phytochemical screening and anti ... physicochemical evaluation and other parameters, preliminary...

Vol-3, Issue-4, Suppl-2, Nov 2012 ISSN: 0976-7908 Pooja et al

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PHARMA SCIENCE MONITOR

AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

PHYTOCHEMICAL SCREENING AND ANTI INFLAMMATORY ACTIVITY

OF BAUHINIA VARIEGATA LINN VAR. CANDIDA

Pooja*, Manju Vyas Singh

Department of Pharmacognosy, Delhi Institute of Pharmaceutical Sciences and Research, New Delhi-110017, India

ABSTRACT In present investigation, the detailed pharmacognostic study and screening for Anti-inflammatory activity of bark of Bauhinia variegata Linn. var. Candida belonging to the family Caesalpiniaceae is carried out to lay down the standards which could be useful in future experimental studies. The study includes standardization i.e macroscopy, microscopy, physicochemical evaluation and other parameters, preliminary phytochemical screening and HPTLC determination. It is a medium sized, deciduous tree, found throughout India. Bark is considered to be used to treat gastric disorders, blood disorders and as liver tonic in folk medicine. Keywords: Bauhinia variegata, Anti-inflammatory, Kachnar, HPTLC, Standardization. INTRODUCTION

Bauhinia variegata also known as Kachnar (Hindi), Mountain Ebony (English) is a

medium sized deciduous tree distributed in sub-Himalayan tract and outer Himalaya of

Punjab. There are two varieties Purple and White flowered. The present work has been

done on white flowered variety that is Bauhinia variegata Linn var candida. The main

chemical constituent present in bark is Lupeol which is a pharmacologically active

triterpenoid found in variety of plants. It has several medicinal properties, one being

Anti-inflammatory which is supposed to be due to its action on interleukin system.

Literature survey suggests that bark of purple flowered variety exhibits hepatoprotective,

hypolipidemic, antioxidant, immunomodulatory, anti-inflammatory and anti microbial

activities but very less work has been reported on the white flowered variety .This is the

major reason behind selecting this plant for present work. So in present study

pharmacognostic standards of the bark of Bauhinia variegata Linn var candida studied.

These standards are of utmost importance not only for finding out genuity, but also in



detection of adulterants in marketed drugs. The results of the study could be useful in

setting some diagnostic indices for the identification and preparation of a monograph of

the plant.

MATERIALS AND METHODS

Collection

Bark of bauhinia variegata Linn var. Candida were collected from residential area,

Pushp vihar, New Delhi and samples get identitified from NISCAIR, New Delhi. Fresh

plant parts were used for macroscopical examination. Some quantity of sample was air

dried and powdered for microscopical studies. An exhaustive pharmacognostic study was

carried out using standard methodology.

PHARMACOGNOSTIC EVALUATION:

Macroscopic Examination:

In organoleptic evaluation, various sensory parameters of the plant material, such

as color, odour, taste, shape and texture of the stem bark were recorded in table 1.

Microscopic examination:

Transverse Section:

Bark sample of b.v was cut into thin pieces and cleared by warming with chloral

hydrate over the Bunsen burner flame and treated with phloroglucinol and HCl and was

then mounted in dil glycerine covered with a cover slip and observed under the

microscope. (Quality control methods for medicinal plant material by WHO; 1998)

Characteristic structures observed in T.S of bark were shown in fig. 1

Powder microscopy:

2 drops of chloral hydrate reagent was placed on a glass slide. Tip of a needle was

moistened with water and dipped into powdered drug material. A small quantity of

powder was transferred to the slide, mixed well. Cover slip was placed on that and

viewed under microscope. Characteristic structures observed in powder of bark were

shown in table 2 and pictures were shown in fig.

Fluorescence Analysis



Small quantity of powdered plant sample was treated with different reagents(

conc. HNO3, conc. H2SO4, conc. HCl, FeCl3, Aq. KOH) and examined under day light

and UV light( 254nm and 366nm). (Rangasamy Manivannan et. al; 2010). Results were

recorded in table 4.

PHYSICOCHEMICAL STADARDIZATION (Quality control methods for medicinal

plant material by WHO; 1998).

1. Determination of Ash Values

A) Total Ash:

Accurately weighed 2g of powdered drug was taken in a tarred silica dish and

ignited at temperature not exceeding 450°C until it became white (carbon free). Cooled in

desiccators and weighed. Finally, percentage of total ash content was calculated with

reference to air dried drug.

B) Acid Insoluble Ash:

The total ash obtained from 2g of bark and leaf sample were boiled with 25ml of

dilute hydrochloric acid for 5 minutes. The insoluble matter was collected on Gooch

crucible, washed with hot water and ignited to obtain constant weight. The percentage of

amount of acid insoluble ash was calculated with reference to air dried drug. C) Water

soluble Ash:

The total ash obtained from the 2g of drug was boiled with 25ml of water for 5

minutes. The insoluble matter was collected on Gooch crucible, washed with hot water

and ignited to obtain constant weight. The weight of the insoluble matter was subtracted

from the weight of the total ash; the difference of weights represents the water soluble

ash. The percentage of water soluble ash was calculated with reference to air dried drug.

2. Determination of Extractive Values

A) Alcohol Soluble Extractive value

Air dried, coarsely powdered 5 g plant material was placed in a glass stoppered

conical flask and macerated with 100 ml of Alcohol (90% v/v) for 6 hours, with frequent



shaking and then allowed to stand for 18 hours. It was then rapidly filtered through

Whatmann filter paper. 25 ml of filtrate was transferred to flat bottom dish and solvent

was evaporated on a water bath. It was then dried at 105 °C for 6 hours and kept in

desiccators for 30 minutes and weighed. The content of extractable matter in mg per g of

air-dried material was calculated.

B) Water Soluble Extractive Value

Air dried, coarsely powdered 5 g plant material was placed in a glass stoppered

conical flask and macerated with 100 ml of water for 6 hours, with frequent shaking and

then allowed to stand for 18 hours. It was then rapidly filtered through Whatmann filter

paper. 25 ml of filtrate was transferred to an evaporating dish and solvent was evaporated

on a water bath .it was then dried at 105 °C for 6 hours and kept in desiccators for 30

minutes and weighed . The percentage of water soluble extractive matter with reference

to air-dried material was calculated.

3. Loss on drying

An accurately weighed amount of the dried plant material was crushed and taken

in a dried petri dish and again weighed. The sample was heated in an oven at a temp.

1050C and this procedure was repeated until constant weight of sample was obtained.

After drying was completed, the petri dish was allowed to cool to room temperature in

desiccators. The loss on weight in percentage of air-dried material was calculated.

4. Swelling index

1gm of powdered drug was introduced into a glass stoppered measuring cylinder and

25ml of water was added to that. Mixture was shaken thoroughly every 10minutes for 1

hour and then allowed to stand for 3 hours at room temperature. This was done in

triplicate. Volume was measured in ml which was occupied by plant material. Mean

value of the three determinations was calculated.

5. Foaming index

1gm of coarsely powdered plant material was taken in a 500 ml conical flask containing

100 ml of boiling water. Mixture was boiled for 30 minutes, cooled and filtered in a



100 ml volumetric flask and sufficient water was added through filter to dilute up to the

volume. This decoction was poured into 10 test tubes in successive portions in 1ml,

2ml, 3ml, 4ml etc. upto the 10 ml and volume was adjusted in each tube with water up

to 10 ml. Tubes were shaken for 15 seconds and then allowed to stand for 15 minutes

and height of foam was measured.

Foaming index = 1000/ a

Where, a= the volume in ml of the decoction used for preparing the dilution in the tube

where foaming to a height of 1 cm is observed.

6. Determination of haemolytic activity

Preparation of the erythrocyte suspension

Sufficient volume of blood was freshly collected from a rat in EDTA coated tube, shaken

immediately and stored at 2-4°C. 1 ml of blood was placed in a 50-ml volumetric flask

with phosphate buffer pH 7.4 TS and carefully diluted to volume. This diluted blood

suspension (2% solution) can be used as long as the supernatant fluid remains clear and

colourless. It must be stored at a cool temperature.

Preparation of Sample solution

Serial dilutions of the plant material extract were prepared with phosphate buffer pH 7.4

and blood suspension (2%) using four test-tubes.

PREPARATION OF SAMPLE DILUTIONS

Material

Tube no.

1 2 3 4

Plant material extract (ml) 0.10 0.20 0.50 1.00

Phosphate buffer pH 7.4 TS (ml) 0.90 0.80 0.50 -

Blood suspension (2%) (ml) 1.00 1.00 1.00 1.00

As soon as the tubes were prepared, they were gently inverted to mix, avoiding the

formation of foam. Shaken again after a 30-minute interval and allowed to stand for 6

hours at room temperature. The tubes were examined to observe at which dilution the



total haemolysis has occurred, indicated by a clear, red solution without any deposit of

erythrocytes, was recorded.

7. Determination of Heavy metal analysis

5gm of sample was extracted with 100ml of ethanol by cold maceration method and

filtered. Determination of heavy metals i.e. lead, arsenic and cadmium was carried out

with the help of Atomic Absorption Spectroscopy (AAS) method from NIPER,

Chandigarh.

8. Determination of Total Phenolic Content

A. Preparation of Test Solution

Accurately 0.5gm powdered drug was taken and extracted with 75ml of 50% methanol

by cold maceration for 2hrs with intermittent shaking. Solution was filtered and volume

was made up to 100ml.

B. Procedure

From the above extract, 0.1ml was pipetted out into a 25ml volumetric flask and 10ml

of distilled water was added followed by 1.5 ml of folin ciocalteu reagent. After 5mins

4ml of 20% sodium carbonate solution was added and volume was made up to 25 ml

with distilled water and incubated for 30mins. After 30mins absorbance was recorded at

766nm.

9. Determination of percent of tannins

Accurately weighed 1g of the powdered drug sample (W) was taken in a 250 ml glass

stoppered flask. 100 ml of water was added to it and the flask was shaken for 1 hour and

was left overnight. The solution was filtered through Whatman filter paper no.1 and first

20 ml of the filtrate was discarded. Rest 10 ml of the filtrate was transferred to a 1 L

conical flask. To it 750 ml water and 25 ml of indigosulphuric acid was added. It was

then titrated with 0.1 N KMnO4 and shaken till a golden yellow end point was reached

(T2). Blank titration (T1) was also performed (Samant, 1971).

Each ml of 0.1 normal KMnO4 = to 0.00 4157 gm of tannins.



Results were recorded in table

10. Phytochemical screening

Weighed amount of powdered bark of B.V was extracted out in Soxhlet

apparatus with petroleum ether (60-80°C), chloroform, ethanol and water separately for

6hrs. Extracts were concentrated in rotary vacuum evaporator. Percentage yield of each

of the extract was calculated. Results were given in table. The extracts were used for

preliminary phytochemical screening by performing specific chemical tests viz., Keller

killani test for glycosides, Dragondorrf’s and Mayer’s test for alkaloids, Shinoda and

Lead acetate test for flavonoids, Frothing test for saponins, Ferric chloride test for

tannins, Liebermann- Burchard’s test for steroids, Fehling test for carbohydrates,

Salkowski test for terpenoids and Ninhydrin test for amino acids and proteins.

11. High Performance Thin-layer Chromatography (HPTLC)

HPTLC of chloroform extract of bark was carried out along with lupeol (standard)

solution in chloroform by using n-hexane: ethyl acetate (8:2) as solvent system. Sample

and standard solution were spotted on pre coated silica gel aluminium plate 60F-254

(10cm×10cm with 250μm thickness) using Camag Linomat IV sample applicator and

100μl Hamilton syringe. Plats were developed in Camag development chamber followed

by detection in Camag TLC scanner III with CATS software.

PHARMACOLOGICAL STUDY

Anti-inflammatory activity

Carrageneenan induced paw edema method (Winter et al; 1962)

Thirty six albino Wistar rats of either sex weighing between 150-200gm were divided

into six groups. Group I, II and III received the ethanol extract of the bark of Bauhinia

variegata at the doses of 50mg/kg, (oral) 100mg/kg (oral) and 200mg/kg(oral)

respectively. Group IV received Diclofenac sodium 5mg/kg and served as standard (C.S.

Shreedhara; 2009). Group V received Lupeol 7mg/kg.GroupVI received normal saline

and served as control. One hour after the administration (as per the experimental

protocol), 0.1ml of 1% carrageenan solution was injected beneath the sub-plantar surface



of the right hind paw of rats of all groups. For the assessment of the anti-inflammatory

activity, the volume of the paw was measured with the help of mercury plethysmometer

at 0hr, 1hr, 3hrs and 5hrs after the carrageenan treatment. Percentage inhibition of paw

oedema volume was calculated.

Permission for conducting the experiment on animals was obtained from the ethical

committee of DIPSAR.

RESULTS AND DISCUSSION:

In present study the stem bark of Bauhinia variegata Linn var. Candida was evaluated

for its pharmacognostic and phytochemical aspects along with HPTLC determination and

anti inflammatory activity which revealed the following results.

PHARMACOGNOSTIC EVALUATION

1. Macroscopic Examination: The results of macroscopical evaluation were given

in table 1.

Outer Surface Inner Surface

Figure 1

TABLE 1: MACROSCOPIC / ORGANOLEPTIC CHARACTERS

S. No Parameters Observation of Bark 1 Colour Fresh bark Dried bark

Dark brown outside, reddish pink inside

Blackish brown

2 Odour Characteristic 3 Fracture Fibrous 4 Shape Curved 5 Texture Hard, longitudinally striated



Microscopical Investigation:

A transverse section of bark showed presence of dark colored cork cells with sclereids

present in groups. Cortex was many layered. Starch grains were scattered all over the

surface. Xylem was transversed by well developed uniseriate medullary rays having

scattered phloem.

(i) Transverse section:

Figure 2

(ii) Powder microscopy

Bark: Bark powder shows presence of cork cells, phloem fibres and abundant prismatic

and rosette calcium oxalate crystals.



Figure 3

TABLE 2: FLUORESCENCE ANALYSIS

Plant Sample Chemical Treatment

Day Light UV Light 254nm 366nm

Bark Powder

Conc. HNO3 Yellowish Brown Yellowish Green Dark Green Conc. H2SO4 Brown Fluorescent Green Yellowish Green Conc. HCl Brown Yellowish Green Dark Brown FeCl3 Yellowish Green Fluorescent Green Dark Green Aq. KOH Brown Yellowish Brown Dark Brown

TABLE 3: PERCENTAGE YIELD DETERMINATION

Weight of Drug Taken (gm)

Solvent Used For Extraction

Color of Extract % Yield (w/w)

50 Petroleum Ether Light Yellowish Brown

1.2

50 Chloroform Light Brown 11.6 50 Ethanol Dark Reddish

Brown 8.56

50 Water Brown 19.68



TABLE 4: PHYSICOCHEMICAL STANDARDISATION

PARAMETER VALUE 1. Total Ash 12.9% 2. Acid-insoluble Ash 0.8% 3. Water-soluble Ash 4.3% 4. Water soluble Extractive 12% 5. Alcohol soluble Extractive 16.7% 6. Loss on Drying 9.7% 7. Swelling Index Zero 8. Foaming Index Zero 9. Heavy metal Analysis for Lead,

Arsenic and Cadmium Within limits

10. Total Phenolic Content 34.85 mgGAE/g 11. Tannin Content 0.914% 12. Microbial content Determination Free from microbial contamination

TABLE 5: PRELIMINARY PHYTOCHEMICAL SCREENING

Phytochemical compound

Petroleum Ether Extract

Chloroform Extract

Ethanol Extract Aqueous Extract

Carbohydrates - - + + Glycosides - - - - Saponins - - + + Terpenoids + + + - Alkaloids - - - + Steroids + + - - Flavanoids - - + + Tannins - - + + Amino acids and Proteins

- - - +

HPTLC Profile

Solvent System- n-Hexane: Ethyl acetate (8:2)



Figure 4

Spot 1- Chloroform extract of Bark

Spot 2- Standard Lupeol in Chloroform

Figure 5 Standard Lupeol peak at Rf 4.5



Figure 6 lupeol peak in bark chloroform extract (0.45)

TABLE 6: HPTLC PROFILE

S.N. Sample Rf AUC 1 Std. lupeol solution 0.45 66.5 2 Bark solution 0.45 2345.2

HPTLC determination showed Rf value 0.45 in standard lupeol solution and bark sample.

PHARMACOLOGICAL STUDIES

TABLE 7: DETERMINATION OF MEAN PAW EDEMA VOLUME

Concentration (mg/kg

Mean Paw Edema Volume (ml) At 0hr After 1hr After 3hrs After 5hrs

50 0.09 0.065 0.067 0.087 100 0.105 0.082 0.084 0.093 200 0.112 0.089 0.092 0.1 Diclofenac sodium

0.102 0.08 0.083 0.09

Lupeol 0.135 0.107 0.122 0.109



TABLE 8: DETERMINATION OF PERCENTAGE INHIBITION OF PAW

EDEMA VOLUME

Concentration (mg/kg)

Percentage inhibition

After 1hr After 3hrs After 5hrs

50

30 36.8 33.2

100 35.6 41 38.3

200 37 44.4 39.6

Diclofenac sodium 39.5 48 42

Lupeol 22 31 28.8

Figure 7 Concentration vs. Percentage Inhibition

Results of anti inflammatory activity showed that bark of B.V Linn var. candida has

potential to be used as an anti inflammatory agent in future as the data obtained for

decrease in paw edema volume with drug extract in concentrations 200 mg/kg has the

highest activity 44.4 % after 3hrs which is comparable to that of the standard diclofenac



sodium solution (5mg/kg) 48% after 3 hrs. Anti inflammatory activity of the sample is

supposed to be due to the presence of flavonoids and terpenoids (lupeol)

CONCLUSION

Bauhinia variegata Linn var. candida is extensively used in the traditional system of

medicine for treatment of number of diseases and considered as an important medicinal

plant. As per literature survey very less work has been reported on this variety. In present

work the results of investigation could serve as a basis for proper identification,

collection and investigation of the plant. Parameters determined in quantitative

microscopy can be useful to differentiate closely related species. Presence of various

phytoconstituents can serve as basis for screening of different pharmacological activities,

investigation and further research.

REFERENCES

1. C.K. Kokate; Practical Pharmacognosy; 4th edition (1994); 7-9.

2. C.S. Shreedhara, V.P. Vaidya et al. Scrrening of Bauhinia Purpurea Linn. For

Analgesic and Anti-Inflammatory Activities. Indian Journal of Pharmacology

2009, 41(2), 75- 79.

3. Gayathri Gunalan, A. Saraswathyand K. Vijayalakshmi. Preliminary

Phytochemical and Pharmacognostical Analysis of Bauhinia variegata Linn.

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4. Kirtikar and Basu ; Indian medicinal plants(2000); vol. 4, (3), 1257-1260.

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8. Rangasamy Manivannan et. al; Pharmacognostical and preliminary phytochemical

studies of Bauhinia tomentosa (Linn) flower; Journal of Pharmacy

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For Correspondence: Pooja Email: [email protected]

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