Materials and Methods

Materials and Methods

For any scientific experiments, materials are resources available for relevant
experiment while the methods are established scientific procedures for selected experiments.
The present study was carried out through in three phases:
 Pharmacognostical Study
 Phytochemical Study
 Pharmacological Study
PHARMACOGNOSTICAL STUDY
The methods adopted for this study were taken as suggested by Wallis (1985), API,
Quality control methods for medicinal plant material, published by W.H.O., Trease and
Evans(1934) etc.
Collection of samples:
The bark of [Alstonia Scholaris(L.)R.Br.] (Family- Apocynaceae) was collected from
college campus of A.L.N. Rao Memorial Ayurvedic Medical College, Koppa while dried
fruit of [Garcinia Indica(Dupetit-Thouars)Choisy] (Family-Guttiferae) was collected from
Umbalebillu (District Shimoga) during September 2016.
Taxonomical Validation1,2
The taxonomical characters of grown plants of both species were matched with
various floras for distinguished identifying structures. Taxonomical verification was done by
noted botanist and visiting professor Prof. Radhakrishna Rao, at the Dept. of Dravyaguna
A.L.N. Rao memorial Ayurveda medical college and in Quality Control Laboratory at A.L.N.
Rao memorial Ayurvedic medical from modern aspects by Dr. Prashant Kumar Jha.
Preservation of samples:
Both of these species were washed properly and then were preserved in a solution of
formalin-aceto-alcohol (FAA) for detailed Microscopic study. For macroscopical studies
samples of both species were air-dried under net in sun. Air-dried samples were coarse
powdered for kashaya and Hima preparation for animal studies while fine powders were
 Materials and Methods

taken for phytochemical and powder studies of drugs.

Macroscopic study:
It includes the observations based on organoleptic characters like shape, size, taste,
odour, colour, touch, texture and fracture. Importance of identification is well mentioned in
Ayurvedic texts for better therapeutic effects by applying Panchendriya pareeksha.
Microscopic Study :
Barks’ Microscopy:
Free hand transverse sections of [Alstonia Scholaris(L.)R.Br.] and[Garcinia
Indica(Dupetit-Thouars)Choisy] were taken. They were cleared with chloral hydrate and
stained with phloroglucinol+HCl, saffranine green, iodine, sudan solution etc. to observe the
nature of cellular bodies and ergastic materials. This was further mounted in glycerine.
Photomicrographs were taken by using Sony digital camera attached to BESTO RCM-20XL
microscope with the help of Quality Control Department, A.L.N. Rao Memorial Ayurveda
Medical College, Koppa.
Powder Microscopy:
Powder of both drugs was studied microscopically and microscopic characters of the
powder were photographed by using Sony digital camera attached to BESTO microscope.
PHYTOCHEMICAL STUDY PHYSICO-
CHEMICAL PARAMETERS3,4
Physico-chemical and phytochemical parameters including qualitative tests,
quantitative estimation of total phenol, total flavonoids and thin layer chromatography were
done for bark of both species.
Loss on Drying (LOD)
10 gram of drug sample was taken in a pre weighed dried petri dish. It was dried in an
oven at 105ºC until reaching a constant weight. The petre dish was taken out, self cooled and
weighed immediately. The weight loss i.e. loss on drying was calculated and expressed as %
w/w.
Ash Value (AV)
3 gram accurately weighed sample was taken in a pre weighed dried crucible. It was
 Materials and Methods

incinerated in a muffle furnace up to 450ºC. The crucible was taken out, self cooled and
weighed immediately. From the weight of the ash, the ash value was derived with reference
to the air dried drug. It was calculated and expressed as % w/w.
Acid Insoluble ash
Ash obtained from total was boiled for 5 minutes with 25 ml of dilute hydrochloric
acid. Insoluble matter was collected on an ashless filter paper after filtering, The ashless filter
paper was taken in crucible and was ignited for constant weight at 450°C. Percentage of acid
insoluble ash was calculated with reference to the air dried drug.
Water Soluble Ash:
The process was repeated as done for acid insoluble ash, only in place of acid hot water
was used and soluble ash was determined after incineration.
Water Soluble Extractive (WSE)
5 g of the sample was weighed accurately. To it 100 ml of distilled water was added and
was kept covered for 24 hours. It was stirred intermittently for first six hours. Next day, it was
filtered. Filtrate was taken in pre-weighed evaporating dish. The evaporating dish was placed on a
water bath for evaporation of the water. After evaporation of the water it was allowed for cooling
and was kept in desssicator. Then it was weighed immediately. From the weight of the residue
obtained, the percentage of water soluble extractive was calculated and expressed as % w/w.
Alcohol Soluble Extractive (ASE)
The method adopted for this experiment was same as that of water-soluble extract but
by using methanol instead of water. Percentage of methanol soluble extract was calculated
and expressed as % w/w.
QUALITATIVE TESTS5,6

Alkaloids: With Dragendorff’s reagent: The alcoholic extract of the samples was taken in a
watch glass, solvent was evaporated. It was added with 2N HCl and few drops of
Dragendorff’s reagent which gave orange brown precipitates.
Tannins:The aqueous extract of test samples was taken in test tubes, by adding 5% lead
acetate solution, it gave precipitate which turned red on addition of KOH solution. Addition
of excess KOH dissolved the precipitate.
 Materials and Methods

Triterpenoids: Liebermann-Burchard reaction: A chloroform solution of test drugs was

taken in a test tube. On addition of acetic anhydride and conc. H 2SO4 the solution turned
green, orange, blue or purple red color.
Carbohydrate: Fehling Test: To 2 ml of solution of both drugs, equal volume of Fehling A
and Fehling B mixture were added. It was place in boiling bath for 5 minutes. A red
precipitate was formed.
Flavonoid: Shinoda Test: To dry powder of both species, 5 ml. of 95% ethanol was added. It
was followed by few drops conc. HCl and 0.5 g magnesium turnings. Pink colour observed
was positive test.
Saponins: Foam test: The dry powder was vigorously shaken with water. Persistent foam
indicated the presence of saponins.
Cardiac glycosides: Keller Killiani test: To 2 ml. aqueous extract of both species glacial

acetic acid was added. It was followed by one drop of 5% FeCl 3 and conc. H2SO4.
Appearance of reddish brown colour at junction of the two liquid layers and changing of
upper layer bluish green was positive result.
FLUORESCENCE TESTS
Fluorescence tests were done using alkalies and acid for powder of both drugs. They
were observed under visible light and under long UV.
QUANTITATIVE ESTIMATION
Determination of Total Phenolic Content7
The amount of total phenolics in hydro-alcoholic extracts of bark of Alstonia
Scholaris and dried fruit of Garcinia Indica was determined with the Folin-Ciocalteu reagent.
Gallic acid (Merck) was used as a standard and the total phenolics were expressed as mg/g
gallic acid equivalents (GAE). For this purpose, the calibration curve of gallic acid was
drawn. 1ml of standard solution of concentration 0.01, 0.02, 0.03, 0.04 and 0.05 mg/ml of
gallic acid were prepared in methanol. Concentration of 0.1 and 1mg/ml of plant extract were
also prepared in methanol and 0.5ml of each sample were introduced into test tubes and
mixed with 2.5ml of a 10 fold dilute Folin- Ciocalteu reagent and 2ml of 7.5% sodium
 Materials and Methods

carbonate. The tubes were covered with parafilm and allowed to stand for 30 minutes at room
temperature before and the absorbance was at read at 760 nm. UV-Visible spectrophotometer
of Systronic 106 was used for determination.

Concentration (mg/100ml) Absorbance

0.5 0.236

1 0.345

1.5 0.402

2 0.472

2.5 0.573

Determination of the Total Flavonoid8:

It was determined by Chang et al. method. Flavonols in hydro-alcoholic extracts of
barks of Alstonia Scholaris and dried fruit of Garcinia Indica were expressed as quercetin
equivalent. Quercetin (Merck) was used to perform the calibration curve. Standard solutions
of 2, 4, 6, 8, and 10 mg per 100 ml were used in 70 % solution of ethanol in water (V/V).
Hydroalcoholic extracts of barks were obtained using the powder of barks. Hydro-alcoholic
extracts consisted of 70% absolute alcohol and 30% water.
1 ml of both samples (hydro-alcoholic extracts and quercetin) was mixed with 3 ml 95 %

ethanol (V/V), 0.2 ml 10 % aluminum chloride (m/V), 0.2 ml of 1 mol L–1 potassium acetate and
5.6 ml water. A volume of 10 % (m/V) aluminum chloride was substituted by the same volume of
distilled water in blank. After incubation at room temperature for 30 minutes, the absorbance of
the reaction mixture was measured at 420 nm. UV-Visible spectrophotometer of Systronic 106
 Materials and Methods

was used for determination.

Sample Concentration

Number (mg/100ml) Absorbance

1 2 0.236

2 4 0.451

3 6 0.662

4 8 0.869

5 10 1.12

Thin layer Chromatography (TLC) (Reference: Quality Control Methods for Medicinal
Plants By WHO)
Materials: Pre coated TLC plates (silica) of thickness 0.20 mm, 20 x20 cm, applicator, glass
chamber, oven, solvents, spraying reagents.
Sample Preparation: Hydro-alcoholic (70 % Methanol and 30% Water) extracts of bark and
dried fruit reference compounds were used. Gallic acid, Quercetin, Rutin, Catechin,
Hyperoside, and Chlorogenic acid of Sigma brand were used. They were dissolved in Hydo-
alcohol with specified ratio of 7:3 for spotting.
Method: Dried TLC chamber was taken and was saturated for 30 minutes with respected
solvent systems for specific kashaya and hima. Vacuum grease was smeared on the lid so that
chamber became air tight. A piece of tissue paper was kept immersed in the chamber for
perfect saturation of solvent system.
Now the preheated pre coated TLC plates were taken and spotted with the help of
 Materials and Methods

applicator 1 cm away from the base, space of 2cm was maintained between each spots. The
spotted plate was dried and gently immersed in TLC chamber in such a way that solvent had
uniform linear contact with the plate. Now the chamber was closed. The chromatogram was
developed at room temperature by allowing the solvent to ascend the specified distance.
Now, the plate was removed from chamber. The position of the solvent front was marked.
All the plates were viewed under long UV without using spraying agent except Pippali. For
Pippali, Anisaldehyde Sulphuric acid was used as spraying agent.
Solvent System : Ethyl acetate : Formic acid : Acetic acid : Water (10:1.1: 1.1: 2.6)
Spraying Agent : Iodine
Observation: The centre of each spot was marked with a needle. The distance from centre of
each spot to the point of application was measured and was recorded.
Resolution factor (RF value) = a/b
Where, a = Distance between the point of application and the centre of the spot of the
material being examined.

PHARMACOLOGICAL STUDY9,10,11,12
Animals Procurement and Standard Setup for Experiment
1. Male Wister strain albino rats weighing 150-250g were used for the study.
2. The animals were obtained from the animal house attached to the A.L.N Rao Memorial
Ayurvedic Medical College, with Reg. No. -191/ CPCSEA,IAEC Approval no:
A.E.D.G.03/11.
3. They were maintained on “Amrut” brand animal pellet feed of Pranav Agro Industries
and plain tap water given ad libitum.
4. Animals were exposed to natural day and night cycles with ideal laboratory conditions in
terms of ambient temperature (22 ± 3ºC) and humidity (50-60%).
5. The experiments were carried out after obtaining permission from “Institutional Animal
Ethics Committee”.
Pilot study: Maximum dose of cholesterol powder in rat is around 25g/kg/day. Average
weight of rat is around 200 gm. Hence, dose of cholesterol comes as:
25×200/1000 = 5mg
 Materials and Methods

A pilot study was conducted to fix the dose of the rat. For that 5mg of cholesterol powder
was mixed with 0. 5ml of vanaspati ghee by making it in lukewarm state, the 5ml of saturated
solution was administered with the help of gastric tube, but on the next day the rat was not
survived because of excess dose of vanaspati ghee. Therefore, dose of ghee was reduced to
2ml and was mixed with same 5mg of cholesterol powder in lukewarm state, it was observed
that this particular dose was suitable for rat and easily digestible.
Autopsy finding: On autopsy, for 5mg cholesterol in 0. 5 ml vanaspati ghee, it was found
that ghee regurgitated back and entered the airways of rat and to lungs, due to these, rat by
choking.
Dose Fixation and Schedule:
• As, per FDA approved The Jounal of Korean Oriental Medicine formula for
conversion of Human dose to rat dose is:
Rat dose (mg/kg) = HED(mg/kg) × Conversion factor
• Whereas, Conversion factor for rat is 6.17 (As per reference for specified weight of rats)
Conversion of the dose obtained above to dose in mg/kg/day by multiplying with suitable
conversion factor based on the average weight of the animal.
Dose of Kashaya for human being = 2pala = 2×48 =96ml/60kg body weight
So, HED = 1.6ml/kg body weight
This can be calculated as per formula that is:
Animal dose = 1.6 × 6.17 = 9.872/kg body weight As, the average weight of rat taken is
in grams so, 9.872×210/ 1000 = 2.07ml/210 gm body weight of rat 2ml of freshly prepared
kashaya was given for experiment.
Preparation of Kashaya and Hima.
Fehling method was applied for removal of bark, then it was cut into small pieces and
was allowed to dry in shade. When bark of the drug got dried then they are pounded well and
make to coarse powder form by passing through the sieve no. 2000/355 according to W.H.O.
guidelines. Then powders of the bark were stored in air tight container in dark.
Test Drug:
To prepare freshly twaka kashaya of the drug Saptaparna [Alstonia
 Materials and Methods

Scholaris(L.)R.Br] as mentioned in classics (Sarangadhara) i.e., 1 pala (48gms) of powdered

drug was taken with 16 parts of water. It was reduced to 1/8 th part.and to prepare freshly
hima of the drug Vrkshamla[Garcinia Indica(Dupetit-Thouars)Choisy] as mentioned in
classics (Sarangadhara) i.e.,dried fruit of Vrkshamla(1 part) will be soaled in water(6
parts)overnight,and on next day it is strained to obtained Hima.
The mentioned methods were adopted to prepare kashaya bark of drugs Saptaparna
[Alstonia Scholaris(L.)R.Br] Fresh kashaya was prepared under mandaagni as per told in
Sharangdhara every day to give to rats. The dose given was 0.9ml as obtained by dose
conversion formula. The drug was administered in morning hours to trial group-2.and the
Vrkshamla[Garcinia Indica(Dupetit-Thouars)Choisy] fresh hima was prepaired 1part will be
soaled in water 6 part.the dose given was 1.7ml and the drug was administered in morning
houars to trial group-1.
EXPERIMENTAL STUDY DESIGN AND PROTOCOL
Table no. 4.1: Showing consolidated Study design and Protocol.

1
Sample 18 Albino rats of either sex will be randomly selected.

2 Inclusion
Healthy, active rats each weighing in between 150-200 g.
Criteria

3 Exclusion Pregnant, diseased rats weighing below 150 g or above 200 g and
Criteria rats under trial for other experiments will be excluded.

No.of Dose/200g
Groups Albino Drugs Form Body Purpose
4. Groupings
Rats Weight
Control 6 Distilled - 2 ml To serve as
 Materials and Methods

Group Water Prophylacti

c
Control
To bring
about
Trial Vrkshamla
6 Hima 1.7 ml Shaulyahar
Drug 1 Fruit
a
Effect
To bring
about
Trial Saptaparna
6 Kashaya 0.9 ml Shaulyahar
Drug 2 Bark
a
Effect
Hima preparation:- Dried fruit of Vrkshamla (1 part) will be
soaled in water (6 parts) overnight, and on next day it is strained to
obtain Hima14.
Procedure Kashaya preparation:- Bark of Saptaparna (1 part) will be taken
along with water (4 parts), then will be boiled to get until water
and
remains upto 1/4th part. Then it will be strained to get Kashaya14.
observation Animal Experimentation:15 Initially body weights of all the
animals will be taken and the animal will be fed on the
hyperlipidemic diet except control (Group-1) where these will be
kept on pellet feed and water, whereas Group-2, 3, received
5. hypercholesterolemic diet. Whereas Group-2 & 3 received
Vrkshamla hima and Saptaparna bark kashaya respectively and
continued for 30 days. The hyperlipidemic diet includes
Hydrogenated vegetable oil (Vanaspathi, ghee and Cholesterol extra
pure power) made in to suspension form. The suspension will be
administrated in the dose of 0.5ml / 1 ml and 200 Gram body
weight of rats respectively daily for 30 days (at evening hours) to all
the rats. On the 30th day final weight of all the rats will be taken and
on the 31st day after overnight fasting, rats will be anaesthetised by
giving mild dose of Ketamine subcutaneous. Then the blood will be
collected by Retro-orbital puncture and sent to laboratory for
biochemical estimations.
 Materials and Methods

6. Duration 31 days
Statistical
7. Anova followed by post hoc.Scheffe test was done.
Test
Observatio
8. Subjective Criteria : Weight (B.T. & A.T.) are taken
n Objective Criteria : HDL, LDL, Total Cholesterol, Triglycerides

Collection of blood:
According to guidelines for Collection of Blood from Laboratory Animals following
procedure is opted for the collection of blood:
Under general anesthesia the rats were grasped so that its back rested on the palm with its
head toward thumb. The thumb was placed just lateral to the animal's trachea so that the jugular
vein on the same side as the eye from which occluded blood was collected. The fur on the
animals head was drawn into the palm, this caused the animal’s eye to proptose (bulge) slightly.
A 50 µL sterile micro hematocrit tube was directed into the medial canthus (junction of eyelids
closest to the animal's nose) of the eye rotating slightly as the tube was directed to a point directly
behind the globe. Sufficient pressure was applied to cut through the fibrous layer that surrounded
the sinus. Blood flowed through the tube once the sinus had been penetrated. After blood
collection, the tube was removed and the eyelids were closed and a dry cotton pad was applied
over the eye with gentle pressure to prevent retro-orbital hemorrhage.
Blood was not collected from the same eye more than 2 times. An antibiotic
ophthalmic ointment was applied following bleeding.
Mode of Drug Administration:
Oral route was selected for administration of drug to respective group of animals by
using syringe with an attached gastric tube.

Criteria for assessment of results:

Blood cholesterol levels were checked before and after treatment. Blood was drawn
from the rats by retro-orbital method.
These values were subjected to statistical analysis to evaluate the
 Materials and Methods

hypercholesterolemic activity.

REFERENCES

1. Evans William Charles, Trease And Evans' Pharmacognosy, Edition, 14.

Publisher, Harcourt Brace & Co, 1997. ISBN, 0702024015, p.p 96-98
2. Indian Pharmacopoeia, Vol. I. New Delhi: Government of India, Ministry of
Health and Family Welfare, 1996
3. Quality standards for indian medicinal plants, Vol. I; pg. no95, 168,169,37,
Vol.III; pg. no7, 357, Vol. IIX; pg. no. 325, Vol. IX, pg. no. 175
4. The Ayurvedic Pharmacopoeia of India, Part I, Vol. III Ist ed. New Delhi:
Government of India, Ministry of Health and Family Welfare, Department of
Indian Systems of Medicine & Homeopathy; 2001; p.110-111.
5. Evans William Charles, Trease And Evans' Pharmacognosy, Edition, 14.
Publisher, Harcourt Brace & Co, 1997. ISBN, 0702024015, p.p 96-98
6. Quality standards for indian medicinal plants, Vol. I; pg. no95, 168,169,37,
Vol.III; pg. no7, 357, Vol. IIX; pg. no. 325, Vol. IX, pg. no. 175
7. Theodore Cooke, Flora of the Presidency of Bombay. Gamble J.S- Flora of
Presidency of Madras, Vol.I, Adlard & Son Ltd., London,1928.
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10. CPCSEA (Committee for the Purpose of Control and Supervision on
Experimentson Animals) Guidelines, India.Santosh et al. (2012). ‘A Study of
Anti- hyperlipidemia, Hypolipidemic, andAndti-antherogenic Activity of Fruit of
 Materials and Methods

Emblica officinalis (Amla) in High Fat FedAlbino Rats’. International Journal of

Medical Research and Health Sciences. Volume 2 Issue 1 Jan-Mar 2013. Zarei A
et al. (2013)
11. Effects of Melissa officinalis on thyroid hormones. ZahedanJournal of
Research in Medical Sciences. 15 (8): 6-12.
12. Madhavi Jagtap et al.(2011) ‘A comparative evaluation of cardioprotective
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isoproterenol induced myocardial infarction in hyperlipidaemic rats’. Indian
Journal of Natural Products and Resources. Volume 2(3), September
2011,pp.335-344.