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 International Journal of Medical Sciences and Pharma Research 

Open Access to Medical Science and Pharma Research

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Phytochemical screening and Antioxidant Potential of Alstonia scholaris Linn leaf Extracts

Atul Bopche*,  Jagdish Rathi, Sikil Ghoshi, Sonu Rajpoot,   Shivraj Noriya, Sikesh Kumar Shah, Shubham Raj

NRI Institute of Pharmaceutical Sciences, Sajjan Singh Nagar, Opposite Patel Nagar, Raisen Road Bhopal, MP, 462022, India

INTRODUCTION

Plants have been the basis of traditional medicines from time immemorial throughout the world and continue to provide new targets for remedies for many afflictions of mankind. The past couple of decades have seen considerable change in opinion regarding ethno-pharmacological therapeutic applications of phytochemicals. A great deal of effort therefore still focuses on identifying and using these phytochemicals as source of novel therapeutic molecules. Antioxidants are radical scavengers which protect the human body against free radicals that may cause pathological conditions such as ischaemia, asthma, arthritis, inflammation, neuro-degeneration, Parkinson’s disease, mongolism, ageing process and perhaps dementia¹. Antioxidant based drugs or formulations for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer have appeared during the last three decades. This has attracted a great deal of research interest in natural antioxidants. The plant kingdom has been described as a reservoir of many novel biologically active molecules of medicinal value². Recently there has been a surge of interest in the therapeutic potential of medicinal plants as antioxidants in reducing free radical-induced tissue injury. A. scholaris is popularly known as Saptaparni or Devil’s tree. It is widely distributed in dried forests of India, Western Himalayas and Western Ghats as well as in the southern India³. It is a medium to large tree, about 40 m high with a corky grey to grey-white bark. The outer blaze is cream to yellow with abundant, milky latex that flows rapidly when cut. Leaves are in whorls of 4-8 in the upper exiles, upper surface is dark green, the lower green-white. The tip of the leaf is rounded or shortly pointed and tapered towards the base⁴. Greenish white flowers are umbrellately branched. They are 7-10 mm long.  A. scholaris is a well-known remedy for the treatment of various types of disorders in the folk and Ayurvedic system of Indian medicine. It has been reported to possess antimalarial, antimicrobial, free radical scavenging and antioxidant, anti-diabetic, analgesic and anti-inflammatory, anticancer and cytotoxicity, radioprotective, CNS activity, immunostimulating, antifertility, antidiarrheal, bronchodilatory, anti-tussive and anti-asthmatic  activities⁵. The objective of the present study was to screen the phytochemicals and assess the antioxidant activity of the solvent extracts of leaf of A. scholaris. Free radical scavenging ability of the extracts was tested using antioxidant assays, viz., DPPH assay. 

MATERIAL AND METHOD

Plant material

Leaves of A. scholaris were collected from botanical garden of Vindhya Herbals Bhopal, Madhya Pradesh. Plant material (leaves part) selected for the study were washed thoroughly under running tap water and then were rinsed in distilled water; they were allowed to dry for some time at room temperature. Then the plant material was shade dried without any contamination for about 3 to 4 weeks. Dried plant material was grinded using electronic grinder. Powdered plant material was observed for their colour, odour, taste and texture. Dried plant material was packed in air tight container and stored for phytochemical and biological studies.

Chemical reagents

All the chemicals used in this study were obtained from Hi Media Laboratories Pvt. Ltd. (Mumbai, India), Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA), SD Fine-Chem. Ltd. (Mumbai, India) and SRL Pvt. Ltd. (Mumbai, India).All the chemicals and solvent used in this study were of analytical grade.

Defatting of plant material

50 gram shade dried powder of leaves of A. scholaris was extraction with petroleum ether using maceration method. The extraction was continued till the defatting of the material had taken place. 

Extraction by soxhletion method 

Marc of leaves of A. scholaris was exhaustively extracted with methanol solvent by soxhletion method. The extract was evaporated above their boiling points. The resultant content was filtered with whatman filter paper no.1 and kept for evaporation of solvent to get the dry concentrated extract. The dried crude concentrated extract was weighed to calculate the extractive yield then transferred to glass vials (6 ×2 cm) and stored in a refrigerator (4°C), till used for analysis⁶. 

Phytochemical screening 

Phytochemical screening to detect the presence of bioactive agents was performed by standard procedures^{7, 8}. After the addition of specific reagents to the solution, the tests were detected by visual observation of color change or by precipitate formation.

Total phenol determination

The total phenolic content was determined using the method of Dutta et al⁹., 2011  A volume of 2ml of each extracts or standard was mixed with 1 ml of Folin Ciocalteau reagent (previously diluted with distilled water 1:10 v/v) and 1 ml (7.5g/l) of sodium carbonate. The mixture was vortexed for 15s and allowed to stand for 10min for colour development. The absorbance was measured at 765 nm using a UV/visible spectrophotometer. The total phenolic content was calculated from the standard graph of gallic acid and the results were expressed as gallic acid equivalent (mg/100mg).

Total flavonoids determination

The total flavonoid content was determined using the method of Dutta et al⁹., 2011  1ml of 2% AlCl₃ solution was added to 3 ml of extract or standard and allowed to stand for 15 min at room temperature; the absorbance of the reaction mixture was measured at 420 nm using UV/visible spectrophotometer. The content of flavonoids was calculated using standard graph of quercetin and the results were expressed as quercetin equivalent (mg/100mg).

Antioxidant activity 

DPPH radical scavenging assay 

DPPH scavenging activity was measured by modified method of Dutta et al⁹., 2011. DPPH scavenging activity was measured by the spectrophotometer. Stock solution (6 mg in 100ml methanol) was prepared such that 1.5 ml of it in 1.5 ml of methanol gave an initial absorbance. Decrease in the absorbance in presence of sample extract at different concentration (10-100 µg/ml) was noted after 15 minutes. 1.5 ml of DPPH solution was taken and volume made till 3 ml with methanol, absorbance was taken immediately at 517 nm for control reading. 1.5 ml of DPPH and 1.5 ml of the test sample of different concentration were put in a series of volumetric flasks and final volume was adjusted to 3 ml with methanol. Three test samples were taken and each processed similarly. Finally the mean was taken. Absorbance at zero time was taken for each concentration. Final decrease in absorbance was noted of DPPH with the sample at different concentration after 15 minutes at 517 nm. The percentage inhibition of free radical DPPH was calculated from the following equation: % inhibition = [(absorbance of control - absorbance of sample)/absorbance of control] × 100%. Though the activity is expressed as 50% inhibitory concentration (IC50), IC50 was calculated based on the percentage of DPPH radicals scavenged. The lower the IC50 value, the higher is the antioxidant activity.

RESULTS AND DISCUSSION

The leaves of A. scholaris were collected from the botanical garden of Vindhya Herbals Bhopal, MP, India. Air-dried and extracted by soxhletion extraction process. The crude extracts so obtained after each of the soxhletion extraction process were concentrated on water bath by evaporation the solvents completely to obtain the actual yield of extraction. The yield of extracts obtained from the leaves of the plants was founds to be 6.34% w/w. The results of qualitative phytochemical analysis of the crude powder leaves of A. scholaris were shown in Table 1. Methanolic extracts of leaves sample of A. scholaris showed the presence of phenols, diterpines, flavonoids, proteins, carbohydrates and saponins. Total phenolic compounds (TPC) was expressed as mg/100mg of gallic acid equivalent of dry extract sample using the equation obtained from the calibration curve: y = 0.015x - 0.001, R²= 0.999, where X is the gallic acid equivalent (GAE) and Y is the absorbance. Total flavonoids content was calculated as quercetin equivalent (mg/100mg) using the equation based on the calibration curve: y = 0.035x + 0.009, R²=0.999, where X is the quercetin equivalent (QE) and Y is the absorbance. The total phenolic and flavonoids estimation of methanolic extracts of leaves of A. scholaris showed the content values of 0.876 and 0.757 respectively Table 2. DPPH radical scavenging assay measured hydrogen donating nature of extracts^{10, 11}. Under DPPH radical scavenging activity the inhibitory concentration 50% (IC₅₀) value of A. scholaris methanolic leaves extract was found to be 77.67μg/ml as compared to that of ascorbic acid (26.72μg/ml). A dose dependent activity with respect to concentration was observed Table 3.

Table 1: Phytochemical evaluation of A. scholaris leaves extracts

+Ve = Positive, -Ve= Negative

Table 2: Results of total phenol and flavonoids content

Table 3: % Inhibition of ascorbic acid and hydroalcoholic extract using DPPH method

CONCLUSION 

The results obtained from the leaves of A. scholaris Linn extract revealed that has highest phytochemical constituents and potential antioxidants activity. The present study suggested that the purified methanolic extract of A. scholaris could be a potential source of antioxidants and phytochemical and thus useful as therapeutic agent against oxidative stress related degenerative disease and disorders in future this study extended to isolation, characterization of bioactive compound.

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