ESTIMATION OF QUERCETIN CONTENT IN THREE DIFFERENT SPECIES OF EUPATORIUM BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

ESTIMATION OF QUERCETIN CONTENT IN THREE DIFFERENT SPECIES OF EUPATORIUM BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

V. Nithya and M. Kamalam ^*

Department of Botany, PSGR Krishnammal College for Women, Coimbatore - 641004, Tamil Nadu, India.

ABSTRACT: Three different species of Eupatorium namely E. glandulosum, E. odoratum and E. triplinerve belongs to the family Asteraceae are selected for the analysis of quercetin content. Leaves are extracted with ethanol and water and used for the analysis of quercetin by HPTLC technique using the mobile phase containing toluene: ethyl acetate: formic acid: methanol (5.5:4:1:0.5). Determination of quercetin content was performed by densitometric scanning under 254 nm, and the quercetin was detected at the R_f value of 0.54. The quantity of the quercetin content in plant extract was estimated by the calibration curve obtained from the standard quercetin. The result showed that the ethanolic extracts of E. glandulosum showed a high amount of quercetin (17.44 mg/g) followed by E. odoratum (13.4 mg/g) and E. triplinrve (9.29 mg/g).

HPTLC, Eupatorium glandulosum, Eupatorium odoratum, Eupatorium triplinerve, quercetin

INTRODUCTION: Plants produce a high diversity of secondary metabolites which are helpful in the defense mechanism of plants played against abiotic stress, and many of them have some medicinal importance. Quercetin is a flavonol, proved to be a potent antioxidant among poly-phenols ^{1, 2, 3}. It possesses antiviral, antibacterial, anticarcinogenic and anti-inflammatory effects ^{4, 5}. As per the literature, quercetin was reported in E. glandulosum ⁶, E. perfoliatum ⁷ and E. cannabinum ⁸. In the present study, three different species of Eupatorium such as E. glandulosum, E. odoratum and E. triplinerve have been selected for the estimation of quercetin using High-Performance Thin Layer Chromatography.

E. glandulosum belongs to the family Asteraceae is a native of Mexico, introduced as an ornamental shrub in several countries. In India, the tribes of Nilgiris use the leaves of this plant to heal wounds and small injuries ⁹. E. odoratum is a fast growing perennial shrub, native of Central and South America has spread in tropical and subtropical regions of the world. The tribes of Indonesia used the leaf extract to cure skin diseases, poison bites, wounds, burns, cough, diabetes, diarrhea, fever, inflammation, and rheumatism. The boiled roots are used to cure urinary disorders ^{10, 11, 12}.

E. triplinerve commonly called as Ayapana is an ornamental, erect, perennial herb having aromatic leaves. In tribal medicine, it is used to cure a fever with convulsions, pneumonia, indigestion, and cough ¹³. Hence, to consider the medicinal importance of the above-said plants, the present study is undertaken with the objective of estimation of the quantity of quercetin content in three different species of Eupatorium using HPTLC technique.

MATERIALS AND METHODS:

Collection of Plant Material: The leaves of E. glandulosum, E. odoratum, and E. triplinerve were collected from Nilgiri Hills of Western Ghats, Coimbatore and Kanjikode Kerela respectively and certified by Botanical Survey of India, Coimbatore, Tamil Nadu. The plant materials are maintained in BSI Coimbatore under Voucher no: BSIR/RC/ 5/23/2017/Tech/338, BSIR/RC/5/23/2017/Tech/339 and BSIR/RC/5/23/2017/Tech/340. The leaves were washed thoroughly, dried, powdered and stored in air tight container for the study.

Preparation of Standard Solution: Quercetin (1mg/10ml) was prepared by dissolving 1 mg of quercetin in 10 ml of methanol in a standard flask.

Preparation of Plant Extracts: The leaf powder was defatted with petroleum ether and extracted with Ethanol (70 ºC) and water (100 ºC) in a Soxhlet apparatus. The extract was then dried and dissolved in a required amount of methanol.

Chromatography and Detection of Quercetin: Chromatography was performed on a 10 × 20 cm precoated HPTLC Silica gel 60 F₂₅₄ plates (E-Merck, Mumbai, India). Aliquots of each of the extracts were separately applied (Samples and standard) to the plate as 6 mm wide band with an automatic TLC applicator Linomat-5 applicator (CAMAG, Switzerland), 5 mm from the bottom. The mobile phase consisted of Toluene: Ethyl acetate: Formic acid: methanol (5.5:4:1:0.5) was used per chromatography. The twin glass chamber was saturated with the mobile phase for about 30 minutes. The plate was developed up to 10 cm in twin glass horizontal developing chamber at the room temperature. Plates were air dried, and scanning was performed on a Camag TLC Scanner at 254 nm.

Calibration curve of Quercetin: The quercetin compound was determined by using a calibration curve established with a standard concentration ranging from 40 to 320 ng/spot. A stock solution of standard quercetin (l mg/ml) was prepared in methanol. The different volumes of stock solution 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8 and 3.2 ml were spotted on HPTLC plate to obtained concentration 40, 80, 120, 160, 200, 240, 280 and 320 ng/spot, respectively (bandwidth 6 mm, distance between tracks 7 mm) using automatic sample spotter. Peak areas were recorded for quercetin, and the calibration curve was obtained by plotting peak area against the concentration of quercetin.

RESULTS AND DISCUSSION: HPTLC fingerprinting of plant species is not only helps in the identification and quality control of a species but also to provide basic information useful for the isolation, purification, characterization, and identification of marker chemical compounds of the species. In the present study, methanol and water extracts of leaf powder of E. glandulosum, E. odoratum and E. triplinerve are used for analysis of quercetin content. Different concentrations of standard quercetin and leaf extracts were applied on HPTLC plates and developed in a solvent system consisting of toluene: ethyl acetate: formic acid: methanol (5.5: 4: 1: 0.5) and dried in air and scanned densitometrically Fig. 1, 2a and 2b. The calibration curve of quercetin was found to be linear in the range of 4 to 32 mg/spot. A good linear relationship of standard quercetin was found to be R² = 0.998 concerning the concentration and peak area Fig. 3 and 4. The regression equation was found to be Y = 1268x + 1578 with respective to concentration.

FIG. 1: DENSITOMETRIC CHROMATOGRAM OF QUERCETIN AND LEAF EXTRACTS (3D VIEW)

The total number of peaks was found to be 10, 12 and 12 in ethanol extracts of E. glandulosum, E. odoratum, and E. triplinerve respectively Fig. 5, 6 and 7. The water extracts of E. glandulosum, E. odoratum and E. triplinerve showed 9, 7 and 10 peaks respectively Fig. 8, 9 and 10. The R_f value of standard quercetin was determined as 0.54 Fig. 3. In the plant samples, the ethanol and water extracts showed the peak at the R_f values of 0.54 and 0.55 Fig. 5, 6, 7, 8, 9 and 10.

A- Ethanol extract of E. glandulosum; B- Aqueous extract of E. glandulosum; C- Ethanol extract of E. odoratum

D- Aqueous extract of E. odoratum; E- Ethanol extract of E. triplinerve; F- Aqueous extract of E. triplinerve

G-N- Different concentration of quercetin

FIG. 5: HPTLC CHROMATOGRAM OF ETHANOL EXTRACT OF EUPATORIUM GLANDULOSUM

PEAK TABLE

FIG. 6: HPTLC CHROMATOGRAM OF AQUEOUS EXTRACT OF EUPATORIUM GLANDULOSUM

PEAK TABLE

FIG. 7: HPTLC CHROMATOGRAM OF ETHANOLIC EXTRACT OF EUPATORIUM ODORATUM

PEAK TABLE

FIG. 8: HPTLC CHROMATOGRAM OF AQUEOUS EXTRACT OF EUPATORIUM ODORATUM

PEAK TABLE

Among the selected three plants, the ethanol extract of E. glandulosum showed the maximum amount of quercetin (17.44 mg/g) followed by E. odoratum (13.40 mg/g) and Eupatorium triplinerve (9.29 mg/g). Water extracts showed a minimum amount of quercetin (2.67 mg/g) in E. triplinerve Table 1 and 2.

FIG. 9: HPTLC CHROMATOGRAM OF ETHANOLIC EXTRACT OF EUPATORIUM TRIPLINERVE

PEAK TABLE

FIG. 10: HPTLC CHROMATOGRAM OF AQUEOUS EXTRACT OF EUPATORIUM TRIPLINERVE

PEAK TABLE

Apart from quercetin, all the leaf extracts showed many numbers of peaks at different R_f values which shows the presence of various other compounds.

Similarly, the ethanolic extract of Calamus rotang showed the R_f value of quercetin at 0.54 ^14, but in contrast, a polyherbal syrub zymodyne and methanolic leaf and flower extract of Moringa oleifera showed the presence of quercetin at the R_f value of 0.86 and 0.35 respectively ^{15, 16}. The HPTLC analysis of an aqueous extract of Eruca sativa was found to be 17.94 ¹⁷ which is equal to E. glandulosum.

TABLE 1: PEAK TABLE OF QUERCETIN IN LEAF EXTRACTS

As per the literature, quercetin possesses biological and therapeutic effects including anti-cancer, anti-oxidative, anti-microbial and anti-inflammatory, cardioprotective and hepatoprotective activities ^{18, 19, 20}. Hence, from the above findings, it is confirmed that the leaf possesses a good amount of quercetin content.

TABLE 2: QUERCETIN CONTENT IN PLANT EXTRACTS

CONCLUSION: The quantitative estimation of quercetin was analyzed in three different species of Eupatorium leaves by HPTLC fingerprinting technique. The quercetin content was found to be maximum in the ethanolic extract of all the three plants than water extract. The ethanolic extract of E. glandulosum leaves showed maximum quercetin content than other two species studied. Since, the leaves possess the promising amount of quercetin, it can be used for curing various ailments.

ACKNOWLEDGEMENT: We are grateful to thank the Principal, PSG College of Pharmacy for providing facilities to complete this work.

CONFLICT OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Nithya V and Kamalam M: Estimation of quercetin content in three different species of Eupatorium by high performance thin layer chromatography. Int J Pharm Sci & Res 2019; 10(1): 303-08. doi: 10.13040/IJPSR.0975-8232.10(1).303-08.

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