1.2 Cinnamomum tamala Fr. Nees.:

Cinnamomum tamala, belonging to family Lauraceae, is also known as Indian Cassia. Due to its aroma, the leaves are kept in clothes and also chewed to disguise bad mouth odour. The dried leaves are used as a common spice in Indian cooking. It is also used in Indian system of traditional medicines. "Ayurveda" describes the use of leaves of Tejpatta in the treatment of ailments such as anorexia, bladder disorders, and dryness of mouth, coryza, diarrhea, nausea and spermatorhea^[8]. The main chemical constituents of Cinnamomum tamala leaves are α-pinene, camphene, myrcene, limonene, eugenol, p-cymene, methyl eugenol, and eugenol acetate and methyl ether of eugenol^[10]. Eugenol (4-hydroxy-3-methoxy allylbenzene) is one of the main constituents of cinnamon oil^[11-12].The leaves of Cinnamomum tamala have been reported to possess antidiabetic, antioxidant ^[9,13], antidiarrhoeal ^[14], antihyperlipidemic ^[15], antioxygenic ^[16], anti-inflammatory ^[17], acaricidal ^[18], hepatoprotective ^[19], gastro protective ^[20], antibacterial and immunomodulatory activities ^[21] carminative, anti - flatulent, diuretic, and in cardiac disorders ^[22]. Tejpatta is commonly used in Ayurvedic pharmacy in asava and arista preparation to augment the fragrance and to promote the appetite and digestion. Parts of Cinnamomum tamala are used in many Ayurvedic preparations e.g. Sudarshan choorna, Chandraprabhavati Citrakadi taila, Kasisadi taila and Vajraka taila. The leaf extracts are used as clarifiers in dyeing procedures with myrobalans or kamala^{[7, 23}^].

1.3 Trigonella foenum gracecum:

Trigonella foenum graecum belonging to family fabaceae are very important for their therapeutic potentials. Trigonella foenum-graecum is commonly known as maithray (Bangla, Gujarati), methi or mithi (Hindi, Nepali, Marathi, Urdu and Sanskrit), menthyada soppu (Kannada), ventayam (Tamil), menthulu (Telugu), hilbeh (Arabic), ulluva (Malayalam) and shambalîleh (Persian) ^[24]. Trigonella foenum-graecum is one of the oldest known medicinal plants in the recorded history^[25-27].

In traditional medicines it is used as an aphrodisiac, astringent, demulcent, carminative, stomachic, diuretic, emmenagogue, emollient, expectorant, lactogogue, restorative, and tonic Trigonella foenum-graecum is used for a variety of health conditions, including digestive problems, bronchitis, tuberculosis, fevers, sore throats, wounds, arthritis, abscesses, swollen glands, skin irritations, diabetes, loss of appetite, ulcers and menopausal symptoms, as well as in the treatment of cancer. It is used to reduce blood sugar level and to lower blood pressure. Trigonella foenum-graecum is used for treating sinus and lung congestion, and loosens and removes excess mucus and phlegm. Trigonella foenum-graecum is known to contain alkaloids, flavonoids, salicylate and nicotinic acid. Many compound were identified as kaempferol 3-O-β-d-glucosyl (1,2)-β-d-galactoside (lilyn), kaempferol triglycoside, quercetin, glucose and galactose, 3,7-disubstitution of quercetin, quercetin 3-O-β-d-glucosyl(1,2)-β-d-galactoside 7-O-β-d-glucoside, a new glycoside of quercetin. The Trigonella foenum-graecum has also been suggested to possess Cholesterol-lowering effects, Glucose-lowering effects, Anti-inflammatory effects, Antioxidant effects and antitumor actions^[^28]. Trigonella foenum-graecum is used as main ingredient in Mustakarista, Mrtasanjivani sura an ayurvedic formulation^[7].

1.4 Foeniculum vulgare:

Foeniculum vulgare commonly known as fennel is a traditional medicinal plant of Apiaceae family. Foeniculum vulgare fruits have been used as traditional herbal medicine in Europe, India and China. It is native to southern Europe and the Mediterranean area ^[29]. The chemical constituents from the fennel include essential oil, fatty acid, phenylpropanoids, monoterpenids, sesquiterpenes, coumarins. It also contains triterpenoids, tannins, flavonoids, cardiac glycosides, saponins, and other types of compounds. The Foeniculum vulgare plant and seeds are used in diseases of chest, spleen, kidney and diuretic problems^[30]. Foeniculum vulgare is used as main ingredient in Panchsakar churna an ayurvedic formulation ^[7].

1.5 Trachyspermum ammi Linn.:

Trachyspermum ammi is also known by the names Bishop’s Weed, Carum copticum, Ajwain, Carom ajowan. Native to Southern India, this plant is grown throughout the country in Madhya Pradesh, Andhra Pradesh, Gujarat, Maharashtra, Uttar Pradesh, Rajasthan, Bihar and West Bengal. It is also grown in Pakistan, Afghanistan, Iran and Egypt. In India the major producing states are Rajasthan and Gujarat where Rajasthan produces about 90% of India's total production. Trachyspermum ammi seeds consist of moisture, protein, fat, minerals, fiber, carbohydrates, calcium, phosphorus, iron, carotene, thiamin, riboflavin and niacin. The thymol from the essential oil of Trachyspermum ammi sprague seeds has shown the activities of strong germicidal, anti-spasmodic, aphrodisiac and fungicidal activities. In India the seeds are used as a household remedy for indigestion and abdominal colic, and used in poultices to relieve asthma and arthritis. A liquid preparation made by boiling ground-up ajwain 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 antiseptic^[31]. It has been shown to possess anti-aggregatory effects^[32], anthelmintic^[33], anti hyperlipidaemic^[34] antifilarial^[35], insecticidal^[36], kidney stone inhibitory; molluscicidal^[37-39] mosquito repellent ^[40]; and nematicidal activities ^[41]. Seeds possess stimulant qualities of capsicum, bitter property of chiretta and anti spasmodic qualities of as a foetida ^[42]. 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 thymine which contains p-cymene (50- 55%), β-pinene (4 - 5%), limonene with γ-and β-terpinenes (30 - 35%)^[43].

In Ayurvedic system of medicine it is mainly used in the treatment of respiratory, gastro-intestinal disorders and inflammatory conditions^[44]. Oil is used to treat renal stones, psoriasis and to expel hookworms. Yavani sadava and Agnimukha curna are the important ayurvedic formulation of Trachyspermum ammi ^[7].

1.6 Nigella sativa Linn.:

The seeds of Nigella sativa (Ranunculaceae), commonly known as black seed or black cumin, mostly found and cultivated in Punjab, Himachal Pradesh, Bihar, West Bengal, Assam, Maharashtra and also cultivated in Syria, Lebanon, Israel and Southern Europe. Nigella sativa used in folk (herbal) medicine all over the world for the treatment and prevention of a number of diseases and conditions that include asthma, diarrhoea and dyslipidaemia^[7]. The seeds contain both fixed and essential oils, proteins, alkaloids and saponin. The seed oil has anti-inflammatory, analgesic, antipyretic, antimicrobial and antineoplastic activity^[45]. The seeds are considered as bitter, pungent, aromatic, appetizer, stimulant, diuretic, emmenagogue, galactagogue, anthelmintic, acrid, thermogenic, carminative, anodyne, deodorant, digestive, constipating, sudorific, febrifuge, expectorant, purgative, abortifacient. They are used in ascites, cough, jaundice, hydrophobia, fever, paralysis, conjunctivitis, piles, skin diseases, anorexia, dyspepsia, flatulence, abdominal disorders, diarrohoea, dysentery, intrinsic hemorrhage and amenorrhea. Seed oil is a local anesthetic^{[46- 47]}. Important Ayurvedic formulations are Narayana curna, Kankayana gutika ^[7].

1.7 Coriandrum sativum:

Coriandrum sativum is an annual herb in the family Apiaceae. It is also known as Chinese parsley or, particularly in the Americas, cilantro. Coriander is native to southern Europe and North Africa to southwestern Asia. It is a soft, hairless plant growing to 50 cm tall medicinal properties of coriander are carminative, diuretic, tonic, stimulant, stomachic, refrigerant, aphrodisiac, analgesic, and anti-inflammatory. Bioactive components of Coriandrum sativum are quercetin, caffeic acid, cineole, geraniol, borneol, 1, 8-cineole, aterpinene, β-carotene, β-pinene, β-sitosterol, cinnamic acid, ferrulic acid, 2-terpinene, kaempferol, monene, myrcene, p-coumaric acid, pcymene, quercetin, rutin, vanillic acid^[48]. Important ayurvedic formulations are Dhanyapancaka kvatha curna ^[7].

1.8 Piper nigrum:

Piper nigrum is the most important cultivated species due to its economic value. Geographically, it is confined to Western-Ghats of South India^[49]. Piperine is an active component in Piper nigrum and contributes to its pungency. Secondary metabolites from Piper nigrum play defensive role against infections by microbes, insects and animals. Another important component of pepper volatile oil is piperine, which is a famous odorants. Piper nigrum is anti-microbial, anti-mutagenic, a free-radical scavenger, immuno-modulator, anti-tumor, anti-depressant, anti-apoptotic, anti-metastatic, anti-thyroid, hepatoprotective, immunostimulator, anti-diarrheal and anti-spasmodic. Piper nigrum was reported to treat pulmonary diseases, fever, cold, colic disorder and gastric conditions. The following biologically important phytochemical have been extracted from Piper nigrum plants alkaloids, amides, propenyphenols, lignans, neolignans, terpenes, steroid, kawapyrones, piperolides, chalcones, dihydrochalcones, brachy-amide^[50], dihydropipericide, 4-dihydroxy-6(N-ethyamine), benzamide, (2E,4E)-N-eicosa-dienoyl pereridine, N-transferuloyltryamine, N-formylpiperidine, guineensine, (2E,4E)-N-5[(4-Hydroxyphenyle)-pentadienoyl] piperidine, (2E,4E)-N isobutyldecadienamide), (2E,4E)-N-isobutyleicosadienamide, (2E,4E,8Z)-N isobutyleicosatrienamide, (2E,4E)-N-isobutylocta-dienamide, piperamide, piperamine piperettine, pipericide, piperine, piperolein, trichostachine, sarmentine, sarmentosine, tricholein, retrofractamide. The major components of the essential oil obtained from the aerial parts of Piper nigrum were glulol, -pinene, -caryophyllene and –terpinene ^[51]. The important Ayurvedic formulations are Maricadi gutika, Maricadi taila, Trikatu curna ^[7].

1.9 Thin layer chromatography:

Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures ^[52]. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. The term chromatography refers to several related techniques for analyzing, identifying or separating mixtures of compounds. In, TLC, capillary action allows a liquid (mobile phase) to ascent a solid (stationary phase) coated on a support plate. A sample of the compound mixture is applied near the bottom of a dry TLC plate, in characterizing and isolating a number of compounds such as acids, alcohols, glycols, amides, alkaloids, vitamins, amino acids, antibiotics, food stuffs etc.

2. MATERIALS AND METHODS

Plant material:

The samples of spices were collected from local market of New Delhi, India. Powders of the samples were used for HPTLC profile and were carried out on collected spices samples.

Table 1. Samples of spices and their part used

S. No	Sample Code	Common Name	Botanical Name	Part Used
1.	S1	Tejpatta	Cinnamomum tamala	Leaf
2.	S2	Saunf	Foeniculum vulgare	Seeds
3.	S3	Jeera	Cuminum cyminum	Seeds
4.	S4	Methi	Trigonella foenum-graecum	Seeds
5.	S5	Ajwain	Trachyspermum ammi	Seeds
6.	S6	Dhaniya	Coriandrum sativum	Seeds
7.	S7	Kali Mirch	Piper nigrum	Seeds
8.	S8	Kalonji	Nigella sativa	Seeds

3. RESULTS AND DISCUSSION:

Thin layer chromatographic technique was used to separate the chemical compounds present in the drug. Various solvent systems were checked to separate the maximum number of active chemical compounds in the drug. 2g of the sample was soaked in 25 ml ethanol for 24 hrs and extracted through cold extraction. The extract was filtered through Whatman No. 1 filter paper and used for HPTLC profile. 5 µl (S1, S4) and 8 µl (S2, S3, S5, S6, S7, S8) of extracts were applied on (E. Merck) Aluminium plate pre-coated with silica gel 60F₂₅₄ of 0.2 mm thickness using Linomat IV applicator. The application positions X and Y were 10 mm and 10 mm, respectively, to avoid edge effects. Linear ascending development to a distance of 80 mm with toluene : ethyl acetate : formic acid (7 : 3 : 0.1v/v/v) as mobile phase was performed in a twin-trough glass chamber (10 cm × 10 cm) previously saturated with mobile phase vapour for 20 min. The plates were dried in air and photo documented at λ 254 nm and λ 366 nm and after derivatization with anisaldehyde-sulphuric acid reagent under visible light.

HPTLC profile of the ethanol extract of sample solutions developed in the mobile phase of toluene : ethyl acetate : formic acid (7 : 3 : 0.1v/v/v) (Figure. 1-3) the R_f and color of the bands given in Table 1-4 and photo of the HPTLC profile also given in figure 1-3.

Figure 1. HPTLC profile of various spices samples at ʎ 254nm (Toluene: Ethyl acetate: Formic acid :: 7: 3: 0.1 v/v/v)

Table 2. HPTLC profile of various spices samples at ʎ 254 nm

S. No	Name of sample	R_f	Color
1	S 1	0.04, 0.59, 0.73	Grey black
2	S 2	0.09, 0.15, 0.18, 0.38, 0.47, 0.59, 0.71	Grey black
3	S 3	0.11, 0.19, 0.42, 0.67	Grey black
4	S 4	0.18	Grey black
5	S 5	0.09, 0.64, 0.69	Grey black
6	S 6	0.09	Grey black
7	S 7	0.18, 0.69	Grey black
8	S 8	0.09, 0.18, 0.22, 0.35, 0.42,0.54	Grey black

Figure 2. HPTLC profile of various spices samples at ʎ 366 nm

(Toluene: Ethyl acetate: Formic acid :: 7: 3: 0.1 v/v/v)

Table 3. HPTLC profile of various spices samples at ʎ 366 nm

S. No.	Name of Sample	R_f	Color
1.	S 1	0.13, 0.34, 0.37	Light red
		0.18	Red
		0.22	Pink
		0.49, 0.55, 0.59, 0.70, 0.77	Fluorescent red
2.	S 2	0.09, 0.28, 0.34, 0.41, 0.50,0.55, 0.63	Blue
		0.15, 0.19, 0.22	Fluorescent blue
		0.25, 0.59, 0.68	Red
3.	S 3	0.11, 0.15, 0.19	Violet
		0.67, 0.73	Red
		0.77	Light blue
4.	S 4	0.11,	Pink
		0.13, 0.14, 0.68	Red
		0.18, 0.22, 0.59, 0.73	Fluorescent red
		0.83	Light red
5.	S 5	0.09, 0.22, 0.25	Light blue
		0.13	Light red
		0.19, 0.73	Red
		0.34	Blue
		0.68	Fluorescent red
6.	S 6	0.09	Blue
		0.13, 0.19, 0.83	Light blue
		0.68, 0.73	Red
7.	S 7	0.15,0.18, 0.65, 0.73	Blue
8.	S 8	0.09, 0.14, 0.18, 0.28, 0.34	Blue
		0.13, 0.55	Red
		0.19, 0.22, 0.38, 0.41	Green
		0.61	Fluorescent blue
		0.66, 0.73	Light blue

Figure 3. HPTLC profile of various spices samples after derivatization with Anisaldehyde sulphuric acid reagent under visible light (Toluene: Ethyl acetate: Formic acid :: 7: 3: 0.1 v/v/v)

Table 4. HPTLC profile of various spices samples after derivatization with Anisaldehyde sulphuric acid reagent under visible light

S. No.	Name of Sample	R_f	Colour
1.	S 1	0.13, 0.49, 0.55	Light blue

		0.59	Green
		0.70, 0.85	Violet
		0.77	Light green
2.	S 2	0.15	Blue
		0.19, 0.28, 0.47, 0.55, 0.71, 0.80, 0.85	Violet
		0.38	Green
		0.63	Orange
3.	S 3	0.04, 0.50	Blue
		0.09	Yellow
		0.14, 0.22, 0.35, 0.44, 0.55, 0.67,0.77, 0.85	Violet
		0.65	Orange
4.	S 4	0.04, 0.11, 0.13, 0.53, 0.85	Violet
4.	S 4	0.18	Green
5.	S 5	0.04, 0.49, 0.53	Blue
		0.65	Orange
		0.85	Violet
6.	S 6	0.04, 0.22, 0.83	Violet
6.	S 6	0.47	Blue
7.	S 7	0.04, 0.08, 0.15, 0.53, 0.59, 0.73, 0. 85	Violet
7.	S 7	0.47	Blue
8.	S 8	0.04, 0.13, 0.22,0.28, 0.59	Blue
		0.18, 0.38, 0.41, 0.64	Green
		0.47, 0.55, 0.68, 0.77, 0.85	Violet

4. CONCLUSION:

Development of standard procedure through HPTLC is a new approach which may lead to proper standardization of different spices and ayurvedic drugs based on fingerprinting characteristics. This investigation shows that these particular characteristics may be used as standardization tool for Ayurvedic formulations and its ingredients more effectively and most accurately. HPTLC Fingerprinting technique is widely employed in pharmaceutical industry in process development, identification and detection of adulterants in herbal product and helps in identification of pesticide content, mycotoxins and in quality control of spices and herbs. Further, the combination of qualitative fingerprinting and quantitative multi component analysis is a novel and rational method to address the key issues of quality control of spices/herbal medicines.

It has been concluded from this study that HPTLC profile is highly essential for spices/herbal drugs or plant parts used for the preparation of ayurvedic formulations. The periodic assessment is essential for quality assurance and safer use of spices/herbal drugs.

5. ACKNOWLEDGEMENT:

The authors are very grateful to Director General, CCRAS, New Delhi and Programme Officers, CCRAS, New Delhi for providing encouragement and facilities for carrying out this work.

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Received on 19.06.2013

Modified on 10.07.2013

Accepted on 17.07.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(4): July- August 2013, 188-193