Analysis of of Quercetin in Camellia Sinensis Extracts

ABSTRACT

A precise and feasible reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the novel analysis of of Quercetin in aqueous and alcoholic extracts of Camellia sinensis extracts with internal standard has been developed. The analysis was carried out on a Phenomenax-C₁₈ column (250 × 4.6mm, 5μm). reversed-phase column, using a mixture of acetonitrile and 0.1% orthophosphoric acid in water at 60:40 ratio as the mobile phase; Propyl paraben was used as the internal standard. The linearity range of the Quercetin was found to be in 1-25 µg/ml and the retention times were found to be 3.3 and 5.6 min for Quercetin and Propyl paraben respectively. The accuracy was good and recovery values for Quercetin ranged from 100 to 102% respectively. The proposed novel method is precise and feasible for the determination of Quercetin with internal standard in a short analytical runtime. The method seems to be suitable for routine analysis in quality control laboratories and research institutes.

Keywords: Quercetin, HPLC, Propyl paraben, Validation.

Introduction

Quercetin (Fig. 1) chemically 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one is a plant pigment (flavonoid) found in many plants and foods, such as red wine, onions, green tea, apples, berries, Ginkgo biloba. It is used for treating conditions of the heart and blood vessels including “hardening of the arteries” (atherosclerosis), high cholesterol, heart disease, and circulation problems (1-3). It is also used to treat inflammation, asthma, gout, viral infections, chronic fatigue syndrome (CFS), preventing cancer, and for treating chronic infections of the prostate (4-7). Quercetin is also used to increase endurance and improve athletic performance. Tooth decay is most common infectious oral disease that afflicts 95% of the human beings. The flavonoids Quercetin found in green tea can stop the formation of dental plaques and tooth decay.

Fig.1 : Chemical structure of Quercetin

Some analytical methods were developed for analysis of quercetin by spectrophotometric (8-10), HPLC (11-13), HPTLC (14-16), electrochemical detection (17). In spite of its wide economical importance, a rapid and efficient method forits identification and quantification is necessary. To the best of our knowledge, there is no published method for the determination of quercetin in Camellia sinensis extracts with propyl paraben as internal standard.

The aim of the present study was to develop a rapid, economical, precise and accurate reversed-phase HPLC method for quantifying quercetin in aqueous and alcoholic extracts of Camellia sinensis.

Materials and Methods

Acetonitrile (HPLC grade, MERCK), Water (HPLC grade, Thomas Baker) and orthophosphoric acid (HPLC grade), Quercetin and Propyl paraben are procured from local analytical laboratories.

Instrumentation and chromatographic conditions

HPLC is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying & purifying the individual components of the mixture. The system consisted of UFLC-Shimadzu make LC-20AD with PDA detector. The data acquisitionwas performed by LC solutions software. The chromatographic separation was achieved on Phenomenax-C₁₈ column (250 × 4.6mm, 5μm) column. The elution was isocratic with mobile phase of 0.1% orthophosphoric acid in water and acetonitrile (60:40, v/v). The flow rate was 1.0 mL/ min and yielded a backpressure of about 57 bar. The column temperature was maintained at 40°C, the detection was monitored at a wavelength of 262 nm and injection volume was 10 µL. UFLC is suitable for separation of Quercetin with propyl paraben as internal standard. The chromatographic conditions are shown in Table 1.

Table 1: The chromatographic conditions of the developed method

Plant material (Camellia sinensis)

Dried leaves of Camellia sinensis i.e Green tea are collected from Doddabetta Tea Factory, Ooty, Tamilnadu and identified by Dr. K. Mruthunjaya, Asst. Professor, Department of Pharmacognosy, JSSCP, Mysore. The procured leaves were used for the preparation of alcoholic and aqueous extract.

Extraction Methodology:

Alcoholic Extract of Camellia sinensis

Alcoholic extract was prepared by hot reflux extraction method on a water bath temperature maintained at 78°C for 2 hours. The dried coarsely powdered leaves of Camellia sinensis (550g) were extracted in 3 batches (150g each). The obtained extract was filtered and solvent was distilled out up to required concentration and combined extract was spray dried to get alcoholic extract of Camellia sinensis and stored in a dessicator.

Aqueous Extract of Camellia sinensis

Aqueous extract was prepared by hot reflux extraction method on a water bath temperature maintained at 80°C for 2 hours. The dried coarsely powdered leaves of Camellia sinensis (550g) were extracted in 3 batches (150g each). The obtained extract was filtered and solvent was distilled out up to required concentration and combined extract was spray dried to get aqueous extract of Camellia sinensis and stored in a dessicator.

Drying of Extract:

Prepared extracts were dried using Spray dryer. Parameters maintained for Spray drying are Inlet temperature maintained at 70-80°C, Outlet temperature maintained at 50-60°C, aspiration rate 1200 rpm per min, feed pump 2ml per min respectively.

PREPARATION OF SOLUTIONS

Preparation of mobile phase

The content of the mobile phase was prepared from Acetonitrile, filtered and degassed mixture of and water and orthophosphoric acid in the ratio of 60:40 v/v.

Preparation of standard solutions

About 10 mg of pure standards of Quercetin and Propyl paraben were accurately weighed and dissolved separately in two 10 mL standard flask and dissolved in methanol to get standard stock solutions of concentration of 1 mg/mL (1000 µg/mL). From the prepared stock solution an intermediate stock solution was prepared which contains 100 µg/mL of Quercetin. From this intermediate stock solution serial dilutions were made to obtain final concentrations of the solutions in the range of 1, 5, 10, 15, 20 and 25 µg/mL of Quercetin. Propyl paraben solution was made to a concentration of 25µg/mL and used as an internal standard throughout the methodology.

Preparation of Camellia sinensis extracts sample solutions

100 mg of extract sample was weighed and transferred in to 10ml volumetric flask and dissolved in a mixture of 5ml of 6M HCl and 5ml of mobile phase and sonicated for 15min. The obtained solution was filtered through a 0.45 µ pore size filter into a HPLC vial and loaded to the instrument.

Method development and optimization

The chromatographic conditions were optimized by changing the mobile phase compositions; buffer used in the mobile phase column stationaryphase and organic solvent. Finally a mixture of 0.1% orthophosphoric acid in water, acetonitrile and C₁₈ column were used. A typical chromatogram obtained by using the aforementioned mobile phase and column are shown in fig. 2 of blank chromatogram and standard drug quercetin chromatogram is illustrated in Fig. 3.

Method validation

When a method has been optimized it must be validatedbefore put into practical use. By following the ICH guidelinesfor analytical method validation e Q2 (R1), the system suitability

Test was performed and the validation characteristics elinearity, accuracy, precision, specificity, limits of detectionand quantitation were addressed.

Preparation of calibration curve

Aliquots of quercetin (containing 10- 250 µg/ml) were pipetted into series of 10ml volumetric flask from 100µg/ml of stock solution followed by addition of propyl paraben containing 250µg/ml and made up to 10ml with HPLC grade methanol to obtain the final concentrations of 1, 5, 10, 15, 20 and 25 µg/ml of quercetin and 25µg/ml of propyl paraben solutions were prepared. The responses were measured at 262 nm. The calibration curve was established by plotting the peak areas of Quercetin versus their concentrations.

Assay Procedure

The column was equilibrated for at least 30 min, with the mobile phase flowing through the system with a flow rate of 1ml/min. Detector was set at a wavelength of 262 nm. Two sets of the sample solutions were prepared containing aqueous and ethanolic extracts with mobile phase mixture. The retention time of Quercetin and Propyl paraben in bulk drug in two replicate samples were found to be 3.3 and 5.6 mins. The peak area of the Quercetin in sample was obtained and the regression equation of the drug concentration over the peak areas was calculated. The RP-HPLC method was used to estimate the amount of Quercetin in aqueous, ethanolic extracts of Camellia sinensis. The typical chromatograms of alcoholic and aqueous extracts are shown in fig. 4 and 5 respectively. It is found that alcoholic and aqueous extract contains 7.35 µg and 4.31 µg per 100 grams of the extract respectively.

System suitability

The system suitability test ensures the validity of the analyticalprocedure as well as confirms the resolution betweendifferent peaks of interest. A data from six injections ofstandard solutions were utilized for calculating system suitability parameters like %RSD (0.19), tailing factor (1.2), theoretical plates (10728) and resolution (7.8).

Linearity and range

To assess the linearity, calibration plots of quercetin in each dilution were constructed in the concentration range 1-25µg/mL the correlation coefficients of quercetin was 0.9998 respectively.

Accuracy and precision

The accuracy and precision of the developed method was evaluated and results are expressed as percent recoveries 99.3 -101.7%. The precision study of intra-day and interday relative standard deviation was less than 2%, stating that method is more precise.

Specificity

The specificity test demonstrated that the used excipients did not interfere with the peak of the main compound. The results showed that the developed method was selective for determination

Of Quercetin in Camellia sinensis extracts.

Sensitivity

The limit of detection and limit of quantitation decide aboutthe sensitivity of the method. Tests for the procedure wereperformed on samples containing very low concentrations of analytes based on the visual evaluation method. In thismethod, LOD (signal to noise ratio of 3:1) is determined by theanalysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected. Accordingly, the LOQ (signal to noise ratioof 10:1) is determined by the analysis of samples with known concentration of analytes and by establishing the minimumlevel at which the analyte can be quantified with acceptableaccuracy and precision (RSD <2%). The LOD and LOQ values were found to be 0.02 and 0.05 µg/mL for Quercetin.

RESULT AND DISCUSSION

We developed and validated a simple and efficient reversed phase HPLC method for analysis of Quercetin in Camellia sinensis extracts. Method conditions were optimized by changing the mobile phase compositions; buffer used in the mobile phase, stationary phase and organic solvent. Finally a mixture of 0.1% orthophosphoric acid in water and acetonitrile and C₁₈ column were used. In this study UFLC instrumentation with PDA detection, which is readily available in most analytical and pharmaceutical laboratories, was used. The analytical method was validated as per current International Conference on Harmonization (ICH) guidelines.

In addition, in the present study,an internal standard was used to provide higher accuracy and precision of several substances tested, propyl paraben was chosen as the most appropriate internal standard. This substance is stable and does not interfere with the excipients present in of samples and composition of the diluent.Indeed, in the developed method, Propyl paraben was adequately separated from Quercetin. Moreover, its elution time was shorter, which resulted in a short run time of less than 10 min. In the accuracy of the method, recovery is in the range of 98.3-101.7% and standard deviation was less than 2% proves that method is more accurate and precise. The LOD and LOQ values of quercetin are 0.03 µg/ml and 0.05 µg/ml respectively. The described HPLC method was successfully applied for the determination of Quercetin in Camellia sinensis plant extracts.

Fig. 2: Blank chromatogram of the method

Fig. 3: Chromatogram of Quercetin (25 µg/ml) and Propyl paraben (Internal Standard)

Table 2: Recovery studies of the method

Table 3: Intraday and Inter day study precision of the method

Fig. 4: Assay of Quercetin in Camellia sinensis Alcoholic Extract

Fig. 5: Assay of Quercetin in Camellia sinensis Aqueous Extract

Table 5: Assay of quercetin in alcoholic and aqueous extract

Conclusion

The proposed method is simple, accurate, precise, specific andlinear over the analysis ranges and was able to simultaneous determination of quercetin with internal standard in a short analytical run time. Hence the method can easily and conveniently applied for routine analysis in qualitycontrol laboratories and research institutes.

Acknowledgements

Authors extend their thanks to the JSS College of Pharmacy, JSS University, Mysore for providing the facilities to carry out the research work.