A BRIEF STUDY ON FLAVONOIDS CONTENT IN COMMERCIALLY AVAILABLE TEA

Flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Over 4,000 flavonoids have been identified, many of which occur in fruits, vegetables and beverages (tea, coffee, beer, wine and fruit drinks). The flavonoids have aroused considerable interest recently because of their potential beneficial effects on human health-they have been reported to have antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor and antioxidant activities.

In present study, determinations of the total flavonoid content and quantitative estimation of rutin in methanolic extract of commercially available tea samples were performed by two methods – Using Spectrophotometer and RP-HPLC. The colorimetric method based on the formation of complexes with aluminium chloride was applied to determine the total flavonoids content. Presence of rutin was identified using chemical test and thin layer chromatography (TLC).

Both the methods were validated for accuracy, precision and specificity. Validation revealed that the methods are specific, accurate, precise, reliable and reproducible. The regression data for the calibration plots showed a good linear relationship with r0.9999 (Colorimetric method) and r0.9985 (RP-HPLC method) for rutin. Recovery studies were carried out to check the accuracy of the methods at three different concentrations and average recovery was between 85.0 and 99.0% for rutin. Total flavonoid content and the amount of rutin present in Tea samples were found to be 6.23% (w/w) and 10.34% (w/w), equivalent to rutin respectively.

Colorimetric Method:

 Principally, the procedure is related to the formation of complex between flavonoid and AlCl3 that produces a yellow colored solution. The absorbance is measured colorimetrically at maximum wavelength of 415 nm.

A standard curve is prepared by using the following concentration of 0.05, 0.10, 0.20, 0.50 mg/mL of Rutin as standard with a correlation coefficient of 0.9999.

RP-HPLC Method:

 Quantification of rutin was done by RP-HPLC method using C-18 column (250mm × 4.6 mm, 5μm) with an isocratic mobile phase, methanol: water: acetic acid (45:54:1 v/v/v) at a flow rate of 1.0 ml/min. The wavelength of maximum absorption (λmax) selected for quantification of rutin was 257 nm. HPLC was performed at ambient temperature.

A standard curve is prepared by using the following concentration of 5.0, 10.0 , 25.0, 50.0 and 100.0  mg/L of Rutin as standard with a correlation coefficient of 0.9985.

Extraction of Flavonoids:

 The flavonoids were extracted with methanol from finely ground tea leaves at room temperature. 0.5 gm sample is taken in an amber colored tube with 50 mL of 70% MeOH. The mixture is shaken and stored at room temperature. After 24 h, infusions are filtered through Whatman No 1 filter paper and re-extracted with equal volume of solvents. After 48 h, the process is repeated. The combined extracts are collected (total volume 100mL) and stored in a refrigerator at 40C.

Linear curve of rutin concentration (mg/mL) vs. absorbance

for determination of total flavonoids content by Spectrophotometer

 

Linear curve of rutin concentration (mg/mL) vs. absorbance

for determination of total flavonoids content by HPLC with UV/Vis Detector

Concentration of Flavonoids in Tea extracts expressed in terms of Rutin equivalent (mg of RU/g of extract

Rutin present as the major flavonoid in various plant / plant products can be selected as the phytochemical marker for chromatographic fingerprints and for routine quality control, thus for standardization of those samples. The method can also be applied as a quality control tool for various formulations containing rutin.

Contributed by : Mr. Bamdev Sinha under guidance of Prof. Barun Gupta

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