Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

[Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 1)].

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.

Quality, safety and efficacy profiling of ginseng adventitious roots produced in vitro.

Ginseng (Panax ginseng C. A. Meyer, Family Araliaceae) is one of the major medicinal and nutraceutical plants, which is native to oriental region. It is used worldwide as a popular herbal medicine because of its pharmacological effects like anti-oxidative, anti aging, anti-cancer, adaptogenic, and other health-improving activities. Chief components of ginseng identified till date are ginsenosides, a group of saponins with triterpenoid structure. Ginseng is cultivated under controlled conditions, and for harvesting of fully grown roots of the plant, the cultivation takes long duration of about 5-7 years and cultivated ginseng roots are inferior in quality and ginsenoside content. Wild Mountain ginseng is superior in quality and ginsenoside content but is scarce in nature. Therefore, for obtaining the useful compounds of this plant at commercial scale, cell and organ cultures especially adventitious roots have been established by using superior clones of wild mountain ginseng, ginseng biomass is produced by applying large scale bioreactors. In this paper, an effort has been made to shed light on the scientific literature and to decipher the evidences for quality, safety, and efficacy of ginseng adventitious roots produced from in vitro cultures.

Establishment of an enzyme-linked immunosorbent assay and application on determination of ginsenoside Re in human saliva.

This work describes an immunochemical approach for the quality control of Panax ginseng and a pharmacological study of ginsenoside Re, a major bioactive constituent in P. ginseng, using an enzyme-linked immunosorbent assay. A hybridoma secreting monoclonal antibody against ginsenoside Re was produced by fusing splenocytes immunized with a ginsenoside Re-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line. The method, at an effective measuring range of 7.8-500 ng ·â€ŠmL(-1) of ginsenoside Re, successfully detected ginsenoside Re in Chinese traditional herb prescriptions. The results demonstrate that we generated a novel and reliable assay system for measuring ginsenoside Re in Chinese medicines more efficiently. Futhermore, we determined the ginsenoside Re concentrations in the saliva of six healthy adults after the oral administration of a ginseng capsule to study the pharmacokinetics of ginsenoside Re in human saliva.

A novel strategy with standardized reference extract qualification and single compound quantitative evaluation for quality control of Panax notoginseng used as a functional food.

Root of Panax notoginseng (Burk.) F.H. Chen (Sanqi in Chinese) is one of traditional Chinese medicines (TCMs) based functional food. Saponins are the major bioactive components. The shortage of reference compounds or chemical standards is one of the main bottlenecks for quality control of TCMs. A novel strategy, i.e. standardized reference extract based qualification and single calibrated components directly quantitative estimation of multiple analytes, was proposed to easily and effectively control the quality of natural functional foods such as Sanqi. The feasibility and credibility of this methodology were also assessed with a developed fast HPLC method. Five saponins, including ginsenoside Rg1, Re, Rb1, Rd and notoginsenoside R1 were rapidly separated using a conventional HPLC in 20 min. The quantification method was also compared with individual calibration curve method. The strategy is feasible and credible, which is easily and effectively adapted for improving the quality control of natural functional foods such as Sanqi.

Influence of sulphur-fumigation on the quality of white ginseng: a quantitative evaluation of major ginsenosides by high performance liquid chromatography.

White ginseng was reported to be sulphur-fumigated during post-harvest handling. In the present study, the influence of sulphur-fumigation on the quality of white ginseng and its decoction were quantitatively evaluated through simultaneous quantification of 14 major ginsenosides by a validated high performance liquid chromatography. Poroshell 120 EC-C18 (100mm×3.0mm, 2.7µm) column was chosen for the separation of the major ginsenosides, which were eluted with gradient water and acetonitrile as mobile phase. The analytes were monitored by UV at 203nm. The method was validated in terms of linearity, sensitivity, precision, accuracy and stability. The sulphur-fumigated and non-fumigated white ginseng samples, as well as their respective decoctions, were comparatively analysed with the newly-validated method. It was found that the contents of nine ginsenosides detected in raw materials decreased by about 3-85%, respectively, and the total content of the nine ginsenosides detected in raw materials, decreased by almost 54% after sulphur-fumigation. On the other hand, the contents of 10 ginsenosides detected in decoctions of sulphur-fumigated white ginseng were decreased by about 33-83%, respectively, and the total content of ginsenosides was decreased by up to 64% when compared with that of non-fumigated white ginseng. In addition, ginsenoside Rh(2) and Rg(5) could be detected in the decoctions of sulphur-fumigated white ginseng but not in that of non-fumigated white ginseng. It is suggested that sulphur-fumigation can significantly influence not only the contents of original ginsenosides, but also the decocting-induced chemical transformation of ginsenosides in white ginseng.

[Systematic study on QAMS method for quality control of Panax notoginseng].

OBJECTIVE: To establish a quantitative method of multi-components by single marker (QAMS) for determining ginsenoside Rg1, Rb1, Rd, Re and notoginsenoside R1 for the purpose of the quality control of Panax notoginseng. METHOD: The relative correction factors (RCFs) between the five active saponins were determined by HPLC-DAD. With any of the five consituents as reference, a QAMS method was established for detect the quantitation of the other four consituents. The durability of the method was evaluated with five different HPLC instruments, five different Cis18 chromatographic columns and four detective wavelengths. Subsequently, the new QAMS method was used to determine the contents of five saponins contained in 43 batches of notoginseng samples, and compare with external standard methods, in order to evaluate the accuracy of the QAMS method. RESULT: When the five saponins were taken for reference, there was no significant difference between the contents of Rg1, Rb1, Rd, Re and R1 contained in the 43 batches of medicines calculated by the QAMS method (Wf) and the content determination result of the external standard method (Ws). The ratio of their results was (Ws/Wr) (94.02 +/- 2.11)%-(99.75 +/- 0.79)%, suggesting that the method was highly accurate. Their relative correction factors showed good durability, ranging between 0.42%-3.7%, 0.52%-3.5% and 0.79%-4.9%, respectively, with different chromatographic columns, different instruments and different detective wavelengths. The relative retention value method could be adopted for accurately position the chromatographic peak of the five consituents, with their values ranging between 0.18%-13%. CONCLUSION: An accurate, rapid and highly durable QAMS method is established for simultaneous determination and location of five saponins, so as to provide reliable basis for the application of the QAMS method in quality control of traditional Chinese medicines.

Current evaluation of the millennium phytomedicine--ginseng (I): etymology, pharmacognosy, phytochemistry, market and regulations.

The dawning of this millennium broke new ground in life science and technology, presented us genomic and proteomic revolution, nanotechnology innovation, and high performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) used for separating and identifying new chemical entities at pico-, or even femto-concentrations. Applications of these high technologies to the traditional Chinese medicine (TCM) opened a new chapter in the ancient medicine, and prompted us to re-evaluate the thousand-year-old phytomedicine- ginseng from current perspectives. We, therefore, collected the latest information (mostly within 10 years) on ginseng, and condensed the information into two parts of this review serial. The present part covers etymology of ginseng, its pharmacognosy (natural origin, physical appearance, chemical properties, and specie identification), its cultivation and processing-related metabolic changes in active ingredients, standardized analytical methods used for quality control of various ginseng products, modern analytical methods used to identify and classify more than 100 chemical entities (many were recently unfolded) derived from ginseng species and their metabolites. The global markets and production of ginseng and relevant government regulations are herein updated to exchange information and understandings about current people's uses and cultivation of ginseng. The second part of the review serial will classify all these 100 chemical entities separated from various ginseng species into different groups based on their structural similarities, and summarize bioactivities of these entities. The second part of the review serial will also focus on recent findings of ginseng pharmacology and its clinical trials for various diseases, and brief side effects of ginseng.

Simultaneous quantification of 14 ginsenosides in Panax ginseng C.A. Meyer (Korean red ginseng) by HPLC-ELSD and its application to quality control.

A new method of high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous quantification of 14 major ginsenosides, which are the marker compounds of Panax ginseng C.A. Meyer (Korean red ginseng). Various types of ginseng samples were extracted, and the amounts of the 14 ginsenosides (Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rk1, Rg5, and Rh2) were determined by reverse-phase HPLC-ELSD using digoxin as an internal standard. The mobile phase consisted of a programmed gradient of aqueous acetonitrile. Calibration curves for each ginsenoside were determined for the quantification. The method was validated for linearity, precision, accuracy, limit of detection, and limit of quantification. This quantification method was applied to several finished ginseng products including white ginseng, red ginseng powder, and red ginseng concentrate. The amounts of the 14 ginsenosides in the various ginseng samples could be analyzed simultaneously. This validated HPLC method is expected to provide a new basis for the quality assessment of ginseng products.

[Comparative study on quality of Tongrentang red ginseng and Korean red ginseng--determination of ginsenosides and polysaccharides].

OBJECTIVE: To establish methods for quantitative determination of ginseng saponins, ginsenoside Rg1, Re, Rb1 and polysaccarides and compare the qualities of Tongrentang Red Ginseng and Korean Red Ginseng. METHOD: Macroreticular resin-colorimetric method was developed to determine ginseng saponins and a new HPLC method with gradient eluents was established for determination of ginsenoside Rg1, Re, Rb1. For ginseng polysaccharides, phenol-oil of vitriol colorimetric method was developed and some factors were also optimized. RESULT: The content of ginseng saponins in Tongrentang Red Ginseng was not lower than that of Korean Red Ginseng. Ginsenoside Rg1 and Rb1 in Tongrentang Red Ginseng were higher than those in Korean Red Ginseng, while Ginsenoside Re was slightly lower than that of Korean Red Ginseng. However, the amount of Ginseng Polysaccharides in Tongrentang Red Ginseng was greater than those in Korean Red Ginseng. CONCLUSION: The contents of ginseng saponins and ginsenoside Rg1, Re, Rb1 in Tongrentang Red Ginseng were not lower than that in Korean Red Ginseng. The methods for determination of ginsenosides and ginseng polysaccharides were quite accurate and reliable to the quality control of Ginseng.

[Determination of notoginsenoside R1 in radix notoginseng by HPLC-ELSD].

OBJECTIVE: To establish a method for the determination of notoginsenoside R1 in Radix Notoginseng. METHOD: The HPLC-ELSD method was used for the determination. Chromatographic conditions: Shimpack CLC-ODS column(6.0 mm x 150 mm), acetonitrile-water(30:70) as the mobile phase, and Shimadzu LC-6A with SEDEX 55 ELSD detector. RESULT: The recovery rate is 101.57% and relative standard deviation 1.98%. CONCLUSION: The method is reliable for the quality control of Radix Notoginseng.