Head-to-Head Comparison of Ultra-High-Performance Liquid Chromatography with Diode Array Detection versus Quantitative Nuclear Magnetic Resonance for the Quantitative Analysis of the Silymarin Complex in Silybum marianum Fruit Extracts

Abstract

Quantitative nuclear magnetic resonance (qNMR) spectroscopy is known as an excellent alternative to chromatography-based mixture analysis. NMR spectroscopy is a non-destructive method, needs only limited sample preparation, and can be readily automated. A head-to-head comparison of qNMR to an ultra-high-performance liquid chromatography with diode array detection (uHPLC-DAD)-based quantitative analysis of six flavonolignan congeners (silychristin, silydianin, silybin A, silybin B, isosilybin A, and isosilybin B) of the Silybum marianum silymarin complex is presented. Both assays showed similar performance characteristics (linear range, accuracy, precision, and limits of quantitation) with analysis times below 30 min/sample. The assays were applied to industrial S. marianum extracts (AC samples) and to extracts locally prepared from S. marianum fruits (PL samples). An assay comparison by Bland-Altman plots (relative method bias AC samples, -0.1%; 2SD range, ±5.1%; relative method bias PL samples, -0.3%; 2SD range, ±7.8%) and Passing-Bablok regression analysis (slope and intercept for AC and PL samples not significantly different from 1.00 and 0.00, respectively; Spearman's coefficient of rank correlation, >0.99) did show that qNMR and uHPLC-DAD can be used interchangeably to quantitate flavonolignans in the silymarin complex.

Influence of flavonoids on mechanism of modulation of insulin secretion

“…Silibinin is the major pharmacologically active compound of Silymarin, an isomeric mixture from the Silybum marianum that containing at least six flavonolignans. [ 97 98 99 100 ] Extracts and flavonoids from S. marianum have been reported in the literature as hypoglycemiant and applied for pancreas recovery for diabetics. [ 101 102 ] This activity can be associated to Silibinin.…”

Silybum marianum pericarp yields enhanced silymarin products

“…The advantages of the qNMR method over the HPLC method are: (1) no standards are needed to calculate response factors; (2) structural information of both silymarin and non-silymarin constituents is readily available; (3) the same spectra may be used to determine relative quantities of silymarin marker compounds as demonstrated by Napolitano et al [21] and Cheilari et al [29]; (4) less solvent is employed for analysis; and (5) the ppm intervals that were chosen are likely to also include resonances from minor flavonolignans in addition to the known silymarin marker compounds.…”

Silymarin Enriched Extract (Silybum marianum) Additive Effect on Doxorubicin-Mediated Cytotoxicity in PC-3 Prostate Cancer Cells

“…This procedure was repeated 3 times, resulting to 0.6 g of SEE. SEE contains significant amount of silymarin (> 60 %), which is a mixture of at least 6 flavonolignans, namely silychristine, silydianin, silybin A, silybin B, isosilybin A, and isosilybin B. Silychristine and silibinin (silybin A and silybin B) are the main ingredients, as they are present in more than of 13 % and 30 % of the extract, respectively [29]. In order to isolate the 2 above-mentioned flavonolignans, an amount (0.5 g) of SEE was subjected to silica flash column chromatography using CH 2 Cl 2 /MeOH of increasing polarity as mobile phase, and after TLC analysis, 80 aliquot-combining fractions were collected.…”

“…The 13th fraction (28.3 mg), eluted with CH 2 Cl 2 /MeOH 95/5, was chromatographed by preparative TLC and CH 2 Cl 2 /MeOH 90/10 as mobile phase, resulting in isolation of silychristine (11.2 mg). All compounds were identified by means of spectral data (MS, 1 H NMR, 13 C NMR, COSY, HMQC, HMBC, 2D-NMR) and direct comparison with the respective literature data [29].…”