ANTIOXIDANT ASSAY OF THE ETHANOLIC EXTRACT OF THREE SPECIES OF RATTAN FRUITS USING DPPH METHOD Heriad Daud Salusu1,2,*, Farida Aryani2, Abdul Rasyid Zarta2, Edy Budiarso3, Irawan Wijaya Kusuma4, Enos Tangke Arung4 1 Graduate School of Forest Science, Faculty of Forestry, Mulawarman University, Samarinda 75119, Indonesia. 2Laboratory of Wood Properties and Product Analysis, State Agriculture Polytechnic of Samarinda, Samarinda 75131, Indonesia 3Laboratory of Wood Preservative, Faculty of Forestry, Mulawarman University. Samarinda 75119, Indonesia 4Laboratory of Forest Product Chemistry Faculty of Forestry, Mulawarman University. Samarinda 75119, Indonesia *Corresponding author: [email protected] ABSTRACT The bioactivity of some species of rattan fruit has been previously studied and it was found that it has potential antioxidant agents. This study was conducted to determine the antioxidant content in three species of edible rattan fruit, namely Calamus manan Miq., Calamus ornatus Bl. and Calamus caesius Bl by using the DPPH method, whereby the test was carried out on the pericarp, flesh, and seeds of each fruit. The research findings revealed that phytochemical compounds, i.e. flavonoids, tannins, and triterpenoids are found almost in all parts of the fruit, while steroid is not found in any of the three species of the fruit. The antioxidant activity found in the fruit of C. caesius Bl is strong, meanwhile the antioxidant activity in the fruit of C. manan Miq only strong in its pericarp and seeds, but it is moderate in its flesh. The antioxidant activity found in the fruit of C. ornatus Bl. is strong in its pericarp and seeds, but it is moderate in its flesh. Therefore, these three species of rattan fruit are potential as antioxidant agents. Keywords: antioxidant, calamus, flesh, pericarp, seed Submitted on: 19 May 2018 Accepted on: 26 September 2018 DOI: https://doi.org/10.25026/jtpc.v4i4.170 INTRODUCTION edible fruit that in several places this fruit Rattan is a typical tropical plant can also be useful for health. Ripa [1] whose stem is utilized as raw materials to stated that the fruit of Calamus rotang L produce a variety of crafts and furniture. was potential for an anti-diarrheal agent. Besides its stem, rattan also produces According to [2], rattan fruit can serve as a J. Trop. Pharm. Chem. 2018. Vol 4. No. 4. 154 p-ISSN: 2087-7099; e-ISSN: 2407-6090 Antioxidant Assay of the Ethanolic Extract of Three Species of Rattan Fruits using DPPH Method medicine to relieve cough, diabetes, and rattan which has better antioxidant abdominal disorders. Furthermore, he properties. stated that rattan fruit contains Vitamin A and C, and minerals such as Ca, Fe, Mg, Zn MATERIALS AND METHOD and K. Uddin [3] stated that the fruit of Calamus tenuis Roxb is used to treat Plant Materials diarrhea. The fruits of the three species of The three species of fruit analyzed rattan, i.e. C. manan Miq., C. caesius Bl., in this study are the species of rattan which and C. ornatus Bl. each separated from the commonly grow in the forest area of East pericarp, flesh and seeds, then each part of Kalimantan. They are C. manan Miq., C. the fruit that has been separated dried at caesius Bl. and C. ornatus Bl., all of which room temperature before they were bear edible fruit. Until recently, the fruit of processed to produce rattan fruit powder. the three species of rattan has not been Then, the fruit powder was extracted by utilized maximally, except for its stems using maceration method with ethanol as a which have been known as having a high solvent. The powder sample was soaked in commercial value. The information about the ethanol for 48 hours with the ratio of 1: the benefits of rattan fruit is not sufficiently 10 (powder: solvent). The result of available. The knowledge and experiences maceration was evaporated using rotary about its benefits are limited only to the evaporator at the temperature of 30-40 °C local people living around the forests. They so that rough extract was obtained. make use of this fruit to relieve pains in a toothache and to treat a diarrhea. This Reagents information can be used as a preliminary Ethanol, ethyl acetate, DPPH (1,1- guide to developing rattan fruit especially diphenyl-2-picrylhydrazyl), DMSO with regard to benefits for human health. (dimethyl sulfoxide), distilled water, Some species of rattan fruit that have been chloroform, sulfuric acid, hydrochloric studied include C. erectus Roxb which acid, acetate lead solution, potassium contains bio-activity which is potential as iodide, acetic acid, sodium hydroxide, antioxidant and antidiabetic agents because alcohol, and ascorbic acid. of its high content of phenol [4]. Moreover, Ahmed [5] in his study on C. tenuis Roxb. Phytochemicals screening found that the phytochemical content in the rattan fruit is related to its bio-activity Alkaloids assay [6] potentials, that is, antioxidant and An amount of 1 ml of dragendorff’s cytotoxic. solution was added into 5 ml of extract and Based on the background above, added with 2 ml of HCl. If the color of the this study intended to screen the solution turned into orange or red color, it phytochemical compounds and to indicated that the extract contained determine antioxidant capacity of the three alkaloids. species of rattan fruit, namely C. manan Miq, C. caesius Bl., and C. ornatus Bl by Flavonoids assay [6] using DPPH method. The parts of the fruit An amount of 1 ml of extract was assayed were its pericarp, flesh and seeds. mixed with some drops of dilute sodium The results of the study will show the part hydroxide (NaOH 1%). An intense yellow of the fruit that has a better antioxidant colour was appeared in the test tube. It content and also will know the type of became colourless when on addition of a J. Trop. Pharm. Chem. 2018. Vol 4. No. 4. 155 p-ISSN: 2087-7099; e-ISSN: 2407-6090 Antioxidant Assay of the Ethanolic Extract of Three Species of Rattan Fruits using DPPH Method few drop of dilute acid that indicated the served as free radicals and ascorbic acid presence of flavonoids. was used as a positive control. Referring to the values of Saponin assay [7] absorbance in the extracted sample, the Saponin was assayed by pouring 10 antioxidant activities were determined ml of hot water in the reaction tube based on the percentage of relative containing 1 ml of the tested sample which inhibition using the following equation: had been dissolved into acetone. Then, the A A Relative Inhibition Activity (%) 0 1 100% solution was cooled and shaken for 10 A0 seconds. The formation of a steady foam which occurred for 10 minutes with the where A0 is the absorbance of DPPH, and height of 1-10 cm and would not disappear A1 is the absorbance of extract sample. The when it was given 1 drop of HCl 2N, antioxidant activities were determined indicated the extract contained saponins. based on the value of IC50 (inhibitory concentration), i.e. the amount of Tanin Assay [6] concentrated solution of sample needed to Tannin was assayed by putting 10 inhibit 50 % of DPPH, which was ml extract solution into a reaction tube and calculated by using regression equation adding acetate lead solution (CH3COO)2Pb where the extract concentration served as 1%. Tannins were positive if there was its abscissa (axis X) and the value of the yellow sediment found in the reaction. relative inhibition served as its ordinate (axis Y). The value of IC50 was calculated Triterpenoid and Steroid Assay [7] at 50% relative inhibition using the Triterpenoids and Steroids were equation as follows: Y = b + aX identified by using the mixture of acetic acid anhydride and concentrated sulfuric RESULT AND DISCUSSION acid. This is commonly known as Liebermann-Burchard reaction. Ten drops Phytochemical Contents of acetic acid anhydride and 2 drops of Phytochemical is a compound which concentrated sulfuric acid were added in gives plants their distinctive smells, tastes series into 1 ml of the tested sample which and colors. The Phytochemical compound had been dissolved in acetone. The tested also gives a variety of potential benefits for sample was shaken and left for several human health. According to [9] some minutes. Red and purple colors found in the phytochemical components in fruit such as reaction showed the presence of tannin, flavonoids, and triterpenoids serve triterpenoids, while green and blue colors as inhibitors against free radicals and indicated the presence of steroids. function as antioxidant agents. In this research, phytochemical testing was Antioxidants Analysis carried out on ethanol extracts on pericarp, Antioxidants were analyzed by flesh and seeds of the three species of rattan using a method suggested by [8] with a fruit. Ethanol does not hydrolyze modification. The absorbance of the compounds and it is easy to evaporate extracted sample toward free radicals was because of its low boiling point and it is assayed by using Spectrophotometer at a good for thermolabile compounds. [7] room temperature (25 °C) with the wave reported that the use of ethanol was good length of 517 nm and using DPPH (1.1- for initial extraction. Flavonoids, alkaloids, diphenyl2-picrylhydrazyl) solution, which tannins, saponins, triterpenoids, and J. Trop. Pharm. Chem. 2018. Vol 4. No. 4. 156 p-ISSN: 2087-7099; e-ISSN: 2407-6090 Antioxidant Assay of the Ethanolic Extract of Three Species of Rattan Fruits using DPPH Method steroids are the phytochemical compounds flesh, and seeds of the three species of which were assayed in this research. The rattan fruit. assays were conducted to the pericarp, Table 1. Phytochemical contained in fruit on three species of rattan Samples Flavonoid Alkaloid Tannin Saponin Triterpenoid Steroid C. manan Miq.