Development of Analytical Method for Estimation of Biomarkers in Hepasave Tonic: A Polyherbal Formulation

Abstract

The Polyherbal formulation, HEPASAVE SYRUP, is an Ayurvedic Proprietary Medicine, used particularly as a Powerful Hepato-protective, an Antioxidant, and a Bitter tonic. The formulation composed of Amalaki fruit i.e. Phyllanthus emblica Ab., Haritaki fruit i.e.,Terminalia chebula Ab., Bibhitaka fruit i.e., Terminalia bellerica AB., Vasa leaf i.e.,Adhatoda vasica Ab., Bhunimba herb i.e., Andrographis paniculata, Katuka root i.e.,Picrorrhiza kurroa, Nimba stem bark i.e., Azadirachta indica Ab, Amruta stem i.e.,Tinospora cordifolia, Sarpankha herbTephrosia purpurea Ab., and also a Flavored syrup base. The quality control of formulation is required to be set in terms of developing HPTLC and HPLC method to ascertain the quality of formulation. Few Biomarkers have been selected for the study which includes AG, GA, KT and VS. A couple of HPTLC and HPLC methods have been reported for the simultaneous estimation of Andrograpolide and Gallic acid in combination with other biomarkers, and a couple of such methods have been reported for the simultaneous estimation of Andrographolide and Kutkin, together with other biomarkers. New, simple and rapid analytical methods like, HPTLC and RP- HPLC developed for the simultaneous estimation of Andrographolide (AG), Gallic acid (GA), Kutkin (KT) and Vasicine (VS) in HEPASAVE can be applied to the routine quality control studies. A new, simple, accurate, sensitive, precise, reproducible and robust High-performance thin layer chromatography (HPTLC) method was developed using Mobile phase Toluene: Ethyl acetate: Formic acid: Methanol (3: 3: 0.8: 0.4, v/v/v/v) and sample Ethyl acetate extract and was further utilized for the HPTLC study. The band dimensions were kept 6 mm in length, 10 mm from the bottom edge, 10 mm from the side edge; the application rate was 0.1µL/s, chamber saturation time utilized was 35 min at room temperature (25 ± 2 °C and 40% RH), the run length and time were kept at 55 mm for 25 min, Densitometric scanning was carried out using Deuterium/Tungsten lamp, in the absorbance mode at 254 nm for Quantitative evaluation (CAMAG winCATS software). The scanning speed of 20 mm s–1 was employed and a slit dimension of 5 mm x 0.1 mm was employed. The results obtained from scanning showed different spots of AG, GA and KT obtained at Rf values of 0.72 min, 0.61 min and 0.17 min, respectively. Vasicine peak was not obtained by the proposed HPTLC method. The data for calibration plots showed good linear relationship for Andrographolide with r2 = 0.995, Gallic acid with r2 = 0.9932 and Kutkin with r2 = 0.9917 in the concentration range of 200ng to 800ng, 80ng to 320ng and 2000ng to 6000ng, respectively with respect to peak area. The present method was validated by linearity, accuracy, precision according to ICH guidelines. The limits of detection and quantitation were determined. The limit of detection for AG, GA and KT were found to be 2.3111, 0.8950 and 0.0095, respectively and the limit of Quantitation was found to be 7.0034, 2.7120 and 0.0287, respectively. Statistical analysis of the data showed that the method is reproducible and selective for the quantitation of Andrographolide, Gallic acid and Kutkin simultaneously. The amount of AG, GA and KT in the formulation were quantified and were found to be 1.27 %w/v, 1.15 %w/v and 0.014 %w/v, respectively. The amount of Andrographolide, Gallic acid and Kutkin in the Ethyl acetate extract were found to be 7.6 %w/v, 6.92 %w/v and 0.22 %w/v, respectively.

Following the HPTLC method, the simultaneous estimation of Andrographolide, Gallic acid and Kutkin in Polyherbal formulation by RP- HPLC was carried out in a C18 PUROSPHERE STAR Hyber column with 250 × 4.5 mm i.d., 5 µm particle size. The column temperature and sample temperature was kept at 25ºC. The diluent used was Water : ACN (75:25), pH 3.45 maintained by 0.1% solution of o- phosphoric acid. The mobile phase used for the separation of the biomarkers was Water : ACN (75:25), pH 3.45 maintained by 0.1% solution of o- phosphric acid, flown at 1.0 ml/min. The injection volume employed was 20 µL, run time was kept 35 min and the detection wavelength was 254 nm. The blank used for the sample solutions was methanol. Prepared standard Andrographolide, Gallic acid and Kutkin solutions as well as the final Ethyl acetate extract interjected to HPLC column as per earlier mentioned chromatographic conditions. The chromatographic peaks of AG, GA, KT in formulation extract were compared with retention time of standard AG, GA and KT. Peak areas were recorded of prepared standard solutions. From the peak area of standard AG, GA and KT obtained from the RP- HPLC conditions, the amounts of these Biomarkers in extract as well as in the formulation were computed simultaneously. The amount of Andrographolide, Gallic acid and Kutkin in the Formulation were found to be 0.56 %w/v, 0.554 %w/v and 0.44 %w/v, respectively. The amount of Andrographolide, Gallic acid and Kutkin in the Ethyl acetate extract were found to be 3.354 %w/v, 3.28 %w/v and 1.8 %w/v, respectively.

Hence, we can conclude that an ICH- validated HPTLC method and an RP- HPLC method was developed for the first time for the quantitation of AG, GA and KT simultaneously.