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Pharmacognostical and Physiochemical Parameters of

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Current Research in Pharmaceutical Sciences 2013; 3 (3): 87-91

ISSN 2250 – 2688 Received: 01/09/2013 Revised: 17/10/2013 Accepted: 23/10/2013

Baby Chauhan, Gopal Kumar and Mohammed Ali Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India

Pharmacognostical and Physiochemical Trachyspermum ammi (L.) Sprague (Fruits)

Parameters

of

Baby Chauhan, Gopal Kumar and Mohammed Ali ABSTRACT Trachyspermum ammi (L.) Sprague, syn. Carum copticum Benth. et Hook., commonly known as ajwain or Bishop’s weeds; belongs to the family 'Apiaceae'. its fruits yielded 2% to 4% brownish essential oil, with thymol as the major constituent (35% to 60%).The present study deals with the Pharmacognostical examinations like morphological and histological characters of fruits of T. ammi besides physiochemical, fluorescence and phytochemical analysis. The HPTLC fingerprintings of T. ammi (fruits) petroleum ether, dichloromethane, chloroform, methanol, ethanol extract developed in solvent system (toluene: ethylacetate; 93:7) and spotted thymol peaks at UV 366 nm. These observations will enable to standardize the botanical identity of the drug in its crude form. Data evolved in this exploration could be used in laying down pharmacopoeial principles for the drug studied, as standardization of herbal medicines is completely essential. Keywords: Trachyspermum ammi (L.) Sprague, thymol, pharmacognostical examinations, standardization.

1. INTRODUCTION Trachyspermum ammi (L.) Sprague, syn. Carum copticum Benth. et Hook., commonly known as ajwain or Bishop’s weeds is an erect, aromatic, annual herb with striate stem 1, white flowers and small brownish fruits. It belongs to the family 'Apiaceae'. Ajwain is grown in Iran, Egypt, Afghanistan and India2 (largely in Uttar Pradesh, Bihar, Madhya Pradesh, Punjab, Rajasthan, Bengal, Tamil Nadu and Andhra Pradesh). The fruits possess characterstic aromatic odour and pungent taste due to presence an essential oil mainly composed of thymol (50%), αcadinol, δ-cadinene, β-caryophyllene and carvacrol3, 4. They are used as antispasmodic, stimulant, tonic and carminative and to treat gastric discomfort 5, 6 . It inhibits the bacterial resistant microbial pathogens and is useful as a plant based antibiotic. The present study provides the pharmacognostic evaluation, i.e. anatomical and microscopic characteristics, physic-chemical parameter, preliminary phytochemical screening and high performance thin layer chromatography (HPTLC) fingerprinting profiles for this plant.

2.

MATERIALS AND METHODS

Correspondence Gopal Kumar Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India E mail: [email protected]

2.1 Plant material The fruits of T. ammi were collected from the local market of Khari Baoli, Delhi and identified by Prof. M. P. Sharma Department of Botany, Jamia Hamdard, New Delhi. A sample of 87 plant material was deposited in the herbarium of the Phytochemistry Reasearch Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi with a voucher specimen number PRL/ JH / 11/ 03.

Current Research in Pharmaceutical Sciences 2013; 3 (3): 87-91

2.2 Pharmacognostical studies The dried powder of the plant (70 g) was extracted with water for 72 hrs. It was filtered and dried on water bath. The percentage yield of the aqueous extract was 3 % w/w. 2.2.1 Macroscopical character The fruits are about 1.5-3.0 mm long and 1.2-2.8 mm wide, ovoid, mainly occur as entire cremocarps with pedicel attached or detached with bifid stylopod and glabrous cremocarps. Dorsal surface convex with five equally distinct, longitudinal primary ridges; at the summit curved stylopodium, commissural surface flat, showing darker and light coloured longitudinal bands, former representing the position of vittae and vascular bundles ; odour aromatic; taste is slightly bitter giving a sensation of warmth to tongue 7.

(A) Endocarp in surface view (B) Detached protuberance (C) Microrosette crystals (D) Striated cuticle in surface view (E) Pitted fibres (F) Endosperm containing microrosette crystal of Ca-oxalate

Figure 1.3: Powder analysis of T.ammi fruits

2.2.2 Microscopical characters

2.3 Preliminary Phytochemical Screening

Epicarp is composed of polygonal cells. In the mesocarpic region, reticulate and lignified parenchyma are seen at vascular strands. Endocarp consists of narrow elongated cells having a parquetry arrangement. Tracheids show helical thickening. Polyhedral, thick walled endosperm contains aleurone grains and oil globules. Vittae are six in number, four on the dorsal surface at the mesocarpic region below the secondary ridges and two on the commissural surface of the mericarp. Vittae long, slender composed of thin walled polygonal cells and is lined by an epithelium of small polygonal tubular cells; 10-15 separate, septum transverse or curved.

The phytochemical screening involves testing of different plant extracts for their contents of different classes of compounds 8, 9, 10 . Preliminary phytochemical screening of the extract for different types of chemical constituents should be followed by qualitative chemical tests to give general idea regarding the nature of chemical constituents present in the crude drugs (table 1.1) 11, 12. 2.4 Physicochemical parameters 2.4.1 Fluorescence analysis Many herbs fluorescence when cut surface or powder is exposed to UV light and this can help in their identification method. Powdered drug (40 mesh) was treated with different reagents and examined under UV light (254 and 366 nm) (Table 1.2). 2.4.2 Determination of ash value

Figure 1.1: Trachyspermum ammi (L.) Sprague, fruits

This parameter is used for determination of inorganic materials, e.g., carbonates, silicates, oxalates and phosphates. Total ash – The ground drug (2 g) was incinerated in a silica crucible at a temperature not exceeding 4500 C until free from carbon. It is then cooled and weighed to get the total ash content (Table 1.3). Acid insoluble ash – The ash was boiled with 25 ml of dilute HCl (6N) for 5 minutes. The insoluble matter collected on ash-less filter paper, washed with hot water and ignited at a temperature not exceeding 4500C to a constant weight (Table 1.3).

Figure 1.2: T.S. of T.ammi fruits

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Current Research in Pharmaceutical Sciences 2013; 3 (3): 87-91

Water soluble ash – The ash was dissolved in distilled water, the insoluble part collected on an ash-less filter paper and ignited at 4500C to a constant weight (Table 1.3).

S.No.

5

Chemical treatment Powder such 1N NaOH water 1N NaOH methanol Iodine water 1 N HCl

6

Conc. HNO3

_

7

Conc. H2SO4

Saponins

_

8

50% KOH

8

Mucilage

_ 9

50% H2SO4

9

Resins

_

10

50% HNO3

10

Lipids / fats.

+

Table-1.1. Preliminary phytochemical screening of methanolic extract T. ammi (fruits)

1

S. No.

2

Constituents T. ammi _

1

Alkaloids

2

Carbohydrates

_

3

Glycosides

+

4

Tannins

_

5

Flavonoids

+

6

Proteins and free amino acids

7

3 4

as in in in

Day light

At 254 nm

At 366 nm

Light brown Yellowish brown Yellowish brown Black

Brown

Fluorescent yellow Yellow

Brown

Greyish yellow Yellow

Reddish brown Brown Yellowish brown Brown

Reddish brown Table-1.3: Ash values of T. ammi

Greyish yellow Brown Brown

Greyish yellow Greyish yellow Yellow Yellow

Pale green Fluorescent blue Brown Fluorescent blue Light green Fluorescent blue Light green Fluorescent blue

+ Present, - Absent Values (% w/w) 2.4.3

Determination of extractive values

S.No

These values provide an indication of the extent extractive values and are determined according to the method described in pharmacopoeia.

1 2

Individual extractives - The air-dried coarse drug powder (5 g) was macerated with solvent (100ml) in a closed flask for 24 hours, shaking frequently during every six hours and allowing to stand for 24 hours. It was filtered rapidly, taking precaution against loss of the solvent. The filtrate was evaporated to dryness in a tared flat bottom dish, dried at 1050C, to constant weight and weighed (Table 1.4).

3

2.4.4

Ash Values

mean (n=3) ±SD

Total ash

8.5±0.006

Water soluble ash

7.7±0.01

Acid insoluble ash

0.17±0.01

Loss on drying

This parameter determines the amount of moisture as well as the volatile components present. The powdered drug sample (10 g) was placed on a tared evaporating dish, dried at 1050C for 6 hours and weighed. The drying was continued until two successive reading matches each other or the difference between two successive weighing was not more than 0.25% of constant weight. The loss of drying (LOD) was found to be 4.5±0.036 % w/w [mean (n=3) ±SD].

Successive soxhlet extractives - The powdered material of the drug (5 g) was packed in a Soxhlet apparatus and subjected to successive extraction with different solvents like petroleum ether, benzene, chloroform, methanol and water. The extracts were evaporated to dryness and their constant extractive values were recorded (Table 1.4). Table 1.2: Fluorescence behaviour of powdered drug of T. ammi

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Current Research in Pharmaceutical Sciences 2013; 3 (3): 87-91

2.5 Table-1.4: Individual and suceessive extractive values of T .ammi fruits Individual extractive value

Values (%w/w) mean(n=3)±SD

Successive extractive value

Values (%w/w) mean(n=3)±SD

Petroleum Ether extract Chloroform extract Methanol extract Acetone extract Hydroalcohol ic extract Water extract

5.4±0.03

Petroleum Ether extract Chloroform extract Methanol extract Acetone extract Hydroalcoh olic extract Water extract

5.4±0.02

5 ±0.01 6.2±0.02 4.4±0.01 6.6± 0.01 15.2±0.03

2.5.1

HPTLC fingerprinting Preparation of plant material

The powdered plant material (15 g) was extracted individually with 100 ml each of methanol, Petroleum ether, Dichloromethane, Chloroform, Ethanol by sonicator for 30 minute and filtered. Each extract was concentrated under reduced pressure separately.

2.8±0.01 2.5.2

TLC Fingerprinting

7.6±0.006 Sample spots were separated on TLC plates using solvent system of toluene: ethyl acetate (93:7) as developing solution 13. Various visualisation techniques were used to obtain best TLC fingerprint, like UV radiation at 254 nm, UV 366 nm, iodinesation and spray reagents, e.g., anisaldehyde, vanillin and sulphuric acid 14, 15 .

3.6±0.02 12 ±0.04 19.4±0.05

2.5.3

HPTLC Scanning

The developed plate were scanned under the Camag HPTLC scanner IV for the densitometric observation. The plate was scanned with UV 254 and 366 nm using Toulene:Ethyl acetate (19:1) as a solvent system. 3. RESULT AND DISCUSSION

A

B

C

D

In the present study the pharmacognostic evaluation, i.e., anatomical and microscopic characteristics, physic-chemical parameters, preliminary phytochemical screening and high performance thin layer chromatography (HPTLC) fingerprinting profiles were performed.

E

Figure-1.4: HPTLC fingerprinting of Petroleum ether extract (A); Dichloromethane (B); Chloroform extract (C); Methanol extract (D); Ethanol extract (E) at UV 366 nm

A transverse section of the fruits showed the presence of polygonal cells, reticulate and lignified parenchyma, tracheids, aleurone grains, oil globules and vittae (Figure 1.2). The powder microscopy of the T. ammi showed the presence of thin walled parenchymatous cells in groups; pitted fibers and endosperm containing microrosette crystal of Caoxalate. The measurements of various cell and tissues are provided respectively (Figure 1.3).

F

Preliminary phytochemical test of the methanol extract of the drug indicated the existence of glycosides, flavonoids and terpenoids (Table 1.1).

G

Figure-1.5: 3-D display of the chromatogram of methanolic extract of T. ammi (fruits) at UV 254 nm (F) and 366 nm (G).

The fruit powder was studied for its physico-chemical constants which included ash values, fluorescence analysis, individual and successive extractive values and LOD. It exhibited

90

Current Research in Pharmaceutical Sciences 2013; 3 (3): 87-91

5.

higher hydroalcohlic and water extractive contents (Table 1.2, 1.3 and 1.4).

Bentely L S, Trimen H. Medicinal Plants. Asiatic Publishing House, New Delhi, India. (1999) 320-322.

HPTLC fingerprinting was performed for the different extracts, e.g., petroleum ether, dichloromethane, chloroform, methanol and ethanol of T. ammi (fruits). The best solvent system for T. ammi (fruits) extracts was found to be toluene: ethyl acetate (93:7) which showed better separation and high resolution.

6.

Krishnamoorthy V, Madalageri MB. Bishop weed (Trachyspermum ammi): an essential crop for north Karnataka. J. Med. And Aromat. Plants Sci. 1999; 21(4): 996- 998.

7.

The HPTLC fingerprintings of T. ammi (fruits) petroleum ether, dichloromethane, chloroform, methanol and ethanol extracts developed in the solvent system (toluene: ethylacetate; 93:7) were scanned at two wavelengths UV 254 nm and UV 366 nm. At UV 254 nm, 9 peaks in the petroleum ether; 10 peaks in dichloromethane, 11 peaks in chloroform, 6 peaks in methanol, 7 peaks in ethanol were observed in chromatogram. Major peaks with high intensity of compounds were observed at Rf values 0.91, 0.74, 0.63, 0.59 in petroleum ether extract; 0.81, 0.7, 0.63, 0.54 in dichloromethane, 0.78, 0.70, 0.63, 0.54 in chloroform; 0.68, 0.59, 0.39, 0.31 in methanol extract; 0.71, 0.60, 0.48, 0.42 in ethanol extract.

The Ayurvedic Pharmacopoeia of India . Government of India, Ministry of Health and Family Welfare, Department of Ayush. (2011) 170-71.

8.

M Ali. Textbook of Pharmacognosy, CBS Publishers and Distributors, New Delhi. (1997).

9.

Esposito AM, Brown P, Tejeira I, Buitrago R, Barris L, Sanchez C, Gupta M.P.

Pharmacological Screening of Panamanian Medicinal

Plants. J. Crude Drug Res. 1985; 23 (1): 17-25.

10. Samualsson G, Kyeremater G and Farah MH. Preliminary chemical

4. CONCLUSION

characterization of pharmacologically active compounds in aqueous plant extracts. J. Ethanopormacol. 1985; 14 (2): 193-201.

The pharmacognostic study of the fruits of Trachyspermum ammi (L.) Sprague has shown certain microscopic feature and preliminary phytochemical data of diagnostic values. Physico-chemical constants such as solubility, ash values, individual or successive extractive values and other parameters including fluorescence analysis and preliminary phyto-chemical studies are important parameters for standardizations of the drug.

11. Finar IL. Organic Chemistry, Stereochemistry and the Chemistry of Natural Products. ELBS, Longman Singapore Publication (P) Ltd., Singapore. (1975) 656-657.

12. Trease GE and Evans WC. Pharmacognosy, Bailliere Tindall, London,

REFERENCES 1.

12th edn. (1985).

Nagalakshmi S, Shankaracharaya NB, Naik JP, Rao LJ.M. Studies on 13. Wagner H, Bladt S, Zgainsk E.M. Plant Drug Analysis: A Thin Layer chemical and tehnological aspects of Ajowan (Trachyspermum ammi Chromatography Atlas, Springer Verleg, Berlin. (1984). (L.) syn. Carum copticum (Hiern) seeds. J. Food Sci. Technol. 2000; 37: 277-281.

2.

3.

4.

14. Ali M. High performance thin layer chromatography for herbal and

Zargari A. Medicinal Plants. Tehran University Publications. Vol. 2,

pharmaceutical product analysis. I J Pharmaceutical Education. 1998;

(1996) 975.

32(1): 15-19.

Balbaa SI, Hilal SH and Haggag MY. The volatile oil from the herb and

15. Mukherjee PK. Quality control of herbal drugs, an application to

fruits of Carum copticum at different stages of growth. Planta Med.

evaluation of botanical HPTLC, printed at syndicate binders, New Delhi;

1973; 23: 311–320.

(2002) 494-95.

Baytop T and Sütlüpinar N. Characteristics of “Nanahan” cultivated in Anatolia and its volatile oil. J. Fac. Pharm. I˙stanbul. 1986; 22: 73–76.

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Pharmacognostical and Physiochemical Parameters of

Current Research in Pharmaceutical Sciences 2013; 3 (3): 87-91 ISSN 2250 – 2688 Received: 01/09/2013 Revised: 17/10/2013 Accepted: 23/10/2013 Baby C...

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