Int J Curr Pharm Res, Vol 11, Issue 4, 128-133Original Article


PRELIMINARY PHYTOCHEMICAL SCREENING AND TO EVALUATE ANTI-OXIDANT PROPERTY ON ROOT EXTRACT OF DILLENIA INDICA (ELEPHANT APPLE)

BIDISHA BORDOLOI*, SUMIT DAS

Department of Pharmaceutical Sciences, Girijananda Chowdhury Institute of Pharmaceutical Science (GIPS), Azara, Hatkhowapara, NH-37, Guwahati, Assam 781017
Email: [email protected]

Received: 19 Apr 2019, Revised and Accepted: 26 Jun 2019


ABSTRACT

Objective: The objective of the present study was to carry out the presence of antioxidants activity on root extract of Dilleniaindica (Family-Dilleniaceae) which is believed to have the protective mechanisms in beneficial health effects.

Methods: Considering its medicinal importance, the plant was chosen for extraction with various solvents such as petroleum ether, chloroform and ethanol which was taken into considerations to determine the phytochemicals analyses present in it. The extracts of the roots were evaluated for antioxidant activity by using different in vitro models like Reducing Power method, TBA Method and DPPH method at different doses (20,50,100,200,400μg/ml). The ic50 values of each extract on different activity were carried out.

Results: The study shows that petroleum ether, chloroform and ethanolic extract of this plant showed potent antioxidant activity against the standard drug (Ascorbic acid). But chloroform extract of the roots shown most significant anti-oxidant activity as compared to petroleum ether and ethanol.

Conclusion: The root part of the plant shows active anti-oxidant activity that can be consumed by mankind.

Keywords: Phytochemical screening, Pharmacognostic evaluation, Anti-oxidant activity


INTRODUCTION

In the last few decades, traditional knowledge on primary healthcare has been widely acknowledged across the world. It is estimated that 60% of the world population and 80% of the population of developing countries rely on traditional medicine, mostly plant drugs, for the primary health care needs. The herbal products today symbolize safety in contrast to the synthetics that are regarded as unsafe to human and environment [1, 2].

Use of plants as a source of medicine has been inherited and is an important component of the health care system in Egypt. Ayurveda, Siddha, Unani and Folk (tribal) medicines are the major systems of indigenous medicines. Among these systems, Ayurveda is the most developed and widely practiced in India. Today this system of medicine is being practiced in countries like Nepal, Bhutan, Sri Lanka, Bangladesh and Pakistan. This ancient system of medicine, believed to be more than 5000 y old, is based on two separate theories about the natural laws that govern good health and longevity, namely yin and yang, and the five elements (wu xing). Chinese medicine was systematized and written between 100 and 200 BC.

The Dillenia indica is comprised of about 100 species of evergreen and deciduous trees or shrubs of disjunct distribution in the seasonal tropics of Madagascar through South and South East Asia, Malaysia, North Australia. Mucilage from their fruits is used in drug formulations. Species from this have been widely used in medicinal folklore to treat cancers, wounds, jaundice, fever, cough, diabetes mellitus, and diarrhea as well as hair tonics. It also produces edible fruits and are cultivated as ornamental plants. Their extracts and pure compounds have been reported for their antimicrobial, anti-inflammatory, cytotoxic, ant diabetes, antioxidant, antidiarrheal, and antiprotozoal activities [3, 4].

MATERIALS AND METHODS

Collection of plant material

The roots of Dillenia indica were collected in the month of Feb, 2019 from Sivasagar, Assam.

Authentication of plants material

The plant is authenticated by Department of Botany Guwahati University.

Drying and grinding of plant material

After authentication of the plant, the roots were shade dried and grinded. The material which is retained on the sieve was used for the extraction purpose.

Extraction of active constituents

The process of removing or extracting or separating of active constituents from the crude drugs by using suitable solvents is called extraction. The active constituents that have been extracted from crude drugs are known as extractives and the preparation so obtained are as extracts. About 400g of the powder of the roots of Dilleniaindica was weighed and extracted successively with the soxhlet apparatus. At first, it was extracted with petroleum ether and then according to their polarity lower to higher with ethanol and chloroform, respectively. The extract collected was filtered and evaporated using distillation.

Preliminary phytochemical screening

The chemical evaluation of different extracts was done by using a various standard procedure of different metabolites like alkaloids, tannins, saponins, glycosides, carbohydrates, flavonoids, proteins, ascorbic acid (vit c), phenolics. The results are shown in table 1.[6]

Pharmacognostic evaluation

Determination of loss on drying

Procedure

The results are shown in table 2.

Determination of moisture content

Procedure

The results are shown in table 3.

Determination of foaming index

Procedure

The results are shown in table 4.

Determination of swelling index

Procedure

The results are shown in table 5.

Determination of ash values of a crude drug

Determination of fluorescence powder drug analysis

In the present study, dry root powder was used. The fluorescent analysis of the root powder of the plant Dillenia indica was carried out. The root of the plant after drying were then blended using a electric blender. This fine powder was analyzed for the fluorescent.

The results are shown in table 7 [5-6].

Chemical studies

Determination of % yield: It is done by following the formula

% of yield = Practical yield ×100

Theoretical yield

The results are shown in table 8.

In vitro anti-oxidant studies

Reducing power assay method

Procedure

Taking 1 ml of methanolic extract (100-400 μg/ml), standard dilutions (20-400 μg/ml) and control sample (1 ml distilled water instead of sample solution) was mixed with2.5 ml phosphate buffer solution (pH 6.6) and 2.5 ml potassium ferricyanide (1%). Then the final mixture was properly mixed and incubated at 50 °C for 20 min. After incubation, the reaction mixture was rapidly cooled and mixed with 2.5 ml of 10% trichloroacetic acid. It was then centrifuged at 3000 rpm for 10 min. About 2.5 ml of the supernatant was taken, and 2.5 ml distilled water and 0.5 ml of ferric chloride (0.1%) were added, mixed well and allowed to stand for 10 min. The absorbance was measured at 700 nm [7, 8].

The results are shown in table 9.

Table 1: Dertermination of phytochemical screening

Chemical test Pet. ether Chloroform Ethanol
Alkaloid -ve +ve -ve
Tannins -ve +ve +ve
Saponins +ve +ve +ve
Glycoside -ve -ve +ve
Carbohydrates -ve -ve -ve
Flavonoids -ve +ve -ve
Proteins and amino acid -ve -ve +ve
Vitamin C(Ascorbic acid) +ve +ve +ve
Phenolics +ve +ve +ve

*(+ve) and (-ve) symbol indicates the presence and absence of respective plant constituents.

Thiobarbituric acid method

Procedure

The test was conducted according to the method of Kikuzaki and Nakatani.

To 2.0 ml of the Sample solution, 1.0 ml of 20% aqueous trichloroacetic acid (TCA) and 2.0 ml of Aqueous thiobarbituric acid (TBA) solution were added. Then the final sample concentration was 0.02% w/v. The mixture was placed in a boiling water bath for 10 min. After cooling, it was then centrifuged at 3000 rpm for 20 min. The absorbance of the supernatant was measured at 532 nm. Antioxidant activity was recorded based on the absorbance of the final day of the FTC assay. Both methods (FTC and TBA) described antioxidant activity by percent inhibition: [9-10]

The results are shown in table 10.

1,1-Diphenyl-2-picryl hydrazil assay (DPPH assay)

Procedure

The radical scavenging activity was determined by the use of DPPH free radical assay. Take 50 μL of various concentrations of plant extracts in methanol were added to 5 ml of 100 μL Solution of DPPH in methanol. After 30 min incubation absorbance was read against blank taken as methanol at 517 nm and the % inhibition was calculated from the following equation below [11, 12].

The results are shown in table 11.

RESULTS

Phytochemical screening

Phytochemical screening was carried out for petroleum ether, chloroform and ethanolic extract of Dillenia indica for the presence of different phytoconstituents like flavonoid, phenolic, carbohydrate, glycoside and proteins.

Pharmacognostic evaluation

Table 2: Determination of loss on drying

Wt. of porcelain Initial wt. of the drug Wt. of empty porcelain+drug before drying Wt. of empty porcelain+drug after drying
118.32 gm 0.50g/500 gm 118.82 gm 118.80 gm

Table 3: Determination of moisture content:

Wt. of the drug

Initial wt. of the drug+pet dish

(gm)

Constant wt. after drying

(gm)

Loss on drying

(gm)

Moisture content
0.50 gm 11.82 gm 118.80 gm 0.02 gm 4%

Table 4: Determination of foaming index

Concentration 1 μg/ml 2 μg/ml 3 μg/ml 4 μg/ml 5 μg/ml 6 μg/ml 7 μg/ml 8 μg/ml 9 μg/ml

10

μg/ml

Test Results -ve -ve -ve -ve -ve -ve -ve +ve +ve +ve

Table 5: Determination of swelling index

Wt of coarse powder Initial wt. of the powder Wt. of empty porcelain+drug before drying Wt. of empty porcelain+drug after drying
1-2 gm 0.50g/500 gm 118.82 gm 118.80 gm

Table 6: Determination of ash values of a crude drug

Wt. of drug (gm) Wt. of crucible+drug (gm) Wt. of total ash (gm) % of total ash Wt. of acid insoluble ash value (gm) % of acid insoluble ash Wt. of water soluble ash value (gm) % of water soluble ash
Crucible 1 2 22.19 1.6 1.8 22.1 0.18 - -
Crucible 2 2 27.85 1.9 8.5 - - 23.8 0.07

Table 7: Determination of fluorescence powder drug analysis

Chemical test Daylight Short UV (254 nm) Long UV (365 nm)
Powder+1N NAOH in methanol MOON YELLOW HAUSER LIGHT LEMONETE
Powder+1N NAOH in H20 RED HAUSER MEDIUM LEMONATE
Powder+1N HCLin methanol GEROGIA CLAY OLIVE GREEN AVOCADO
Powder+1N HCL in H20 YELLOW ORCHE SOFT SAJE DARK CHOCOLATE
Powder+1N HNO3 in methanol GEROGIA CLAY HAUSER LIGHT AVOCADO
Powder+1N HCLin H2O SOFT SAFE LEMONATE DARK CHOCOLATE
Powder+5% Iodine COCOA OLIVE GREEN AVOCADO
Powder+50% KOH COUNTRY RED HAUSER MEDIUM HAUSER LIGHT
Powder+5% FeCl3 DARK CHOCOLATE GREEN ARBOR GREEN

DETERMINATION

Table 8: The % of the yield of different extracts of Dillenia indica roots

S. No. Extracts % Yield

1.

2.

3.

Petroleum ether

Chloroform

Ethanol

7.1

2.4

4.7

In vitro anti-oxidant studies

Reducing the power method

Table 9: In vitro antioxidant activity of ascorbic acid (Stand.), pet ether, chloroform and ethanol extract of Dillenia indicia by RP method

S. No. Extracts 20μg/ml 50 μg/ml 100 μg/ml 200 μg/ml 400 μg/ml
01 Ascorbic acid (Stand.) 34.32±0.001 48.66±0.002 56.23±0.001 66.62±0.05 74.88±0.01
02 Pet. Ether Extract 33.43±0.002 42.65±0.003 50.27±0.012 59.55±0.001 64.86±0.016
03 Chloroform Extract 19.23±0.020 23.36±0.018 26.22±0.022 34.86±0.012 48.26±0.010
04 Ethanol Extract 25.56±0.002 16.19±0.012 18.38±0.019 21.37±0.020 25.39±0.023

Fig. 1: Reducing power method of different extract of Dillenia indica

Thiobarbituric acid method

Table 10: In vitro antioxidant activity of ascorbic acid (Stand.), pet ether, chloroform and ethanol extract of Dillenia indicaby TBA method

S. No. Extracts 20μg/ml 50 μg/ml 100 μg/ml 200 μg/ml 400 μg/ml
01 Ascorbic acid (Stand.) 34.32±0.001 48.66±0.002 56.23±0.001 66.62±0.05 74.88±0.01
02 Pet. Ether Extract 15.55±0.021 26.09±0.017 33.023±0.023 37.151±0.013 44.024±0.040
03 Chloroform Extract 14.228±0.012 17.506±0.013 27.028±0.014 36.228±0.007 44.007±0.024
04 Ethanol Extract 18.470±0.016 24.330±0.016 34.123±0.024 50.220±0.024 64.125±0.026

Fig. 2: TBA method of different extract of Dilleniaindica

DPPH assay method

Table 11: In vitro antioxidant activity of ascorbic acid (Stand.), Pet ether, chloroform and ethanol extract of Dillenia indicia by DPPH ASSAY method

S. No. Extracts 20μg/ml 50 μg/ml 100 μg/ml 200 μg/ml 400 μg/ml
01 Ascorbic acid (Stand.) 34.32±0.001 48.66±0.002 56.23±0.001 66.62±0.05 74.88±0.01
02 Pet. Ether Extract 11.23±0.02 16.06±0.008 18.04±0.020 22.36±0.025 29.32±0.001
03 Chloroform Extract 33.36±0.005 46.09±0.006 50.61±0.016 61.56±0.002 78.38±0.002
04 Ethanol Extract 25.36±0.001 30.44±0.003 36.33±0.003 46.32±0.025 52.01±0.030

Fig. 3: DPPH scavenging activity of different extract of Dilleniaindica

Table 12: IC50 value from reducing power method, tba method and DPPD scavenging activity of Dilleniaindica

S. No. Activity Extract IC50
01 Reducing Power Ascorbic acid(stnd) 2.38
Pet. Ether 2.98
Chloroform 5.81
Ethanol 62.1
02 TBA Ascorbic acid(stnd) 2.38
Pet. Ether 5.76
Chloroform 5.83
Ethanol 4.10
03 DPPH Ascorbic acid(stnd) 2.38
Pet. Ether 10.23
Chloroform 2.62
Ethanol 4.72

DISCUSSION

Phytochemical screening

The phytochemical screening of powdered roots of Dillenia indicaindicates the presence of active constituents. Solvent selection was made for root and extraction was performed. From this study, it may be concluded that petroleum ether, chloroform and ethanolic extracts of this root of plant (Dillenia indica) have various phytoconstituents which was shown in (table 1).

Pharmacognostic evaluation

The pharmacognostic evaluation of the roots of this plant Dillenia indica gives us a brief idea about the various potential pharmacognostic activities in the plant.

Anti-oxidant activity

The antioxidant activity of this roots of Dillenia indica by using different models gives us a positive response that the roots of the plant shows active anti-oxidant properties which was compared with reference standard Ascorbic acid.

CONCLUSION

The current study shows that petroleum ether, chloroform and ethanolic extract of Dillenia indica have significant anti-oxidant property. Dillenia indica is widely available and also cultivated in different region in the world. The scientific research suggests a huge biological potential of this plant. A detailed study on the pharmacognostical, phytochemical and antioxidant properties of the root have been discussed and also provided details evidence for use of this root in different diseases. These results also justify the use of roots in traditional medicines.

ACKNOWLEDGMENT

I consider it a great privilege andhonor to have had the opportunity to undergo the Project work in Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati, Assam. Hence, I would like to offer my heartiest thanks to Dr. Sumit Das; Assistant Professor, Department of Pharmaceutical Chemistry, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati, Assam, for his valuable guide and suggestion and the Principal for conveying the work.

AUTHORS CONTRIBUTIONS

All the author have contributed equally

CONFLICT OF INTERESTS

Declare none

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