INTRODUCTION:

Quality control of polyherbal formulation is a basic requirement to ensure their safety and effectiveness⁴. Qualitative and quantitative analyses of marker components is a potentially cost-efficient aspect of quality control as any change in quality of the marker compound may also represent the quality of overall product, which would also directly affect the other phytoconstituents of the product⁵. HPTLC is the important analytical method used for marker profiling of botanicals. A poly-herbal formulation available in tablet form was used for estimation of phyllanthin. The marketed tablet contains plants like Phyllanthus niruri, Eclipta alba, Cichorium intybus, Boerhaavia diffusa, Embelia ribes, Berberis aristata, Picrorhiza kurroa. The major lignans present in Phyllanthus niruri Phyllanthin and Hypophyllanthin has been reported to exhibit antihepatotoxic activity.

Phyllanthin

Molecular formula: C₂₄H₃₄O₆

Molecular weight: 418.53

It is a (+) 3, 4, 3¹, 4¹, 9, 9¹, Hexa Methoxy-8, 8` bytyro lignin. Phyllanthin is a white crystalline powder which is soluble in Methanol, Chloroform, acetone, ether and sparingly soluble in petroleum ether, almost in soluble in water².

As no analytical method has so far been reported for quantification of the phyllanthin in tablets, so an attempt has been made to estimate the phyllanthin content by HPTLC.

MATERIALS AND METHODS:

The marker compound (standard) Phyllanthin was purchased from SPIC, Chennai. The polyherbal tablets were procured from local market of Wardha, Maharashtra, India. HPTLC plates silica gel 60 F₂₅₄ [E. Merck] were used as a stationary phase. Toluene-Ethyl acetate (9:1) used as a mobile phase for estimation. A Camage HPTLC system comprising of Camag linomate V semiautomatic sample applicator, Camag TLC scanner 3, Camag Win CATS software, Camag twin trough chamber sonicator and magnetic stirrer were used during study. All chemicals used were of HPLC grade and were purchased from Loba chemicals, Mumbai, India.

Preparation of standard stock solution:

Methanolic solution of phyllanthin (1mg/ml) was used for application on TLC plate. The five standard levels of standard phyllanthin were applied in triplicate on TLC plate. It was further diluted with methanol to obtained required concentration of 5, 10, 15, 20, 25 μg/μl. All solutions were stored at room temperature; these solutions were shown to be stable during the period of study¹.

5gm of tablet powder was macerated in 50 ml methanol (4×60 min.). Maceration was done with magnetic stirrer to ensure complete extraction. The solution was filtered through Whatman filter paper no.42 to a 100 ml volumetric flask. The filtered methanolic extracts from all the four extracts were combined, and concentrated under reduced pressure to about 1ml and then diluted to 5ml with methanol. TLC plates were prewashed with methanol and activated prior to use. Toluene-Ethyl acetate [9:1 V/V] was used as mobile phase for the detection. Samples were applied as bands using Camag linomate V semiautomatic sample applicator and migration distance allowed was 80 mm, drying of plate done for 3 min at 60⁰ C temperatures. The chromatogram was scanned at 200 nm and average peak areas of 3 samples were calculated (Fig. 2), with Camag TLC scanner III, using Camag Win CATS software⁵. The results are reported in Table 3 and the amount of the drug was determined.

Optimization of mobile phase and flow rate were performed based on peak parameters such as height, area, tailing factor, Rf value, run-time and resolution. The mobile phase Toluene-Ethyl acetate [9:1 V/V] was found to be satisfactory and gave symmetrical and well resolved peaks for Phyllanthin (Fig. 1). The wavelength 200 nm was selected for the determination based on maximum absorption of Phyllanthin and best detector response at this wavelength⁶.

Fig 1: Peak for phyllanthin in single track of Standard applied

Fig 2: HPTLC Fingerprint of polyherbal tablets showing presence of phyllanthin

The calibration curve for phyllanthin was obtained by plotting the peak area of phyllanthin versus their concentration over the range of 5-80 μg/μl for phyllanthin and was found to be linear (Fig. 3) with (r² = 0.9989) reported in Table 1. The detection limit for phyllanthin was 60 ng/spot. The quantitation limit for phyllanthin was 180 ng/spot which suggested that a nanogram quantity of the drug can be estimated accurately. The validation and system suitability parameters are shown in Table 2. Recovery studies were carried out by adding known amount of standard phyllanthin to the drug solution and analyzed to estimate drug content. (Table 4).

Table no. 1: Observation for standard calibration graph for phyllanthin in methanol

Sr. no.	Conc. (µg/µl)	Peak Area
1	0	0.00
2	5	4489.7
3	10	8913.4
4	20	16426.8
5	40	33153.6
6	80	70307.2

Table no. 2: Results of Validation studies

SST and other Parameters

Phyllanthin

*Rf value

λ max

Linearity range

LOD

LOQ

% recovery

Co-relation coefficient

Specificity/Selectivity

Stability of sample solution

0.83 ± 0.04

200 nm

5-80 μg/spot

60 ng/spot

180 ng/spot

97.69 S. D. 1.555

0.9989

No interference

≥ 24 hrs

SST: System Suitability Test Calculated at 5% peak height,

LOD: Limit of Detection,

LOQ: Limit of Quantitation

RESULTS AND DISCUSSION:

A solvent system that would give dense and compact spots with appropriate and was desired for quantification of phyllanthin The mobile phase consisting of Toluene-Ethyl acetate [9:1 V/V] gave R_f value 0.83 ± 0.04 and matches to the phyllanthin present in polyherbal tablet. The phyllanthin content of tablet was determined and it was found to be 0.178 % w/w ±0.0016 (Table no. 3).

Table no. 3: Result of Phyllanthin content in polyherbal tablets by HPTLC

Track no.	Sample	Standard	Peak area	Phyllanthin content (µg)	% phyllanthin content (w/w)
1	200µg	------	3086.3	0.355	0.177
2	------	20µg	16426.8	-----	------
3	400µg	------	6294.12	0.725	0.181
4	600µg	------	9311.15	1.073	0.178
5	800µg	------	12345.51	1.423	0.177
6	1000µg	------	15431.88	1.780	0.178
				Mean	0.178
				S. D.	0.0016
				C. V.	0.8978

S. D. – Standard deviation

C.V. – Coefficient of variation

Table no. 4: Percent recovery by HPTLC

Track no.	Sample applied(µg) [A]	Phyllanthin present in A (µg) [B]	Stand. added to A (µg) [C]	Total Phyllanthin applied (µg) [D]	Total phyllanthin recovered [E]	% Recovery (E/D)×100
1	200µg	0.355	----	0.355	0.355	100
2	200µg	0.355	10	10.355	10.016	96.72
3	200µg	0.355	20	20.355	19.793	97.24
4	200µg	0.355	40	40.355	39.067	96.81
					Mean	97.69
					S. D.	1.555

Fig 3: Standard calibration curve of phyllanthin

Table no. 5: Intraday, Interday, Different analyst, LOD and LOQ

Parameter	Phyllanthin
% RSD Intraday	0.191
% RSD Interday	0.231
% RSD Different Analysts	0.103
LOD	25 ng/ml
LOQ	75 ng/ml

RSD: Relative Standard Deviation

LOD: Limit of Detection

LOQ: Limit of Quantitation

CONCLUSION:

The developed method was validated in terms of accuracy, linearity and range, reproducibility and recovery study. Recovery studies were carried out to study accuracy and precision of the method. The result of recovery studies for phyllanthin content was found to be 97.69% S. D. 1.555 (Table no. 4), indicating that the method is free from interference from excipients. From the above results it can be concluded that the HPTLC method is accurate, precise, specific and reproducible and can be used for estimation of phyllanthin in polyherbal tablets.

ACKNOWLEDGEMENT:

The author thanks to principal, Institute of Pharmaceutical Education and Research, Wardha for providing facilities and chemicals for the research work.

REFERENCES:

1. Venkatesh, S., M., Reddy, B., Kasi Vishwanath, C. “Spectrophotometric Determination of Hepatoprotective Principle of Phyllanthus niruri linn.”, Indian J. pharm. Sciences, 2004, 66(3), 336-337.

2. Mukharjee, P. K., Wahile, A., Kumar, V., Saha, B. P., “Marker profiling of Botanicals used for Hepatoprotection in Indian system of Medicine” Drug information J. 2006, (40), 131-139.

3. Sane, R. T., “Standardization, quality control, and GMP for herbal drug”, Indian Drugs 2002, 39(3), 184-189.

4. Agrawal, A., “Critical issues in quality control of herbal products”, Pharma Times 2005, 37(6), 9-11.

5. Aeri, V., Zafer, R., Ahmad, S., Khan, P. I., “Quantitative estimation of Phyllanthin in Phyllanthus niruri and some polyherbal Hepatoprotective formulations by HPTLC”, Indian J. Nat. prod. 2005, 21(1), 12-13

6. Scott, P.W., Liquid Chromatography for the Analyst, 67, Marcel Dekker, New York, 1994; 1-4.

Received on 07.01.2011 Modified on 25.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 815-817