ANTIOXIDANT ASSAY OF THE ETHANOLIC EXTRACT OF THREE SPECIES OF 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 can also be useful for health. Ripa [1] whose stem is utilized as raw materials to stated that the fruit of 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

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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 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

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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 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

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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. Pericarp + + + + + - Flesh + - + + + - Seed + + + - + - C.caesius Bl. Pericarp + + - - + - Flesh + - + + + - Seed + + + - + - C. ornatus Bl. Pericarp + + + - + - Flesh - - + - + - Seed + - + - + - Note : presence (+) absence (-)

The results of the assays on Table 1 detected in the flesh of the fruit. Tannins shows that the fruit extract of C. manan were found almost in all parts of the fruit, Miq contains flavonoids, tannins, and except in the pericarp of C. caesius Bl. triterpenoids which are found in its Saponins were not evenly distributed in the pericarp, flesh, and seeds; alkaloids are three species of fruit. Especially in C. found in its pericarp and seeds; saponins ornatus Bl., saponins are not found at all. are found in its pericarp and seeds, while The findings showed that phytochemical steroids are not detected. The fruit extract compounds are observed and distributed in of Calamus caesius Bl. contains flavonoids three different species of rattan fruit, in and triterpenoids in its pericarp, flesh, and which the parts of the fruit were assayed seeds; alkaloids in its pericarp and seeds, separately. A study conducted by [10] on tannins in its flesh and seeds; saponins in the Lephisanthes amoenea fruit showed a its flesh; but steroids are not detected. The similar result, where flavonoids are found fruit extract of Calamus ornatus Bl. in all parts of the fruit. In addition, the contains flavonoids, tannins, and distribution of phytochemical compounds triterpenoids which are found in the three are mostly found in seeds and pericarp. parts of the fruit; alkaloids are found in its Some previous studies showed that the pericarp; but saponins and steroids are not existence of phytochemical compounds in detected. Generally, flavonoids and plants are influenced by some factors such triterpenoids are identified in the three as adaption to the environmental factor and species of fruit, while steroids are not the growth of each individual species. detected. Alkaloids are commonly found in According to [11], the distribution of the pericarp and seeds, but they are not alkaloid content in various species of plant

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is different, depending on the Antioxidant Capacity environmental factors and the specific The antioxidant capacity were growth of each type of plant. Toxic feature assayed by using DPPH method with in alkaloid is very useful as the defense of ascorbic acid as its positive control. The plant against predators. Konarska and parameter of this method was expressed as Domaciuk [12] stated that tannins are the value of the Inhibition Concentration of mostly accumulated in the pericarp and 50% (IC50) namely the concentration value mesocarp of Viburnum opulus and which could inhibit 50% of free radical Viburnum lantana. Moreover, according to activities (DPPH). DPPH is free radicals [13], there are some factors which which are stable in a room temperature influence the composition and distribution having electrons and hydrogen, and form a of saponins in plants, for example, the stable molecule. The absorbance of free protection level against predator attacks radicals occurs when the antioxidant and the changes of the environment; the compounds which can give hydrogen atom protection on the reproductive organs have been bond to DPPH which results in during the growth of fruit and the the changes of violet colors into yellow formation of tubers. The existence of colors. The absorbance of DPPH was phytochemical compounds in the three measured by using spectrophotometer at species of rattan fruit in this study indicates the wavelength of 517 nm. Antioxidant their potentials as natural antioxidant activity on the pericarp, flesh, and seeds of agents. the three species of rattan fruit and ascorbic acid as a positive control is displayed in Figure 1 to Figure 3.

80

pericarp y = 0.4561x + 45.585 70 seed R² = 0.970 flesh 60

50 y = 1.2402x + 9.0269 R² = 0.9938 40

Inhibition (%) Inhibition 30

20 y = 0.5738x - 0.8957 R² = 0.9916 10

0 0 10 20 30 40 50 60 Concentration (µg/ml)

Figure 1. Antioxidant Activity in the Pericarp, Flesh and Seeds of Calamus ornatus Bl., and Ascorbic Acid

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90 y = 1.3879x + 15.252 80 R² = 0,991. Pericarp 70 Flesh 60 Seed

50 y = 1.3853x - 7.0321 R² = 0.946 40

Inhibition (%) Inhibition 30 y = 0.279x + 2.5147 20 R² = 0.9755 10 0 0 10 20 30 40 50 60 Concentration (µg/ml) Figure 2. Antioxidant Activity in the Pericarp, Flesh and Seeds of Calamus manan Miq., and Ascorbic Acid

90

80 y = 2.1292x + 31.259 70 R² = 0.9242 y = 2.0684x + 27.95 60 R² = 0.9405 50

40 y = 2.5076x + 11.524 R² = 0.9857 Inhibition (%) Inhibition 30 Pericarp Flesh 20 Seed 10

0 0 5 10 15 20 25 30 Concentration (µg/ml) Figure 3. Antioxidant Activity in the Pericarp, Flesh and Seeds of Calamus caesius Bl., and Ascorbic Acid

Tabel 2. IC50 values of fruit of three rattan species and ascorbic acid * Samples IC50 (µg/ml) Antioxidant Category Calamus manan Miq Pericarp 41.17 Strong Flesh 170.19 Moderate Seed 25.03 Strong Calamus caesius Bl. Pericarp 15.34 Strong Flesh 8.80 Strong Seed 10.66 Strong Calamus ornatus Bl. Pericarp 9.69 Strong Flesh 88.82 Active Seed 33.04 Strong Ascorbic Acid 5.42 Strong

* IC50 Value < 50 µg/ml Strong, 50 µg/ml - 100 µg/ml Active, 100 µg/ml - 250 µg/ml moderate, and > 500 µg/ml non active [14]

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Furthermore, in order to identify the compounds which serve as antioxidant effectiveness of sample in inhibiting free agents because their structure contains radical, the value of IC50 was calculated hydroxyl group which is able to give its which was obtained from linear regression hydrogen atom to free radical compounds equation (Figure 1, 2, and 3) which so that it can inhibit a lot of oxidation expressed the correlation between sample reactions [15]. Jing [16] stated that the total extract concentration as an x-axis and free flavonoids and phenol in Rhododendron radical inhibition as a y-axis. The results of anthopogonoides correlated significantly calculation about the value of IC50 with antioxidant activities. A study on displayed on Table 2 showed that the three rattan fruit of C. tenuis Roxb was species of the fruit and the three different conducted by [5] showed that the total parts of the fruit had different antioxidant flavonoids correlated with the increasing activities. The smaller the value of IC50 the antioxidant activities. Moreover, tannins as stronger the antioxidant activities will be antioxidants have been discussed in the [14]. In the fruit of C. manan Miq., the previous research findings. Rebaya [17] power of antioxidant activities was reported that there was a significant successively ordered as follows: seeds > correlation between tannins, flavonoids pericarp > flesh; in C caesius Bl. flesh > and polyphenolic compounds and seeds > pericarp, while in C.ornatus Bl. antioxidant activities in Halimium pericarp > seeds > flesh. If compared with halimifolium. Bizuayehu [18] in their study ascorbic acid as a positive control toward also found that there was a positive the three type of fruit, it was found that correlation between tannins and ascorbic acid > C. caesius Bl .> C. ornatus antioxidant activities. Bl .> C.manan Miq. The antioxidant Triterpenoid is an antioxidant activities which were presented in order compound which serves as an antioxidant from the highest to the lowest activities can agent. Lin [19] stated that the increasing be illustrated as follows: the flesh of C. triterpenoid concentration will also caesius Bl. (8.80 µg/ml) > the pericarp increase the antioxidant activities in of Calamus caesius Bl. (9.69 µg/ml) > the Garcinia lucidum. This indicates that seeds of C.caesius Bl (10.66 µg/ml ) > the triterpenoids play a role in increasing pericarp of C. caesius Bl (15.34 µg/ml) > antioxidant activities. According to [20] the seeds of C. manan Miq (25.03 µg/ml) > triterpenoid content also strongly the seeds of Calamus ornatus Bl. (33.04 contributes to the antioxidant activities of µg/ml) > the pericarp of Calamus manan mistletoe tea (Scurrula atropurpurea Bl. Miq (41.17 µg/ml) > the flesh of Calamus Dans). ornatus Bl. (88.82 µg/ml) > the flesh of Calamus manan Miq. (170.19 µg/ml) CONCLUSIONS AND SUGGESTION respectively. Ascorbic acid as a positive The fruit of C. caesius Bl has strong control has the highest antioxidant antioxidant activities in all parts of the properties with IC50 values 5.42 µg/ml. tested fruit, while the fruits of C. manan Based on the results of the phytochemical Miq dan C. ornatus Bl. have strong assay, antioxidant activities mostly antioxidant activities in its pericarp and influenced by the existence of flavonoids, seeds. Generally, the three species of rattan tannins and triterpenoids compounds, fruit have potentials as natural antioxidant which were detected almost in all species agents based on the DPPH method, but the of rattan fruit in this study. Flavonoids are active compounds which play roles as

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