Validation and Development of HPTLC Method for Simultaneous Estimation of Apigenin and Luteolin in Selected Marketed Ayurvedic Formulations of ‘Dashmula’ and in Ethyl Acetate Extract of Premna integrifolia L

Dashmool (dashmul, dashamula) are specific combination of ten roots, famous Ayurvedic remedy mainly for strengthening body, promotes healthy elimination of toxins, tonic for kidney, liver, tonic in infertility, uterine tonic, used for anorexia, edema, anaemia, potent antioxidant and in disorders of nerves, bones, joints and muscles. It contains ten roots in equal propotion under category as ‘Brihat Panchmul includes; Aegle marmelos, Premna integrifolia, Oroxylum indicum, Stereospermum suveolens, Gmelia arborea While Laghu panchmul includes; Solanum indicum, Solanum xanthocarpum, Uraria picta, Desmodium gangeticum and Tribulus terrestris [1].

P. integrifolia, therefore selected here for biomarking using sophisticated analytical techniques HPTLC using bioactive markers like Apigenin and Luteolin.
This research is first attempt for standardisation of 'dashmul' using two flavonoids simultaneously, along with raw plant extract.

Plant material and marketed formulation
Stem powder of P. integrifolia: Agnimanth are distributed through flora of Trimbakeshwar forest area of Nashik District. Few of them identified, spotted stem branches were collected and herbarium was prepared. These was deposited to Botanical Survey of India, Pune for identification purpose. Certificates were issued as i. The HPTLC analysis was performed using above chromatographic condition, using Linomat 5 syringe 5 µl, 10 µl of AS and 5 µl, 10 µl of LS applied on pre coated TLC Silica gel 60 F 254 plates as band length of 8 mm, sample were air dried, mobile phase Toluene: Ethyl acetate: Formic acid (6:4:0.3) poured to CAMAG twin trough chamber,   saturation allowed for 10 min., spotted plates were developed till 90 mm, dried with dryer, scanned over TLC scanner 5 (1.14.26) with absorption/remission mode at scan speed 20 mm/s, spectral scan done in between 200-400 nm with 100 nm/s speed, spectra of apigenin and luteolin over layed and iso-absorptive wavelength noted, again plates were rescanned at 347 nm in detection scanner mode. Retention Factor (Rf), AUC for both standard AS and LS noted, used the data for further detection.

Fingerprinting and specificity of apigenin and luteolin standard in PI-ET and in DF1, BF4, KF7
The HPTLC analysis for fingerprinting and specificity of Apigenin and Luteolin standard done using developed parameter as above, applying on track-AS, PI-ET, DF1, BF4, and KF7, LS, plates developed as above and scanned in detection (347 nm) and spectral mode (200-400 nm). Rf obtained for AS and LS, identified and marked in PI-ET, DF1, BF4, KF7. The identified bands spectral scan over laid for confirmation of specificity. The purity of the bands was confirmed at start, middle and end position of chromatogram. The plate was derivatized with NP-PEG reagent and chromatogram visualized at 366 nm in florescence mode as shown in Figures 1a-1c.

Concentration T (ng/band) Average Concentration a (n=3) Intraday (ng/band) Intra day % RSD Average Concentration a (n=3) Inter day (ng/band)
The Limit of detection (LOD) and limit of quantification (LOQ) were calculated using equation: 3.3×Standard deviation of the Y -intercept LOD = Slope of the caliberation curve 10×Standard deviation of the Y -intercept LOQ = Slope of the calibration curve

Accuracy (% recovery):
The accuracy of the method was established by performing recovery experiments over known concentration on Extract -PI-ET and formulation DF1 at 80%, 100% and 120% spike of AS and LS separately in triplicate for each experiment and analyzing it with %RSD. As per Table 4a.
Intermediate precision: Precision study for Standard AS and LS done for three concentration as 40, 80, 120 ng/band, done in triplicate for intraday and inter day precision.
Precision study over recovery method for PI-ET and DF1 done separately in triplicate for 80%, 100% and 120% spike of AS and LS triplicate for intraday and inter day precision with 3 × 3 model for each experiment and analyzing it with %RSD.
Further result obtained were subjected for one way analysis of variance and with-day mean square compared to between -day mean square by F test (Tables 4b and 4c).

Repeatability precision:
The repeatability of the method was assessed by three concentration 40,80,120 ng/band in triplicate for each AS and LS done. The percentage relative standard deviation was expressed as Tables 3a and 3b.
Robustness: Mid concentration of 80 ng/band in triplicate of both standards subjected for robustness study using variability like wavelength 347-5 nm, slit width change as 6 × 0.3 mm, scan speed change as 40 mm/s.

Results and Discussion
In fingerprinting and specificity study with developed chromatographic condition in mobile phase Toluene: Ethyl Acetate: formic acid (6:4:0.3), Rf-obtained 0.39 and 0.29 for standard apigenin and standard luteolin respectively. There is single spot over track for standard. Track of PI-ET, DF1, BF4, KF7 shows spots with similar Rf for Apigenin and luteolin. Spectral scan of this selected spots gave specific overlay for Apigenin and Luteolin. Upon derivatization with NP-PEG reagent Spot of apigenin and luteolin appears bright yellow visually in day light, parrot green and bright yellow in florescence mode at 366 nm respectively.
That confirms presences and helped for fingerprinting of Apigenin and luteolin bioactive flavonoids in PI-ET and in selected formulations.
The calibration curve of Standard Apigenin and Standard Luteolin was found to be linear in range of 40-120 ng/band for both, with good regression coefficient 0.9983, 0.9997 for Apigenin and luteolin respectively. Table 1 summaries the validation parameter.
Estimation analysis for Extract PI-ET and formulation as shown in Table 2 indicates high %value for PI-ET extract due to highly purified flavonoid fraction from extract. Variability observed between manufactures and even within batches.
Resolution of extract and formulations are better while estimating apigenin and luteolin simultaneously as shown in Figures 3a-3l Accuracy study with %recovery at three concentration level performed in PI-ET and in one of formulation DF1 shows result as per Table  4a Precision study with repeatability performed over low, mid high concentration over both standard expressed as %relative standard deviation inters and intraday as per Tables 3a and 3b.
Accuracy with% recovery in PI-ET and DF1 at 80, 100, 120 %level with spike of standard and luteolin subjected for intraday and inter day, results at each level subjected to one way analysis of variance and the F value for each level were determined as per Tables 4b and 4c. F value as ratio of BMC/WMC, compared with tabulated F (2,6) value which is 5.14 and all calculated values are below it, therefore there was no significant difference between intra and inter day variability, suggesting good intermediate precision of the method.

Conclusion
Hence with regards to the method development, validation and simultaneous estimation, it can be concluded that, • HPTLC method is simple, precise, rapid and selective for simultaneous estimation of bioflavonoid Apigenin and Luteolin in Dashmul-P. integrifolia stem and ethyl acetate extract and marketed formulations.
• PI-ET shows high %of Apigenin and luteolin due to highly purified fraction of extract.
• The result of this study though shows great variance in %content of this bioflavonoids between manufactures and even in batches but method is precise and accurate through validation therefore can be method of analysis qualitative and quantitative for quality control parameter for herbals.
• HPTLC method development for standardization of Ayurvedic 'Dashmula formulation' gives high throughput results.