INTRODUCTION:

A great deal of research has been carried out to evaluate scientific basis for the claimed hepatoprotective activity of herbal agents as in the form of formulation. The selected plant materials; Cassia fistula (family- Caesalpinaceae), Coccinia indica (family- Cucurbitaceae) and Vigna mungo (family- Papilionaceae) reported to have hepatoprotective activity. High Performance Thin Layer Chromatography (HPTLC) is emerging as a versatile, high throughput and cost-effective technology that is uniquely suited to assessing the identity and quality of botanical materials^[1-12].

MATERIALS AND METHODS:

Plant materials

All the three plants crude materials Cassia fistula (leaf material), Coccinia indica (leaf material) and Vigna mungo (seed material) were collected in and around of Nilgiri District, Tamilnadu and authenticated by Dr. S. Rajan, Field Botanist, Medicinal Plant Collection and Survey Unit, Department of Ayush, Emerald, Ooty, (T.N.), India.

Preparation of formulation

Polyherbal tablet formulation contains aqueous commercial extracts of Cassia fistula, Coccinia indica and Vigna mungo were obtained from Amsar Pvt. Ltd., Indore, (M.P.) and prepared by wet granulation (non-aqueous) method using suitable excipients like Starch, Sodium benzoate, Poly vinyl pyrrolidine, Aerosil, Primlose, Sodium starch glycolate, Starch, Talc and Magnesium stearate [Table 1].

Physical evaluation

The prepared polyherbal tablet formulations were subjected to determination of various physical parameters like disintegration time, hardness, thickness, friability and weight variation test as per the standard procedures ^[8].

Method development of HPTLC

Standard preparation

5 mg of aspartic acid and vitexin were dissolved in 5 ml of methanol individually at (1mg/ml concentration).

Formulation preparation: 2000 mg of crushed tablet formulation was dissolved in 10 ml of methanol and slightly warmed on water bath and filtered through whatman filter paper, and the same solution was used for HPTLC analysis (200 mg/ml concentration).

Chromatographic conditions for determination of aspartic acid

Stationary phase : Precoated Silica gel F ₂₅₄Plates (MERCK)

Mobile phase : n-Butenol: Glacial acetic Acid: Water(50: 10:40)

Saturation : 60 minutes

Development chamber: CAMAG twin trough development chamber

Applicator : CAMAG Linomat IV applicator

Scanner : CAMAG Scanner III CATS (4.06),

Switzerland

Mode of scanning : Absorption (deuterium)

Detection wavelength : 544 nm

Volume applied (standard) : 5 µl

Volume applied (sample) : 7 µl

Chromatographic conditions for determination of vitexin

Stationary phase : Precoated Silica gel F ₂₅₄Plates (Merck)

Mobile phase : Ethyl acetate: Formic acid: Glacial acetic Acid: Water (100: 11: 11: 26)

Saturation : 60 mins

Development chamber : CAMAG twin trough development chamber

Applicator: CAMAG Linomat IV applicator

Scanner : CAMAG Scanner III CATS (4.06),

Switzerland

Mode of scanning : Absorption (deuterium)

Detection wavelength : 366 nm

Volume applied (standards) : 2 µl

Volume applied (samples) : 10 µl

Procedure

Before spotting, the plates were pre-washed with methanol. Standard and samples solutions were applied to the plates as sharp bands by means of CAMAG Linomat IV applicator. The spots were dried in a current of air. The mobile phase (20 ml) was poured into a twin trough glass development chamber was left to equilibrate for 30 minutes and the plate was placed in the chamber ^{[10, 11]}. The plate was then developed until the solvent front had travelled at a distance of 75 mm above the base of the plate. The plate was then removed from the chamber and dried in a current of air. Detection and quantification was performed with CAMAG Scanner III at a wavelength of 544 nm and 366 nm ^{[11, 13-17]}.

Linearity

Linearity was performed by applying standard solution at different concentration range from 100 to 500 ng/spot and 500 to 2500 ng/spot aspartic acid and vitexin respectively, on 20 x 20 cm HPTLC plates, pre coated silica gel G F ₂₅₄Plates (Merck) in the form of sharp 7 mm bands; the distance between two adjacent band was 8 mm. the plates were developed in a solvent system of n-Butenol: Glacial acetic Acid: Water (50: 10: 40) and Ethyl acetate: Formic acid: Glacial acetic Acid: Water (100: 11: 11: 26) for aspartic acid and vitexin respectively, up to a distance 75 mm, at room temperature. The plates were dried in air. The detector response for aspartic acid and vitexin were measured for each band at wavelength of 544 nm and 366 nm respectively, using CAMAG TLC Scanner and win Cat software. The peak area of aspartic acid and vitexin were recorded for each concentration. The linearity curve of aspartic acid and vitexin were obtained by plotting a graph of peak area of aspartic acid and vitexin vs applied concentration of aspartic acid and vitexin (ng) respectively^[17-20].

Method validation

The method was validated for precission, repeatability and accuracy. The precission was checked by repeated sacannig of same spot of aspartic acid (250 ng) and vitexin (1500 ng) respectively, three times each and was expressed as relative standard deviation (% RSD). The repeatability of the method was confirmed by analyzing 100 ng, 250 ng and 500 ng/spot and 500 ng, 1500 ng and 2500 ng/spot of standard aspartic acid and vitexin respectively, solution (n = 3) and was expressed as % RSD. The precision of the method was studied by analyzing aliquots of standard solution of aspartic acid (100 ng, 250 ng and 500 ng/spot) and vitexin (500 ng, 1500 ng and 2500 ng/spot) respectively on the same day (intra-day precision) and on different days (inter-day precision) and the results were expressed as % RSD. Study the accuracy, the recovery experiment was performed by the method of standard addition. The recovery of the added amount of standard was analyzed at three different levels. Each level of addition was repeated three times on three different days and the recovery of the add amount of standard was calculated. Limit of detection and limit of quantitation was also calculated by the proposed method. ^{[15, 16]}

RESULTS AND DISCUSSION

Formulation development

The prepared formulations was subjected to determinations of various physical evaluations like disintegration time, hardness, thickness, friability and weight variation test and pass the Indian Pharmacopoeia standards (Table 2).

HPTLC Estimation

The amount of aspartic acid and vitexin present in polyherbal commercial extracts tablet formulation was estimated by using HPTLC technique by comparing with the peak area of standard and sample. The results are given in table 3. The results reveals that the R_fof the sample polyherbal formulation (tablet) was matching with the standard R_fof marker compound aspartic acid and vitexin and the amount of marker compound present in the sample was calculated. The content of aspartic acid and vitexin were found to be 0.2624 % w/w and 0.1239% w/w in polyherbal commercial extracts tablet formulation respectively. (Fig. 1, 2, 3 and 4).

Validation

The calibration curve was linear in the range of 100 to 500 ng/spot and 500 to 2500 ng/spot aspartic acid and vitexin respectively and the correlation coefficient was determined. The correlation coefficient was found to be 0.9899 and 0.9982 for aspartic acid and vitexin respectively. The limit of quantification was found to be 120 ng and 450 ng for aspartic acid and vitexin respectively and the limit of detection was 40 ng and 150 ng respectively. The method was validated in terms of precission and reproducible expressed as % RSD which were found to be less than 2% and less than 2% for aspartic acid and vitexin respectively. The recovery values obtained were 99.49 to 100.24 % and 98.82 to 105.15 % showing accuracy of the method for aspartic acid and vitexin respectively. The average percentage recovery was found to be 99.88 % and 101.44 % for aspartic acid and vitexin respectively given in table 4.

Table 1: Composition of polyherbal commercial extracts tablet formulation

S. No	Ingredients	Per Tablet (mg)
1	Cassia fistula	450
2	Coccinia indica	125
3	Vigna mungo	175
4	Starch (diluent)	140
5	Sodium benzoate (preservative)	1
6	Aerosil	15
7	Iso propyl alcohol	q. s.
8	Poly vinyl pyrrolidine (binding agent)	15
9	Primlose(super disintegrating agent)	20
10	Aerosil (super disintegrating agent)	10
11	Sodium starch glycolate (super disintegrating agent)	20
12	Starch (disintegrating agent)	14
13	Talc (glidant)	10
14	Magnesium stearate (lubricating agent)	5
	Total weight	1000

Table 2: Physical evaluation of polyherbal commercial extracts tablet formulation

S.No	Quality control tests	Formulation lab extracts
1	Physical appearance	Light brown coloured tablets
2	Weight variation test	1000 ± 3.75
3	Hardness test	4.2 kg/sq.cm
4	Friability test	0.78 %
5	Thickness	6.92 mm
6	Disintegration time test	12.8 min

Table 3: Estimation of aspartic acid and vitexin in polyherbal commercial extracts tablet formulation by HPTLC

No.

Marker compounds

Standard R_fvalues

Sample

R_fvalues

Amount of Marker

Compound

(%w/w)

Aspartic acid

0.26

0.25

0.2624

Vitexin

0.75

0.76

0.1239

Table No. 4. Validation parameters for quantification of aspartic acid and vitexin by HPTLC

Parameters	Aspartic acid	Vitexin
Precission (% RSD)	< 2 %	< 2%
Linearity	100 to 500 ng/spot	500 to 2500 ng/spot
Limit of detection	40 ng/spot	150 ng/spot
Limit of quantification	120 ng/spot	450 ng/spot
Accuracy	99.49 to 100.24 %	98.82 to 105.15 %

Fig. 1. HPTLC Chromatogram of standard aspartic acid

Fig. 2. HPTLC Chromatogram of standard vitexin

Fig.3. HPTLC Chromatogram of Polyherbal commercial formulation (Track No. 11) for aspartic acid

Fig. 4. HPTLC Chromatogram of lab formulation (Track No. 9) for Vitexin

CONCLUSION:

The developed HPTLC method was simple accurate, precise, economic and can be utilised for estimation of aspartic acid and vitexin in polyherbal tablet formulation could be used as a valuable analytical tool in the routine analysis. Aspartic acid and vitexin can be used as one of the appropriate analytical markers present in the various medicinal plants.

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Received on 20.07.2012 Modified on 18.08.2012

Asian J. Research Chem. 5(8): August, 2012; Page 1057-1060