HPTLC Estimation of Qurecetin in Tylophora indica and Tephrosia purpurea

 

Praveen Patidar*, Anis Shaikh, Darshan Dubey and Kamlesh Dashora

Institute of Pharmacy, Vikram University, Ujjain (M.P.).

 

 

ABSTRACT:

Tylophora indica (Family- Asclepiadaceae) having anti-asthmatic, anti-inflammatory, sedation decreased motor activity and also useful in allergic rhinitis patients. Tylophora indica contains tylophorine, α-amyrin, querceitin, and other major alkaloids like tylophorinine, tylophorinidine and dehydrotylophorine. Tephrosia purpurea Linn (Family- Fabeaceae) having anti-hepatotoxic activity, CNS depressant activity and it also show anti-allergic activity. The plant Tephrosia purpurea contains rutin, quercetin, rotenoids, rotenone, tephrosin and lupeol. The powdered aerial part of Tylophora indica  and Tephrosia purpuria were extracted with 95% ethanol by hot continuous extraction method individually. The prepared extracts were subjected to HPTLC analysis. The marker compounds were estimated by using pre-coated HPTLC aluminium silica gel 60 F254 plates (MERCK) and compared with standard. The methanolic solution of standard quercetin 5 µl (1 mg/ ml), Tephrosia purpuria extract and Tylophora indica extract 8 µl each (10 mg/ ml) were applied as 7mm band width using C AMAG Linomat IV applicator. The Mobile phase is ethyl acetate: formic acid: glacial acetic acid: water (100:11:11:26). The detection was carried out at 366 nm. The amount of quercetin was estimated by the comparing the peak area of standard and the same was present in the extracts. The content of quercetin in extracts was found to be 4.30% w/w and 1.56% w/w for Tylophora indica extract and Tephrosia purpuria extract respectively. This estimation technique is very much useful for the estimation of quercetin present in the various medicinal plants.

 

KEYWORDS: Quercetin, HPTLC, Tylophora indica and Tephrosia purpuria.

 

 

INTRODUCTION:

Tylophora indica belonging to family- Asclepiadaceae having anti-asthmatic, anti-inflammatory, sedation decreased motor activity and also useful in allergic rhinitis patients. Tylophora indica contains tylophorine, α-amyrin, querceitin, and other major alkaloids like tylophorinine, tylophorinidine and dehydrotylophorine. Tephrosia purpurea Linn belonging to family- Fabeaceae having anti-hepatotoxic activity, CNS depressant activity and it also show anti-allergic activity. The plant Tephrosia purpurea contains rutin, quercetin, rotenoids, rotenone, tephrosin and lupeol. The aim of this study is to estimate qurecetin by HPTLC method which is present in plants, Tylophora indica and Tephrosia purpurea [1-6].

 

EXPERIMENTAL METHODS:

Collection of plants

The plant material of Tylophora indica and Tephrosia purpurea were collected from Erode and Madurai district, Tamil Nadu. All the collected plant materials were identified and authenticated by Botanical Survey of India (BSI), Coimbtore (TN).


 

Fig. 1. HPTLC chromatogram of Standard Quercetine (track 2)

 


Extraction

The powdered aerial parts of Tylophora indica and Tephrosia purpurea were extracted with 95 % ethanol by hot continuous soxhlet extraction method, individually.

 

Method Development of HPTLC

Standard preparation

10 mg of standard quercetin was dissolved in 10 ml  of methanol and used for HPTLC quantification at concentration of 1mg/ml.

 

Sample Preparation (extracts)

100 mg of extract was taken and extracted with 10 ml of methanol and slightly warmed. This solution was filtered by whatman filter paper to get a clear solution used for HPTLC analysis at a concentration of 10 mg/ml.

 

Chromatographic Condition [7-8]

Stationary phase

:

Precoated Silica gel F 254 Plates (MERCK)

Mobile phase

:

Ethyl acetate: Formic acid: Glacial acetic Acid: water

(100:  11:   11: 26)

Saturation

:

60 minute 

Development chamber

:

CAMAG twin trough development chamber

Applicator

:

CAMAG Linomat IV applicator

Scanner

:

CAMAG Scanner III CATS (4.06), Switzerland

Mode of scanning

:

Absorption (deuterium)

Detection wavelength

:

365 nm

Volume applied standard

:

5 µl

Volume applied samples

:

8 µl each

 

RESULT AND DISCUSSION:

Before spotting, the plates were pre-washed with methanol. Standard and samples solutions were applied to the plates as sharp bands by means of CAMAG Linomat IV applicator. The spots were dried in a current of air. The mobile phase (20 ml) was poured into a twin trough glass development chamber was left to equilibrate for 30 minutes and the plate was placed in the chamber. The plate was then developed until the solvent front had travelled at a distance of 75 mm above the base of the plate. The plate was then removed from the chamber and dried in a current of air. Detection and quantification was performed with CAMAG Scanner III at a wavelength of 544 nm.

 

Tephrosia purpurea extract:

Totally 4 peak observed in the Tephrosia purpurea extract, In this fourth peak with Rf value of 0.97 is matching with standard Rf value of 0.94 and confirmed the presence of quercetin. The amount of quercetin was found to be 1.56% w/w (Fig. 1 and 2).

 

Tylophora indica extract:

Totally 6 peak observed in Tylophora indica extract, in this sixth peak with Rf value of 0.97 is matching standard quercetin with Rf value of 0.94. The amount of quercetin was estimated by comparing the peak area of standard and the same present in the sample. The content of quercetin was found to be 4.30 % w/w (Fig. 1 and 3).

 


 

Fig. 2. HPTLC chromatogram of Tephrosia purpurea extract (track 7)

 


 


 

Fig. 3. HPTLC chromatogram of Tylophora indica extract (track 4)

 


 

CONCLUSION:

By HPTLC quantification the extract Tylophora indica extract shows more percentage of quercetin when compared to Tephrosia purpurea. This estimation technique is very much useful for the estimation of flavonoidal compounds like quercetin present in the plant materials, extracts and their formulations. 

 

BIBLIOGRAPHY:

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2.        Md. Sarfaraj Hussain, Sheeba Fareed, Mohammad Ali, Hyphenated chromatographic analysis of bioactive gallic acid and quercetin in Hygrophila auriculata (K. Schum) Heine growing wildly in marshy places in India by validated HPTLC method. Asian Pacific Journal of Tropical Biomedicine. 2012:  S477-S483.

3.        M. Ali and K. K. Bhutani, Alkaloids From Tylophora indic. Phytochemistry. 28 (12); 1989: 3513-3517.

4.        T. Ganguly1, L. P. Badheka2 and K. B.Sainis, Immunomodulatory effect of Tylophora indica on Con A induced lymphoproliferation. Phytomedicine. 8(6); 2001: 431–437.

5.        V.U. Ahmad, Z. Ali, S.R. Hussaini, F. Iqbal, M. Zahid, M. Abbas, N. Saba, Flavonoids of Tephrosia purpurea. Fitoterapia. 70; 1999: 443-445.

6.        A.S. Damre, A.B. Gokhale, A.S. Phadke, K.R. Kulkarni, M.N. Saraf, Studies on the immunomodulatory activity of flavonoidal fraction of Tephrosia purpurea.  Fitoterapia. 74; 2003: 257–261.

7.        Seth P. D. HPTLC.  First edition, CBS Publishers and distributors, New Delhi. 1996.

8.        Wagnar H and Bladt S. Plant Drug Analysis. Second edition, Springer-Verlag Publication, Berlin, 1996.

 

Received on 11.10.2012

Modified on 20.10.2012

Accepted on 25.10.2012

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 5(1): January–February 2013, 26-29