Quantitative Estimation of Immunomodulatory Flavonoid Quercetin by HPTLC in Different Leafy Vegetables Available in West Bengal

Introduction: Leafy vegetables are commonly consumed medicinal plants used in various metabolic and infectious diseases. Additionally, their antiviral, antioxidant, anticancer, and antihypertensive activities have been reported in the literature. Objectives: To determine the quantity of immunomodulatory flavonoid, quercetin in different types of leafy vegetables available in West Bengal by using a simple validated HPTLC method. Materials and Methods: The chromatographic analysis was performed by using aluminium-backed silica gel 60 F 254 plates with Toluene – Ethyl acetate – Formic acid 5:4:0.2 (%v/v) as the mobile phase. The method was validated according to the ICH guidelines. Results: The total flavonoid content (TFC) in the four leafy vegetables studied varied between 58.10 to 12.28 mg QE/g. Well-separated and compact spots (R f ) of quercetin (0.55±0.03) were detected. The regression equation obtained was y = 0.0011x - 0.0569, with a correlation coefficient ( R ²) of 0.9939. The linearity range (µg/spot) was 60-160. The LOD/LOQ (ng/spot) was 6.09/18.47. Murraya koenigii (0.1992±0.037 %w/w) contained the maximum amount of quercetin compared to Ipomoea aquatic (0.1501±0.039%w/w), Coriandrum sativum (0.1430±0.061%w/w) and Trigonella foenum-graecum (0.1201±0.055%w/w) in methanolic extract. Conclusion: This study revealed that the validated HPTLC method was simple, accurate and sensitive for separating and quantifying quercetin in different leaf vegetables. Quercetin content was highest in Murraya koenigii (0.1992±0.037 %w/w), and least in Trigonella foenum-graecum (0.1201±0.055 %w/w). The developed method might be used further in standardizing and quality control of secondary metabolites in herbal formulations.

amounts.Immunomodulators are substances which are synthetic or biological in nature, that help to modulate, suppress or stimulate both the adaptive and innate immune systems. [8]Immunomodulatory flavonoids from 40 leafy vegetables, belonging to 26 different plant families were reviewed. [12]Some of the flavonoids possessing immunomodulatory activity were identified; one among them being 'Quercetin' .Quercetin is a flavanonol glycoside found abundantly in leafy vegetables and possess immunomodulatory activity. [13]Quercetinits a bio flavonoids is used for anticancer, [14] anti-mutagenic, [15] anti-oxidative, [16] anti-inflammatory, [17] anticancer/ chemo-preventive, neuroprotective, [18] antihypertensive, and blood glucose-lowering activities. [19]Several analytical methods for the determination of quercetin have been reported in different literature.Several high-performance liquid chromatography (HPLC) [20] methods were developed for the quantification of quercetin either combination or in alone with other flavonoids in leafy vegetable juices or in pharmaceutical formulations.In order to simultaneously determine of kaempferol, naringenin, rutin, and quercetin, an LC-MS/MS method [21] was created.There hasn't been a single method published for the quantitative detection of quercetin by high performance thin layer chromatography (HPTLC) in several species of leafy vegetables.Among the 40 different leafy vegetables reviewed, quantification of quercetin is already reported for 16 of them. [11]Others have reported qualitative analysis for the presence of quercetin.As a result, the objective of the current study was to create a quick, accurate, and reliable HPTLC densitometric technique for estimating the quantity of the the immunomodulatory flavonoid quercetin (Figure 1) from the leafy vegetables of West Bengal to aid in the treatment of immune disorders that are frequently seen around the world.

Chemicals
Standard quercetin was purchased from Loba Chemie Pvt. Ltd.All the solvents used were of HPLC grade and other chemicals used were of analytical reagent grade.

Sample preparation for analysis of quercetin in methanolic extract of Leafy vegetables
The air-dried leaves of four different leafy vegetables were coarsely powdered, separately, then exhaustively extracted by maceration with methanol for 7 days.The solvent was evaporated to dryness under reduced pressure by use of a rotary vacuum evaporator and each of the residues were separately dissolved in methanol in 50 ml volumetric flasks. [22]termination of Total Flavonoid Content

Preparation of Standard Quercetin for Calibration Curve
The extracts' total flavonoid concentration (TFC) was assessed using a colorimetric aluminum chloride test. [23]A stock solution (100 mg/ml) was made by dissolving 100 milligrams of quercetin in one ml of methanol.This standard solution was serially diluted to produce solutions with concentrations of 20, 40, 60, 80 and 100µg/ml.To this, add 4 ml of distilled water and 0.3 ml of 5 percent NaNO 2 in the test tube.Five minutes later, mix with 0.3ml of 10% AlCl 3 .1 M NaOH in 2 ml was added to the mixture after 6 min.The mixture was immediately diluted with distilled water to a level of 10 ml.A spectrophotometer was used to measure the absorbance at 510 nm.

Preparation of Samples for Total Flavonoid Content
The extracts were produced as 100 mg/ml stock solutions in methanol, which were then diluted to create various 0.5 mg/ml concentration solutions.The extracts were processed using a method similar to that described for quercetin, and a spectrophotometer set at 510 nm was used to detect absorbance.The total quantity of flavonoids present was calculated using the average absorbance value obtained from three measurements.Using a linear equation based on the standard calibration curve, the flavonoid concentration was represented as quercetin equivalent (mg QE/100g).

Chromatographic Conditions
On 10cm × 10cm aluminum-backed plates coated with 0.2mm layers of silica gel 60 F 254 , densitometric HPTLC examination was carried out.The "CAMAG manual TLC Sampler 4" sample applicator, which is equipped with a CAMAG micro liter syringe, was used to apply samples to the TLC plates as 6mm bands.150 L/s was applied at a continuous rate. [24]oluene-Ethyl acetate-Formic acid 5:4:0.2(% v/v/v) [25] was used as the mobile phase to carry out linear ascending development of the plates to a distance of 80 mm in a CAMAG manual Developing Chamber 2 that had been previously saturated with the mobile phase developed for 30 min at 22°C.

Method Validation
Precision: Instrumental precision, intra-assay precision, and intermediate precision of the method were determined. [26]Instrumental precision was measured by replicate (n=3) application of the same quercetin standard solution (concentration 100mg/ml).Intra-assay precision was evaluated by analysis of three replicate applications of freshly prepared standard solutions of same concentration, on the same day.Intermediate precision was evaluated by analysis of three replicate applications of standard solution of same concentration on three different days.Limit of Detection and Limit of Quantification: The limit of detection (LOD) and limit of quantification (LOQ) values were determined as the amounts for which the signal-to-noise ratios were 3:1 and 10:1, respectively.Recovery Studies: The accuracy of the method was established by performing recovery experiments at three different levels using the standard addition method.The values of percentage recovery and average value of percent recovery for quercetin were calculated.Specificity: The specificity of the method was ascertained by analyzing the standard quercetin 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 quercetin was assessed by comparing the spectra at three different levels, viz.peak start, peak apex, and peak end positions of the spot.

Quantification of quercetin in different varieties of methanolic extract of leafy vegetables
The test samples were applied on the plates and chromatograms were obtained under the same conditions as for the analysis of standard quercetin.The area of the peak corresponding to the R f value of quercetin standard was recorded and the amount present was calculated from the graph.

Total Flavonoid Content Determination (TFC)
A colorimetric method based on aluminium chloride was used to measure the flavonoid content of a few leafy vegetable extracts.Equivalents of quercetin (QE) per gram of weight of dry extract were calculated using the calibration curve of quercetin (20-100 g/ml), and the results are shown in (Figure 2) as y = 0.0049x + 0.4664, R 2 = 0.9671.(Table 1).The content of flavonoid in methanol extracts ranged from 58.10 to 12.28 mg QE/g.The three plants with the greatest levels of flavonoids were C. sativum L., M. koenigii, and T. foenum-graecum (58.1±20.4,49.1±5.77,and 42.07±16.34mg QE/g, respectively), whereas I. aquatica had the lowest levels (12.28±1.5 mg QE/g).
The quantity and location of free hydroxyl (-OH) groups affect the efficacy of flavonoids, the secondary metabolites with immunomodulatory characteristics. [27]The methanol extract of C. sativum L. had a TFC of 60.87 mg QE/g dry weight, whereas the methanolic extract of M. koenigii had a TFC of 9.75 mg QE/g dry weight, according to D.T. Abeysinghe et al. [28] and Hasna Bouhenni et al. [29] The flavonoid concentration of leafy vegetables was shown to be considerably influenced by biological, environmental, genetic, seasonal, and yearly fluctuations, according to publications in the literature.

Calibration curve
The calibration plot shown in Figure 3 indicates the response is a linear function of concentration range 60-160 µg/ml quercetin.The correlation coefficient, intercept and the slope were 0.9939, -0.0569 and 0.0011 respectively.The linear regression data for the calibration curve of quercetin is shown in Table 2.

Recovery Studies
Results from recovery studies, listed in Table 3, were within acceptable limits (90.03 to 119.04 %), indicate the accuracy of the method was good.

Precision
Results from determination of repeatability and intermediate precision, expressed as SD (%) are show in Table 4. RSD was in the range 2.1 -9.59 for repeatability and 1.44-3.44 for intermediate precision.These low values indicated that the method is precise.

Limit of Detection and Limit of Quantification
LOD and LOQ of the proposed method was found to be 6.09 and 18.4 µg/spot, for quercetin, which indicated that the proposed method can be used in a wide range for detection and quantification of quercetin effectively.

Method Development
The mobile phase composition was optimized to establish a suitable and accurate densitometric HPTLC method for analysis of quercetin.The mobile phase Toluene-Ethyl acetate-Formic acid 5:4:0.2(%v/v/v) resulted in a sharp, symmetrical, and well resolved peak at Rf value of (0.55±0.03) (Figure 4).

Quantification of Quercetin in the Methanolic Extract of Different Varieties of Leafy Vegetables
Quercetin peaks from the methanolic extracts of different varieties of leafy vegetables were identified by comparing their single spot at R f = 0.55±0.03(Figures 5-8) with those obtained by chromatography    of the standard under the same conditions.The quercetin content in methanolic extracts of four different varieties of leafy vegetables was quantified by using the standard concentration and AUC as presented in Table 5.

DISCUSSION
For the identification and measurement of chemical compounds found in various green vegetables and plants, HPTLC is a useful method for quality evaluation. [30]The retention factor (Rf) values produced from it can be used to identify chemicals as they are specific to each chemical in a given solvent system.To measure the herbal extract, the TLC process was improved.Rf = 0.55±0.03for quercetin was the value for the mobile phase, which was composed of toluene, ethyl acetate, and formic acid in the ratio (5:4:0.2%v/v/v) (Figure 1).The HPTLC technique created for quercetin quantification in the current work was shown to be userfriendly, precise, repeatable, and sensitive, and it can be used to analyze methanolic extracts of four distinct kinds of leafy vegetables.The maximum amount of quercetin was found to be present in Murraya koenigii (0.1992mg/100gm dry weight) and minimum amounts were present in Trigonella foenum-graecum (0.1221mg/100gm dry weight).Also, the reported HPTLC method would find wide applications in the standardization and quality control of herbal raw materials as well as formulations.

CONCLUSION
TLC is a crucial tool for standardising natural medicines and nutritious meals.It is not possible to identify herbal preparations using a single approach because of their chemical complexity brought on by the       presence of various bioactive ingredients.For the simultaneous quantitative detection of the immunomodulatory flavonoid quercetin in various green vegetable extracts, the presented HPTLC approach is an appealing easy, quick, and selective method.This technique might be used extensively for the direct, routine analysis and quality control of associated extracts and medications.By using the approach described, bioactive components of additional plants with similar characteristics might be found and utilised to select genotypes appropriate for further development into cultivars tailored to the industry of producing natural health products; Additionally, statistical evidence shows that the technique is reliable and selective for the detection of quercetin, with the added benefits of quick turnaround, little sample preparation, and low cost.The comparative quercetin content analysis among the four commonly consumed leafy vegetables will be helpful for the nutritional choice in case of immunity boost up.However further study can be investigated to accomplish the quercetin content analysis for the remaining such common edible plants which will provide the nutritional indicator to enhance the immunity among common people of the society.