Abstract. Keywords: Voacanga foetida (Bl.) rofle, Chemical profile, GC-MS, Lombok Island

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1 Proceedings of The 9 th Joint Conference on Chemistry ISBN Chemical Constituent of DCM Extract and Neutral-Acid Fraction ofvoacanga foetida (Bl.) Rolfe Leaves from Three Locations of Lombok Island on The Basis of GC-MS Analysis Surya Hadi a,lely Kurniawati a, Baiq Mariana a, Handa Muliasari a, Sri Rahayu a Abstract In our research group, Kumbi (Voacanga foetida (Bl.) rofle) has been investigated for several uses, such as anti-microbial medicine, bio fuel, and papers, this article is part of the activitiesto study chemical profile of the plants collected from three locations of Lombok Island. The study reported the chemical identifications of DCM extract and neutral-acid fraction base on Gas Chromatography-Mass Spectroscopy (GC-MS). The Sample preparation used solvent of dichloromethane (DCM) and followed an acid-base extraction. Chemical profile of three DCM extracts the plant leaves tended to be different with half of the metabolites was similar. On the other hand, the pattern of the chemical composition in the neutral-acid fraction of three leaves extracts was similar, which 1,2 benzene dicarboxylic acid as major constituents. Keywords: Voacanga foetida (Bl.) rofle, Chemical profile, GC-MS, Lombok Island a Study Program of Chemistry, Faculty of Mathematics and Natural Sciences, University of Mataram, JalanMajapahit No 62 Mataram 83125, Indonesia Corresponding author address: sur_hadi88@yahoo.com Introduction Voacanga foetida(bl.) Rolfe, locally in Lombok called kumbi, is a plant belonging to the family of Apocynaceae and is distributed all over Indonesia, including in Lombok Island. Kumbi thrives in tropical regions at an altitude of about 400 m above sea level. The leaves of kumbi have been used ethnomedically for the treatment of wounds, itches and swellings (Hadi et al., 2010). Kumbi leaves has been recently starting to be develop as antibacterial and antifungal drugs, biofuel and paper raw materials by utilizing the compounds of primary and secondary metabolites (Dini, 2014; Qibtiyah, 2014; Novianti, 2014; Hikmah, 2014). These metabolites will vary depending on the growth location of kumbi, due to the differences of biotic and abiotic environment such as climate, soil conditions, water resources, and predator and pathogen existence (Ramakrishna and Ravishankar, 2011; Julsing et al., 2006; Karlovsky, 2008). Exploration of chemical constituent of kumbi leaves in a variety of growth locations is needed as reference for the greater utilization in the future. This article provides information of the compounds containing in kumbi leaves that grown at three locations: North Lombok (Rempek Village), Central Lombok (Barabali Village) and West Lombok (Suranadi Forest); based on analysis using GCMS. Materials and Methods Materials The leaves of Voacanga foetida (Bl.) Rolfe was collected in three locations of Lombok Island: North Lombok Rempek Village, Central Lombok Barabali Village and West Lombok - Suranadi Forest. The sample was prepared and analysed at the Laboratory of Chemical Analysis, Faculty of Mathematics and Natural Sciences, the University of Mataram, Lombok. Instrumentation Instrument used in this studyis GC-MS Shimadzu QP2010ULTRA. The injection temperature was on 260 C.Initialcolumn temperature was started at a temperature of40 Cfor 5minutesand programmed to increase30 Cmin -1 heated until260 Cfor 2minutes. MeanwhileMS is conditionedat temperatures of transfer line at 260 Candion sourceat 200 C. Ion was obtained by electron ionization mode. The RTX-5MS capillary column was length 30m, diameter0.25mm andfilm thickness0.25 µm. Helium is usedasgas carrierwithflow rate of 30mLmin -1. Molecularmass rangeof ionswas identifiedat35-500m/z. Procedure Finely-powdered, air-dried old leaves (180 g) were extracted with dichloromethane (DCM) (3 x 1 litre) with occasional swirling. After filtration, the solvent Green Chemistry Section 4: Organic Chemistry,Surya Hadi, et al. P a g e 345

2 ISBN was removed under reduced pressure at 40 C yielding a dark-green DCM extract. The acid-neutral fraction was then separated from base materials by initial extraction with aqueous acetic acid (5% v/v) to ph 3-4, followed by DCM extractionof the aqueous acid extract with ratio of 1:2. The aqueous fraction was separated and evaporated under reduced pressure to get acidneutral fraction. The DCM extracts and acid-neutral fraction were filtered using 0.45 µm filter before analysed by GCMS. Results and Discussion DCM extract of kumbi leaves from three locations of Lombok Island GC-MS analysis result of DCM extract of kumbi leaves growing in three locations of Lombok Island is presented in Figure 1-3. The same compounds identified at three locations are summarized in Table Proceedings of The 9 th Joint Conference on Chemistry 1, while the different compounds of kumbi leaves at three locations are shown in Table 2. Based on the GC-MS analysis of DCM extracts of kumbi leaves growing at three locations, the compounds of kumbi leaves were identified mostly in Central Lombok (CL), followed by West Lombok (WL) and North Lombok (NL). The same six compounds identified in three growth locations have different percent of abundant. The same compounds are belongs to the group of fatty acids, fatty acid esters, phytosterols, and terpenoids. The identified fatty acids were saturated fatty acids, namely palmitic acid and stearic acid. Palmitic acid is the major component of DCM leaves extract of kumbi in the region of NL (22.27%) and WL (14.06%), whereas in CL it is found in small amounts (5.62%). Stearic acid was identified in the three growth locations with percent abundant of 4 to 6.9%. Figure1. Chromatogram of kumbi leaves DCM extract from North Lombok (Rempek Village) Figure 2.Chromatogram of kumbi leaves DCM extract from Central Lombok (Barabali Village) Figure 3.Chromatogram of kumbi leaves DCM extract from West Lombok (Suranadi Forest) 346 P a g e Green Chemistry Section 4: Organic Chemistry, Surya Hadi, et al.

3 Proceedings of The 9 th Joint Conference on Chemistry ISBN Table 1. The same compounds identified in kumbi leaves DCM extract from three locations of Lombok Island Compound Abundant (%) NL CL WL Palmitic Acid Tricyclodecanedimethanol(C AS)4,8-Bis Phytol ac-6-et-1,1,4,4-me4- tetralin Stearic Acid Hexanedioicacid,bis(2- ethylhexyl) ester(cas) 2-propanol,1,1-oxybis (CAS) dipropanol 1-propanol,2-(2- hydroxypropoxy) 1,2-benzenedicarboxylic acid diethyl Neophytadiene ,12-octadecadienoic acid (Z,Z)-methyl allyl-6-methoxyphenol NL = North Lombok; CL = Central Lombok; WL = West Lombok The compounds of DCM leaves extract of kumbi with the highest percentage in CL is Tricyclodecane dimethanol (alcohol group) (18.50%), while in WL is terpenoid alcohol (phytol) (16.94%). The distribution of compounds appear more diverse in CL and WL; in the form of fatty acid esters and some secondary metabolites (terpenoids and alcohol groups) that identified with percent abundant of below 6%. Table 2 shows the different compounds identified in DCM extracts of kumbi leaves growing at three locations. Six compounds were identified only in NL, ten compounds were identified in CL and eight compounds in WL. The major compounds which is only found in NL is oleic acid (15.20%) and (S, S, S, S)- bicyclopentyl 1,1'-2,2'-dicarboxaldehide (6.38%). in CL, the compounds identified were Stigmas CL-5-en-3-ol (7.83%) and Heptadecene- (8) -carbonic acid (6.42%) which is a class of sterols; and compound with percent abundant of 11.65% that is Cyclopentadecanone, 2- hydroxy is only found in WL. The other different compounds are present in very small amount (<5%). The existence of similar compounds with different percentage and the compounds that are only found in one growth location indicate that the differences of growth location significantly affect the content of compounds in plants. Assuming the age of the plants collected at three locations are the same, then the growing environmental factors such as climate, temperature, soil type, and the presence of predators and pathogens has greatly contributed to the formation of primary and secondary metabolites of kumbi. Thus, the consistency and reproducibility of the compounds of kumbi need to be studied before the use can be ascertained (Aziman et al., 2012; Cock, 2011; Karlovsky, 2008). The major compounds are generally primary metabolites fatty acids, such as palmitic acid and oleic acid; and some types of fatty acid esters. Fatty acids and fatty acid esters are found in many plants and are known to have antibacterial and antifungal activities (Agoramoorthy, 2007; Vital et al., 2011). Palmitic acidis saturated fatty acid that commonly used for cosmetics, food and soap; and is known to has antifungal and anticancer activities (Warsinah et al., 2011; Hideki et al., 2002). Palmitic acid together with oleic acid and stearic acid are also known has insecticidal activities (Affene et al., 2012; Gutierrez et al., 2011). The other compounds are including in secondary metabolites, the compounds derived from primary metabolites (Ramakrishna and Ravishankar, 2011). The major compounds identified are terpenoids alcohol (phytol) and sterols. These compounds are known to have biological activities such an antibacterial, antioxidant, and anticancer (Ranganathan, 2014; Komiya and Hibasam-netrali, 2001). Neutral-acid fraction of kumbi leaves from three locations of Lombok Island The results of GC-MS analysis of neutral-acid fraction of kumbi leaves from three locations of Lombok Island are shown in Figure 4-6, and the resume present in Table 3. GC-MS analysis indicate that there are seven different types of compounds identified. Six compounds appear in almost every growth location, while another one, Cyclopentadecanone 2-hydroxy, only presents in CL with abundance of 2.27%. Table 2. The different compounds identified in kumbi leaves DCM extract from three locations of Lombok Island Green Chemistry Section 4: Organic Chemistry,Surya Hadi, et al. P a g e 347

4 ISBN Proceedings of The 9 th Joint Conference on Chemistry Compound Location NL CL WL Abundant (%) 7-octadecenoid acid, methyl ester 1.84 (S,S,S,S)1,1'-bicyclopentyl-2,2'-dicarboxaldehide octadecenoid acid, methyl ester (CAS) octadecenoid acid (Z)-(CAS) oleic acid Hexanedioic acid, dioctyl ester (CAS) 1.86 Lycopersen 3.30 Ethane,1,1,2-trichloro-(CAS)1,1,2 trichloro 0.53 Bis(1-methyl-2-hydroxyethyl) 0.42 Veridiflorol 0.25 Hexadecanoic acid, methyl ester (CAS) 0.30 Hexadecanoic acid, ethyl ester (CAS) 1.18 Stigmas-5-en-3-ol, (3.beta.,24S)-(CAS) 7.83 Heptadecene-(8)-carbonic acid 6.42 Octadecanoic acid, ethyl ester (CAS) ,19-cyclolanost-24-en-3-ol (3.beta) ,6,10,14,18,22-tetracosahexaene,2, propanol,1,1-oxybis (CAS) dipropanol propanol,2-(2-hydroxyproxy)-(CAS) allyl-6-methoxyphenol napthaleamine,N-ethyl-(CAS) octadecanoic acid, methyl ester 0.86 Cyclopentadecanone,2-hydroxy Decanal,o-methyloxime (CAS) DECANAL 1.11 Ergost-5-en-3-ol,(3.beta.,24R) 5.78 exist NL = North Lombok; CL = Central Lombok; WL = West Lombok Figure 4.Chromatogram of Neutral-acid fraction of kumbi leaves from North Lombok (Rempek Village) 348 P a g e Green Chemistry Section 4: Organic Chemistry, Surya Hadi, et al.

5 Proceedings of The 9 th Joint Conference on Chemistry ISBN Figure 5.Chromatogram of Neutral-acid fraction of kumbi leaves from Central Lombok (Barabali Village) Figure 6.Chromatogram of Neutral-acid fraction of kumbi leaves from West Lombok (Suranadi Forest) Table 3. The identified compounds in Neutral-acid fraction of kumbi leaves from three locations of Lombok Island Compound 2-Hexadecene-1-ol-3,7,11,15- tetramethyl Hexadecanoic acid (CAS) Palmitic acid Abundant (%) NL CL WL octadecenoic acid (Z)-(CAS) Hexanedioicacid,bis(2- ethylhexyl) ester(cas) 1,2-benzenedicarboxylic acid,bis(2-ethyl) Cyclopentadecanone,2- hydroxy Octadecenoic acid(cas) stearic acid NL = North Lombok; CL = Central Lombok; WL = West Lombok The major compound 1,2-benzenedicarboxylic acid, bis (2-ethyl) is identified in all locations with abundance of above 90%. The present of 1,2-benzenedicarboxylic acid in neutral-acid fraction is in a very high number while in DCM extract in small concentration (~2%), it may be persisted in insoluble part of the DCM extract. This compound is a member of fatty acids which are the dominant compounds found in plants that used for plant growth and development. Another fatty acid that appear in each location is palmitic acid, with ratio abundance for the region of NL, CL and WL are 1.51%, 4.26% and 2.27%, respectively. Conclusions GC-MS analysis showed that the chemical profile of DCM extract kumbi leaves from three locations of Lombok Island tended to be different with half of the metabolites was similar. This indicated that different growth location significantly effecting the metabolites in kumbi leaves. On the other hand, the pattern of chemical composition in the neutral-acid fractions of three leaves extracts was similar, which 1,2- Benzenedicarboxylic acid as major constituent. Acknowledgments We would like to thank for MENRISTEK (Ministry of Research and Technology) for funding this research and to Mr. RuruHoniar in assisting of GC-MS analysis. References Affene, Z.B.C., Chaieb, I., Haouala, F., and Skhiri, F.H. (2012).Effects of Wild Rose (Rosa sp.)crude Seed Extracts on Two Legume Aphids.Bulletin UASVM Horticulture, 69(1), Agoramoorthy, G., Chandrasekaran, M., Venkatesalu, V., and Hsu, M.J. (2007). Antibacterial and Antifungal Activities of Fatty Acid Methyl Esters of The Blind-Your- Green Chemistry Section 4: Organic Chemistry,Surya Hadi, et al. P a g e 349

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