PROCEEDING of Aceh International Pharmacy Conference 2013 Syiah Kuala University, Indonesia, September 13-15, 2013

Table of Contents

No Title Page Welcome remarks from Rector of Syiah Kuala University Welcome remarks from Dean of Mathematics and Natural Sciences Faculty, Syiah Kuala University Welcome remarks from Chairperson of AIPC2013 ORAL PRESENTATIONS 1. Measuring Pharmacological Influences on the Gastrointestinal Tract with In 1 Vitro Methods (Karl-Herbert Schäfer) 2. Alternative medicine: Special Emphasis on the Indian Example (Philip 2 Stokoe) 3. Quality Control for Drugs derived from Herbs, and its recent method of 3 Validation (Gunawan Indrayanto) 4. Herbal Products: Evidence-Based or Products of Quackery? 4 (Ibrahim Jantan) 5. Development of Retention Index and its Application in Herbal Medicines 5 (Rinaldi Idroes) 6. Antityrosinase and Antimicrobial Activities from Thai Medicinal Plants 6 (Sukanya Dej-adisai, Imron Meechai, Jindaporn Puripattanavong, Sopa Kummee) 7. Simplex Characterization and Isolation of Volatile Oil In Fresh and and 7 Dried of Citrus x jambhiri Lush Peel and Analysis It’s Components By GC-MS, (Shanty Hutabarat, Panal Sitorus, Effendy De Lux Putra) 8. Basic and Applied Research in The Field of Herbal Medicine (Chaidir). 8 9. System Biology in Herbal Medicine: Concept, Development and Aplication 10 on JAMU (Latifah K Darusman) 10. Botanical Ingredients in Cosmetics: Formulation and Safety Assessment 11 (Endang Lukitaningsih) 11. Bioactivity Screening of Natural Products from West Sumatran Marine 13 Invertebrates (Dian Handayani) 12. Characterization of Chemical Compounds By Gas Chromatography - Mass 14 Spectroscopy On Artocarpus elasticus (Bendo) leaves As Antidiabetic (Rosnani Nasution, Nurdin Saidi, Marianne) 13. Strategy Analysis of Smallholder Cocoa Development Models in Aceh 20 Province (Ashabul Anhar, Agussabti, Yusya Abubakar, Romano, Indra, T. Saiful Bahri, Akhmad) 14. Cytotoxic Activities from Stem Bark Extracts of Calotropis gigantea L 28 (Kartini Hasballah, Murniana and Ichsan) 15. The Quality of Spermatozoa Tends to Increase in Male Local Kacang Goats 36 after Supplementation of Powder of Katuk Leaf (Sauropus androgynus, L. Merr.) At Different Doses (Teuku Reza Ferasyi, Hamdani Budiman, Juli

iii

PROCEEDING of Aceh International Pharmacy Conference 2013 Syiah Kuala University, Indonesia, September 13-15, 2013

Melia, Razali, Azhari, Husnul Rizal, Rina Aulia Barus, and Agik Suprayogi) POSTERS 16. Antibiotic Production From Thermophilic Jaboi Sabang Local Isolate : 43 Using TSB Medium (Febriani, Ramayanti, T.M. Iqbalsyah, Khairan, and Frida Oesman) 17. Pharmacy Students Perception on Complementary Alternative–Medicine 48 (CAM) : A Pilot Study (Khairunnisa, Marianne and Lia Laila) 18. The Effect of Garlic Extract, Aspirin and The Combination of Garlic Extract 53 and Aspirin on Bleeding Time in Mice (Mus musculus) (Rosalia, Rinidar, Sumarti) Analysis of Volatile Oils Components in Fresh and Dried of Citrus x 19. jambhiri Lush Peel By GC-MS (Shanty Hutabarat, Panal Sitorus, 58 Effendy De Lux Putra) 20. Evaluation of Radioprotective Potential of Ginseng on Micronuclei in 70 Gamma Ray Irradiated Human Blood Lymphocyte (Yanti Lusiyanti, Zubaidah Alatas and Mukh Syaifudin) 21. Potential of Celery (Apium graveolens L.) For Reducing Triglyceride 77 Levels Serum In Rat Male With Electrical Stress Induced (Yusni ) 22. Key to the Seven Species of Lacessititermes Homlgren (Isoptera, 81 Termitidae) from the Leuser Ecosystem, Sumatra (Novita and Syaukani) 23. Detection Morphine and Cannabinol in opioid abusers hair by GC-MS 85 (Muhammad Taufik and Mahmudi) 24. The Temperature Effect on Dead Time and Retention Index Determination 89 in Capillary Gas Chromatography (Frida Oesman, Rinaldi Idroes, Mahmudi and Athailah) 25. Possibility Application of Kovats Retention Index in Supercritical Fluid 91 Chromatography (Rinaldi Idroes, Frida Oesman, Mahmudi and Athailah)

PROCEEDING of Aceh International Pharmacy Conference 2013 Syiah Kuala University, Indonesia, September 13-15, 2013

Characterization of Chemical Compounds By Gas Chromatography - Mass Spectroscopy On Artocarpus elasticus (Bendo) leaves As Antidiabetic

Rosnani Nasution1, Nurdin Saidi1, Marianne2

1Faculty of Mathematics and Natural Sciences, Syiah Kuala University, Darussalam, Banda Aceh, 23111, Indonesia 2Faculty of Pharmacy, University of Sumatera Utara, Medan, Indonesia Email: [email protected]

ABSTRACT

Results with GC-MS characterization hexane and ethyl acetate leaf extract Artocarpus elasticus obtained several compounds, including, the compound is a precursor compound of terpenoids / steroid that is neopyhitadiene and squalene. Antidiabetic testing of these extracts can lower blood sugar levels male Swiss Webster mice were performed with a glucose tolerance test. The extract of ethyl acetate can lower blood sugar in mice is greater than hexane extracts (p>0.05).

Key words: Hexane extract, ethanol extract of A. elasticus leaf, blood glucose, glucose Tolerance

Introduction

Chemical compounds that exist in the Family Moraceae Artocarpus genus, generally in the form of compounds terpenoids, steroids, and flavonoids (Jagtab and Bapat, 2010). Terpenoid compounds terpenes and steroids are derived from isoprene compounds [(in this case the active isoprene is acetyl coenzyme A, with various reactions to be mevalonic acid and this mevalonic acid react to be isopentenyl pyrophosphate (IPP) and dimetilallil pyrophosphate (DMAPP)]. Subsequent investigation showed that mevalonic acid, reacts to farnesil pyrophosphate, two molecules of farnesil pyrophosphate combine to produce squalene (atom C30) with adverse outcomes HOPP. squalene in the next lane is going to be a steroid (Achmad, 1987). In previous studies the authors have obtained the steroid compounds from the leaves of Artocarpus camansi that is betasitosterol propionate, the determination of its structure by using instruments such as IR, UV, 1H-NMR, 13C-NMR, COSY, HMBC, HSQC, DEPT, etc. (Rosnani at al.,2013), while the results of previous studies of the stem bark of Artocarpus elasticus contains triterpenoids (Esti, 2009). From the reaction of steroid biosynthesis, there is a relationship between the terpenoids in plant A. elasticus and steroids in the leaves of A. camansi that called chemotaxonomic in Moraceae family, but research of the chemical compounds from the leaves of A. elasticus have not done, whereas the GC-MS in this research, results of its, suggest a link compounds (in this case squalene) which is a steroid precursor. Beside that, in research that has been done by the authors that Artocarpus camansi leaves can lowering blood sugar levels male Swiss Webster mice

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(Rosnani, 2013). Based on this, strategic to do research of chemical compounds in the leaves A.elasticus and its antidiabetic activity.

Material and Methods This research was conducted at the Research Laboratory of the Department of Chemistry at the Syiah Kuala University in 2012, while the characterization by GC-MS performed in UPI Bandung. Sample of Artocarpus elasticus have taken from Langkahan, North Aceh District, determined in Bogoriense Bogor. The air dried and coarsely powdered leaves (1 kg) was macerated with n-hexane, and the hexane extract was concentrated to get a dark brown viscous mass (15 g). Then the grout was macerated with the solvent of ethyl acetate, to get ethylacetate extract (30 g). Both of these extracts were characterized by GC-MS and tested their antidiabetic against male mice aged 3 months with glucose tolerance test. The mice were divided into 4 groups, and test preparation are given as follows: group I ( negative control) was given 1% CMC-Na, group II (comparison, positive control) given glibenclamide 0.45 mg/kg bw, group III-IV (test) respectively given hexane/ethylacetate extract of the leaves A. elasticus doses of 50, 100 mg/kg bw. Thirty minutes later, each group was given glucose 30 mg/kg bw. After 30 minutes of glucose loading, blood glucose levels of mice were measured, and measured again at minute 60, and 90. The data obtained were processed statistically using the program Statistical Package for the Social Sciences) SPSS 18. Test for normality using the Kolmogorov-Smirnov test and nonparametric test using Kruskal-Wallis and Mann Whitney U with significance level of p <0.10 against the control and p <0.05 for comparison (glibenclamide), to determine the existence of significant differences between the negative control group, treatment and comparison (positive controle).

Result and Discussion The results of characterization by GC-MS, in the ethyl acetate extract there are three (3) chemical compounds, those are: acetic acid, ethyl acetate ester, and neophytadiene. From hexane extracts obtained 2 compounds, namely: cyclopentadiene, and squalene, (with 85% similiarity). MS spectrum of the compound with 87% similarity with neophytadiene (2,6,10-trimethyl-14,14- ethylene-pentadecne), in Figure 1 below.

Figure 1. MS spectrum of compound in ethyl acetate extract

Neophytadiene spectrum in Figure 2

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Figure 2. MS Neophytadiene spectrum (Library)

MS spectrum of the compound with 85% similarity with methyl squalene found in Figure 3 below

Figure 3. MS spectrum of compound in the hexane extract

Based on data available at the MS Library is used to characterize the hexane extract is known that the spectrum of compounds derived from the hexane extract is thought to methyl squalene, with 85% similarity. Spectrum of methyl squalene in Figure 4 below

Figure 4. Compounds spectrum metilsqualen (Library)

The same peaks can be seen on both of the spectrum, such as the peak, base peak at 69 m/e, as well as other peaks, such as: 41, 55, 81, 95, 107 (109), 121, 137, 147 (149), 163, 175, 189 (190), and 231 (232). Bioassay results against ethyl acetate and hexane extracts in Table 1 below.

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Table 1. Glucose tolerance test using hexane extract / ethylacetate of Artocarpus elasticus leaves with various concentration

Group Average Blood Glucose Levels (mg / dL) at minute 30 p 60 p 90 p CMC-Na (control) 305 ± - 230,67 - 213,67 - 61,73 0,002^ ± 22,37 0,000 ± 13,01 0,000 Ethyl acetate Extract Leaf 242,67 0,004* 195,67 0,186 170,33 0,033* Artocarpus elasticus 50 mg/kg bw ± 28,20 0,987# ± 41,10 0,000 ± 16,50 0,000 Ethyl acetate Extract of leaves 221 ± 0,002* 189,67 0,011* 166,33 0,004* Artocarpus elasticus 100 mg/kg 17,35 1,000# ± 15,28 0,006 ± 10,50 0,002 bw N-hexane extract of leaves 189 ± 0,170 180 ± 0,524 151,67 0,193 Artocarpus elasticus 50 mg/kg bw 10,54 0,112# 17,58 0,000 ± 36,50 0,000 N-hexane extract of leaves 174 ± 0,039* 146 ± 0,368 130 ± 0,135 Artocarpus elasticus 100 mg/kg bw 7,00 0,414# 28,93 0,000 27,84 0,000 Glibenklamid 0,45 mg/kg bw 174 ± 0,002* 54 ± 0,000* 38 ± 0,000* (control +) 7,00 - 2,00 - 2,00 - Description: P = number of meaningfulness, confidence level of 95%, The first line was compared with control The second line compared to the comparator *= Significant contrast to controls (p <0.05) # = Not significantly different to the control + (p ≥ 0.05) ^ = Significant contrast to the control + (p <0.05)

The measurement of blood glucose 30 minutes after administration of glucose , EEADK mice given 50 mg/kg bw showed an average blood glucose levels were lower and significantly different compared to controls who were given CMC-Na 1 % ( p < 0.05 ), and no significantly different to the positive control (glibenclamide ) ( p < 0.05 ), it means both extracts can lowering the blood sugar levels of mice. After 60 did not show significant differences with the negative control , and the positive controle, and at 90 minutes the blood sugar levels significantly different from the control but significantly different from the positive controle, showing this extract still affect the relative decrease in blood sugar, but little activity due to significantly different to positive controle. At a concentration of 100 mg/kg bw, at 30 minutes after administration of glucose, ethyl acetate extracts have activity lowers blood sugar levels significantly different from the negative control and not significantly different to the positive controle, it means the extracts active states in the first 30 minutes. In the next 60 minutes ethylacetate extract significantly different to negative control, but also different from the positive controle , which shows the 60 minute activity lowers blood sugar levels are smaller, likewise the same thing in the next 90 minutes. Hexane extract with a concentration of 50 mg/kg bw of mice after 30 min of glucose showing activity significantly different from negative controls and not significantly different from positive controle. However, at 60 and 90 minutes, activity -minute blood sugar levels are not

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significantly different from negative controls and significantly different from the positive controle, it means the hexane extract was not active at the time 60 and 90 minutes.

Hexane extract (EHAE) with a concentration of 100 mg/kg bw mice at 30 min after glucose administration showed activity significantly different from control and not significantly different from the comparison stating hexane is actively lowering blood male mice, but it did not show any activity at minute 60 and 90.

Curves which showed a decrease blood sugar levels above is demonstrated in Figure 3 below. Based on Figure 3 it can be seen that the four samples tested the hexane extract and ethyl acetate extract with a concentration of 50 and 100 mg/kg bw mice show activeness in lowering blood sugar levels of mice. Due to the lowered blood sugar is under the control (dark blue line)

Figure 3 . Glucose tolerance test using hexane and ethyl acetate extracts of leaves of Artocarpus elasticus with various concentration

Figure 3. Glucose tolerance test using hexane and ethyl acetate extracts of leaves of Artocarpus elasticus with various concentration

However, the activity of hexane extract and ethyl acetate extract in lowering blood sugar levels are under active glibenclamide as a comparison or positive controle ( brown line ). The relationship between chemical compounds that exist in the hexane and ethyl acetate extracts with biological activity are as follows. In the possibility of active hexane extract lowers blood sugar levels are squalena compounds as the active betasitosterol preqursor lowering blood sugar levels (Rosnani, 2013) , and the active compounds in the ethyl acetate extract in lowering blood sugar levels is neophytadiene ( 2,6,10 - trimethyl - ethylene - 14 ,14 - pentadecne ), which is a possible precursor to squalene , because these compounds are in the same leaf.

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Conclusion Hexane leaf extract of Artocarpus elasticus contains compounds similar squalena, while the ethyl acetate extract contains compounds similiar with neophytadiena. Hexane and ethyl acetate extracts of leaves of Artocarpus elasticus have the ability to lower blood glucose levels, doses of 50 and 100 mg/kg bw . However, the ethyl acetate extract more active than hexane extract

Acknowledgement The author is thankful to the head of Department FMIPA UNSYIAH, Nature Materials Chemistry Laboratory, UNSYIAH, Banda Aceh

References U.B. Jagtap, and V.A. Bapat. 2010. Artocarpus: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology 129, (2010) 142–166. Achmad, S.A., 1987. Topic books Organic Natural Products Chemistry, The Open University, Publishers Karunika, Jakarta Rosnani, Tonel, B., Pandapotan, N., Nurdin, S., 2013. Isolation and structure elucidation of steroid from the leaves of Artocarpus camansi (kulu) as antidiabetic, Dissertation, USU Medan. Esti Wahyu Widowati, (2009) chemical content at stem bark of Bendo (Artocarpus elasticus. Reinw)./ Journal / Kaunia / Volume II, No. 1, April 2006 /.