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Phytochemical screening and antioxidant property of methanolic extract of Bauhinia variegata leaves | Advance Pharmaceutical Journal

Research Articles

2016  |  Vol: 1(4)  |  Issue: 4 (September- October)
Phytochemical screening and antioxidant property of methanolic extract of Bauhinia variegata leaves

Aditi Singhal*, Rupa Mazumder, Avijit Mazumder, G.S. Chakraborthy

Department of Pharmaceutical Technology,

Noida Institute of Engineering and Technology,

Knowledge Park-2, Greater Noida -201306, U.P., India.

 

*Author for correspondence

Aditi Singhal

Department of Pharmaceutical Technology

Noida Institute of Engineering and Technology,

Plot no.-19, Knowledge Park-2, Greater Noida -201306, U.P., India.

Contact:- +91-9643155815


Abstract

Objective: Aim to present study was studies the phytochemical screening and Antioxidant property of methanolic extract of Bauhinia Variegata leaves. Materials and methods: Phytochemical screening of methanolic extract was performed to detect the presence of chemical constituents. Methanol extract of B.variegata leaves was selected for phyto-chemical screening and also determine the anti-oxidant property in the leaves. Results: Phytochemical screening of methanolic extract reveals the presence of Flavanoids, Proteins, Resins, Tannins, Alkaloids and also shows the anti-oxidant property by using DPPH and Ferric chloride reducing method. Conclusion: Study performed on the plant extract sample indicates the presence of active constituents like proteins, flavonoids, resins, tannins, alkaloids and carbohydrates. The antioxidant property of the extract shows that there is an increase with the amount of concentration increases. This shows that the methanolic extract of Bauhinia variegata leaves shows the greater amount of antioxidant property.

KeywordsBauhinia variegate, methanolic extract, Formulation, Antioxidant, DPPH, Ferric chloride reducing method


Introduction

Bauhinia variegata Linn belonging to family Caesalpiniaceaeis commonly called as Kachnar in Hindi. It is a medium-sized tree found in India, Burma and China. It is mainly used for the asthma, abdominal distention, diarrhoea and also for skin diseases. This plant posses antioxidant property. Tannins, alkaloids, flavonoids and phenolic compounds are found as bioactive constituents. Apart from the above phytoconstituents fatty acids such as oleic acid, linolinic acid, stearic acid, palmitic acid and myristic acid are also present in this plant. This plant also possesanti-inflammatory, antibacterial activity and immunomodulatory property for various inflammatory diseases. Somewhat it is having an antitumor activity (Sawhnay and Amin, 2012). The preservative effect of different species and herbs shows the presence of antioxidant and antimicrobial constituents. A large number of phenolic compounds with high antioxidant property in the plant extracts have also been found. Antioxidant property is found due to the presence of phenolic compounds like flavonoids, phenolic diterpenes and phenolic acids which play a vital role in absorbing and neutralizing free radicals. Both water soluble and fat soluble vitamins are present in the plant extract which helps in increasing the levels ofcalcium and potassium. The medicinal value of this plant is very useful for treating human body. The barks of Bauhinia help in reducing hypertension and the level of cholesterol in mankind (Saha et al 2011).

Authentication of Plant

The plant was collected from the herbal garden of NIET and the plant was authenticated. The authenticated plant was preserved in the Department of Pharmacognosy for further reference.

Extraction procedure

The leaves of Bauhinia variegata were shad dried and powdered with a mechanical grinder and stored in an airtight container. The dried powder (100gm) was extracted with methanol in soxhlet apparatus for 72 hours. The solvent was distilled off to 1/3rd volume and it was transferred on a previous tarred vessel over water bath maintaining a temperature of 35˚C to obtain a semi solid mass. The semisolid mass was kept in a desiccator for drying. The crude extract was found to be 3.6%w/v.

Calculation

Practical yield = 4.5 gm

% yield = Practical yield × 100 / Theoretical yield

4.5 × 100 / 125 = 3.6%

Phytochemical screening of extract

1.      Alkaloids

a.      Dragendroff’s test- To 1mg of methanolicextract 5ml of distilled water was added, 2M Hcl was also added. To this add 1ml of dragendroff’s reagent.Formation of orange or orange red color precipitate occurs indicates the presence of alkaloids.

b.      Hager’s test- To the 1mg of methanolic and ethanolic extract added 1 or 2 drops of Hager’s reagent. Formation of yellow precipitate occurs.

c.       Wagner’s test-1mg of methanolicextract acidified with 1.5% v/v Hcl, after this few drops of Wagner’s reagent was added. Yellow or brown precipitate occurs.

d.      Mayer’s test- To the 1mg ofmethanolicextract, add few drops of Mayer’s reagent. White or pale yellow color precipitate occurs.

2.      Carbohydrates

a.      Benedict’s test- 1mg of extract was shaken with 10ml of distilled water, filtered and to the concentrated filtrate add few drops of Benedict’s solution boiled for few seconds, formation of brick red color precipitate indicates the presence of carbohydrates.

b.      Anthrone test- 1mg extract wasshaken with 10ml of distilled water, filtered and to the concentrated filtrate add few drops of Anthrone’s reagent boiled for few seconds, formation of blue or green color indicates the presence of carbohydrates.

c.       Fehling’s test-1mg of methanolicextract was shaken with 10ml of distilled water, filtered and to the concentrated filtrate add few drops of equal parts of both Fehling solution A and B, boiled for few seconds, formation of brick red color precipitate indicates the presence of reducing sugars.

d.      Molisch’s test- 1mg of methanolicextract wasshaken with 10ml water, filtered and the filtrate wasconcentrated, to the filtrate add 2 drops of freshly prepared 20% alcoholic solution of α- naphthol wasadded. 2 ml of conc. sulphuric acid was also added. Red-violetring appear, indicating the presence of carbohydrates which disappear on the addition ofexcess of alkali.

3. Proteins

a. Biuret’s test- To 1ml of hot methanolic aqueous extract, added 5-8 drops of 10% w/v sodium hydroxide solution and added1 or 2 drops of 3% w/v copper sulphate solution. Formation of violet red color occurs.

b. Millon’s test- 1ml of aqueous extract dissolved in 1 ml of distilled water and added 5-6 drops of Millon’s reagent. Formation of white precipitate, which turns red on heating, indicated the presence of proteins.

4. Flavonoids

a. Shinoda’s test- 1mg of methanolicextract was dissolved in 5ml of ethanol and to this adds 10 drops of dilute hydrochloric acid followed by a small piece of magnesium. Formation of pink, reddish or brown colour occurs indicates the presence of flavonoids.

b. Alkaline reagent test-To 1mg of extract adds 10% ammonium hydroxide solution, yellow fluorescence indicating the presence of flavonoids.

5. Glycosides

a. Molisch’s test- 2 mg of methanolicextract was shaken with 10ml of water, filtered and the filtrate was concentrated. To this filtrate add 2-3 drops of Molisch’s reagent, mixed and added 2ml of concentrated sulfuric acid carefully through the side of the test tube. Reddish violet ring appear, indicating the presence of glycosides.

6. Triterpenoids

a. Liebermann - Burchard’s test-2 mg of dry extract was dissolved in acetic anhydride, heated to boiling, cooled and then added1 ml of concentrated sulphuric acid along the sides of the test tube. Formation of a pink colour indicates the presence of triterpenoids.

7. Resins

One ml of methanolic extracts dissolved in acetone and solution was poured in distilled water. Turbidity indicates the presence of resins.

8. Saponins

In a test tube add 5 ml of methanolic extract, a drop of sodium bicarbonate solution was added. Test tube was shaken vigorously and left for 3 minutes. Formation of honeycomb like froth indicates the presence of saponins.

9. Tannins

To 1-2 ml of methanolic extract, few drops of 5% w/v FeCl3 solution were added. A green color indicated the presence of gallotannins, while brown color indicates the presence of pseudotannins.

10. Starch- 0.01 g of Iodine and 0.075 g of potassium iodide were dissolved in 5 ml of distilled water and 2-3 ml of methanolic extract was added. Formation of blue color indicated the presence of starch.

Results

Table 1. Phytochemical screening of methanolic extract 

Tests

Results

 Alkaloids

·         Wagner’s test

·         Hager’s test

·         Mayer’s test

 

+ve

-ve

-ve

Carbohydrates

·         Anthrone’s test

·         Molisch’s test

·         Fehling’s test

 

-ve

+ve

-ve

Proteins

·         Biuret’s test

·         Millon’s test

 

+ve

+ve

Flavanoids

·         Shinoda’s test

·         Alkaline reagent test

 

+ve

+ve

           Glycosides

·         Molisch’s test

 

-ve

Triterpenoids

·         Libermann-Burchard test

 

-ve

Resins

+ve

Saponins

-ve

Tannins

+ve

Starch

-ve

Evaluation of Antioxidant Activity

The stock solution of 1 mg/ml was prepared by weighing 10 mg of extract and dissolving it in a 10 ml of methanol. 6 ml was taken from this stock solution and diluted upto 60 ml with methanol, various dilutions were prepared from the stock solution and then prepared several dilutions (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/ml respectively) the absorbance was measured at 517 nm.

Ten mg in 10 ml stock solution. 6 ml from this stock solution and make up the volume upto 60 ml with methanol. 1ml of the prepared solution was taken and it was diluted upto 10 ml with methanol similarly various concentration were prepared for sample 10 to 100 µg/ml solution.

By using DPPH method: The antioxidant for methanolic extract was determined by the help of using DPPH radical scavenging method. Antioxidant activity was performed by using DPPH radical scavenging method. DPPH (1,1-diphenyl-2-picryl hydrazyl) was used as source of free radicals which is a light sensitive chemical compound. 0.1mM concentration of DPPH was prepared in methanol. The methanol and DPPH mixture was used as a control (Lee et al, 2005; Doman et al, 2003; Gray et al 2002). Various concentrations of ascorbic acid was prepared and used as standard (100, 200, 300, 400, 500, 600 µg/ml) similarly was prepared in methanol. Different dilutions (100, 200, 300, 400, 500, 600 µg/ml) of test drug was prepared. From the above test solutions, 1ml was discarded and the remaining 1ml was mixed with 1ml of 0.1mM DPPH solution. The mixture was allowed to stand for 30 minutes in dark condition. After 30 minutes the absorbance of the above mixture, control and standard were taken at 517nm through U.V. Spectrophotometer. The % inhibition was calculated by the formula:

% Inhibition= [( Acontrol- Atest/standard) / Acontrol)] × 100

Table 2. DPPH assay method

S. No.

Ascorbic acid (Test)

Plant extract

0

0

0

100

0.4294

0.2188

200

0.4581

0.2678

300

0.4677

0.3160

400

0.4708

0.3549

500

0.5112

0.3973

600

0.5349

0.4296

 

 

 

 

 

 

 

 

 

 

Figure 1. DPPH radical scavenging activity

 

 

By using Ferric chloride reducing method

Preparation of Standard solution

100 mg of ascorbic acid was dissolved in 100 ml of distilled water. Dilution of this solution with distilled water was prepared to give the concentrations of 100, 200, 300, 400, 500 and 600 μg/ml.

Preparation of test sample

20 mg of methanolic extract was dissolved in 20 ml of methanol. Dilutions of this solution with methanol were prepared to give the concentrations of 100, 200, 300, 400, 500 and 600 μg/ml.

Preparation of Reagents

·         Phosphate buffer- 0.2M

Phosphate buffer pH 6.8 was prepared according to I.P (1996).

·         1% Potassium ferricyanide solution

1gm of potassium ferricyanide was dissolved in 100 ml of distilled water.

·         10% Trichloroacetic acid solution

10 gm of TCA dissolved in 100 ml of distilled water.

·         0.1% Ferric chloride solution

0.1  gm of FeCl3 dissolved in 100 ml of distilled water.

Method of Determination

·         Ascorbic acid is used as standard and different concentrations (100, 200, 300, 400, 500 and 600 µg/ml) of standard and test solutions were prepared.

·         To each of the volumetric flask 2.5ml of 0.2M phosphate buffer pH 6.8 and 2.5ml of potassium ferricyanide was added.

·         The mixture was incubated for 20 min at 50˚C in an oven.

·         To incubate solution 2.5 ml of 10% w/v TAA was added and centrifuged for 10 min at 300 rpm. 2.5 ml of solution was taken without disturbing.

·         In above 2.5 ml solution added 2.5 ml of distilled water and 0.5 ml of 1% freshly prepared FeCl3 solution was added.

·         Absorbance was observed at 517 nm.

·         % reducing power was calculated by the Eq.

Control absorbance – Sample absorbance / Control absorbance × 100

Table 3. Ferric chloride reducing activity method

Conc. (µg/ml)

Ascorbic acid

(% inhibition)

Plant extract

(% inhibition)

0

0

 

0

100

0.4498

0.37841

200

0.4682

0.4037

300

0.5067

0.4564

400

0.5408

0.5099

500

0.6011

0.5625

600

0.6408

0.6068

Figure 2. Ferric chloride reducing activity method

 

 

Result and discussion

Study performed on the plant extract sample indicates the presence of active constituents like proteins, flavonoids, resins, tannins, alkaloids and carbohydrates. The antioxidant property of the extract shows that there is an increase with the amount of concentration increases. This shows that the methanolic extract of Bauhinia variegata leaves shows the greater amount of antioxidant property. By this experiment it is clear that in the future a lot of formulations will be prepared even the plant extract is not having any side effect to the skin.From the anti-oxidant activity determination method of extract, it was clear that the extract (sample solution) was showing the anti-oxidant power by comparison with ascorbic acid (test solution), using FRAP reagent as a control sample, as the concentration increases the % inhibition also increases, that was indicating that the extract was having an anti-oxidant property.

References

Sawhnay SS, Amin MM. 2012. Phytochemical screening and Antioxidant property of Bauhinia variegate. International Journal of Research in Phytochemistry and Pharmacology, 2(1): 21-24.

Saha S, Subrahmanyam EVS, Kodangala C, Shastry SC  2011. Isolation and characterization of triterpenoids and fatty acid ester of triterpenoid from leaves of Bauhinia variegate. Der Pharma Chemica, 3(4): 28-37.

Lee CH, Yang L, Xu JZ, Yeung SYV, Huang Y, Chen ZY. 2005. Relative antioxidant activity of soybean isoflavones and their glycosides. Food Chemistry, 90: 4; 735-741.

Lo KM, Cheung PCK. 2005. Antioxidant activity of extracts from the fruiting bodies of Agrocybe aegerita var. alba. Food Chemistry, 89:4; 533-539.

Dorman HJD, Peltoketo A, Hiltunen R, Tikkanen MJ. 2003. Characterization of the antioxidant properties of de-odorized aqueous extracts from selected Lamiaceae herbs. Food Chemistry, 83:2; 255-262.

Gray DA, Clarke MJ, Baux C, Bunting JP, Salter AM. 2002. Antioxidant activity of oat extracts added to human LDL particles and in free radical trapping assays. Journal of Cereal Science, 36:2; 209-218.

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