MATERIALS AND METHODS

Whole plants of Asteracantha longifolia Nees. were collected in winter (October-November 2007) during flowering season from Thane Maharashtra and from different regions viz. Patalganga, Mahad, Karjat, Kolhapur, Dehradun and Nazimabad. Herbaria of the whole plant were authenticated from National Institute of Science Communication (NISCOM), New Delhi and National Botanical research Institute (NBRI), Lucknow. The plants were collected, washed and dried in an oven at 45°C for four days, powdered and sieved through BSS mesh No. 85. Standard β-sitosterol (99% purity) and Lupeol (97% purity) were procured from Sigma-Aldrich Chemie Gmbh (Aldrich Division, Steinheim, Federal Republic of Germany). The solvents toluene, ethyl acetate and methanol of analytical grade purchased from Qualigens Fine Chemicals, Mumbai, India were used for the analysis.

A TLC scanner with computer system and Cats 3 Version Software were obtained from Camag (Muttenz, Switzerland). The source of radiation was mercury lamp. Camag Linomat IV was used as applicator. Separation was done on silica gel F₂₅₄ HPTLC precoated plate procured from Merck (Darmstaldt, Germany).

Different parts of the plant were powdered separately, similarly plants collected from different regions were powdered and used for analysis. Thousand milligram of dried powder was accurately weighed and placed in a stoppered tube and 10 mL of methanol was added, the sample was vortexed for 1-2 min and left to stand overnight at room temperature (28±2°C). The contents of the tube were filtered through Whatmann No. 41 paper (E. Merck, Mumbai, India) and the filtrate was used for experimental work.

Chromatography
Procedure: Chromatography was performed on silica gel F₂₅₄ HPTLC pre-coated plate. Samples (10 μL) were applied on the plates as band of 7 mm width with the help of a Camag Linomat IV sample applicator at the distance of 14 mm from the edge of the plates. The mobile phase constituted of toluene-ethyl acetate-methanol, 15 + 3 + 1.5 (v/v). The plates were developed to a distance of 80 mm in a Camag twin-trough chamber previously equilibrated with mobile phase for 30 min. The chromatographic conditions had been previously optimized to achieve the best resolution and peak shape. After development, plates were derivatised in Liebermann-Burchard reagent and heated at 105°C for 15 min and densitometric evaluation of the plates was performed at 366 nm in fluorescence/ reflectance mode using mercury lamp with a Camag Scanner II in conjunction with Cats 3 Version Software. The chromatographic plate of Lupeol and β-sitosterol standard with different morphological parts and geographical regions of Asteracantha longifolia Nees. are shown in Fig. 1 and 2 shows phytochemical fingerprint of Asteracantha longifolia Nees. with Lupeol and β-sitosterol standards.

Fig. 1:	The chromatographic plate of standard Lupeol and β-sitosterol with Asteracantha longifolia Nees. Methanolic extracts of-
	T1: T2: T3: T4: T5: T6: T7: T8: T9: T10: T11: T12:	Asteracantha longifolia Nees. (Root) Asteracantha longifolia Nees. (Stem) Asteracantha longifolia Nees. (Leaf) Asteracantha longifolia Nees. (Seed) Asteracantha longifolia Nees. (Thane) Standard β-sitosterol + Lupeol Asteracantha longifolia Nees. (Patalganga) Asteracantha longifolia Nees. (Mahad) Asteracantha longifolia Nees. (Karjat) Asteracantha longifolia Nees. (Kolhapur) Asteracantha longifolia Nees. (Dehradun) Asteracantha longifolia Nees. (Nazimabad)

Fig. 2:	The chromatographic plate of standard Lupeol and β-sitosterol with Asteracantha longifolia Nees.
	T1: T2: T3: T4:	Methanolic extract of whole plant powder of Asteracantha longifolia Nees. β-sitosterol Standard β-sitosterol + Lupeol Lupeol

Assay procedure: The solution of 0.5 mL of 50 ppm (50 μg mL^-1) Lupeol was mixed with 0.5 mL of 50 ppm (50 μg mL^-1) β-sitosterol and 10 μL of plant extract were spotted on a HPTLC plate. The amount of Lupeol and β-sitosterol present in the plant extract was calculated by comparison of area measured for the sample to that for the respective standard.

The assay procedure described earlier was repeated three times. The results of assay are given on Table 1a, b and 2a, b.

RESULTS AND DISCUSSION

Different parts of Asteracantha longifolia Nees. are prescribed in different indications and the whole plant is used as a tonic for general debility. Since different parts of same plant have different therapeutic applications, it is important to distinctively identify the plant part constituent of the plant powder. In this study HPTLC fingerprint patterns have been therefore, evolved for powders of different plant parts of Asteracantha longifolia Nees. Presence of Lupeol and β-sitosterol standards were detected and quantitated accurately using silica gel F₂₅₄ HPTLC pre-coated plates with the mobile phase toluene-ethyl acetate-methanol, 15+ 3+ 1.5 (v/v). Developed chromatograms showed distinct phytochemical variations in morphological parts as well as geographical regions of collection of plant material (Fig. 1). It showed variation in content of Lupeol, which was found to be maximum in roots collected from Thane region (0.25%) and in the whole plant powder it was maximum in the plants collected from Kolhapur (0.237%). β-sitosterol was found to be maximum in leaves collected from Thane region (0.069%) and in whole plant collected from Mahad region (0.067%) (Table 1a, b, 2a, b). Lupeol content is found to be maximum in the roots while β-sitosterol content is maximum in the leaves of Asteracantha longifolia Nees. This also justifies the therapeutic use of roots as a diuretic and the use of leaves in the management of female reproductive dysfunction (Joshi, 2000; Warrier, 1995). The HPTLC technique thus developed demonstrates variations in phytochemical markers which help to identify the difference in phytochemical constituents due to the geographical region of collection and due to the constituent part of the plant in medicinal plant raw material.

Table 1a:	Results of assay of β-sitosterol in different parts of Asteracantha longifolia (Nees.)

Table 1b:	Results of assay of Lupeol in different parts of Asteracantha longifolia (Nees.)

Table 2a:	Results of assay of β-sitosterol in Asteracantha longifolia Nees. collected from different regions

Table 2b:	Results of assay of Lupeol in Asteracantha longifolia Nees. collected from different regions

CONCLUSION

In this study it has been demonstrated that HPTLC fingerprint can be employed to identify the source of the plant powder to the specific part of the plant that is constituent in the powder. Further, quantitation of two phytochemical markers namely Lupeol and β-sitosterol using HPTLC from the powder of different plant parts has also been established. The results indicate that the plant parts show specific variations in their content of these phytochemical markers. Distinctive phytochemical variations have also been demonstrated in the whole plant powder sourced from various geographical regions of India. HPTLC fingerprint, as standardized in this study can therefore, be successfully applied to identify the region of collection of the plant. Moreover, the phytochemical markers Lupeol and β-sitosterol, also show region specific variation in their concentrations. Lupeol content is maximum in the whole plant powder sourced from Kolhapur while β-sitosterol content is maximum in whole plant powder sourced from Mahad. Therefore, as a diuretic, the whole plant from Kolhapur may be recommended while as a uterine tonic the whole plant from Mahad may be recommended.

High performance thin layer chromatography (HPTLC) as reported in this study provides a chromatographic fingerprint of phytochemicals and is suitable for confirming the identity and purity of medicinal plant raw materials.