Determination of Quercetin a Biomarker in Hepatoprotective Polyherbal Formulation through High Performance Thin Layer Chromatography

Methods: Polyherbal hepatoprotective formulation was developed by using five bioactive fractionated extracts of three plants namely Butea monosperma, Bauhinia variegate and O. gratissimum. All three plants contain quercetin. Chromatographic separation was performed on aluminium foil plates coated with 200 μm silica gel 60F254 Linear ascending development with toluene:ethyl acetate:formic acid, 5:4:0.1 (v/v/v) was performed at room temperature (25 ± 2°C) in a twin-trough glass chamber saturated with mobile phase vapor. Compact bands (Rf=0.38) were obtained for quercetin. Spectro densitometric scanning was performed in fluorescence mode at 380 nm. The method was validated for precision, recovery, specificity, detection and quantification limits.


Introduction
Nature still obliges as the man's primary source for the cure of his ailments. Research in preventive medicine showed the importance of functional nutrition in reducing the risk factor of certain chronic diseases. Innate defense system of the human body may be insufficient for the damage caused by continued oxidative stress [1]. Quercetin and other flavonoids, have the structure to act as powerful antioxidants, and have often proven so in vitro. Quercetin, being a major constituent of the flavonoid intake, could be a key in fighting several chronic degenerative diseases [2]. Growing scientific evidence has shown adverse side effects, like liver damage and mutagenesis, of synthetic antioxidant [3]. Therefore, recently there has been an upsurge of interest in natural products as antioxidants, as they inhibit the free radical reactions and protect human body from various diseases, such as cancer and diabetes. Recent studies showed that a number of plant products including polyphenolic substances (e.g., gallocatechins, delphinidin, cyanidin, gallic acid, ellagic acid, pelargonidin and sitosterol) and various plants or herbal extracts exert potent antioxidant actions, which are very well known for their healing powers [4].
Stem bark powder is used to apply on injury caused due to axe. Stem juice is applied on goitre of human being. Paste of stem bark is applied in case of body swellings. Bark is acrid, bitter, appetizer, aphrodisiac and laxative, anthelmintic, useful in fractures of the bones, diseases of theanus, dysentery, piles, hydrocele, cures ulcers and tumors. Bark is useful in biliousness, dysmenorrhea, liver disorder, gonorrhea and it also purifies the blood. The ash of young branch is prescribed in combination with other drugs in case of scorpion sting [5].
O. gratissimum is associated with chemo-preventive, anticarcinogenic, free radical scavenging, radio protective and numerous others pharmacological use [10]. O. gratissimumis used to treat different diseases, e.g., upper respiratory tract infections, diarrhea, headache, ophthalmic, skin diseases, pneumonia, and also as a treatment for cough, fever and conjunctivitis [11,12]. Earlier reports have shown the smooth muscle contracting lipid soluble principles, and antimutagenic activity in organic solvent extracts of O. gratissimum leaves [12,13]. This medicinal plant has also potential role as antibacterial [14,15], antifungal [16,17,18], antimicrobial [19,20] and anthelmintic [21]. The aqueous leaf extract and seed oil showed anti-proliferative and chemo-preventive activity on HeLa cells. Nangia-Makker et al. reported that, aqueous extract of O. gratissimum leaves inhibits tumor growth and angiogenesis by affecting tumor cell proliferation, migration, morphogenesis, stromal apoptosis and induction of inducible cyclooxygenase (COX-2) [22]. Ursolic acid was determined in dichloromethane and ethyl acetate fractions of methanolic extract of O. gratissimum in previously published report [23].
A limited number of study have been used for the determination of quercetin in Butea monosperma bark [24] and Ocimum gratissimum leaf and Bauhinia vareigata bark [25]. The quercetin concentrations were also determined by UV spectropho-tometry [26], liquid chromatography coupled with different types of detectors [27][28][29][30][31]. Even though these analytical procedures are suitable for the detection of quercetin in samples originating from plants, they have limitations with respect to their applications in the determination of quercetin in plant samples. The reported colorimetric method lacks sensitivity and is tedious and time-consuming. Even though high performance liquid chromatography (HPLC) is a method of choice, it is limited by extensive sample clean-up and requires expensive solvents and longer periods of column stabilization. In comparison to HPLC, HPTLC is a versatile analytical technique that requires less expensive instrumentation and expertise. The present study was carried out to develop a rapid, sensitive and accurate analytical method for estimation of quercetin in bioactive fractions of plant extracts and its pharmaceutical dosage form (hepatoprotective tablet formulation) for the routine analysis of a large number of plant extract samples and their formulations.

Reagents and standards
Quercetin was purchased from Yucca enterprises, Wadala, Mumbai and methanol AR grade from S.d. fine-Chem Ltd., Mumbai.

Plant materials
Polyherbal hepatoprotective tablet was prepared by using fractions obtained from alcoholic extracts of Butea monosperma, Bauhinia variegata stem bark and O. gratissimum leaves ( Figure 1). All these ingredients were collected from Maliba Pharmacy College campus and were authenticated by Prof. Minoo H. Parabia, Department of Bioscience, Veer Narmad South Gujarat University, Surat. Voucher specimen (No: MPC/13032010/01, 02 and 03) has been deposited in the Department of Bioscience.

Extraction and fractionation procedures:
The dried and powdered material of each plant (500 g) was extracted with methanol at room temperature for three weeks with shaking and stirring. Combined methanolic extracts were evaporated to dryness under reduced pressure below 40°C and then dissolved in distilled water and subjected to solvent-solvent fractionation.
Bauhinia variegate L.: Methanolic extract was fractionated with hexane, ethyl acetate (EtOAc) and n-butanol (n-ButOH) in the order of their increasing polarity to obtain respective fractions [18].
Ocimum gratissimum L.: Alcoholic extract was fractionated with hexane, dichloromethane (DCM) and ethyl acetate (EtOAc) in the order of their increasing polarity to obtain respective fractions [33].
Each fraction was concentrated to dryness under reduced pressure and below (

Establishment of qualitative and quantitative phytoprofile of fractionated extracts
Qualitative phytochemical analysis: Each fraction was subjected to various qualitative chemical tests using reported methods to determine the presence or absence of metabolites viz., alkaloids, tannins, flavonoid, steroid, terpernoids and phenolic compounds, etc. [34].
Chemical test for flavonoids: Chemical tests were performed for flavonoids according to Macdonald et al. [35].

Quantitative phytochemical analysis
Determination of total phenols: Each sample was mixed with 1 mL Folin-Ciocalteu reagent and 0.8 mL of 7.5% Na 2 CO 3 . The resultant mixture of was measured at 765 nm after 2 hr at room temperature. The mean of three readings was used and the total phenolic content was expressed in milligram of gallic acid equivalents/1 g extract. The coefficient of determination was found to be r 2 =0.992 [36]. sample and standard solution were measured at 415 nm. The mean of three readings was used and the total flavonoid content was expressed in milligram of quercetin equivalents/1 g extract. The coefficient of determination was r 2 =0.99020 [37].

Sample preparation
Preparation of standard solutions of quercetin: Stock solution of quercetin was prepared by dissolving 50 mg quercetin in 100 mL of methanol (500 µg/mL). Standard solutions of concentration 0.5, 1.0, 1.5, 2.0 and 2.5 in µg/mL were prepared by dilution of the stock solution with methanol.

Samples preparation from each plant extracts fractions:
Accurately weighed 100 mg of each, acetone fraction of Butea monosperma, ethyl acetate and n-butanol fractions of Bauhinia variegata and dichloromethane and ethyl acetate fractions of Ocimum gratissimum was transferred to separate 10 mL volumetric flask and dissolved in 10 mL of methanol. These solutions were sonicated for 10 minutes and filtered through Whatman No. 1 filter paper to get solution containing 10 mg/mL each.

Sample preparation from polyherbal tablet:
Polyherbal tablets equivalent to about 100 mg of mixture of fractionated extracts of Butea monosperma, Bauhinia variegata and Ocimum gratissimum was weighed and transferred to 10 mL volumetric flask containing 10 mL methanol to get solution containing 10 mg/mL. The resulting solution was centrifuged at 3000 rpm for 5 min and supernatant was analyzed for quercetin content [38].

Instrumentation and chromatographic conditions
HPTLC was performed on 15 cm × 10 cm aluminum backed plates coated with silica gel 60F254 (Merck, Mumbai, India). Standard solution of quercetin and sample solution were applied to the plates as bands 6.0 mm wide, 9.2 mm apart, and 15.0 mm from the bottomedge of the same chromatographic plate by use of a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100 μL Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed at room temperature (28 ± 2°C), with toluene: ethyl acetate: formic acid, 5:4:0.2 (v/v/v), as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. After development, the plates were dried with a hair dryer and then scanned at 380 nm with a Camag TLC Scanner with WINCAT software, using the deuterium lamp. The method was validated according to the ICH guidelines [11].

Calibration curve of quercetin
Different volumes of stock solution (500 µg/mL) were spotted on the TLC plate to obtain concentration 0.5, 1.0, 1.5, 2.0 and 2.5 µg /spot of quercetin, respectively. The data of peak areas plotted against the corresponding concentration.

Method Validation
The proposed method was validated as per ICH guidelines [39]. Samples were prepared as per the earlier adopted procedure given in the experiment.

Linearity and range
Linearity is expressed in terms of correlation coefficient of linear regression analysis. The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 0.5, 1.0, 1.5, 2.0 and 2.5 µg /spot of quercetin respectively. The calibration curve of absorbance vs. concentration was plotted and correlation coefficient and regression line equations were determined.

Precision
Result of precision should be expressed as relative standard deviation (% R.S.D) or coefficient of variance (% C.V.).

Repeatability
Standard solutions were applied by Linomat 5 automatic sample applicator. Sample was spotted seven times for repeatability studies. The peak area obtained with each solution was measured and % C.V. was calculated.

Intraday precision
Mixed solution containing (1.0-2.0 µg/spot) of quercetin was analyzed three times on the same day and % C.V. was calculated.

Interday precision
Mixed solution containing (1.0-2.0 µg/spot) of quercetin was analyzed on three different days and % C.V. was calculated.

Accuracy
It was determined by calculating the recovery of quercetin by standard addition method.

Recovery studies
The accuracy of the method was established by performing recovery experiments at three different levels using the standard addition method. In 1 µl (1 µg/mL) of samples, known amounts of quercetin (0.5, 1.0 and 1.5 µg/spot) standard were added by spiking. The values of percent recovery and average value of percent recovery for quercetin were calculated.

Limits of detection and limit of quantization
The LOD and LOQ were estimated from the set of 5 calibration curves. The LOD and LOQ may be calculated as

Specificity
The specificity of the method was ascertained by analyzing the standard drug and extract. The spot for quercetin in the sample was confirmed by comparing the R f values and spectra of the spot with that of the standard. The peak purity of the quercetin was assessed by comparing the spectra at three different levels, viz. peak start, and peak apex and peak end positions of the spot.

Optimization of mobile phase
Various ratios of solvents were tried as a mobile phase and optimum mobile phase was selected was toluene:ethyl acetate:formic acid, (5:4:0.1 v/v/v). This mobile phase allowed good resolution, dense, compact and well-separated spots at R f value 0.38. Wavelength 380 nm was used for quantification of the drug. Since there is only one peak seen, is shown in Figure 3.

Quantification by HPTLC Method development
In HPTLC chromatogram, all tracks for standard quercetin at wave length 380 nm were shown in Figure 2. The R f value of standard quercetin was found to be 0.38 and peak area was 9726 (Figure 3).

Method validation
Linearity and range: The linearity was determined for both drugs at five different concentration levels. The linearity of quercetin was in the range of 0.5-2.5 µg/spot and calibration curves are shown in Figure  3. Correlation co-efficient for calibration curve of quercetin was 0.9843.
The regression line equation for quercetin is as follows: y=9076x+6315 (Figure 4).

Precision
Repeatability: The data for repeatability are shown in Table 1. The % C.V for repeatability was found to be 0.5 (24341 ± 125) ( Table 1) Intra-day precision: The data for intra-day precision for quercetin are shown in Table 2. The % C.V of quercetin was found to be in range of 0.69%-0.97% (Table 2).

Inter-day precision:
The data for inter-day precision for quercetin are shown in Table 3. The % C.V of quercetin was found to be in range of 0.77%-1.50% (Table 3).
Accuracy: Accuracy of the method was confirmed by recovery at three level of standard addition. Percentage recovery for quercetin was found to be in range of 97.33%-99.11%. The results are shown in (Table 4).

Limits of detection (LOD) and limit of quantitation (LOQ):
Limit of detection and quantitation were determined by equation LOD=3.3 × (SD/s) and LOQ=10 × (SD/s) LOD and LOQ results are shown in (Table 5).

Estimation of Quercetin in Fractionated Extracts of Butea monosperma, Bauhinia variegata and Ocimum gratissimum and Polyherbal Formulation
The peak purity was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot. Good correlation (R 2 =0.9843) was obtained between the standard and the samples in the range of 0.5-2.5 µg/spot.  (Table 6). Acetone fraction of Butea monosperma showed eight peaks; the fourth peak R f value (0.39) was coinciding with standard R f value ( Figure 5). The concentration of quercetin was found to be 0.395 (µg/10 mg).
Ethyl acetate fraction of Bauhinia variegata showed eight peaks; the fourth peak R f value (0.38) was coinciding with standard R f value of quercetin ( Figure 6). The concentration of quercetin was found to be 0.174 (µg/10 mg). n-butanol fraction of Bauhinia variegata showed six peaks, the third peak R f value (0.38) was coinciding with standard R f value (Figure 7). The concentration of quercetin was found to be 0.138 (µg/10 mg). Dichloromethane fraction of Ocimum gratissimum showed nine peaks, the third peak R f value (0.38) was coinciding with standard R f value (Figure 8). The concentration of quercetin was found to be 0.322 (µg/10 mg). Ethyl acetate fraction of Ocimum gratissimum showed seven peaks; the third peak R f value (0.39) was coinciding with standard R f value (Figure 9). The concentration of quercetin in ethyl acetate fraction of Ocimum gratissimum was found to be 0.673 (µg/10 mg) [39,40]. Polyherbal tablet of formulation showed eighteen peaks, the R f value (0.38) of seventh peak was coinciding with standard R f value. The HPTLC densitogram is shown in Figure 10. The concentration of quercetin was found to be 0.113 (µg/10 mg) [38].

Summary of validation parameters
The detailed summary of validation parameters is described in (Table 7)