Phytochemical Screening and Antioxidant Activity of Ethanolic Extract of Cawat Hanoman Stem ( Bauhinia aculeata L.) using DPPH Method

The free radical is an unstable molecule because contains one or two unpaired electrons. The antioxidant substance is a simple way to decrease the illness caused by free radicals. Cawat hanoman ( Bauhinia aculeata L.) was known to contain tannin components one of the benefits as an antioxidant. This research aims to determine the antioxidant activity of the B. aculeata stem tested by qualitatively used thin-layer chromatography (TLC) and quantitatively using the DPPH method. Bauhinia aculeata stem was extracted using a maceration extract method with 96% ethanol. Antioxidant activity test was done qualitatively by eluent of ethyl acetate : methanol : purified water (6 : 2 : 1) using TLC and quantitatively using the DPPH method. The result of antioxidant activity from 96% ethanol extract of B. aculeata stem qualitatively showed the presence of yellow spots on a purple background at TLC after syringed DPPH 0.5 mM and quantitative test that resulted in an IC 50 of 21.862 μg/mL. These results indicate that 96% ethanol extract of B. aculeata has very strong antioxidant activity.


INTRODUCTION
Free radicals are unstable molecule because contains one or more electrons unpaired (Phaniendra et al., 2015). It tends to react with the molecules around it, if this reaction continues to occur in the body will cause negative effects that is the onset of degenerative diseases and cell damage (Liochev, 2013). Therefore, the body needs substance antioxidants that are able to ward off free radicals (Lobo et al., 2010). Natural antioxidants come from plants, like polyphenols compounds which has a hydroxyl group on molecular structure. Polyphenols compounds with hydroxyl groups has catch free radicals (Tungmunnithum et al., 2018). Nowadays ample evidence from copious studies exists of polyphenols activity as antioxidative, antiinflammatory and other various biological effects that exert in the prevention of various pathologies including cardiovascular diseases and cancer (Mojzer et al., 2016).
One of the endemic plants from Borneo Island especially in South Kalimantan is cawat hanoman (Bauhinia aculeata L.). Empirically this plant is used to increased male stamina. Previous research has been proven the 96% ethanolic extract of B. aculeata stem have polyphenols compounds and proven efficacious as an aphrodisiac . Bauhinia aculeata contains phenolic compounds such as flavonoids and tannins (Margaretta et al., 2011

Abstract
The free radical is an unstable molecule because contains one or two unpaired electrons. The antioxidant substance is a simple way to decrease the illness caused by free radicals. Cawat hanoman (Bauhinia aculeata L.) was known to contain tannin components one of the benefits as an antioxidant. This research aims to determine the antioxidant activity of the B. aculeata stem tested by qualitatively used thin-layer chromatography (TLC) and quantitatively using the DPPH method. Bauhinia aculeata stem was extracted using a maceration extract method with 96% ethanol. Antioxidant activity test was done qualitatively by eluent of ethyl acetate : methanol : purified water (6 : 2 : 1) using TLC and quantitatively using the DPPH method. The result of antioxidant activity from 96% ethanol extract of B. aculeata stem qualitatively showed the presence of yellow spots on a purple background at TLC after syringed DPPH 0.5 mM and quantitative test that resulted in an IC50 of 21.862 μg/mL. These results indicate that 96% ethanol extract of B. aculeata has very strong antioxidant activity.
research report that methanolic extract of B. variegata barks showed IC50 value was 6.48 µg/ml, indicating that B. variegata extract has a very strong antioxidant activity (Sharma et al., 2015).
Based on previous studies, B. aculeata stem is also expected to have antioxidant potential. This research aims to determine the antioxidant activity of the B. aculeata stem. Maceration method was chosen to withdraw the compound in B. aculeata stem used 96% ethanol as solvent . The antioxidant assay in this research used 2,2-Dyphenyl-1-Picrylhyrazil (DPPH) method.

Tools and materials
The tools used in this research was analytical balance

Plant determination
Determination is carried out to identify and ascertain the identity of the plant species used. Determination is done using all parts of the plant carried out at the Department of Pharmacy Biology, Faculty of Pharmacy, Gadjah Mada University, Yogyakarta.

Extraction
A total of 300 g of B. aculeata stems simplicia were extracted by maceration method using ethanol 96% with a simplicia : solvent ratio of 1 : 5. The liquid extract obtained was then concentrated with a vacuum rotary evaporator. The thick extract is then evaporated again using water bath to obtain a fixed weight (Pandey et al., 2011).

Phytochemical screening
Phytochemical screening including alkaloids, phenolic, flavonoids, saponins, steroids/triterpenoids, and tannins test. The test method used was a standard method with some modifications.

Alkaloids test
A total of 0.2 g extract was dissolved with 5 ml HCl 10% and filtered. The filtrate used as test solution.

Dragendorff test
As much as 1 ml filtrate were treated with 3-5 drops Dragendorff reagent (solution of potassium bismuth iodide). Formation of red precipitate indicates the presence of alkaloids (Tiwari et al., 2011).

Mayer test
As much as 1 ml of filtrate were treated with 3-5 drops Mayer reagent (potassium mercuric iodide).
Formation of a yellow precipitate indicates the presence of alkaloids.

Wagner test
As much as 1 ml of filtrate were treated with 3-5 drops Wagner reagent (potassium iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids.

Phenolic test
Ferric chloride test: As much as 0.3 g of extract was added with 3-4 drops FeCl3 solution. Formation of bluish black color indicates the presence of phenols (Tiwari et al., 2011).

Flavonoids test
As much as 0.5 g of extract added with 10 ml purified water, then added with 0.1 g magnesium powder, a few drops HCl 5 N, and 2 ml amyl alkohol solvent. The solution was shaken vigorously, then wait until it separated. The positive result showed the formation of orange or yellow in amyl alcohol layer .

Saponins test
Foam test: As much as 0.5 g of extract was shaken for ± 1 minute with 5 ml of purified water. Foam produced that persists for at last 10 minutes indicates the presence of saponins .

Steroids and triterpenoids test
Liebermann-Burchard test: As much as 0.5 g extracts were treated with 2 ml chloroform, shaken, and filtered.
The filtrates were added with 1 ml of acetic anhydride, and 0.5 ml of concentrated sulphuric acid. The steroids positive results if there a formation of blue or green ring and the triterpenoids positive results give the formation of red or purple color .

Tannins test
Gelatin test: As much as 0.2 g extract was added with 2-3 drops 1% gelatin solution containing sodium chloride.
Formation of white precipitate indicates the presence of tannins (Tiwari et al., 2011).

Qualitative test of antioxidant activity
Ethanolic extract of B. aculeata stem was spotted on the TLC plate silica gel GF254. Extract was spotted on silica plate sized 1.5 cm x 10 cm by the upper limit and lower limit of 1 cm so that developer within 8 cm (Mustarichie et al., 2017). The extract has been diluted was eluted in chamber with mobile phase of EtOAc : MeOH : H2O (6 : 2 : 1). After the plate was eluted, then observed in visible light, ultraviolet (UV) with wavelength of 254 nm, 366 nm, and sprayed with DPPH 0.5 mM. The sample shows antioxidant activity characterized by the appearance of yellow spots against a purple background on the TLC plate (Muthia et al., 2019).

Quantitative test of antioxidant activity
A total of 1.98 mg of DPPH was dissolved with methanol until 50 ml, then shaken until homogeneous and placed in dark bottle. As much as 2 ml ethanolic extract of B.
aculeata stem with concentrations of 10, 20, 30, 40, and 50 μg/ml were reacted with 2 ml of 0.1 mM DPPH then incubated for 30 minutes in dark room at room temperature (25°C). Each absorbance sample was measured at a wavelength of 515 nm using a UV-Vis spectrophotometer. As a reference quercetin was used with the same treatment, with quercetin concentrations of 1, 2, 3, 4, and 5 μg/ml (Muthia et al., 2019).

Data analysis
The antioxidant activity of the ethanol extract of B.

RESULTS AND DISCUSSION
Determination was the first step carried out in this research, the aim was to find out and ascertain the  Table I.  that contribute hydrogen atoms to DPPH so that they are reduced to a more stable form (Pratiwi et al., 2013). This study uses quercetin as a positive control because quercetin is an isolate from natural ingredients which is included in the flavonoid group and is proven as a powerful antioxidant compound (Ningsih et al., 2017).