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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 1

Indukaka Ipcowala College of Pharmacy, P.O.Box-53, Po.Vitthal Udyognagar, Dist. Anand-388121, Gujarat, India. 2 A. 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 0

(21±1 C) 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 Figure 2 T.S. of seed of Amomum subulatum Roxb.

c

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. Percentage

No. Determination

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

Brownish Black

Creamish black

Sodium hydroxide in methanol Brownish 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. No.

Extracts

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 y = 0.0915x - 0.0072 R2 = 0.9996

1 0.9

1.2 1 ABSORBANCE

0.8 ABSORBANCE

y = 0.0961x + 0.0114 R2 = 0.9948

0.7 0.6 0.5 0.4 0.3 0.2

0.8 0.6 0.4 0.2

0.1 0

0 0

5

10

CONCENTRATION (MCG/ML)

15

0

2

4

6

8

10

12

CONCENTRATION (MCG/ML)

equivalent. a

b

Figure 4 Calibration curve of standard gallic acid and quercetin

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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

Groups

Mean ± SEM

Normal

4.16±0.6009

Model control

10.83±0.4014***a

Methanol extract (MEOH) 100 mg/kg

12±0.6831***b

300 mg/kg

11.16±0.401***b

Ethyl acetate extract 200 mg/kg

10.16±0.542***b

400 mg/kg

11.16±0.4014***b

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 www.pharmasm.com

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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 Groups

Mean ± SEM

Model control

9.66±0.6146

Methanol extract (MEOH) 100 mg/kg

1±0.2582***a

300 mg/kg

0.33±0.210***a

Ethyl acetate extract 200 mg/kg

1±0.258***a

400 mg/kg

0.33±0.210***a

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.

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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. REFERENCES 1. Wealth of India: Council of Scientific and Industrial Research, New Delhi, Publication and Information Directorate, CSIR, Vol. A, 1985: 227-229. 2. Reviews on Indian Medicinal plants: Indian Council of Medical Research, New Delhi, India, Vol. 2, 2004: 215- 218. 3. Chatterjee A, Pakrashi SC: Treaties on Indian Medicinal Plants: National Institute of Science Communication and Information Resources, New Delhi, India, Vol. 3, 2003: 146-147. 4. Brain KR, Turner TD: The practical evaluation of Phytopharmaceuticals: WrightScientechnica Bristol, 1975: 4-9. 5. Trease GE, Evans WC: Trease and Evans Pharmacognosy: Harcourt brace & Co. Asia, Pvt. Ltd. and W.B. Saunders Company Ltd., Edition 15, 2002: 542-543. 6. Ayurvedic Pharmacopoeia-I: Controller of

Publication, Civil lines, Delhi, India,

Edition 1, Vol. 2, 158-159. 7. Chase CR, Pratt R, Fluorescence of powdered vegetable drug wirh particular reference to development of a system of identification: Journal of American Pharmaceutical Associaton 1948; 38: 324-331 8. Mukherjee PK: Quality Control of Herbal Drugs: Business Horizon Pharmaceutical Publication, New Delhi, 2008: 187-191, 214-215. 9. Indian Pharmacopoeia: The Indian Pharmacopoeia commission, Ghaziabad, Vol. I, 2007: 78 10. WHO/ PHARM/92.559/ rev.1: Quality control methods for Medicinal Plant Materials, Organization Mondiale De La Sante, Geneva, 1992: 9, 31, 28, 61. 11. Ayoola GA, Ipav SS, Sofidiya MO, Coker HA and Odugbemi TO: Phytochemical screening and free radical scavanging activities of the fruit and leaves of Allanblackia floribunda Oliv. International journal of health research 2008; 1(2): 8793. 12. Kokate C, Purohit A, Gokhale S: Practical Pharmacognosy: Vallabh Prakashan, New Delhi, India, Edition 10, 1999: 107-111.

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13. Wagner H, Bladt S: Plant drug analysis- A Thin Layer Chromatography Atlas: Springer publication, Edition 2: 5, 210, 306,318-320, 322-326. 14. Stahl E: Thin layer Chromatography- A Laboratory Handbook: Springer International, New York, Editon 2, 1969: 460-482. 15. Shahidi F, Chavan UD, Bal AK and Mackenzie DB: Chemical composition of beach pea (Lathyrus maritimus Linn.) plant parts: Food Chemistry 1999; 64: 39-44. 16. Okwu GA, Josiah C: Evaluation of Chemical composition of two Nigerian medicinal plants: African Journal of Biotechnology 2006; 5(4). 17. Agrawal SS, Paridhavi M: Herbal Drug Technology: University Press, Hyderabad, India, 2007: 557 – 560. 18. Skoog, Holler, Nieman: Principles of Instrumental Analysis: Thomson brooks, Bangalore, Edition 5: 543-550. 19. Vogel G: Drug discovery and evaluation: Springer publication, New York, Edition 2, 2002: 696, 716.

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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