Characteristic of 70% EthanolExtract from Cyclea barbata Miers leaves and Antioxidant Activity using DPPH Method

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1 Proceedings of The 9 th Joint Conference on Chemistry ISBN Characteristic of 70% EthanolExtract from Cyclea barbata Miers leaves and Antioxidant Activity using DPPH Method Yunahara Farida a, Erlindha Gangga a, Kartiningsih a, Elisa a, Teguh a Abstract Cycleabarbata L. Miers is a plant that has been traditionally utilized by Indonesian community as a refreshing drink, colds,fever and antiinflammation. The purposed of the study wasstandardized of 70% ethanol extract and the antioxidant activity using DPPH method from CycleabarbataL.Miers leaves. The characteristic of 70% ethanol extractshowed the non specific parametershasbrown viscous extract, bitter taste and aromaticodor, water contentof 6.07± 0.07%, loss on drying of 7.47±0.28%, total ash content of 8.30±0.1%, total acid insoluble ash content of 0.12±0.0%, ethanol residual content of 0.70±0.16%, heavy metalcontaminant (Cd: mg/kg, Pb 0.03 mg/kg), total number of bacteria of 2.94 x 10 3 colony/g, and fungi (molds/yeasts) of 8.63 x 10 2 colony/g, ethanol residual content of 0.70±0.16%, whereas characteristic of specific parameter showed dilute ethanol content of 71.34±0.72%, dilute water content of 67.14±0.17% and assay of total flavonoid of 9.93±0.2%, the phytochemical screening was estimated that revealed the presence alkaloids, flavonoids, steroids/triterpenoid, tannin, saponins, and coumarins. The ethanol extract of Cyclea barbataleavescontain flavonoid as antioxidant activity with IC 50 value of 49.45±0.64µg/mL. The ethanol extract of Cyclea barbata leaves meets requirements standards that include specific and non specific parameter as a raw material for medicine. a Faculty of Pharmacy, Pancasila University, Jl. Srengseng Sawah, Jagakarsa, South Jakarta 12640, Indonesia Corresponding author address: yunahara_farida@yahoo.com Introduction Cyclea barbata is a plant used for health by some communities in Indonesia as medication for fever, flatulence, stomach peptic ulcer, and cancer (Sunarto, 1995; Pitojo, 1997). Cyclea barbata leaf is able to kill four types of cancer cells namely blood cancer (leukemia), cervical cancer, lung and breast cancer. This is because Cyclea barbata leaf contain compounds that have antioxidant activity. Cyclea barbata leaves contain bisbenzylisoquinoline alkaloids derivatives such as limacine, thalrugosine, homoaromoline, tetrandrine, cycleapeltine, cycleabarbatine, berbamine, cycleanorine, daphnandrine, coclaurine (Shudansu et al., 2003, Guinaudeau H et al., 1993) which can show antimalarial activity (Angerhofer et al., 1999; Guinaudeau et al., 1993; Lin LZ et al., 1993) and antioxidant activity (Chalid SY, 2003; Katrin et al., 2012). The former research has been studied toward the activity of antioxidant enzyme on liver of C3H mice which were transplanted with cell tumor mammary and the result showed that the extract of Cyclea barbata leaves infusion did not influence tumor latent period but decrease tumor size (Chalid, 2003). Katrin (2012) has been carried out the antioxidant activity of Cyclea barbata extract and fraction using the different degree polarity solvent, the methanol extract showed the highest antioxidant, while the chemical compunds test that showed an antioxidant positif result were detected alkaloid and flavonoid. Viewing the potential of Cyclea barbata leaves, it is necessary to standardize the extract using 70% ethanol to obtain the raw materials or extracts are top quality, safe and beneficialthen the extract was tested its antioxidant activities using the DPPH method to investigate free radical scavenging effect indicated by the colour change of the 1,1-diphenyl-2-pikrilhidrazil (DPPH) in which a free radical has reacted with plant extracts containing antioxidants. Methodology Materials. Simplisia powdered from CycleabarbataL.Miers leaves, 70% ethanol, methanol, DPPH, hydrochloric acid, chloroform, ammonia 30%, Dragendorf reagent, Mayer reagent, magnesium powder, amylalcohol, ferri chloride 1%, Stiassny reagent, sodium hydroxide 1N, ether, anhydrous acetic acid, sulphuric acid, nitric acid, Karl Fischer reagent, Nutrient Agar, Potato Dextrose Agar, phosphate buffer (ph 7.2). Green Chemistry Section 4: Organic Chemistry,Yunahara Farida, et al. P a g e 369

2 ISBN Equipment. UV-Vis Spectrophotometer, Atomic Absorption Spectrophotometer, Absorbance Microplate Reader Elx800, Gas Chromatography, Analytical balance, waterbath, rotary evaporator, electric cooker, blender, dessicator, furnace, refrigrator, oven, micropipette, pipette volume, petridish, crucible, ash free filter paper, weighing bottle, aluminium foil, Erlenmeyer, volumetric flask 5 ml, filter paper, Methods Principle of Research. This research was done by collecting Cyclea barbata L.Miers leaves,then the driedpowderedleaves was extracted by kinetic maceration with 70% ethanol and the crude ethanolic solution was subsequently concentrated using rotary vacuum evaporatorto obtain thick extracts. Furthermore, the extracts was examined by the phytochemical screening, characterization/standardization of extracts including specific and non specific parameters as well as antioxidant activityassay using radical scavenger DPPH. Preparation of extract: The powdered leaf (1.5 kg) was extracted by maceration with 70% ethanol for five days. The filtrate was evaporated using rotary vacuum evaporator. The extracts were stored in desiccators and used for subsequent experiments. Phytochemical Screening. Phytochemical screening were determined from extractto identify compounds such as flavonoids, saponins, tannins, quinones, steroid/triterpenoid, coumarins, and volatile oils based on the method of Farnsworth (1996) Non Specific Parameters Determination Water/moisture content.50 mg of the sample extract was weighed, then the extract is placed in a beaker containing solvent and then titrated with Karl-Fischer reagent (a solution that contain iodine) white any water remains in the sample, the iodine reacts with it and the solution remains colourless (HI), but once all the water has been used up any additional iodine is observed as a dark and brown colour (I 2), the volume iodine solution required to titrate the water is measured and can be related to the moisture content using pre-prepared calibration curve. Loss on drying.1-2 g of the sample extract was accurately weighed in a dried and tared flat weighing bottle and dried at 105 C for 5 hours. The percentage was calculated with reference to initial weight Total Ash Content.The total ash usually consists of carbonates, phosphates, silicates and silica. Sulphates present in the drug on long storage get converted in to carbonates and oxide on treatment of drug with conc. H 2SO 4 the carbonates and oxides get reconverted to sulphate which is stable at high temperature. Proceedings of The 9 th Joint Conference on Chemistry 2-3 g of the sample extract was weighed and ignited in a suitable tared crucible of silica. The powdered drug is ignited by gradually increasing the heat until free from carbon and cool. Thereafter we kept it in desiccator and weighed the total amount of ash. The % of the total ash with reference to the air dried sample of crude drug was calculated. Acid insoluble ash content.total ash was washed with 25 ml of dilute sulphuric acid and transferred into 100 ml beaker then boiled for 5 minutes and filtered through an ash less filter paper. The residue was washed with hot water and then the crucible was ignited, cooled and kept in desiccator. The % acid insoluble ash with reference to air dried sample of crude drug was calculated. Water soluble ash content.total ash was washed with 25 ml of distilled water in to 100 ml beaker. Wire gauze was placed over a bunch burner and was allowed to boil it for 5 min. It was filtered through an ash less filter paper and the residue was washed with hot water. The crucible was ignited on flame, cooled and weighed. The % of water soluble ash with reference to the dried sample of crude drug was calculated. Heavy metal contamination (Pb and Cd). About 1.0 g of the well homogenized sample extract was added 1.0 ml sulphuric acid P, then chared on top of the heater until the haze is gone and incandescented in the furnace to ashes.about 5 ml of concentrated nitric acid and 2 ml of 30 % hydrogen peroxide were added. The tubes were closed with the cap and filtered after completion of digestion process. The solution was transferred into a 25 ml standard flask and diluted to the mark with distilled water. This is then taken for analysis by Atomic Absorption Spectrometry (AAS). Microbial contamination.weigh1 g of the sample extract accurately, put into 10 ml volumetricflask, add phosphate buffer (ph 7.2) up to 10 ml. Total Plate Number(Bacteria, molds/yeasts).afterthe samples were prepared into the first tube obtain (a 10-1 dilution), thentake 1 ml liquid sample using a sterile pipette and included in 9 ml of phosphate buffer solutionto obtain a 10-2 dilution.subsequent dilutions were made to from each dilution, pipette 1 ml into a petri dish sterile, then pour as much as 15 to 20 ml of Nutrient Agar (NA) with a temperature of 45±1 C for bacteria and ml of Potato Dextrose Agar (PDA) for molds/ yeasts, and then shaken and rotated on a flat surface in order to obtain bacteria and molds/yeasts colonies spreadly growing. Once a petri dish freezes, petri dish reversed and incubated for hours (2 days) in an incubator with temperature of C for Nutrient Agar, and 25 C for PDA for 1-7 days. Colonies of bacteria, molds/yeasts and fungi growing in a petri dish is then observed and counted Etanol residual.residual solvents determined with gas liquid chromatography. Gas chromatography instrument equipped with a flame ionization 370 P a g e Green Chemistry Section 4: Organic Chemistry, Yunahara Farida, et al.

3 Proceedings of The 9 th Joint Conference on Chemistry ISBN detectorand a glass column 30 cm x 0.32 mm stationary phase flowed TR-WAX with a particle size of mesh, and nitrogenascarrier gas. The column was conditioned overnight at temperature of 235 C, set the carrier gas flow 20 ml/min, the injector and detector temperature are 200 Cand 160 C respectively.one gram extract is diluted with distilledwaterup to 50 ml, then pipetted1.0 ml into 10 ml flask, diluted with distilled water up to the mark (sample solution). for standard solution, pipette 1.0 ml of absolute ethanol diluted with distilled water up to 50 ml, then pipetted 1.0 ml into 10 ml flask, diluted with distilled water up to the mark (standard solution). Each performed triplo and injected ± 5 µl of sample solution andstandard solution into the instrument. Specific Parameters Determination Organoleptic.Consistency, colour, smell of the ethanol extract was visually observed, also using nose and tongue. Content of dilute ethanol.5 g of extract was macerated with 100 ml of alcohol in a closed flask for 24 h., shaking frequently during six hours and allowed to stand for 18 h. It was then filtered rapidly, taking precautions of loss of solvent. 20 ml of the filtrate was evaporated to dryness in a tared flat bottomed hallow dish at 105 C and obtained a constant weight.the percentage of dilute ethanol was calculated with reference to the air dried drug and represented as % value Content of dilute water.5 g of extract was macerated with 100 ml of chloroform water in a closed flask for 24 h., and then shaken for six hours and allowed to stand for 18 h. It was then filtered rapidly, taking precautionsofloss of solvent. 20 ml of the filtrate was evaporated to dryness in a tared flat bottomed hallow dish at 105 C to constant weight and weighed. The percentage of dilute water was calculated with reference to the air dried drug and is represented as % value Assay of total flavonoid. Unless otherwise stated, carefully weighed amount of 200 mg of crude extract, put in a round bottom flask, added consecutive 1 ml solution of HMT (hexamethylentetramine), 20 ml of acetone and 2 ml P hydrochloric acid solution P, refluxed for 30 minutes. Then the residue is filtered using cotton, enter the filtrate into a 100 ml volumetric flask. Refluxed again with 20 ml of acetone residue for 30 min, filtered and the filtrate was mixed into 100 ml volumetric flask. Pipetted 20 ml into a separating funnel, add 20 ml of water and extracted three times, each time using 15 ml of ethyl acetate P. Ethyl acetate phase included in the flask add 50 ml of ethyl acetate P up to the mark. Dilution test solution. Pipetted 10 ml of test solution into 25 ml volumetric flask, was added a solution of glacial acetic acid 5% v/v in methanol P up to the mark. Test solution with a solution of aluminium chloride. Pipetted 10 ml of test solution into 25 ml volumetric flask, add 1 ml solution of aluminium chloride and a solution of glacial acetic acid 5 % v/v in methanol P up to the mark. Measurement. Measure 30 min after the addition of aluminium chloride solution using a spectrophotometer at a wavelengthof 428 nm. Calculated as the total flavonoid content of flavonoids comparison DPPH radical scavenging assay The free radical scavenging of the extract was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (Brand Williams et al., 1995). All extracts were prepared in various concentrations from μg/ml in methanol solution. Into each flask, test solution and reference solution (Quercetine) was added 1 ml of 0.4 mm DPPH solution and methanol pro analysis to 5.0 ml and then homogenized. Flask covered with aluminium foil. Without inhibition of DPPH solution (blank solution), the test solution and reference solution (positive control) immediately incubated at room temperature for 30 min. The absorbance was measured by spectrophotometerat wavelength of 517 nm. The ability to scavenge DPPH radical was calculated by the following equation: DPPH radical scavenging activity (%)= [(Abs control- Abs sample) / (Abs control)] x 100. Results and Discussion According to Katrin (2012) the chemical compound of Cyclea barbata leaves were alkaloids and flavonoids. Flavonoids are polyphenolic compounds to act as antioxidants depends on their molecular structure. The position of hydroxyl groups are important for their antioxidant and free radical scavenging activities because the hydroxyl group of phenolic compound to eliminate radicals and they contribute directly to antioxidant effect of the system (Duh, 1994).Theresult of the phytochemical screening of Cyclea barbata L.Miers of 70% ethanol detected the presence of alkaloids, flavonoids, tannins, saponins, steroids/triterpenoids,and coumarins.the result showed that Cyclea barbatacontains flavonoids as antioxidant. The result of phytochemical screening showed in Table 1. Table 1. Phytochemical screening of Cyclea barbata leaves No Secondary metabolites Result 1 Alkaloids + 2 Flavonoids + Green Chemistry Section 4: Organic Chemistry,Yunahara Farida, et al. P a g e 371

4 ISBN Tannins + 4 Saponins + 5 Steroids/triterpenoid + 6 Coumarins + 7 Essential oil - Presence of individual phytochemicals is represented by +, while absence is indicated by - Characteristic of non specific parameters showed loss on drying of Cyclea barbata extract was less than 10%, thus there is less chances of microbial growth. The result of ethanol residual in the extract of 0.70±0.16% (less than maximum residual solvent in the extract is les than 1%). Total number of bacteria of Cyclea barbata leaf extract was 2,94 x 10 3 colony/g, it was below standard limit 1 x 10 5 colony/g. while total number of fungi of Cyclea barbata leaf extract was 8.63 x 10 2 colony/g. Result showed that microbialcontaminant in Cyclea barbata leaf was below the standard limit. Heavy metal enter the biological cycle through the roots and leaves of plants and are enriched in various plant organs. They can directly affect plant growth and an excess dietary intake of contaminant plants could also be dangerous for the health of humans and animals (Rai, et.al. 2011). Result of contaminant heavy metal Cd mg/kg and Pb 0.03 mg/kg were less than limit allowed(cd < 0.3 mg/kg; Pb < 10 mg/kg).non specific parameters results as seen in Table 2 Table 2. Characteristic of Non Specific Parameters No Non specific parameters Result 1 Water content 6.07± 0.07% 2 Loss on drying 7.47±0.28% 3 Total ash content 8.30±0.1% 4 Total acid insoluble ash 0.12±0.0% 5 Ethanol residual 0.70±0.16% 6 Heavy metal contaminant Cd Heavy metal Contaminant Pb Cd: mg/kg Pb: 0.03 mg/kg 7 Total number of bacteria 2.94 x 10 3 colony/g Total number of fungi (yeast/molds) 8.63 x 10 2 colony/g Characteristic of Specific parameter showed organoleptic of Cyclea barbata extract hasbrown viscousextract, bitter taste and aromatic odor. Content of dilute alcohol and content of watershowed chemical compounds suspected to have a role in determining the pharmacological effect, thus the higher the percentage, the better the extract. The results showed that content of ethanol greaterthan content of water (Table 3). The results of totalflavonoid of 70% ethanol extract was 9.93±0.2%. These results were in line with its antioxidant activity. Proceedings of The 9 th Joint Conference on Chemistry Table 3. Characteristic of Specific Parameters No Specific parameters Result Organoleptic Colour Taste Smell Content of dilute ethanol Content of dilute water Assay of total flavonoid brown viscousextract bitter taste aromatic odor 71.34±0.72%, 67.14±0.17% 9.93±0.2% Antioxidant activity of 70% ethanol extract Based on the IC 50 value, an extract was chategorized as very active if its IC 50 values less than 50 µg/ml, active if the IC 50 values µg/mL, so the 70% ethanolic extract of Cyclea barbata leaves was an very active antioxidantwith IC 50 of 49.45±0.64µg/mL while quercetin as positive control was 1.07 µg/ml. The result showed that Cyclea barbata leaves havea potential antioxidant.acccording to Chalid there is a relationship between antioxidant status and imune system, antioxidant status and the prevention and treatment of tumors. Conclusions The ethanol extract of Cyclea barbata leavescontain flavonoid as antioxidant activity. The ethanol extract of Cyclea barbata leaves meets requirements standards that include specific and non specific parameter as a raw material for medicine. Acknowledgments We are grateful to Directorate General for Higher Education, Ministry of National Education of Republic Indonesia, for Research Grant of Hibah Bersaing (2013) for financial support References 1. Angerhofer, C.K., H. Guinaudeau, V. Wongpanich, J.M. Pezzuto, G.A. Cordell. (1999). Antiplasmodial and Cytotoxic Activity of Natural Bisbenzyliquinoline Alkaloids, J.Nat. Prod, 62(1): AOAC International. (2003). Official Methods of Analysis.17 th ed. AOAC Int., Gaithersbersburg, MD. 372 P a g e Green Chemistry Section 4: Organic Chemistry, Yunahara Farida, et al.

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