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.

A Two-Step Orthogonal Chromatographic Process for Purifying the Molecular Adjuvant QS-21 with High Purity and Yield.

QS-21 is a triterpene glycoside saponin found in the bark of the Chilean soap bark tree Quillaja saponaria. It is a highly potent vaccine adjuvant that is included in two approved vaccines and has shown promise in numerous other vaccine candidates in the research and clinical pipelines. One major hurdle to the widespread use of this adjuvant is the difficulty of obtaining it in high yield and purity. Previously reported purification approaches either showed suboptimal purity and/or yield, lacked efficiency, or had strict requirement on the composition of the starting material. Here, we report the development of a new two-step orthogonal chromatographic process, consisting of a polar reversed-phase (RP) chromatography step followed by a hydrophilic interaction chromatography (HILIC) step, for purifying QS-21 from a commercially available Quillaja saponaria bark extract with high yield and > 97% purity. This process makes available a simple and efficient method for obtaining highly pure QS-21 from saponin-enriched bark extract.

[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.

Analytical methods and biological activities of Panax notoginseng saponins: Recent trends.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng (Burk.) F. H. Chen, also called Sanqi, is a widely used traditional Chinese medicine, which has long history used as herbal medicines. It is currently an important medicinal material in China, holding the first place in the sale volume of the whole patent medicines market in China, and the market size of the single species has exceeded 10 billion yuan. In addition, P. notoginseng is an important constituent part of many famous Chinese patent medicines, such as Compound Danshen Dripping Pills and Yunnan Baiyao. P. notoginseng saponins (PNSs), which are the major active components of P. notoginseng, are a kind of chemical mixture containing different dammarane-type saponins. Many studies show that PNSs have been extensively used in medical research or applications, such as atherosclerosis, diabetes, acute lung injury, cancer, and cardiovascular diseases. In addition, various PNS preparations, such as injections and capsules, have been made commercially available and are widely applied in clinical practice. AIM OF THE REVIEW: Since the safety and efficacy of compounds are related to their qualitative and quantitative analyses, this review briefly summarizes the analytic approaches for PNSs and their biological effects developed in the last decade. METHODOLOGY: This review conducted a systematic search in electronic databases, such as Pubmed, Google Scholar, SciFinder, ISI Web of Science, and CNKI, since 2009. The information provided in this review is based on peer-reviewed papers and patents in either English or Chinese. RESULTS: At present, the chromatographic technique remains the most extensively used approach for the identification or quantitation of PNSs, coupled with different detectors, among which the difference mainly lies in their sensitivity and specificity for analyzing various compounds. It is well-known that PNSs have traditionally strong activity on cardiovascular diseases, such as atherosclerosis, intracerebral hemorrhage, or brain injury. The recent studies showed that PNSs also responded to osteoporosis, cancers, diabetes, and drug toxicity. However, some other studies also showed that some PNSs injections and special PNS components might lead to some biological toxicity under certain dosages. CONCLUSION: This review may be used as a basis for further research in the field of quantitative and qualitative analyses, and is expected to provide updated and valuable insights into the potential medicinal applications of PNSs.

[Continual improvement of quantitative analytical method development of Panax notogineng saponins based on quality by design].

This study is aimed to propose a continual improvement strategy based on quality by design (QbD). An ultra high performance liquid chromatography (UPLC) method was developed to accomplish the method transformation from HPLC to UPLC of Panax notogineng saponins (PNS) and achieve the continual improvement of PNS based on QbD, for example. Plackett-Burman screening design and Box-Behnken optimization design were employed to further understand the relationship between the critical method parameters (CMPs) and critical method attributes (CMAs). And then the Bayesian design space was built. The separation degree of the critical peaks (ginsenoside Rg1 and ginsenoside Re) was over 2.0 and the analysis time was less than 17 min by a method chosen from the design space with 20% of the initial concentration of the acetonitrile, 10 min of the isocratic time and 6%•min⁻¹ of the gradient slope. At last, the optimum method was validated by accuracy profile. Based on the same analytical target profile (ATP), the comparison of HPLC and UPLC including chromatograph method, CMA identification, CMP-CMA model and system suitability test (SST) indicated that the UPLC method could shorten the analysis time, improve the critical separation and satisfy the requirement of the SST. In all, HPLC method could be replaced by UPLC for the quantity analysis of PNS.

Primary constituents of blue cohosh: quantification in dietary supplements and potential for toxicity.

Dietary supplements containing dried roots or extracts of the roots and/or rhizomes of blue cohosh (Caulophyllum thalictroides) are widely available. This botanical has a long history of use by Native Americans and its use continues to the present day. The primary constituents of blue cohosh are its alkaloids and saponins. The structures of the alkaloids magnoflorine, baptifoline, anagyrine, and N-methylcytisine have been known for many years. The last 10 years have seen a great increase in isolation and identification of the large number of saponins present in blue cohosh. Important developments in nuclear magnetic resonance techniques have contributed substantially to the increase in elucidation of the structures of the complex saponins. Several authors have described quantitative methods for both the alkaloids and saponins in blue cohosh. Such methods have made it possible to quantify these constituents in dietary supplements containing this botanical ingredient. Concentrations of both alkaloids and saponins vary substantially in dietary supplements of blue cohosh. The nicotinic alkaloid, N-methylcytisine, a potent toxicant, has been found in all dietary supplements of blue cohosh analyzed. The teratogenic alkaloid anagyrine has been found in some but not all dietary supplements.

LC-MS/MS method for determination of hederacolchiside E, a neuroactive saponin from Pulsatilla koreana extract in rat plasma for pharmacokinetic study.

A simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was applied to pharmacokinetic study of a neuroactive oleanolic-glycoside saponin, hederacolchiside E from SK-PC-B70M, a standardized extract of Pulsatilla koreana in rat. Rat plasma samples were pretreated by protein precipitation with acetonitrile, eluted from C(18) column, and analyzed using electrospray ionization (ESI)-MS/MS in negative ion mode. Digoxin was used as an internal standard. The standard curves were linear (r>0.997) over the concentration ranges of 2-500 ng/mL. The intra- and inter-day precisions were measured to be below 9% and accuracy between 90 and 111% for all quality control samples at 2, 20, 100, and 500 ng/mL (n=5). The lower limits of quantification (LLOQ) for hederacolchiside E was 2 ng/mL and the limit of detection (LOD) 0.5 ng/mL using 20 microL of plasma sample. Subsequently, hederacolchiside E was determined in rat plasma samples after oral administration of SK-PC-B70M. The mean maximum plasma concentrations of hederacolchiside E were 0.07, 0.13, and 0.36 microg/mL and the mean areas under the plasma concentration versus time curve 0.56, 1.27, and 6.46 microg h/mL at doses of 100, 200, and 400 mg/kg, respectively, which indicated non-linear pharmacokinetic pattern. In conclusion, this method was successfully applied to the pharmacokinetic study of hederacolchiside E after an oral administration of SK-PC-B70M to rats.

[Quality estimation on material medica of Rhizoma Paridis from Sichuan & Yunnan province].

OBJECTIVE: To provide valuable reference for comprehensive qualitative estimation and reasonable utilization of material medica Rhizoma Paridis. METHODS: Rhizoma Paridis collected from different region of southwest Sichuan & northwest Yunnan. The texture and the content of parissaponin I, II, VI, VII were observed a nd measured b y HPLC. RESULT: The difference of target compounds' content among samples from different producing area is significant; the eligibility rate of material medica is only 25%; the texture of material medica has certain relativity with content of parissaponin.

Simultaneous analysis of seven astragalosides in Radix Astragali and related preparations by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry.

A method has been developed for the qualitative and quantitative analysis of pharmacologically active astragalosides isolated from several species of the genus Astragalus by high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. Seven astragalosides in Radix Astragali and their commercial pharmaceutical preparations were analyzed using the developed method. The extracted ion current chromatograms were obtained from the total ion current chromatogram using the m/z of [M+Na]+ ions produced by target compounds for peak determination. The limits of detection and limits of quantification were in the range of 0.10-0.22 ng and 0.22-0.52 ng in full scan mode, respectively. All calibration curves showed good linear regression (r2 > or = 0.9965) within the test range. The overall intra- and inter-day precision was less than 2.86% for peak area and the accuracy was higher than 92.9% on using ginsenoside I as internal standard. The assay was successfully utilized to analyze the major biologically active astragalosides in six samples of Astragalus membranaceus (Fisch.) Bge var. mongholicus (Bge.) Hsiao. and eight commercial preparations. The overall results demonstrate that this method is simple, selective, and suitable for the quality control of Chinese medicine and their preparation in the low nanogram range.

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle.

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.

[Experimental studies on the quality of ostracean powder].

In this paper, the contents of astragaloside IV, calcium carbonate, amino acids and trace elements in Ostraacean Powder have been measured. These studies present a scientific evaluation on the quality of Ostracean Powder.

Saponins as anticarcinogens.

Saponins, which are present in plants, have been suggested as possible anticarcinogens. They possess surface-active characteristics that are due to the amphiphilic nature of their chemical structure. The proposed mechanisms of anticarcinogenic properties of saponins include direct cytotoxicity, immune-modulatory effects, bile acid binding and normalization of carcinogen-induced cell proliferation. However, the anticarcinogenic effects of saponins from commonly consumed plant foods have not been studied. Soybeans are one of the most important sources of dietary saponins. They are the main protein supplier in many vegetarian diets. Our results showed that soybean saponins at the concentration of 150-600 ppm had a dose-dependent growth inhibitory effect on human carcinoma cells (HCT-15). Viability was also significantly reduced. Soybean saponins did not increase cell membrane permeability in a dose-dependent fashion, whereas gypsophilla saponin, a nondietary saponin, increased permeability with increasing concentrations. Electron microscopy indicated that soybean and gysophilla saponins alter cell morphology and interact with the cell membrane in different ways.

The evidence for soybean products as cancer preventive agents.

There is much evidence suggesting that compounds present in soybeans can prevent cancer in many different organ systems. The evidence for specific soybean-derived compounds having a suppressive effect on carcinogenesis in animal model systems is limited, however. There is evidence that the following isolated soybean derived products suppress carcinogenesis in vivo: a protease inhibitor, the Bowman-Birk inhibitor, inositol hexaphosphate (phytic acid) and the sterol beta-sitosterol. Other compounds that may be able to suppress carcinogenesis in animals are the soybean isoflavones. Soybean compounds reported to have other types of anticarcinogenic activity include soybean trypsin inhibitor, saponins and genistein. There is much evidence to suggest that diets containing large amounts of soybean products are associated with overall low cancer mortality rates, particularly for cancers of the colon, breast and prostate. It is believed that supplementation of human diets with certain soybean products shown to suppress carcinogenesis in animals could markedly reduce human cancer mortality rates.