Screening Phytochemical, Antioxidant Activity and Vitamin C Assay from Bungo perak-perak (Begonia versicolar Irmsch) leaves

Ermi Abriyani*, Lia Fikayuniar

Department of Pharmacy, Buana Perjuangan University of Karawang, West Java, Indonesia.

*Corresponding Author E-mail: ermi.abriyani@gmail.com

ABSTRACT:

The study of screening phytochemical and activity antioxidant has been done and this is the first report on Bungo Perak-perak (Begonia versicolar Irmsch) leaves about screening phytochemical, antioxidant activity and vitamin C assay. The extraction using maceration method on Begonia versicolar Irmsch leaves with three type solvent are n-hexane, ethyl acetate and methanol. Antioxidant activity assay from n-hexane, ethyl acetate, and methanol extracts at various concentration (31.25µg/ml, 62.5µg/ml, 125µg/ml) using the DPPH (1,1-diphenyl-2-dipicryl-hydrazin/DPPH•) methods and as reference solution is ascorbic acid. Absorbance was measured at a wavelength of 515nm in a uv-visible spectrophotometer. Determination of vitamin C assays was tested by iodometry methods. The results of phytochemical from Begonia versicolar Irmsch are indicated the sample had contains alkaloids, flavonoids, phenolic, terpenoids, steroids, carbohydrate, saponin and reducings sugar. The results of activity antioxidants are ethyl acetate 69.82µg/ml, and methanol extracts 8.67µg/ml. Result of vitamin C assays from Begonia versicolar Irmsch are 3.256mg from 20grams sample. This research was showed Begonia versicolar Irmsch can be a good source of antioxidants and have vitamin C and good of phytochemicals that supports traditional herbal medicine.

KEYWORDS: Begonia versicolar Irmsch, phytochemicals, antioxidants activity, vitamin C assay.

INTRODUCTION:

The genus of Begonia is one of of the plants spread in four large islands in Indonesia that are Sumatera island, Kalimantan island, Sulawesi island and Papua island. There are 200 types of begonias in Indonesia and the most widely distributed on Papua Island². Begonia can be used as a traditional herbal medicine which is to increase high body resistance, juice for infants is believed to prepare the development of bones and teeth, increase body weight, reduce body temperature during fever¹, wounds, laxatives, cough ingredients, proven as anti-tumor has conducted by herbalist³.

In several study of genus Begonia are conducted as follows the in vitro study of antioxidants from Begonia malabarica Lamk. and Begonia floccifera Bedd⁴, anti-bacterial Begonia floccifera Bedd flower of methanol extract⁵, hypoglycemic and anti-hyperglycemic effects of Begonia malabarica Lamk. from methanol extract⁶, antimicrobial studies on extracts of water, methanol and chloroform from Begonia malabarica⁷ and rhizome of Begonia sinensis has antimicrobial sterol extract⁸. In previous studies found some chemical content of this Begonia plant¹⁸. Vitamin C content from leaves of Begonia floccifera bedd. and Begonia malabarica Lamk.². Begonia evansiana Andr has a content cucurbitacin B, cucurbitacin D, 23,24-dihidrocucurbitacin D, 23,24-dihidrocucurbitacin F, 2-O-beta-glucopiranosil-cucurbitacin B and 2-O-beta-glucopiranosil-cucurbitacin D¹¹, in β-sitosterol, β-amyrin, daucosterol, stigmasterol, stigmasterol-3-O-β-D-glucopiranoside and 4’, 5’, 7 ’-trihydroxiflavone-6-O-β-D-glucopiranoside⁴. In other types, Begonia Linn. found two rutin compounds and quercetin. The content of alkaloids, triterpenes, flavonoids, saponins, tannins in Begonia fallax, Begonia cordifolia, Begonia malabrica¹³, tamarixetin glycoside in Begonia versicolar Irmsch¹⁰. This is the first report on screening phytochemical and antioxidant activity from Begonia vesicolar Irmsch.

Antioxidants are chemical substances that can react with electrons as free radicals by donating one or more electrons so that they can inhibit the occurrence of free radical reactions. Free radicals are an atom or group that has one or more unpaired electrons¹⁴, unstable and very reactive¹⁵. Sources of free radicals can come from pollution, baked goods, packaged foods, cigarette smoke, additives and others. Unpair electrons that are not stable can form chain reactions and produce abnormal compounds that can damage cells in the body to form an abnormal tissue¹⁶ or the antioxidants levels and the oxidants in tissue are occurred at balance conditions²⁴. Antioxidants can overcome the reaction of these free radicals¹⁷. Potential source of natural antioxidants is plants²³ or plants that contain phenolic compounds, flavonoids, terpenoids and alkaloids or that have health promoting³². The crudes extract of sample from the plants could be subjected for phytochemical screening²⁸ to revealed the secondary metabolites compounds and antioxidants assays.

MATERIALS AND METHODS:

The leaves of Bungo Perak-perak (Begonia versicolar Irmsch) as a sample were collected from West Sumatera Province, Indonesia. This plant was identified in ANDA Herbarium, Andalas University, West Sumatra, Indonesia with identification number 188/K-ID/ ANDA/IX/2013. Samples of Begonia versicolar Irmsch leaves were dried at room temperature for two weeks. The dried samples of Begonia versicolar Irmsch are powdered and stored in a closed container.

Screening of Phytochemical:

The Phytochemical were chemicals that may effect of health that are contained the chemicals compounds that occur naturally in plants²⁵. The screening of phytochemical to showed the secondary metabolites^29-31 and also used the application of Parekh and Chanda¹⁹, Harborne^20,Onyechi²⁶_. The dried powder sample of 0.1 gram of Begonia versicolar Irmsch leaves was put into a test tube, then macerated with methanol which had been heated (above the water bath) for 15 minutes. The maceration in methanol was filled in to test tube and allow all the methanol to evaporate until dryness. Then added chloroform and distilled water with a ratio of 1: 1 each of 5mL, shaken well, then transferred into a test tube, left for a moment to form two layers of chloroform-water. The lower chloroform layer was used to examine triterpenoid and steroid compounds while the water layer was used as a test for flavonoid, phenolic and saponin compounds.

Flavonoid:

Some of the layers of water from extracts are taken and transferred using a pipette into the test tube, then concentrated hydrochloric acid is added and added magnesium powder, the presence of orange, pink to red indicated of a positive (+) of flavonoids.

Phenolics and tannins:

The layers of water from the extract are taken and transferred using pipette into the test tube. Then ferric cloride reagent was added, a dark blue, green, black that indicated of phenolic or tannins.

Saponins:

The layers of water was shaken strong in test tube along 30 seconds, persistent of frothing that indicates saponins.

Triterpenoid dan Steroid (Lieberman Buchard):

The chloroform layer were taken to three holes of drop plate, let to dry. Drop the concentration of sulphurric acid to each of holes drop plate and a single drop of acetat anhydride, green or bluish green indicates of presence the steroids and red or magenta indicates triterpenoids.

Alkaloids (Mayers):

Two until four grams leaves was smoothed with mortal stampler, add a little sand and 10mL chloroform – ammonia solution 0,05N. the filtrate was filtered with cotton and enter into test tube, add 10 drops sulphurric acid 2 N and shake slowly. The mixture allowed until two layers formed that are the acid layers and chloroform layers. The acid layers was taken with glass pippete drops and entered into test tube. The acid layers was added the mayers reagent, white precipite indicated to presence the alkaloids groups

Carbohydrates (Molisch):

Molisch Assays to knowed the carbohidrates in sample. 0,1grams sample of methanol extract diluted in test tube, add the α-naftol in ethanol and shake homogeny. Thus, add concentration sulphurric acid slowly across the wall of test tube and until two layers formed. There are purple layers or look like purple ring indicated to presence the carbohydrate.

Reducing sugars (Fehling):

Fehling test used to determine whether there were any reducing sugars in sample. 2mg extract methanol was dilluted with methanol in test tube and put on water bath in 10 minutes, add about 3mL aquadest and shaken. The filtrate was test with fehling A and B. the existence of the precipitate of brownish red is revealed of reducing sugars.

Extraction:

250grams powdered of Begonia vesicolar Irmsch was extracted with three solvents are n-hexane, ethyl acetat and methanol. The extraction was maceration methods. The maceration were 6 times for 5 days with n-hexane, ethyl acetate and methanol respectively. The extract was evaporate with rotary evaporator and stored for next treatment.

Antioxidant assays with DPPH:

DPPH (1,1-diphenyl-2-picryl-hydrazyn/DPPH•) is a free radicals which stable and used widely to estimate activity of scavenging the radical components. This method is based on the reducing DPPH in methanol in with the antioxidant donor hydrogen that formed non-radical of DPPH-H²¹. The free radical activity of all extract in accordance with previous method²¹. The treatment of antioxidant activity assay of Begonia versicolar Irmsch was based on a few modification of Velusami Kapalnadevi method²². The DPPH was dilute on 0,1mM concentration in methanol. All of extracts were diluted with methanol and variation concentration 31.25µg/ml, 62.5µg/ml, 125µg/ml. The extract was 2ml, add 2ml DPPH and incubated 30 minutes at room temperature. The absorbance was measured at 515nm on UV-Vis spectrophotometer. The reference standar is ascorbic acid that dissolved in methanol. The lowest absorbance from the mixture of sample and DPPH were indicated on scavenging activity antioxidant are higest. The ability of DPPH scavenging effect (inhibition) was calculated by apply the equation:

% inhibition = (A_control – A_sample) / A_control x 100

Where, A_control is the absorbance of” reference standar (control), A_sample is the absorbance of sample (three extracts = n-hexane extract, etyl acetat extract and methanol extract).

Thus, the calculation of IC₅₀is the concentration of sample to able reduce 50% of DPPH radical activity. The value of IC₅₀ obtained by plot the line between 50 percent of power inhibition by concentration of the sample.

Vitamin C assay by iodometric methods:

Ascorbic acid or vitamin C in Begonia versicolar Irmsch was determine with iodine titration.

The sample of Begonia versicolar Irmsch leaves was mashed or formed slurry. Then 20grams slurry dilute to 100ml with distilled water in volumetric flask and shake until homogeny. The mixture from dilute of slury was taken 10ml into erlenmeyer and add 1 ml of 1% starch. Then, titrate with 0.1 N iodine solution and the end point from titration was the appearance of the dark blue stable or the mixture of solution like blue starch-iodine color and determination of vitamin C followed equation of stoichiometric.

RESULT AND DISCUSSION:

Screening phytochemical of bungo perak-perak (begonia versicolar Irmsch) leaves:

Phytochemical including non-chromatographic assay that characterized mono and polyherbal of the product³⁰. The result from qualitative analysis can showed by phytochemicals^27,28 and can revealed the secondary metabolites of the plants²⁹. The result of screening phytochemical from Bungo perak-perak (Begonia versicolar Irmsch) were revealed of presence the bioactive components. The bioactive components from sample of Begonia versicolar Irmsch were summarized in table 1.

Tabel 1. The results oh screening phytochemical from bungo perak-perak (Begonia versicolar Irmsch)

S. No	Chemical compounds	Results
1.	Alkaloids	+
2.	Flavonoids	+
3.	Phenolics	+
4.	Tannins	+
5.	Terpenoids	+
6.	Saponins	+
7.	Steroids	+
8.	Carbohydrates	+
9.	Reducing Sugars	+

(+) = identified

(-) = not identified

Antioxidant activity from extracts of Begonia versicolar Irmsch leaves:

Determination antioxidant activity of sample Begonia versicolar Irmsch that assays to different solvents of polarity. The antioxidan activity assay was carried out by spectrophotometry using the DPPH method. This method was chosen because it is simple, easy, fast, and sensitive which requires only a small quantity of sample. The based on this method was of the hydrogen atom from the phenolic group in the sample have ability to capture DPPH free radicals. The reduced intensity of purple from the DPPH solution means that there is a reaction between the hydrogen donor radical from the sample and the radical from the DPPH molecule so that yellow will form 1,1-diphenyl-2-pikril hydrazine. The results of the antioxidant assays was seen that the greater the concentration from sample, the stronger the yellow color can be seen so that the spectrophotometer will read the smaller absorbance and the activity of the sample in receiving hydrogen radical from DPPH is also greater. The measurement of absorbance is obtained from the absorbance of the remaining DPPH which does not react with the sample.

Fig 1. Calibration curve of percentage inhibition and IC₅₀ of etyl acetat extract Begonia versicolar Irmsch

Fig 2. Calibration curve persentage inhibition and IC₅₀ of methanol extract Begonia versicolar Irmsch

The absorbance value of each sample was analyzed by regression calculation with the percentage of inhibition determined by linear regression. Next, it is plotted to determine 50% activity so that an effective concentration value is obtained. specifically that a compound is a very strong anti-oxidant if the IC₅₀ value is less than 50ppm, strong for IC50 is 50-100ppm, moderate if it is worth 100-150ppm, and weak if the IC₅₀ value is 151-200ppm (Endang Hanani et al, 2005). Based on the data obtained the activity of samples from methanol extract showed IC₅₀ values of 8.67ppm, which meant that is provided very strong antioxidant activity. Abriyani, Ermi (2014) reported that methanol extract contained tamarixetin glycoside that is quercetin methylated at position O-4’, It is derives of quercetin compound. While the ethyl acetate extract showed a fairly strong activity of 69.82 ppm and the hexane extract did not have antioxidant activity. It is caused of the hexane extract does not contain compounds that can donate radical hydrogen.

Vitamin C Assays by titrimetric methode:

Based on the titration results of iodine solution with thiosulphate solution 0.1 N, iodine concentration of 0.07 N was obtained. Then iodine solution was used as titer of vitamin C, which would be determined (milligrams of vitamin C in gram samples). In titrating vitamin C which was carried out three times, the average volume of iodine used was 0.53 ml. determination of vitamin C 3.256 mg of vitamin C is obtained in 20 grams of sample.

CONCLUSION:

The research of screening phytochemical and antioxidant assay for bungo perak-perak (Begonia versicolar Irmsch) from leaves heve been conducted. The results of screening of phytochemical from Begonia versicolar Irmsch were alkaloids, phenolic, tannins, flavonoid, triterpenoid, steroid and saponins. The Begonia versicolar Irmsch has carbohydrate and reducings sugar. Antioxidant activity assays revealed that methanol extract has very strong activity antioxidant. The Begonia versicolar Irmsch leaves also contain vitamin C.

ACKNOWLEDGMENT:

The Author greatest thank to Buana Perjuangan University of Karawang for support of this research.

CONFLICT OF INTEREST:

Autor states that have no conflict of interest in research and the publication towards this article.

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Received on 21.01.2020 Modified on 18.02.2020

Asian J. Pharm. Res. 2020; 10(3):183-187.

DOI: 10.5958/2231-5691.2020.00032.5