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Development, validation and application of RP-HPLC method for quantitative estimation of wedelolactone in different accessions and plant parts of Eclipta alba (L.)

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Abstract

In the current study, a simple, rapid reversed phase high performance liquid chromatographic (RP- HPLC) method was developed and validated for evaluation of wedelolactone in Eclipta alba, an indigenous medicinal herb commonly known as Bhringaraj. Wedelolactone was quantified in the dried root, stem, leaves, flowers and whole plant of E. alba by a gradient RP-HPLC procedure with buffer (1 mM potassium dihydrogen phosphate—KH2PO4) and acetonitrile as mobile phase and separation was carried out on CAPCELL PAK C18 (SHISEIDO) column at a flow rate of 1.5 mL/min and UV detection at 351 nm using photodiode array detector (PDA). The method was validated as per ICH Q2 (R1) (International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use) guidelines for various analytical method validation parameters such as linearity, accuracy, specificity, precision, and sensitivity (limit of detection and limit of quantification). The precision of the method was confirmed by a relative standard deviation < 2.0% (n = 6), and wedelolactone recovery was observed between 99.5 and 103.6%. The LOD was found to be 0.084 µg/mL and the LOQ was 0.25 µg/mL, and the response was linear from 2.5 to 140 µg/mL with excellent correlation coefficient (R2) of 0.9997. The study also revealed that variation for the content of wedelolactone among accessions and between different plant parts, the highest content is found in the leaves. The developed method is precise, sensitive, accurate, reproducible and can be used for the quantitative estimation of wedelolactone in E. alba plant material.

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Fig. 1
Fig. 2 Few of the accessions used in the study
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Abbreviations

RP-HPLC:

Reverse Phase High Performance Liquid chromatography

PDA:

Photodiode array detector

LOD:

Limit of detection

LOQ:

Limit of quantification

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Acknowledgements

The authors are grateful to the director, ICAR-IIHR, Hesaraghatta, Bangalore for providing necessary facilities and encouragement.

Funding

The authors are very thankful to the financial assistance provided from National Medicinal Plants Board, Department of AYUSH, Ministry of Health and Family Welfare, Govt. of India (Project No: Z.18017/187/CSS/R&D/KR-01/2018–19-NMPB-IVA entitled ‘Collection, characterization and genetic improvement of Eclipta alba’.

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Authors and Affiliations

  1. Division of Flower and Medicinal Crops, ICAR- Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bangalore, Karnataka, 560089, India

    B. Anil Kumar, K. Hima Bindu, M. R. Rohini &  Shivakumar

  2. Division of Basic Sciences, ICAR- Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bangalore, Karnataka, 560089, India

    V. K. Rao

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  1. B. Anil Kumar
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Correspondence to K. Hima Bindu.

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Kumar, B.A., Rao, V.K., Bindu, K.H. et al. Development, validation and application of RP-HPLC method for quantitative estimation of wedelolactone in different accessions and plant parts of Eclipta alba (L.). J. Plant Biochem. Biotechnol. 31, 788–802 (2022). https://doi.org/10.1007/s13562-021-00721-w

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