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Vol-1, Issue-1, 2010 Shukla et al
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PHARMA
SCIENCE
MONITOR
AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES
PHARMACOGNOSTICAL, PRELIMINARY PHYTOCHEMICAL
STUDIES AND ANALGESIC ACTIVITY OF AMOMUM
SUBULATUM ROXB
S.H. Shukla*1, H. A. Mistry2, V.G. Patel2 and B.V. Jogi2
1Indukaka Ipcowala College of Pharmacy, P.O.Box-53, Po.Vitthal Udyognagar, Dist. Anand-388121,
Gujarat, India.
2A. R. College of Pharmacy and G. H. Patel Institute of Pharmacy & Institute of Science & Technology for
Advanced Studies & Research, Vallabh Vidyanagar-388120, Gujarat, India
ABSTRACT
The present study deals with the pharmacognostic, preliminary phytochemical studies
and pharmacological activity of seeds of Amomum subulatum Roxb. The present paper
highlights the macroscopic and microscopic characters of seeds, physic-chemical
evaluation, preliminary phytochemical studies and analgesic activity of the seeds. These
observations would be of immense value in the botanical identification and
standardization of t he drug in crude form and would help distinguish t he drug from its
other species. Phytochemical standardization parameters such as moisture content,
total ash, water soluble and acid insoluble ash, alcohol soluble and water soluble
extractives were determined. Preliminary identification of phytoconstituents was
performed. GC-MS study of the volatile oil obtained from t he seeds was carried out and
six compounds were separated. The mass fragmentation studies revealed the presence
of 1, 8- cineole and caryophyllene in the volatile oil. Methanolic extract and ethyl
acetat e extract of seeds of plant were investigated for the analgesic activity using hot
plate method and writhing method.
Keywords: Amomum subulatum, Pharmacognosy, Phytochemical, GC-MS, Analgesic
activity
INTRODUCTION
Amomum subulatum Roxb. (Family: Zingiberaceae) commonly known as Large
cardamom, is a tall perennial herb found in Eastern Himalayas and sub-Himalayan
region of West Bengal, Assam and Sikkim. The seeds are aro matic pungent, stimulant,
stomachic, alexipharmic and astringent. They are prescribed for treatment of
indigestion, vomiting, biliousness, abdominal pain and rectal diseases. A decoction of
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the seed is used as a gargle in affections of the t eeth and gums. The seeds are also used
as antidote to snake bite and scorpion sting. The pericarp is used in headache and heals
stomatitis. In Ayurvedic and Unani medicine, large cardamom are used as preventive as
well as a curative for throat trouble, congestion of lungs, inflammation of eyelids,
digestive disorders and in the treatment of pulmonary tuberculosis. The seeds contain
mainly essential oil, flavonoids, carbohydrates and fats [1-3].
Inspite of numerous medicinal uses attributed to t his plant, there is no
pharmacognostical report on the microscopical and other physicochemical standards
required for the quality control of the crude drugs. Hence the present investigation
includes macroscopical and microscopical evaluation, determination of physicochemical
constants, preliminary phytochemical screening and analgesic activity of A. subulatum.
MATERIALS AND METHODS
Plant material
Fruits were collected from the local market of Bilimora, District Navsari, Gujarat
and its identification was confirmed by Dr. Geetha K. A., Senior Scientist, Directorate of
Medicinal and Aromatic Plants Research, Boriavi, Anand, Gujarat. A voucher specimen of
the plant (no.HAM/AS-1/1/ARGH-09) was deposited in the herbarium of t he
Department of Pharmacognosy, A. R. College of Pharmacy, Vallabh Vidyanagar, Gujarat.
Pharmacognostic Studies
The macroscopy of the seeds were studied by comparing their macroscopical
characters mentioned in the literature. The free hand cut transverse sections of t he
seed were taken and various parts of it were observed under the microscope for further
confirmation and identification of the plant. Further, the histological examination of the
clear powder mounts of the seed was carried out using reported methods [4-6].
Physicochemical parameters
Different physicochemical parameters such as moisture cont ent and total solids
content, ash values and extractive values of the crude drug powder was carried out
using reported methods by subjecting the seed powder to various det erminations. The
fluorescence and general behaviour of the powder of seeds under visible and UV lights
(254 and 365 nm) were carried out [7-10].
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Preliminary phytochemical screening
20 gm of the air-dried powdered plant material was extracted successively using
solvents like petroleum ether, toluene, chloroform, ethyl acetate, methanol and water
in a Soxhlet’s extractor. The extracts were then subjected to various qualitative tests
using reported methods to determine the presence of various phytoconstitue nts.
Various extracts were also subjected to Thin Layer Chromatography in order to separate
and detect the presence of different phytoconstituents. Determination of total phenolic
and total flavonoid content were studied according to Folin-ciocalteu method and
colorimetric aluminium chloride met hod respectively [11-16].
GC-MS
Volatile oil was extracted from the powder of seeds using Clevenger’s apparatus
and it was subjected to GC-MS studies [17, 18].
Pharmacological studies
Animals
Albino Swiss mice weighing 25-30 g were housed at ambient temperatur e
(21±10C) and relative humidity (55±5%) with fixed 12 h light /dark cycles in animal house
of A. R. College of Pharmacy, Vallabh Vidyanagar. Animals were fed with a standard
pellet diet (Pranav Agro Ltd., Baroda) and were provided with water ad libitum.
Preparation of extracts
1) Methanol extract- Defatted powdered seeds of A. subulatum (50 g) were soaked in
met hanol for 48 hours, filtered and evaporat ed to dryness. The doses taken for
screening were 100 mg/kg and 300 mg/kg.
2) Ethyl acet ate extract- Defatted powdered seeds were soaked in ethyl acet ate for 48
hours, filtered and evaporated to dryness. The doses taken for screening were 200
mg/kg and 400 mg/kg.
Hot plate method
Groups of 6 mice in each group of either sex weighing 18 to 22 g were used.
Group I – Normal group with no treatment
Group II – Model Control group which received the standard drug
Group III, IV – Methanol extract at dose of 100 mg/kg and 300 mg/kg respectively
Group V, VI – Ethyl acetate extract at dose of 200 mg/kg and 400 mg/kg respectively
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The animals are placed on the hot plate and the time until either licking or
jumping occurs is recorded by a stop-watch. The lat ency is recorded before and after 20,
60 and 90 min following oral or subcutaneous administration of the standard or the test
compound.
Writhing method
Groups of 6 mice in each group of either sex weighing 20 to 25 g were used.
Acetic acid in a concentration of 0.6% is injected intraperitoneally (10 ml/kg body
weight).
Group I – Normal group with no treatment
Group II – Model Control group which received acetic acid
Group III, IV – Methanol extract at dose of 100 mg/kg and 300 mg/kg respectively
Group V, VI – Ethyl acetate extract at dose of 200 mg/kg and 400 mg/kg respectively
Test animals were administered the extract prior to acetic acid administration.
The mice were placed individually into glass beakers and observed for a period of t en
min and the number of writhes was recorded for each animal. For scoring purposes, a
writhe is indicated by stretching of the abdomen with simultaneous stretching of at
least one hind limb [19].
Statistical analysis
Results were expressed as mean ± standard error of the mean (SEM). Any
significant difference between mean was assayed by the Student- Newman- Keuls
Multiple Comparision test. 95% level of significance (p<0.001) was used for the
statistical analysis. Statistical analysis was performed using GraphPad In Stat software.
RESULTS
Macroscopy
Seed were 0.4 cm long, 0.3 cm wide, irregularly ovoid with 3 flatt ened faces
covered externally with a colorless, membranous aril. The colour of the seed was brown
to dark brown, odour aromatic and taste aromatic pungent.
Microscopy
Seed showed a very thin membranous aril composed of several layers of
collapsed cells cont aining oil globules and prismatic crystals of calcium oxalat e.
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Testa consisted of single layered epidermis of rectangular cells followed by 1-2
layers of collapsed, thin-walled parenchymatous cells, beneath which a single layered
large rectangular cells containing oil globules were present, which were internally
surrounded by several layers of flattened, thin-walled, parenchymatous cells.
Perisperm consisted of polygonal, thin-walled, parenchymatous cells containing
round to oval starch grains, and cluster crystals of calcium oxalate. Perisperm was
surrounded externally by thick-walled, sclerenchymatous, radially elongated dark brown
beaker cells. Perisperm enclosed the endosperm and embryo, both composed of
polygonal, thin-walled, parenchymatous cells.
Figure 1
Macroscopy of seeds of Amomum subulatum Roxb.
a b c
Figure 2
T.S. of seed of Amomum subulatum Roxb.
a) Whole section showing Testa, Perisperm and Embryo.
b) Testa and Perisperm- Epidermis (EP), Collapsed cells (CP), Rectangular cells (RC),Parenchyma cells
(PA)
c) Embryo- Rectangular cells (RC), Oil globules (OG), Parenchyma cells (PA)
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Powder study
Organoleptic characters:
Appearance : Coarse powder
Colour : Light brown
Odour : Aromatic
Taste : Aromatic
a b c d
Figure 3
Powder study of seeds of Amomum subulatum Roxb. a) Fragments of test a, b) Perisperm
cells, c) Cluster crystals of calcium oxalate and d) St arch grains
Physicochemical parameters
Different physicochemical parameters for t he purpose of standardization such as
total solids, moisture content, ash value (total ash, water soluble ash, acid insoluble ash)
and extractive values (water soluble, ethanol soluble and ether soluble) for seeds of A.
subulatum were det ermined and given in Table 1.
Fluorescence analysis
The powder of seeds were subjected to fluorescence analysis as per the standard
procedure and shown in Table 2.
Preliminary phytochemical screening
The extracts were subjected to preliminary phytochemical analysis to determine
the presence of various phytoconstituents and results are tabulat ed in Table 3.
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TABLE 1: PROXIMATE ANALYSIS OF SEEDS OF AMOMUM SUBULATUM
ROXB.
No. Determination Percentage
w/w
1. Moisture content 8.6
2. Total solids 91.4
3. Total ash value 5
4. Acid-insoluble ash value 1.5
5. Water-soluble ash value 3.5
6. Alcohol soluble extractive value 4.88
7. Water soluble extractive value 40
8. Ether soluble extractive value 4
TABLE 2: FLUORESCENCE ANALYSIS OF AMOMUM SUBULATUM SEED
POWDER.
Reagent Day light UV 254 UV 365
1M sodium hydroxide Brown Dark brown Dark brown
1% picric acid Brown Greenish brown Brown
Acetic acid Pinkish brown Greenish brown Brown
1M Hydrochloric acid Brown Dark brown Brown
Dilute nitric acid Brown Blackish brown Brown
5% iodine Creamish black Creamish brown
Brown
5% ferric chloride Yellowish brown Yellowish green Black
Methanol Brown Brown Brownish Black
50% nitric acid Brown Green Brown
1M sulphuric acid Light brown Greenish brown Brown
dil. Ammo nia Brown Black Black
10% potassium dichromat e Brownish Orange Greenish Brown Dark brown
Sodium hydroxide in met hanol
Brownish Black Brownish Black Creamish black
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TABLE 3: THE RESULTS OF CHEMICAL TESTS PERFORMED WITH THE
EXTRACTS OBTAINED BY SUCCESSIVE SOLVENT EXTRACTION OF
SEEDS OF AMOMUM SUBULATUM ROXB.
Extracts
No. Chemical tests
PE T C A M W
1. For Alkaloids * * - * - -
2. For Carbohydrates * * * * + +
3. For Glycosides * * * * - -
4. For Steroids and Triterpenoids * * * * - -
5. For Flavonoids * * * + + -
6. For Tannins * * * - - -
7. For Fixed oils and Fats + + * * * *
8. For Proteins and Amino acids * * * * - -
9. For Phenolics * * * + + -
*: not done +: positive -: negative
Total phenolic content has been reported as gallic acid equivalent with reference
to standard curve (Figure 4a). Total phenolic content in seeds was found to be 0.00366
% w/w calculated as gallic acid equivalent. The total flavonoid cont ent has be en
calculated as quercetin equivalent with reference to standard curve (Figure 4b). Total
flavonoid content in seeds was found to be 0.0361% w/w calculated as quercetin
equivalent.
a b
Figure 4
Calibration curve of standard gallic acid and quercetin
y = 0. 0915x - 0.0072
R
2
= 0.9996
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 5 10 15
CONCENTRATION (MCG/ML)
ABSORBANCE
y = 0.0961x + 0.0114
R
2
= 0.9948
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
CONCENTRATION (MCG/ML)
ABSORBANCE
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GC-MS analysis of volatile oil
Volatile oil separated by Clevenger’s apparatus was subjected to GC-MS for the
separation and identification of constituents present in the oil. Six compounds were
separated at different retention time. The mass fragmentation pattern of all the
compounds separated was matched with t he st andard compounds by NIST library. Out
of the six compounds separated co mpound 3 separated at retention time 8.71 and
compound 6 separated at ret ention time 16.94 were found to be 1, 8- cineole and
caryophyllene respectively.
TABLE 4: RETENTION TIME (RT) AND % AREA OF ISOLATED
COMPOUNDS.
Compound
Retention time % area
1 6.05 1.62
2 7.26 2.85
3 8.71 68.02
4 11.59 3.78
5 11.90 14.04
6 16.94 5.79
Pharmacological activity
In the preliminary pharmacological screening, methanolic extract at dose 100
mg/kg and 300 mg/kg and ethyl acetate extract at dose 200 mg/kg and 400 mg/kg
of seeds of plant were investigated for the analgesic activity using hot plate method and
writhing method.
The analgesic effect of the ethyl acetate and methanol extract using the hot
plate and writhing method are shown in Table 5, 6 and Figure 5, 6
The hot plate method is used specifically to screen the central nervous syste m
acting analgesic activity of drug. In this method both the extracts of plant showed
significant (p<0.001) analgesic action 60 min after its administration.
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TABLE 5: RESULTS OF THE ANALGESIC EFFECT OF A. SUBULATUM
SEEDS USING HOT PLATE METHOD
SEM-Standard Error of Mean, ***- p<0.001, a- all groups compared with normal group,
b- all groups compared with model control group
Figure 5
Graphical representation of the analgesic effect using the Hot plate method.
The acetic acid induced writhing response is used to screen peripheral acting
analgesics. The abdominal constrictions observed in this study are because of irritation
of peritoneal cavity caused by administration of acetic acid. In the present study, the
Groups Mean ± SEM
Normal 4.16±0.6009
Model control 10.83±0.4014***a
Methanol extract (MEOH)
100 mg/kg
300 mg/kg
12±0.6831***b
11.16±0.401***b
Ethyl acetat e extract
200 mg/kg
400 mg/kg
10.16±0.542***b
11.16±0.4014***b
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analgesic action of both the extracts can be attributed to the blockage of release of
endogenous mediators of pain i.e. prostaglandins. It suggests t hat the plant has some
inhibitory action on cyclooxygenase pathway which is involved in synthesis of
prostaglandins biosynthesis.
TABLE 6: ANANLGESIC EFFECT OF A. SUBULATUM SEEDS USING
WRITHING METHOD
SEM-Standard Error of Mean, ***- p<0.001, a- all groups compared with normal group,
Figure 6
Graphical representation of the analgesic effect using the Writhing method.
Groups Mean ± SEM
Model control 9.66±0.6146
Methanol extract (MEOH)
100 mg/kg
300 mg/kg
1±0.2582***a
0.33±0.210***a
Ethyl acetat e extract
200 mg/kg
400 mg/kg
1±0.258***a
0.33±0.210***a
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From the results it can be inferred that both methanolic and et hyl aceta te
extract of seeds of A. subulatum possessed significant (p<0.001) analgesic activity.
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For Correspondence:
Dr. S.H. SHUKLA
Indukaka Ipcowala College of Pharmacy, P.O.Box-53, Po.Vitthal Udyognagar, Dist.
Anand-388121, Gujarat, India.
Email: shshukla@rediffmail.com