International Journal of Minor Fruits, Medicinal and Aromatic . Vol. 6 (1) : 13- 19, June 2020

Phytochemical composition and anti-oxidant properties of ovoideum thwaites (Gal Siyambala) leaves V.P. Bulugahapitiya*, T.N. Rathnaweera and H.C. Manawadu Department of Chemistry, Faculty of Science, University of Ruhuna, Wellamadama, Matara, Sri Lanka *Email: [email protected] Received : 18.11.2019 ; Revised : 30.12.2019 ; Accepted : 05.01.2020

ABSTRACT Dialium ovoideum thwaites() is an endemic to Sri Lanka, found in the semi-dry zone of the country. The various parts of the plant have being used for treating many health disorders in the traditional medicinal system of Sri Lanka. The aim of this study was qualitative and quantitative determination phytochemicals and evaluation of anti-oxidant properties of the leaves. Methanolic extract of D. thwaites leaves was prepared by macerating and then subjected to phytochemical screening using standard procedures. The results of phytochemical screening showed the presence of alkaloids, flavonoids, saponins, steroids, glycosides, tannins and coumarins. Quantitative determination was done for alkaloids, flavonoids and saponins using gravimetric method, and for tannins using colorimetetric method. It was found that the leaves contains 2.05% (w/w) of alkaloids, 3.58%(w/w) of flavonoid, 2.07 %(w/w) of saponins and 370.4 mg TAE / g of tannin. The anti-oxidant properties of leaves were evaluated as total phenolic content(TPC) using Folin-Ciocalteau reagent and colorimetric method as garlic acid equivalent, using free radical scavenging assay (DPPH assay) with ascorbic acid as standard anti-oxidant and using FRAP assay as total anti-oxidant capacity. Results indicates that total phenolic content is 189.7 mg GAE/ g,IC50value for DPPH assay is131 mg/mL whereas 31.0mg/mL is for ascorbic acid standard, and the FRAP value gives as977 μ mol Fe2+ /g, showing higher anti-oxidant properties of leaves. Keywords : Dialium ovoideum thwaites, phytochemical analysis, anti-oxidant, DPPH Assay, FRAP Assay

INTRODUCTION higher plants on earth which is about 250,000 Since the beginning of mankind, humans have whereas only 6% of them have been screened for been relying on plants to fulfill their vital their biological activity and only 15% has been requirements hence plants have played a salient analyzed phytochemically (Fabricant and role as a good source of medicine (Gurib-Fakim, Farnsworth, 2001) even though phytochemicals 2006 and Komolafe, 2014, Sabitha Rani et al., serve as the base for their potent medicinal activity. 2019). In the modern medical aspects many plants Antioxidant activity is one such property in which or plant-based materials have gained wide attention the world is keener today and plants are very on developing/extracting potential drug candidates popular in this aspect due to their innate ability in especially for the treatment of non-communicable biosynthesizing a wide range of antioxidants which diseases such as diabetes mellitus and cancer etc prevent or delay cell damages caused by oxidative (Bhowmik, 2019). According to a report of World stress (Kasote et al., 2015). Many of such Health Organization 80% of the world’s population unexplored plants including endemic and native in developing countries still rely on plant products plants are being used in different traditional for their primary health care (Tanveer et al., 2017 medicine systems all over the world (Gülçin et al. and Aqil et al., 2010). Furthermore it has been 2010). Sri Lankan Ayurvedic system is such a reported that 61% of novel drugs which have been traditional system which has a history of about 2500 developed between 1981 and 2002 have been based years and is based on a series of prescriptions on natural products and has been reported to be handed down from generation to generation (De successful in areas of infectious diseases and cancer Alwis, L. 1997). Ayurveda uses about 550 to 700 (Bhalodia and Shukla, 2011). species out of over 3000 species Nevertheless extent of such discoveries is not present and a quarter of which is endemic to Sri satisfactory compared to the estimated number of Lanka (De Alwis, 1997). IJMFM&AP, Vol. 6 No. 1, 2020 13 Phytochemical composition and anti-oxidant properties

Dialium ovoideum thwaites (Fabaceae, local Phytochemical screening name; Gal siyambala) is an endemic plant to Sri Screening for Alkaloids Lanka used in Ayurvedic system for different Mayer’s, Wagner’s and Dragendroff’s Tests applications such as treating skin infections, as an - About 5 g of powdered plant material was mixed antidote to treat snake bites etc. Since no adequate with 8 mL of 1% HCl and was boiled in a water scientific study has been reported so far on D. bath for 5 minutes. The solution was cooled and thwaites, this study was aimed at investigating the filtered, the filtrate was tested with few drops of phytochemical profile and antioxidant capacity of Mayer’s, Wagner’s and Dragendroff’s reagents leaves of D. thwaites. Furthermore while (Bulugahapitiya, 2013; Ezeonu and Ejikeme, 2016; ethnomedicinal plants are being scientifically Abulude, 2007). For the conformation, 6 g of investigated, it is equally important to produce powdered plant material moistened with water and ready to use products by incorporating them in mixed with 1 g of Ca(OH)2. The paste was mixed order to give the maximum benefit to the society. well with 5.0 ml of diethyl ether followed by Thus the ultimate objective of this study was to evaporating of ether. The residue was mixed with 5.0 ml of 1% H SO , filtered and the filtrate was produce such a value added product by making use 2 4 presence of important phytochemicals and anti- tested Draggendroff’s reagent. oxidants. Screening for Flavonoids MATERIALS AND METHODS Alkaline reagent Test - About 500 mg of methanolic crude was dissolved in 2 ml of MeOH. Chemicals and Instruments Few drops of 10% NaOHwas added followed by The solvent, methanol was distilled prior to few drops of 10% HCl until the colour changed to maceration while all other chemicals such as colourless (Bulugahapitiya, 2013). chemicals for phytochemical screening, Folin- Lead acetate Test - About 500 mg of dried Ciocalteu’s (FC) reagent, gallic acid, tannic acid, powder was mixed with 2 ml of MeOH and few DPPH, FeSO .7H O, TPTZ were analytical grade 4 2 drops of 1% Pb(CH3COO)2 (Bulugahapitiya, 2013). thus were used without any further purifications. For vacuum evaporation the rotary Screening for Tannins evaporator,model Heidolph WB 2000 was used Ferric chloride Test - About 100 mg of dried while UV spectrophotometer, model SHIMADZU powder was mixed with 2 ml of MeOH and 1.0 ml UV-1601was used for the spectrometric analysis. of 2% FeCl3 was added. (Bulugahapitiya, 2013) Alternatively, about 0.15 g of methanolic extract Plant Material was mixed with 30.0 ml of distilled water and was The leaves of D. thwaites were collected from boiled for 10 minutes. The solution was filtered Wellawaya area, Monaragala district of Sri Lanka and 5.0 mL of the filtrate was mixed with few drops of 0.1% FeCl solution (Ezeonu and Ejikeme, and were authenticated. The leaves were washed 3 and blot dried followed by standing for 21 days in 2016). shade for complete drying as the leaves are watery Screening for Saponins in nature. The dried leaves were then ground to Froth Test - About 2 g of powdered plant fine powder using a blender and stored in sealed material was mixed with 15.0 ml of distilled water. 0 zip-lock bags at 4 C until usage. The mixture was boiled and filtered; the filtrate was mixed with 5.0 mL of distilled water and was Extraction of phytochemicals shaken vigorously. Then the formed froth was For the preparation of extract, 160 g of dried mixed with 3 drops of olive oil and was again powder was macerated with 600 ml of methanol shaken vigorously (Bulugahapitiya, 2013). for 4 days with frequent agitation. Then the macerated solution was filtered and concentrated Screening for Terpenoids using the rotary evaporator to obtain a solid In the first method about 2 g of powdered plant methanolic crude. material was defatted with petroleum ether and the

IJMFM&AP, Vol. 6 No. 1, 2020 14 Bulugahapitiya et al. residue was extracted with 10.0 ml of was heated in a water bath at 55oC with constant

CHCl3followed by drying. To 5.0 ml of the above stirring for 4 hours, non-polar matter was extracted extract, 0.25 ml of acetic anhydride followed by 2 off with diethyl ether. The solution was dissolved drops of conc. H2SO4were added. In the second in 12.0 mL of n-butanol, after evaporating n-butaol, method, about 1 g of dried plant material was mixed the saponin was weighted out (Ezeonu and with 5 drops of Cu(CH3COO)2 solution Ejikeme, 2016). (Bulugahapitiya, 2013). Quantification of flavanoids Screening for Glycosides 1g of powdered plant material was first Keller-killani Test- About 1 g of powdered extracted off with 20.0 ml of 80% methanol, after plant material dissolved in 3 ml of glacial acetic discarding the supernatant, the residue was acid and few drops of 5% FeCl3 was added. The extracted with ethanol. After evaporating ethanol, mixture was poured in to a test tube containing 2.0 extracted flavonoids was dried until constant ml of conc. H2SO4 (Bulugahapitiya, 2013). weight (Ezeonu and Ejikeme, 2016). Alternatively in to 1 g of powdered plant material, Quantification of tannin 5.0 ml of CHCl3 were added followed by 5.0 ml of An amount of 0.8967 g of methanolic crude was 10% NH3 (Ezeonu and Ejikeme, 2016. extracted into 10.0 ml of PET ether, 10.0 mL of Screening for Coumarins acetone: water (7:3 v/v). Filtration followed by About 1 g of powdered plant material was mixed evaporation of excess solvent, it was dissolved in with 2 ml of MeOH. The mouth of the test tube 25.00 ml of MeOH and methanolic crude was was covered with a filter paper soaked in 1 N prepared. The stock solution (15,000 μg/ml) of NaOH. The tube was placed in a boiling water bath tannic acid and then the concentration series was for few minutes. The filter paper was then removed prepared to measure the absorbance at 725 nm and immediately observed under UV light using the UV spectrophotometer. The test solution (Bulugahapitiya, 2013). prepared using 1.00 ml of extract, 1.00 ml of 7.5%

Na2CO3, 0.5 ml of FC reagent and 7.50 mL of Screening for Steroids distilled water. Absorbance was measured at same About 1 g of powdered plant material mixed wave length (Ezeonu and Ejikeme, 2016). with 20.0 ml of ethanol, covered and was allowed to stand for 2 hours. The solution was filtered and Evaluation of anti-oxidant capacity the second mixture was prepared by mixing 2.0 ml Determination of total phenolic content (TPC) of acetic anhydride with 2.0 ml of conc. H2SO4. Total phenol content was determined using 5.0 ml of the sample extract prepared above was Folin-Ciocalteau reagent and following mixed with the prepared acid solution colorimetric method. Amount of 0.3 ppm plant (Bulugahapitiya, 2013;Ezeonu and Ejikeme, 2016). extract and 200 ppm standard gallic acid stock solution was prepared in MeOH followed by Quantitative Determination of Phytochemicals solution series of 20, 40, 60, 80, and 100 ppm. Quantification of alkaloids Folin-Ciocalteau reagent was used for 1g of powdered plant material was extracted complexation (2.50 ml) and 80% MeOH was used into 10% acetic acid in EtOH, filtered and as the blank. After incubation period of 1 h at room concentrated. 10 ml of Conc. NH3added and filtered temperature, the absorbance was measured at 765 after 24h, dried at 400C until constant weight. The nm using the UV spectrophotometer (Valko et al., percentage alkaloids was calculated. (Ezeonu and 2007). Ejikeme, 2016) DPPH assay Quantification of saponins DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical 1g of powdered plant material was extracted scavenging assay was performed using the methods into 20% aqueous of ethanol. Resulting solution reported with slight modifications (Alam et al.,

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2013). The stock solution of plant extract of 10,000 glycosides, coumarins and terpenoids in the leaves. ppm and ascorbic acid of 1000 ppm was prepared The quantification gave the amounts given in the in MeOH. For the test 100 μL of the extract with table 1 below. Accordingly leaves contains different concentration, 3.90 ml of DPPH solution significant amount of alkaloids, flavonoids and were mixed. After 30 minutes of incubation at room saponins which can be directly related to many temperature, the absorbance was measured at 517 medicinal values of D. thwaites (Chew et al., 2011). nm using the UV spectrophotometer. The Table 1.: Quantitative analysis of measurements were triplicated and the antioxidant phytochemicals for methanolic activity was measured as percentage inhibition of extract DPPH as described below where A and A are the 0 Phytochemical class Quantity absorbance of the control and the sample Alkaloids (w/w)% 2.05 respectively. The IC50 values were obtained by plotting the percentage inhibition vs concentration. Flavanoids (w/w)% 3.58 Percentage inhibition = A0 –A × 100% Saponins (w/w)% 2.07 A0 Tannins mg TAE/g 370.40 FRAP assay The evaluating of anti-oxidant capacity of leaves was done as total phenolic content, DPPH The Ferric reducing anti-oxidant power (FRAP) assay and FRAP assay. The total phenolic content assay was performed which assessed the reduction (TPC) was expressed in gallic acid equivalents of ferric tripyridyltriazine (Fe (III)-TPTZ) complex which was 189.7 mg GAE/g (Figure 1), which was to ferrous tripyridyltriazine (Fe (II)-TPTZ) by relatively a higher amount. This test is based on measuring the absorbance at 593 nm. A 1200 ppm the oxidation of phenolic groups by FeSO .7H O stock solution and a dilution series 4 2 phosphomolybdic and phosphotungstic acids was prepared in water while the same plant extract (Folin-Ciocaltue) and yielding a blue colour with series above was used. For the test solution 100 a broad maximum absorption at 765 nm, where μL of extract or standard, 3.00 mL of working gallic acid used as the reference. Phenolic FRAP reagent were mixed. Absorbance was compounds are known to act as anti-oxidants and 0 measured after 30 minutes of incubation at 37 C hence it can quench the harmful free radicals (Abulude, 2007). generated in the cells via oxidizing of phenolic Results and Discussion group of the substance. This property is attributed to the ability of acting against non-communicable The methanolic extract of D. thwaites showed diseases (NCDs) as there is a strong correlation the presence of eight common phytochemicals such between harmful free radicals and NCDs (Chew et as alkaloids, flavonoids, tannins, saponins, steroids, al., 2011; Kukula and Widelski, 2017).

Figure 1. Standard Gallic acid curve for TPC determination

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Free radical scavenging capacity is a good resulting colour is proportional to the remaining measurement for the ability quenching harmful free concentration of DPPH after reaction with the radicals generated in the cells. The DPPH assay antioxidant. The results of DPPH assay of this study measures the free radical scavenging ability of plant is given in figure 2 and the IC50 values were 131.0 substances and also it is a method of measuring ppm and 31.0 ppm for leaves of D. thwaites and reducing power of plant compounds, it is expressed standard (ascorbic acid) respectively, implying that as IC50 value (antioxidant concentration needed to higher anti-oxidant capacity of leaves. Anti-oxidant scavenge 50 % of DPPH radical). In this assay, capacity depends on the maturity stage of the plant antioxidants react with DPPH radical, forming (Dudareva et al., 2004). reduced form of DPPH and the intensity of the

Figure 2. % Inhibition Vs concentration plot for leaves of D. thwaites

The FRAP value in Ferric Reducing Antioxidant 977 μ mol Fe2+ /g.This further supports the anti- Power (FRAP) assay was calculated using the oxidant capacities given in DPPH assay standard curve drawn (figure 3) and the value was

Figure 3. FeSO4.7H2O standard curve for FRAP assay

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