ISOLATION AND IDENTIFICATION OF THE FLAVONOID “QUERCETIN” FROM TRIDAX PROCUMBENS LINN.

Received on 12 November, 2013; received in revised form, 12 January, 2014; accepted, 23 March, 2014; published 01 April, 2014

ISOLATION AND IDENTIFICATION OF THE FLAVONOID “QUERCETIN” FROM TRIDAX PROCUMBENS LINN.

Nikita Sanghavi, Rashmi Srivastava and Yashwant Malode*

MET Institute of Pharmacy, Bhujbal Knowledge Center, MET Complex, Bandra (W), Mumbai 400050, Maharashtra, India

ABSTRACT: Tridax procumbens (Linn.) is an important medicinal plant belonging to the family Asteraceae. It is known for a number of pharmacological activities like antidiabetic, anticancer, anti-inflammatory, wound healing, hepatoprotective and antioxidant activity.Flavonoid “Quercetin” was isolated from the flowers of this plant. Dried flowers were Soxhlet with petroleum ether, chloroform, and methanol successively. The methanolic fraction so obtained was successively extracted with petroleum ether, diethyl ether and ethyl acetate. The ethyl acetate fraction was hydrolyzed with 7% H₂SO₄ and extracted with ethyl acetate to obtain crude Quercetin. The crude product was recrystallized with dilute ethanol to get pure Quercetin.  The purified material was subjected to various chromatographic and spectral techniques such as UV, IR, HPTLC, HPLC, NMR etc. and was identified as “Quercetin”. This study is also of practical importance because quercetin is an important constituent of Tridax procumbens and it has many uses such as in cancer, diabetes, inflammation, & in viral infections etc.

Tridax procumbens, Flavonoid, Quercetin, Isolation, Soxhlet

INTRODUCTION:Ayurveda, the ancient healing system of India, has been steadily gaining importance and acceptance as the dangers and shortcomings of modern medicines are getting more apparent ¹.

Man is able to obtain from them a marvelous assortment of industrial chemicals. Plant based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds, etc ². The beneficial medicinal effects of plant materials typically result from the combinations of secondary products present in the plant ³.

Tridax procumbens L. (family – Asteraceae) is commonly known as Jayanthi in Ayurveda/Sanskrit and Coat buttons/Tridax daisy in English. It is native of America and widely distributed throughout the tropics and sub-tropical countries such as India, Brazil, Mexico, Australia, Indonesia, Sri Lanka, Bangladesh, Africa etc ⁴. The plant has number of chemical constituents like alkaloids, tannins, flavonoids like luteolin, quercetin, keampherol, saponins, carotenoids, β-Sitosterol, n-hexane, and various acids like fumaric, lauric, myristic, palmitic, stearic, arachidic, benenic, palmitoloic, linoleic acid etc ⁵.

It is known for a number of pharmacological activities like antidiabetic, anti-inflammatory, wound healing, hepatoprotective and antioxidant activity. It has antimicrobial activity against both gram-positive and gram-negative bacteria. It is also known to have hypotensive effect and an immunomodulating property.

It is use in bronchial catarrh, dysentery and diarrhoea. Studies also suggest that it is used to stimulate hair growth and prevent hair fall. It shows anticancer activity and is being studied for prostate cancer and skin cancer ^{6, 7}.

The present study deals with the isolation and identification of the flavonoid Quercetin from Tridax procumbens L. Flavonoids are a group of about 4000 naturally occurring polyphenolic compounds, found universally in foods of plant origin ⁸. They are usually subdivided according to their substituents into flavanols (kaempferol, quercetin), anthocyanins, flavones, flavonones and chalcones. These flavonoids display a remarkable array of biochemical and pharmacological actions viz., anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral and anticancer activities ^{9, 10}.

Quercetin belongs to this group of plant pigments called flavonoids which is known to have antioxidant, anticancer, anti-inflammatory and antiviral activity. It is also useful for a variety of cardiovascular diseases ^{11, 12}.

MATERIALS AND METHODS:

Chemicals: Quercetin standard was procured from Yucca Enterprises (Mumbai). All other chemicals, solvents and reagents were obtained from SD Fine chemicals (India).

Plant Material: Flowers of Tridax procumbens were handpicked from a private farm in the eastern region of Nashik and were authenticated at the Botany department of KTHM College, Nashik.

EXPERIMENTAL: The flowers of Tridax procumbens L. were shade dried and grinded to obtain a coarse powder. 100 gm. of the coarse powder was successively soxhlet extracted with 750 ml. each of petroleum ether, chloroform and methanol respectively. Each of the solvent was used for extraction for a period of 24 hrs. The methanolic fraction was concentrated to obtain a semisolid consistency (13 gm). 2 gm of this fraction was successively extracted with 50 ml of petroleum ether (fraction І), 50 ml of diethyl ether (fraction ІІ) and 50 ml of ethyl acetate (fraction ІІІ) with the help of a separating funnel.

Each extraction was repeated three times to ensure complete extraction in each case. This was done for the entire methanolic extract. Fraction І and ІІ were rejected because of the presence of fatty acids and free flavonoids respectively. Fraction ІІІ was used for further processing as it contained Quercetin in its glycoside form. Fraction ІІІ was concentrated and hydrolyzed using 7% H₂SO₄ (10 ml/ gm extract) for 5 hrs. The hydrolyzed fraction was filtered and extracted with ethyl acetate (1:1/ thrice) by using a separating funnel. It was then concentrated to get the crude Quercetin which was later crystallized with 10% ethanol to get pure Quercetin. Quercetin was subjected to various spectral and chromatographic techniques.

Characterization of Quercetin:

UV Spectroscopy:

Sample preparation: Sample solution of the strength 1000.0 ppm was prepared by dissolving accurately weighedpowder of isolated Quercetin (10.0 mg) with ethanol (10.0 ml) in a standard volumetric flask. From this, a stock solution of 10.0 ppm was prepared.

Preparation of standard solution: Solution of standard Quercetin of the strength 1000.0 ppm was prepared by dissolving the accurately weighed standard (10.0 mg) with ethanol (10.0 ml) in a standard volumetric flask. From this, a stock solution of 10.0 ppm was prepared.

Infrared Spectroscopy (IR): The sample was prepared by mixing the isolated fraction of Quercetin with KBr (1:100) and subjected to Fourier Transform Infrared Spectroscopy (FTIR) using Diffused Reflectance Spectroscopy (DRS) assembly.

 Thin Layer Chromatography (TLC): The precoated TLC plate (3.0×8.0 cm) were activated in hot air oven at 105⁰ C for 30 min and cooled to room temperature. Quercetin was dissolved in ethanol and was applied 1 cm. above the edge of the plate along with the standard Quercetin. This plate was developed in an air tight chromatography chamber containing about 8.5 ml of solvent mixture of ethyl acetate, toluene, formic acid (4:3.5:0.5).

The developed plates were air dried and visualized under UV. These TLC plates were also subjected to spraying reagents for flavonoids such as ferric chloride and alc. Aluminum chloride.

High Performance Thin Layer Chromatography (HPTLC):

Sample preparation: Sample solution of the strength 1000.0 ppm was prepared by dissolving accurately weighedpowder (10.0 mg) with methanol (10.0 ml) in a standard volumetric flask. From this, a stock solution of 10.0 ppm was prepared.

Preparation of standard solution: Solution of standard Quercetin of the strength 1000.0 ppm was prepared by dissolving the accurately weighed standard (10.0 mg) with methanol (10.0 m) in a standard volumetric flask. From this, a stock solution of 10.0 ppm was prepared. Optimized HPTLC conditions are given in Table 1.

TABLE 1: OPTIMIZED HPTLC CONDITIONS

High Performance Liquid Chromatography (HPLC): The purified material was also subjected to HPLC studies. Optimized HPLC conditions are given in Table 2.

TABLE 2: OPTIMIZED HPLC CONDITIONS

RESULTS: The λ_max of isolated sample and the standard Quercetin are same (i.e. 255 and 372 nm) (Figure 1, 2). The characteristic IR peaks were found to be matching with those of their respective standard reference compound of Quercetin (Figure 3, 4). The TLC plate developed under UV light shows spots with the standard quercetin. R_f value (0.43) of Quercetin isolated from sample coincided with the R_f value of standard Quercetin (Figure 5). HPTLC overlay at 254 nm and spectrum scan of isolated and standard Quercetin are comparable (Figure 6, 7). When isolated Quercetin was subjected to HPLC, it showed retention time of 8.4 min which coincided with that of standard Quercetin (Figure 8, 9). H1 NMR spectra of both reference and isolated compounds are matching (Figure 10, 11). Results of tests carried on standard and isolated Quercetin are summarized in Table 3.

FIGURE 1: UV SPECTRA OF STANDARD QUERCETIN

FIGURE 2: UV SPECTRA OF ISOLATED QUERCETIN

FIGURE 3: IR SPECTRA OF STANDARD QUERCETIN

FIGURE 4: IR SPECTRA OF ISOLATED QUERCETIN

FIGURE 5: TLC PLATE SHOWING PRESENCE OF QUERCETIN 1) STD., 2) ISOLATED

FIGURE 6: OVERLAY AT 254 NM OF STANDARD AND ISOLATED QUERCETIN IN HPTLC

FIGURE 7: SPECTRUM SCAN OF STANDARD AND ISOLATED QUERCETIN IN HPTLC

FIGURE 8: HPLC OF STANDARD QUERCETIN

FIGURE 9: HPLC OF ISOLATED QUERCETIN

FIGURE 10: H¹-NMR SPECTRA OF STANDARD QUERCETIN

FIGURE 11: H¹-NMR SPECTRA OF ISOLATED QUERCETIN

DISCUSSION: Quercetin is an important constituent of Tridax procumbens which has number of uses such as anticancer, antidiabetic, hepatoprotective, antioxidant, etc. was isolated from flowers of Tridax procumbens using simple, rapid and convenient isolation procedure. The yield was found to be 0.072%.

The isolated fraction was characterized using sophisticated method of analysis from physicochemical, spectral and chromatographic studies, the structure of standard and isolated Quercetin was confirmed.

TABLE 3: RESULTS OF TESTS CARRIED ON STANDARD AND ISOLATED QUERCETIN

ACKNOWLEDGMENT: Authors acknowledge the valuable help rendered by Botany Department, KTHM College, Nashik for the authentication of plant sample, Herbal Research Lab, Ramnarain Ruia College, Mumbai for the HPTLC studies, SAIF, IIT Bombay for NMR studies. Authors also acknowledge the constant support rendered by Dr. Abha Doshi, principal MET Institute of Pharmacy, Mumbai for providing all the necessary facilities for the project.

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    How to cite this article:

    Sanghavi N, Srivastava R and Malode Y: Isolation and identification of the flavonoid “Quercetin” from Tridax procumbens linn..Int J Pharm Sci Res 2014; 5(4): 1454-59.doi: 10.13040/IJPSR.0975-8232.5(4).1454-59

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