PHYTOCHEMICAL SCREENING AND ANTI ... physicochemical evaluation and other parameters, preliminary...
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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
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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
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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
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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
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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
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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.
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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
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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
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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.
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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
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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)
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Figure 4
Spot 1- Chloroform extract of Bark
Spot 2- Standard Lupeol in Chloroform
Figure 5 Standard Lupeol peak at Rf 4.5
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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
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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
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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.
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For Correspondence: Pooja Email: [email protected]