acceptability and market demands. These oils are preferred over other oils for blending for their micronutrient content and economic reasons. Rice bran oil, for example, is well known for its hypocholesterolemic action both in animals and in humans.

With the fatty acid composition resembling groundnut oil, rice bran oil has limited PUFA, which provides additional health benefits. Rice bran oil contains oleic acid (38.4%), linoleic acid (34.4%), and linolenic acid (2.2%) as unsaturated fatty acids. Palmitic acid (21.5%) and stearic acid (2.9%) are the saturated fatty acids in rice bran oil. Its unsaponification matter (4.2%) consists of several micronutrients including totaltocopherols (81.3mg/g),γoryzanol (1.6mg/g) and squalene (320 mg/g). The MN of rice bran oil namely γ-oryzanol and phytosterols help to reduce the triglyceride and elevate the HDL. In studies on the hypocholesterolemic action of rice bran oil (Seetharamaiah and Chandrasekhra 1989), addition of oryzanol at 0.5% level to the diet containing rice bran oil has been observed to significantly decrease the serum total cholesterol. Similarly unconventional oil in India, the palm olein oil, is a rich source of β-carotene and tocotrienols.

Dietary tocotrienols reduce the concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2, and platelet factor 4 in pigs with inherited hyperlipidemias (Qureshi et al. 1991b). These and other facts have prompted researchers to use unconventional oil for health, economic and industrial benefits as the presence of a large amount of MN and antioxidants in selected unconventional

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oils confer nutritional benefits and oxidative stability to the blends prepared from these oils. In 1984, Suzuki et al have demonstrated that rice bran oil (RO) in combination with safflower oil (SFO) that has high PUFA, can significantly bring down the serum cholesterol to normal values within 8 days. A study in human volunteers feeding a blend of RO: SFO could effectively bring down the cholesterol below 120 mg/dl in a week. Safflower oil is rich in PUFA (75%) and there are several reports available on the adverse effects of PUFA (Grundy 1975). SFO when fed to animals or human reduces serum total cholesterol but at the same time it lowers the HDL levels. On the other hand, rice bran oil does not only significantly reduce total cholesterol, triglycerides, LDL, and VLDL levels, but also significantly increases the HDL, which is an important benefit of blending SFO and RO. Hypothesizing the health benefits of the constituents (MN plus fatty acids) present in rice bran oil and palm olein oil, and the traditional acceptability of mustard oil, we prepared the MN rich binary edible vegetable oil blends that were expected to have nutritional and health benefits particularly with regard to the serum lipid and MN composition. The oil blend was fed to rats in diet strictly in accordance to the procedure described in the methods section earlier, and the serum lipid profile was studied following a six-month or a one-month treatment period.

Long-term feeding Serum cholesterol and triglyceride The results of the six-month study of feeding the oil/blend mixed in diet to rats have been summarized in Table 2. As shown in Table 2, total cholesterol in control animals receiving normal diet was 54 mg/dl. The level of cholesterol in rats receiving the MP group it was 52 mg/dl.

Incidentally, this blend was the richest as far as the MN level of all the blends used in this study is concerned, and had a fatty acid composition close to the recommended international standards. As described earlier, presence of unsaturated fatty acids in diet is a very effective way of lowering serum cholesterol. An intriguing hypothesis to explain this decrease takes into account the kinks in unsaturated fatty acids; the lipid esters containing PUFA require more space in lipoproteins and thereby sterically exclude cholesterol. Because a PUFA occupies a greater spatial area, the LDL can carry fewer lipids and their lipid content is thereby lowered. PUFA would cause a drop in the lipoprotein concentration and the VLDL synthesis that would be responsible for the reduced LDL synthesis (Lewis et al. 1961). Further, the unsaturated fats cause plasma cholesterol to be transferred to the tissue pools, and the cholesterol transferred into the liver could cause a temporary depression of lipoproteins (Spritz 1965).

Lipid lowering action of rice bran oil has been extensively studied (Rukmini and Raghuram 1991). In fact, edible grade crude palm oil (Elaeis guineensis) is one of the richest natural sources of β-carotene (7.5 mmol/L). In the present study, we have used refined palm olein oil that retains substantial quantity of β-carotene.

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The mustard oil based blends particularly the MP blend could cause a reduction in the level of cholesterol particularly in the group of rats with cholesterol overload (1% cholesterol mixed in the blend). In a six-month study on MP fed blends, the cholesterol level in 46 in MPC. These values were well below the levels seen in the normal control rats (54 mg/dl) and also lower than the values observed in mustard oil alone treated animals (47 mg/dl).

The values in MP groups alone were 52 mg/dl, respectively. The MP blend (without cholesterol supplementation) appears to be not effective in bringing down the cholesterol level. However, in cholesterolsupplemented group, there was some lowering of cholesterol in MPC fed rats in the six-month study. A possible explanation might be a feedback effect due to a long-term feeding of cholesterol on cholesterol biosynthesis involving control via HMG Co-A reductase (Yung et al. 2010) Lipoprotein LDL, VLDL and HDL play important role in CHD. Accumulation of oxidized low-density lipoproteins in macrophages and smooth muscle cells causes foam cell formation - an initial step in atherosclerosis (Ozer et al.

1995). The results of the analysis of serum for LDL, VLDL and HDL are presented in Table 2. In the six-month study, the LDL values in all the groups were higher than the rats fed normal diet. This was expected as the gravimetric analysis for determining the lipid content of the normal diet showed that the amount of oil in normal diet (as extracted by hexane) was less when compared to the groups of rats supplied with the single oil or the blend. To the blend fed as well as the single oil fed rats, 10% oil mixed with fatfree diet was supplied. In the normal control rats (with usual diet), the LDL level was 12 mg/dl. In palm olein oil and mustard oil fed rats, the values were - respectively - 15, and 19 mg/dl. Significantly, the mustard oil based blends performed much better, and more interestingly, in the palm olein oil containing blend of mustard oil (MP blend), the value was lowest (9 mg/dl). In MPC fed blends, the values were 36 mg/dl, respectively. This increase of LDL in cholesterol-supplemented rats needs some explanation. In this group, cholesterol-supplementation resulted in lowering of the LDL value (Table 2). The hypolipidemic action of rice bran oil seems to be an obvious reason.

With regard to the values for VLDL, palm olein oil treated rats showed lowest VLDL levels (approximately, 15 mg/dl in each group). In single vegetable oil fed rats, VLDL was highest in mustard oil group (33 mg/dl).

Among blends, the MP blend also showed higher VLDL value (33 mg/dl). On the contrary, the value in MPC blend was 20 mg/dl. As shown above, palm olein oil group had the lowest LDL as well as VLDL (15 mg/dl). Thus, the MN present in palm olein oil might have contributed towards the low value of VLDL in the MPC group. HDL-cholesterol is an important scavenger of surplus cholesterol transporting it from the cell membrane to the liver where it is metabolized or converted into the bile acids. In mustard oil and palm olein oil alone treated rats, the values were 32 and 33 mg/dl respectively. In all groups in this study (except for the cholesterol loaded groups), the HDL levels were higher than the value reported from the group fed control diet (29 mg/dl). As expected, in the group of rats supplied with extra cholesterol (1% mixed in the blend), the HDL values were lower in comparison to the increased values observed following the non-cholesterol blend feeding. Respectively, the values were 26 mg/dl for MPC. The obvious reason for this is the increased cholesterol load.

Briefly, six-month study results suggest that in comparison to the control values. On the other hand, the MP blend resulted in a decrease in HDL and did not affect the cholesterol level. MP blends also resulted in increase in triglycerides. In cholesterol-supplemented groups, the three blends could lower the total cholesterol, but had no beneficial effects on HDL. Triglycerides decreased in MPC rats.

Body weight The effect of feeding fat on body weight is well established. Results presented in Table 2 depict the effect of a six-month feeding study of various oils (mixed with diet) on the animal body weight. The increase in the body weight of rats fed palm olein oil and mustard oil alone was 80 and 59 mg/dl, respectively, after six months. In MP it was 79 mg/dl. In ‘C‘ supplemented groups viz. MPC, the increase in body weight was also least. In MPC it was 66 mg/dl. Presence of mustard oil in the blend appears to have a beneficial effect on health with regard to the body weight increase. This has implication in patients with life-style diseases - diabetes and cardiovascular disease.

Table 2. The effect of long-term feeding of the MN rich blend on serum cholesterol and triglyceride levels in rats and the animal body weight.

Serum(mg/dl) Lipoprotein (mg/kg) Body weight(g) Cholesterol Triglyceride LDL VLDL HDL IBW FBW Weight gain Control 12.0 ± 4.50 24.9 ± 4.27 29.0 ± 10.1 n.a n.a n.a 54.17 ± 19.00 124.6 ± 21.3 MO 19.2 ± 10.7 33.3 ± 10.2 32.0 ± 06.0 265.0 420.0 155.0 (59%) 48.68 ± 06.70 166.4 ± 14.1 PO 14.8 ± 9.70 15.2 ± 5.2 33.6 ± 9.12 275.8 495.2 219.4 (80%) 42.25 ± 03.20 75.7 ± 30.23 MP 8.71 ± 5.00 33.4 ± 5.2 23.5 ± 4.35 278.3 498.2 219.9 (79%) 52.50 ± 10.80 166.6 ± 51.30 22.2 ± 5.30 19.7 ± 5.3 25.78 ± 9.6 305.8 510 204.2 (66%) MPC 46.40 ± 13.50 98.2 ± 38.20 Note: 1.5 mL of oil was administered to each rat, which corresponds to 10% oil in diet. ‘C’ represents the blend containing 1% cholesterol. Male rats were used in this study for six months. The values in bracket indicate percent increase. IBW: Initial body weight, FBW: Final body weight.

Oil

KHAN et al. â&#x20AC;&#x201C; Serum lipid profile and retinol after vegetable oil treatment

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Serum cholesterol and triglyceride The results of one-month study are presented below.

These data do not truly reflect the picture produced by the six-month study protocol; although in this study also the level of serum cholesterol was reduced following palm olein oil feeding, the blend fed rats reported values that do not show much variation. Serum cholesterol could be reduced to a significantly low level (Table 3). In fact, a study in normocholesterolemic human volunteers (Sanders and Reddy 1992) has shown that consumption of realistic amounts of rice bran oil does not contribute to a reduction in plasma cholesterol concentration in men consuming the oil for three-weeks.

Feeding mustard oil for one month could slightly decrease serum total cholesterol (79 mg/dl versus 74 mg/dl). In MP rats, the value was 77 mg/dl that increased to 88 mg/dl in MPC rats (Table 3). The possible reason for the discrepancy could be the short duration of treatment that did not allow the physiological system to adjust to the new dietary habit that ensured that 10% oil blend must be supplied in diet.

The triglyceride levels measured in rats fed blend for one month were found to be lowest in palm olein oil rats.

In MO rats the value was comparable to the control, which was not the case in the six-month study. In six-month study, lowest triglyceride values were observed in the MPC blends (Table 3). In one-month study, the values in these groups were also less than the value in mustard oil group (214 mg/dl) but only marginally. In case of triglyceride levels, the cholesterol-supplemented rats reported lesser values when compared to the animals fed with the blend without cholesterol (Table 3). For MPC group, the values for triglycerides were 209 mg/dl. In MP groups, triglyceride values were 284 mg/dl, respectively. The results suggest that one-month feeding of blends could bring down the level of triglycerides in animals supplemented with cholesterol, which has nutritional and health implications as described above.

In mustard oil and palm olein oil alone rats, the values were almost similar (35 mg/dl). Among the blends, mustard based blend showed significant rise in HDL in one-month study; here, the values were 64 mg/dl for MP, respectively.

In rats fed blend mixed with 1% cholesterol for one month, HDL values were 32 mg/dl, respectively, for MPC. Comparison of one-month and six-month study data on serum lipid profile showed that while in one-month study HDL decreased in palm olein oil alone and mustard oil alone rats, in six-month study, HDL increased in all groups (palm olein and mustard oil fed groups). Cholesterol did not change in mustard oil fed rats; it decreased in rats given palm olein oil for one-month. Triglyceride was low in all groups (mustard oil and palm olein oil) after one-month treatment, and remained low in palm olein oil rats following six-month treatment; it increased in mustard oil fed rats. In six-month study, cholesterol, however, decreased in all groups receiving single oil. However, after six-month MP caused a slight decline in HDL. MP groups after one month, but after six months, the level was unaffected in MP. In six-month study, triglyceride increased in MP. Triglyceride also in one month feeding was unchanged in MP. However, in six-month feeding study, cholesterol decreased in this group; triglyceride remained unchanged. In MPC group of rats, HDL decreased after one-month period and cholesterol increased. After sixmonth, HDL in this group was normal (with respect to control) and cholesterol reduced. Triglyceride decreased in MPC following six-month feeding. However in rats receiving cholesterol-supplemented blends in diet for one month, triglyceride was low in all. All values discussed here are in comparison to the control rats fed normal diet in which no oil was mixed. The results suggest superiority of the blends over single edible vegetable oils. An important point to be noted while comparing the values of similar parameters in one-month and six-month study experiment is the difference in actual values for the same parameter.

These differences were due to the kits that were supplied by different manufacturers. The two studies were performed on different times.

Lipoprotein With respect to the level of VLDL in one-month study, the highest level was observed for the MP blend (Table 3).

For palm olein oil it was 22 mg/dl; the control value for VLDL was 48 mg/dl. Among all the blends, the VLDL value was lowest in MPC blend. In MPC rats, VLDL levels were 42 mg/dl, respectively. In MP rats, these values were higher and were reported to be 45 mg/dl (respectively).

VLDL synthesis has been associated with LDL. A depressed VLDL synthesis is concluded to be responsible for the reduction of LDL synthesis (Lewis et al. 1961). The data were consistent with others. In the blend fed rats (except for the MPC rats), the LDL was around 41 mg/dl.

This is interesting because in cholesterol supplemented rats, VLDL values were more in comparison to the MP groups. In case of LDL, the values in different groups were quite similar (with the exception of MPC). In MPC, depressed VLDL could easily be associated with decreased LDL.

Body weight The results depicting the change in the body weight of rats fed the oil and the blends are presented in Table 3.

When compared to the increase in body weight in rats fed mustard oil alone mixed in fat free diet (50 mg/dl). Among the groups exhibiting highest increase in body weights following oil administration in diet for one month were MPC group (67 mg/dl) and the palm olein oil group (61 mg/dl. In other blend fed rats the values were comparable among themselves. Overall, like the results displayed for the six-month study.

As described earlier in this text, the blend of palm olein oil and mustard oil, palm olein oil have been found effective in influencing the serum lipid profile of rats fed the oil mixed in diet. The blend also appeared quite effective in bringing down the serum lipid profile values when the rats were loaded with cholesterol (1% in the blend) in a one-month or a six-month long feeding study. It not only consistently bring down the cholesterol,

Short-term feeding

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Table 3. The effect of short-term feeding of the MN rich blend on serum cholesterol and triglyceride levels in rats and the animal body weight.

Serum(mg/dl) Lipoprotein (mg/kg) Body weight (g) Cholesterol Triglyceride LDL VLDL HDL IBW FBW Weight gain Control 78.93 ± 17.24 238.1 ± 40.00 23.87 ± 8.3 47.63 ± 7.9 38.54 ± 15.9 n.a n.a n.a MO 74.00 ± 11.60 214.0 ± 37.50 22.14 ± 3.7 42.80 ± 7.5 35.75 ± 11.9 236.2 354.0 117.8 (50%) PO 60.89 ± 19.22 29.0 ± 33.36 22.37 ± 4.6 45.80 ± 2.3 34.62 ± 07.0 216.3 348.7 132.4 (61%) MP 77.01 ± 13.37 225.9 ± 44.56 42.50 ± 9.2 45.18 ± 2.4 64.37 ± 15.83 268.0 395.0 115.0 (47%) MPC 88.21 ± 06.18 209.2 ± 43.80 23.87 ± 8.3 41.90 ± 4.1 32.08 ± 06.32 305.8 510.0 204.2 (67%) Note: 1.5 mL of oil was administered to each rat, which corresponds to 10% oil in diet. ‘C’ represents the blend containing 1% cholesterol. Male rats were used in this study for one month. The values in bracket indicate percent increase. IBW: Initial body weight, FBW: Final body weight.

Oil

triglycerides and other biochemical changes, but also found to be the best edible oil blend with respect to the increase in body weight in both long-term as well as short term studies.

The blends capable of lowering the cholesterol and triglycerides have health benefits. Diet influences the risk factors for CHD, which include the elevated levels of serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), and serum triglycerides and reduced levels of high-density lipoprotein cholesterol (HDL-C). The fat content in diet readily modulates these risk factors. A high intake of saturated fatty acids and cholesterol in diet may lead to hypercholesterolemia, largely through an increase in LDL-C. On the other hand, polyunsaturated fatty acids have a hypocholesterolemic effect in human. A large number of studies contributing to the polyunsaturated hypothesis have used diets that provide dietary fat calories derived almost entirely from the polyunsaturated fatty acids. Contribution of energy from saturated fatty acids was hence almost negligible. This created an artificial dietary environment that is seldom applicable in daily life.

Although the hypocholesterolemic effect of the polyunsaturated can often be observed in a high fat diet, its effects are less clear in a low-fat diet. The monounsaturated, long regarded as neutral in its effect on serum and lipoprotein cholesterol, can also lower cholesterol.

Beside this, as described elsewhere, MN such as oryzanol, tocotrienols etc exert direct effects on cholesterol synthesis.

Presence of these micronutrients in blends can account for their beneficial effects on serum lipid profile (Amos 2007).

Effect of blend feeding on serum retinol Retinol is an important lipid soluble micronutrient in animals that can be measured in serum. The molecule is a form of vitamin A that is formed in the body using βcarotene as a precursor. Its level in the blood is dependent on the type of food consumed. Crude palmolein oil is one of the richest natural sources of β-carotene and is cheaper than the conventional edible vegetable oils in India, although most of the carotenoids are lost during refining.

Nutritional evaluation of crude palm oil has been done in rats (Manorama and Rukmini 1991), and no adverse effects could be observed as judged by the growth rate, feed— efficiency ratio, protein—efficiency ratio, net protein utilization, digestibility, fat absorption, nitrogen balance, phosphorous and calcium retention, serum enzymes and

haematology. These parameters were comparable to the control values. Palm oil feeding could cause an increase in serum retinol that has been well documented. In this study, we have tested serum retinol in rats that were fed the three binary blends prepared using mustard, palm or rice bran oil in specific proportions. Table 4 depicts the results of a study on serum retinol estimated in rats receiving the oil at a dose level of 1.56 g per day. Measurement of serum retinol after one month feeding revealed higher levels in all groups in comparison to the control rats (30 μg/dl). While in palm olein oil, it was 45 μg/dl. Lower value in palm olein oil rats could be due to the use of refined palm oil; losses of β-carotene due to refining process have been reported. This statement is supported from the β-carotene level in palm olein that has been discussed in earlier reports on the MN level in various oils. Unpredictably the values in palm oil rats were similar to the mustard oil fed rats (44.46 and 45.50 μg/dl) following one-month feeding.

When the blends were compared, MP reported the highest increase (46 μg/dl); in MPC also, the value was 46 μg/dl.

Overall, one-month study results suggest that MP or MPC rats had the highest retinol level in all blends, and the blend was found to be effective in both normo- and hypercholesterolemic rats. Cholesterol seems to have no effect on serum retinol level in blend fed animals.

Table 4: Serum retinol level in rats receiving oil blend in diet for various duration of time.

Group

30 days

180 days

240 days

Control 30.30 ± 5.62 37.00 ± 5.68 37.19 ± 6.63 MO 44.46 ± 4.88 42.73 ± 6.16 48.28 ± 8.82 PO 45.50 ± 10.4 37.17 ± 6.70 37.17 ± 6.60 MP 45.89 ± 16.9 43.81 ± 2.07 43.81 ± 2.07 MPC 46.10 ± 8.05 47.98 ± 9.72 47.98 ± 9.72 Note: Values are expressed as mg/dl (Mean ± S.D.). The oil was fed to rats at a dose level of 1.56 g (1.75 mL) per animal per day that is equivalent to 10% daily oil requirement. The group denoted by superscript C has 1% cholesterol dissolved in oil.

Following six-month feeding study, retinol level increased in control rats from 30 μg/dl to 37 μg/dl. This value sustained when measured following 8 months feeding study. In palm olein oil group, retinol level was brought down to 37 μg/dl from 45 μg/dl reported in one-

KHAN et al. – Serum lipid profile and retinol after vegetable oil treatment

month study. Of particular interest is the fact that after sixmonths, retinol in control and palm olein fed rats was exactly similar. A simple but valid explanation can be the loss of β-carotene in the palm olein rats. It should be noted that the same batch of palm olein was used throughout this study. The higher retinol level observed in 30 days study could be due to higher levels of β-carotene in fresh oil; βcarotene deteriorated on subsequent storage. Interestingly, in mustard fed animals the level reported in one-month study were maintained for six-months, and following 240 days (8 months) feeding, slight increase in retinol could be found. In MP, the value decreased slightly. In cholesterolsupplemented rats, initial values were maintained till the end of the study, and cholesterol does not seem to affect the serum retinol level in blend fed rats.

CONCLUSION The present study demonstrated the substantial hypolipidemic action of PO blends which may be due to their minor components. These minor components appear to play a major role in reducing the level of circulating lipids in rats. They appear to lower TC, LDLC, TG levels.

The cholesterol lowering effect of PO has been attributed to tocotrienols and other components of the unsaponifiable matter. Tocotrienols inhibit endogenous synthesis of cholesterol through the HMGCOA reductase. It can be suggested that PO blends should be considered for edible purposes in spite of the high unsaponifiable matter, which may confer some benefit. Comparison of two blends among themselves revealed a maximum increase in MP rats; in MPC also, the value was similar. Overall, onemonth study results suggest that MP or MPC rats had the highest retinol level in all blends, and the blend was effective in both normo- and hypercholesterolemic rats.The effect of feeding the individual blend in diet to rats and to measure serum lipid profile and serum micronutrient retinol.. The blends of PO with these oils not only bring about a favorable circulating lipid profile but may also result in an economic advantage of lower prices as PO is cheaper oil than SFO and SNO in the current retail market in India.

REFERENCES AHA Medical/Scientific statement. 1990. Circulation 81: 1721-1733.

Amos LM. 2007. Enzimes from yeast adjuncts in proteolysis during Cheddar cheese ripening. [Dissertation]. University of the Free State, Bloemfontein, South Africa.

115

Ausman LM, Rong N, Nicolosi RJ. 2005. Hypocholesterolemic effect of physically refined rice bran oil: Studies of cholesterol metabolism and early atherosclerosis in hypercholesterolemic hamsters. J Nutr Biochem 16: 521-529.

Elson CE, Qureshi AA. 1995. Coupling the cholesterol- and tumorsuppressive actions of palm oil to the impact of its minor constituents on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Prostaglandins Leukot Fatty Acids 52:205-207.

Grundy SM. 1975. Effects of PUFA on lipid metabolism in patients with hypertriglyceridimia. J Clin Invest 55: 249-256.

ICMR. 1990. Nutrient requirements and recommended dietary allowances for Indians. ICMR. New Delhi.

Kamat JP, Sarma HD, Devasagayam TP, Nesaretnam K, Basiron Y. 1997.

Tocotrienols from palm oil as effective inhibitors of protein oxidation and lipid peroxidation in rat liver microsomes. Mol Cell Biochem 170: 131-137.

Lasserre M, Mendy F, Spielmann D, Jacotot B. Effects of different dietary intake of essential fatty acids on C20:3 omega 6 and C20:4 omega 6 serum levels in human adults. 1985. Lipids 20: 227-233.

Lewis B, Pilkington TR, Hodd KA. 1961. A mechanism for the action of unsaturated fat in reducing cholesterol. Clinical Science (London) 20: 249-256.

Manorama R, Brahmam GN, Rukmini C. 1996. Red palm oil as a source of beta-carotene for combating vitamin A deficiency. Plant Foods Hum Nutr 49: 75-82.

Manorama R, Rukmini C. 1991. Nutritional evaluation of crude palm oil in rats. Am J Clin Nutr 53 (4 Suppl): 1031s-1033s.

Ozer NK, Boscoboinik D, Azzi A. 1995. New roles of low-density lipoproteins and vitamin E in the pathogenesis of atherosclerosis.

Biochem. Mol Biol Intl 35: 117-124.

Qureshi AA, Qureshi N, Hasler-Rapacz JO, Weber FE, Chaudhary V, Crenshaw TD, Gapor A, Ong AS, Chong YH, Peterson DM. et al 1991a. Dietary tocotrienols reduce the concentrations of plasma cholesterol, apolipoprotein B, thromboxane, B2, and platelet factor 4 in pigs with inherited hyperlipidemias. Am J Clin Nutr 53:1042S1046S. Qureshi AA, Qureshi N, Wright JJ, Shen Z, Kramer G, Gapor A, Chong YH, DeWitt G, Ong A, Peterson DM. et al. 1991b. Lowering of serum cholesterol in hypercholesterolemic humans by tocotrienols (palmvitee). Am J Clin Nutr 53 (4 Suppl): 1021S-1026S. Rukmini C, Raghuram TC. 1991. Nutritional and biochemical aspects of the hypolipidemic action of rice bran oil: a review. J Am Coll Nutr 10: 593-601.

Sanders TA, Reddy S. 1992. The influence of rice bran oil on plasma lipids and lipoproteins in human volunteers. Eur J Clin Nutr 46: 167172.

Seetharamaiah GS, Chandrasekhara N. 1989. Studies on hypocholesterolemic activity of rice bran oil. Atheroscelrosis 78: 219223.

Snedecor GW, Cochran WG. 1990. Statistical Methods. The Iowa State University Press. Ames, IA Spritz N, Mishkell C. 1965. Effect of fatty acid saturation on the distribution of plasma lipids and lipoproteins, a hypothesis for the lipid lowering effect of unsaturated fatty acids. J Clin Invest 48: 7884.

Suzuki S, Tezuka T, Oshima S, Kuga T, Mitani M. 1984. Oils and Fats (Japan) 37: 59.

Tomeo AC, Geller M, Watkins TR, Gapor A, Bierenbaum ML. 1995.

Antioxidant effects of Tocotrienols patients with hyperlipidemia and carotid stenosis. Lipids 30: 1179-1183.

Yung LC, Liang YW, Tsung CL, Lucy S H. 2010. Cholesterol-lowering effect of phytosterol-containing lactic-fermented milk powder in hamsters. Food Chem 119: 1121-1126.

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 116-120 November 2010

Expression of vimetin protein and neurofilamen on forelimb buds of black-6 mice on gestation day 12 induced by 2-methoxyethanol by RTPCR YULIA IRNIDAYANTIâ&#x2122;Ľ Department of Biology, Faculty of Mathematics and Natural Sciences, State University of Jakarta. Jl. Pemuda No. 10 Rawangun, Jakarta Timur 13220, Indonesia. Tel: +92-21-4894909. email: irnidayanti@yahoo.com Manuscript received: 31 Augustus 2010. Revision accepted: 16 October 2010.

Abstract. Irnidayanti Y. 2010. Expression of vimetin protein and neurofilamen on forelimb buds of black-6 mice on gestation day 12 induced by 2-methoxyethanol by Real Time RT-PCR. Nusantara Bioscience 2: 116-120. The aim of this study was to investigate impact of 2-methoxyethanol, a major industrial chemical of plastic. Gene expression analysis is increasingly important in biological research, while real-time reverse transcription PCR (RT-PCR) is becoming the method of choice for high-through put and accurate expression profiling of selected genes. Pregnant black-6 mice were injected intraperitoneally to 7.5 mmol/kg of 2- methoxyethanol on gestation day (GD) 10. Embryo were obtained on gestation day 12. Forelimb buds of embryo was collected and then put in the tube, which containing RNA-latter solution. To identify gene expression changes in forelimb bud caused induction 2-methoxyethanol, Real Time PCR were using in this research. For the experiments the real-time RT-PCR Light Cycler technology was used. The results suggested that injection of 2- methoxyethanol, in prenatal period especially on gestation day 12, the expression of vimentin in forelimb buds of mice treatment increase than control mice. Meanwhile the expression of neurofilament tended to decrease, indirectly is not caused by the injection of 2methoxyethanol.

Key words: vimentin, neurofilament, 2-methoxyethanol, limb bud, black-6 mice.

Abstrak. Irnidayanti Y. 2010. Ekspresi protein vimetin dan neurofilamen pada tunas anggota depan mencit black-6 umur kebuntingan 12 hari akibat induksi 2-metoksietanol secara Real Time RT-PCR. Nusantara Bioscience 2: 116-120. Tujuan penelitian ini adalah untuk mengetahui dampak dari 2-metoksietanol, bahan kimia utama industri plastik. Analisis ekspresi gen semakin penting dalam penelitian biologi, dimana real-time reverse transcription PCR (RT-PCR) menjadi metode yang dipilih untuk mendapatkan profil ekspresi dari seleksi gen secara akurat. Mencit black-6 bunting diinjeksi 2-metoksietanol secara intraperitoneal dengan 7,5 mmol/kg pada umur kebuntingan 10 hari. Embrio diperoleh pada umur kebuntingan 12 hari. Tunas anggota depan embrio dikumpulkan dan dimasukkan ke dalam tabung, yang mengandung larutan RNA-latter. Untuk mengetahui perubahan ekspresi gen tunas anggota depan yang disebabkan oleh induksi 2-metoksietanol digunakan RT-PCR dalam penelitian ini. Dalam percobaan digunakan teknologi RT-PCR Light Cycler.

Hasil penelitian menunjukkan bahwa injeksi 2- metoksietanol, pada periode pralahir terutama pada umur kebuntingan 12 hari, ekspresi vimentin pada tunas anggota depan mencit perlakuan meningkatkan dibandingkan kontrol. Sementara itu ekspresi neurofilamen cenderung menurun, secara tidak langsung tidak disebabkan oleh injeksi 2- metoksietanol.

Kata kunci: vimentin, neurofilamen, 2-metoksietanol, limb bud, mencit black-6.

INTRODUCTION The compound of 2-methoxyethanol (2-ME) or ethyleneglycol methyl ether is one of the glycol ether compounds derived from the compounds of phthalate esters. This compound is widely used as the basic material of plastic. Plastic is very useful in everyday life. People use plastics in their everyday life. Plastic is generally used for a variety of human activities, such as household appliances, packing materials, bottles, food containers, toys, water pipes and even used for the purposes of health such as blood storage for transfusion.

The waste of the compounds is often wasted in the environment and being the cause of pollutants, particularly

in the aquatic environment or the river (Miller et al. 1983).

The compounds were known to be toxic or teratogenic in several mammalian species (Feuston et al. 1990). Some previous reports also mentioned that, some people have been poisoned with 2-ME through penetration into the skin and bronchial tube (Dugard et al. 1984). Approximately 100,000 people were poisoned by 2-ME per year, of which allegedly were women who were still in the fertile period or able to give birth (Scott et al. 1989). The teratogenic of 2-ME in experment animal is caused by the metabolism of 2-ME in the hepatic cells transforming into methoxyacetic acid (MAA), by the help of a catalyst namely alcohol dehydrogenase (Brown et al. 1984; Moslen et al. 1995).

Our previous studies mentioned that, 2-ME causes abnormalities in fetal mice whose dams was given 2-ME or

IRNIDAYANTI â&#x20AC;&#x201C; Expression of vimetin protein and neurofilamen

MAA, whose main disorders that appear were abnormalities the skeletons, the disorder on the axial skeleton, due to the damage of embryonic somit tissues which in turn led disorders on the spines and ribs, spina bifida; and exencephaly (Darmanto et al. 1994; Darmanto 1998). Some researchers have reported that after giving MAA with a single dose of 10 mmol/kg body weight on gestation day 11 of in mice JCL: ICR (Rasjad et al. 1991), AJ (Sudarwati 1993), Swiss Webster, (Suripto et al. 1996) , showed that 94% of limb bud of the fetus experiment was abnormalities.

The nature of both Embryo toxic and teratogenic MAA compound has been shown in mammals, especially in embryos both pre and post implantation periods. This embryo toxic characteristic is similar to that caused by the compound 2-ME (Darmanto et al. 1994).

Limb bud is a good model to study the pattern of certain growth and also to understand the possibility of developmental disorders caused by specific teratogens (Ruyani et al. 2008). The results of the research conducted

by Rasjad et al (1991), shows that the distribution pattern of defects caused by MAA, a metabolite of 2methoxyethanol, is caused by the difference in the number and distribution of dead cells that occurs in the mesoderm of the limb plate. The result of microscopic observations showed that signs of limb bud abnormality that experienced necrosis of mesenchymal cells and the AER (apical ectoderm Ridge), which was observed for 2 hours after being given MAA at gestation days to 10.5, then after 6 hours showed hyperplasia on AER. AER itself plays a role in the formation mechanism of the disorder, because there is a degenerative change in structure and a more rapid depreciation in the fetus that was being treated compared than control (Sudarwati, 1995). The results of the research

by Mebus and Welsch (1989) that MAA treatment may interfere with the availability of purine and pyrimidine bases, which are expected to affect DNA and/or RNA synthesis, which in turn influence normal cellular proliferation and differentiation in the developing mouse embryos In the process of formation of a normal limb bud, there are several phases: proliferation, migration, differentiation and cell death phase. When the embryo is gestation days 10, it is an early stage of initial bud formation, in which cells undergo proliferation by forming AER. In addition, there is also migration of miogynic cells on the buds. The muscular structure of the buds is partly from the miotom somites. The migration of miogynic cells begins soon after the formation of buds. The bud cells have the ability to aggregate forming two entities of muscles namely dorsal muscles and premuscular muscles, which are formed at the early stages of miogynic differentiation (Ewan and Everett, 1997). Miogenic cells accumulated in the buds expressed vimentin proteins (Hayasi et al. 1993).

The Results from Vaittinen et al. (2010), indicate that vimentin proteins play roles in miogenesis, both functional roles in the construction and restoration of skeletal muscle fibers. The presence of vimentin proteins related with cellular function of cells during embryonic development.

So we can say that vimentin is expressed in the in early

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phase of organogenesis, which is characterized by the aggregation and condensation of prekartilago (Viebahn et al. 1988). Vimentin role tansports kinase into the cell nucleus. The kinase activates the neurons and affect gene expression in such a way that neurons can respond to the damage. This proves that vimentin is required in the repair of scar tissue through the migration of cells and tissue formation (Moon et al. 2004).

The expansion of those aggregated cells will stop when it reaches the form of condensation of pre-cartilages. The form of pre-cartilages condensation will trigger cartilage differentiation, which eventually forms cartilage (Lee et al.

1998). This development process includes the activities of cells namely cell migration, cell adhesion, intracellular signaling and cell proliferation. The process of proliferation and migration of these miogenic cells are influenced by fibronectin. This protein helps facilitate the migration of miogenic cells, as a place of attachment and the guidance of the migration.

Neurofilament are a group of proteins that guide cells during the organogenesis process, if the expression level of these compounds are disturbed by the compound 2-ME, it is assumed that there will be abnormalities of limb bud.

However, the abnormality that occurs is in the form of polydactyl, caused by dead cell, which is not the only mechanism that causes the appearance of disorder. Another research states that a failure in the functioning of the longitudinal and circular smooth muscle in cases of stenosis pyrolus, is known associated with the unexpressed ncam and neurofilamen or indicating the absence of innervations on the smooth muscle tissues (Kobayashi et al.

1995). The development of the embryo limb buds would be disrupted, with a decrease of the expression neurofilamen protein (Nicholas et al. 2003). Based on these results there might be any of a certain kind of disturbed protein expression that was induced by 2-ME compound. In this study, it is invertigated the expressions of these proteins induced by 2-ME compound in the critical period of the development of the buds, in real time RT-PCR. It is expected to know the proteins that are expressed during the formation of bud members and it is also expected to reveal the role of proteins in causing abnormalities limb buds.

MATERIALS AND METHODS Experimented animals Black-6 mice were used in this study were brought from Charite-Universutats Medizin Berlin. vaginal plug detected the following morning was defined as day 0 of gestation (Rugh, 1968). The pregnant mice were killed by cervical dislocation at gestation days 10 (GD-10). The sample of the buds members were isolated under the microscope, and then put into the tube containing the RNA-latter, for the analysis of RNA and DNA. Test RNA was conducted with RNeasy Kits Real Time RT-PCR. Materials, dosage and samples collection 2-ME in liquid form (Product Number: 135â&#x20AC;&#x201C;07762) was produced by Wako Pure Chemical Industries, Ltd. Japan.

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2-ME diluted with sterilized distilled water was administered by peritoneally injection at a dose of 7.5 mmol/kg body weight on GD-10. Control mice were given sterile distilled water with the same dose. On the GD-12, the mice were killed by cervical dislocation, and then the fore limb buds were isolated and put in tube with the latter RNA solution, then stored for further analysis at a temperature of-200C.

complete series of targetted cDNA, followed by Oligonucleotide primers. The Primers used in this study were synthesized into Biotez Berlin-Buch GmbH, Berlin, Germany. Information on primers can be seen in Table 1.

Table 1. Sequencing orimer position (f = forward; r = reverse), % contens of G/C dan referency primer product Primer

Real Time RT-PCR The total RNA forelimb buds tissue was extracted with the RNeasy kit according to the manufacture’s protocols. cDNA was synthesized from the total RNA using the Qiagen One Step RT-PCR Kit (Cat. No. 210 210). PCR reactions using enzymes AidTM H Minus M-MuLV RT (Cat. No. 130 125 486) at a temperature of 95oC, 7 min, 45 cycles of PCR (20 sec, 95oC, 60oC, 20 sec, 72oC, 30 sec), 42oC, during 1 hour 15 minutes, 70°C elongation then followed with the temperature of 70oC, for 5 minutes.

Quantitative analysis performed by Real-Time PCR. Analysis of Polymerase chain reaction (PCR) was done by adding each cDNA 9:00 µl of treatment or control forelimb buds and 1 µl of Primary-Mix into each different tube. In our experiments, Primary-Mix consists of 4 primary types of vimentin and NFh, Nfm, and the NFl. In buds of both the control and treated, then was added by the component of reactions that was aqua, SYBER Green, 2x Bioline buffer. Then reaction of Real Time RT-PCR showed the

Vim-f Vim-r Nef h-f Nef h-r Nef m-f Nef m-r Nef l-f Nef l-r

Sequence (5´3`) CTG AGG CTG CCA ACC GGA ACA A CCT CGC CTT CCA GCA GCT TCC AGG AGA TAA CTG AGT ACC GGC G CCA AAG CCA ATC CGA CAC TCT TC GTG GTT CAA ATG CCG CTA CGC C GAG GCC CGG TGA TGC TTC CTG TGG CCT TGG ACA TCG AGA TTG CA GCT TCT CCT TCA GAG GGG GGC

GC (%) 59,1

011701.3:1376F22

66,7

011701.3:1682R21

54,5

010904.3:1071F22

52,2

010904.3:1349R23

59,1

008691.2:1055F22

66,,7

008691.2:1432R21

52,2

010910:1221F23

66,7

010910:1489R21

TIB reference no.

Table 2. DNA cycle conditions forelimb buds proteins UK-12-day embryo induced by 2-ME in real-time reverse transcriptionpolymerase chain reaction (RT-PCR).

IRNIDAYANTI â&#x20AC;&#x201C; Expression of vimetin protein and neurofilamen

In Table 2, it is shown that the number of copies of genes for vimentin expression is very high compared with vimentin protein gene in the control group. The number of the copies of genes is around 272,989, relatively higher compared than control that is 37,127. While the number of the copies of neurofilamen low protein in the treatment group was lower than the control group, that is 38,269 for the group of treatment and 47,927 for that of control. While the medium and high neurofilament are very low in the treatment group that are 9416 and 849, while in the control are 2327 and 200.

RESULTS AND DISCUSSION Results of DNA copies by the real time RT-PCR is from the mice black -6 using the primers of vimentin and high, medium and low neurofilamen low (figure 1). In the picture above, it is shown that the expression of vimentin in the early development of limb buds is very high. Other proteins such as nf-low look the opposite that is relatively decrease, when compared with the control group.

Figure 1. The number of copies of genes for DNA protein embryonic forelimb buds on GD-12, the results of real time RTPCR between forelimb buds of the control and treated with 2ME.

Discussion The limb buds firstly appear on the 10th day of gestation, which seem a group of mesenchyme cells that grow like forelimb bud. Mesenchyme cells that cover the primordium limb buds will form the cube-shaped cells derived from ectoderm layer. This form is known as the apical ectoderm Ridge (AER).On the 12th gestation day, the limb buds turn themselves in the shape of polygonal or rather pentagonal. The role of proteins in the development of limb buds, can directly work on the cells or through an intermediary by changing the configuration of the substrate. In the process of formation of a normal limb bud, there are several phases: proliferation, migration, differentiation and cell death phase. In embryo on the 10th days gestation, is the early stage of limb buds formation, in which cells undergo proliferation by forming AER. In

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addition, there is also a migration of miogenyc cells in the limb buds. Miogenyc cells accumulated in the linb buds, express vimentin proteins (Hayasi et al. 1993).

From the results of this study it is shown that vimentin protein expression in treatment group increased compared to the control one. Under normal circumstances, the expression of vimentin protein in embryonic period on GD10 is very high (Irnidayanti, 2009), then gets decreased on the GD-12. But in this study, after injected with 2-ME, the expression of vimentin protein appears the opposite, that it gets increased on GD-12. These data are supported by Vaittinen et al (2001), that the expression of vimentin increases maximum for 3-5 days after the post injury on the mioblas. This means that after being given with 2-ME, there occurs damage on limb buds tissue. In the damaged tissue, it can be seen the raising of the expression of vimentin protein. The high expression of vimentin protein is needed to repair the damaged tissue induced by 2-ME. Based on the research by (Viebahn, 1988) showed that the existence of protein expression appear to be associated with cellular functions during embryonic development.

Vimentin, which is an intermediate filament, acts as transduction signal (Helfand et al. 2005). Vimentin can interact with the MAP (mitogen-activated protein) kinase, which is found on the tip of the injured axon. Vimentin plays role in transporting the kinase into the body and toward nucleus of cells. The Kinase activates and affects the expression of the neuron gene in such a way that the neurons can respond to the damage. Moon et al (2004) stated that vimentin is a lot of expressed in damaged tissue.

Therefore it is suggested that the high expression of vimentin protein in embryo on GD-12 is related with the process of proliferation and migration of cells needed for repairing thelimb buds tissue, as a result of being given with 2-ME. In this study, the giving of 2-ME was done on the 10th days gestation and the observations were made on the 12th. 48 hours after being given with the 2-ME, which is not the end of the recovery effort, it leads to a very high expression of vimentin and it is indispensable to the process of proliferation in repairing the tissues. The results of the Ruyaniâ&#x20AC;&#x2122;s research (2008) states that after giving 2ME in Swiss Webster mice on the 10th day with a dose of 10 mmol/kg body weight, the protein profile of the forelimb buds changed. The metabolite result of 2-ME, the MAA, directly effects on gene expression which then also affects the existence of certain proteins. Thus the high expression of vimentin protein is required for tissue repairing due to being given with 2-ME and are associated with functions of the cells during the development stage The expression of Neurofilamen-low protein in the treatment group, seemed lower compared to that in the control group. The expression of the three sub-units of neurofilamen varied, both among the population of neurons and the axons in the development stage. The expressions of the three subunits are associated with various functions of the cells during the development stage (Viebhan, 1988). As it is already known that, neurofilmen plays role in the morphogenesis process of the neurons (Matus, 1988; Robinson and Anderton, 1988; Riederer, 1990; Riederer, 1992), mainly to maintain the rigidity of the cell. Beside

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that it also serves to guide the intracellular transport into the axons and dendrites. Together with other cytoskeleton proteins, neurofilamen functions role to establish and maintain the cells’ shape and to facilitate the transportation of particles and organelles within cytoplasm (Liu et al.

2004). Given the nerve supply and muscle into the limb buds began to occur in the embryo on the 13th day gestation, the expression of neurofilamen-low protein also appears lower in the control group. From the data obtained, it is shown that the expression of neurofilamen-low protein got decreased after being given with 2-ME on the 10th day of pregnancy , that was apparently not caused by disruption of RNA and DNA synthesis by 2-ME. The low expression neurofilamen-low protein was not caused by the compound 2-ME, considering the fact that the innervation of nerves occured on the 13th day of pregnancy. While neurofilamen medium and neurofilamen-high protein began to appear expressed, which tended to increase compared to that of the control group.

CONCLUSION A single dose of 2-methoxyethanol 7.5 mmol/kg body weight given intraperitoneally on gestation day 10, causes an increase in vimentin protein expression. And the decrease expression in neurofilamen protein in the forelimb buds of mice embryo is not caused by 2-methoxyethanol.

ACKNOWLEDGEMENT This research was funded by the Sandwich Like Program of the Directorate General of Higher Education, 2008. To that end, the authors thank.

REFERENCES Darmanto W. 1998. Effect of 2-methoxyethanol to somite formation and axial bone abnormalities in mice. Proceeding of 7th Scientific Meeting.Hiroshima. Japan. [Indonesia] Darmanto W, Sudarwati S, Sutasurya LA. 1994. Effects of methoxyacetic acid on prenatal development of mice. Environ Med 38: 25-28.

Dugard PH, Walker M, Mawdsley SJ, Scott RC. 1984. Abssoption of some glycol ethers through human skin in vitro. Environ Health Perspect 57: 193-197.

Ewan KBR, Everett AW. 1997. Migration of myogenic cells in the developing limb. Basic Appl Myol 7 (2): 131-135.

Feuston MH, Kerstetter SL, Wilson PD. 1990. Teratogenicity of 2methoxyethanol applied as a single dermal dose to rats. Fundam Appl Toxicol 15: 448-456.

Hayashi K, Hagiwara Y, Ozawa E. 1993. Vimentin expression pattern is different between the flank region and limb regions of somatopleural mesoderm in the chicken embryo. Develop Growth Differ 35: 301309.

Helfand BT, Chou YH, Shumaker DK, Goldman RD. 2005. Intermediate filament proteins participate in signal transduction. Trends Cell Biol 15 (11): 568-570.

Irnidayanti Y. 2009. Comparison of the expression of cDNA extra celular matrix protein between brain E-10 days black-6 mice, hLN-405, rF98 and cell line mHT-22 [Research Sandwich Program]. Humbold University. Berlin.

Kobayashi H, O’Brain DS, Puri P. 1995. Immunochemical characterization of neural cell adhesion molecule (NCAM), nitric oxide syntethase, and neurofilament protein expression in pyrolic muscle of patients with pyrolic stenosis. J Pediatr Gastroenterol Nutr 20 (3): 319-325.

Lee YS, Stott NS, Jiang RB, Widelitz, Chuong CM. 1998. Early events during precartilago condensation in limb bud micromass culture.

Cells and Materials 8: 19-32.

Liu Q, Xieb F, Siedlak SL, Nunomurac A, Hondaa K, Moreiraa PI, Zhua X, Smitha M.A. Perrya G. 2004. Neurofilament proteins in neurodegenerative diseases. Rev Cell Mol Life Sci 61: 3057-3075.

Mebus CA, Welsch F, 1989. The possible role of one-carbon moieties in 2-methoxyethanol and 2-methoxyacetic acid induced development toxicity. Toxicol Appl Pharmaco 99: 98-109.

Miller RR, Hermann EA, Langvardt PW, McKenna J, Schwetz, BA. 1983.

Comparative Metabolism and Disposition of Ethylene Glycol Monomethyl Ether and Propylen Glycol Monomethyl Ether in Male Rats. Toxic and App Pharmac 67: 229-237.

Moon C, Ahn M, Kim S, Jin JK, Sim KB, Kim HM, Lee MY, Shin T. 2004. Temporal patterns of the embryonic intermediate filaments nestin and vimentin expression in the cerebral cortex of adult rats after cryoinjury. Brain Res 1028: 238-242 Moslen MT, Kaphalia L, Balasubramanian H, Yin YM, William WA. 1995. Species differences in testicular and hepatic biotransformation of 2-methoxyethanol. Toxicol 96: 217-224.

Nicolas MA, Cai L, Brown DD. 2004. Thyroid hormone controls the developments between the spinal cord and limbs during Xenopus laevis metamorphosis. Proc Nat Acad Sci USA 101 (1): 165-170.

Rasjad C, Yamashita K, Datu AR Yasuda M. 1991. Pathogenesis of limb malformation in mice induced by methoxyacetic acid. Hiroshima J Med Sci 40(3):101-107.

Riederer BM. 1992. Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development. Histochem J 24: 783-790.

Rugh R.. 1968. The mouse: its reproduction and development. Burgess.

Minneapolis.

Ruyani A, Sudarwati S, Sutasurya LS, Sumarsono SH. 2008. Changes in protein profile of the front limb buds of mice (Mus musculus) Swiss Webster treated with methoxyacetic acid (MAA). Bandung Institute of Technology. Bandung. [Indonesia] Scott WJ, Fradkin R, Wittfoht W, Nau H. 1989. Teratologic potential of 2metoxyethanol and transplacental distribution of its metabolite, 2methoxyacetic acid, in non-human primates. Teratology 39: 363-373.

Sudarwati S, Surjono TW, Yusuf AT. 1993. Effect "methoxyacetic acid" (MAA) to the development of mice extremity (Mus musculus) strains A/J. J Matematika & Sains 1: 11-19. [Indonesia] Sudarwati S, Surjono TW, Yusuf AT. 1993. Abnormalities of the early development of the front extremity of mice A/J strain induced by methoxyacetic acid (MAA). J Matematika & Sains, Suplement H: 6070. [Indonesia] Suripto S, Surjono TW, Kamal A. 1996. Influence of methoxyacetic acid on DNA content of embryonic limb mice (Mus musculus) Swiss Webster. [Research Report]. Bandung Institute of Technology.

Bandung. [Indonesia] Vaittinen S. Lukka R, Sahlgren C, Hurme T, Rantanen J, Lendahl U, Eriksson JE, Kalimo H. 2001. The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle. J Neuropathol Experiment Neurol 60 (6): 588-597.

Viebahn C, Lane EB, Ramaekers FCS. 1988. Keratin and vimentin in early organogenesis of the rabbit embryo. Cell Tissue Res 253: 553-562.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 121-125 November 2010

Induced mutations by gamma ray irradiation to Argomulyo soybean (Glycine max) variety 1

DIANA SOFIA HANAFIAH1,♥, TRIKOESOEMANINGTYAS2, SUDIRMAN YAHYA2, DESTA WIRNAS2 Departement of Agriculture, North Sumatra University (USU). Jl. Nazir Alwi No. 4 Kampus USU Medan 20154, North Sumatra, Indonesia; Tel. +6261-8215170, Fax. +62-61-8201920; ♥email: dedek.hanafiah@yahoo.co.id 2 Departement of Agronomy and Horticulture, Bogor Agricultural University (IPB), Kampus IPB Darmaga, Bogor 16680, West Java, Indonesia.

Manuscript received: 3 June 2010. Revision accepted: 15 July 2010.

Abstract. Hanafiah DS, Trikoesoemaningtyas, Yahya S, Wirnas D. 2010. Induced mutations by gamma ray irradiation to Argomulyo soybean (Glycine max) variety. Nusantara Bioscience 2: 121-125. Induced mutation by gamma ray irradiation is one way to increase genetic variability of plants. This research used gamma ray irradiation on low doses (micro mutation). The aim of this research was to know the respons of doses level by micro mutation on gamma ray irridation to the growing and development of Argomulyo variety of soybean [Glycine max (L) Merr]. The seeds were irradiated by gamma ray micro mutation doses, namely 0 gray, 50 gray, 100 gray, 150 gray, and 200 gray. Variations that were obtained of each characters at generation M1 and M2 influences plants growth and development either through qualitative and quantitative that finally will influence plant’s production. The average highest genetic variation at M2 generation of soybean was on 200 Gray doses. Results of the research indicated that gamma ray irradiation on 200 Gray doses effectively caused of plant variation genetic.

Key word: induced mutation, micro mutation, gamma ray irradiation, Argomulyo soybean variety.

Abstrak. Hanafiah DS, Trikoesoemaningtyas, Yahya S, Wirnas D. 2010. Mutasi induksi irradiasi sinar gamma pada varietas kedelai Argomulyo (Glycine max). Nusantara Bioscience 2: 121-125. Induksi tanaman dengan irradiasi sinar gamma merupakan salah satu cara untuk meningkatkan keragaman genetik tanaman. Penelitian ini menggunakan irradiasi sinar gamma pada tingkat atau dosis rendah (mutasi mikro). Tujuan penelitian ini untuk mengetahui respon pemberian tingkat irradiasi mikro sinar gamma pada benih kedelai.

Benih kedelai [Glycine max (L) Merr] yang diuji adalah kedelai varietas Argomulyo yang diirradiasi sinar gamma dengan dosis 0 Gray, 50 Gray, 100 Gray, 150 Gray dan 200 Gray. Keragaman yang diperoleh dari setiap peubah amatan yang diperoleh pada generasi M1 dan M2 menunjukkan bahwa perlakuan irradiasi dapat mempengaruhi pertumbuhan dan perkembangan tanaman secara kualitatif dan kuantitatif, yang akhirnya akan mempengaruhi produksi tanaman. Variasi fenotipe pada tanaman kedelai generasi M2 tertinggi rata-rata terjadi pada perlakuan 200 Gray. Hasil penelitian menunjukkan bahwa irradiasi sinar gamma pada dosis 200 Gray efektif menyebabkan terjadinya keragaman genetik tanaman.

Kata kunci : mutasi induksi, mutasi mikro, irradiasi sinar gamma, kedelai varietas Argomulyo.

INTRODUCTION Indonesia is one of soybean importers [Glycine max (L.) Merr] in the world, especially from the United States.

This need has not been adequated by domestic production; moreover there is a tendency of decrease of the production of local farmers recently. The Temporary Figures (ASEM/TF) of soybean production in 2009 is 972.95 thousand tons of dry beans. The Forecast Figures I (ARAM I/FF) of soybean production in 2010 is estimated at 962.54 thousand tons of dry beans. Compared to production in 2009 (ASEM), there is a decrease of 10.41 thousand tons (1.07%) (CSB/BPS 2010). Domestic soybean production can only meet 20-30% of the national soybean demand.

The rest of 70-80% is filled by importing soybean (Sudaryanto and Swastika 2007; Purna et al. 2009). This is an opportunity as well as a challenge for Indonesian farmers to increase soybean production in the country. One of the attempts to increase national soybean production is

done through improving desired plant traits, including productivity.

Soybean is a self-pollinated plant, which will form solid lines, or no segregation. The population is composed by lines, with a genetic diversity of a very thin intra lines or almost zero, and the diversity of a visible inter lines. New genetic diversity would appear in nature as a result of mutation or the occurrence of cross inter strains, even with a small degree, therefore the soybean genetic diversity is low (Jusuf 2004).

Soybean is not a local native plant of Indonesia; therefore, it has a poor genetic diversity. It is estimated that the central area of distribution of the Glycine genus is in Asia, where most endemic species are still alive. In China, several species of wild soybean can be found now, and become the source of genes from the cultivated soybean, where there are wild relatives of soybean species called G. ussuriensis. Exploration of the source regions of genes related to the provision of data and germ plasma resources

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is needed for improving the varieties and breeding as well as adaptation to the cultivated soybean (Leppik 1971).

An assembling of the new varieties requires basic population that has a high genetic diversity which can be obtained through the introduction, cross over, mutation, and genetic transformation. Argomulyo soybean is one of the variety development introduced from Thailand, in addition to other introduced varieties such as Bromo, Krakatau and Tambora (Hidajat et al. 2000; Arsyad et al.

2007). Increasing genetic diversity of soybean crop will facilitate the selection efforts to obtain plants with desirable traits, such as plant characters for resistance to drought stress.

Mutation induction with radiation is the most frequently used method to develop direct mutan varieties, where improvement by acclimatization, selection, hybridization and also laborious have proven to be time consuming and also with limited genetic variation. Mutation breeding of plants is useful to improve the character if the character you want is not located in a plant germplasm of a species, and also for generating variability in the existing varieties (Van Harten 1998; Yaqoob and Rashid 2001; Khan and Goyal 2009).

Induction of plants with gamma ray irradiation is one way in improving plant genetic diversity. Gamma ray irradiation at low dose levels or (micro mutation) is less influencing changes in quantitative characters of plants and chromosomes compared with the macro mutation using gamma ray irradiation at high doses. Mutation induction can be done on the plants by mutagenic treatment of certain materials of plant reproductive organs such as seeds, stem cuttings, pollen, root rhizome, tissue culture and others. If a natural mutation process is very slow, the acceleration, frequency and spectrum of mutations can be induced by mutagen treatment of certain materials (BATAN 2006).

Micro mutation performed on Argomulyo soybean varieties aims at improving the quantitative plant characters that ultimately aims at increasing production and development of plant adaptation to marginal lands. The purpose of this research is to study the response of microlevel gamma irradiation on the growth and development of soybean plants first generation (M1) and knowing the genetic diversity in the second generation (M2).

MATERIALS AND METHODS This research was conducted from February to June 2009. Irradiation treatment was performed at the Center for Biological Resources and Biotechnology, Bogor Agricultural University (IPB), Bogor and field research was conducted at the University Farm of Bogor Agricultural University, at Darmaga, Bogor, West Java.

Soybean seed varieties of Argomulyo were radiated with the gamma ray dose of 50 Gray, 100 Gray, 150 Gray and 200 Gray (micro dose) derived from 137Cs using IBL 437C Irradiator type H (CIS Bio International, France) with a dose rate of 2.23 Gray/min . A total of 200 seeds (M1) in each treatment dose planted with a spacing 40 x 20 cm2 and the influence of gamma irradiation on morphological

characters of plants including flowers, leaves, plant height, number of branches, number of productive pods, the number of empty pods and number of seeds per plant were then evaluated. For the M1 generation, in each plant at each treatment dose, 10 pods per plant then were harvested (restricted bulk) and grown as an M2 generation. Then 2000 seedlings of M2 seeds were planted for each dose treatment with spacing of 40 x 20 cm2, and the diversity in agronomic characters including plant height, number of branches, number of pods, number of empty pods and seed weight per plant were evaluated. Genetic variation in M2 generation was calculated with the formula:

σ2 = σ2M2 σ2p σ2g

(∑ x 2 ) − [(∑ x) 2 / n] n −1

= σ2p; = σ2g + σ2e; = σ2p-σ2e =σ2M2-σ2M0,

σ2 = variance n = number of members of the population σ2p = phenotypic variance σ2g = genotypic variance σ2e = environment variance σ2M2 = M2 population variance σ2M0 = M0 population variance (Argomulyo population as a control) Heritability values calculated using the following formula: h2 = σ2g/σ2p (Singh and Chaudhari 1977) Criterion of heritability: h2> 0.5 = high heritability values h2 = 0.2 to 0.5 = value of heritability is h2 <0.2 = low heritability values Genetic variation is determined based on the coefficient of genetic variation (CGV) by using the method proposed

by Singh and Chaudhari (1977) as follows: CGV = (

σg x

) x100%

= genotypic standard deviation σg x = character means CGV the highest absolute value determined from the relative value of 100% CGV.

RESULTS AND DISCUSSION Performance of M1 plants micro-mutations result of gamma ray irradiation.

The results showed that the M1 derivative of soybean varieties with micro mutation Argomulyo gamma ray irradiation of each variable observation showed the higher the dose of irradiation, the lower the parameters that were observed became (Table 1). Irradiation dose significantly

HANAFIAH et al. â&#x20AC;&#x201C; Mutations of Argomulyo soybean variety with gamma rays

affected the plant height (Table 1). At a dose of 200 gray, the plant heights were shorter compared with other irradiation doses. The height of Argomulyo varieties that were irradiated with gamma ray at doses of 50 gray, 100 gray and 150 gray was higher than the control, but when the dose of the irradiation was raised the average height of the plant tended to decrease. Sakin (2002) stated that gamma ray irradiation treatment increased the average plant height compared with controls. This study is similar to the research conducted by Tah (2006) and Sing et al.

(2001) who observed the effect of high dose treatment on plant derivatives M1 mungbean [Vigna radiata (L.) Wilczek], where plant height decreased due to the treatment of gamma ray irradiation dose at 10 kR, 20 kR, 30 kR and 40 kR, with the highest decline occurred at a dose of 40 kR (1Gray=10 kR).

Research conducted by Shakoor et al. (1978) suggested that the treatment in the range of 10-30 kR doses was not significantly different but the dose of 40 kR caused plants to become stunted. Similar results were found in rice seed (Cheema and Atta 2007; Shah et al. 2008), where germination percentage decreased when irradiated with gamma rays, but its reduction is not proportional with the increase of the dose.

The effect of irradiation dose on the number of branches of M1 plants Argomulyo soybean varieties was not significantly different (Table 1). The numbers of branches generally formed were two, which was not much different between the branches of control plants and that of irradiation produced ones. The maximum effect of dose occurred at 50 Gray with increase as much as 25.5% compared with the controls plants.

The number of branches at 150 Gray dose of irradiation is less than 200 Gray, where there were unproductive branches, no pods formed and flowers did not develop.

This is consistent with the research conducted by Ganguli and Bhaduri (1980) who claimed there was a reduction in productive branches due to gamma ray irradiation and the numbers of main branches were more than the control plants at each treatment dose. Research conducted by Tah (2006) states that the influence of gamma ray irradiation dose on the number of branches at a dose of 30 kR increased by 30.55% compared with controls. With the increasing number of productive branches, number of pods per plant will increase.

Effect of gamma-ray irradiation makes the pod number greater than the control, with the varying increase (Table

123

1). The highest number of pods obtained at 150 Gray dose of irradiation, the increase is as much as 27.33% compared with controls. Number of pods showed an increase in M1 plants, especially at 150 Gray dose of irradiation, which formed more pods on main stem, while the number of branches is only a few. Many pods formed on the main stem. In plants with radiation dose of 200 Gray irradiation, the number of pods formed is a few, many flowers did not grow and the flowers did not grow to form pods (experienced sterility and abnormality development).

Increase in the number of pods at M1 also occurred in the study conducted by Tah (2006), where an increasing number of pods as a result of gamma ray irradiation reached 15-23% and reached its maximum at the dose of radiation 30 kR. Number of empty pods were not significantly different at each dose of irradiation (Table 1).

The highest numbers of empty pods of M1 plants occurred at 200 Gray dose of irradiation, where the pods were empty, because the seeds failed to form due to the disruption on plant growth.

Irradiation dose significantly affected the number of seeds per plant (Table 1). At a dose of 200 Gray the number of seeds per plant was smaller compared with other irradiation doses. The number of seeds per plant of Argomulyo soybean varieties that were irradiated with gamma rays at doses of 50 gray, 100 gray, gray and 150 were higher than in control plants, but when the average dose of irradiation was increased the average plant height then decreased.

Figure 1 shows that the gamma-ray irradiation affects the diversity of phenotypes on the M1 derivative by its characteristic morphology. This is indicated by qualitative changes, such as leaf shape changes from oval (normal) to elongate, there are unifoliat and bifoliat leaves on the first stem together with trifoliate leaf on a single plant, flowers color changes from purple to white, flower Rasim does not develop into pods, leaves still green even though the pods were ripe harvest.

These qualitative changes occured in some plants from dose irradiation treatment 150 and 200 Gray. The same things happened on mutation induction with gamma ray irradiation in soybean (Manjaya and Nandawar 2007) and green beans (Sangsiri et al. 2005) that indicated a change in shape and color of leaf and flower shapes and colors and also cause sterility in plants.

Table 1. The mean observation variables at different irradiation doses Variables observations Plant height (cm) Number of branches (fruit) Number of pods (fruits) Number of empty pods (fruit) Number of seeds Note: statistics analysis performed by t test

0 34.20BC 1.75A 27.65B 0.35A 70.60BC

50 38.00A 2.35A 36.25A 0.35A 88.35A

Irradiation dose (Gray) 100 150 37.88A 36.42AB 2.30A 1.90A 35.75A 38.05A 0.35A 0.35A 78.05AB 83.4AB

200 30.47C 2.20A 29.20B 0.45A 56.35C

Â

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A

B

C

D

E

F

Figure 1. Growth abnormality of Argomulyo variety which is caused by the gamma ray irradiation: A. Trifoliate and oval leaves (normal), B. Bifoliat leaves and elongated, C. Unifoliat leaves and elongated, D. Rasim flowers and buds (not develop), E. Purple flowers (normal), F. White flowers.

Genetic variation and heritability of M2 generation resulted from gamma-ray irradiation Genetic variation and heritability of Argomulyo M2 soybean variety plants at different irradiation doses can be seen in Table 2. Seeds which are harvested from M1 plants are planted as seed M2, and M2 plants are expected to show segregation at the genetic locus that has mutation. Genetic variety can be observed in M2 generation. In this study, observations focused on agronomic characters like plant height, yield components and yield.

The average plant height tends to increase with increasing dose of irradiation (Table 2). At a dose of 200 Gray, the average plant height is lower than the average plant height at the other doses. The average number of productive branches reaches the highest value at a dose of 200 Gray and lowest values found at a dose of 150 Gray.

The highest average number of productive nodes is reached at dose 200 Gray and the lowest average value is at 50 Gray. The highest average number of productive pods is reached at dose 200 Gray and the lowest average value is at 150 Gray. The highest average value of empty pods is at 200 Gray and the lowest is at 50 Gray. The highest average value of the seeds weight is at 50 Gray dose.

The variance of morphology (phenotype) which increases with the increase of irradiation dose occurs at observation variables such as plant height, number of productive nodes and number of productive pods. For all these characters, the highest phenotypic variance comes in a dose of 200 Gray. Jamil and Khan (2002) reported promising fluctuation in germination (%), plant height,

number of grains per plant, grain yield in the wheat through gamma irradiation. Gamma ray irradiation dose recommended by the IAEA (International Atomic Energy Agency) for the soybean crop is at 200 Gray irradiation, which is useful for improving quantitative characters of plants (Srisombun et al. 2009). For the other characters, the variance of phenotypes did not increase proportionally in line with the increase in the doses of irradiation, and they are the number of productive branches per plant and seed weight.

The results shows that low dose irradiation (micro dose) can produce the variance of characters desired. Sakin (2002) who observed mutation quantitative characters of wheat at low doses of gamma ray irradiation found the same thing. The advantage of using low-dose irradiation allows mutations to happen in the minor genes that can be observed in coming generation without harmful mutations.

Sangsiri et al.(2005) reported many adverse mutations such as albino and leaf shape changes in green beans after irradiation treatment at a dose of 500 Gray.

The variance of agronomic characters in soybean after being irradiated by gamma rays is genetically controlled.

High genetic diversity is very important in the selection process, because the genetic response to selection depends on the level of genetic diversity (Hallauer 1987). Previous researchers have reported hereditary changes in the desirable characters in crop plant by using gamma rays as a physical mutagen, which has been used to develop 64 % of the radiation-induced mutant varieties (Ahloowalia et al.

2004). High expectation value of wide heritability was

HANAFIAH et al. – Mutations of Argomulyo soybean variety with gamma rays

found in the observation variable such as plant height and number of productive pods. Selection for improvement of these two characters can be done to produce genotypes with the desired plant height and production. For other characters, the estimated expectation value of heritability range from low to moderate. Sakin (2002) who observed wheat found that heritability for some mutant population depends on the characters observed.

Table 2. Genetic variation and heritability of M2 plants at different irradiation doses.

Character

50

Dose irradiation 100 150

200

Plant height

x 32.76 12.953 σ2p σ2g 7.092 0.547 h2 CGV (%) 8.129 Number of productive branches x 2.73 σ2p 2.572 1.924 σ2g h2 0.748 CGV (%) 50.785

33.14 13.492 7.630 0.565 8.334

34.73 24.824 18.962 0.764 12.536

34.43 28.060 22.198 0.791 13.683

2.81 1.055 0.406 0.385 22.645

2.48 1.237 0.588 0.475 30.810

3.01 0.951 0.302 0.317 18.276

x 17.24 15.587 σ2p σ2g 1.105 0.071 h2 CGV (%) 6.097 Number of productive pods x 39.08 111.368 σ2p σ2g 36.013 0.323 h2 CGV (%) 15.355 Number of empty pods

18.00 23.611 8.611 0.365 16.302

17.28 25.468 12.468 0.490 20.431

19.02 24.507 14.507 0.592 20.024

38.13 220.605 145.251 0.658 31.601

38.07 191.209 115.855 0.606 28.269

43.19 209.976 134.622 0.641 26.862

x

1.40 2.783 0.131 0.047 25.882

2.53 5.792 3.140 0.542 70.015

2.60 4.866 2.214 0.455 57.237

3.05 7.705 5.053 0.655 73.578

x

10.93 10.775 0.033 0.003 1.664

9.85 16.458 5.716 0.347 24.257

9.47 16.574 5.832 0.352 25.494

10.17 12.116 1.374 0.113 11.521

σ2p σ2g h2 CGV (%) Weight of seeds per plant σ2p σ2g h2 CGV (%)

CONCLUSION Phenotypic variations that occur on M1 plants were caused by changes due to gamma ray irradiation, which affects plant growth and development. The highest average genetic variation in M2 generation plants occurred in the

125

treatment of 200 Gray. Gamma ray irradiation at a dose of 200 Gray effectively leads to genetic diversity in plants.

REFERENCES Ahloowalia B, Maluszynski M, Nichterlein K. 2004. Global impact of mutation-derived varieties. Euphytica, 135: 187-204.

Arsyad DM, Adie MM, Kuswantoro A. 2007. The engineering of soybean variety specific agroecology. In: Sumarno, Suyamto, Widjono A, Hermanto, Kasim H (eds). Soybean: Production technique and development. Research centre and food plants development, Bogor.

[Indonesia] National Atomic Energy Agency [BATAN]. 2006. Mutation in plant breeding.

Central Statistic Bureau [CSB/BPS]. 2010. Rice, corn and soybean production (the temporary figures 2009 and the forecast figures I).

CSB No.18/03/Th.XIII. Cheema AA, Atta BM. 2003. Radiosensitivity studies in Basmati rice.

Pakistan J Bot 35 (2): 197-207.

Ganguli P, Bhaduri P. 1980. Effect x-rays and thermal neutrons on dry seeds of Greengram (P. aureus). Gentica Agraria 34: 257-276.

Hallauer AR. 1987. Maize. In: Fehr WR (ed). Principles of cultivar development crops species, 2: 249-294. Machmillan, New York.

Hidajat JR, Harnoto, Mahmud M, Sumarno. 2000. The technology of soybean seed production. Research centre and food plants development, Bogor. [Indonesia].

Jamil M, Khan UQ. 2002. Study of genetic variation in yield components of wheat cultivar bukhtwar-92 as induced by gamma radiation. Asian J. Plant Sci., 1: 579-580.

Jusuf M. 2004. Exploration method, inventarisation, evaluation and conservation of germ plasma. Research center of Biotechnology, IPB. Bogor.

Leppik EE. 1971. Assumed gene centers of peanuts and soybeans. Econ Bot J 25 (2): 188-194.

Khan S, Goyal S. 2009. Mutation Genetic Studies in Mungbean IV. Selection of Early Maturing Mutants. Thai Journal of Agricultural Science, 42(2): 109-113.

Manjaya JG, Nandanwar RS. 2007. Genetic improvement of soybean vanety JS 80-21 through induced mutations. Plant Mut Rep 1 (3): 3640.

Purna I, Hamidi, Prima. 2009. The efforts to increase soybean production.

Secretariat state of Republic Indonesia, Jakarta.

Sakin MA. 2002. The use of induced micro mutation for quantitative characters after EMS and gamma ray treatments in durum wheat breeding. Pakistan Journal of Applied Sciences 2(12): 1102-1107.

Sangsiri C, SorajjapinunW, Srinives P. 2005. Gamma radiation induced mutations in mungbean. Sci Asia 31: 251-255.

Shah TM, Mirza JI, Haq MA, Atta BM. 2008. Radiosensitivity of various Chickpea genotpes in M1 generation. Pakistan J Bot 40 (2): 649-665.

Singh, A., M. Diwakar, J. Singh and J. Singh, 2001. Mutagenic responses of mungbean Vigna radiata L. Wilczek. J. Applied Biol., 3: 75-79.

Shakoor A, Ahsan-ul-haq M, Sadiq M. 1978. Induced variation in mungbean. Env Exp Bot 18: 169-175.

Srisombun S, Benjamas K, Chitima Y, Jeeraporn K. 2009. Soybean variety improvement for high grain protein content using induced mutation. IAEA/RCA project RAS/5/045, Feb 16-20, 2009, Vietnam.

Sudaryanto T, Swastika DKS. 2007. Soybean economy in Indonesia. In: Sumarno, Suyamto, Widjono A, Hermanto, Kasim H (eds). Soybean: Production technique and development. Research centre and food plants development, Bogor. [Indonesia] Tah, PR. 2006. Studies on gamma ray induced mutations in mungbean [Vigna radiata (L.) Wilczek]. Asian J of Plant Sci 5 (1): 61-70.

Van Harten AM. 1998. Mutation breeding, theory and practical application. University of Cambridge, Cambridge, UK. Yaqoob M, Rashid A. 2001. Induced mutation studies in some mungbean (Vigna radiata L.) Wilczek cultivars. J. Biol. Sci., 1: 805-808.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 126-134 November 2010

Teratogenic test of Pandanus conoideus var. yellow fruit extract to development of rat embryo (Rattus norvegicus) LINTAL MUNA1,♥, OKID PARAMA ASTIRIN², SUGIYARTO² ¹ Office of Education Pati District. Jl. P. Sudirman No. 1B Pati 59113Central Java, Indonesia. Tel./Fax. +62-295-381421. ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia.

Manuscript received: 2 Augustus 2010. Revision accepted: 10 October 2010.

Abstract. Muna L, Astirin OP, Sugiyarto. 2010. Teratogenic test of Pandanus conoideus var. yellow fruit extract to development of rat embryo (Rattus norvegicus). Nusantara Bioscience 2: 126-134. This experiment was performed to examine the effect of Pandanus conoideus Lam. var. yellow fruit extract on the percentage of the living foetus, the death intrauterus, heavy and long of foetus, foetus morphology, and skeleton structur of foetus. The experiment was used by 25 pregnant mice that randomly were divided into 5 groups, they contained 5 mice. Each group was given with the different dose. The P1 group (control) was given 1 mL sesame oil, the group P2, P3, P4 and P5 were respectively given the yellow fruit extract 0,02 mL, 0,04 mL, 0,08 mL and 0,16 mL. The P. conoideus var. yellow fruit extract was given orally on day 5 to 17 of gestation (organogenesis periode). Observation was carried out on day 18 of gestation by caesarean section to take the foetus from the uterus. Foetus morphology was observed after taking foetus from uterus, whereas observation of skeleton structure was made wholemount preparat with dual colourization, they are Alcian blue and Allizarin Red-S. The result was analyzed with one way anova. Results showed that giving yellow fruit extract didn’t influence to the percentage of the living foetus, the death intrauterus, heavy and long of foetus. The effect of giving yellow fruit extract to the maternal were abnormality skeleton (lordosis) of foetus in the dose 0.16 mL and obstacled to the ossification of foetus.

Key words: Pandanus conoideus var. yellow fruit, teratogenic, white rat.

Abstrak. Muna L, Astirin OP, Sugiyarto. 2010. Uji teratogenik ekstrak Pandanus conoideus varietas buah kuning terhadap perkembangan embrio tikus putih (Rattus norvegicus). Nusantara Bioscience 2: 126-134. Penelitian ini betujuan untuk mengkaji pengaruh pemberian ekstrak Pandanus conoideus Lam. var. buah kuning terhadap persentase fetus hidup, kematian intrauterus, berat dan panjang fetus, keadaan morfologi fetus, serta struktur skeleton fetus tikus putih. Dalam penelitian ini digunakan 25 tikus bunting yang dibagi menjadi lima kelompok secara acak, sehingga masing-masing kelompok terdiri dari lima ekor tikus. Setiap kelompok diberi dosis yang berbeda. P1 (kontrol) diberi 1 mL minyak wijen, P2, P3, P4 dan P5 diberi ekstrak masing-masing: 0,02 mL, 0,04 mL, 0,08 mL dan 0,16 mL. Ekstrak tersebut diberikan secara oral pada kebuntingan hari ke 5 sampai hari ke 17 (fase organogenesis). Pengamatan dilakukan pada hari ke 18 dengan cara bedah sesar untuk mengambil fetus dari uterus. Morfologi fetus diamati setelah fetus dikeluarkan dari uterus, sedangkan untuk pengamatan struktur skeleton dibuat preparat wholemount dengan pewarnaan ganda Alcian blue dan Allizarin Red-S. Hasil percobaan dianalisis dengan ANAVA satu jalur. Hasil penelitian menunjukkan bahwa pemberian ekstrak tidak berpengaruh terhadap persentase fetus hidup, kematian intrauterus, serta berat dan panjang fetus (P≥0,05). Pemberian ekstrak pada induk mengakibatkan kecacatan skeleton (lordosis) fetus pada dosis 0,16 mL dan menghambat osifikasi fetus.

Kata kunci: Pandanus conoideus var. buah kuning, teratogenik, tikus putih.

INTRODUCTION Cancer is a disease characterized by uncontrolled cell growth. Cancer is the second cause of death in the world after cardiovascular disease (Ganiswara 2001; Foye 1996).

Cancer cells in the organs of the body will grow and develop in an abnormal, rapid and uncontrolled with the cells shape, nature and movement different from its origin and cause damage to the form and function of organs (Dalimartha 2004). Uncontrolled growths of cancer cells push the normal cells around them, because cancer cells can metastasize to other body parts (Harkness 1989).

Treatments to suppress or cure these diseases are surgery, radiation, and treatment with chemical compounds (Harkness 1989; Alatas 2005). Anti-cancer drug or

sitostatica are medications that can stop the growth of malignant cells or even kill normal cells (Tjay and Rahardja 2002). Anti-cancer drugs are teratogenic and can not only affect the cancer cells, but can affect normal cells (Foye 1996; Ganiswara 2001). The high cost and side effects by the presence of cancer therapy (Harkness 1989; Harmanto 2001), encourage communities to use anti-cancer drugs from plants.

In Indonesia, there are no less than 2039 species of medicinal plants from tropical forests. This situation makes Indonesia as one of the world's storehouse of biodiversity important for pharmaceutical or medicinal ingredients for human health (Zuhud 2009). One of the plants that acts as anti-cancer is a red fruit (Pandanus conoideus Lam) varieties of P. conoideus var. yellow fruit. These plants

MUNA et al. – Teratogenic test of Pandanus conoideus extract

contain various useful chemical compounds (Budiman 2000). According to Mun'im (2006) mampung red fruit extract inhibits tumor growth. Atsirin (2009) and Pratiwi (2009) independently proved that the extract of yellow varieties of P. conoideus fruit can inhibit the growth of T47D breast cancer cells. Hidayati (2010) proved that this plant extract could inhibit Hela cell growth. P. conoideus var. yellow fruit is a endemic plant of Papua, which has the tocopherol and beta-carotene content that act as antioxidants. As an antioxidant, both compounds were able to ward off free radicals and are thought to help the healing process of cancer (Budi and Paimin 2005).

Gysin et al. (2002) revealed that α-tocopherol could inhibit the growth of DU145 prostate cancer cells by 50%, LNCaP, prostate cancer cells as much as 48%, and 50% in adenocarcinoma colon cancer cell (Caco-2). Consuming 30-60 mg of beta-carotene daily for 2 months will make the body have natural killer cells and more T cells and lymphocyte-helpers which are more active. The increase in natural killer cells is essential to fight cancer cells and control free radicals that disturb health badly (Budi and Paimin 2005). The same is stated by Russell (2002) that the cancer risks are lower in people who consume vegetables and fruits that contain high carotenoids.

Tocopherol is a form of vitamin E, when consumed excessively; it can cause poisoning (Almatsier 2002). And, beta-carotene is an vitamin A. The results of experiments on animals showed the occurrence of birth defects as a result of hipovitaminosis and hipervitaminosis of vitamin A during pregnancy (Pergament 1996).

Anti-cancer drug is used by all cancer patients with including pregnant women, while pregnant women are particularly vulnerable to drugs, especially during organogenesis. Some of drugs that are not allowed to eat by pregnant women are anti-cancer drug, because the anticancer drugs are capable of stopping cell division (Nogrady 1992) and drugs that reach the fetus can cause miscarriage, malformation and death of the fetus (Suryawati 1990).

This study aims to examine the effect of the extract by oral administration of external abnormalities in the form of percentage of live fetuses, intrauterine death, fetal weight and length, and the state of fetal morphology, and internal abnormalities of fetal skeleton structure of the white rat (R. norvegicus).

MATERIALS AND METHODS Material Test animals used were white rat (R. norvegicus), with zero day to 2.5 months of pregnancy and an average weight of 200 g, pellets Br 2 (PT. Japfa Comfeed Indonesia, Sidoarjo) as the daily feed, extracts of P. conoideus var. yellow fruit, water to drink and sesame oil as a solvent.

Sesame oil is packed by PT. Heinz ABC Indonesia, Jakarta).

Study design This research was conducted with experimental methods used 25 white rats which were divided into 5 groups with completely randomized design (CRD). Each

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treatment consisted of 5 replicates. The treatment is as follows: P1: Control = 1 mL of sesame oil P2: The dose of 0.02 mL of extract of P. conoideus var. yellow fruit oil and 0.98 mL of sesame oil/200 g BW P3: The dose of 0.04 mL of extract of P. conoideus var. yellow fruit oil and 0.96 mL of sesame oil/200 g BW P4: The dose of 0.08 mL of extract of conoideus var. yellow fruit oil and 0.92 mL sesame oil/200 g BW P5: The dose of 0.16 mL of extract of P. conoideus var. yellow fruit oil and 0.84 mL sesame oil/200 g BW Extracts P. conoideus var. yellow fruit and sesame oil given orally in the morning and afternoon on gestation day of 5 to 17.

Procedures Pre-treatment. Twenty-five white adult female rats (R. norvegicus) in estrus cycle were gathered in one cage with 10 male of white rats. The following day the female rats were examined in way of vaginal plug (vaginal plug), if there was vaginal plug or after viewed microscopically with vaginal smear method and the spermatozoa was found, then that day was considered as the first day of gestation. Furthermore, female rats were separated from white male rats and they were divided into 5 groups, each group consisted of 5 female rats.

Preparation of the animals test. The 2.5 months pregnant rats with an average weight of 200 g were kept in a cage, each cage contained five mice with the same treatment group. The total number of white rats used was 25. They were divided into five treatment groups. Before being used in the study, rats were acclimatized for 4 days, and they were given by food and drink.

Extraction. Preparation of extracts of P. conoideus var. yellow fruit refers to Budi and Paimin (2005) as follows: the fruit that is mature was selected, the maturity was marked with a yellow fruit color and the distance between bulges which were rare. The fruit was cleft and the pith was discarded, then it was cut and washed. Fruit flesh was boiled for 1-2 hours, when it was soft, it was removed and cooled. Fruit meat was kneaded until the seeds were separated. Water was added up to 5 cm above the surface of the material (3:1). This material was squeezed again until the seeds seemed white and clean of meat. The result was the digest of P. conoideus var. yellow fruit which look like coconut milk. The digest was refined to separate juice from seeds. The refined digest was cooked in ± 40ºC for 56 hours as it was stirred. If yellow oil was appeared on the surface, the flame is turned off and continued the stirring for 10 minutes to cool it quickly. Juice was removed and was left alone for 1 day until there was the formation of 3 layers, namely the pulp (bottom layer), water (middle layer), and oil (top layer). Oil was taken slowly with a spoon and moved into a transparent container, and then it was left alone for 3 hours until the oil, water and waste were completely separated. With a spoon, the oil was moved again carefully into the container.

Determination of dose usage. The recommended dosage of P. conoideus var. yellow fruit is 2-3 times of one tablespoon daily (Budi and Paimin 2005), where one

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tablespoon is equivalent to about 15 mL (Wiryanta 2007).

That dose is for people with 70 kg of bodyweight. If the dose is applied on white rats weighing 200 g, it is obtained: X/200g = 15 mL/70 kg Ă&#x2020; X = 0.043 mL = 0.04 mL. The determination of dose is also based on the research by Pratama (2009) who uses the extract with a concentration of 0.03125 mL to T47D breast cancer cells. The results of these studies indicate that with this concentration, not all T47D breast cancer cells die. While the use of the same oil solvent is based on the research by Mun'im (2006) using 1 mL of sesame oil on the control. The results show that there is no abnormality in the fetus due to the use of 1 mL of sesame oil.

Treatment of the animals test. Previously, all rats were weighed to determine their initial weight. The extract was given orally in each treatment group from day 5th to the 17th day of gestation, respectively. On the 18th day, the surgery was done. Before the surgery process, all rats were weighed to determine the final weight. Observations were carried out by taking a fetus from the uterus and then it is cleaned from the placenta and the mucous membrane enveloped it. Fetus external observations began with the count and record the number of implantation which consists of the number of living fetus, the number of dead fetuses, and the number of fetuses that were resorption.

Furthermore, the body weight, the body length, and the fetal morphology were observed including: body shape, number of extremities, skull, tail, etc that were considered abnormal. The observation was also done on the internal skeleton system (bone shape, the amount of bone and the process of reinforcement). To study the fetal skeleton structure, a wholemount preparations with dual staining method of Allizarin red-S and Alcian blue is made (Inouye 1976).

Wholemount preparations making process was as follows: Fetuses were fixed into 95% alcohol for 3 days.

Viscerasi is the disposal process of skin, fat tissue and internal organs of the fetus. This process was done very carefully as not to damage the fetus nor change the fetal organ position. White rat fetuses were put into acetone for 1 day to dissolve fat. Fetuses were stained on day 4th using a double staining namely Allizarin red-S and Alcian blue for 1-3 days at 37 Âş C temperature. Fetuses were washed with running water for several times until they were clean.

Fetuses were clarified with 1% of KOH solution in water for 2 days until the tissues that wrap the body become transparent and the ones that have red or blue color were only bone tissues. Fetus transferred into 20% of glycerin solution in 1% of KOH for 1-4 days. In a row, fetuses were inserted in a solution of glycerol 50% and 80% in KOH 1% respectively for 1 hour, and then were stored in 100% of glycerin for later observation. Observations on the results of ossification were based on of the dye absorption of skeleton. True normal bone turns red and inhibited growth bone will be blue or no color at all as a dyeing reaction of Allizarin Red-S. The photographing of fetal was performed at the time of abnormalities observation, both external (abnormalities of morphology, hemorrhage, and resorption) and internal (ossification abnormalities) using a digital camera.

Collection of data. The quantitative data was obtained by observing the number of implantation consists of the number of live fetuses, number of dead fetuses, fetal weight, and fetal body length. The qualitative data was obtained by observing the morphology of the fetus (eyes, ears, knuckles, skulls, tails and others that are considered abnormal) and its skeleton system (bone shape, number of bones, and the process of reinforcement).

Data analysis Quantitative data were analyzed using single lane of analysis of variance (ANOVA) with significance level of 5% to know the real differences among the treatments. If the analysis of variance obtained significant results, it continued with Duncan multiple range test (DMRT) to know the difference. For the observation of external and internal abnormalities (the abnormalities of ossification results) a descriptive analysis was conducted.

RESULTS AND DISCUSSION Pandanus conoideus var. yellow fruit is one of the alternative medicines to cope with cancer (Budi and Paimin 2005). Anti-cancer drug is used by all cancer patients, including pregnant women, while pregnant women are particularly vulnerable to drugs, especially during organogenesis (Briggs et al. 2008). Anti-cancer drugs are teratogenic; not only affects the cancer cells, but can affect the normal cells around it (Ganiswara 2001; Foye 1996).

Fetal tissue grows rapidly, the cells divide rapidly so it is very vulnerable to anti-cancer drug. In addition, the drugs consumed by the parent will move to the fetus through the placenta, namely the same path in which the nutrients needed for growth and development of the fetus go through. Drugs that reach the fetus can cause miscarriage, malformation or even death of the fetus (Suryawati 1990).

White rat fetal external abnormalities In this study, external abnormalities were observed in a morphometry way by seeing the occurrence of reproduction of white rats parent by counting the number of live fetuses, the number of intrauterine death (dead fetuses and resorption), measuring weight and length of fetal body and observing the abnormalities in the form of hemorrhage and deformity in some parts of fetal body (Table 4).

Fetal morphometry Fetal weight and length are important parameter to be observed in teratogenic research. Wilson (1973) states that the decrease in fetal body weight and length is the lightest form of an effect of teratogenic compound. Fetal body weight and length is a parameter that is sensitive enough to determine the effect of foreign compounds on the growth of a fetus. There were changes in fetal body weight and length ranging from the control group until the group treated with the highest dose (Table 4).

Based on statistical analysis, weight and average length of fetuses between treatment groups were not significantly

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different (P> 0.05). The result of variance analysis is supported by the analysis of correlation between the dose with fetal weight and between fetal length with doses. R value between the dose with body weight was 0.265 (weak correlation). Probability value was > 0.05 (0.200> 0.05), then H0 was accepted. It means that the relationship between doses with fetal body weight was not significant at the level of 95%. While the value of r between dose with fetal length is 0.123 (the correlation was very weak).

Probability value was > 0.05 (0.559> 0.05), it could be concluded that the relationship between dose with fetal length is not significant at the level of 95%.

Beta-carotene and tocopherol in a certain amount are needed by the body because it is a vitamin that is important for the body, but if there's too much, it would be toxic (Almatsier 2002). The results of Azmanâ&#x20AC;&#x2122;s study (2001) said that, vitamin A and E effect weight gain of normal rats and rats with treatment of diovariectomy (removal of two ovaries). In this study, weight gain primarily is due to the increase of fat mass. Tocopherol and beta-carotene are fat soluble vitamins that are lipophilic, allowing it to easily cross the placenta.

Tocopherol and beta-carotene are absorbed in the intestines along with the fat or oil consumed. One of the reasons why the extract is taken orally is because tocopherol and beta-carotene are not water soluble, which means it also not soluble in blood plasma. In order for these vitamins can be transported into the blood circulation, it must bind to proteins (lipoproteins), which is then absorbed by the lymphatic system. From the lymphatic system, these vitamins along with VLDL (Very Low Density Lipoprotein) go into the blood circulation. Some go to the needed part and the others go to the liver through the ductus toracicus which later merged with triglyceride-rich VLDL and HDL (High Density Lipoprotein) which is rich in phospholipids, cholesterol and esters. VLDL and HDL are synthesized by the liver. Then E vitamin will be back into the blood vessels and then converted to LDL (Low Density Lipoprotein) by the help of the enzyme of lipoprotein lipase in the blood. Furthermore, the vitamins in LDL transported to adipose tissue.

There are three types of entry of drugs through the placenta, namely: Type 1, drugs with a balanced concentration between mother and fetus; Type II, drugs that have a concentration in fetal plasma is higher than the concentration in maternal plasma or an excessive transfer is happened. This may occur because of the flowing out transfer of fetal drug is slower; Type 3, drugs that have a concentration in fetal plasma is lower than the concentration in maternal plasma or an incomplete transfer is happened (Nindya 2001).

Fetal body weight tends to increase due to the tocopherol contained in P. conoideus var. yellow fruit.

Tocopherol is stored in the liver and fat tissue, so that if fetuses are deficient in E vitamin, the tocopherol may be used again soon. In addition, if there is damage to fetal cells, then the fetus can immediately make the recovery, because the fetal cells are still actively dividing, so damage to the cell can be easily replaced with another normal cell.

Normal levels of A vitamin in plasma is 100-120

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units/dL. The requirements of A vitamin in pregnant women is > 200 RE (Dewoto 2007). The content of A vitamin in P. conoideus var. yellow fruit is 240 ppm (240 mg/L), so that meet nutrient adequacy rate for pregnant women. While the requirement of E vitamin for pregnant woman is 10 mg. The content of E vitamin on P. conoideus var. yellow fruit is 10,400 ppm (10,400 mg/L). E vitamin acts as protective of fatty acids from free radical oxidation (Almatsier 2002), so the use of high concentration of E vitamin has no effect.

Average normal fetal body length on day 17th of gestation is 19.31 mm, whereas on day 18th is 20-23 mm (Kauffman 1992). Average length of fetuses in this study is 25 cm, so it can be said that the length of fetuses in this study was normal. Besides influenced by the external factors namely the inclusion of A and E vitamin in the body of the fetus, growth is also influenced by genetic factors (Wilson 1973). The decrease or increase in fetal body weight and length are associated with the genes contained in each individual and the room for its growth.

PKH (2009), states that the consumption of fat will affect the production of progesterone which is essential for implantation and as nutrients that are essential for the formation of early embryos. Number of embryos in the uterus also affects the availability of space for embryonic development and blood supply.

Fetuses which are derived from one uterine sac with a little amount of implantation have relative heavier and longer size than the fetus from the uterus with a lot of implantations. This relates to nutrition received by the fetus. The fewer the number of implantation in the uterus, the availability of nutrients for the fetus are met, so that the fetal weight and length will increase (Zahrah 2008). This can be seen in the control group with the largest number of implantation compared with treatment group. The control group tends to have average lowest body weight and length. The treatment group of 0.08 mL and 0.16 mL of extract that has the same number of implantation had nearly the same weight.

Percentage of intrauterine death and fetal life Fetal death or resorption is a form of intrauterine death.

Intrauterine death occurred due to the inability of cells to repair (recovery) to replace damaged cells with normal cells. This is probably because of the large number of damaged cells, so there is no balance between damaged cells with normal cells. Fetal cells are able to do recovery causing fetal survival. Based on the analysis of variance with a level of 95%, there are no significant differences for the percentage of live fetuses, fetal death and resorption between the control group and treatment group. This is evidenced by the value (P> 0.05). Fetal death in this study was at a dose of 0.02 mL of extract. Fetus is categorized into fetal death, when the fetus is fully developed and there are no signs of Autolisis, and it gives no respond to a touch (Hutahean 2002). The provision of P. conoideus var. yellow fruit on the parent does not affect fetal death, because the dosages are very small. In addition, A and E vitamin contained in P. conoideus var. yellow fruit are necessary for fetal growth. Fetal death in this study is likely

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caused by the process of cell division and disrupted cell differentiation, so the fetus can no longer continue its development or can be caused by severe functional abnormalities so that the fetus can not survive. In addition, the dead fetus in the womb has not finished its development, so it has smaller size compared to fetuses born alive (Setyawati 2009).

Fetuses which experience resorption are marked by a clump of red or yellow-brown color that does not respond when touched (Hutahean 2002). Resorption is a manifestation of the death of the conceptus (Lu 1995) that may occur due to improper body morphology with various disabilities and ended with the death (Rugh 1968).

Figure 9. The morphology of the fetus inside the mother of the control and that fed by stem extracts. The arrows show where resorption. (A) The uterus containing a normal fetus, (B) The uterus containing the fetus resorption, (C) The form resorption.

In this study, resorption presents in all treatment groups. Treatment group with the highest number resorption is found in 0.02 mL doses of extract (Table 4).

In the early stages of proliferation, the embryo will respond to die or grow normally, because at this stage, cell differentiation does not happen yet, so there is no selective effect of a teratogenic agent. This is because the cell is still totipotent, whereas if it occurs during cell intensive differentiation, mobilization and organogenesis, it will cause malformations or birth defects, but if it happens after the phase of organogenesis, it will cause abnormal function. If the effects of teratogens can not be handled by the embryo, it can cause embryonic death followed by abortion or resorption on rodensia if it occurs in early pregnancy, and fetal death in late pregnancy.

In addition, genetic plays an important role in the death in the phase of pre-implantation (resorption). Embryo death in pre-implantation stage often occurs due to inbreeding marriage. Before implantation, the embryo is more susceptible to the influence of genetic mutations and chromosomal abnormalities, followed by early embryonic

death (PKH 2009). Abnormalities of chromosomes can be distinguished on the number of chromosome abnormalities and chromosome structure. This can occur because of failure of the spread of chromosomes or arrangement of chromatin in cells that occur during meiosis and mitosis of the egg or sperm cells that produce 2 forms poliploid cells.

Aneuploid is chromosome abnormalities in animals that may occur due to the reduction of the normal chromosome number (2n-1), while poliploid is the addition of a normal chromosome number (2n+1). The abnormalities caused resorption.

Morphology of the fetus-hemorrhage There are 4 forms of embryo developmental disorders, namely death, deformity, growth inhibition and impaired function (Hutahean 2002). Overall, the fetal organs are fully developed in this study (body components are complete), although there are fetuses with less size than the others. In this research, there are three types of external abnormalities, such as: transparent skin, growth inhibition and humpbacked body (Table 5).

Transparent skin or which is often referred to hemorrhage is an event of blood discharge from the cardiovascular system which is accompanied by accumulation in tissues (Price and Wilson 1984 in Widiyani and Sagi 2001). Hemorrhage is a form of external abnormalities that often occur as an effect of a teratogen. In this study, Hemorrhage is found on the head, the neck, the back and the stomach.

Fetuses with transparent skin can be found in all treatment groups, including the control group (Figure 11).

This possibility occurs because the extract is given repeatedly in high enough doses, so there is high concentration in the blood, resulting in osmotic imbalance.

In normal circumstances, the embryo develops in the amniotic fluid that is isotonic with body fluids. The entry of foreign substances in tissue can alter the osmotic pressure. Osmotic imbalance can cause pressure and viscosity of liquid in different parts of the embryo, between blood plasma and extra capillary space or between extraembryonic and intra-embryonic fluid. This difference causes the blood vessels rupture and hemorrhage occurred (Wilson 1973).

In cases of hemorrhage, there is no difference between control (sesame oil provision) with treatment group (the provision of a mixture of sesame oil and extract). If the red blood cells are at hipotonic solution, ie solution in which concentration of dissolved substance outside the cell is lower than inside the cell, then the red blood cells are lysis (rupture). This is due to the absence of cell wall which may hinder the process of red blood cell lysis (Zulti 2008).

Table 5. Percentage of external fetal abnormality of R. norvegicus after the provision of extracts to the parent.

Types of external abnormalities 0 mL Number of fetuses 58 Hemorrhage/transparent skin 14 (24.14%) Barriers to growth 0 (0%) Body of humpback 0 (0%) Total 14 (24.14%)

0.02 mL 52 15 (28.85%) 1 (1.92%) 0 (0%) 16 (30.77%)

Dose extract 0.04 mL 44 4 (9.09%) 0 (0%) 3 (6.82%) 7 (15.91%)

0.08 mL 46 12 (26.09%) 0 (0%) 0 (0%) 12 (26.09%)

0.16 mL 44 6 (13.64%) 0 (0%) 0 (0%) 6 (13.64%)

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14 15 Figure 14. The development of fetal skeleton R. norvegicus as a result of the provision of 0.02 mL of P. conoideus var. yellow fruit extract on the stem. The arrows indicate the area which experiences delayed ossification. A) Skeleton control group: A1. Perfect ossification, A2. Transparent skin, perfect ossification. B. Skeleton of 0.02 mL extract treatment groups: 1. Ossification perfect, B2.

Delayed ossification of os interparietal, B3. Skeleton of stunted fetuses, delayed ossification in the frontal os, os parietal, os interparietal, cervical vertebrae, lumbar vertebrae, sacral vertebrae, tibia and fibula, B4. Skeleton of normal fetuses, delayed ossification of os parietal (a), os interparietal (b), and cervical vertebrae (c), B5. Skeleton of delayed ossification in the cervical vertebrae.

Figure 15. The development of fetal skeleton R. norvegicus as a result of the provision of 0.04 mL of the extract on the parent. A. Skeleton of the normal control group. B. Skeleton of groups of 0.04 mL extract: B1. Normal Skeleton, B2. Skeleton of delayed ossification in os interparietal

16 17 Figure 16. The development of fetal skeleton R. norvegicus from the treatment of 0.08 mL of the extract on the parent. A. Control. B. Skeleton 0.08 mL extract treatment groups: B1. Normal fetus, ossification perfect, B2. Fetus transparent skin, perfect ossification, B3.

Normal fetus, but delayed ossification of interparietal os (a) and cervical vertebrae (b), B4. Skeleton lordosis.

Figure 17. The development of fetal skeleton R. norvegicus from the provision of 0.16 mL of the extract on the parent. A. Skeleton control group. B. 0.16 mL extract skeleton groups: B1. Ossification perfect, B2. Skeleton delayed ossification in the lumbar vertebrae, B3. The delay in ossification of the cervical vertebrae (a), clavicula (b) and lumbar vertebrae (c).

Fetal morphology-growth retardation In this study, fetal which experienced growth inhibition is only in the treatment group of 0.02 mL of extract (Table 6). According to Ritter (1977), compounds with low doses of teratogens are capable of causing the death of some cells and can also cause cell turnover, because the fetal cells have high regeneration ability. If one or a group of cells are damaged by the interference of toxic agents, the normal cells around it will divide and replace the damaged cells.

The replacement of fetal damaged cells will be maintained during the period of organogenesis in order to form a normal morphology. If that fails or does not reach the target in the phase of organogenesis, it will cause fetal malformations, forming morphologically normal, but small.

Growth inhibition in the fetus may be caused by disruption of cell division, namely the nucleic acid and protein synthesis are disrupted and then the damaged cells can not be repaired. Fetal growth occurs because of cell proliferation by mitosis and proliferation rapidity is a function of growth velocity (Herbold 1985). In this study, growth inhibition on fetuses is caused by cell death due to the provision of extract as an anti-cancer which is given at the time these cells begin to actively divide, resulting in inhibition of cell division. If a foreign substance is given continuously, in the process of time, these cells will die. If

more and more cells die, it will be difficult for fetus to develop.

Fetal morphology-humpback body In this study, Humpback body was only found in group of 0.04 mL of extract treatment (Figure 13). Normal embryogenesis ended with the formation of new individuals that shape and structure is the same as its parent, but abnormal embryogenesis will end with the formation of individuals who vary (Wilson 1973).

Abnormal shape in the form of humpback body in this study is likely caused by a deformity of the vertebrae (spine) which is caused by the death of bone cells making up some vertebrae, resulting in bone growth rate of one to another is not the same, so the bone bends. Fitrianna (2009) states that the vertebrae are formed on day 12th . In this study, the extract is given to the parent from day 5th to day 17th. Presumably this cell death is caused by the extract given to the parent. Given that the immune response against the teratogens substance of each individual is different (Wilson 1973), the fetus with a low immune system is unable to repair cells, especially spine constituent cells which are damaged or die by the presence of substances such teratogens.

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perfect or not the ossification process, and the presence or absence of abnormalities in the formation of skeleton. Based on the observation, the results show the presence of delays in the process of ossification in all treatment doses ranging from lowest to highest dose (Table 6).

11 The development consists of increasing bone size, maturity and age.

Changes from the development of membranous and cartilaginous hard bone are called bone maturation. There are five periods of bone formation, namely: (i) the embryonic period: mandible, maxilla, humerus, radius, 10 12 13 ulna, femur, and fibia, (ii) fetal period: Figure 10. Morphologically normal fetuses of R. norvegicus. Note: 1. Pinnae, 2. Eye, scapula, illium, fibula, (iii) cartilage: 3. Vibrisae, 4. Mouth, 5. Anterior limb, 6. Posterior extremity, 7. Tail epiphisis on the limb, carpal, tarsal, and Figure 11. Comparison of fetal normal skin and fetal transparent skin after sesamoids; (iv) adolescent bones: administration of extract on the parent. Arrows indicate hemorrhage. (A) Fetal normal scapula, ribs, hip/waist, (v) adult bone skin, (B1) Fetus transparent skin doses of 0 mL; (B2) Fetus transparent skin dose of (Jessop 1988). Abnormalities of 0.02 mL; (B3) Fetus transparent skin dose of 0.04 mL; (B4) Fetus transparent skin dose skeletal malformations found in this of 0, 08 mL, (B5) Fetus transparent skin dose of 0.16 mL of extract. research is a form of lordosis skeleton Figure 12. Comparison of normal fetuses (A) with an experienced fetal growth which is found in 0.16 mL of extract inhibition (B) effect of extract to the parent. treatment group and delayed Figure 13. Comparison of normal fetuses (A) and fetal body humpbacked after ossification process found in all administration extract to the parent. treatment groups. However, the treatment group with deformity Internal fetal abnormality of white rats skeleton is mostly found in 0.02 mL of extract treatment The observation of internal abnormalities, namely the group (Table 6). observation on the development of fetal skeleton of R. norvegicus wistar strain is by making the wholemount Table 6. Percentage of deformity of fetal R. norvegicus skeleton preparation. The preparations were made using double as the result of the provision of the extract to the parent. staining, ie Allizarin Alcian Blue and Red-S. Alcian Blue and Red Allizarin-S is a special chemical substance for Dose of extract of fetal Amount of Amount Amount P. conoideus var. observed defects staining bone tissue (Inouye 1976). Alcian Blue will of parent of fetus yellow fruit fetus percentage perform affinity with the matrix in cartilage tissue, so the 5 58 20 0% cartilage will be stained in blue. While Allizarin Red-S will P1 0 P2 0.02 mL 5 52 12 33.33% perform affinity with the matrix in bone tissue, so the bones P3 0.04 mL 5 44 18 5.56% will be stained in red. The internal abnormalities observed P4 0.08 mL 5 46 21 9.52% included bone structure and results of its ossifications.

P5 0.16 mL 5 44 9 22.22% Skeleton comes from the mesoderm. In the mesoderm, In this study, the bones experiencing delays in differentiation occurs including mesoderm of head, body and tail which in this level of development is called ossification were found in the Cranium, cervical vertebrae, embryonic Mesenchyme. Mesenchyme develops into clavicula, lumbar vertebrae, sacral vertebrae, and tibia and mesoderm structures of the body, including connective fibula. The delay in ossification is blue in the fetal skeleton tissue namely cartilage (cartilage) and bone (Sagi 1997). and indicates that these bones are still cartilage (cartilage).

The results shows that 0.02 mL of the extract treated Calcification process occurs in two ways, namely intramembrane and endochondric ossification. Intra-membrane group experienced delayed ossification compared to other ossification is the process of bone formation of groups. This is most likely influenced by a lack of nutrients mesenchymal tissue into bone tissue, such as the formation to the mothers of white rat, so that the metabolic processes of lamellar bone. While ossification endokondral the bone are inhibited. It is indicated by the presence of very light formation process that occurs in which mesenchymal cells fetal weight compared to fetal weight of other groups, first differentiate into cartilage (cartilage tissue), then namely 0.7 g. The most important nutrient for growth and transformed into bone tissue, such as the formation of long development of bone is calcium (Dewoto 2007). According Setiyohadi (2009), calcium holds two important bones, vertebrae and pelvis.

According Loegito et al. (1995) in Ekawati (2002), physiological roles in the body. In bone, calcium salts play there are three benchmarks to determine the growth and a role in the calcification process, so the bones become development of the skeleton, namely: number of harder. Hardening of the bone serves to sustain weight loss. components of skeleton and their ossification level, the While in the extracellular fluid and cytosol, calcium plays a

MUNA et al. â&#x20AC;&#x201C; Teratogenic test of Pandanus conoideus extract

role in various biochemical processes of the body in the form of calcium ions.

Norazlina et al. (1999) states that the consumption of 1% of calcium in rats with low E vitamin can increase bone mineral density. The body normally contains 1100 g of calcium for body weight of 70 kg. If the dose is converted in rats weighing 200 g, then there are 3.14 g of calcium in the body of rats. Yuliati et al. (2007) revealed that the addition of 27 mg/200 g BW/day can increase the thickness of the trabecular bone. Histologically, the bone is divided into 2, namely cortical bone and trabecular bone. Cortical bone has a mechanical and protective role, whereas trabecular bone is metabolical. Bone thickness indicates the strength of the bone, because in thick bone, there is an abundant amount of minerals.

Calcium in P. conoideus var. yellow fruit was 15.3 mg/100 g of material. The dose is very small compared to the intake of calcium that is used in research by Yuliati et al. (2007). Calcification is the deposition of calcium salts and phosphorus in bone matrix. Vitamin D is also needed in this process. Vitamin D is a prohormone, so that if the body does not get enough sunlight, D vitamin needs to be met through food. The conversion of D vitamin into active vitamin D3 (calcitriol) occurred in the kidney. In the intestine, calcitriol can increase the absorption of calcium and phosphorus. The calcitriol synthesis is regulated by calcium and phosphorus levels in the blood. When blood calcium levels are low, parathyroid hormone will stimulate the kidney to produce calcitriol. Role of vitamin D3 in the process of bone calcification is by making the calcium and the phosphorus available in the blood to be precipitate in the bone matrix. Because the amount of calcium used is not sufficient with the levels of calcium in the blood, the calcification process is inhibited. As a result, the bones still have the quality of cartilage.

There are two primary metabolisms in bone formation that are vulnerable to nutritional deficiencies, namely: the synthesis protein process to form the organic matrix of bone tissue consisting of collagen and non collagen protein.

The next process is the bone calcification, in this stage; minerals such as calcium and phosphorus are precipitated in the bone matrix. If there are obstacles in the formation of organic matrix, then there will be obstacles in the process of calcification of bone resulting in decreased levels of bone minerals, including calcium and phosphorus. The occurrence of bone calcification resistance will lead to obstacles in the formation of osteoclast cells (Setiyohadi 2009).

Bone regeneration was influenced by two cells, namely osteoclasts and osteoblasts. Osteoclasts play role in bone reconstruction using acids and enzymes (bone resorption); while Osteoclasts play a role in the formation of new bone to replace the old bone which is dismantled by Osteoclasts (Rebecca and Brown 2007). Osteoclasts are motile cells.

These cells will do resorption on bone to form lacuna, then they will move to another part of the bone. At the time of osteoclast cells move, resorption of bone does not occur.

But when the osteoclast cell stops moving, then the process of resorption happens. The increase of bone resorption causes a reduction in the amount of bone.

Santoso (2004), in his study, states that a teratogen

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agents can affect the thickness of cells from the femur of mice. The layers on the femur of mice become thinner and thinner, and some even die. Teratogen agents are accumulated in several organs, especially the organ that is undergoing calcification which would result in abnormalities in fetal development. This is caused partly because the fetus does not have enzymes that can metabolize these toxic agents perfectly.

The presence of fetal skeleton with perfect ossification is due to internal factors, namely the hormone that maintains bone mass. Rebecca and Brown (2007) states that the hormone is one of the factors that influence the bone to be strong or not. Hormones are natural substances produced by specialized cells in the body. Hormones circulate in the bloodstream and can affect cell activity at various places in the body. In addition, the hormone can also help limiting the amount of bone resorption, because it regulates calcium levels in the blood. This is clarified by the study of Masyita (2006), that lack of estrogen can cause osteoporosis in mice. Osteoporosis occurs due to loss of bone mass and increased bone absorption. Estrogen is assessed to affect the process of bone destruction by inhibiting cytokine production. Estrogen plays an important role in the process of homeostasis, namely supporting the secretion of calcitonin, as an inhibitor resorption bone and can increase levels of D3 vitamin which serves to increase the degree of calcium absorption in the intestine. Low cytokines may decrease osteoclast activity that plays a role in the overhaul of the bone. Cytokines are proteins that play a role in the process of bone resorption. There are two kinds of cytokines, namely IL-1 and IL-6. IL-1 plays a role in stimulating bone resorption, replication of bone cells and increase synthesis of IL-6. IL-6 also plays a role in undergoing resorption bone by cells to activate osteoclasts.

Synthesis of IL-6 would be inhibited by estrogen, so the intake of estrogen can reduce bone resorption (Norazlina et al. 2007). There is a relationship between estrogen with E vitamin, namely E vitamin which is a fat soluble vitamin may be converted into cholesterol, while cholesterol is the precursor of estrogen that its construction is through a series of enzymatic reactions.

Shuid et al. (2010) stated that E vitamin has potential as an anabolic agent in bone and can improve bone strength.

In addition, E vitamin can improve bone structure.

Anabolic agents are agents that can improve bone strength by increasing bone mass. In addition, E vitamin also acts as anti-osteoporotic agents (Nazrun et al., 2010). Tocopherol contained in P. conoideus var. yellow fruit is E vitamin, which plays a role in bone formation. Tocopherol along with calcium and vitamin D plays a role in bone metabolism process. Calcium plays a role in increasing bone mineral density, thus reducing resorption activity on bone. E vitamin can increase the density of calcium and D3 vitamin is required for calcium absorption in intestine and for calcium storage in bones (Norazlina et al. 1999).

Xu et al. (1995) stated that E vitamin can stimulate the growth of trabecular bone. Lack of E vitamin can reduce the transport of calcium in intestine. E vitamin intake of 10-30 mg/day is sufficient to maintain normal levels in the blood (Dewoto 2007). Norazlina et al. (1999), states that

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the use of E vitamin by 60 mg/kg BW/day can increase the calcium content in rat femur. The content of tocopherol in P. conoideus var. yellow fruit is 10,400 ppm (10,400 mg).

Excess tocopherol will not be discarded, because tocopherol is stored in the liver and adipose tissue in the form of glycerol and it can be reused by the body any time.

CONCLUSION Morphometry of white rats fetus changes from control to highest dose treatment group. There are fetuses that have: mortality and growth inhibition at doses of 0.02 mL extract; resorption in all treatment groups, ie at a dose of 0.02 mL, 0.04 mL, 0.08 mL, and 0.16 mL extract; or transparent skin presents in the control and in all treatment groups. The extract can cause fetal abnormalities in the skeleton structure of the white rat. Abnormalities in the form of lordosis skeleton is found at a dose of 0.16 mL of the extract and the occurrence of ossification barriers can be found in all treatment groups.

REFERENCES Alatas Z. 2005. Teratogenic effects of ionizing radiation. Bul Alara 6 (3): 133-142. [Indonesia] Almatsier S. 2002. The basic principle of nutrition science. Gramedia.

Jakarta. [Indonesia] Astirin OP, Harini M, Handajani NS. 2009. The effect of crude extract of Pandanus conoideus Lamb. var. yellow fruit on apoptotic expression of the breast cancer cell line (T47D). Biodiversitas 10 (1): 44-48.

Azman A, Kholid BAK, Ima-Nirwana S. 2001. The effects of vitamin E on bodyweight and fat mass in intactand ovariectomized female rats.

Med J Islamic Acad Sci 14 (4): 125-138.

Briggs GG, Freeman RK, Yaffe SJ. 2008. Drugs in pregnancy and lactation; a reference guide to fetal and neonatal risk. 8th ed.

Lippincott Williams & Wilkins. Philadelphia.

Budi IM. 2000. Study nutrients and physical-chemistry properties of various types of red fruit (Pandanus conoideus Lam.) oil traditionally extracted in Jayawijaya, Irian Jaya Province. [M.Sc.-Thesis].

Graduate Program, Bogor Agricultural University. Bogor. [Indonesia] Budi IM, Paimin FR. 2005. Red fruit. Penebar Swadaya. Jakarta. [Indonesia] PKH. 2009. Causes of early embryonic death in cattle. Center for Animal Health. http://www.vet-klinik.com. [Indonesia] Dalimartha S. 2004. Early detection of cancer and anticancer simplisia.

Penebar Swadaya. Jakarta. [Indonesia] Dewoto HR. 2007. Pharmacology and therapy: vitamins and minerals. 5th ed5. Faculty of Medicine, Indonesian University. Jakarta. [Indonesia] Foye WO. 1996. The principles of medicinal chemistry. Gadjah Mada University Press. Yogyakarta. [Indonesia] Ganiswara SG. 2001. Pharmacology and therapy. Gaya Baru. Jakarta. [Indonesia] Gysin R, Azzi A, Visarius T. 2002. α-Tocopherol inhibits human cancer cell cycle progression and cell proliferation by down regulation of cyclin. FASEB J 16: 1952-1954. [Indonesia] Harmanto N. 2001. Healthy with a traditional herb medicine heirloom crown god of the gods. Agro Media Pustaka. Jakarta. [Indonesia] Herbold B. 1985. Micronucleus test on the mouse to evaluate for mutagenic effects. Report No. 14102E (Study Nos. T6019042 & T8019675). Institute of Toxicology, Pharmaceutical Division, Bayer AG, Wuppertal, Germany. Submitted to WHO by Bayer AG, Leverkusen, Germany (report No. 74164).

Hidayati MN. 2010. Cytotoxicity test of P. conoideus Lam. var. yellow fruit in vitro against Hela cell growth and chemical profile of the most active. [Thesis]. Department of Biology, FMNS, Sebelas Maret University. Surakarta. [Indonesia] Hutahean S. 2002. The principles of toxicology development test.

Laboratory of Animal Structure, Department of Biology, FMNS, North Sumatra University. Medan. [Indonesia] Inouye M. 1976. Differential staining of cartilage and bone in fetal mouse skeleton by Alcian Blue and Allizarin Red S. Congenital Anomalies J 16 (3): 171-173.

Jessop NM. 1988. Theory and problem of zoology. B&JO Entreprise. Singapore.

Kauffman MH. 1992. The atlas of mouse development. Academic Press.

New York.

Lu FC. 1995. Basic toxicology: principles, target organs and risk assessment. UI Press. Jakarta. [Indonesia] Masyita D. 2006. Microscopic structure of mandibular bone in ovarectomic rats and feeding ratio phosphate/calcium high. Media Kedokteran Hewan. 22 (2): 112-117. [Indonesia] Mun‘im A, Retnosari A, Heni S. 2006. Constraints test tumorigenesis Red Fruit (Pandanus conoideus Lam.) juice against female White Rats induced by 7,12 Dimetilbenz (a) Antrasen (DMBA). Majalah Ilmu Kefarmasian 3 (3):153-161. [Indonesia] Nazrun AS, Norazlina M, Norliza M and N S Ima. 2010. Comparison of the effects of tocopherol and tocotrienol on osteoporosis in animal models. Intl J Pharmacol 6 (5): 561-568.

Nindya S. 2001. Changes of pharmacokinetics drugs in Pregnant Women and its clinical implication. Cermin Dunia Kedokteran 133: 40-43.

[Indonesia] Nogrady T. 1992. Medicinal chemistry: The biochemical approach.

Bandung Institute of Technology. Bandung. [Indonesia] Norazlina M, Ima-Nirwana S, Khalid BAK. 1999. Effect of palm vitamin E, vitamin D and calcium supplementation on bone metabolism in vitamin E deficient rates. Med J Islamic Acad Sci 12 (4): 89-96.

Norazlina M, Lee PL, Lukman HI, Nazrun AS, Ima-Nirwana S. 2007.

Effect of vitamin E supplementation on bone metabolism in nicotinetreated rats. Singapore Med J 48 (3): 195-199.

Pergament E, Schechtman A, Curell C. 1996. Vitamin A and pregnancy.

ITIS Newslett 4 (6). http://fetal-exposure.org.

Pratiwi AP. 2008. Cytotoxicity test of mixture extract of Pandanus conoideus Lam. var. yellow fruit and lauric acid of VCO to T47D breast cancer cells in vitro. [Thesis]. Department of Biology, FMNS, Sebelas Maret University. Surakarta. [Indonesia] Rebecca F, Brown P. 2007. Simple guides to osteoporosis. Penerbit Erlangga. Jakarta.

Ritter EJ. 1977. Altered biosynthesis In: Wilson JG, Fraster FC (eds).

Hand book of teratology. Plenum Press. New York.

Rugh R. 1968. The mouse, its reproduction and development. Burgess Publising. Minneapolis.

Russel RM. 2002. Betacarotene and lung cancer. Pure Appl Chem 74 (8): 1461-1467.

Sagi M. 1997. Comparative embryology of vertebrates. Faculty of Biology, Gadjah Mada University. Yogyakarta. [Indonesia] Santoso HB. 2004. Skeleton anatomical abnormalities of mice due to defects of caffeine. Bioscientiae 1: 23-30. [Indonesia] Setiyohadi B. 2009. The role of calcium and vitamin D on bone metabolism. Subsections of Rheumatology, Department of Medicine Faculty of Medicine/RSCM. Jakarta. [Indonesia] Setyawati I. 2009. Morphology of fetal mice (Mus musculus L.) after administration of bitter leaf extract (Andrographis paniculata Nees. J Biologi 13 (2): 41-44. [Indonesia] Shuid AN, Zulfadli M, Norazlina M, Norliza M, Ima-Nirwana S. 2010.

Vitamin E exhibits bone anabolic actions in normal male rats. Bone Miner Metab J 28: 149-156.

Suryawati S. 1990. Use of drugs in pregnancy. Laboratory of Clinical Pharmacology, Faculty of Medicine, Gadjah Mada University.

Yogyakarta. [Indonesia] Takekoshi S. 1964. The mechanism of vitamin A induced teratogenesis. J Embryol Exp Morph 12 (2): 263-271.

Tjay TH, Rahardja K. 2002. Properties of essential drugs, the use and side effects. PT. Elex Media Komputindo. Jakarta. [Indonesia] Wilson JG. 1973. Environment and birth defects. Academic Press. New York.

Wiryanta BTW. 2007. Wonders of red fruit; testimony of those who healed. http://www.deherba.com/khasiat-buah-merah.html. [Indonesia] Xu H, Watkins BA, Seifert MF. 1995. Vitamin E stimulates trabecular bone formation and alters epiphyseal cartilage morphometry. Calcif Tissue Int 57: 293-300.

Zahrah S. 2008. Teratogenic effects of water extract of sarang semut (Myrmecodia pendens Merr. & Perry) in rats (Rattus norvegicus L.) organogenesis phase of wistar strain. [Thesis]. Department of Biology, FMNS, Sebelas Maret University. Surakarta. [Indonesia] Zuhud EAM. 2009. Development of pharmaceutical ethnoforestry in Indonesia. Plant Biodiversity Conservation Section, Faculty of Forestry, Bogor Agricultural University. Bogor. [Indonesia] Zulti F. 2008. Mechanism of transport through the membrane. http://www.crayonpedia.org. [Indonesia]

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Vol. 2, No. 3, Pp. 126-134 November 2010

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 135-140 November 2010

Histological study of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura larvae YAYAN SANJAYA1,♥, DADANG MACHMUDIN², NANIN DIAH KURNIAWATI² ¹Biology Program, Educational University of Indonesia (UPI). Jl. Setia Budhi No. 229, Bandung 40154, West Java, Indonesia; Tel./Fax.: +62-22-201383; email: yayan229@yahoo.com Manuscript received: 21 Augustus 2010. Revision accepted: 8 November 2010.

Abstract. Sanjaya, Machmudin D, Kurniawati ND. 2010. Histological study of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura larvae. Nusantara Bioscience 2: 135-140. The effect of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura Fab. larvae has been carried out. The method was used Probit analysis, and based on LD 50 the virus was infected to know body weight and post infection damage.The damage of histological structure caused by SlNPV (0, 315, 390, 465, 540 dan 615 PIB/mL) was investigated after 0, 12, 24, 72 and 96 hours post infection. The histological material was prepared by using parafin method after fixation with Bouin Solution, then slice into 7 um and colored with Hematoxilin-Eosin. The result showed that the exposure SlNPV cause decreasing food consumption especially on 540 PIB/mL give average rate as amount of 0.1675 mg. The descriptive obsevation on structural intact of peritrophic membrane histology caused by SlNPV infection shows a tendency to decrease, while in control, there was no damage at all. The longer the exposition of virion in the midgut lumen the more damage on peritrophic membrane occurred. The severest damage occurred 96 hour after infection. The result prove that haNPV virion can destroy hystological structure of midgut.

Key words: SlNPV, Spodoptera litura, LD50, consumption rate, peritrophic membrane.

Abstrak. Sanjaya, Machmudin D, Kurniawati ND. 2010. Kajian histologis infeksi SlNPV terhadap berat badan dan kerusakan membran peritrofik larva Spodoptera litura. Nusantara Bioscience 2: 135-140. Pengaruh infeksi SlNPV pada berat badan dan kerusakan membran peritrofik larva Spodoptera litura Fab. telah dilakukan. Metode yang digunakan adalah analisis probit, dan berdasarkan LD 50 virus yang terinfeksi untuk mengetahui berat badan dan kerusakan pasca infeksi. Kerusakan struktur histologi yang disebabkan oleh infeksi SlNPV (0, 315, 390, 465, 540 dan 615 PIB/mL) diamati setelah 0, 12, 24, 72 dan 96 jam pasca infeksi. Preparasi histologis dibuat dengan metode parafin setelah fiksasi dengan larutan Bouin, kemudian diiris setebal 7 um dan diwarnai dengan HematoxilinEosin. Hasil penelitian menunjukkan bahwa paparan SlNPV menyebabkan penurunan konsumsi pangan terutama pada 540 PIB/mL dengan rata-rata 0.1675 mg. Pengamatan deskriptif pada struktur histologi membran peritrophic yang terkena infeksi SlNPV menunjukkan kecenderungan kerusakan, sementara pada kontrol tidak ada kerusakan sama sekali. Semakin lama paparan virion di dalam lumen midgut maka semakin tinggi terjadinya kerusakan membran peritrofik. Kerusakan paling parah terjadi 96 jam setelah infeksi. Hasilnya membuktikan bahwa virion haNPV dapat menghancurkan struktur histologi midgut.

Kata kunci: SlNPV, Spodoptera litura, LD50, tingkat konsumsi, membran peritrophic.

INTRODUCTION One of the main concepts of IPM is to maintain populations of pests insect so they will not exceed the economic threshold and keep other animals so as not to interfere, so that the balance of ecosystems is maintained (Bonning and Hammock 1996). These organisms are pathogenic naturally against insect larvae with a specific target so as not to interfere with insect species and other non-target species. In addition, these agents are very virulent, easily spread in the population and can be persistent in the long term if environmental conditions allow (Teakle et al. 1994).

Some viruses that attack the plant are able to kill insect pests, such as members of the genus Baculovirus often called Nuclear Polyhedrosis Virus (NPV) (Yamagishi et al.

2003 Maramorosch 2007). Moscardi (1994) states that the

application of baculovirus is quite effective as an insect control agent for pest Lepidoptera. NPV can attack several leaf-eating Lepidoptera larvae which destroyed many crops. Until now about 700 viruses have been isolated and identified from insects and other arthropods animals. These arthropod viruses mostly belong to six genera namely virus Baculovirus, Poxivirus, Iridivirus, enterovirus and Rhabdovirus (Cristian 1994). According Barbehenn and Marin (1994), Baculovirus specifically works to and infects several species of insects, usually from the same family.

NPV can suppress the Spodoptera exigua caterpillar attack (SeNPV) which attacks the onion leaves for up to 84% (Sutarya 1996). The use of NPV in tomato plants suppresses Heliothis sp. caterpillars attack for up to 65% and save the lost crop yields up to 83% (Novizan 2004).

This virus is a deadly pathogen because it can damage the peritrophic membrane in the area of middle intestine of

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Lepidoptera insect species. Granados and Corsaro (1990) argued that when the virus infects the middle of the insect intestine, the histological structure of the peritrophic membrane which plays very vital role in the digestive process is estimated to be defective, so that the digestive process disrupted and eventually reduce the weight of larvae.

Researches on the presence of peritrophic membrane against pathogen attack in several Lepidoptera larvae have been reported. Among these are the defense mechanisms of Trichoplusia ni larvae against viral infection by forming peritrophic membrane (Wang and Granados 1998).

Glossina morsitans morsitans larvae also form a membrane-peritrophic against Trypanosoma infection (Lehane and Msangi 1991). According to Utari (2000), the integrity of peritrophic membrane histological structure on Helicoverpa armigera larvae due to HaNPV infection decreases with increasing doses of infection.

Research on NPV infection of some Lepidoptera insect larvae has been reported, but research on the effect of NPV on peritrophic membrane area in the middle intestine of larvae of S. litura is rare. This study aims to determine the effect of SlNPV infection on body weight and the damage to peritrophic membrane on the instar-5 larvae of S. litura.

MATERIALS AND METHODS Determination of Lethal Dose 50 (LD50) This study used 5 doses of treatment with 1 type of dosage control. The dose was determined based on the range of LD50 with 95% confidence level. This experiment used 6 test larvae for each treatment with 4 replicates; observations were made until the pre-pupa stage. Stages of viral insecticides are not different from the methods performed on the initial study.

Spodoptera litura Nuclear Polyhedrosis Virus (SlNPV) is given to the test larvae through the feeding method with food that has been poured with virus suspension for as much as 10 μL of doses that have been determined.

Previously, the larvae are given no food for 24 hours. The next day, all larvae were given fresh food without SlNPV suspension. For control, larvae fed with no virus suspension addition, but distilled water.

Every day for 8 days, the death larvae observed in each treatment is accounted. The time needed by the virus to interact with its host takes several days from the onset of infection until the larvae die.

Observation of larval weight This observation Is aimed to determine whether there is a larval weight change due to SlNPV infection in the larvae of Spodoptera litura. This observation began with separating the last four instars larvae that had stopped eating. If skin had changed, then the body weight of larvae was weighed and then separated individually in each zalp bottle with a piece of food that had been given with virus suspension. The observation in this phase was ended when the mortality observations ended. Furthermore, the final weights of larvae were weighed again.

Histological preparations The making of an incision across the membrane peritrophic is based on Utari (2000) which has been modified. The middle intestine of S. litura instar-5 larvae was taken at the time of 0, 24, 48, 72, 96 hours after infection using an infection dose based on LD50 values of SlNPV delivered from research, with a sharp razor blade.

To obtain a good piece, larvae were snared in Bouin's solution on the bearing candles. Part of front intestine and rear colon is pinned with a needle. About five minutes after soaking, the dead larvae were removed from the bearing candles, and their middle colons were taken. Furthermore, the middle intestines were fixed in Bouin's solution for 24 hours. The next day, the dehydration process was made with graded alcohol, namely: 70%, 80%, 90%, 96%, and 100%, respectively, each for three hours.

The Organs purification was done by soaking the object in alcohol 100%, i.e. xilol. Organs that have been clear then infiltrated in paraffin at a temperature of 48°C for 30 minutes, 52°C for 60 minutes, and 56°C for 90 minutes.

Furthermore, organs were put into paraffin until frozen, then were sliced crosswise for as thick as 8 um. Incision ribbon was affixed to the glass objects that have been given Mayer's albumin adhesive. Then HE staining was performed by immersing the object for 40 minutes in pure xilol, alcohol 100%, 96%, and 80% respectively for 3 minutes, 70% alcohol for 30 minutes, dye HE for 2 min, water 6 minutes, alcohol 70% + 3 drops of HNO3 twice dyeing, alcohol 70% and 80% respectively for 3 minutes, eosin Y for 25 minutes, alcohol 96% as much as 3 times dyeing, alcohol 100% as much as two times each for 6 minutes, pure xilol for 10 minutes, then they were given with glue and covered with glass cover.

Peritrophic membrane observation In observation of this study, data analysis was done descriptively. The result of cross-sectional and longitudinal incision of the intestine was observed under the microscope. Observation of peritrophic membrane structure on the value of LD50 was performed at 0, 24, 48, 72 and 96 hours after infected by SlNPV. Determining the level of damage from SlNPV infection was done descriptively based on the presence or absence of peritrophic membrane damage, regenerative cells and basal membrane.

Data analysis To determine the doses that can cause death on insect by 50% (LD50), the number of individual larvae of S. litura who died of infection are recorded every day SlNPV and its repetition until the pre-pupa stage. Furthermore, mortality data were analyzed using Probit analysis of the Polo-PC (Utari 2000). The calculation of the weight of larvae used ANOVA test by meeting the requirements that it should be normal and homogeneous. Homogeneity test used Bartlet test and continued with normality test.

If in the control treatment larval mortality was found, the mortality data corrected using Abbot's formula (Busvhine 1972), namely:

SANJAYA et al. – SlNPV influence on body weight and peritrophic membrane

Pt =

Po − Pc X 100% 100 − Pc

Pt : Percentage of dead insects after corrected (%) Po : Percentage of dead insects for each treatment insects Pc : Percentage of dead insects in the control

RESULTS AND DISCUSSION The result of observation on Lethal Dose (LD) of instar5 S. litura larvae is listed in Table 1.

Tabel 1. The value of LD10, LD50 and LD90 of the instar-5 larvae of S. SlNPV litura at different doses.

LD

LD values

95% Fiducial limit

10 277 156.26-341.63 50 438 365.06-491.06 90 692 595.39-1011.18 Note: The fiducial limits of 95% indicate the range of the upper and lower limit of the SlNPV insecticide obtained by Probit PoloPC analysis with 95% of confidence level.

From the daily observed mortality data, it is found out that the early symptoms on S. litura larvae which were infected by SlNPV appear within 24 hours after treatment (day 1st ) with LD 50 values of 438/Inclusion Body (OB) and has a range from 365.06 – 491.06 / OB. As stated by Rohrmann (1994) that in order to create an effect in the host, the amount of inclusion body needed is about 50thousand per insect. From the number of doses administered during the treatment which was 315 – 615 PIB / mL, it can be seen that the amount has been met the dosage range that can cause the virus to infect its host insect within the body. This is demonstrated by the response of larvae mortality occurred.

In addition to the amount of PIB, mortality of larvae of S. litura can also be influenced by the stage of larval development, temperature and insect species (Christian 1994). According Dibyantoro (1996), the optimum temperature for larval development are 23-24°C with relative humidity of 60-65%, while based on the results of measurements during the study at the Laboratory of Animal Structure-UPI Bandung, it is obtained the temperature range between 24-26°C with relative humidity of 59 - 67%.

Environmental factors are still within the normal range for larval development so it is unlikely if these factors affect the mortality of larvae of S. litura.

In addition to the number of incoming PIB, temperature and insect species can affect the mortality of larvae, the development stage factors are also contributed.

According Gothama et al. (1990); Laoh et al. (2003), the organs of young larvae are still weak, especially the middle intestine which is the primary target of pathogen attack, so the NPV is more easily penetrate these organs and damage its vulnerable cells. While in advanced instar larvae, the sensitivity of larvae would be reduced along with the development of body, weight and age of larvae. The organs

137

and tissues of larvae grow and create differentiation.

Intestinal wall, peritrophic membrane and integument become thicker and stronger, so they are more difficult to be penetrated by the NPV. The mechanism of infection to cause death in larvae begins with swallowing polyhedra that enter with food into the body of insects which will then be digested in the middle intestine of the insect. Then the membrane that covers polyhedra will be dissolved in the middle intestine of the insect because of the alkaline conditions in that area.

The next step, the virion will do the fusion with the plasma membrane and penetrate the peritrophic membrane or epithelial cells of the middle intestine which is the primary target of NPV infection. In the cytoplasm, virions will release the nucleocapsid. The remaining Nucleocapsids will then enter the nucleus while releasing its DNA and form virogenic stroma. In this condition, these virions replicate or reproduce themselves in the host cell nucleus.

Furthermore, these infected cell nucleuses grow, and then lysis and release new virions derived from viral replication.

When the infection occurs continuously, this will damage the entire intestinal tissue and the conditions in the hemolymphs tissues seem turbid because they are full of NPV fluid resulted from replication of newly formed virions within hemosol (body cavity) and other tissues such as epidermis cells, fat cells and trachea (Bedjo et al. 2005).

Infected tissue is filled with virions that cause the cells to lysis. Finally, in the advanced stage, larvae will die after the majority of their tissues are infected by NPV. From the observation of symptoms caused by insect larvae after being given the treatment, it appears that there was a change in the body after the insects were infected by SlNPV. The description of changes in the initial symptoms until the larvae died in all treatments is relatively the same, except the controls. At first the larval body was red, especially on the abdomen and shiny, then swell, finally the larvae will be lazy to move and its appetite decreased (Wigglesworth 1984). Feels soft to the touch and it excretes fluid from its body which is turbid and smelly. These symptoms are relatively similar to the symptoms caused on Helicoverpa armigera larvae infected with NPV (Laoh et al. 2003). On the other hand, the deaths in the controls showed no symptoms which are mentioned earlier.

Morphological condition of larvae remains the same, but the larvae are reluctant to eat, it appears from the remaining food that is different from the other larvae.

In addition to the ability of the NPV as virus for insect in killing its host as a direct response of its biological activity, it turns out that the impact or consequences arise indirectly to the infected larvae were also seen in larvae that successfully passed to live with the response of "debilitating effect" (Pawana 2000). This impact is the changes of host quality, such as the level of sensitivity to other pathogens, the decrease of reproductive capacity, fertility, sex ratio and a smaller body size.

Insects as the host of baculovirus certainly have the potential to perform self-defense against pathogen attack (Blum 1985). Besides, they can also develop a tolerance level (Sanjaya 2000). In adult insects, adverse effects due to pathogen attacks are treated with the apoptosis response.

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Response can prevent the virus to multiply and prevent the spread of infection throughout its body.

In addition to the larvae that die due to viral infection, there are larvae stays alive although they were given the same treatment with SlNPV insecticide. This can occur because of several factors, including the ability to perform selfdefense against pathogen attack. According to Blum (1985), a low dose of single cell pathogens such as viruses and bacteria will be responded by the phagocytosis immune, whereas in high doses, the response that occurs is the formation of nodules. Hemocyte that functions as a phagocyte is plasmosit (Wigglesworth 1984). Even in its development, the individual host can release itself from the effect of NPV infection with the mechanism of "maturation resistance" that is in line with the increasing age, the increasing resistance or the decreased sensitivity to the NPV would be obtained (Kurnia et al. 2002).

Gambar 1. Pengaruh dosis SlNPV terhadap berat larva instar 5 Spodoptera litura.

Observation of larval weight of S. litura Based on the observation of larval weight of S. litura that has been done, it seems that there is a correlation between the effects of SlNPV infection and weight larvae. The more and more doses provided to the test larvae, the consumption rate of larvae decreases causing the weight decrease on test larvae (Figure 1).

After the calculation of the weight of S. litura larvae, the result is obtained that the SlNPV infected larvae looses its body weight. This decrease can be caused by the transfer of energy, where the energy that would be used for metabolic activity and growth is converted to counter attack pathogens entering the body (Pawana 2000). The results also show that larvae treated with a dose of SlNPV, on next day, when given a new food that is not contaminated, it tends to be quiet and does not eat the food directly. If it is done continuously, the larvae will die. The larvae rejection is presumably because SlNPV consumption through food has led to the change of food quality.

Changes in the quality of food, of course, can inhibit the eating activity of larvae and even the larvae can be inert. In low doses, the larvaeâ&#x20AC;&#x2122;s feed consumption is still normal so the rejection response is rare. This is because the number of pathogens coming to the body is still below the threshold to be able to cause infection in the larval body. In this condition, the presence of peritrophic membrane as one of the body's defense mechanism still functions optimally in defending the attack of pathogens.

At higher doses, the larvae begin to show the response of inertia to eat the provided food. It is assumed that when the body has been attacked by pathogens, and the body will respond with her body's defense system. But at a certain time, larval body's defense system will decline. It is mentioned before that the protease enzyme secretion from intestinal epithelium has a central role in virus resistance (Bolognesi et al. 2002). Larvaeâ&#x20AC;&#x2122;s activity still runs optimally when the body is not susceptible to interference, but when pathogen attacks, the activities will be disrupted

resulting in the metabolic process to be not optimum. Thus, if most of the tissues have become infected, then along with the decrease in the body's defense system, the rate of feed consumption will decline; even larvae will stop eating at all that causes a decrease in weight of larvae and the larvae eventually die.

According to Blum (1985) the main target of NPV infection is the area of the middle intestine of insects which are the main digestive organ because it serves as an absorber of nutrients and secretion of digestive enzymes.

Therefore, if the intestine is damaged then the activity of digestion will be disrupted. Thus, the metabolism becomes inhibited. Based on this, allegedly baculovirus infection can reduce the weight of larvae.

Histology of peritrophic membrane From the results of earlier studies, it is obtained that doses which are capable of causing 50% death of S. litura larvae (LD50) is 465 PIB / mL. At the LD50 value, a postinfection incision for 0, 12, 24, 48, 72 and 96 hours is made (Table 2 and Figure 2).

Table 2. The damage level to the intestinal area instar-5 larvae of S. litura.

SlNPV infection time at a dose of 438 PIB / Middle Ml n (%) intestinal regions 0 24 48 72 96 Peritrophic - 3/10 (30) 5/21 (24) 5/9 (55) 3/5 (60) membrane Regenerative 6/20 (30) 7/15 (47) 7/11 (64) cells Alkaline 3/19 (16) 5/15 (33) membrane Note: from the list, it can be grouped into 4 categories as follows: A. no damage (0%), B. minor damage (10-29%), C. medium damage (30-59%), D. major damage (60-79%).

SANJAYA et al. â&#x20AC;&#x201C; SlNPV influence on body weight and peritrophic membrane

139

MP MP

MP SR MB

SR SR MB MB

A

C

B

MP SR

MP SR

MB MB

D

E

Gambar 2. Kerusakan struktur membran peritrofik akibat infeksi SlNPV pada selang waktu tertentu. A. 0 juam, B. 24 jam, C. 48 jam, D. 72 jam, E. 96 jam. Keterangan: MP: membran peritrofik, SR : sel regeneratif, MB: Membran basal.

Based on histological observations of the middle intestine of S. litura larvae at 0 hour, it turns out that peritrophic membrane structure still appeared intact; constituent tissues of the middle intestine was still in normal condition. Results of histological incision on abdomen of S. litura larvae after treatment for 24 hours show the damage to the outermost layer of the middle intestine of larvae which was peritrophic membrane. When compared to the controls, the profile of membrane peritrophic in this treatment begins to disintegrate towards the lumen of middle intestine. Furthermore, after 48 hours of SlNPV infection, the spread of the virus in the body of the insect larvae begins to enter the deeper areas, where the cells making up the middle intestine (regenerative cells) begin to experience degradation so it seems to move toward the intestinal lumen. After 72 hours of treatment, the tissue damage level in the middle intestine began to spread, until reaching the basal membrane. Damage of these cells is estimated by SlNPV infection in the body of the test larvae which occurs due to PIB consuming process.

After 96 hours of treatment, the intestine making up tissue was increasingly unclear, making it difficult to find the constituent parts. It is presumably caused by the process of viral infection which has entered the advanced phase.

Viruses that have undergone replication in the mid intestine region began to be released into the hemosol and will attack other parts of the body.

Based on the results above, at 0 hours it can not be found any damages caused by SlNPV on the peritrophic

membrane structure of S. litura instar-5 larvae. Intestinal epithelium was still seemed intact with its constituent cells, which consists of a collection of columnar cells and was arranged densely at their ends and there were regenerative cells at the side of epithelium basal and had a direct boundary with basal membrane (Levy et al. 2004). These conditions clarify that with the integrity of the membrane profile peritrophic the metabolic activity is still going well since the middle intestine to optimize its function as a place of absorption and secretion of enzymes (Kikhno 2002).

Mentioned by Wang and Granados (1998) who studied the existence peritrophic on Trichoplusia ni membranes that peritrophic membrane proteins composed of Insect intestinal mucin, which is the largest protein that was conceived by the membrane peritrofik.

On further observation due to infections that contributed SlNPV digested with food, then the membrane structure peritrophic at 24 hours after infection began to experience damage as shown in Table 2 by 10%. This confirms the existence of peritrophic membrane also serves as protection against damage to the intestine was strong by food particles (Day and Waterhouse 1953). The damage increases with the length of time of infection. In early infection, pathogen attack will be responded by the insect's defense system with the existence of morphologically peritrophic membrane that serves as a protection against pathogen attack (Terra 2001). Funakoshi and Aizawa (1989) stated that given the infection process constant, the function of peritrophic membrane untenable.

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One of the factors that influence the success of the virus in the histological structure of membrane damage due to viruses peritrophic accelerating factor produce a virus that make virus capable of infecting insect cells and damage membranes peritrophic (Engelhard and Volkman 1995; Lehane 1997). Thus peritrophic membrane will be easier to be penetrated by NPV virions, which in turn will attack the cells next to it. The damage was observed after 48 hours in which the regenerative cells (damaged by 30%) began to disintegrate towards the middle of the gut lumen. With the disruption of the cell, then the middle gut function as a producer of enzymes will be disrupted. Mentioned that one of the enzymes secreted by the gut middle is a protease that acts as an anti-virus (Bolognesi et al. 2002). Therefore, when their activities disrupted the presence of pathogens is the metabolic process will not proceed smoothly.

Based on the observation of the advanced stage of infection at 72 and 96 hours after infection, it is found that peritrophic membranes become less and less intact. As proposed by Rohrmann (1994), that the NPV virions takes several days to express their interactions. Thus, the longer the contact time between NPV virions with host cells, the level of damage caused higher. The damage that occurs in this advanced stage causes the ability of epithelial in shaping peritrophic membrane is disturbed. This is consistent with the statement of Patton (1963) that the peritrophic membrane is the secretion from the epithelium of middle intestine.

CONCLUSION Number of isolates SlNPV dose having an effect on the death of the instar-5 larvae of S. litura by 50% (LD50) is 438 PIB / mL. SlNPV infection was also influential in lowering feed intake of larvae of S. litura larvae; causing the weight loss that the largest is in the 540 PIB / mL at 0.1675 mg. Histological Structure of peritrophic membrane after infected by SlNPV appears damaged in line with increasing time of SlNPV infection given, whereas the greatest damage occurs during 96 hours after infection.

REFERENCES Barbehenn RV, Marin M. 1994. Peritrophic envelope permeability in herbivorous insect. J Insect Physiol 41: 303-311.

Bolognesi R, Terra WR, Ferreira C. 2002. Functions of insect peritrophic membrane. ESA-Entomological Society of America. Fort Launderdale, USA. Bonning BC, Hammock BD. 1996. Development and recombinant Baculovirus for insect control. Ann Rev Entomol 41: 191-210.

Busvine JR. 1972. A critical review of the technique for testing insecticides. 2nd ed. Commonwealth Agricultural Bureaux. London.

UK. Cristian P. 1994. Recombinant Baculovirus insecticides: catalyst for change of heart? In: Biopesticides Opportunities for Australian Industry. Symposium on Biopesticides, June 9-10 1991, Brisbane, Australia.

Day MF, Waterhouse DF. 1953. Insect physiology. Chapman & Hall.

London.

Dibyantoro AL. 1996. Biology of armyworm Spodoptera litura F and usability microbiota in integrated pest control efforts armyworm.

Indonesian Vegetable Research Institute. Lembang, Bandung.

[Indonesia] Engelhard EK, Volkman LE. 1995. Developmental resistance in fourth instar Tricholupsia ni orally inoculated with Autographa californica M. Nuclear Polyhedrosis Virus. J Virol 209: 381-389.

Funakoshi M, Aizawa K. 1989. Viral inhibitory factor produced in the hemolymph of the silkworm, Bombyx mori, infected with a Nuclear Polyhedrosis Virus. J Invert Pathol 54: 151 -155.

Gothama AAA, Indrayani AA, Tukimin. 1990. Sensitivity of four-instar larvae of Helicoverpa armigera Hubner on Nuclear Polyhedrosis virus and Bacillus thuringiensis Berliner in the cotton. Penelitian Tanaman Tembakau & Serat 5: 82-91. [Indonesia] Granados RR, Corsaro NG. 1990. Baculovirus enhancing protein and their implications for insect control. Proceeding of 5th Youth International Colloquinon in Invertebrate Pathology & Microbial Control, Adelaide, Australia, 1990 Kikhno. 2002. Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus. J Gen Vir 83: 3013-3022.

Kurnia NT, Anggraeni, Laksanawati A. 2002. Response of S. litura F. to SlNPV infection. Proceedings of the 26th National Seminar on Biology. Bandung, 25-26 Juli 2002. [Indonesia] Laoh JH, Puspita F, Hendra. 2003. Vulnerability of Spodoptera litura F. larva against Virus Nuclear Polyhedrosis. J Natur Indonesia 5 (2): 145-151. [Indonesia] Lehane MJ, Msangi AR. 1991. Lectin and peritrophic membrane development in the gut of Glossina morsitans and a discussion of their role in protecting the fly against Trypanosoma infection. J Med Vet Entomol 5: 495-501.

Lehane MJ. 1997. Peritrophic matrix structure and function. Ann Rev Entomol 42: 525-550.

Levy SM, Falleiros AMF, Gregorio EA, Arrebola NR, Toledo LA. 2004.

The larval midgut of Anticarsia gemmatalis (Hubner) (Lepidoptera: Noctuidae): light and electron microscopy studies of the epithelial cells. Braz J Biol 64 (3B): 633-638.

Maramorosch K. 2007. Viruses, vectors, and vegetation: an autobiography. Adv Vir Res 70: 1-31.

Blum MS. 1985. Fundamentals of insect physiology. John Willey & Sons.

New York.

Moscardi F. 1994. Assesment of the appplication of Baculovirus for control of Lepidoptera. Ann Rev Entomol 44: 247-249.

Novizan. 2004. Membuat dan memanfaatkan pestisida ramah lingkungan.

Agro Media Pustaka. Tangerang Pawana G. 2000. Response of Helicoverpa armigera Hubner to sublethal NPV infection and its impact on reproductive rate. [Thesis]. Bandung Iinstitute of Technology. Bandung. [Indonesia] Patton RL. 1963. Introductory insect physiology. Toppan Co. Tokyo.

Rohrmann GF. 1994. Nuclear Polyhedrosis Virus. In: Encyclopedia of virology. Academic Press. London.

Sanjaya Y. 2000. Changes in national levels of Helicoverpa armigera Hubner tolerance infected by Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV). [Thesis]. Bandung Iinstitute of Technology. Bandung. [Indonesia] Sutarya R. 1996. Testing of Spodoptera exigua Nuclear Testing Polyhedrosis Virus in relation to the nature of persistence to control Spodoptera exigua Hubn. J Hort 6 (2): 167-171. [Indonesia] Teakle RE, Jensen RE, Mulder, JC. 1985. Susceptibility of Heliothis armiger (Lepidoptera: Noctuidae) on sorghum to a Nuclear Polyhedrosis Virus. J Econ Entomol 78: 1373-1378 Terra WR. 2001. The origin and functions of the insect peritrophic membrane and peritrophic gel. Arch Insect Biochem Physiol 47: 47-61.

Utari E. 2000. Effect of HaNPV infection against peritrophic membrane damage and nutrition index instar V larvae of Helicoverpa armigera (Hubner). [Thesis]. Bandung Iinstitute of Technology. Bandung.

[Indonesia] Wang P, Granados RR. 1998. Observation on the presence of the peritrophic membrane in larval Trichoplusia ni and its role in limiting Baculovirus infection. J Invert Pathol 72: 57-62.

Wigglesworth VB. 1984. Insect physiology. Toppan Company. Tokyo, Japan Yamagishi J, Isobe R, Takebuchi T, Bando H. 2003. DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines. Arch Virol 148 (3): 587-597.

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 141-145 November 2010

Hitherto unreported Agaricus species of Central India ALKA KARWA, MAHENDRA KUMAR RAIâ&#x2122;Ľ Department of Biotechnology, SGB Amravati University, Amravati 444602, Maharashtra, India. Tel: +91-721-2662207/8, Extension-267. Fax: +91 721 2660949, 2662135. email: pmkrai@hotmail.com Manuscript received: 24 July 2010. Revision accepted: 6 October 2010.

Abstract. Karwa A, Rai MK. 2010. Hitherto unreported Agaricus species of Central India. Nusantara Bioscience 2: 141-145. Melghat forest region from Central India was surveyed for occurrence of medicinal and culinary mushrooms during the years 2005-2008. Out of total 153 species, ten species of Agaricus were recorded from different localities. Of these, seven species namely Agaricus bitorquis, A. subrufescens, A. augustus, A. placomyces, A. essettei, A. basioanolosus and Agaricus sp. nov (a new species) are being reported for the first time from the region. The commercial button mushroom Agaricus bisporus lacks good breeding characters due to its bisporic nature. These wild cousins of the button mushroom can definitely prove to be a good source of genetic manipulations to the existing strains and also to develop new strains with improved characters.

Key words: Agaricus, Central India, commercial, edible, Melghat.

Abstrak. Karwa A, Rai MK. 2010. Spesies Agaricus dari India Tengah yang belum dilaporkan sampai sekarang. Nusantara Bioscience 2: 141-145. Kawasan hutan Melghat di India Tengah disurvei untuk mengetahui keberadaan jamur yang berkhasiat obat dan kuliner selama tahun 2005-2008. Dari total 153 spesies jamur, sepuluh spesies Agaricus ditemukan di berbagai lokasi yang berbeda. Dari jumlah tersebut, tujuh spesies yaitu Agaricus bitorquis, A. subrufescens, A. augustus, A. placomyces, A. essettei, A. basioanolosus dan Agaricus sp. nov. (spesies baru) baru pertama kali dilaporkan keberadaannya di kawasan ini. Jamur komersial Agaricus bisporus tidak memiliki karakter perkembangbiakan yang baik karena secara alamiah bersifat bispora. Kerabat liar dari jamur ini dapat digunakan sebagai sumber manipulasi genetik pada strain yang ada dan juga untuk mengembangkan strain baru dengan karakter yang lebih baik.

Kata kunci: Agaricus, India Tengah, komersial, dimakan, Melghat.

INTRODUCTION Agaricus bisporus (J.E. Lange) Imbach (Agaricaceae) commonly known as commercial white button mushroom is the most extensively cultivated mushroom worldwide and comprises about 32% of world mushroom production.

However, modern approaches to breeding this economically important fungus have been largely ignored.

The previous attempts for genetic improvements in this commercially important mushroom has little success due to low genetic diversity amongst commercially cultivated white strains and non-inclusion of wild collections in the breeding programs. Thus, limited availability of genetic variation significantly slowed down the progress of genetic improvement in this strain of commercially important button mushroom (Hawksworth 1991; Singer 1989). On the other hand, there is a plethora of wild species in the genus Agaricus in many Indian forests. Many of them are collected and consumed by the local people, mostly by the tribes. There is a great need to bring into cultivation the other possible wild isolates of this much-preferred genus.

The new species entering the commercial market can promise an improved productivity, shelf life and quality as compared to the currently cultivated button mushroom.

Collection of wild germplasm of Agaricus is the first phase for initiating the breeding program.

India being the 6th mega spot of biodiversity has innumerable mushroom species and their ethnomycological importance. One third of fungal diversity of the globe exists in India (Butler et al. 1960, Bilgrami et al. 1981; Sarbhoy et al. 1996; Doshi and Sharma 1997; Manoharachary 2001; Maria and Sridhar 2002; Manoharachary et al. 2005). Melghat is a Reservation Forest for tigers in Amravati District, Maharashtra State, India. The biodiversity of this region is unique due to its varying biogeographical and physicochemical environment. This region has intermingling forests of highly valuable and endangered medicinal plants, as well as a variety of edible and medicinal mushrooms few of which are consumed by local tribes. Biodiversity of edible fungi has been reported from different parts of India by several workers but this region remained unexplored and the fungal treasure of the region yet unnoticed by eminent mycologists of the country.

The aim of the present study was to explore the region for the existence of the valuable and neutriceutically important wild mushrooms and their conservation. Six different zones in the region were surveyed from July 2005 to December 2008 for the availability of wild edible and medicinal mushrooms. In this paper we report a total of 10 wild species of Agaricus of which seven species are hitherto unreported.

2 (3): 141-145, November 2010

142 MATERIAL AND METHODS

Ten species of wild Agaricus mushrooms were collected from different localities in Melghat region of Central India in Amravati District, Maharashtra State (Figure 1) during 2005 to 2008. Repeated visits and periodical surveys of the localities revealed a plethora of wild mushrooms out of which the genus Agaricus seemed to spring out in all the localities throughout the monsoons (June to September).

Mushrooms were collected from the non reserved region of the forest like roadsides, landscapes, grasslands, pastures.

Mushrooms were digitally photographed using a Sony DSC R1 Professional Camera. The collected specimens were brought to the lab, cleaned, and microscopical examinations of the hymenium, basidiospores, and cuticle were performed. Taxonomic identifications were made based on their morphological, microscopic and staining studies according to the methods given by Brietenbach and Kranzlin (1991) and Phillips (1991, 2006).

RESULTS AND DISCUSSION Results During the present study, a total of 153 species of mushrooms were identified and keyed to 47 genera belonging to 26 families. Of these, species of the genus Agaricus were found to be more abundant compared to other collected mushroom species. Table 1 illustrates a list of the wild Agaricus mushrooms that are identified till date.

After interaction with local people and the tribals inhabiting the region we came to know that though some of these species of Agaricus are eaten in some or the other parts of the world, they are not utilized here for food. The wild Agaricus mushrooms (Figure 2) and their description is as follows:

free, white in young, pink to dark brown to blackish in older ones. Stipe: central, equal, 4-10 cm long, 1-2 cm thick, white to pale brown, annulate, annulus white membranous prominent. Basidia: 2 spored, spores brown, ellipsoid, 7x5.5 µm2. Spore print: sepia to brown.

Agaricus bitorquis (Quel.) Sacc.

Found solitary as well as in groups on roadside and soil rich in manure. Pileus: 10.5 cm in diameter, convex in young, plane in old fruit bodies. Yellow to brownish towards center with a small depression in center. Gills: white in young, pink to brown to blackish in old, crowded, free, broad. Stipe: 5-7 cm long, 2 cm thick, white, thick annulus in middle of stipe. Basidia: 4 spored, spores brownish purple, ellipsoid, 7.5x6 µm2. Spore print: dark brown.

Agaricus arvensis Schaeff.

Found solitary or scattered and in fairy rings in grazing lands. Pileus: approximately 10 cm in diameter, convex in young, flattened in old, white smooth surface, light brown towards center. Gills: free, crowded, white in young, pink to chocolate brown in old, moderately broad. Stipe: thick, cylindrical, central, 8-10 cm long, white, hollow, large annulus. Basidia: squat, broad, 4 spored, spores small and brown, oval 6x4 µm2. Both pileus and stipe bruises yellow on handling.

Agaricus augustus Fr. Found solitary or scattered in grazing lands and gardens. Pileus: sub-globose in young and convex to flat in old, 3-7 cm in diameter, yellow-beige coloured smooth surface, reddish towards center. Gills: white in young, pink in old, narrow, free crowded. Stipe: thick, smooth, cylindrical, 5 -10 cm long, cream coloured, hollow, annulus thin, close to pileus. Basidia: broad, 4 spored, spores: light brown, oval 4x6 µm2.

Agaricus essettei Bon Found solitary as well as in tufts in grasslands and forest. Pileus: light to dark brown, 3-8 cm in diameter, surface scaly with dark brown center, sub-globose when young and expanded rounded when old. Gills: pink to brown with age, narrow to moderately broad, crowded, free. Stipe: thick, cylindrical, stuffed, bulbous base, light Table 1. Species of the genus Agaricus collected from Melghat region of Central brown, scaly, annulate. Basidia: 4 spored, India. spores brown, subelliptical 4x5.5 µm2. Both pileus and stipe bruise yellow with handling.

Agaricus bisporus Lange Found scattered on pastures, lawns and on scattered manure. Pileus: 5-10 cm diameter, convex in young, flattened in old fruit bodies. White to pale-brown, finely scaly surface, margin entire. Gills: prominent, crowded,

Accession Name of the Period of collection number mushroom MGCC 98 Agaricus arvensis July 2005-August 2008 MGCC 62 Agaricus augustus July 2005-August 2008 MGCC 03 Agaricus bitorquis July2005-September 2008 MGCC 07 Agaricus bisporus July2005-September 2008 MGCC 77 Agaricus essettei June 2005-July 2008 MGCC 37 Agaricus placomyces June2005-July 2008 MGCC 63 Agaricus silvaticus Aug2005-September 2008 MGCC 136 Agaricus silvicola July2005-September 2008 MGCC 33 Agaricus sp.nov.

June2005-Aug MGCC 55 Agaricus subrufescens Aug2005-August 2008 MGCC = Mushroom germplasm culture collection.

Population Abundant Moderate Moderate Abundant Rare Rare Moderate Rare Moderate Moderate

Agaricus silvaticus Schaeff.

Found solitary in bushy and grassy places. Pileus: 6-10 cm in diameter, cream to perfect beige coloured, convex when young, expanded and gibbous in old.

Surface fibrillose. Gills: crowded, free, moderately broad, red to narrow towards apex, hollow, annulus thin ring. Basidia: 4 spored, spores small, globose, 2.5x2.8 µm2, brown.

KARWA & RAI-Unreported Agaricus species of Central India

Agaricus silvicola (Vittad.)

Found solitary or in tufts in shady places of the forest.

Pileus: 6-10 cm in diameter, convex in young, expanded to nearly plane in old, white, smooth, with distinct umbo.

Gills, crowded, white to pink to dark brown with age, broad. Stipe: 6-10 cm long, cylindrical, uniform, white, slightly bulbous base, white prominent annulus. Basidia: 4 spored, spores brown, elliptical, 5x6.5 Âľm2.

Maharastra State INDIA

143

Agaricus sp. nov.

Found solitary in grasslands and forest. Pileus: 5-9 cm in diameter, light brown surface covered with dark brown scales throughout, dense at center, conical to convex in young, more expanded convex in old. Gills pink to brown to black with age, not auto deliquescent, broad. Stipe: 5-9 cm long, light brown, less scaly, equal, cylindrical, stuffed, light brown annulus. Basidia: 4 spored, spores brown, spherical 5x5 Âľm2.

MELGHAT FOREST

Amravati District MAHARASTRA

MELGHAT FOREST

Figure 2. Locations of mushroom collection (highlighted in circle) in Melghat forest, Amravati District, State of Maharastra, India

2 (3): 141-145, November 2010

144

A

B

E

F

I

C

G

D

H

J

Figure 3. Plates of ten different Agaricus species collected from wild in Central India. Note: A. Agaricus arvensis, B. Agaricus augustus, C. Agaricus bitorquis, D. Agaricus bisporus, E. Agaricus essettei, F. Agaricus silvaticus, G. Agaricus silvicola, H. Agaricus placomyces, I. Agaricus sp.nov. J. Agaricus subrufescens.

Agaricus subrufescens Peck.

Found in tufts on ground or lawns or pastures in shades.

Pileus: 3-10 cm in diameter, hemispherical to somewhat conical when young, convex to broadly expanded old, light yellow to brown surface with brown scales. Gills: crowded, free, white to chocolate brown with age, narrow. Stipe: 1015 cm long, hollow, white, annulated, shiny, swollen base.

Basidia: 2-3 spored, spores dark brown elliptical, 6x3.5 µm2. Found solitary or scattered in grazing lands and gardens. Pileus: subglobose in young and convex to flat in old, 3-7 cm in diameter, yellow-beige coloured smooth surface, reddish towards center. Gills: white in young, pink in old, narrow, free crowded. Stipe: thick, smooth, cylindrical, 5 -10 cm long, cream coloured, hollow, annulus thin, close to pileus. Basidia: broad, 4 spored, spores: light brown, oval 4x6 µm2. Dark brown. Stipe: 7-12 cm long, cylindrical, slightly

Agaricus placomyces Peck.

Found solitary and scattered on soil. Pileus: 10 cm in diameter, ovate when young, convex to plane in old, white with dark brown center, brown scales. Gills: not crowded, free, white to pink to dark brown with age. Stipe: 7-10 cm long, cylindrical, stuffed, white to pale brown, tapering towards apex, large white annulus towards pileus. Basidia: 4 spored. Spores brown, ellipsoid, 5x3 µm2, spore print brown.

Discussion Though India has rich macro fungal biodiversity, most traditional knowledge about mushrooms come from the Far East countries like China, Japan, Korea, and Russia where mushrooms like Ganoderma, Lentinus, Grifola and others were collected and used since time immemorial. Most of

KARWA & RAI-Unreported Agaricus species of Central India

the mushrooms grow abundantly in nature and their commercial harvest is being undertaken for the benefit in these countries. Therefore, systematics of wild mushrooms has received more attention than other threatened aspects like conservation. However, the ecological data available on some of the genera is still not enough.

Besides extensive surveys and records from Punjab, Kerala and Western Ghats published during the last decade (Pradeep et al. 1998; Atri et al. 2000). In his book Purkayastha described identification of wild Indian mushrooms (Purkayastha and Chandra, 1985). Lakhanpal (1996, 1997) and his students from India extensively surveyed the Himalayan ranges during 1980’s to 1990’s and reported a wide range of wild mushrooms including some highly medicinal species. What is noteworthy is the component of macro fungi that dominates the Central India, and the Genus Agaricus in particular, that has been neglected.

Guzman (1983) of Mexico reported many wild edible and medicinal mushrooms along with Psilocybins (1983).

Wasser et al. (2004) and Chang and Buswell (2003) studied nutraceutical and therapeutic properties of wild mushrooms including Agaricus. Stamets (2000) is the sole name in USA since the last 3 decades who is dedicated to study wild mushrooms and their applications in various fields of medicine as well as societal and ecological development.

However, research on wild mushrooms in Central India has been greatly neglected by mycologists. Here we summarize that there is an urgent need to explore the Central Indian forests and other regions so that a complete inventory of wild mushrooms can be developed and conservation of the important species can be sought for.

Out of the wild species of Agaricus mentioned in this paper, seven species namely Agaricus bitorquis, A. subrufescens, A. augustus, A. placomyces, A. essettei, A. basioanolosus and Agaricus sp. nov. are being reported for the first time from the region and are promising sources for genetic improvement of the available commercial white button mushroom.

CONCLUSION Out of total 153 species mushroom in Melghat forest, ten species of Agaricus were recorded from different localities. Of these, seven species namely Agaricus bitorquis, A. subrufescens, A. augustus, A. placomyces, A. essettei, A. basioanolosus and Agaricus sp. nov. (a new species) are being reported for the first time from the region. These wild Agaricus can definitely prove to be a good source of genetic manipulations to the existing strains of the commercial button mushroom Agaricus bisporus and also to develop new strains with improved characters.

145

ACKNOWLEDGEMENTS Dr. Alka Karwa is thankful to Department of Science and Technology, Government of India, New Delhi for sponsoring the post-doctoral fellowship to carry out this study.

REFERENCES Atri NS, Kaur A, Saini SS. 2000. Taxonomic studies on Agaricus from Punjab plains. Indian J Mushroom 18: 6-14.

Bilgrami KS, Jamaluddin, Rizvi MA. 1979. The fungi of India. Part I (List and Reference). Today and Tomorrow’s. New Delhi.

Bilgrami KS, Jamaluddin, Rizvi MA. 1991. The Fungi of India Part III (List and References) Today and Tomorrow’s. New Delhi.

Brietenbach J, F Kranzlin (eds). 1991. Fungi of Switzerland. Vol. 3.

Boletes and Agarics, 1st Part. Mykologia Lucerne. Switzerland.

Butler EJ, Bisby GR. 1960. The fungi of India. Revised by Vasudeva RS. ICAR. New Delhi.

Chang ST, Buswell JA. 2003. Medicinal mushrooms-a prominent source of nutriceuticals for the 21st century. Curr Topics Nutraceut Res 1: 257-280.

Doshi A, Sharma SS. 1997. Wild mushrooms of Rajasthan. In: Rai D, Verma (eds) Advances of mushroom biology and production.

Mushroom Society of India. Solan, India Guzmán G. 1983. The genus Psilocybe. J. Cramer. Berlin.

Hawksworth DL. 1991. The fungal dimension of biodiversity: magnitude and significance and conservation. Mycol Res 95: 641-655.

Lakhanpal TN. 1996. Mushrooms of Indian Boletaceae. In: Mukherji KG (ed). Studies in Cryptogamic Botany Vol. I. APH Publishing Corp.

New Delhi.

Lakhanpal TN. 1997. Diversity of mushroom mycoflora in the NorthWest Himalaya. In: Sati SC, Saxena J, Dubey RC (ed). Recent researches in ecology, environment and pollution. Today and Tomorrow’s. New Delhi.

Manoharachary C, Sridhar K, Singh R, Adholeya A, Suryanarayanan, Seema TS, Johri BN. 2005. Fungal biodiversity: distribution, conservation and prospecting of fungi from India. Curr Sci 89 (1): 58-71.

Manoharachary C. 2001. Biodiversity, conservation and biotechnology of fungi. Presidential Address, Section-Botany, The 89th Session of Indian Science Congress, Indian Science Congress Association, Lucknow, India, January 3-7, 2002.

Maria GL, Sridhar KR. 2002. Richness and diversity of filamentous fungi on woody litter of five mangroves along the west coast of India. Curr Sci 83: 1573-1580.

Philippoussis AN, Diamantopoulou PA, Zervakis G.I. 2003. Correlation of the properties of several lignocellulosic substrates to the crop performance of the shiitake mushroom Lentinula edodes. World J Microbiol Biotechnol 19: 551-557.

Phillips R. 1991. Mushrooms of North America. Little, Brown and Company. Boston.

Phillips R. 2006. Roger's mushrooms. http://www.rogersmushrooms.com Purkayastha RP, Chandra A. 1985. Manual of Indian edible mushrooms.

Jagmander. New Delhi.

Sarbhoy AK, Agarwal DK, Varshney JL. 1996. Fungi of India 19821992. CBS Publishers and Distributors. New Delhi.

Singer R. 1989. The Agaricales in modern taxonomy. 4th ed. J. Cramer, Weinheim.

Stamets P. 2000. Growing gourmet and medicinal mushrooms. Ten Speed Press. Toronto.

Wasser SP, Didukh M.Y, Nevo E. 2004. Dietary supplements from culinary-medicinal mushrooms: a variety of regulations and safety concerns for the 21st century. Int J Med Mushr 6: 231-248.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 3, Pp. 146-156 November 2010

Diversity of secondary metabolites from Genus Artocarpus (Moraceae) ALIEFMAN HAKIM♥ Study Program of Chemistry Education, Faculty of Teacher Training and Education, Universitas Mataram. Jl. Majapahit 62 Mataram, West Nusa Tenggara, Indonesia. Tel./Fax.: +62-370-623873, Fax: +62-370-634918. Email: ♥aliefmanhakim27@gmail.com Manuscript received: 28 Augustus 2010. Revision accepted: 4 October 2010.

Abstract. Hakim A. 2010. The diversity of secondary metabolites from Genus Artocarpus (Moraceae). Nusantara Bioscience 2:146-156.

Several species of the Artocarpus genus (Moraceae) have been investigated their natural product. The secondary metabolites successfully being isolatad from Artocarpus genus consist of terpenoid, flavonoids, stilbenoid, arylbenzofuran, neolignan, and adduct Diels-Alder. Flavonoid group represent the compound which is the most found from Artocarpus plant. The flavonoids compound which are successfully isolated from Artocarpus plant consist of the varied frameworks like chalcone, flavanone, flavan-3-ol, simple flavone, prenylflavone, oxepinoflavone, pyranoflavone, dihydrobenzoxanthone, furanodihydrobenzoxanthone, pyranodihydrobenzoxanthone, quinonoxanthone, cyclopentenoxanthone, xanthonolide, dihydroxanthone.

Key words: Artocarpus, Moraceae, flavonoid, Diels-Alder, secondary metabolites.

Abstrak. Hakim A. 2010. Keanekaragaman metabolit sekunder Genus Artocarpus (Moraceae). Nusantara Bioscience 2:146-156.

Beberapa spesies dari genus Artocarpus (Moraceae) telah diteliti kandungan bahan alamnya. Metabolit sekunder yang berhasil diisolasi dari genus Artocarpus terdiri dari terpenoid, flavonoid, stilbenoid, arilbenzofuran, neolignan, dan adduct Diels-Alder. Kelompok flavonoid merupakan senyawa yang paling banyak ditemukan dari tumbuhan Artocarpus. Senyawa flavonoid yang telah berhasil diisolasi dari tumbuhan Artocarpus memiliki kerangka yang beragam seperti calkon, flavanon, flavan-3-ol, flavon sederhana, prenilflavon, oksepinoflavon, piranoflavon, dihidrobenzosanton, furanodihidrobenzosanton, piranodihidrobenzosanton, kuinonosanton, siklolopentenosanton, santonolid, dihidrosanton.

Kata kunci: Artocarpus, Moraceae, flavonoid, Diels-Alder, metabolit sekunder.

INTRODUCTION One family of plants in tropical forests that has the potential as a source of bioactive chemicals and the number is relatively large is the Moraceae. Moraceae Family consists of 60 genera and includes 1400 species. The main genus of the Moraceae family is Artocarpus which is composed of 50 species and spread from South Asia, Southeast Asia to the Solomon Islands, Pacific Islands, North Australia and Central America (Kochummen 1987; Verheij and Coronel 1992). On the island of Kalimantan, there are 25 species, of which 13 species of them are endemic, but only two species are utilized, namely: Artocarpus heterophyllus and A. integer (Verheij and Coronel 1992).

In Indonesia, Artocarpus is known as the jackfruit that have characterized by a tall tree with white latex in all parts of plants, hard wood, fleshy fruit with lots of seeds. All parts of Artocarpus has been used extensively by the community for various purposes such as wooden sticks used for building materials and its fruit as a food ingredient. In addition, Artocarpus can also be used as traditional medicine, such as leaves of A. communis Frost which is burned and mixed with coconut oil plus turmeric can be used to cure skin diseases. The flowers is used to

cure toothache, while its root is used to stop bleeding (Kochummen 1987; Heyne 1987).

Based on literature studies, it is known that some species of Artocarpus produce many compounds of terpenoid, flavonoids, and stilbenoid classes. The uniqueness of the structure of secondary metabolites in Artocarpus produce a broad physiological effects, such as anti-bacteria (Khan et al. 2003), anti-platelets (Weng et al.

2006), anti-fungal (Jayasinghe et al. 2004), anti-malarial (Widyawaruyanti et al . 2007; Boonlaksiri et al. 2000) and cytotoxic (Ko et al. 2005, Judge et al. 2002, Shah et al.

2006), so the research on bioactivity of secondary metabolites anti-malarial from Artocarpus can provide benefits in the search for new drugs of the natural material compound, as well as provide a scientific explanation of the use of these plants in traditional medicine.

This information produces the consequences of the need for sustainability investigation of the chemical content of the Artocarpus genus. This article provides a review of researches that have been done on Artocarpus located in Indonesia.

Extraction, isolation, and purification The method for extracts making consists of four phases, namely the manufacture of powder, the process of extraction, solvent separation and extract concentration

HAKIM â&#x20AC;&#x201C; Secondary metabolites of genus Artocarpus

phase. The dried Artocarpus is mashed then is extracted by maceration at room temperature for 24 hours with methanol, then filtered. The extraction process is repeated until the less colorful supernatant is obtained. Solvent separation is performed using a rotary evaporator and then is concentrated in a water bath so a thick extraction is produced.

Then, the obtained extraction underwent TLC (Thin Layer Chromatography) treatment using various eluents.

This stage was carried out to determine the chemical components in the extract. Furthermore, the TLC chromatogram was used as a basis for conducting separation/fractionation by vacuum liquid chromatography (VLC).

The main fractions obtained from the VLC then were analyzed again by TLC. Fractions that had same spots (Rf) were pooled and analyzed again by TLC. Purification process on main factions were done repeatedly by radial chromatography while were monitored by TLC so pure isolates are obtained. TLC chromatogram is used to test the purity of an isolate, in which the pure isolates must show a single stain on three different eluent systems, besides the purity test can also be done by measuring its melting point.

Structural determination The structures of pure isolates obtained are determined by spectroscopic methods: (i) Analysis by UV-Vis to determine the presence or absence of double bond conjugation in the structure of these compounds. (ii) Analysis by infrared to know what types of functional groups possessed by these compounds, such as whether the existing O atoms in these molecules exist as clusters of alcohol, ether ketones, aldehydes and so on. (iii) The sample was analyzed by 1H and 13C NMR (Nuclear Magnetic Resonances). (iv) analysis of this structure should be comprehensive of all existing data, to avoid any error in the determination of the structure of a compound.

SECONDARY METABOLITES OF ARTOCARPUS The content of secondary metabolites from Moraceae family has long been studied and in recent years there are many research groups which examine secondary metabolite of Artocarpus species (Nomura et al. 1998; Sultanbawa et al. 1989; and Judge et al. (1999, 2006). The researches have found many secondary metabolites belonging to the group of terpenoid, flavonoids, stilbenoid, arilbenzofuran, neolignan, and Diels-Alder adducts compounds.

Terpenoid Terpenoid compounds with a sikloartan frame are succeeded to be isolated from Artocarpus plants among others, cycloartenol (1) that have been successfully obtained from A. champeden (Achmad et al. 1996) and A. altilis (Altman and Zito 1976). Other terpenoid compounds that have been isolated from this same plant are cycloeucalenol (2), 2,4-methylencycloartenone (3), and cycloartenone (4) (Achmad et al. 1996) which also has been isolated from A. heterophyllus (Dayal and Seshadri

147

1974). Compounds of (24R) and (24S)-9.19-cyclolanost-3on-24 0.25-diol (5) have been isolated from A. heterophyllus (Barik et al. 1997). Glutinol compound (6) is so far the only pentacyclic triterpenoid compound with a glutan frame which is isolated from Artocarpus i.e. A. champeden (Achmad et al. 1996).

Flavonoids The content of flavonoid compounds with a variety of frameworks such as chalkon derivatives, flavanones, flavan-3ol, simple flavone, prenylflavone, oxepinoflavone, pyranoflavone, dihydrobenzoxanthone, furanodihydrobenzoxanthone, pyranodihydrobenzoxanthone, quinonoxanthone, cyclopentenoxanthone, xanthonolide, dihydroxanthone, and cyclopentenoxanthone have been isolated from Artocapus plants.

Chalcone Chalcone compounds are found as chalcone and dihydrochalcone. Prenylation of chalcone by isoprenoid groups and geranyl can be found in ring A or B but can not be found on CÎą which is comparable to C3 on flavone.

Some Diels-Alder aduct compounds are also came from chalcone. It is noted that most chalcone compounds found are derived from the leaf. Canzonol compound C (7) and artoindonesianin J (8) was isolated by Ersam (2001) from the stem bark of A. bracteata.

Another class of chalkon compounds is dihydrochalcone. These compounds have some cytotoxic activities. Some dihydrochalcone compounds are successfully isolated by Wang et al. (2007) from Artocarpus altilis, namely 1-(2,4-dihydroxyfenyl)-3-(8hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-yl-5benzopyran )-1-propanone (9), 1-(2,4-dihydroxyfenyl)-3{4-hydroxy-6 ,6,9-trimethyl-6a, 7,8,10 a-tetrahydro-6Hdibenzo (b, d) pyran-5-il}-1-propanone (10), 2-geranyl-2 ', 3,4,4'-tetrahydroxydihydrochalcone (11).

Flavanones Flavanones compounds are found in all parts of Artocarpus. Several compounds have been isolated, among others, by Djakaria (1999) that is artocarpanone (12) from the root timber of A. champeden. Judge et al. (2001) isolates artoindonesianin E (13) and heteroflavanone A. (14) from the stem bark of A. champeden, compound 14 also been isolated from the root bark of A. champeden by Nomura et al. (1998), while Jayasinghe et al. (2006) from the fruit of A. nobilis isolates 8-geranyl-4 '0.7dihydroxyflavanon (15), 3'-geranyl-4',5,7-trihydroxyflavanon (16) and isonimfaeol-B (17). Compounds 15 and 17 are reported to have strong antioxidant activity.

Compounds flavanones have oxygenation pattern in ring B is unique is there monohydroxide at position 4 '; dioxygenation 2', 4 ', 3', 4 'or trioxygenation 2', 4 ', 6'.

Flavan-3-ol Compounds with a flavan-3-ol framework found are not prenylated. The three compounds of flavan-3-ol are afzelecin (18) and catechin (19) from the root bark of A. reticulatus (Udjiana 1997), where compound 19 had

Â

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2 (3): 146-156, November 2010

previously been isolated from A. integra by Yamazaki et al.

(1987), and afzelecin ramnoside (20) which is isolated from the bark of A. reticulatus by Murniana (1995). By considering the structure of these compounds (18, 19, 20), significant differences in oxygenation patterns are found if they are compared with the structure of simple flavonoids.

In the flavan-3-ol oxidation in ring B is monohydroxy or 3', 4''dihydroxy.

Simple flavone There are only a few simple flavonoids which are not prenylated, two of them are artocarpetin (21) and norartocarpetin (22) which were isolated from the root

wood of A.heterophyllus by Lin et al. (1995). Compound 21 is also successfully obtained from A. hirsutus (Venkataraman 1972) and A. integrifolia (Dave et al.

1962), while compound 22 is successfully isolated from the bark of A. scortechinii by Ferlinahayati (1999). This compound is believed to be a precursor for the biosynthesis of prenylated flavonoids.

These simple flavonoids have characteristic of ring B oxygenation patterns in position of dihydroxy 2 ', 4'. This fact is interesting because some prenylated flavones that are found have patterns of monooxygenation at C4' and trioxygenation at C2', C4 'and C6'.

H

H

H

HO

HO

O

(2)

(1)

(3)

OH H

OH

H

HO

O

O

(4)

(6)

(5)

OH

OH HO

HO

OH O OH

OH O

(7)

(8)

OH O

OH O

OH O HO

HO

HO

H

OH

OH

OH

O

OH

O H

(9)

(10)

(11)

HAKIM – Secondary metabolites of genus Artocarpus

HO H3CO

H3CO

OH OR

O

149

OCH3

OH

O

O

HO

OCH3 OH O

OH O

(12)

OH O

R=H R=CH3

(13) (14)

(15)

OH OH OH HO

O

HO

O

OH O

OH O

(16)

(17)

OH HO

O

R1

OH

(18) R1=H;

R2= H

(19) R1=H;

R2=ramnosid

RO

O

(20) R1=OH; R2=H

OR2

(21) R=CH3 (22) R=H

OH

OH

OH O

OH OH H3CO

O

HO

OH

O

O

OH

O

OH

OH

OH O

OH

O

O OH O

OH O OH O

(23)

(24)

(25)

(26)

HO 3'

HO 8

HO

B

O

A 3

5 15

OH O

(27)

HO

OH 5'

H3CO

O

OH

OH

HO

OH O

O

O

OH 9

OH O

(28)

OH O

(29)

OH OH O

(30)

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2 (3): 146-156, November 2010 OH

OH

OH O

OH O

OH O

OH HO

O

OH

HO

OH

OH O

OH O

OH O

(34)

OH

O

HO

O

O

OH

(33)

(32)

(31)

O

OH

OH

OH

HO

OH

O

O

O

HO

O

O

HO

OH

OH

(35)

(36)

OH OH HO

H3CO

OH

O

O

O

O

O

O

O OH O

(38)

(39)

OH

OH HO

O

OH

O O

O OH O

OH

OH

(37)

O

OH O

OOH

OH O

HO

O O

OH O OH O

(40)

Prenyl flavon Flavonoids are prenylated either by force or by geranyl isoprenoid which have been isolated from Artocarpus quite a lot. The prenylation is especially in ring A (C6 and C8) and C3 positions. Prenylated flavonoids are intermediate compounds for further biosynthesis. Prenyl flavone compounds are found in the bark or stem of both wood and root. Flavonoids compounds have been prenylated only at C6 or C8 that have been isolated, among others cycloartocarpin A (23) by Lin et al. (1995) from the root timber of A. heterophyllus. Kijjoa et al. (1996) isolates artocarpesin (24) of stem wood of A. elasticus. Wang et al.

(2004) isolates artocamin C (25) from the root of A. chama, these compounds are reported to be active as anticancer. cycloaltilisin (26) that are isolated from the bud covers of

(41)

(42)

A. altilis by Patil et al. (2002) are reported as an inhibitor of cathepsin.

Prenylated flavonoids on the C6 or C8 is found to have monooxygenation patterns at C4 'or dioxygenation at C3', C4 'or C2', C4 '. Cromen ring formation is a common matter in this class of compounds.

Another prenylflavon compound is class of 3-prenyl flavone. This prenylation process on C3 gives a lot of modifications to the structure of the flavonoids found in the genus of Artocarpus. The diversity of compound structure modification results also depends on the oxygenation pattern of ring B. Oxygenation pattern of flavonoids with 2', 4' and 5' produces more structural modifications.

Compounds of 3-prenyl flavone with mono or dihydroxy in ring B which have been isolated, among others, are cudraflavone C (27) which is isolated from A. scortechinii

by Judge (2008). Compound 27 had previously been

HAKIM â&#x20AC;&#x201C; Secondary metabolites of genus Artocarpus

isolated from A. communis by Han et al. (2006). Artocarpin (28) is isolated from the root timber of A. heterophyllus by Lin et al. (1995). Kijjoa et al. (1996) from the stem wood of A. elasticus managed to isolate artelastisin (29) and artelastofuran (30). Compound 30 is also been isolated from A. scortechinii by Judge (2009).

The other 3-prenyl flavonoids compounds is the pattern of oxygenation at C2 ', C4' and C5 '. This group of compounds has the highest level of oxidation. Many compounds have been reported to exist mainly in the subgenus Artocarpus. Some of them have been isolated, among others are artonin E (31) from the stem bark of A. scortechinii by Ferlinahayati (1999), artonin E (31) is also isolated from the root bark of A. nobilis (Jayasinghe et al.

2008), A. lanceifolius (Cao, et al. 2003), A. kemando (Seo et al.), and A. communis (Aida et al. 1997). Artonin V (32) is isolated from the root bark of A. altilis by Hano et al.

(1994). Ko (2008) isolates artelastoheterol (33) from the root bark of A. elasticus.

Geranyl group attached to C3 is also found in the flavonoids isolated from the Artocarpus genus. Like the other 3-prenyl flavonoids, the oxygenation pattern in ring B of these compounds is also dioxygenation or trioxygenation. Several compounds found, among others, is artoindonesianin L (34) of A.rotunda root bark (Suhartati et al. 2001). The compound is reported to have cytotoxic activity. Chan (2003) from the root bark of A. communis isolates artocommunol CB (35) and artocommunol CD (36).

Oxepinoflavone Compounds with oxepinoflavone framework are derived from 3-prenylflavone, where clusters of prenyl experiencing oxidative cyclization with hydroxy group at C2' form a heptagon ring. Compounds that have been found are not much. Oxepinoflavone compounds mostly have a pattern of 2', 4' dioxygenation on ring B. Compounds with oxepinoflavone structures among others are artelastinin (37) which is isolated from the stem wood of A. elasticus (Kijjoa et al. 1998), Artoindonesianin B (38) which is isolated from the root bark of A. champeden by Judge et al. (1999) has cytotoxic properties. Chan et al.

(2003) from the root bark of A. communis isolate artocommunol CC (39).

Pyranoflavone The pyranoflavone framework differs from oxepinoflavone in terms of ring formed by cyclization prenyl group at C3 to hydroxyl at C2 '. Pyranoflavone forms a hexagon ring. Some piranoflavone compounds with dioxygenation in ring B which have been isolated by Chen et al (1993) from the stem wood of A. altilis, among others are isocyclomorusin (40), isocyclomullberin (41), cyclomulberin (42).

Dihydrobenzoxanthone In dihydrobenzoxanthone, C6' in ring B bound directly to the carbon from the group of prenyl hexagon forms a ring. It is quite interesting that dihydrobenzoxanthone is only formed from the flavone with ring B which is

151

oxygenated with pattern of 2', 4' and 5'. This is because the two hydroxy groups at C2' and C5' activate C6' which is located at the ortho position of hydroxy group. Class of dihydroxanthone compounds which have been isolated are artobiloxanthone (43) which are isolated from the stem bark of A. scortechinii by Ferlinahayati (1999). Compound 43 is also isolated from A. nobilis (Sultanbawa et al. 1989, Jayasinghe et al. 2008). Syah et al. (2002) is succeeded in isolating artoindonesianin S (44) and artoindonesianin T (45) of stem wood of A. champeden.

Furanodihydrobenzoxanthone Furanodihydrobenzoxanthone compounds are derived from dihydrobenzoxanthone experiencing further cyclization at the end of prenyl with hydroxy group at C5', then they form a furan ring. Several compounds are reported, namely artonin M (46) which is isolated from A. rotunda by Suhartati et al. (2001) and has cytoxic characters. Cycloartobiloxanthone (47) is isolated from A. scortechini (Ferlinahayati 1999), A. nobilis (Jayasinghe et al. 2008), A. heterophyllus (Uno, 1991). Hakim et al.

(1999) isolate artoindonesianin A (48) from the root bark of A. champeden, these compounds have cytotoxic properties against P-388murine leukemia cells.

Pyranodihydrobenzoxanthone Pyranodihydrobenzoxanthone is probably derived from dihydrobenzoxanthone experiencing cyclization to form Annex ring. Only one compound is that reportedly ever recovered from A. lanceifolius namely artoindonesianin Z2 (49) by Hakim et al. (2006).

Quinonoxanthone Quinonoxanthone is derived from dihydrobenzoxanthone experiencing rearrangement on two hydroxy groups at C2' and C5' to form quinone ring. This class of compounds that is artomunoxantentrion (50) is isolated from the root bark of A. communis by Shieh et al. (1992).

Artonin O (51) from the root bark of A. rotunda which are isolated by Suhartati et al. (2001) are cytotoxic, these compounds are very interesting because they have experience of prenylation in ring B. Cyclopentenoxanthone Compounds with a cyclopentenoxanthone framework is derived from xanthone experiencing rearrangement so that the ring B turned into a pentagon. The reported compounds are artoindonesianin C (52) which is isolated from the stem bark of A. scortechinii (Armin 1999) and root bark of A. teysmanii (Makmur 2000). This compound has been reported to have activities as anti-mycobacterial.

Xanthonolide The isolated xanthonolide compounds namely artonol B (53) is derived from the stem bark of A. scortechinii (Armin 1999) and from the root bark of A. rigidus (Namdaung et al. 2006). The compound is reported to have cytotoxic properties.

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Dihydroxanthone Dihydroxanthone is derived from xanthonolide experiencing bond disconnection as to form compounds with a more stable structure. So far only one compound had

HO O

been reported namely artonol A. (54) which were isolated from the stem bark of A. scortechinii by Armin (1999).

O

OR2

HO

OH OR1

OH

O

OH

OH O

OH O

(43)

(44) R1=R2=CH3 (45)R1=H, R2=CH3

HO O

OH

O

O

O

O

OH O

HO

O

O

(48)

OH

O

O O

O

O

OH O

(47)

HO

OH

O

OH O

(46)

HO

OH

HO

OCH 3

O

O O

HO

O

OH O

OH OH O

H

OH O

(49)

OH O

(50)

(51)

O

O

O

COOMe

O

O

OH

O

O

O

O

O

O

OH O OH O

OH O

(53)

(52)

(54)

OH

OH HO

OH OH HO

(55)

OH

OH

OH HO

OH

(56)

OH

(57)

HAKIM – Secondary metabolites of genus Artocarpus

153

HO

HO

OH

O

OH MeO

OH OCH3

OH

O

HO

OH

(59)

(58)

HO

OCH3

HO

O

(60)

OH HO

O

OH

O

OCH3

OH

O

HO

OH OH

OH

(61)

(62)

(63)

O

(64)

OCH3 OH

O

O

O HO OH

R

O

HO OCH3

(65) R=OMe (66) R=H

HO

OH

HO

HO

OH O O

HO OH

HO

OH

HO

OH O

O

OH

OH

HO

HO

HO

OH O O

O

(68)

(67)

STILBENE As in flavonoids, prenylated stilbene is also found. The prenylations are found on the two rings like the pattern on chalcon. Some stilbene compounds which have been isolated, among others, oxyresveratrol (55) from the stem bark of A. nitida (Yuliani 1997) and from the bark of A. reticulatus (Murniana 1995). Boonlaksiri et al. (2000) isolate stilbene compounds which are anti-malarial from aerial parts of A. integer which is 3,4-trans-4-isopentenyl-3, 5.2 ', 4'-tetrahydroxy stilbene (56), 3,5-trans-4-(3-methylE-but-1-enil)-3,5,2 ', 4'-tetrahydroxystilbene (57) and 4methoxy-2 ,2-dimethyl-6-(2 (2,4-dihydroxy) phenyl-trans-

HO

OH O O

OH

OH

HO

OH HO

HO

OH

HO

OH

(69)

OH

(70)

etenil) cromen or also known as artocarben (58) (Boonlaksiri, 2000) which also have been isolated from A. incisus (Shimizu, 1997). Artoindonesianin N (59), the first derivative stilben reported containing methoxy group, were isolated from the bark of A. gomezianus (Judge, 2002).

ARYLBENZOFURAN As with stilben, the arylbenzofuran compounds found are also prenylated in both rings. These compounds include artohetetophyllin A (60) which were isolated from A.heterophyllus by Zong et.al (2009), 3-(γ, γ-

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dimethylpropenil) morasin M (61) of bark and twigs of A. dadah (Su et al. 2002). Puntumchai et al. (2004) successfully isolate two anti-microbacterial compounds from root of A. lakoocha named lakoochin A (62) lakoochin B (63). From the bark of A. tonkinensis, it is found artotonkin (64) (Lien et.al 1998)

NEOLIGNAN Neolignan derivative compounds were also reported by Su et al. (2002) namely dadahol A (65) and dadahol B (66) which were isolated from the twigs of A. medicine. This group of compound has never been reported to be isolated from other species of Artocarpus. The discovery of

neolignan compounds which is a combination of the two compounds of arylpropanoid is very interesting.

DIELS-ALDER ADDUCTS COMPOUNDS From the root bark of A. heterophyllus, Hano et al.

(1990) isolate the two compounds of Diels-Alder adducts namely artonin C (67) and D (68). Artonin X (69) and kuwanon R (70) are isolated from the bark of A. heterophyllus by Kazuki et al. (1995). Of the framework, it can be seen that this compound comes from two chalcone which experienced a reaction of Diels-Alder adducts. It is interesting that the two units of chalcon making up two compounds are derived from the same compound.

Gambar 1. Biogenesis of several progeny compound of flavonoid from Artocarpus

HAKIM – Secondary metabolites of genus Artocarpus

FLAVONOIDS BIOGENESIS AND ITS PROGENY IN ARTOCARPUS Flavonoid compounds have been isolated from the Artocarpus genus comprise chalcone, flavanones, flavan-3ol and flavone. The flavone compounds are especially the prenylated one. Prenylated flavone with oxygenation pattern of ring B on C2', C4' and C5' can produce a more complex flavone derivatives especially xanthone compounds. Experimental information about the biosynthesis of these compounds from xanthone group of Artocarpus does not exist, but its presence in the secondary metabolites of flavone derivatives are found together in the Artocarpus genus. This reinforces allegations regarding biogenesis path, which usually begins from the 3prenylflavon and produce dihydrobenzoxanthone as an intermediates precursor.

Several hypotheses about the biogenesis of this Artocarpus flavonoids have been reported in the literature, ranging from flavanones derivatives such as artocarpanon that have a pattern of 2', 4'-dioxygenation in ring B followed by a series of related framework establishment of flavone derivatives such as norartocarpetin, followed by prenylation and hydroxylation reactions. 3-prenylflavon compound is the primary precursor for all types of flavonoid derivatives in Artocarpus. Through isoprenoid cyclization on the C3 with oxygen at C2', the pyranoflavone or oxepynoflavone is formed. Isoprenyl group at C-3 of compounds with 2', 4', 5' trioxygenation can bind to the C6' to form dihydrobenzoxanthone, subsequent cyclization of isopropyl with oxygen at C-5' forms the furanodihydrobenzoxanthone or pyranodihydrobenzoxanthone framework.

Xanthone compounds as flavone derivatives resulting from several stages of degradation reaction or rearrangement of B ring framework of the original flavone, such as found out in dihydroxanthone, xanthonolide and cyclopentenoxanthone.

The origional path of cyclopentenocromene framework is still indefinable because it has not been found between the corresponding compounds. General description of flavonoid compounds biogenesis and their derivatives, which are found in the genus of Artocarpus are shown in Figure 1.

CONCLUSION Secondary metabolites have been isolated from Artocarpus comprises of terpenoid, flavonoids, stilbenoid, arylbenzofuran, neolignan, and Diels-Alder adducts compounds. Flavonoid compounds are most abundant class of compounds from Artocarpus. Flavonoids which are succesfully isolated from Artocarpus consists of a variety of frameworks such as derivatives of chalcone, flavanones, flavan-3-ol, simple flavone, prenylflavone, oxepinoflavone, pyranoflavone, dihydrobenzoxanthone, furanodihydrobenzoxanthone, pyranodihydrobenzoxanthone, quinonoxanthone, cyclopentenoxanthone, xanthonolide, and dihydroxanthone. Terpenoid compounds isolated from

155

Artocarpus have cycloartan framework. The simplest stilbene compounds of the Artocarpus genus are the resveratrol compound isolated from A. caplasha.

REFERENCES Achmad SA Hakim EH, Juliawaty LD, Makmur L, Suyatno, Aimi N, Ghisalberti EL. 1996. A new prenylated flavone from Artocarpus champeden. J Nat Prod 59: 878-879.

Aida M, Yamaguchi N, Hano Y, Nomura T. 1997. Constituents of the Moraceae plants. 30. Artonols A, B, C, D, and E, five new isoprenylated phenols from the bark of Artocarpus communis forst.

Heterocycles 45(1): 163-175.

Altman LJ, Zito SW. 1976. Sterols and triterpenes from the fruit of Artocarpus altilis. Phytochemistry 15: 829–830.

Armin AN. 1999. 5’Hidroksikudraflavon dan beberapa senyawa turunan santon dari tumbuhan Artocarpus scortechinii King [Thesis]. Institut Teknologi Bandung. Bandung Barik BR, Bhaumik T, Kundu AK, Kundu AB. 1997. Triterpenoids of Artocarpus heterophyllus. J Indian Chem Soc 74: 163-164 Boonlaksiri C, Oonanant W, Kongsaeree P, Kittakoop P, Tanticharoen M, Thebtaranonth Y. 2000. An antimalarial stilbene from Artocarpus integer. Phytochem 54 415-417.

Cao S, Butler MS, Buss AD. 2003. Flavonoids from Artocarpus lanceifolius. Nat Prod Res 17(2): 79-81.

Chan SC, Ko HH, Lin CN. 2003. New prenylflavonoids from Artocarpus communis. J Nat Prod 66: 427-430.

Chen CC, Huang YL, Ou JC, Lin CF, Pan TM. 1993. Three new prenylflavones from Artocarpus altilis. J Nat Prod 56: 1594-1597.

Dave KG, Telang SA, Venkataraman K. 1962. Flavonoid pigments of the heartwood of Artocarpus integrifolia. Tetrahedron Lett 9-14.

Dayal R, Seshadri TR. 1976. Colourless compounds of the roots of Artocarpus heterophyllus. Isolation of new compound artoflavone., Indian J Chem 12: 895-898 Djakaria YK. 1999. Artocarpanone, A flavanone derivative from root trunk of Artocarpus champeden Spreng [Dissertation]. Bandung Institute of Technology. Bandung. [Indonesia] Ersam T. 2001. Senyawa kimia mikromolekul beberapa tumbuhan Artocarpus hutan tropika Sumatera Barat [Dissertation]. Bandung Institute of Technology. Bandung. Indonesia] Ferlinahayati. 1999. Norartokarpetin dan flavonoid terisoprenilasi dari kulit batang Artocarpus scortechinii King [Thesis]. Bandung Institute of Technology. Bandung. Indonesia] Hakim A. 2008. Kudraflavon C dari kayu batang Artocarpus scortehinii King. Nasional Seminar, Mataram University. Mataram. [Indonesia] Hakim A. 2009. A Prenylated Flavone From The Heartwood Of Artocarpus scortechinii King (Moraceae). Indo. J. Chem. 9 (1): 146150.

Hakim EH, Aripin A, Fahriyati A, Kau MS, Achmad SA, Makmur L, Ghisalberti EL, Nomura T. 1999. Artoindonesianins A and B, two new prenylated flavones from the root of Artocarpus champeden. J Nat Prod 62: 613-615.

Hakim EH, Aripin A, Achmad SA, Aimi N, Kitajima M, Makmur L, Mujahidin D, Syah YM, Takayama H. 2001, Artoindonesianin E, a new flavanone derivative from Artocarpus champeden. Proc ITB (Indonesia) 33: 69-73.

Hakim EH, Asnizar, Yurnawilis, Aimi N, Kitajima M, Takayama H. 2002.

Artoindonesianin P, a new prenylated flavone with cytotoxic activity from Artocarpus lanceifolius, Fitoterapia 73: 668-673 Hakim EH, Achmad SA, Juliawaty LD, Makmur L, Syah YM, Aimi N, Kitajima M, Takayama H, Ghisalberti EL. 2006. Prenylated flavonoids and related compounds of the Indonesian Artocarpus (Moraceae). J NatMed 60: 161-184.

Han AR, Kang YJ, Windono T, Lee SK, Seo EK. 2006. Prenylated Flavonoids from the Heartwood of Artocarpus communis with Inhibitory Activity on Lipopolysaccharide-Induced Nitric Oxide Production. J Nat Prod 69 (4): 719-721.

Hano Y, Aida M, Nomura T. 1990. Two new natural diels-alder type adducts from the root bark of Artocarpus heterophyllus. J Nat Prod 53: 391-395.

156

Â

2 (3): 146-156, November 2010

Hano Y, Inami R, Nomura T. 1994. Constituents of Moraceae plants. 20. a novel flavone, artonin V, from the root bark of Artocarpus altilis. J Chem Res Syn 9: 348-349.

Heyne K. 1987. Useful Plants of Indonesia II. Penerbit Badan Litbang Kehutanan. Jakarta. Indonesia] Khan MR, Omoloso AD, Kihara M. 2003. Antibacterial activity of Artocarpus heterophyllus. Fitoterapia 74: 501-505.

Ko HH, Lu YH, Yang SZ, Won SJ, Lin CN. 2005. Cytotoxic prenylflavonoids from Artocarpus elasticus. J Nat Prod 68: 16921695.

Kochummen KM. 1987. Moracea in tree flora of Malaya. Vol. 2. Forest Research Institute. Kepong, Malaysia.

Jayasinghe L, Balasooriya BAIS, Padmini WC Hara N, Fujimoto Y. 2004.

Geranyl chalcone derivatives with antifungal and radical scavenging.

Phytochem 65: 1287-1290.

Jayasinghe L, Rupasinghe GK, Hara N, Fujimoto Y. 2006. Geranylated phenolic constituents from the fruits of Artocarpus nobilis.

Phytochem 67: 1353-1358.

Jayasinghe ULB, Samarakoon TB, Kumarihamy BMM, Hara N, Fujimoto Y. 2008. Four new prenylated flavonoids and xanthones from the root bark of Artocarpus nobilis. Fitoterapia 79: 37-41.

Kazuki S, Aida M, Hano Y, Nomura T. 1995. A diels-alder-type adduct from Artocarpus heterophyllus. Phytochemistry 40. 754-756 Kijjoa A, Cidade HM, Pinto MMM, Gonzalez MJTG, Anantachoke C, Gedris TE, Herz W. 1996. Prenylflavonoids from Artocarpus elasticus. Phytochem 43 691-694.

Kijjoa A, Cidade HM, Gonzalez MJTG, Afonso CM, Silva AMS, Herzt W. 1998. Further prenylflavonoids from Artocarpus elasticus.

Phytochem 47: 875-878.

Lien TP, Ripperger H, Porzel A, Sung TV, Adam G. 1998. Constituents of Artocarpus tonkinensis. Pharmazie 53 : 353-355.

Likhitwitayawuid K, Sritularak B. 2001. A new dimeric stilbene with tyrosinase inhibitiory activity from Artocarpus gomezianus. Journal of Natural Product 64: 1457-1459.

Lin CN, Lu CM, Huang PL. 1995. Flavonoids from Artocarpus heterophyllus. Phytochem 39 1447-1451.

Makmur L, Syamsurizal, Tukiran Achmad SA, Aimi N, Hakim EH, Kitajima M, Takayama H. 2000. Artoindonesianin C, a new xanthone derivative from Artocarpus teysmanii. J Nat Prod 63 243-244.

Murniana 1995. Several compounds of secondary metabolites from bark of Artocarpus reticulatus [Thesis]. Bandung Institute of Technology.

Bandung. [Indonesia] Nomura T, Hano Y, Aida M. 1998. Isoprenoid substitued flavanoids from Artocarpus plants (Moraceae). Heterocycles. 47: 1179-1205.

Patil AD, Freyer AJ, Killmer L, Offen P, Taylor PB, Votta BJ, Johnson RK. 2002. A new dimeric dihydrochalcone and a new prenylated flavone from the bud covers of Artocarpus altilis: potent inhibitors of cathepsin K. J Nat Prod 65: 624-627.

Puntumchai A, Kittakoop P, Rajviroongit S, Vimuttipong S, Likhitwitayawuid K, Thebtaranonth Y. 2004. Lakoochins A and B, new antimycobacterial stilbene derivatives from Artocarpus lakoocha.

J Nat Prod 67: 485-486.

Namdaung U, Aroonrerk N, Suksamrarn S, Danwisetkanjana K, Saenboonrueng J, Arjchomphu W, Suksamrarn A. 2006. Bioactive constituents of the root bark of Artocarpus rigidus subsp. rigidus.

Chem Pharm Bull 54: 1433-1436.

Seo EK, Lee D, Shin YG, Chai HB, Navarro HA, Kardono LBS, Rahman I, Cordell GA, Farnsworth NR, Pezzuto JM, Kinghorn AD, Wani

MC, Wall ME. 2003. Bioactive prenylated flavonoids from the stem bark of Artocarpus kemando. Arch Pharm Res 26 (2): 124-127.

Shieh WL, Lin CN. 1992. A quinonoid pyranobenzoxanthone and pyranodihydrobenzoxanthone from Artocarpus communis.

Phytochem 31 364-367.

Shimizu K, Kondo R, Sakai K. 1997. A stilbene derivative from Artocarpus incisus. Phytochem 45: 1297-1298 Su BN, Cuendet M, Hawthorne ME, Kardono LBS, Riswan S, Fong HHS, Mehta RG, Pezzuto JM, Kinghorn AD. 2002. Constituents of the bark and twigs of Artocarpus dadah with cyclooxygenase inhibitory activity. J Nat Prod 65: 163-169.

Suhartati T, Achmad SA, Aimi N, Hakim EH, Kitajima M, Takayama H, Takeya K. 2001. Artoindonesianin L, a new prenylated flavone with cytotoxic activity from Artocarpus rotunda. Fitoterapia 72 912-918.

Sultanbawa MUS, Surendrakumar, Sivagnanasundram. 1989. Two pyranodihydrobenzoxanthones from Artocarpus nobilis. Phytochem 28(2): 599-605.

Syah YM, Achmad SA, Ghisalberti EL, Hakim EH, Makmur L, Mujahidin D. 2002. Artoindonesianins Qâ&#x20AC;&#x201C;T, four isoprenylated flavones from Artocarpus champeden Spreng. (Moraceae).

Phytochem 61 949-953.

Syah YM, Juliawaty LD, Achmad SA, Hakim EH, Ghisalberti EL. 2006 Cytotoxic prenylated flavones from Artocarpus champeden, Journal Natural Medicine 60: 308-312.

Udjiana SS. 1997. Several compounds of flavan and stilbene derivatives from root bark of Artocarpus reticulatus [Thesis]. Bandung Institute of Technology. Bandung. [Indonesia] Uno Y, Aida Y, Shiina M, Nomura T, Kagei K. 1991. Isolation of flavone derivatives from Artocarpus heterophylla. Jpn. Kokai Tokkyo Koho 8: 197-200 Venkataraman K. 1972. Wood phenolics in the chemotoxonomy of the Moraceae. Phytochem 11 : 1571-1586 Verheij EWM, Coronel RE (eds). Plant Resources of South Asia No. 2 Edible Fruits and Nuts. Prosea Foundation. Bogor.

Wang Y, Xu K, Lin L, Pan Y, Zheng X. 2007. Geranyl flavonoids from the leaves of Artocarpus altilis. Phytochem 68: 1300-1306.

Wang YH, Hou AJ, Chen L, Chen DF, Sun HD, Zhao QS, Bastow KF, Nakanishi Y, Wang XH, Lee KH. 2004. New isoprenylated flavones, artochamins A-E and cytotoxic principles from Artocarpus chama. J Nat Prod 67: 757-761.

Weng JR, Chan SC, Lu YH, Lin HC, Ko HH, Lin CN. 2006. Antiplatelet prenylflavonoids from Artocarpus communis. Phytochem 67: 824829.

Widyawaruyanti A, Subehan, Kalauni SK, Awale S, Nindatu M, Zaini NC, Syafruddin D, Asih PBS, Tezuka Y, Kadota S. 2007. New prenylated flavones from Artocarpus champeden, and their antimalarial activity in vitro. J Nat Med 61: 410-413.

Yamazaki M, Okuyama E, Matsudo T, Takamaru T, Kaneko T. 1987.

Principles of indonesian herbal drugs having an antiulcerogenic activity. I. Isolation and identification of (+/-)-catechin from Artocarpus integra merr. Yakugaku Zasshi 107(11) : 914-916 Yuliani E. 1997. Stilbene derivatives compounds from wood of Artocarpus nitida Trec [Thesis]. Bandung Institute of Technology.

Bandung. [Indonesia] Zong P, Zheng, Sibao C, Shiyun W, Xia CW, KaWC, Jia JW, Dajiang Y, Mingfu W. 2009. Chemical Components and Tyrosinase Inhibitors from the Twigs of Artocarpus heterophyllus. J Agric Food Chem 57: 6649-6655.

A-1

Authors Index Ade R Ali S Astirin OP Buntaran W Estikomah SA Farooqi H Faturrahman Hakim A Hanafiah DS Hesni MA Irnidayanti Y Julisaniah NI Jupri A Kamrani E Karwa A Khan HN Khan JA Kurnia AI Kurnianingsih R Kurniawati ND Kusmiyati Latief AS Listiawati A Machmudin D Mahajoeno E Moradi M Muhadiono Mukarlina Mulyani S

89 109 55, 126 55 1 109 38 146 121 34 116 73 73 34 141 109 109 48 73 135 7 43 62 135 48, 55 34 43 62 62

Muna L Pangastuti A Pattiselanno F Pramudya B Purnomo DW Purwanto E Rai MK Ranjbar MS Sadsoeitoeboen MJ Sajidan Sanjaya Y Saragih EW Sarkono Sasmita P Setyawan AD Shokri MR Sobir Sofyan Y Sugiyarto Suhartanto MR Sunarpi Suranto Sutarno Syarief R Tribadi Trikoesoemaningtyas Widiastuti A Wirnas D Yahya S

126 1 78 43 83 48 89, 141 34 78 14 135 78 38 67 96 34 23 38 126 23 73 14 1 43 14 121 23 121 121

A-2

Subject Index 2-methoxyethanol Agaricus agroforestry alternative energy anatomy Anodonta woodiana Argomulyo soybean variety Artocarpus biodiversity bioethanol black-6 mice candy/candied cassava Central India cheese coconut water colchicinamide colchicine commercial conservation

consumption rate coronary heart disease Coscinaraea monile Cu cuscus Cyphastrea chalcidicum Dayak diabetes Diels-Alder diets double haploid lines edible exposure extract

farmers' profits fermentation fertilizing first record flavonoid gamma ray gamma ray irradiation Garcinia mangostana genetic variability

116, 117, 120 141-145 96, 97, 101, 103-107 7 14, 16, 19-22, 24, 25, 28, 31-33, 51, 65 48-53 121-124 146, 147, 150, 151, 154156 86, 95-97, 99-107, 126, 134, 141, 144, 145, 7-13, 15 116, 117, 120 55-61 7-22, 61, 85, 102, 103 141-142, 144-145 1-6, 115 62-66 89, 90 89-95 75, 77, 92, 94, 95, 110, 141, 145 62, 66, 78, 79, 87, 88, 95-97, 99, 100, 103-107, 125, 134, 141, 145 135, 138 109, 111 34-37 48-53 78-82 34-37 96, 97, 99-107 109, 112 146, 147, 154-156 78-82, 109, 114, 67, 68, 70, 72 16-18, 33, 109, 111, 113-115, 141, 145, 156 29, 48-52, 93-94, 135 3, 6, 9, 16, 24, 73-77, 89, 92, 101, 103, 112, 118, 126-132, 134, 146, 147 43, 44, 46, 56 1, 2, 6, 7, 9-13, 39, 41, 42, 61 43-46 34 146-148, 150, 151, 153156 23-33, 121-125 24-32, 121-125 23, 33 23, 33, 70, 121

growth

habitat

habitat selection hypertension identification iles-iles induced mutation isolation Kalimantan lactic acid bacteria Lactobacillus paracasei ssp. paracasei LD50 limb bud lipid profile Lycopersicum esculentum Manihot esculenta Melghat micro mutation micronutrient Moraceae morphologic variation mustard oil NAA neurofilament nutrient contents oil blend palm olein oil Pandanus conoideus var. yellow fruit Paraphalaenopsis serpentilingua peritrophic membrane Persian Gulf photoisomerization polyploidy production

promising protein

2, 3, 8, 12, 13, 19, 23, 25-29, 32, 33, 35, 38-42, 56, 58, 61-66, 73-77, 9092, 94, 95, 101, 103, 104, 114, 117, 120-134, 138 14, 22, 34, 35, 40-42, 48, 50, 78-85, 87, 88, 97, 100, 104 83, 84, 88 109 1, 2, 5, 22, 35, 37-39, 41, 42, 77, 142, 145, 156 7-13 33, 121, 125 6, 38, 39, 42, 77, 90, 95, 146, 155, 156 62, 66, 96-106, 146, 1, 6, 38-42 38, 41, 42 135-138, 140 116-120 109-115 55 14-16, 18, 22 141-143, 145 121, 122, 125 109, 111, 114, 115 146, 147, 155, 156 14 109-113 62-66 116, 117, 119, 120 78, 80, 81 109-111, 113, 114 109-114 126-129, 131-134 62-64, 66 135-140 34-37 89, 90 89, 90, 94 1, 2, 7-13, 15, 22, 23, 42, 43, 46, 47, 55, 56, 61, 68, 73-75, 77, 89, 92, 94, 95, 100-103, 105, 106, 120-122, 125, 128, 129, 133, 134, 141, 145, 155 7, 67, 124, 145 1-6, 10, 14-16, 21, 22, 38, 48-53, 64, 75, 79-82,

A-3

protein band pattern Qeshm ratooning retinol Rhizopus oryzae rice plants ripening Rusa timorensis seaweed secondary metabolites shifting cultivation SlNPV Spodoptera litura stable

89, 91, 93, 99, 109, 114120, 125, 129, 131, 133, 139, 140 14, 16, 22 34-37 43, 44, 47 109, 110, 114, 115 1-6 24, 69, 72-77 1-6, 115 83, 87, 88 40, 73-77 91, 92, 95, 146, 147, 155, 156 96, 97, 99-107 135-140 135-140 8, 44, 46, 56, 67-69, 71,

sugar sugarcane plant temperature

teratogenic tomato toxicity uniform vimentin Wanagama I Forest white rat

72, 90, 152, 3, 7, 8, 10-13, 15, 41-43, 45, 47, 55-61 43-37 1-6, 8-13, 15-19, 22, 30, 34, 37, 39, 50-52, 57, 85, 91, 118, 128, 136, 137, 147, 116, 117, 120, 126, 128, 130, 134 55-61, 74, 77, 135 52, 53, 89,92, 93, 120, 134 67-71, 143, 9, 35, 50, 52, 56 116-120 83-88 126-128, 132, 134

A-4

List of Peer Reviewer Abd Fattah N. Abd Rabou

Department of Biology, Faculty of Science, Islamic University of Gaza, Palestine

Ahmad Dwi Setyawan

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Aluh Nikmatullah

Biology Program, Faculty of Mathematics and Natural Sciences, University of Mataram, Mataram 83125, West Nusa Tenggara, Indonesia.

Artini Pangastuti

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Edi Purwanto

Faculty of Agriculture, Sebelas Maret University, Surakarta 57126, Indonesia

Edwi Mahajoeno

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Ehsan Kamrani

Department of Marine Biology, School of Basic Sciences, University of Hormozgan, P.O. Box 3995, Bandar Abbas, Iran.

Irnanda Aiko Fifi Juuna

Department of Soil Sciences, Faculty of Agriculture and Agriculture Technology, State University of Papua, Manokwari 98314, West Papua, Indonesia.

Mahendra Kumar Rai

Department of Biotechnology, SGB Amravati University, Amravati 444602, Maharashtra, India.

Okid Parama Astirin

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Rajni Kaul

Director General Basic Medical Science, Indian Council of Medical Research (ICMR), Ansari Nagar, New Delhi 110029, India

Rizal Syarief

Faculty of Agriculture Technology, Bogor Agricultural University, Darmaga Bogor 16680, West Java, Indonesia

Sajidan

Faculty Teacher Training and Education, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Savita Yadav

Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Skyler J. Hackley

University of California, Santa Cruz, CA, USA and Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA, USA

Sobir

Faculty of Agriculture, Bogor Agricultural University, Darmaga, Bogor 16680, West Java, Indonesia

Sugarjito

Zoology Division, Research Center for Biology, Indonesian Institute of Sciences, Cibinong-Bogor 16911, West Java, Indonesia

Sugeng Budiharta

Purwodadi Botanic Garden, Indonesian Institute of Sciences, Pasuruan 67163, East Java, Indonesia

Sugiyarto

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Suranto

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Sutarno

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Trikoesoemaningtyas

Departement of Agronomy and Horticulture, Bogor Agricultural University, Darmaga, Bogor 16680, West Java, Indonesia

Yayan Sanjaya

Biology Program, Educational University of Indonesia. Bandung 40154, West Java, Indonesia

A-5

Table of Contents

Vol. 2, No. 1, Pp. 1-53, March 2010 Ripening for improving the quality of inoculated cheese Rhizopus oryzae SOLIKAH ANA ESTIKOMAH, SUTARNO, ARTINI PANGASTUTI Comparasion of iles-iles and cassava tubers as a Saccharomyces cerevisiae substrate fermentation for bioethanol production KUSMIYATI

1-6 7-13

Variation of morphological and protein pattern of cassava (Manihot esculenta) varieties of Adira1 and Cabak makao in Ngawi, East Java TRIBADI, SURANTO, SAJIDAN

14-22

Diversity analysis of mangosteen (Garcinia mangostana) irradiated by gamma-ray based on morphological and anatomical characteristics ALFIN WIDIASTUTI, SOBIR, MUH RAHMAD SUHARTANTO

23-33

First record of two hard coral species (Faviidae and Siderastreidae) from Qeshm Island (Persian Gulf, Iran) MAHDI MORADI, EHSAN KAMRANI, MOHAMMAD R. SHOKRI, MOHAMMAD SHARIF RANJBAR, MAJID ASKARI HESNI

34-37

Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina) as a potential candidate of probiotic SARKONO, FATURRAHMAN, YAYAN SOFYAN

38-42

Productivity of sugarcane plants of ratooning with fertilizing treatment A SUTOWO LATIEF, RIZAL SYARIEF , BAMBANG PRAMUDYA, MUHADIONO

43-47

Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana) and its relation to Cu and protein content in the body shell AHMAD INTAN KURNIA, EDI PURWANTO, EDWI MAHAJOENO

48-53

Vol. 2, No. 2, Pp. 55-107, July 2010 Effect of various sugar solution concentrations on characteristics of dried candy tomato (Lycopersicum esculentum) WAWAN BUNTARAN, OKID PARAMA ASTIRIN, EDWI MAHAJOENO

55-61

The effect of coconut water and naphthalene acetic acid (NAA) application on the in vitro growth of Paraphalaeonopsis serpentilingua from West Kalimantan MUKARLINA, AGUSTINA LISTIAWATI, SRI MULYANI

62-66

Evaluation of uniformity, variability, and stability of agronomic traits of doubledd haploid rice lines resulting from anther culture PRIATNA SASMITA

67-72

Effect of seaweed extracts on growth and yield of rice plants SUNARPI, AHMAD JUPRI, RINA KURNIANINGSIH, NUR INDAH JULISANIAH

73-77

The diet of cuscus (Spilocuscus maculatus) in natural and captivity habitat EVI W. SARAGIH, MARIA JUSTINA SADSOEITOEBOEN, FREDDY PATTISELANNO

78-83

A Habitat selection model for Javan deer (Rusa timorensis) in Wanagama I Forest, Yogyakarta DANANG WAHYU PURNOMO

84-89

Review: Colchicine, current advances and future prospects RAVINDRA ADE, MAHENDRA KUMAR RAI

90-96

A-6 Review: Biodiversity conservation strategy in a native perspective; case study of shifting cultivation at the Dayaks of Kalimantan AHMAD DWI SETYAWAN

97-108

Â

Vol. 2, No. 3, Pp. 109-156, November 2010 Serum lipid profile and retinol in rats fed micronutrient rich edible vegetable oil blend HAMID NAWAZ KHAN, HUMAIRA FAROOQI, SHAKIR ALI, JAFAR SALAMAT KHAN

109-117

Expression of vimetin protein and neurofilamen on limb bud of black-6 mice on gestation day 12 induced by 2-methoxyethanol by RT-PCR YULIA IRNIDAYANTI

116-120

Induced mutations by gamma ray irradiation to Argomulyo soybean (Glycine max) variety DIANA SOFIA HANAFIAH, TRIKOESOEMANINGTYAS, SUDIRMAN YAHYA, DESTA WIRNAS

121-125

Teratogenic test of Pandanus conoideus var. yellow fruit extract to development of rat embryo (Rattus norvegicus) LINTAL MUNA, OKID PARAMA ASTIRIN, SUGIYARTO

126-134

Histological study of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura larvae YAYAN SANJAYA, DADANG MACHMUDIN, NANIN DIAH KURNIAWATI

135-140

Hitherto unreported Agaricus species of Central India ALKA KARWA, MAHENDRA KUMAR RAI

141-145

Review: Diversity of secondary metabolites from Genus Artocarpus (Moraceae) ALIEFMAN HAKIM

146-156

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| Nus Biosci | vol. 2 | no. 3 | pp. 109‐156| November 2010 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

Serum lipid profile and retinol in rats fed micronutrient rich edible vegetable oil blend HAMID NAWAZ KHAN, HUMAIRA FAROOQI, SHAKIR ALI, JAFAR SALAMAT KHAN

109‐117

Expression of vimetin protein and neurofilamen on limb bud of black‐6 mice on gestation day 12 induced by 2‐methoxyethanol by RT‐PCR YULIA IRNIDAYANTI

116‐120

Induced mutations by gamma ray irradiation to Argomulyo soybean (Glycine max) variety DIANA SOFIA HANAFIAH, TRIKOESOEMANINGTYAS, SUDIRMAN YAHYA, DESTA WIRNAS

121‐125

Teratogenic test of Pandanus conoideus var. yellow fruit extract to development of rat embryo (Rattus norvegicus) LINTAL MUNA, OKID PARAMA ASTIRIN, SUGIYARTO

126‐134

Histological study of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura larvae YAYAN SANJAYA, DADANG MACHMUDIN, NANIN DIAH KURNIAWATI

135‐140

Hitherto unreported Agaricus species of Central India ALKA KARWA, MAHENDRA KUMAR RAI

141‐145

Review: Diversity of secondary metabolites from Genus Artocarpus (Moraceae) ALIEFMAN HAKIM

146‐156

Published three times in one year PRINTED IN INDONESIA

ISSN 2087‐3948 (print)

ISSN 2087‐3956 (electronic)