Figure 1. Structure of Andrographolide

Figure2. Overlay spectra of andrographolide in kalmegh extract and pharmaceutical dosage form with the standard showing purity profile

Preparation of test solution- Twenty tablets (each 300 mg tablet contains 40 mg of kalmegh extract standardized for 10% AGD) were weighed accurately and ground to fine powder. The powdered drug of 375mg equivalent to 50mg of kalmegh extract was extracted with methanol and sonicated for 30 minute and the volume was diluted to 100 mL. The resulting solution was filtered through Whatman No. 41 filter paper and used as a test solution for the quantification study.

Similarly the Kalmegh extract test solution was prepared by accurately weighing about 50 mg kalmegh extract into 100 mL of volumetric flask.

Chromatographic conditions:

Chromatography was performed on 20cm × 10cm aluminium backed TLC plates coated with 250 µm layer of silica gel 60F₂₅₄ (E. Merck, Darmstadt, Germany, Supplied by Merck India, Mumbai). Before the chromatographic development the plates were activated by heating at 105-110ºC for 10-15 min. Samples were applied as 6mm wide bands, under a continuous flow of nitrogen, with the help of Camag (Muttenz, Switzerland) Linomat V sample applicator fitted with a 100µL syringe. A constant application rate 150 nL s^-1 was used. The plates were then conditioned 45 min in a presaturated chamber (20×10cm) with the mobile phase toluene: ethyl acetate: formic acid: methanol (50:30:05:2.5 v/v/v/v). Plate was then placed in the mobile phase and ascending development was performed to a distance of 8cm from the point of application at 25 ºC. After development the plate was then air dried and densitometric scanning was performed at 226 nm with TLC scanner 3 operated in reflectance mode and controlled by WinCATS software (V 1.1.3.0). The slit dimension was 5×0.45 mm with the scanning speed of 20 mm s^-1. The concentrations of the compound chromatographed were determined by the intensity of reflected light. Evaluation was based on the linear regression of peak areas.

Figure3. Three-dimensional overlay chromatograms showing the specificity of the method in presence of blank methanol, Placebo, Standard, Kalmegh extract and formulation

Calibration plot:

A fresh stock solution of 100 µg mL^-1 AGD was prepared in methanol. Standard solution containing AGD was applied 1-8 µL as 6mm band to furnish a concentration of 100-800ng per band. Each amount was applied to the plate six times.

METHOD VALIDATION:

Precision:

The repeatability of sample application and the measurement of the peak areas were determined by performing six replicate measurements of the same band using 500 ng per band of AGD.

Limit of detection and quantification:

To estimate the limit of detection (LOD) and limit of quantification (LOQ), blank methanol was applied to a plate for six times by a method described above. The signal to noise ratio (S/N) was determined. LOD and LOQ were regarded as the amount for which S/N was 3:1 and 10:1, respectively.

Table2. Method validation parameters for the estimation of Andrographolide by HPTLC

Parameters	Andrographolide
Method Precision	% RSD -1.52
System precision (n=7)	% RSD -1.38
Limit of detection	30ng
Limit of quantification	100ng
Specificity	Specific
Linearity range	100-800ng

Table3. Linear regression equation and R_F value for Andrographolide

Compound	R_F value	Regression equation	Correlation coefficient
Andrographolide	0.34	y=142.24x + 138.05	0.993

Robustness:

Following the introduction of small changes in the mobile phase composition (±0.1 mL for each component), the effect on the result was examined. Mobile phases having different compositions, e.g. toluene: ethyl acetate: formic acid: methanol (50:30:5.1:2.5 v/v/v/v), (50:30:4.9:2.5 v/v/v/v), (50:30:05:2.6 v/v/v/v), (50:30:05:2.4 v/v/v/v) were tried. The amount of mobile phase was varied over the range of ± 5%. The saturation time was also varied in the range of ± 5%. The robustness of the method was determined at three different concentration levels at 400, 500 and 600 ng spot^-1 for AGD (Table 4).

Specificity:

The specificity of the method was ascertained by analyzing standard, drug sample of equivalent concentration (500ng per spot) and placebo. The band for the AGD in the sample was confirmed by comparing R_F value and spectra of the band from the samples and standard. The peak purity of AGD was assessed by comparing the spectra at three levels i.e., peak start, peak apex and peak end.

Recovery studies:

Recovery study was performed to confirm accuracy and suitability of the method. The Kalmegh placebo (identical formulation without active ingredient) was prepared in-house containing Picrorhiza kurroa, Boerrhavia diffusa, Phyllanthus niruri, microcrystalline cellulose, cross carmilose sodium, cross povidone, aerosol, and magnesium stearate. The placebo samples prepared in house were spiked with 4, 5 and 6mg of standard AGD and methanol was added to give 80, 100 and 120 µg/mL solution then 5 µL of each solution was applied on TLC plate to give 400, 500 and 600ng of AGD per spot. The mixtures were analyzed by proposed method. The experiments were conducted in triplicate.

Analysis of the plant extract and pharmaceutical dosage form:

10 µL of standard, Kalmegh extract, Pharmaceutical dosage form and placebo were spotted on the 20×10 cm plate, developed and scanned by the proposed chromatographic conditions. The analysis was repeated in triplicate. The AGD content was calculated from the peak areas recorded.

Figure 4. The Image of TLC plate at short wavelength UV light. Lanes: 1-4 standard andrographolide, 5-6 plant extract, 7-8 formulation and 9-10 kalmegh placebo

RESULTS AND DISCUSSION:

The composition of mobile phase for TLC was optimized by testing different solvent mixtures of varying polarity and the best results were obtained by using toluene: ethyl acetate: formic acid: methanol (50:30:05:2.5). The scanning wavelength selected was 226nm as is the absorption maxima of AGD.

The selected mobile phase produced highly symmetrical peaks showing good resolution for AGD. Peak purity was assessed by comparison of overlay spectra of standard and test peak at the start, apex and end, found satisfactory (Fig.2). The R_F value of AGD in standard and sample track was found to be 0.34 ± 0.03 and identity of the peak was checked by adding respective amount of standard to the placebo, the recovery percentage of AGD was in the range of 96%-99% (Table 1) calculated by the addition of AGD standard into the placebo. The specificity of the method was determined by comparing the three dimensional overlay of chromatogram of standard, extract, formulation and placebo (Fig. 3). The limit of detection and limit of quantitation for AGD was 30ng and 100ng per spot (Table 2). The calibration curve is linear in the working range of 100-800ng (Table 2) with correlation coefficient and regression equation 0.993 and y=142.24x + 138.05 (Table 3) respectively for AGD.

Table4. Robustness testing for HPTLC

Parameters	% RSD
Mobile phase composition	1.86
Saturation time	1.57
Amount of mobile phase	1.50

The method reported here is very simple, rapid and suitable for the quantitation of AGD from kalmegh extract and its pharmaceutical dosage form. Assay can be performed without pretreatment of sample for both plant extract as well as its pharmaceutical dosage form on a single plate at a scanning wavelength 226nm. A single compound can easily be quantified in presence of three other plant extracts as well as tablet excepients from the formulation containing Kalmegh extract without compromising the accuracy.

REFERENCES:

1. Mishra SK, Sangwan NS and Sangwan RS. Andrographis paniculata (Kalmegh): A Review. Pharmacog Rev. 2007; 1, 2: 283-298.

2. Handa SS and Sharma A. Hepatoprotective activity of andrographolide against galactosamine and paracetamol intoxication in rats. Indian J Med Res. 1990; 92: 284-292.

3. Singha P K, Roy S and Dey S. Protective activity of andrographolide and arabinogalactan proteins from Andrographis paniculata Nees. against ethanol-induced toxicity in mice. J Ethnopharmacol. 2007; 1, 11: 13-21.

4. Murti KRS. “Shrangadhar Samhita”. Chaukhamba Orientalia Publication, India. 2007.

5. Dua VK et al. Anti-malarial activity of some xanthones isolated from the roots of Andrographis paniculata. J Ethnopharmacol. 2004; 95, 2-3: 247-251.

6. Poolsup N et al. Andrographis paniculata in the symptomatic treatment of uncomplicated upper respiratory tract infection: systematic review of randomized controlled trials. J Clin Pharm Ther. 2004; 29, 1: 37-45.

7. Habtemariam S. Andrographolide inhibits the tumour necrosis factor--induced upregulation of ICAM-1 expression and endothelial-monocyte adhesion. Phytother Res. 1998; 12, 1: 37-40.

8. Pholphana N et al. Determination and variation of three active diterpenoids in Andrographis paniculata (Burm.f.) Nees. Phytochem Anal. 2004; 15, 6: 365-371.

9. Vijaykumar K et al. Estimation of andrgrapholide in Andrographis paniculata herb, extract and dosage form. Int J Appl Sci Eng. 2007; 5: 27-39.

10. Raina AP, Kumar A and Pareek SK. HPTLC analysis of hepatoprotective diterpenoid andrographolide from Andrographis paniculata. Indian J Pharm Sci. 2007; 69: 473-475.

11. Akowuah GA et al. HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata J food Comp Anal. 2006; 19: 118-126.

Received on 06.05.2009 Modified on 13.07.2009

Asian J. Research Chem. 2(3): July-Sept., 2009, page 314-317

Amount of andrographolide added in mg	Amount of andrographolide recovered in mg	Percent recovery	Average recovery
4.03	3.99	99.00
5.01	4.82	96.20	97.34
5.99	5.80	96.82