HYPERICUM WIGHTIANUM

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Balaperiasamy et al. World Journal of Pharmaceutical Research

PRELIMINARY PHYTOCHEMICAL ANALYSIS OF

HYPERICUM WIGHTIANUM WALL. EX WIGHT &ARN

*Balaperiasamy Bˡ, Panneerselvam Aˡ, Thajuddin N², Jayapal Jˡ

1PG& Research Department of Botany & Microbiology, AVVM Sri Pushpam College,

Poondi - 613 503, Thanjavur Dist. Tamilnadu, India. 2Department of Microbiology, Bharathidasan University, Tiruchirappalli - 620 024,

Tamilnadu, India.

ABSTRACT

Kodaikanal hills of south India has been known for its rich biological

diversity. Numerous medicinally important plants are available here.

These medicinal plants have been used by its people for a long time.

The aim of the present study was to investigate the presence of

phytochemicals and to determine total phenolic, alkaloid and flavonoid

contents of the plant. Solvent used were water and methanol. Total

phenolic content of the aqueous extract of the plant was determined by

Folin-ciocalteus reagent method and total flavonoid content was

determined by Aluminium chloride method. Protein, carbohydrate,

phenol, tannin, Flavonoid and saponins were detected in the extracts of

the plant. From the present study it is concluded that aqueous (AEHW)

and methanol (MEHW) extracts of Hypericum wightianum contain

medicinally important bioactive compounds and it can be used in the

treatment of different diseases.

Key words: Hypericum wightianum, Phytochemicals, Phenol, alkaloid, flavonoid.

INTRODUCTION

The use of herbs and medicinal plants as the first medicines is a universal phenomenon.

Every culture on earth, through written or oral tradition, has relied on the vast variety of

natural chemistry found in healing plants for their therapeutic properties [1].Knowledge of

the chemical constituents is desirable for the discovery of therapeutic agents as well as

discovering new source of economic materials, such as herbals, tannins, oil, gums and

World Journal of Pharmaceutical research

Volume 3, Issue 1, 726-738. Research Article ISSN 2277 – 7105

Article Received on 16 October2013 Revised on 19 November 2013, Accepted on 12 December 2013

*Correspondence for

Author:

Dr. Balaperiasamy B

PG& Research Department of

Botany & Microbiology,

AVVM Sri Pushpam College,

Poondi, Thanjavur Dist.

Tamilnadu, India.

[email protected]

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precursors for the synthesis of complex substances. Medicinal plants will continuously

provide a source for generating novel drug compounds. Plants may become the base for the

development of a new medicine or they may be used as phyto-medicine for the treatment of

diseases [2]. Medicinal plants contain some organic compounds which provide definite

physiological action on the human body and these bioactive substances include tannins,

alkaloids, carbohydrates, terpenoids, steroids and flavonoids [3].

These compounds are synthesized by primary or rather secondary metabolism of living

organisms. Secondary metabolites are chemically and taxonomically extremely diverse

compounds with obscure function. They are widely used in the human therapy, veterinary,

agriculture, scientific research and countless other areas [4]. Plant products have been part of

phytomedicines since time immemorial. This can be derived from barks, leaves, flowers,

roots, fruits, seeds etc. [5]. Knowledge of the chemical constituents of plants is desirable

because such information will be value for synthesis of complex chemical substances [6].

According to many report, phytochemical constituents are found to be bioactive. Several

studies confirmed presence of these phytochemicals contribute medicinal as well as

physiological properties to the plants studied in the treatment of different ailments.

MATERIAL AND METHODS

Chemicals and reagents

All the chemicals used in the study were analytical grade and procured from Merck India Pvt.

Ltd.

Plant material and extraction

The aerial parts of Hypericum wightianum were collected from Kodaikanal hills in the month

of June 2013. The plant was authenticated by Dr. M. Palanisamy, Scientis ‘C’ In-charge,

Botanical Survey of India, Coimbatore, Tamil nadu, India. A voucher specimen was

preserved in our laboratory for future reference (voucher No. BSI/SRC/5/23/2013-

14/Tech.850, Dt.21 August 2013). The plant material was shade dried, pulvarized (500 g) and

extracted with methanol and distilled water at room temperature for 72 hrs. The extract was

filtered and concentrated to dryness under reduced pressure and controlled temperature (40 –

50oC) in a rotary evaporator. Both the extracts were dark greenish brown solid weighing

MEHW 89.30 gr. (yield, 17.86), AEHW 51.25 gr. (yield, 10.23%) and were preserved in a

vacuum desiccator at 4oC until further use.

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Screening procedure

The extract was tested for the presence of bioactive compounds by using standard Methods

QUALITATIVE PHYTOCHEMICAL ANALYSIS

Methanol and aqueous extracts of H. wightianum were subjected to chemical tests for the

identification of their active constituents.

Test for Carbohydrates and Glycosides

A small quantity of extracts was dissolved separately in 4 ml of distilled water and filtered.

The filtrate was subjected to the following tests to detect the presence of carbohydrates and

glycosides.

Molisch’s test

The filtrate was treated with 2-3 drops of 1 % alcoholic -naphthol and 2 ml of concentrated

sulphuric acid was added along the sides of the test tube. Appearance of brown ring at the

junction of two liquids shows the presence of carbohydrates.

Fehling’s test

The filtrate was treated with 1 ml of Fehling’s solution A and B and heated on a water bath.

A reddish precipitate was obtained which shows the presence of carbohydrates.

Another portion of extract was hydrolyzed with dilute hydrochloric acid for few hours on a

water bath and the hydrolysate was subjected to the following tests to detect the presence of

glycosides.

Legal’s test

To the hydrolysate 1 ml of pyridine and few drops of sodium nitroprusside solution were

added and then it was made alkaline with sodium hydroxide solution. Appearance of pink to

red colour shows the presence of glycosides.

Borntrager’s test

Hydrolysate was treated with chloroform and the chloroform layer was separated. To this

equal volume of dilute ammonia solution was added. Ammonia layer acquires pink colour

that shows the presence of glycosides.

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Detection of Fixed Oils and Fats

Filter paper test

Small quantities of the extracts were pressed separately between the filter paper. Appearance

of oil stain on the paper indicates the presence of fixed oils.

Saponification test

Few drops of 0.5 N alcoholic potassium hydroxide was added to small quantity of extract

along with a drop of phenolphthalein. The mixture was heated on a water bath for 1-2 hours.

Formation of soap indicates the presence of fixed oils and fats.

Detection of Proteins and Free Aminoacids

Small quantities of extracts were dissolved separately in few ml of water and then it was

subjected to the following tests.

Millon’s test

The above-prepared extracts were treated with Millon’s reagent. Red colour formed shows

the presence of proteins and free amino acids.

Biuret test

To the above prepared extracts, equal volume of 5 % sodium hydroxide and 1 % copper

sulphate solution were added. Appearance of violet colour shows the presence of proteins and

free amino acids.

Ninhydrin test

The extracts were treated with Ninhydrin reagent. Purple colour produced shows the presence

of proteins and free amino acids.

Detection of Saponins

The extracts were diluted separately with 20 ml of distilled water and it was agitated in a

measuring cylinder for 15 minutes. The formation of 1cm layer of foam shows the presence

of saponins.

Detection of Tannins and Phenolic Compounds

Small quantities of the extracts were taken separately in water and test for the presence of

phenolic compounds and tannins was carried out with the following reagents.

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1) 5% Ferric chloride solution - violet colour

2) 1% solution of gelatin containing

10% sodium chloride - white precipitate

3) 10% lead acetate solution - white precipitate

Above findings shows the presence of phenolic compounds and tannins.

Detection of Phytosterols

Small quantities of extracts were dissolved separately in 5 ml of chloroform. Then this

chloroform solution was subjected to the following tests to detect the presence of

phytosterols.

Salkowski test

To 1 ml of above prepared chloroform solution, few drops of concentrated sulphuric acid was

added. Brown colour produced shows the presence of phytosterols.

Libermann Burchard test

The above prepared chloroform solution was treated with a few drops of concentrated

sulphuric acid followed by few drops of diluted acetic acid and 3ml of acetic anhydride. A

bluish green colour appeared indicates the presence of phytosterols.

Detection of Alkaloids

Small quantities of extracts were separately treated with few drops of dilute hydrochloric acid

and filtered. The filtrate was used for the following tests.

1) Mayer’s reagent - cream precipitate

2) Dragendroff’s reagent - orange brown precipitate

3) Hager’s reagent - yellow precipitate

4) Wagner’s reagent - reddish brown precipitate Detection of Gums and Mucilages

Small quantities of extracts were added separately to 25 ml of absolute alcohol with constant

stirring and filtered. The precipitate was dried in air and examined for its swelling properties.

No swelling was observed indicates the absence of gums and mucilages.

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Detection of Flavonoids

With aqueous sodium hydroxide solution, Small quantities of extracts were dissolved in

aqueous sodium hydroxide. Appearance of yellow colour indicates the presence of

flavonoids, with concentrated sulphuric acid

To small portion of extract, concentrated sulphuric acid was added. Yellow orange colour

was obtained shows the presence of flavonoids.

Shinoda’s test

Small quantity of the extract was dissolved in alcohol. To that piece of magnesium followed

by concentrated hydrochloric acid were added drop wise and heated. Appearance of magenta

colour shows the presence of flavonoids.

QUANTITATIVE PHYTOCHEMICAL DETERMINATION

Estimation of Glycosides

2.5 g of the plant extracts were weighed separately and 15 ml of hot distilled water was

added. The flask was kept on a water bath till extract was dissolved. To this 25 ml of 80

%v/v alcohol was added, shaken well and 50 ml of 95 %v/v alcohol was added. The resultant

solution was allowed to settle down and filtered on a filter paper and washed with 80 %v/v

alcohol until washings were colourless. Filtrate and washings were transferred to dish and

the alcohol was evaporated to get a syrupy mass. Then it was transferred into a 50 ml

cylinder with stopper and volume was made up with distilled water up to the level of 30 ml. 3

ml of 10 %v/v sulphuric acid was added slowly, with constant shaking and allowed to stand

overnight at room temperature. Supernatant liquid was decanted through a filter paper,

washed the pot 2 or 3 times with cold water passing the washings each time through filter

paper.

The residue in the cylinder and on the filter paper were dissolved with a little dilute alcohol

(45 %), 2 or 3 drops of ammonia (10 %) was added to neutralize the acid. The contents were

evaporated to dryness in a tarred beaker and dried to a constant weight at 105oC. Increase in

weight of beaker represents total glycosides19.

Estimation of Saponins

5 gm of the plant extracts were dissolved separately in 50 ml of 90 % v/v alcohol by refluxing

on a water bath for half an hour. Filtered and the residue was washed thoroughly to take

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maximum quantity of soluble matter (refluxation is repeated until dissolution is not

completed). The alcoholic extract was concentrated to a thick paste. 50 ml of petroleum

ether (40 – 60 oC) was added and refluxed for half an hour. The petroleum ether soluble

portion was discarded by filtering through an extraction thimble. The thimble was transferred

to a soxhlet extractor and refluxed with chloroform for half an hour and soluble portion was

discarded. The same treatment was done with carbon tetra chloride and ethyl acetate and

respective soluble portions were discarded. The residue was dissolved in 10 ml methanol and

poured drop-wise into 50 ml of acetone with constant stirring. The precipitate was collected

and dried at 105 oC to obtain constant weight19.

Estimation of Total Phenol Content

Total phenol is determined in powder crude drugs, extracts and beverages by using the Folin

– Ciocalteu method. This test is based on the oxidation of phenolic groups with

phosphomolybdic and phosphotungstic acids. After oxidation a green – blue complex is

measurable at 750 nm. The total phenol content of a tested material is being related to the

antioxidant activity shown by it.

Chemicals and Reagents used

Folin – Ciocalteu Reagent: Folin – Ciocalteu reagent was diluted with water (1:10) and

used.Sodium carbonate: 202.5g of sodium carbonate was dissolved in 1 litre of distilled water

and used.Methanol: Distilled.

Preparation of test Solution

50 mg of the extracts were dissolved separately in 50 ml of methanol. The solutions were

serially diluted with methanol to obtain lower dilutions.

Preparation of Standard Solution

Gallic acid: 50 mg of gallic acid monohydrate was dissolved in 50 ml of distilled water. It

was serially diluted with water to obtain lower dilutions.

Procedure

About 0.1 ml of each extract (0.1 mg/ml) was mixed with 0.5 ml of Folin-Ciocalteu reagent

and 1.5 ml of sodium carbonate. The mixture was shaken thoroughly and made up to 10 ml

with distilled water. The mixture was allowed to stand for 2 h. The absorbance was measured

at 750 nm using PerkinElmer Lambda 25 UV-VIS Spectrophotometer. Using gallic acid

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monohydrate, standard curve was prepared and linearity was obtained in the range of 2-10

µg/ml. Using the standard curve the total phenol content was obtained. The total phenol

content was expressed as gallic acid equivalent in mg/g or % w/w of the extract26.

Estimation of Total Flavonoids

Flavonoids present in the extracts, food preparations and beverages can be estimated by its

characteristic absorption in the UV region by the specific reaction with aluminium chloride

and potassium acetate.

Chemicals and Reagents

Aluminium chloride 10%: 10 g of aluminium chloride was dissolved in 100 ml of distilled

water.Potassium acetate 1M: 98.1 g of potassium acetate was dissolved in 1000 ml of

distilled water.Methanol:Distilled

Preparation of Test Solution

50 mg of the extracts were dissolved separately in 50 ml of methanol. These solutions were

serially diluted with methanol to obtain lower dilutions.

Preparation of Standard Solution

10 mg of rutin (Sigma Aldrich, USA) was weighed and dissolved in 100 ml of methanol to

get 100 µg/ml solution. It was serially diluted with methanol to obtain lower dilutions.

Procedure

The total flavonoids content of extracts were estimated using aluminium chloride method.

500 µl of each extract was mixed with 1.5 ml of methanol, 100 µl of 10% aluminium

chloride, 100 µl of 1M potassium acetate and 2.8 ml of distilled water. After incubation at

room temperature for 30 min, the absorbance was measured at 415 nm. Using rutin, standard

curve was prepared and from the standard curve the total flavonoids content of extracts were

obtained. The total flavonoids content was expressed as rutin equivalent in mg/g or %w/w of

the extract27.

Estimation of Ascorbic Acid

Principle

Ascorbic acid reduces the 2, 6-dichlorophenol indophenol dye to a colorless leuco-base. The

ascorbic acid gets oxidized to dehydroascorbic acid. Though the dye is a blue colored

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compound, the end point is the appearance of pink color. The dye is pink colored in acid

medium. Oxalic acid is used as the titrating medium.

Solutions and reagents

Oxalic acid 4 %: 4 g of oxalic acid was dissolved in 100 ml of distilled water.

2, 6-dicholorphenol indophenol dye solution: 42 mg of sodium bicarbonate was weighed and

dissolved into a small volume of distilled water. 52 mg of 2, 6-dicholorophenol indophenol

was dissolved in it and made up to 200 ml with distilled water.

Preparation of standard solution: 100 mg ascorbic acid was dissolved in 100 ml of 4% oxalic

acid in a standard flask (Stock solution).

Preparation of working standard: 10 ml of the stock solution was diluted to 100 ml with 4 %

oxalic acid. The concentration of working standard was 100 µg/ml.

Procedure

5 ml of the working standard solution was pipette out into a 100 ml conical flask. To this 10

ml of 4 % oxalic acid was added and titrated against 2, 6-diclorophenol indophenol (V1 ml).

End point was the appearance of pink color which persists for a few minutes. The amount of

dye consumed is equivalent to the amount of ascorbic acid.0.5 g of each extract was

dissolved separately in 4 % oxalic acid, filtered and made up to 100 ml and centrifuged. 5 ml

of the supernatant was pipetted out and treated with 10 ml of 4 % oxalic acid and titrated

against the dye (V2 ml)28 .

Amount of ascorbic acid present was calculated by the following formula

= (0.5 mg/V1 ml) X (V2/5 ml) X (100 ml/wt. of the sample)

RESULTS

The phytochemical screening of the organic extracts of the plant Hypericum wightianum

shows the presence of bioactive phytochemicals. The extracts contain protein, carbohydrate,

amino acid, flavonoid, glycosides, saponins, steroid and tannin. Table 1 and Table 2 shows

the presence of phytochemical constituents in the extracts of the water and methanol.

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Table 1. Preliminary phytochemical (Qualitative) screening of Methanol and Aqueous

extracts Hypericum wightianum.

S. No Phytochemical constituents Methanol extract Aqueous extract

01 Carbohydrates + +

02 Alkaloids - -

03 Glycosides + +

04 Proteins & Amino acids + +

05 Tannins & Phenolic + +

06 Flavonoids + +

07 Terpenoids + -

08 Saponins + +

09 Gums &Mucilages _ -

10 Fixed oils & Fats + -

11 Phytosterols _ -

+ Present - Absent

Table: 2 Quantitative Analysis of Major Phytochemical Constituents Present in

Methanol & Aqueous Extract of H.wightianum

S.No. Phytochemical Constituents

Methanol Extract Quantity/Amount Present*

(per g of extract)

Aqueous Extract Quantity / Amount Present* (per g

of extract)

01 Total Glycosides 143.73 ± 2.63 mg 121.3 ± 4.72 mg

02 Saponins 16.99 ± 1.62 mg 19.36 ± 1.22 mg

03 Total Phenol Content 73.95 ± 0.82 mg of Gallic Acid equivalent

94.51 ± 0.61 mg

Of Gallic acid equivalent

04 Total Flavonoid Content

46.11 ± 3.18 mg Of Rutin equivalent

51.16 ± 2.04 mg

Of Rutin equivalent

05 Vitamin C Content 13.35 ± 1.36 mg 15.5 ± 0.84 mg

*Average of three determinations

Data are expressed as mean ± SEM

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DISCUSSION

Analysis of the plant extracts revealed the presence of phytochemicals such as phenols,

tannins, flavonoids, saponins, glycosides, steroids and alkaloids The phenolic compounds are

2nd largest groups of plant constituent. They possess biological properties such as

antiapoptosis, antiaging, anticarcinogen, antiinflammation, antiatherosclerosis, cardiovascular

protection and improvement of endothelial function as well as inhibition of angiogenesis and

cell proliferation activities [7]. Several studies have described the antioxidant properties of

medicinal plants which are rich in phenolic compounds [8]. Natural antioxidant mainly

comes from plants in the form of phenolic compounds such as flavonoid, phenolic acids,

tocopherols etc. [9,10]. Tannins bind to proline rich protein and interfere with protein

synthesis. Flavonoids are important constituent in regulating control of growth in some plant

and their adversely affect on insect feeding [11]. The ability of flavonoids is due to their

ability to complex with extracellular and soluble proteins and to complex with bacterial cell

wall [12]. They also are effective antioxidant and show strong anticancer activities [13]. The

plant extracts were also revealed to contain saponins which are known to produce inhibitory

effect on inflammation [14]. Saponins have the property of precipitating and coagulating red

blood cells. Some of the characteristics of saponins include formation of foams in aqueous

solutions, hemolytic activity, cholesterol binding properties and bitterness [15]. Steroids have

been reported to have antibacterial properties [16] and they are very important compounds

especially due to their relationship with compounds such as sex hormones. Alkaloids have

been associated with medicinal uses for centuries and one of their common biological

properties is their cytotoxicity [17]. Several workers have reported the analgesic [18]

antispasmodicand antibacterial [19, 20] properties of alkaloids. Glycosides are known to

lower the blood pressure according to many reports [21]. The results obtained in this study

thus suggest the identified phytochemical compounds may be the bioactive constituents and

this plant is proving to be an increasingly valuable reservoir of bioactive compounds that are

medicinally important

CONCLUSION

The study reveals the presence of medicinally valuable constituents in the plant. The

information available from the previous studies in different plants shows the identified

phytochemicals are bioactive. Several studies confirmed the presence of these

phytochemicals contribute medicinal as well as physiological properties to the plants studied

in the treatment of different ailments. Therefore, extracts from this plant could be seen as a

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good source for useful drugs. It is suggested that further work should be carried out to isolate,

purify and characterize the active constituents which are responsible for the bioactivity of

these plants. Also additional work is encouraged to elucidate the possible mechanism of

action of the extracts.

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14.Just MJ, Recio MC, Giner RM, Cueller MU, Manez S, Billia AR, Rios JL.

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extracts of bark of Pansinystalia macruceras.Global J Pure Appl Sci. 2000; 6:83-87.

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HYPERICUM WIGHTIANUM