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Development and validation of a high-performance thin-layer chromatography method for the simultaneous quantification of rosmarinic acid, quercetin, glycyrrhizin and betulinic acid in polyherbal immunostimulant formulation

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Abstract

Quantification of bioactive markers through modern analytical methods is very essential for establishing the authenticity and creditability of prescription and usage of herbal drugs formulations. In the present study, simultaneous quantification of rosmarinic acid (RA), quercetin (Q), glycyrrhizin (G) and betulinic acid (BA), in polyherbal formulation by high-performance thin-layer chromatography (HPTLC) method was developed. Mobile phase of ethyl acetate‒toluene‒methanol‒formic acid (7:1:0.5:0.5, V/V) was used on pre-coated plate of silica gel 60F254 and quantified by densitometric method. Validation of the method was done to demonstrate its selectivity, linearity, precision and accuracy as per the International Council for Harmonization (ICH) guidelines. Intra-day assay and inter-day assay precision of the analytes were less than 2%, and the average recovery rates obtained were in the range of 98.11‒101.14% for all with percent relative standard deviation (%RSD) below 2%. Correlation coefficient shows that the developed method was accurate and precise. This method can be used in the routine analysis of polyherbal formulations.

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Abbreviations

BA:

Betulinic acid

DCP:

Di-calcium phosphate

G:

Glycyrrhizin

HPTLC:

High-performance thin-layer chromatography

ICH:

International Council for Harmonisation

LOD:

Limit of detection

LOQ:

Limit of quantification

Q:

Quercetin

RA:

Rosmarinic acid

RSD:

Relative standard deviation

SD:

Standard deviation

SEM:

Standard error of mean

TLC:

Thin-layer chromatography

UV:

Ultraviolet

WHO:

World Health Organization

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Acknowledgements

The authors are grateful to University Grants Commission (UGC), New Delhi, India for providing senior research fellowship and to the Amsar Pvt. Ltd., Indore, Madhya Pradesh, India for providing Prunella vulgaris and Zizyphus jujube extracts with Certificate of Analysis (COA).

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Authors and Affiliations

  1. Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, 390001, India

    Reshma Jain & S. J. Rajput

  2. Faculty of Pharmacy, DIT University, Mussoorie Diversion Road, Dehradun, Uttarakhand, 248009, India

    Neeraj Kumar Sethiya & Sushil K. Chauddhary

  3. Institute of Bioresources and Sustainable Development (IBSD), Takyelpat, Imphal, Manipur, 795001, India

    Sushil K. Chauddhary

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  1. Reshma Jain
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Correspondence to Neeraj Kumar Sethiya or Sushil K. Chauddhary.

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Jain, R., Rajput, S.J., Sethiya, N.K. et al. Development and validation of a high-performance thin-layer chromatography method for the simultaneous quantification of rosmarinic acid, quercetin, glycyrrhizin and betulinic acid in polyherbal immunostimulant formulation. JPC-J Planar Chromat 35, 571–577 (2022). https://doi.org/10.1007/s00764-022-00212-1

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