Hua-Li Long

Ruggedness and robustness of conversion factors in method of simultaneous determination of multi-components with single reference standard

Single standard to determine multi-components (SSDMC) is a novel and rational method for quality control of botanical products and traditional Chinese medicines (TCMs). However, it is restricted to wide application due to unknown fluctuation in conversion factors when it is performed in different laboratories. To evaluate the fluctuations of conversion factors, we selected Salvia miltiorrhiza as an example to determine three components of tanshinones by SSDMC method. Then ruggedness and robustness test were adopted to comprehensively investigate three kinds of factors that may influence stability of conversion factors, which were related with environmental parametric variables, operational parametric variables and peak measurement parametric variables. Nested-factorial-design was used to perform ruggedness tests. One-variable-at-a-time (OVAT) procedure and Plackett-Burman (PB) design were both used in robustness test. The results showed that stability of conversion factors was principally related with accuracy of wavelength of UV detector, peak measurement parameters and concentration of standard solution. The acceptable range of conversion factors was obtained from robustness test. Our results showed that conversion factors were inevitable to change, but when key parameters were well controlled, the range of its fluctuation was acceptable and the SSDMC method could be used widely in different laboratories.

Comparison of two officinal Chinese pharmacopoeia species of Ganoderma based on chemical research with multiple technologies and chemometrics analysis.

AIM OF THE STUDY To investigate the chemical differences between Ganoderma lucidum (G. lucidum, Chizhi) and Ganoderma sinense (G. sinense, Zizhi). MATERIALS AND METHODS Thirty two batches of commercial Ganoderma samples were collected, including 20 batches of G. lucidum and 12 batches of G. sinense cultivated in different geographical regions. Chemical substances in aqueous extract and alcoholic extract, mainly polysaccharides and triterpenes respectively, were investigated. Determination of polysaccharides was carried out with a high performance liquid chromatography with an variable wavelength detector. Meanwhile, analysis of triterpenes were performed on an ultraviolet spectrophotometer, an ultra performance liquid chromatography and a rapid resolution liquid chromatograph combined with an electrospray ionization mass spectrometer. Chromatograms and spectra for all batches and reference standards of main components were obtained and used for direct comparison. Further discussion was made on the basis of the result of principal component analysis (PCA). RESULTS Significant difference of triterpenes was shown between G. lucidum and G. sinense. In 20 batches of G. lucidum, 12 main components, including eight ganoderic acids and four ganoderenic acids were identified and ten of them were quantitatively determined, with the total content from 0.249% to 0.690%. However, none of those triterpenes was found in either batch of G. sinense. As for constituents of polysaccharides, seven monosaccharides were identified and four main components among them were quantitatively determined. Difference of polysaccharides was not directly observed, but latent information was revealed by PCA and the discrimination became feasible. CONCLUSIONS G. lucidum and G. sinense were chemically different, which might result in pharmacological distinction. Preparations of traditional Chinese medicine (TCM) from Ganoderma should make accurate specification on the origin of species.

A single, multi-faceted, enhanced strategy to quantify the chromatographically diverse constituents in the roots of Euphorbia kansui.

Kansui radix is a famous poisonous traditional Chinese medicine. However, due to its different types of constituents with broad polarity, a variety of UV absorptions and lack of the reference standards, it was difficult to simultaneously determine the main component in kanui radix. A single, multi-faceted, enhanced strategy, exogenous reference standard - single standard to determine multi-components method (ERS-SSDMC), was proposed. Thirteen major components of kansui radix, including three jatrophane diterpenoids, eight ingenane diterpenoids and two triterpenes, among which there were three pairs of isomers, were simultaneously assayed. A C8 column, packed with 2.7μm core-shell particles, was optimized to separate these constituents in 25min on HPLC instrument detected at a program wavelength. Ethyl benzoate employed as single exogenous reference standard. The method was fully validated with respect to linearity (r(2)>0.9995), LOQs (0.1-0.4μg/mL), precision, accuracy (92-114%, RSD<4.4%) and stability. The robustness of the method was performed by Plackett-Burmantest tests which eight primary chromatographic parameters were investigated. It was found that the two factors, wavelength and flow rate, should be strictly controlled. A total of 75 batches of kansui radix and its three different processing products were successfully analyzed and discriminated by applying the proposed method. This work demonstrates an effective strategy for the SSDMC method making the simultaneous assay of complex multi-component TCM system achievable.

TCM-based new drug discovery and development in China.

Over the past 30 years, China has significantly improved the drug development environment by establishing a series of policies for the regulation of new drug approval. The regulatory system for new drug evaluation and registration in China was gradually developed in accordance with international standards. The approval and registration of TCM in China became as strict as those of chemical drugs and biological products. In this review, TCM-based new drug discovery and development are introduced according to the TCM classification of nine categories.

A reproducible analytical system based on the multi-component analysis of triterpene acids in Ganoderma lucidum

Ultra-performance liquid chromatography (UPLC) and Single Standard for Determination of Multi-Components (SSDMC) are becoming increasingly important for quality control of medicinal herbs; this approach was developed for Ganoderma lucidum. Special attention was necessary for the appropriate selection of markers, for determining the reproducibility of the relative retention times (RRT), and for the accuracy of conversion factors (F). Finally, ten components were determined, with ganoderic acid A serving as single standard. Stable system parameters were established, and with successful resolution of those issues, this analytical method could be used more broadly.

Anti-proliferation activity of terpenoids isolated from Euphorbia kansui in human cancer cells and their structure-activity relationship

Euphorbia kansui is a commonly used traditional Chinese medicine for the treatment of edema, pleural effusion, and asthma, etc. According to the previous researches, terpenoids in E. kansui possess various biological activities, e.g., anti-virus, anti-allergy, antitumor effects. In this work, twenty five terpenoids were isolated from E. kansui, including thirteen ingenane- and eight jatrophane-type diterpenoids (with two new compounds, kansuinin P and Q) and four triterpenoids. Eighteen of them were analyzed by MTS assay for in vitro anticancer activity in five human cancer cell lines. Structure-activity relationship for 12 ingenane-type diterpenoids in colorectal cancer Colo205 cells were preliminary studied. Significant anti-proliferation activities were observed in human melanoma cells breast cancer MDA-MB-435 cells and Colo205 cells. More than half of the isolated ingenane-type diterpenoids showed inhibitory activities in MDA-MB-435 cells. Eight ingenane- and one jatrophane-type diterpenoids possessed much lower IC50 values in MDA-MB-435 cells than positive control staurosporine. Preliminary structure-activity relationship analysis showed that substituent on position 20 was important for the activity of ingenane-type diterpenoids in Colo205 cells and substituent on position 3 contributed more significant biological activity of the compounds than that on position 5 in both MDA-MB-435 and Colo205 cells.

A feasible, economical, and accurate analytical method for simultaneous determination of six alkaloid markers in Aconiti Lateralis Radix Praeparata from different manufacturing sources and processing ways

Aconiti Lateralis Radix Praeparata (Fuzi) is a commonly used traditional Chinese medicine in clinic for its potency in restoring yang and rescuing from collapse. Aconiti alkaloids, mainly including monoester-diterpenoidaconitines (MDAs) and diester-diterpenoidaconitines (DDAs), are considered to act as both bioactive and toxic constituents. In the present study, a feasible, economical, and accurate HPLC method for simultaneous determination of six alkaloid markers using the Single Standard for Determination of Multi-Components (SSDMC) method was developed and fully validated. Benzoylmesaconine was used as the unique reference standard. This method was proven as accurate (recovery varying between 97.5%-101.8%, RSD < 3%), precise (RSD 0.63%-2.05%), and linear (R > 0.999 9) over the concentration ranges, and subsequently applied to quantitative evaluation of 62 batches of samples, among which 45 batches were from good manufacturing practice (GMP) facilities and 17 batches from the drug market. The contents were then analyzed by principal component analysis (PCA) and homogeneity test. The present study provided valuable information for improving the quality standard of Aconiti Lateralis Radix Praeparata. The developed method also has the potential in analysis of other Aconitum species, such as Aconitum carmichaelii (prepared parent root) and Aconitum kusnezoffii (prepared root).

Green Quantification Strategy Combined with Chemometric Analysis for Triglycerides in Seeds Used in Traditional Chinese Medicine

Triglycerides are the primary constituents of some seed kernels used in traditional Chinese medicine. Quality control of seed kernels containing multiple components with an environmentally friendly method is indispensable for establishing their quality standards (called monographs) in pharmacopeia. Using coix seeds (Semen Coicis) as an example, a green quantification strategy was proposed by combining C8 core-shell particles with single standard to determine multicomponent technologies to quantify seven triglycerides simultaneously. A core-shell column, namely, Halo C8 (3.0 × 100 mm, 2.7 µm), was used. Methanol was used as the mobile phase at a flow rate of 0.3 mL/min, enabling UV detection of the elutes. Seven triglycerides were well separated in 20 min, and simultaneously quantified using triolein as a single standard. The conversion factor for each standard was set as 1.0 on ELSD, while for the conversion factors at 203 nm, the values increased with the reduction of linoleate. The recovery values were all in the range of 97 - 107% (RSD < 3.0%). The RSD values of precision, including intraday and intermediate precision, were < 3.0% when the total content of triglycerides was calculated. The linearity reached r ≥ 0.9990, and the limit of quantitation reached 40 - 70 ng. Forty-nine batches of coix seeds from four different places of origins and eight batches of adulterants were evaluated and differentiated using principal component analysis. In addition, the validated method was used successfully to quantity seven triglycerides in Semen Persicae, Semen Armeniacae Amarum, and Semen Pruni.

Orientalol L–P, novel sesquiterpenes from the rhizome of Alisma orientale (Sam.) Juzep and their nephrotoxicity on HK2 cells

Two new sesquiterpenes (1–2) and three new nor-sesquiterpenes (3–5) were obtained from the rhizome of Alisma orientale (Sam.) Juzep, one of which (2) possessed an unprecedented cyclopentene and furanone containing natural skeleton. Their structures and relative stereochemistry were elucidated by NMR spectroscopy (1H and 13C NMR, HSQC, HMBC, NOESY) and electronic circular dichroism (ECD) and HR-ESI-MS data analyses. Two plausible biosynthetic pathways for compound 1 and 2 were discussed. The nephrotoxicities of these compounds were evaluated on normal human HK2 cells by high content screening, and neither the MeOH extract nor the compounds possessed potential nephrotoxicity in vitro.

Development of specific and quantitative methods for the quality control of the polysaccharides from sea-tangle and sargassum

Polysaccharides from numerous traditional Chinese medicines have been proven as the bioactive ingredients and are hence used as the quality control markers. However, the assessment criteria always show a poor specificity, due to the lack of systematic comparison among the analogous herbs. In the present study, two similar materials, namely sea-tangle and sargassum, were selected as the model herbs to develop more specific methods for quality control. Two well-established methods, determination of the total polysaccharides content and monosaccharides composition analysis, were both employed. Based upon the quantitative results, the evaluation criteria of the polysaccharides contents of not less than 2.0% and 1.7% were proposed for sea-tangle and sargassum, respectively. Nine identical monosaccharide derivatives appeared on the HPLC chromatograms of the hydrolysis and derivatized solutions of the two drugs. Principal component analysis and orthogonal partial least squares discriminant analysis using the peak areas of monosaccharides derivatives as the variables were performed, and the results indicated that mannuronic acid and xylose with the opposite concentrations in the two drugs were the differential components. A discriminative criterion using the peak area ratio of these two monosaccharides derivatives was proposed for the qualitative identification. In conclusion, a more specific and quantitative quality control method was developed for sea-tangle and sargassum.