Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography / Asian Journal

of Traditional Medicines, 2009, 4 (3 )

Regular Articles

Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thinlayer chromatography
Natural Product Research Laboratory, Department of Pharmaceutical Sciences, Dr H S Gour Vishwavidyalaya, Sagar MP, India

Avijeet Jain *, Santram Lodhi, A K Singhai

Abstract In present work, simultaneous quantitative estimation of two biologically active flavonoidal compounds, quercetin and rutin, in Tephrosia purpurea (TP) leaves was performed using high-performance thin-layer chromatography (HPTLC). TLC aluminum plates precoated with silica-gel RP-18 F 254 S were used with a mobile phase of methanol-water-formic acid (40:57:3, v/v/v) (reported earlier for another herbal drug) and densitometric determination of these compounds was carried out at 254 nm in reflectance/ absorbance mode. The linear regression data for the calibration plots showed a good linear relationship with r=0.9942 and 0.9996 for quercetin and rutin, respectively. A recovery study was carried out to check the accuracy of the method. At two different levels the average recovery of quercetin and rutin was 100.16 % and 99.94 %, respectively. The present method has been repeated with some modification in an earlier reported method and the present method can be used routinely for the quantitative estimation of these two compounds. Key words: HPTLC; quantitative estimation; quercetin; rutin; Tephrosia purpurea

Introduction
The flavonoids, which occur both in the free state and as glycosides, are the largest group of naturally occurring phenols. They are formed from three acetate units and phenylpropane units. They are widely distributed in nature but are more common in young tissues, where they occur in cell sap. They have been used extensively as chemotaxonomic markers and are abundant in the Polygonaceae, Rutaceae,

* Author to whom correspondence should be addressed. Address: Natural Product Research Laboratory, Department of Pharmaceutical Sciences, Sagar MP, India; Email: avijeet_9826275340@rediffmail. com Received: 2008-11-19 Accepted: 2009-04-01

Leguminosae, Umbellifarae, and Compositae. While most are O-glycosides, a considerable number of Cglycosides are also known. Although the original high hopes regarding their therapeutic usefulness were not immediately realized, recent researchers have demonstrated their involvement in the medicinal action of a number of plant drugs. It is very probable that a number of herbal remedies, the constituents of which are still unknown, will be shown to contain active flavonoids. The group is known for its antiinflammatory and anti-allergic effects, antithrombitic and vasoprotective properties, and inhibition of tumor promotion as well as protecting the gastric mucosa. Many flavonoids-containing plants are diuretics or antispasmodics and some flavonoids have

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Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography / Asian Journal of Traditional Medicines, 2009, 4 (3 )

antitumor, antifungal and antibacterial properties as well as antihepatotoxic activity [1]. Quantitative estimation of these compounds is important for current research and a variety of methods are required for this. Consequently, present study was focused on the simultaneous quantitative estimation of two flavonoids, quercetin and rutin, by high performance thin-layer chromatography (HPTLC) in an ethnopharmacologically important herbal species Tephrosia pupurea (TP). TP is a pantropical coastal shrub that grows up to 1 m in height and occurs throughout the Indian subcontinent. Previous phytochemical investigations of this plant have shown the presence of coumarins, flavonoids and rotenoids, flavanones and isoflavanones and quercetin [1]. It is an important component of some preparations, such as Tephroli and Yakrifit, used to treat liver disorders. In the Ayurvedic system of medicine, various parts of this plant are used as remedies for impotence, asthma, diarrhoea, gonorrhoea, rheumatism, ulcers and urinary disorders. The plant has also been claimed to cure diseases of the kidney, liver spleen, heart and blood [2]. The dried herb is effective as a tonic laxative, diuretic and deobstruent. It is also used in the treatment of bronchitis, bilious febrile attacks, boils, pimples and bleeding piles. The roots and seeds are reported to have insecticidal and piscicidal properties and they are also used as a vermifuge. In addition, the roots are reported to be effective in the treatment of leprous lesions while their juice is used to treat skin eruptions. An extract of the pods is effective for the treatment of pain, inflammation and their decoction is used to combat vomiting [3]. The ethanolic extract of this plant has been reported to have anticancer activity against KB cells in culture while the aqueous extract of the seeds has shown significant in vivo hypoglycaemic activity in diabetic rabbits. The ethanolic extracts of TP possess potential antibacterial activity and the flavanoids have been shown to exhibit antimicrobial

activity [4]. This study reports the quantitative estimation of quercetin and rutin in TP leaves for the first time using an HPTLC method which is suitable for routine use. Although quantitative estimation of quercetin and rutin has been reported by HPTLC in a variety of herbal extracts, this is the first time that such a method has been reported for TP with some modification of existing methods.

Experimental
Reagents and materials
TP leaves were collected from the forests of Dr H S Gour Vishwavidyalya, Sagar, MP during the month of September 2007. The standards of quercetin and rutin were purchased from S D Fine Chemicals, India. The TLC plates RP-18 F254 S (57.5 cm) (E. Merck, Germany) were used without any pretreatment. All chemicals and solvents used were of analytical and HPLC grade.

Standard stock solutions and sample preparation

Standard stock solutions of quercetin and rutin (1 mg/ml) were prepared by dilution in methanol and

Fig. 1. RP-TLC plate of Tephrosia purpurea leaves at 254 nm. Track I, II and III III represent quercetin, rutin and the extract. and represent quercetin, rutin and the extract.

Fig. 1. RP-TLC plate of Tephrosia purpurea leaves at 254 nm.

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Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography / Asian Journal of Traditional Medicines, 2009, 4 (3 )

Fig. 2. HPTLC chromatogram of quercetin

Fig. 2. HPTLC chromatogram of quercetin

Fig. 3. HPTLC chromatogram of rutin

Fig. 3. HPTLC chromatogram of rutin

passed through 0.45 Millipore filters. A sample was prepared from a coarse powder of dried TP leaves and the powder (50 g) was refluxed with ethanol in a Soxhlet extractor for 72 h. The extract was then filtered and concentrated under vacuum to obtain the crude extract. A known amount of the extract (200

16

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Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography / Asian Journal of Traditional Medicines, 2009, 4 (3 )

mg) was taken and dissolved in methanol (10 ml) and then 1 ml of this solution was diluted to 10 ml and passed through a 0.45 m Millipore filter.

HPTLC analysis
A Camag HPTLC system equipped with an automatic TLC sampler (Linomat 5), a TLC scanner 3 (WINCATS version 1.2.3) with a UV cabinet and a twin trough glass tank were used. The samples were applied using an automated TLC sampler in 5 mm bands at 10 mm from the X and Y axis and 5 mm spaces were left between adjacent bands.

Calibration and quantification

Standard stock solutions of quercetin and rutin (1 mg/ml) were diluted in methanol to obtain the working solutions (0.10 mg/ml) for quercetin and rutin. The obtained working standard solutions were then applied to RP-TLC plates to prepare fourpoint linear calibration curves. Quercetin and rutin were both applied in volumes of 5, 10, 15, and 20 l. For sample quantification, 5 l quercetin, rutin and

samples were applied to the plates as 5 mm bands at intervals of 5 mm. The experiment was performed in triplicate. The TLC plates were developed in a Camag twin-trough glass tank which was pre-saturated with developing solvent (40:57:3, v/v/v) up to a height of 7 cm. The composition of the developing solvent was selected from the method previously developed [8] . After development, the plate was removed, dried and spots were visualized under UV light at 254 nm. Quantitative evaluation of the plate was performed in the reflectance/absorbance mode at 254 nm under the following conditions: slit width (60.4 mm, Micro), scanning speed (20 mm/s) and data resolution (100 mstep).

Recovery
To determine the recovery, known concentrations of standards were added to a preanalyzed sample of TP leaves. The spiked samples were then analyzed by the proposed HPTLC method and the analysis was carried out in triplicate.

Fig. 4. HPTLC chromatogram of an extract of Tephrosia purpurea leaves

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Fig. 4. HPTLC chromatogram of an extract of Tephrosia purpurea leaves

Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography / Asian Journal of Traditional Medicines, 2009, 4 (3 )

Results and discussion

TLC or HPTLC is primarily used as an inexpensive method for separation, qualitative identification, or semi-quantitative visual analysis of samples. Accordingly, TLC is often described as a pilot method for HPLC [5]. However, recent reviews show that the TLC and HPTLC techniques can be used to solve many qualitative and quantitative analytical problems in a wide range of fields, including medicine, pharmaceuticals, chemistry, biochemistry, food analysis, toxicology and environmental analysis [6]. The use of TLC/HPTLC has expanded considerably due to the development of forced flow (FF) and gradient TLC methods, improved stationary and mobile phase selection, as well as new methods of quantitation methods [7]. To estimate quercetin and rutin in TP leaves TLC was performed on RP-TLC plates using the solvent system methanol-water-formic acid (40:57:3, v/v/v), which produced good separation with Rf values of 0.07 (quercetin) and 0.17 (rutin) for the ethanolic extract and reference compounds. The ethanolic extract was able to resolve 6 compounds in the developing

solvent system. The identity of the bands of quercetin and rutin in the ethanolic extract was confirmed by comparing the UV-Vis absorption spectra with those of standards using a CAMAG TLC scanner 3 (Fig. 1-4). The four-point linear calibration curves of both compounds were found to be linear over the range 500-2000 ng (Table 1). Good recoveries were obtained following enrichment of the sample at two different concentrations. The results showed that the percentage recoveries after sample processing and application were in the range of 100.12 % to 100.21 % (quercetin) and 99.77 % to 100.11 % (rutin) (Table 2). The percentage of rutin in TP leaves was higher than that of quercetin (Table 3).

Conclusions
In conclusion, an HPTLC method has been developed with some modifications and it can be used for the simultaneous quantitative determination of quercetin and rutin in TP leaves; its main advantages are its simplicity, accuracy, and selectivity. This method can also be used for the estimation of these compounds in other herbal preparations and may be

Table 1. Rf and linear regression equation of quercetin and rutin Compound Quercetin Rutin
*

Rf 0.07 0.17

Regression equation y = 6.9601 x + 5.18 y = 6.1496 x + 85

r* 0.9942 0.9997

Correlation coefficient

Table 2. Recovery study of quercetin and rutin by HPTLC (n=3) Compound Quercetin Rutin Amount of compound present in the plant material (mean, mg/100 mg) 473 1913 Amount of standard added (mg) 473 946 1913 3826 Amount of standard found in mixture (mg) 948.00 1420.66 3830.33 5726.00 Recovery (%) 100.21 0.87 100.12 0.44 100.11 1.14 99.77 0.93

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Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography / Asian Journal of Traditional Medicines, 2009, 4 (3 )

Table 3. Amount of quercetin and rutin in Tephrosia purpurea leaves Compound Quercetin Rutin Quantity (mean) (mg/100 mg) 0.473 1.913 Mean SE 0.473 0.004 1.913 0.005 CV (% ) 0.84 0.26

useful for standardization purposes.

Acknowledgments
Authors are grateful to the Indian Council of Medical Research, New Delhi for funding the present research.

[4]

[5]

References
[1] Jain A, Singhai AK, Dixit VK. A Comparative study of ethanol extract of leaves of Tephrosia purpurea Pers and flavonoid isolated for hepatoprotective activity. Indian J of Pharm Sci, 2006, 68(6): 740-3. [2] Kirtikar KR, Basu BD. In: Indian Medicinal Plants. 2nd ed. Allahabad, India: Lalit Mohan Basu. 1956, 98-9. [3] The Wealth of India, In: A Dictionary of Indian Raw

[6] [7] [8]

Materials and Industrial Product. vol. X. NewDelhi, India: Publication and Information Directorate, CSIR. 1976, 151-6. Lodhi S, Pawar RS, Jain AP, Singahi AK. Wound healing potential of Tephrosia purpurea Linn Pers in rats. J Ethnopharmacol, 2006, 108(2): 204-10. Rozylo JK, Janicka M. Different planar techniques for prediction of solute retention in column liquid chromatography. J Planar Chromatogr, 1996, 9(6): 418-24. Weins C, Hauck HE. Advances and developments in thin layer chromatography. LC-GC Int, 1996, 14(6): 455-71. P o o l e C F, P o o l e S K . I n s t r u m e n t a l t h i n - l a y e r chromatography. Anal Chem, 1994, 66(1): 27A-37A. S w a r o o p A , G u p t a P, S i n h a A K . S i m u l t a n e o u s determination of quercetin, rutin and coumaric acid in flowers of Rhododendron arboreum by HPTLC. Chromatographia. 2005, 62(11-12): 649-52.

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