PROCEEDING The 7th AIC-ICMR on Health and Life Sciences The Annual International Conference 2017 Syiah Kuala University “Advancing Knowledge, Research, and Technology for Humanity” ISSN: 2089-208X Banda Aceh, Aceh, Indonesia October 18-20, 2017 The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia Jiringa‘s Pods as a Source of a New Natural Antioxidant Misri Yanty Lubis1,2*, Lamek Marpaung2, Muhammad Pandapotan Nasution3 And Partomuan Simanjuntak4,5 1Department of Agrotechnology, Faculty of Agriculture, Graha Nusantara University, Tor Simarsayang, Padangsidimpuan 22712, Indonesia. 2Department of Chemistry, Faculty of Mathematic and Natural Science, Sumatera Utara University, Padang Bulan, Medan 20155, Indonesia. 3Department of Pharmacology, Faculty of Pharmacy, Sumatera Utara University, Padang Bulan, Medan 20155, Indonesia. 4Department of Pharmacology, Faculty of Pharmacy, Pancasila University, Srengseng Sawah, Jagakarsa, Jakarta 12630, Indonesia. 5Research Centre for Biotechnology, Indonesian Institute of Science, Jln. Raya Bogor Km 46, Cibinong 16911, Indonesia. Abstract This research studied about antioxidant activity various extracts of jiringa (Archidendron jiringa) Jack. I. C. Nielsen) pods by using DPPH method. The IC50 of extracts obtained from linear regression equation on chart concentration vs % inhibition. Pods of jiringa dried at room temperature 1 x 24 h and then macerated with methanol. Filtrat were evaporated with rotary evapoarator to obtained methanol extract. Further, methanol extract dissolved with water and partitioned with ethyl acetate for several times, and then evaporated to obtained ethyl acetate extract. Ethyl acetate extract partitioned with methanol and n-hexane to obtained n-hexane extract and total phenolic. All extracts tested antioxidan activity by using DPPH method and consitution of phenolic compound by using FeCl3. The IC50 values for all extracts in order to evaluate antioxidant activity were calculated from linear regression equation on chart consentration vs % inhibition. Test phenolic compounds with FeCl3 showed positive test for methanol extract, ethyl acetate extract and total phenolic. In the other hand, n- hexane extract showed negative test. The IC50 values of methanol extract, ethyl acetate extract, n- hexane extract and total phenolic were 30.5134; 20.3220; 139.3794 and 11.7987 µg/ml, respectively. Extract pods of jiringa with variaty solvent showed high antioxidant activity, except n-hexane extract. The high antioxidant activity cause of phenolic compounds constitution. Pods of jiringa extracts have high antioxidant activity because it contains phenolic compounds that can be used as a source of a new natural antioxidant. Keywords: antioxidant, jiringa, phenolic, DPPH INTRODUCTION Jiringa (Archidndron jiringa (Jack) I. C. Nielsen) is a leguminous tree that widely distributed in Indonesia, Malaysia and Thailand (Lim 2012). It‘s used as a vegetable (young shoots), food flavouring agent (seeds) (Ashuwini et al. 2012), medicine (leaves and seeds), source of dye for silk (pods) and timber for craft work and firewood (Charungchitrak et al. 2011). The plant usually stands up to 25 meters in height with a smooth, grey coloured bark. 88 Proceedings of the 7th AIC-ICMR on Health and Life Sciences The beans measure up to 3.5 centimeters in diameter and 2.0 centimeters in thickness. Jiringa‘s beans are typically taken together with rice as a side dish either as raw vegetable, roasted, fried or boiled (Muslim and Abdul 2010; Ruzilawati et al. 2012). Jiringa is one of traditional medicine herbs. Seeds have been reported as a source of natural antioxidant (Mohammad 2006) that could destroy excess free radicals and prevent oxidative damage (Fonseca 2017). Seeds are used to treat hypertension and diabetic disease. The old leaves burnt to obtain ashes were used against itching (Mohammad 2006). Jiringa‘s pods is organic waste. If the pods of jiringa is cut with a knife containing iron, it will appear a black bluish or black purple colour. This indicates the presence of phenolic compounds in the pods of jiringa (Khoddami 2013). Phenolic compounds of plant can inhibit oxidation in the human body becouse of it‘s antioxidant potential (Amarowicz and Shahidi 2017). This time, There has been interesting to find natural sources of antioxidant in plants, because of their potential health associated with several degenerative and aging related diseases such as cancer and cardio vascular diseases (Badhani et al. 2015; Baydar et al. 2007; Tomas et al. 2012; Andreata et al. 2009). Synthetic antioxidants may have toxic, carcinogenic and negative effects to humans body. Ascorbic acid is one of the sources of natural antioxidants (Baydar et al. 2007; Tomas et al. 2012; Andreata et al. 2009). We use it as a standard in this reserach. The aim of this research is to investigate the antioxidant activity of various extract jiringa‘s pods and relatied with content of phenolic compounds. Solvent extraction is commenly applied to isolate bioactive compounds from plants (Pai et al. 2017; Pinela et al. 2016; Mokrani and Madani 2016; Krishnan 2016; Xu 2016). Ethanolic extract pods of jiringa showed contrentation phenolics and potent antioxidant activity (Nahdzatul et al. 2012). The antioxidant activity studies of extracts jiringa‘s pods in some of these solvents (methanol, n- hexane and ethyl acetat) have not yet been reported. Figure 1 Jiringa‘s pods 89 The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia MATERIALS AND METHODS Plant Material Jiringa‘s pods (Archidendron jiringa (Jack) I. C. Nielsen) were collected from Namorambe village, Deli Serdang, North Sumatra, Indonesia. The plant material was identified at the Herbarium Bogoriensis of The Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia. Extraction Jiringa‘s pods (Archidendron jiringa (Jack) I. C. Nielsen) were dried and powdered (4,160 g) at room temperature. Further, macerated with methanol (16 liters) for several times. The resultant extracts were concentrated to dryness in a rotary evaporator. Methanol extract (140 g) was dissolved with water for several times. After filtration, the aqueous solution was partitioned with ethyl acetate solvent. Soluble portion of ethyl acetate solvent after concentrated with rotary evaporator was collected (37.88 g). Ethyl acetate extract was partitioned further with mixture of n-hexane-methanol to get extracts. The methanol extracts was considered as total phenolic (13.87 g) and n-hexane extract was obtaned 2.08 g. We use FeCl3 to test phenolic compounds. DPPH Method Antioxidant of methanol extract, ethyl acetate extract, n-hexane and total phenolic were based on reactions with DPPH (1,1-diphenyl-2-picrylhydrazyl) respectively and were compare to standard ascorbic acid. Radical has a deep violet colour do to its unpaired electron and radical scavenging can be followed spectrophotometrically by the loss of absorbance at 515 nm as the pale yellow non-radical form is produced (Musa et al. 2016). DPPH solution 0.4 mM was made from 7.9 mg DPPH (Molecular weight is 394.32) which was dissolved with methanol pro analysis up to 50 ml. Placed in a dark bottle. The blank soluble was prepared from 0.4 mM DPPH of 1 ml into 5 ml tested reaction tube, then added methanol pro analysis until the mark and homogenized. The tube was covered with aluminum foil. The extract was weighed as 5 mg and then dissolved into 10 ml methanol pro analysis (500 µg/ml). This solution is called as the mother liquor. The mother liquor 50, 100, 150, 200 and 250 µl were piped into 5 ml reaction tube to obtain concentrations of 5, 10, 15, 20 and 25 µg/ml. In each tube was added 1 ml of DPPH solution 1 mM and added with 90 Proceedings of the 7th AIC-ICMR on Health and Life Sciences methanol pro analysis up to 5 ml then homogenized. The tube was covered with aluminum foil. Ascorbic acid solution was prepared same with solution test. dry jiringa‘s pods (4,160 g) - Macerated with methanol (16 liters) 1 x 24 h - Concentrated with rotary evaporator Methanol extract Antioxidant (140 g) activity test - Dissolved with water for several times (16 liters) - Partitioned with ethyl acetate for several times Water fraction Ethyl acetate fraction - Concentrated with rotary evaporator Ethyl acetae extract Antioxidant (37.88 g) activity test - Dissolved with methanol - Partitioned with n- hexane for several times n-hexane Ethyl acetate fraction fraction - Concentrated with - Concentrated with rotary evaporator rotary evaporator Total Phenolic Antioxidant n-hexane extract Antioxidant (13.87 g) activity test (2.08 g) activity test Figure 2 Flow diagram to obtained extracts and antioxidant activity test 91 The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia Test solution with several concentrations was incubated in 37 oC