A novel stability indicating HPTLC method for comparative quantitative estimation of quercetin in herbal preparations

This research introduces a novel stability-indicating HPTLC (High-Performance Thin-Layer Chromatography) method for the comparative quantitative estimation of Quercetin in herbal preparations. Through extensive stability studies, the method is validated, providing a robust framework for evaluating Quercetin's stability in diverse herbal formulations. The developed method ensures accurate and reliable quantification of Quercetin, facilitating comparative analysis across various herbal preparations. Additionally, results reveal that Quercetin degraded under most tested conditions, such as acidic, alkaline, oxidative and neutral conditions underscoring the necessity for intervention by experts to eliminate such challenges and ensure product efficacy and quality in the herbal industry


Introduction
Herbal medicine, also known as botanical medicine or phytotherapy, refers to the use of plants or plant extracts for medicinal purposes.This traditional form of medicine has been practiced for centuries across various cultures worldwide and involves utilizing the therapeutic properties of herbs to promote health and treat illnesses.The resurgence of Ayurveda, an ancient system of medicine originating from India, as a prominent force further underscores the popularity of herbal medicine.Ayurveda emphasizes the use of herbs, diet, lifestyle modifications, and spiritual practices to promote health and well-being.As interest in holistic health approaches grows, Ayurveda's principles of personalized medicine and holistic healing are gaining recognition and acceptance worldwide.
Overall, the popularity of herbal medicine is driven by a combination of factors, including a desire for natural remedies, cultural heritage, a focus on wellness and prevention, accessibility, and increasing scientific validation..1.Quercetin Quercetin, derived from the Latin word "Quercetum," meaning Oak Forest, is a flavonol compound that is not synthesized within the human body.It possesses a yellow hue and exhibits poor solubility in hot water, moderate solubility in alcohol and lipids, and insolubility in cold water.Widely recognized as one of the most utilized bioflavonoids for addressing metabolic and inflammatory ailments, quercetin is abundantly present in various dietary sources including fruits (particularly citrus fruits), leafy greens, seeds, nuts, flowers, barks, broccoli, olive oil, apples, onions, green tea, red grapes, red wine, dark cherries, blueberries, and cranberries.Vegetables such as onions and broccoli, along with fruits like apples, cherries, and berries, as well as beverages such as tea and red wine, contain the highest concentrations of flavonols.
Quercetin is purported to offer numerous health benefits, including protection against ailments like osteoporosis, lung cancer, and cardiovascular diseases.Studies have indicated a correlation between a high intake of flavonoids and a reduced risk of cardiovascular diseases.Chronic obstructive pulmonary disease (COPD), the third-leading cause of death in the USA, currently lacks fully effective therapies and may present side effects.However, emerging evidence suggests that quercetin supplementation could offer therapeutic benefits for COPD.Preclinical studies have shown that elevated plasma quercetin levels resulted in a significant reduction in lung inflammation and halted disease progression.Furthermore, quercetin has demonstrated potent anti-inflammatory properties in various animal models and human trials, with reports of reduced expression of inflammatory genes in response to a quercetin-enriched diet.Clinical trials have shown that quercetin supplementation, at a dosage of 1000 mg/day, reduced the incidence of upper respiratory tract infections in middle-aged and older individuals over a 12-week period.

Materials and methods
Equipment: A CAMAG HPTLC system comprising of a Linomat V applicator and CAMAG HPTLC scanner and single pan balance of Shimadzu model was used, for the present study.
Chemicals: Analytical grade Toluene, Ethyl acetate,Methanol, Formic acid, formic acid was obtained from Loba and CDH Chemical Pvt Ltd.Stationary phase used was silica gel G60F254, 20x10 cm TLC plate were obtained from E. Merk Ltd (Mumbai, India).

Organoleptic Evaluation
Organoleptic evaluation involves assessing the drug based on its color, odor, taste, etc., utilizing our sensory organs.
 Appearance: Visual examination was conducted to assess the appearance. Color: The dried sample was placed in a test tube, and its color was observed in sunlight.
 Odor: Evaluation of odor was performed using a freshly prepared sample. Taste: The taste was evaluated using a freshly prepared sample.To conduct the Molisch's test, a solution of Molisch's reagent is prepared by dissolving 10 g of alpha naphthol in 100 ml of 95% alcohol.A small amount of the test residue is mixed with 0.5 ml of water in a test tube, followed by the addition of 2 drops of Molisch's reagent.Then, 1 ml of concentrated sulfuric acid is carefully added from the side of the inclined test tube, forming a layer beneath the aqueous solution.The appearance of a red-brown ring at the interface of the two liquids indicates the presence of sugars.

 Test for Proteins -Xanthoproteic Test
In the Xanthoproteic test, a sample is mixed with 2 ml of water and 0.5 ml of concentrated nitric acid.The development of a yellow color indicates the presence of proteins.

 Test for Alkaloids -Dragendorff's Reagent
For the Dragendorff's reagent test, a solution is prepared by mixing solutions A and B in a 1:1 volume ratio.Solution A contains bismuth subnitrate and tartaric acid, while solution B consists of potassium iodide and water.From this solution, a working standard is prepared.The test filtrate, obtained by filtering a sample dissolved in 1.5% hydrochloric acid, is basified with dilute ammonia.Chloroform extract of this solution is applied to filter paper impregnated with Dragendorff's reagent.The appearance of an orange color indicates the presence of alkaloids.

 Test for Tannins -Ferric Chloride Test
To perform the Ferric chloride test, the test residue is dissolved in water, warmed, and filtered.A few drops of a 5% solution of ferric chloride in 90% alcohol are added to the filtrate.The formation of a dark green or deep blue color indicates the presence of tannins.

 Test for Flavonoids -Shinoda Test
In the Shinoda test, a small amount of the test extract is dissolved in 5 ml of 95% ethanol.To this solution, a few drops of concentrated hydrochloric acid and 0.5 g of magnesium metal are added.The appearance of a pink, crimson, or magenta color within a minute or two indicates the presence of flavonoids.

 Test for Saponins -Foam Test
For the Foam test, a small amount of the test extract is vigorously shaken with a mixture of sodium bicarbonate and water in a test tube.The formation of a stable, characteristic honeycomb-like froth indicates the presence of saponins.

 Solubility
The presence of adulterant in a drug was indicated by solubility studies-Quercetin is soluble in organic solvents such as Ethanol, Methanol and DMSO.

 Melting Point
Melting point determination involves heating a dry, impurity-free sample in a melting point apparatus, observing the temperature range at which it transitions from solid to liquid phase, and comparing it to known values to confirm identity and purity.
The melting point of Quercetin is 314°C-316 °C

Preparation of Standard Solutions
Solution A: Approximately 25 mg of Quercetin was accurately weighed and dissolved in 25.0 mL volumetric flask using methanol, and then the volume was adjusted to mark with the same solvent (Concentration: 1 mg/mL).
Solution B: 5.0 mL of Solution A was accurately measured and further diluted in a 50.0 mL volumetric flask with methanol (Concentration: 100 µg/mL).
Solution C: 10.0 mL of Solution A was accurately measured and further diluted in a 50.0 mL volumetric flask with methanol (Concentration: 200 µg/mL).

Selection of Mobile Phase
Solution B of quercetin was applied to TLC plates, and various solvents of different polarities were tested individually and in combination to optimize plate development for sharp, stable, and distinct peaks.The mobile phase consisting of toluene: ethyl acetate :methanol (4:4:2 v/v) was found to be the most satisfactory and suitable for further experimentation.

Selection of Detection Wavelength for Densitometric Evaluation of Sample Spots
After chromatographic development and plate drying, bands were scanned over a wavelength range of 200-400 nm.
The wavelength selected for detection was 254.0 nm.

Finalized Chromatographic Conditions
The chromatographic conditions were optimized by evaluating various parameters and were kept constant during further experimentation: -Samples were applied as 6 mm wide bands with a spacing of 6 mm using a micro syringe (Hamilton, Switzerland) on pre-coated silica gel aluminum TLC plates 60F_{24} (20 x 10 cm, Merck) with a thickness of 250 µm using a CAMAG LINOMAT V automatic sample applicator.-Slit dimensions of 3.0 x 45 mm and a scanning speed of 20 mm/s were employed in the analysis.
-Linear ascending development was conducted in a twin-trough glass chamber (10 x 10 cm, Camag, Switzerland) using toluene: ethyl acetate:methanol (4:4:2 v/v) as the mobile phase.-Chamber saturation time was 30 minutes, migration time was 15 minutes, and migrating and the distance was 75 mm.After application, TLC plates were dried using a dryer with a current of air.Densitometric scanning was conducted using the CAMAG TLC SCANNER 3 at 254 nm, employing WINCATS software.The scanner utilized a deuterium lamp as a radiation source emitting a continuous UV spectrum ranging from 200 to 400 nm.

Construction and Study of Calibration Curve
Following the general procedure, calibration curves were constructed by spotting 5-35µL of solution B, achieving a concentration range of 50 to 350 ng/band on TLC plates.The plates were developed and scanned under the optimized chromatographic conditions.This process was repeated three times, and the mean peak height and peak area were recorded for different drug concentrations.Calibration curves were then constructed plotting concentration versus peak height and peak area.The resulting calibration curves are depicted in Figure 2.

Validation parameters
Validation of the proposed method was carried out as per the USP guidelines.

 Accuracy
Accuracy of the proposed method was ascertained on the basis of recovery studies.Recovery studies were performed by standard addition method.

Precision
Precision of an analytical method is expressed as S.D. or R.S.D. of series of measurements.It was ascertained by replicate estimation of the drugs by proposed method.The results of estimation are shown in Table 5 3.

Specificity
The specificity of the method was ascertained by analyzing standard drug and sample.The spot of Quercetin in sample was confirmed by comparing the Re and spectra of spot with that of standard.The peak purity was assessed by comparing the spectra at three different levels peak start (S), peak apex (M) and peak end (E).

Linearity
The study was performed by applying different volumes of standard solution on TLC plates, developing and scanning the plates as per the optimized chromatographic conditions.The test results that are directly proportional to the concentration of the analyte within a given range.The response (e.g., peak area or peak height) obtained from each standard solution is then plotted against its corresponding concentration.That is shown in fig. 2 3.

Robustness and ruggednes
Robustness is the measure of capacity of a method to remain unaffected by small but deliberate variations in the method conditions and is an indication of the reliability of the method.To study the robustness of the method, small but deliberate variations in mobile phase composition (±2%) chamber saturation period (±10%), development distance (±10%), time from application to development (0, 10, 15 20 min), time from development to scanning (0, 30, 60, 90 min) were carried out.Results of robustness studies are given in Table 8 and 9

Results of ruggedness studies
The ruggedness of the method was studied under three different parameters.

Intraday variation
The samples were analysed on different times on same day by proposed method.The percent labeled claim was calculated and results of estimation proposed method.

Interday variation
The samples were analysed by proposed method on three different days ( 1 st , 3rd and 5 th day).The percent labeled claim was calculated and results of estimation are shown in

Different analysts
The samples were analysed by three different analysts as per the proposed method.The percent labeled claim was calculated and results of estimation are shown in 11.  [25][26][27][28][29][30][31][32][33][34] In order to ensure the stability indicating property and specificity of the proposed method stress studies were performed.In all following degradation studies, the average peak area Quercetin of was determined by applying 200 ng/band .

Conclusion
In this study, we developed and applied a validated HPTLC (High-Performance Thin-Layer Chromatography) method for the quantitative estimation of Quercetin in pharmaceutical marketed formulations.The chromatographic conditions were meticulously optimized, considering various parameters such as mobile phase composition, linearity range, detection wavelength, band size of the spots applied, chamber saturation time, solvent front migration, and slit width.These optimizations were crucial to ensure the accuracy and reproducibility of the results.
The Rf value obtained for Quercetin was found to be 0.65 ± 0.02, indicating its characteristic migration behavior under the specific chromatographic conditions employed.The linearity of the method was established over a concentration range of 5 -35 µg/mL, with high correlation coefficient values of 0.9998 and 0.9999 obtained for height and area, respectively.These high correlation coefficient values signify the excellent linear relationship between the concentration of Quercetin and its corresponding response in the chromatogram.
The method exhibited high accuracy, with the results showing an accuracy of 99.945 ± 0.321%.This high level of accuracy indicates the reliability of the method in quantifying Quercetin content in the tested formulations.
Furthermore, degradation studies were conducted to assess the stability of Quercetin under various stress conditions.It was observed that Quercetin underwent degradation in all stress conditions tested, highlighting the importance of monitoring its stability in pharmaceutical formulations.
Overall, the developed HPTLC method proved to be robust, sensitive, and suitable for the quantitative analysis of Quercetin in pharmaceutical formulations.The optimized chromatographic conditions, along with the observed accuracy and reproducibility, validate the applicability of this method for routine quality control analysis in the pharmaceutical industry.

Disclosure of conflict of interest
No conflict of interest to be disclosed.

Figure 2 Figure 3 Figure 4 and 5
Figure 2 Calibration curve for Quercetin Conc.Vs Height and Conc.Vs AUC

Table 1
Chemicals and Suppliers

Table 3
Organoleptic Properties of Quercetin Drug  Test for Sugars -Molisch's Test

Table 4
Phytochemical Evaluation Physical Standards are determined for drug.These are rarely constant for crude drug but may help in evaluation, specifically with reference to moisture content, drug gravity, density optical rotation, refractive index, melting point and solubility in different solvents.

Table 8
Results of robustness studies

Table 9
Results and statistical data for intraday study

Table 10
Results and statistical data for interday study

Table 11
Results and statistical data for different analysts