Survey, collection and authentication of plant material
Picrorhiza plants growing wild in their natural habitat were collected from high altitude regions of North Western Himalayas. The plant material was authenticated at the Centre of Plant Taxonomy, University of Kashmir, Srinagar. A voucher specimen has been deposited in the repository of the Indian Institute of Integrative Medicine, Srinagar against voucher specimen (voucher no. IIIM/PK/Srinagar).
Chemicals and reagents used
The standard picroside I and II compounds used in the present investigation were isolated in the Natural Product Chemistry division by routine chromatography techniques. Identity and purity was confirmed by chromatographic methods like (TLC, HPLC) and spectral (IR, 1D- and 2D-NMR). Solvents were of HPLC grade and purchased from Ranbaxy Fine Chemicals Limited (Okhla, New Delhi, India). The structures were confirmed by their UV, MS, 1H NMR and C13 NMR data compared with the authentic data from literature. Acetonitrile of HPLC grade (Aldrich, USA) and Millex syringe filter unit were purchased from Reagent, New Delhi, India. Water for preparation of samples and HPLC–DAD analysis was deionized by a Milli-Q purification system with a 0.2 mm fiber filter (Barnstead, CA, USA).
Moisture analysis
For uniform and standard quantification of biomarkers in plant samples, it is necessary to remove the moisture content in the test sample. To remove the moisture content, the material was dried in hot air oven for 3 h at 50 °C and the process was repeated several times until a uniform dried weight of the sample was obtained. Moisture content was further analyzed in 1.0 g of the dried powdered plant material by the help of Moisture analyzer (Sailorius MA 100).
Preparation of herbal extracts
The rhizomes and leaf samples were taken from all the seven accessions grown at three different locations viz. gene bank, IIIM, Srinagar, field Station Bonera (Pulwama) and Yarikha (Gulmarg), J&K, India. The air dried plant material was finely powdered and stored at 4 °C. A known quantity of finely powdered sample was weighed into a 250 ml conical flask and subjected to sequential cold extraction using methanol and water in the ratio of 1:1 as extraction solvents while stirring at room temperature. Contents of the flask were squeezed through muslin cloth and the filtrate from aqueous extract was filtered using whatman filter paper. The extraction process was repeated thrice (2–3 h stirring each time). The extracts from each of the sample were evaporated under reduced pressure to give residues in different amounts.The yield of the extract was approximately 10 %. Extract was suspended in HPLC grade methanol in preparatory tubes (5 ml) and used for all experiments.
Isolation and characterization of marker compounds
The marker compounds picroside I and picroside II were isolated by column chromatography using different solvent systems and percolated over water bath for 3 h at 25 ± 2 °C (Kita et al. 1971; Singh et al. 2005). Use of the solvents in the increasing order of polarity yielded the extracts which helped in clear baseline separation of compounds during LC-UV-DAD-MS analysis. The aqueous extracts were filtered and dried by evaporation at reduced pressure and temperature (40 ± 2 °C). The quality of the residue was analyzed visually by HPTLC. The dried filtrate (200–250 mg) was dissolved in 50 ml methanol to yield picroside I and II using column chromatography. The residue obtained after solvent evaporation was passed through 900-g silica gel (60–120 mesh) column. The column chromatography of methanolic extract residue in chloroform with increasing polarity of methanol yielded two major marker compounds picroside I [6-0-trans-cinnamoyl catalpol] and picroside II [6-O-vanilloyl catalpol]. The isolated compounds were characterized by recording melting point using DSC, UV, IR and Mass spectra and identified by comparing with the reported data (Singh et al. 1993).
Preparation of stock solutions
Stock solutions of both the reference marker compounds, picroside I and picroside II (1000 mg/ml) were prepared in HPLC grade water. The concentration (μg ml−1) of each compound was 69 for pk-I and 125 for pk-II. The mixed solution was filtered through a Millipore filter (0.2 mm) before injection onto the HPLC system. The solution was prepared by accurately adding appropriate volume of each of the prepared standard solution. 10 µl of this mixed solution was injected in the column which exhibited different retention times for both the marker compounds. All the solutions were stored in refrigerator at 4 °C and kept at room temperature before use. On the same day of analysis, the precision as well as reproducibility of the method was observed. The peaks of the sample solution were identified by comparison of the retention time with those corresponding authentic samples run individually.
Calibration solutions
In order to establish the linear detection range for each compound, individual standard solutions were prepared in mobile phase in 250 ml measuring flask. Aliquots of these solutions were diluted and analyzed to determine method linearity. Limit of quantification (LOQ) values were estimated from serial dilution and analyzed for each sample. Triplicate 10 μl injections were made for each compound to see the reproducibility of the detector response at each concentration level. The peak area of each compound was plotted by running different concentrations to obtain the calibration graph. The five concentrations of each compound were subjected to regression analysis to calculate calibration equation and correlation coefficients.
Analytical HPLC conditions
The HPLC analysis was performed on a ThermoFinnigan HPLC machine equipped with auto sampler, column apartment and UV detector for the visual confirmation of the presence of marker compounds in the extract. Acquiring and analysis of data was controlled by Shemstation software (Agilent Tech, USA). A RP-18 column (3 mm × 150 mm) from E. Merck was used at 30 °C temperature. Separation was done using methanol and water (40:60) at a flow rate of 0.7 ml/min with injection volume of 4 ul for each sample and UV detection was set at 270 nm. Prior to use, solvents were filtered through a 0.22 mm diameter membrane filters. Equal volume of the standard solution were mixed and injected in the HPLC system in volumes of 2, 4, 6, 8 and 10 µl for plotting calibration curves. Solutions were injected in triplicate and the calibration curve was plotted by plotting concentration value of each anlyte. Satisfactory separation was obtained as shown in the chromatograms (Fig. 2). Quantification and linearity of the HPLC method was carried out for picroside content in different samples of P. kurroa. The percentage of the picrosides was calculated by calibration curve using peak height and peak area ratio. ICH guidelines were followed for the validation of the analytical methods for precision, repeatability and accuracy.
Linearity and accuracy
Linearity was determined by analyzing the standard solutions of both the picrosides pk-I and pk-II at five levels. The least square regression equation and correlation coefficient were used for assessing the linearity. Recovery and accuracy was assessed by the addition of known amounts of picroside I and picroside II to the pre-analyzed sample at three different concentration levels. The applicability of the extraction procedure was confirmed by recovery experiments of 99.9 and 100 % and the relative standard deviations (RSD) calculated for all samples proved to be less than 2 %.