Quantitative NMR: an applicable method for quantitative analysis of medicinal plant extracts and herbal products.
BACKGROUND
Quantitative analysis and standardisation of plant extracts or herbal products is a tedious process requiring time-consuming sample preparation and analytical method development for the resolution of analyte peaks from the complex natural extract. Quantitative analysis by HPLC requires a pure authentic standard of the compound being quantified. We report here a quantitative NMR (qNMR) method for quantitative analysis of three medicinal plant extracts and their herbal products without the need of authentic standards. Quantitation can be done by using any commercially available pure sample as an internal reference standard.


OBJECTIVE
To develop a reliable method for standardisation and quantitative analysis of extracts from medicinal plants Eugenia jambolana, Withania somnifera and Aegle marmelos and their herbal products using qNMR.


METHODS
The (1) H-NMR spectra of known amounts of crude plant extracts with internal standards were recorded in deuterated solvents and quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds and the internal reference standard. Anthocyanins [delphinidin-3,5-diglucoside (1), petunidin-3,5-diglucoside (2) and malvidin-3,5-diglucoside (3)] for E. jambolana fruit extract and imperatorin (4) for A. marmelos fruit extract were selected as marker constituents for quantitation and 1,3,5-trimethoxybenzene (TMB) was used as an internal reference standard. Total withanolide content was determined for W. somnifera using 2,4-diformyl phloroglucinol as an internal reference standard.


RESULTS
The (1) H-NMR gave a linear response for the marker constituents, anthocyanins, withaferin A and imperatorin. Using the described method, the amount of anthocyanins in Amberlite(R) XAD7HP and Sephadex enriched extracts of E. jambolana was 3.77% and 9.57% (delphinidin-3,5-diglucoside), 4.72% and 12.0% (petunidin-3,5-diglucoside), 6.55% and 15.70% (malvidin-3,5-diglucoside), respectively. The imperatorin content was 0.424% in A. marmelos fruit and 0.090 % and 0.114% in sharbat and candies. Total withanolides content was 0.191% in the chloroform extract and 0.234% in the capsule extract. These values are in accordance with HPLC results.


CONCLUSIONS
This qNMR technique could be used for NMR fingerprinting and quantitation for the purpose of quality control and standardisation of many plant-based herbal products and medicines and has certain advantages over HPLC.
Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab -, 160062, India.
Siddheshwar K
SK
Chauthe
Ram Jee
RJ
Sharma
Farrukh
F
Aqil
Ramesh C
RC
Gupta
Inder Pal
IP
Singh
D045471:Aegle / Q000737:chemistry; D000872:Anthocyanins / Q000032:analysis; D002182:Candy / Q000032:analysis; D005638:Fruit / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D010936:Plant Extracts / Q000032:analysis / Q000737:chemistry*; D028321:Plant Preparations / Q000737:chemistry*; D010946:Plants, Medicinal / Q000737:chemistry*; D012015:Reference Standards; D015203:Reproducibility of Results; D027842:Syzygium / Q000737:chemistry; D032341:Withania / Q000737:chemistry; D054358:Withanolides / Q000032:analysis
D000872:Anthocyanins; D010936:Plant Extracts; D028321:Plant Preparations; D054358:Withanolides; C065861:malvidin; C017185:delphinidin; C473206:petunidin