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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 the drug in crude form and would help distinguish the 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 the 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 acetate 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 aromatic 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 teeth 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 this 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 the

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 the

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 content 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 determinations. 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 phytoconstituents.

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 method 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 temperature

(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

methanol for 48 hours, filtered and evaporated to dryness. The doses taken for

screening were 100 mg/kg and 300 mg/kg.

2) Ethyl acetate extract- Defatted powdered seeds were soaked in ethyl acetate 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 latency 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 ten

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 flattened 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 containing oil globules and prismatic crystals of calcium oxalate.


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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 testa, b) Perisperm

cells, c) Cluster crystals of calcium oxalate and d) Starch grains

Physicochemical parameters

Different physicochemical parameters for the 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 determined 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 tabulated 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. Ammonia Brown Black Black

10% potassium dichromate Brownish Orange Greenish Brown Dark brown

Sodium hydroxide in methanol 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 content has been

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

R2 = 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)

AB

SO

RB

AN

CE

y = 0.0961x + 0.0114

R2 = 0.9948

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12

CONCENTRATION (MCG/ML)

AB

SO

RB

AN

CE


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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 the standard compounds by NIST library. Out

of the six compounds separated compound 3 separated at retention time 8.71 and

compound 6 separated at retention 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 system

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 acetate 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 that 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 acetate extract

200 mg/kg

400 mg/kg

1±0.258***a

0.33±0.210***a



From the results it can be inferred that both methanolic and ethyl acetate

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: [email protected]

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