INTRODUCTION:
Medicinal plants are used since medieval times for treating ailments. Herbal formulations are used to cure the diseases in both humans and animals. The available herbal formulations in different forms are powder, decoction, fresh juice, vati oil, clarified butter preparations and alcoholic preparations. The herbal medicinal plants and their traditional preparations contain therapeutically important chemical constituents which is responsible for the biological activity or pharmacological activity1.
Marketed polyherbal formulation used as blood glucose regulator and it contains 34 ingredients. Main components present in the tablet are a) Daruharidra - strengthens the natural functionality of the pancreas, b) Vijaysar –Maintaining blood glucose levels and strengthens cells as it is rich in flavonoids, c) Giloy –Strengthens immunity and protects against infections, d) Majeeth – is a powerful antioxidant that protects vital organs from oxidative stress, e) Methika – The Potent source of Micronutrients, f) Gudmar - Delays glucose absorption as it maintains post meals blood glucose levels. This herbal tablet is mainly used for the treatment of diabetes and it has antioxidant, cardio-protective, Anti-hyperglycemic, Antipruritics, Antibacterial, Antimicrobial, Carminative activity2.
High performance thin layer chromatography is an advanced form of thin layer chromatography. The distinctness between HPTLC and TLC is about the separation plates3. HPTLC plates are based on silica gel 60 with smaller particle size than used for classical TLC. This allows a higher packing density and a smoother surface. Hence, sample diffusion is reduced. The particle size of HPTLC is 5-6µm whereas TLC contains particle size of 10-12µm4-18. Hence the study involves development and validation of HPTLC method for the determination of Quercetin and Gallic acid in marketed poly herbal formulation and the validation parameters involved are accuracy, precision, linearity, range, limit of detection and limit of quantification and robustness.
MATERIALS AND METHODS:
Chemicals and solvents:
Reference standards quercetin and gallic acid were purchased from sigma-Aldrich. Marketed poly herbal formulation was procured from local market. All other solvents are of analytical grade.
Equipment:
A Camag HPTLC system with Camag linomat V automatic sample applicator, twin trough development chamber, Hamilton syringe (100µl), Camag TLC scanner-IV, Win CATS software version 1.4.6, Camag TLC plate heater were also used during the study.
Preparation of Standard Solution:
Preparation of standard Quercetin: A stock solution of standard Quercetin was prepared by dissolving 10mg of Quercetin in methanol and making up the volume to 10ml to get a concentration of 1mg/ml.
Preparation of Standard Gallic Acid: Accurately weighed 10mg of standard Gallic acid and transfer into 10ml volumetric flask, dissolved by using methanol and made up the volume up to the mark with the same to get a concentration of 1mg/ml.
Preparation of Sample Solution: Sample solution was prepared in methanol and chloroform.
Preparation of Methanolic Extract: Accurately weighed 3 gm marketed polyherbal formulation powder was dissolved in methanol and made up the volume upto 10 ml to get a final concentration of 3gm/10ml.
Preparation of Chloroform Extract: Accurately weighed 3gm marketed polyherbal formulation powder was dissolved in chloroform and made up the volume to 10 ml to get a final concentration of 3gm/10ml.
Development of HPTLC Technique: HPTLC analysis was performed on 20cm × 10cm aluminium plates coated with silica gel 60F254. Standard solution of Quercetin, Gallic acid and sample solution were applied to the plates as bands of 6mm width. The plates were developed using toluene: ethyl acetate: formic acid: methanol in the ratio of 3:6:1.6:0.4, v/v/v/v as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. The developed plates were dried and then scanned at 254 nm with a Camag TLC Scanner with WINCAT software, using the deuterium lamp. The method was validated based on ICH guidelines.
Method validation:
Accuracy:
The reference standard of quercetin and gallic acid was added to the sample solution at a level of 50%, 100%, 150%. This was further diluted according to the procedure followed in the estimation of formulation. Assay was used for the determination of concentration of drug in the sample solution.
Precision:
Intra-day precision and inter-day precision of the method were determined. Intra-day precision was carried out at a concentration of 0.5-2.5µg / spot of Quercetin and Gallic acid for three times on the same day and the percentage RSD was calculated. Inter-day precision was evaluated by analysis of three replicate applications of standard solution of same concentration on three different days.
Linearity and Range:
A suitable standard solution containing 0.5 to 2.5 µg / spot were prepared from the standard stock solution. The solutions were examined by the assay procedure and the calibration curve was plotted using peak area vs. concentration of the standard solution. From the calibration curve, the slope and intercept were calculated.
Limit of Detection (LOD) and Limit of Quantification (LOQ):
The LOD and LOQ were experimentally verified by the known concentration of a standard solution of Quercetin and Gallic acid until the average response approximately 3 or 10 times the standard deviation of the responses for the 6 replicate determinations.
Robustness:
Small changes are made in the mobile phase composition and detection wavelength, the effects on the results were examined.
RESULTS AND DISCUSSION:
Development of HPTLC Method:
The HPTLC procedure was optimised to quantify the amount of drug present in the sample. Different mobile phases were selected for the optimisation. Standard Quercetin and Gallic acid were spotted on the HPTLC plates and trial was carried out using chloroform, toluene, ethyl acetate, formic acid, methanol (Table 1). Toluene: ethyl acetate: formic acid: methanol (3:6:1.6:0.4, v/v/v/v) was finally selected as the mobile phase for the detection and quantification of Quercetin and Gallic acid.
Table 1: Different mobile phase systems were tried for selecting the best mobile phase.
|
Mobile phase |
Ratio |
Observation |
|
Chloroform: ethylacetate: formic acid |
3:6:1 |
Drug does not migrated |
|
Toluene: ethylacetate: formic acid |
3:6:1 |
Drug does not migrated |
|
Toluene: ethylacetate: formicacid: methanol |
3:6:1.6:0.4 |
Symmetrical peak |
The standards (Quercetin and Gallic acid) and sample were spotted on the plates and the chromatograms were scanned at 254nm. A symmetrical peak with Rf value of 0.85 for Quercetin and 0.75 for Gallic acid were obtained.
Method Validation:
The validation of the method was done and the parameters involved are linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. The calibration curve (Fig 1 and 2) shows that Quercetin and Gallic acid are linear in the range of 0.5 to 2.5µg/spot (Table 2). The correlation coefficient was found to be 0.994 and 0.995 for Quercetin and Gallic acid respectively (Fig 3 to 12). The results of linearity were found to be within the specified limits.
Fig. 1: Calibration curve of Quercet
in
Fig. 2: Calibration curve of Gallic acid
Fig. 3: Standard chromatogram of Quercetin (0.5µg) and Gallic acid (0.5µg)
Fig. 4: Standard chromatogram of Quercetin (1µg) and Gallic acid (1µg)
Fig. 5: Standard chromatogram of Quercetin (1.5µg) and Gallic acid (1.5µg)
Fig. 6: Standard chromatogram of Quercetin (2 µg) and Gallic acid (2µg)
Fig. 7: Standard chromatogram of Quercetin (2.5µg) and Gallic acid (2.5µg)
Fig. 8: Chromatogram of BGR 34 (methanolic extract)
Fig. 9: Chromatogram of BGR 34 (chloroform extract)
Fig. 10: Quercetin on all tracks
Fig. 11: Gallic acid on all tracks
Fig. 12: Image of the developed plate
Table 2: Linearity and range of Quercetin and Gallic acid
|
Concentration (µg/spot) |
Quercetin |
Gallic acid |
||
|
Rf value |
Peak area |
Rf value |
Peak area |
|
|
0.5 |
0.84 |
3413 |
0.75 |
4495 |
|
1 |
0.84 |
5762 |
0.75 |
6529 |
|
1.5 |
0.84 |
7820 |
0.75 |
8515 |
|
2 |
0.84 |
9573 |
0.75 |
10256 |
|
2.5 |
0.84 |
11245 |
0.75 |
11709 |
The accuracy of the method was determined by recovery experiments. The percentage recovery of Quercetin was found to be 95.29%, 99.97%, 100% and 95.95%, 99.93 %, 99.98% for Gallic acid respectively. The average percentage of Quercetin and Gallic acid recovered were 98.42% and 95.95% respectively (Table 3 and 4).
Table 3: Accuracy (Recovery studies) of Quercetin
|
Concentration (µg/spot) |
Average Peak Area* |
% Recovery |
% RSD |
|
1.5 |
7818 |
99.97 |
0.043 |
|
2 |
9573 |
100 |
0.038 |
|
2.5 |
10716 |
95.29 |
0.84 |
Table 4: Accuracy (Recovery studies) of Gallic Acid
|
Concentration (µg/spot) |
Average Peak Area* |
% Recovery |
% RSD |
|
1.5 |
6525 |
99.93 |
0.078 |
|
2 |
8514 |
99.98 |
0.054 |
|
2.5 |
10251 |
95.95 |
0.071 |
