Optimization of analytical method for the quality control of ginseng preparations and comparative analysisof ginsenosides of Wild Panax speciesusing LC-ELSD and LC-Q-TOF-MS

Abstract

Ginseng, the root of the Panax genus plant has been used as an herbal medicine in Asia for over two thousand years for its various health benefits including (but not limited to) antioxidant, anti-carcinogenic, anti-inflammatory, antihypertensive and anti-diabetic effects. The pharmacologically active compounds behind the claims of ginsengs efficacy are ginsenosides. More than 300 different ginsenosides have been identified from several Panax plants. Ginsenoside is triterpene glycoside with four rings and several sugar moieties

they are found exclusively in ginseng plants and are in higher concentration in their roots. Qualitative and quantitative techniques for the analysis of ginsenosides are in demand to ensure quality control of ginseng. Korean Pharmacopeia (KP) and Japanese Pharmacopeia (JP) describe ginseng as it contains not less than 0.10% of ginsenoside Rg1 and not less than 0.20% of ginsenoside Rb1. Chinese Pharmacopeia (CP) describes that ginseng should contains not less than 0.30% of Rg1+Re and not less than 0.20% of Rb1.This kind of standard is appropriate for the quality control of white ginseng and its powder. However, that most ginsenoside lose a part of its sugar moiety and transformed to less polar ginsenoside especially under the heat, in acidic condition, or by fermentation. Heating process converts the naturally occurring ginsenoside Rg1 and Rb1 into artifact ginsenosides such as gisenosides Rg3, Rg5, Rh1, and Rh2. Therefore, gisenoside Rg1 and Rb1 which have been used as quality markers of ginseng, do not express the quality of some ginseng products. Consequently, the official method for the quality control of ginseng is not applicable for the processed ginseng. The aim of this study is to develop a new simple analytical method which can be applied to all kinds of ginseng products including processed ginseng. To achieve this goal, sample preparation method and analytical condition were optimized using design of experiment (DOE) technique. The optimal conditions for the extraction of Rg1 and Rb1, which are the major compounds for White ginseng and Red ginseng were found at 60% aqueous methanol. However, 80% aqueous methanol was better for the efficient extraction of ginsenoside Rk1 and Rg5. The result indicates that the less polar solvent system might be a better choice for the efficient extraction of a ginseng preparation containing a large amount of ginsenoside Rk1 and Rg5. In general, 70% aqueous methanol was superior to 60% or 80% methanol for the extraction of polar and less polar ginsenosides together. The coefficient of correlation (R2) was higher than 0.994 for all calibration curves. The method sensitivity was measured in terms of the limit of detection (LOD), which ranged from 1-7 ng/ml. The limit of quantification (LOQ) were found 2-23 ng/ml. Ginsenoside Rb1 and Rg1 are described in Pharmacopeia as the marker compounds for the quality control of ginseng. However, they are not adequate marker for the certain ginseng products as they are unstable and easily convert to less polar ginsenosides. To solve this problem, group quantitation of ginsenoside was applied. All kinds of ginsenosides were categorized into three groups as follows. Group A: protopanaxatriol (PPT) group includes Rg1 and Re

Group B: protopanaxadiol (PPD) group includes Rb1, Rb2, Rc and Rd

Group C: less polar ginsenosides group includes Rg3, Rk1 and Rg5. The contents of ginsenosides in Group A, B and C were expressed as the contents of ginsenosides Rg1, Rb1, and Rg3, respectively. The group quantitation method was successfully applied for the quantitative analysis of White ginseng and heat-treated ginseng products. The contents of ginsenoside Rg1 and Rb1 in White ginseng were 0.37 mg/g and 1.41mg/g, respectively. The content of PPT group and PPD group in the same sample were 1.23 mg/g and 3.91mg/g, respectively which gives better information about the total content of ginsenosides and, consequently, quality of ginseng. Sum of all ginsenosides was 5.14 mg/g. Due to the steaming process the contents of ginsenoside Rg1 (0.08 mg/g) and Rb1 (0.78 mg/g) in processed ginseng were degraded, which does not meet the requirements of the Pharmacopoeia. The content of group analysis in the same sample were as follows: 0.38 mg/g for PPT group, 3.16 mg/g for PPD group and 1.90 mg/g for less polar gisenosides group. Sum of all ginsenosides was 5.44 mg/g. Group analysis method is applicable not only White and Red ginseng but also other processed ginseng. Furthermore it gives more information about the quality of ginseng products. High performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) and quadrupole time-of-flight mass spectrometry (Q-TOF-MS) were used for qualitative and quantitative analysis of ginsenosides in wild Panax species, namely Panax ginseng, Panax quinquefolius and Panax vietnamensis. Identification of ginsenosides was achieved using Q-TOF-MS and contents were determined by ELSD. The contents of 17 ginsenosides were determined by LC-ELSD. Among them 11 ginsenosides were identified using standards. Those were notoginsenoside R1, majonoside R1, ginsenoside Rg1, ginsenoside Re, majonoside R2, vina-ginsenoside R2, notoginsenoside R2, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd. To identify six other unidentified metabolites, m/z of [M-H] - and fragment ions were measured by LC-Q-TOF-MS and compared to literature values. Total content of ginsenosides in the radix and rhizome of wild P. vietnamensis were 111.06 and 84.44 mg/g, respectively. Total content of ginsenosides in the radix and rhizome of P. ginseng were 10.17 and 38.39 mg/g, respectively. Those in P. quinquefolius were 13.25 and 14.93 mg/g, respectively. Peak patterns between radix and rhizome of P. ginseng and P. quinquefolius was not significantly different. But it was quite different between radix and rhizome of wild P. vietnamensis. Radix contains higher amount of ginsenosides in particular due to variation in protopanaxatriol type ginsenoside contents.