“EVALUATION OF DIFFERENT MARKET SAMPLES OF NAGAKESARA (Mesua ferrea Linn.) BY PHARMOCOGNOSTIC AND ANALYTICAL PARAMETERS.” By Dr. HITHA.M, B.A.M.S Dissertation submitted to the Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka In partial fulfillment of the requirements for the degree of AYURVEDA VACHASPATI (Doctor of Medicine) In DRAVYAGUNA VIGNANA Guide Dr. BHAGYALAKSHMI T.R M.D (Ayu.) Co- Guide Dr. YALLAPPA G.K M.D (Ayu.) DEPARTMENT OF POST GRADUATE STUDIES IN DRAVYAGUNA VIGNANA K.V.G. AYURVEDA MEDICAL COLLEGE AND HOSPITAL SULLIA – 574327 2014 i
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Review of Literature
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 47
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Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 48
4. MATERIALS AND METHODS
Analytical study
PLANT IDENTIFICATION:-
The plant Nagakesara (Mesua ferrea Linn.) is identified on the
basis of its -
1. Synonyms given in classics of Ayurveda.
2. Morphology and family characters of the plant.
AUTHENTICATION:-
Genuine sample is procured from the natural habitat from Jawaharlal
Nehru Tropical
Botanical Garden and Research Institute
(JNTBGRI),Palode,Thiruvananthapuram
during the month of May 2013 and named as Sample G and get
authentified by the
Botanist.
COLLECTION:-
Literary data is collected from the library of K.V.G ayurvedic
Medical
College and hospital, Sullia and Internet.
Collection of the samples from markets of Pune, Kolkatta, Delhi
and
Thrissur was done.These samples were collected during April 2013
to
August 2013.
Sample S1 – Pune
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 49
Sample S2 - Kolkatta
Sample S3 – Delhi
Sample S4 – Thrissur
The word pharmacognosy is formed by combination of
‘Pharmakon’which
means drug and ‘gignosco’ which means ‘to acquire knowledge’.
Therefor,
pharmacognosy can be defined as a branch of bioscience that deals
with the
knowledge and authentication of medical and related products of
crude or primary
type obtaining from both plants and animals in detailed form.
The original and basic approach towards pharmacognosy includes
study of
Morphological system,study of cell structure and organization and
study of tissue
systems,which still hold a key in identification of the correct
species of the plant.
It includes both macroscopic and microscopic study of
samples.
a. Organoleptic study
The macroscopic characters of the drug were observed for colour,
size,
shape, odour, taste, texture.
b.Microscopic study
Microscopic study of the drug was carried out in the Department
of
Botany, Nehru Memorial College, Sullia.
MICROSCOPIC EXAMINATION
Transverse sections of Nagakesara flower,stamens and flower buds
were taken
and photomicrography was done after proper mounting and
staining
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 50
Materials required:
Drug , 1% safranin stain, 50% glycerine, water, a sharp razor
blade, watch
glass, thin painting brush, needles, forceps, glass slides, cover
slips, blotting paper,
dropper, compound microscope.
Procedure:
• The drug was collected washed and soaked in water before carrying
out the
procedure.
• The drug was held between thumb and index finger in the left
hand, with the
help of a sharp razor blade, thin sections were taken and put into
watch glass
containing water.
• A thin uniform and entire section was selected and transferred on
to a clean
glass slide with the help of a brush.
• A drop of safranin stain was put and left for few minutes. Excess
stain was
removed by washing with water.
• Section was mounted with 1-2 drops of 50% glycerine and covered
with a
clean cover glass.
• Excess glycerine was removed by blotting paper and observed
under
microscope93.
POWDER MICROSCOPY:
A pinch of powder was warmed with drops of chloral hydrate on
a
microscopic slide and mounted in glycerine. Slides observed under
microscope and
diagnostic characters were observed and photographed using Zeiss
AXIO trinocular
microscope attached with Zeiss AxioCam camera under bright field
light.
Magnifications of the figures are indicated by the
scale-bars94.
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 51
4.2 ANALYTICAL STUDY
Hand lenses were used for the detection of foreign matter.
For quantitative extraction (Aqueous, Alcoholic, Chloroform
and
Peroleum ether) of all samples, water bath, conical flasks etc. are
used.
Total ash, acid insoluble ash, and moisture content, are determined
by
using silica crucible, oven, desiccator, electronic balance &
muffle
furnace.
All chemicals and reagents used were of A.R. grade for the
qualitative
analysis of extracts.
Methodology
In physical methods quantitative standards like total ash, acid
insoluble ash,
water-soluble ash, moisture content, extractives, foreign matter
and pH are
determined.
DETERMINATION OF ASH VALUE OF CRUDE DRUG:
Procedure:
• Weigh & ignite the flat thin porcelain dish or stared silica
gel.
• Weigh about 2 to 3 gms of the powdered drug into dish
/crucible.
• Support the dish on pipe-clay triangle placed on a ring of retort
stand.
• Spread the drug in an even layer &ignite it by gradually
increasing the heat to
500 to 600oC till vapours almost cease to be evolved until all the
carbon is
burnt off.
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 52
• Weigh the dish & calculate the percentage of total ash with
reference to the
air-dried sample of the crude drug.95
Calculation:
Weight of the drug taken = y gm
Weight of dish + ash (after complete incineration)= z gm
Weight of the ash = (z-x) gm
Y gm of drug gives (z-x) gm of ash
Therefore, 100gm of crude gives 100 (z-x) gm of ash.
Y
Y
Procedure:
Proceed as per the steps mentioned in the procedure for
determination of total
ash value of crude drug. Then
Using 25 ml of dil HCl, wash the ash from the dish used for total
ash
into a 100ml beaker.
Place wire gauze over a bunsen burner & boil for 5
minute.
Filter through an ashless filter paper; wash the residue twice with
hot
water.
Ignite the crucible in the flame, cool & weigh.
Put the filter paper & residue together into the crucible, heat
gently until
vapours cease and then more strongly until all carbon has
been
removed.
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 53
Cool in a desiccator.
Weigh the residue and calculate acid insoluble ash of the crude
drug
with reference to the air-dried sample of the crude drug.96
Calculation: Similar to previous experiment
Weight of residue (acid insoluble ash)= a gm
Y gm of air dried drug gives =a gm of acid insoluble ash
Therefore 100gm of the air dried drug gives - 100 x a of acid
insoluble ash
Y
Acid insoluble ash value of the sample - 100 x a %
Y
Materials:
Total ash, digital balance, muffle furnace, desiccator, ash less
filter paper,
electric bunsen, funnel, silica crucible.
Procedure:
• To the total ash obtained, 25ml of water was added and boiled for
5 minutes.
• It was filtered through an ash less filter paper to separate the
insoluble matter.
• The residue along with the filter paper was taken in a
pre-heated, weighed
silica dish.
• Transformed to muffle furnace and ignited for 15 minutes at the
temperature
not exceeding 450°C.
• The dish was cooled in a desiccator & weighed again.
• Heating was continued till constant weight of the dish was
obtained.
• The weight of the insoluble matter from the weight of the ash was
substracted.
• The percentage of water soluble ash with reference to the air
dried drug was
calculated.97
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 54
DETERMINATION OF WATER SOLUBLE EXTRACTIVE
Procedure:
• Weigh about 5 gm of the powdered drug in a beaker and transfer it
to a dry
250 ml Iodine flask.
• Fill a 100 ml graduated cylinder to the required mark with the
solvent (water +
chloroform). Washout the weighing bottle and pour the washings
together
with the remainder of the solvent into the conical flask.
• Cork (stopper) the flask and set aside for 24 hrs shaking
frequently
(maceration).
• Filter it into a 50 ml Cylinder. When sufficient filtrate has
been collected,
transfer 25 ml of the filtrate to a weighed 25 ml beaker as used
for the ash
value determination.
• Evaporated to dryness on water bath and complete the drying in an
oven at
100OC for about 10 –15 mins.
• Cool in dessicator and weigh
• Calculate the percentage w/w of extractive with reference to the
air- dried
drug.98
Procedure:
• Weigh about 5 gm of the powdered drug in a beaker and transfer it
to a dry
250 ml Iodine flask.
• Fill a 100 ml graduate cylinder to the required mark with the
solvent (90%
alcohol). Washout the weighing bottle and pour the washing,
together with the
remainder of the solvent into the conical flask.
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 55
• Cork (stopper) the flask and set aside for 24 hrs shaking
frequently
(maceration).
• Filter into a 50 ml Cylinder. When sufficient filtrate has been
collected,
transfer 25 ml of the filtrate to a weighed 25 ml beaker as used
for the ash
value determination.
• Evaporated to dryness on water bath and complete the drying in an
oven at
100OC for about 10 –15 mins.
• Cool in dessicator and weigh.
• Calculate the percentage w/w of extractive with reference to the
air-dried
drug.99
The same procedure was repeated for petroleum ether extractive
value.
DETERMINATION OF MOISTURE CONTENT
Powdered drug, digital balance, porcelain dish, desiccator, hot air
oven.
Procedure:
• Accurately weighed 5g of the coarsely powdered drug was taken in
a dried,
weighed porcelain dish.
• Dish was kept in hot air oven at 105oC for five hours.
• Dish was taken out, cooled in a desiccator and weighed.
• Drug was weighed at each one hour interval.
• Drying was continued till constant weight was obtained.
• Percentage of moisture content (loss on drying) with reference to
the air dried
drug was calculated.99
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 56
DETERMINATION OF FOREIGN MATTER
Procedure:
• 100gm of crude drug was taken and spread into thin layer.
• It is examined for the presence of foreign matter like mud,
leaves etc. with the
help of hand lens.
• The foreign matters were separated & the drugs were weighed
again.
Percentage of foreign matter was calculated.100
DETERMINATION OF PH
The pH value of an aqueous liquid may be defined as the common
logarithm
of the reciprocal of the hydrogen ion concentration expressed in
gram per litre for
qualitative indication of the acidity or alkalinity of a
solution.
Procedure: The pH of a given solution can be measured with the help
of an
apparatus called pH meter, consists of a voltmeter connected with
two electrodes.
a. A standard electrode of known potential.
b. A special electrode enclosed in a glass membrane that allows
migration of
H+ ions.
The glass case contains a reference solution of dilute hydro
chloric acid. The
two electrodes are dipped in the solution to be tested. If this
solution has a different
pH from the solution in the probe, an electrical potential results.
Thus the potential
between the standard electrode and the glass electrode varies with
the pH of the
solution under test. This potential is recorded by an inbuilt
potentiometer of the pH
meter. The potentiometer reading is automatically converted
electrically to a direct
reading of the pH of the unknown solution.101
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 57
DETERMINATION OF TOTAL PERCENTAGE OF VOLATILE OIL
Apparatus:
2.A special steelhead contains
Procedure:
10 to 20 gram of powdered drug was taken with 250 to 300 ml of
water in
distillation flask added along with a few pieces of porcelain
Apparatus was arranged properly for the extraction
By closing the side tubes main tube was filled with water through a
pipette
Bunsen burner was used to heat the flask
Flask should be lifted from the furnace and shaken properly in
frequent
intervals till the liquid boils steadily.
Boiling was continued up to the maximum collection of oil.
Flame was adjusted properly to allow the sample for cooling
After the complete draining of liquid in condenser volume of the
oil was
measured102.
EXTRACTION:-
Methanol Extraction:-
Extraction of Flower, Stamens and flower buds of Nagakesara (Mesua
ferrea
Linn) is carried out according to the A.P.I procedures.
Ingredients: - a) Powdered drug – 5 gm
b) Methanol– 100 ml
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 58
Procedure:-
(i) About 5 gm of the powdered drug is weighed in a beaker and
transferred it to a
dry 250 ml Iodine flask.
(ii) 100 ml graduated cylinder is filled to the required mark with
the solvent, 90%
alcohol.
(iii) The flask is stoppered and set aside for 24 hours shaking
with frequently at the
interval of 6 hours (maceration).
(iv) Filter into a 50 ml cylinder after sufficient filtrate has
collected; transfer 25 ml
of the filtrate to a weighed 25 ml beaker.
(v) Evaporated to dryness on water bath and complete the drying in
an oven at
1000C for about 10 –15 minutes.
(vi) Cooled in desiccators and stored in glass bottle use for
HPTLC
Aqueous Extraction (Water extract):-
- Water (95 ml)
Procedure:-
(vii) About 5 gm of the powdered drug is weighed in a beaker and
transferred it to a
dry 250 ml Iodine flask.
(viii) 100 ml graduated cylinder is filled to the required mark
with the Methanol and
add in iodine flask.
(ix) The flask is stoppered and set aside for 24 hours shaking with
frequently at the
interval of 6 hours (maceration).
(x) Filter into a 50 ml cylinder after sufficient filtrate has
collected; transfer 25 ml
of the filtrate to a weighed 25 ml beaker.
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 59
(xi) Evaporated to dryness on water bath and complete the drying in
an oven at
1000C for about 10 –15 minutes.
(xii) Cooled in desiccators and stored in glass bottle use for
HPTLC
Fluorescence Analysis:-
The dry powder of all samples, both extracts of samples were
treated with
Methanol, chloroform and these were observed under U.V. light to
evaluate the
fluorescence.
ALKALOID ESTIMATION:
5gm of the sample was weighed into a 250ml beaker and 200ml of 10%
acetic
acid in ethanol was added and covered and allowed to stand for
4hrs. This was filtered
and the extract was concentrated on water bath to one quarter of
the original volume.
Conc. ammonium hydroxide was added drop wise to the ext. until ppt.
was
complete103.
Procedures:
Preliminary phytochemical tests: are used to detect the presence of
various organic
functional groups, which is the indicative of type of
phytochemicals present in the
plant. These tests indicate the presence of different class of
constituents present in the
extract. Tests were performed as per the methodology mentioned by
Harborne JB,
1973 (Phytochemical Methods. Jackman H. (Ed.), London, p.
70.)104
The following tests have been carried out for Petroleum ether,
Chloroform, Alcoholic
and Aqueous extracts.
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 60
Tests for alkaloids
Dragendroff’s test: To a few mg of extract dissolved in alcohol, a
few drops of acetic
acid and Dragendroff’s reagent were added and shaken well. An
orange red
precipitate formed indicates the presence of alkaloids.
Wagners’s test: To a few mg of extract dissolved in acetic acid, a
few drops of
Wagner’s reagent was added. A reddish brown precipitate formed
indicates the
presence of alkaloids.
Mayer’s test: To a few mg of extract dissolved in acetic acid, a
few drops of Mayer’s
reagent was added. A dull white precipitate formed indicates the
presence of
alkaloids.
Hager’s test: To a few mg of extract dissolved in acetic acid, 3 ml
of Hager’s
reagent was added, the formation of yellow precipitate indicates
the presence of
alkaloids.
Tests for carbohydrates
Molisch’s test: To the extract, 1 ml of α-naphthol solution and
conc. sulphuric acid
were added along the sides of test tube. Violet colour formed at
the junction of the
two liquids indicates the presence of carbohydrates.
Fehling’s test: A few mg of extract was mixed with equal quantities
of Fehling’s
solution A and B. The mixture was warmed on a water bath. The
formation of a brick
red precipitate indicates the presence of carbohydrates.
Benedict’s test: To 5 ml of Benedict’s reagent, a few mg of extract
was added, and
boiled for two minutes and cooled. Formation of a red precipitate
indicates the
presence of carbohydrates.
Anthrone-sulphuric acid test: A few mg of the extract was mixed
with equal quantity
of anthrone and treated with two drops of conc. sulphuric acid. It
was then heated
Materials and Methods
Evaluation of Different Market samples of Nagakesara (Mesua ferrea
Linn.) by Pharmocognostic and Analytical Parameters 61
gently on a water bath. Dark green colour formed indicates the
presence of
sugar/glycoside.
Test for steroids
Libermann-Burchard test: To the extract dissolved in chloroform, 1
ml of acetic acid
and 1 ml of acetic anhydride were added, then heated on a water
bath and cooled. Few
drops of conc. Sulphuric acid was added along the sides of the test
tube. Appearance
of bluish green colour indicates the presence of steroids.
Salkowski test: The extract was dissolved in chloroform and equal
volume of conc.
Sulphuric acid was added. Formation of bluish red to cherry red
colour in chloroform
layer and green fluorescence in the acid layer indicates the
presence of steroids.
Test for saponins: To a few mg of extract, distilled water was
added and shaken.
Stable froth formation indicates the presence of saponins.
Test for tannins: To the extract, a few drops of dilute solution of
ferric chloride was
added, formation of dark blue colour shows the presence of
tannins.
Test for flavonoids
Shinoda’s test: To the extract in alcohol, a few magnesium turnings
and few
drops of conc. hydrochloric acid were added and heated on a water
bath. Formation
of red to pink colour indicates the presence of flavonoids.
Test for phenol
To the extract in alcohol, added two drops of alcoholic ferric chlo