Trachyspermum ammi Linn. seeds consist of moisture, protein, fat, minerals, fiber, carbohydrates, calcium, phosphorus, iron, carotene, thiamin, riboflavin and niacin. Seeds are used to flavor savory dishes, including curries, legumes, breads (naan, pakora, paratha) and pastry snacks, especially in India, Iran, Ethiopia, and Afghanistan. The thymol from the essential oil of Trachyspermum ammi (Linn) Sprague seeds has shown the activities of strong germicidal, anti-spasmodic, aphrodisiac and fungicidal activities. Thymol is also used in toothpaste and perfumery. It is used in a steeped liquid form against diarrhea and flatulence³. In India the seeds are used as a household remedy for indigestion and abdominal colic, and used in poultices to relieve asthma and arthritis. It is customary in India to extract the fruits or the entire herb with diluted alcohol in order to prepare a tincture which is reported to be a powerful germicidal. A liquid preparation made by boiling ground-up ajava seeds in water, taken after intercourse, is thought to prevent implantation of a fertilized egg in the uterus. It relaxes spasms, improves digestion, increases perspiration, and is strongly antiseptic⁴.

It has been shown to possess anti-aggregatory effects⁵; anthelmintic⁶; anti hyperlipidaemic⁷ antifilarial⁸; insecticidal⁹; kidney stone inhibitory; molluscicidal^10-12 mosquito repellent¹³; and nematicidal activities¹⁴.

In Ayurvedic system of medicine it is mainly used in the treatment of respiratory, gastro-intestinal disorders and inflammatory conditions¹⁵. Oil is used to treat renal stones, psoriasis and to expel hookworms. In the Unani system of medicine it is used as an enhancer of body’s resistance¹⁶. Seeds possess stimulant qualities of capsicum, bitter property of chiretta and anti spasmodic qualities of as a foetida¹⁷.

Seeds contain 2.5 - 5% essential oil. The principal constituents of essential oil are phenols- thymol (35 - 60%), carvacrol (11%). The remainder of the oil is called thymene which contains p-cymene (50 - 55%), beta-pinene (4 - 5%), limonene with gamma-and betaterpinenes (30 - 35%)¹⁸. The essential oil distilled from aerial parts (flowers, leaves) of Trachyspermum ammi Linn. grown in Algeria, contains isothymol (50%) which is the dominant constituent before p-cymene, thymol, limonene and γ-terpinene.

MATERIALS AND METHODS:

Plant material:

Three samples of Trachyspermum ammi Linn. were collected from three different locations in Punjab State (Sample S1), (Sample S2) and (Sample S3). Powder of the samples was used for chemical analysis. Physicochemical studies were carried out on collected three samples as per the WHO/AOAC guidelines.^19,20

Analysis of Various Parameters:

Different physicochemical parameters like Total ash, acid insoluble ash, water soluble ash, ethanol soluble extractive value, water soluble extractive value, loss on drying, pH, TLC.

Procedures adopted for Physicochemical Parameter:

Moisture content:

4gm of the sample was taken and heated in an oven at 100- 105°C for 5 hours in a previously weighed 100 ml. cooled in desiccators and weighed. Repeated the procedure till constant weight is obtained. Calculated the percentage of loss in weight of the sample.

Total ash:

2gm of the sample was taken accurately in a previously ignitedd and tarred Silica dish. Made to spread the material evenly and ignited in a muffle furnace by gradually increasing the temperature to 600 °C until it turned to white, indicating the absence of carbon. Then cooled the dish in desiccators and weighed. Wherever the carbon free ash could not be obtained in this manner, cooled the dish and moistened the residue with about 2 ml of water or a saturated solution of Ammonium nitrate. Then dried on a water-bath, and then ignited in the muffle furnace to constant weight. Cooled the dish in a desiccator for 30 minutes, and then weighed. Calculated the percentage of total ash of air dried material.

Acid insoluble ash:

To the dish containing the total ash, added 25ml of 1:5 Hydrochloric acid, covered with a watch glass and boiled gently for 5 minutes. Rinsed the watch glass with 5 ml of hot water and added the washings to the dish. Collected the insoluble matter on an ash-less filter paper (Whatman No. 41) and washed with hot water until the residue is free from acid.

Transferred the filter paper containing the insoluble matter to the original dish, dried and ignited to constant weight. Cooled the dish in a desiccator for 30 minutes, and then weighed. Calculated the percentage of Acid insoluble - ash of the air-dried material.

Water-soluble extractive:

4 gm of the sample was taken in a glass stoppered flask, added 100 ml of distilled water, shaken occasionally for 6 hours and then allowed standing for 18 hours. Filtered the solution and pipette out 25ml of the filtrate in a pre-weighed 100 ml beaker and evaporated to dryness on a water bath. Kept it in an air oven at l05°C for 6 hours, cooled in desiccators for 30 minutes and weighed. Repeated the experiment twice, and taken the average value.

Alcohol-soluble extractive:

4 gm of the sample was taken in a glass stoppered flask, added 100 ml of distilled Alcohol, shaken occasionally for 6 hours and then allowed to stand for 18 hours. Filtered the solution and pipette out 25ml of the filtrate in a pre-weighed 100 ml beaker and evaporated to dryness on a water bath. Kept it in an air oven at 105°C for 6 hours, cooled in a desiccator for 30 minutes and weighed. Calculated the percentage of Alcohol extractable matter of the sample. Repeated the experiment twice, and taken the average value.

Thin layer chromatographic:

4gm of the sample was soaked in 40 ml of rectified spirit (90%) with occasional shaking for 18 hrs, boiled for 10 minutes and filtered. The filtrate was evaporated and concentrated and made up to 10 ml in standard flask. The 20 μl of the solution was applied on (E.Merck) Aluminium plate pre-coated with Silica gel 60 F254 of 0.2 mm thickness using Linomat IV applicator. The plate was developed in Toluene: Ethyl acetate:: 8:2 v/v. After air drying the plate was visualized in UV 254 and 366 nm and visible light the spots appeared.

RESULTS AND DISCUSSIONS:

The analytical results on physico-chemical parameters of the Trachyspermum ammi Linn. are tabulated in Table 1. Deterioration time of the plant material depends upon the amount of water present in plant material. If the water content is high, the plant can easily be deteriorated due to fungus. The loss on drying at 105°C in samples S1, S2 and S3 were found to be 7.9, 15.6 and 9.3 % w/w respectively. Total ash value of plant material indicated the amount of minerals and earthy materials attached to the plant material. Analytical results showed total ash value samples S1, S2 and S3 content were 8.1, 8.5 and 8.2 % w/w respectively. The amount of acid insoluble siliceous matter present in the plant samples were 0.76, 01.0 and 0.8 % w/w respectively. The water-soluble extractive value was indicating the presence of sugar, acids and inorganic compounds. The water soluble extractive value in the drug sample were 19.2 (S1), 18.5 (S2) and 17.9 (S3) % w/w and alcohol soluble extractive value were 16.7 (S1), 10.4 (S2) and 15.2 (S3) % w/w. The alcohol soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, glycosides, flavonoids and secondary metabolites in the plant sample. Other parameters, like pH (10% w/v aqueous solution) value of sample S1, S2 and S3 were 5.8, 5.7 and 5.5 respectively.

Table 1. Analysis of various physico-chemical parameters Trachyspermum ammi Linn.

S. No.	Parameters	Results
S. No.	Parameters	Sample 1	Sample 2	Sample 3
1.	pH (10% w/v aqueous solution)	5.8	5.7	5.5
2.	Total ash value (% w/w)	8.1	8.5	8.2
3.	Acid-insoluble ash (% w/w)	0.76	1.0	0.8
4.	Water-soluble extractive (% w/w)	19.2	18.5	17.9
5.	Alcohol soluble extractive (% w/w)	16.7	10.4	15.2
6.	Loss on drying at 105°C (% w/w)	7.9	15.6	9.3

The solution was applied on Merck Aluminium pre-coated plate with silica gel 60 F₂₅₄ of 0.2 mm thickness. The plates were developed in Toluene: Ethyl acetate (8: 2 v/v). After air dry the plates were visualized in UV 254 and spot not visualized in 366 nm. The plates were dried and then dipped in Vanillin- Sulphuric acid and heated at 105°C till the spots appeared. Then the plates were derivatized in Iodine chamber till the colour of the spots appeared TLC figures showed in Figure. 1 and Table 2, 3, 4.

After Derivatisation with Vanillin sulphuric acid in visible light

Figure 1. TLC profile of various samples of Trachyspermum ammi Linn.; (Toluene : Ethyl acetate :: 8:2 v/v)

Table 2. TLC profile of various samples of Trachyspermum ammi Linn.; (Toluene: Ethyl acetate:: 8:2 v/v) λ 254 nm

S. No	Sample 1		Sample 2		Sample 3
S. No	R_f	Colour	R_f	Colour	R_f	Colour
1.	0.67	Green	0.8	Green	0.77	Green

Table 3. TLC profile of various samples of Trachyspermum ammi Linn.; (Toluene: Ethyl acetate:: 8:2 v/v) After Derivatisation with Iodine in visible light

S. No	Sample 1		Sample 2		Sample 3
S. No	R_f	Colour	R_f	Colour	R_f	Colour
1.	0.41	Yellow	0.36	Yellow	0.41	Yellow
2.	-	-	-	-	0.53	Yellow
3.	0.61	Yellow	-	-	0.61	Yellow
4.	0.76	Yellow	0.76	Yellow	0.76	Yellow

Table 4. TLC profile of various samples of Trachyspermum ammi Linn.; (Toluene: Ethyl acetate:: 8:2 v/v) After Derivatisation with Vanillin sulphuric acid in visible light

S. No	Sample 1		Sample 2		Sample 3
S. No	R_f	Colour	R_f	Colour	R_f	Colour
1.	-	-	0.27	Pale Blue	-	-
2.	-	-	0.33	Pale Blue	0.40	Pale Blue
3.	0.46	Pale Blue	0.46	Pale Blue	0.46	Pale Blue
4.	-	-	0.53	Pale Brown	-	-
5.	0.60	Pale Blue	0.60	Pale Blue	0.60	Pale Blue
6.	0.69	Pale Blue	0.66	Pale Blue	-	-
7.	0.83	Pink	0.77	Pale Blue	0.73	Dark Pink

CONCLUSION:

The analytical results on various physiochemical parameters - loss on drying at 105ºC, Total ash, Acid insoluble ash and extractive values on comparison revealed that extractive value is highest in sample S1. Highest extractive values show the highest active component in sample S1, S2 comparison with sample S3. It was observed that there is variation in the values obtained on analysis of the various samples of Trachyspermum ammi Linn. collected from different places. It has been concluded from this study that estimation of physico-chemical parameters and TLC is highly essential for raw drugs or plant parts used for the preparation of various compound formulations. The periodic assessment is essential for quality assurance and safer use of herbal drugs, so that study can be considered as the Pharmacopoeia standard and helpful to confirm the genuineness of the single plant drug to be used for the preparation of effective Ayurvedic formulation.

ACKNOWLEDGEMENT:

The authors are very grateful to Director General CCRAS, New Delhi for providing necessary help for carrying out this work. Authors acknowledge the secretarial assistance rendered by Ms. Rekha in the preparation of this paper.

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Received on 14.12.2010

Accepted on 25.12.2010

Research Journal of Pharmacognosy and Phytochemistry. 3(1): Jan. - Feb. 2011, 41-44