Exploring lipid markers of the quality of coix seeds with different geographical origins using supercritical fluid chromatography mass spectrometry and chemometrics.

BACKGROUND: Lipids, a group of primary metabolites, could be used as quality markers of Traditional Chinese medicine. PURPOSE: The present study was designed to develop a research method to explore lipid markers of the quality of coix seeds with different geographical origins. STUDY DESIGN: The geographical origins of coix seeds were divided into three regions based on the latitude. A central composite design (CCD test) was used to optimize the chromatographic parameters of supercritical fluid chromatography to obtain optimal lipid profile of coix seed. METHODS: An untargeted method based on ultra-performance convergence chromatography - quadrupole/time-of-flight hybrid mass spectrometry (UPC2-QTOF) was developed. Four chromatographic parameters were optimized using CCD test, and a fusion index established by Derringer function was used to evaluate. The lipid profile of 27 batches of coix seeds were acquired and processed by Progenesis QI software, and the MS/MS spectrums were obtained to identify, simultaneously. The difference lipids were explored by orthogonal partial least squares discriminant analysis (OPLS-DA). The lipids that showed differences depending on their seeds' geographical origin were selected as markers of the quality of coix seeds from the three regions. RESULTS: A Torus 2-PIC (1.7 µm, 100 mm × 3.0 mm) was selected as the optimal column of the untargeted method which the run time was only 8 minutes. From the CCD test, the interaction of chromatographic parameters between column temperature and backpressure was founded which the optimal parameters were 55 °C and 2600 psi, respectively. Thirty-two peaks in the lipid profile of coix seed were tentatively identified, of which 20 were triglyceride, and 12 were diglyceride. Nine features that could potentially be used to distinguish the coix seeds by their geographical origin were identified, most of which were diglycerides, such as OP. CONCLUSIONS: Our findings confirm that UPC2-QTOF combined with chemometrics could be used as an efficient method for exploring potential lipid markers of the quality of herbal medicine.

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.

Characteristic Chromatogram: A Method of Discriminate and Quantitative Analysis for Quality Evaluation of Uncaria Stem with Hooks.

It remains a challenge to establish new monographs for herbal drugs derived from multiple botanical sources. Specifically, the difficulty involves discriminating and quantifying these herbs with components whose levels vary markedly among different samples. Using Uncaria stem with hooks as an example, a characteristic chromatogram was proposed to discriminate its five botanical origins and to quantify its characteristic components in the chromatogram. The characteristic chromatogram with respect to the components of Uncaria stem with hooks with the five botanical origins was established using 0.02% diethylamine and acetonitrile as the mobile phase. The total analysis time was 50 min and the detection wavelength was 245 nm. Using the same chromatogram parameters, the single standard to determine multicomponents method was validated to simultaneously quantify nine indole alkaloids, including vincosamide, 3α-dihydrocadambine, isocorynoxeine, corynoxeine, isorhynchophylline, rhynchophylline, hirsuteine, hirsutine, and geissoschizine methyl ether. The results showed that only the Uncaria stem with hooks from Uncaria rhynchophylla, the most widely used in the herbal market, showed the presence of these nine alkaloids. The conversion factors were 1.27, 2.32, 0.98, 1.04, 1.00, 1.02, 1.26, 1.33, and 1.25, respectively. The limits of quantitation were lower than 700 ng/mL. The total contents of 31 batches of Uncaria stem with hooks were in the range of 0.1 - 0.6%, except for Uncaria hirsuta Havil and Uncaria sinensis (Oliv.) Havil. The results also showed that the total content of indole alkaloids tended to decrease with an increase in the hook diameter. This showed that the characteristic chromatogram is practical for controlling the quality of traditional Chinese medicines with multiple botanical origins.

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).

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla.

Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

An intelligentized strategy for endogenous small molecules characterization and quality evaluation of earthworm from two geographic origins by ultra-high performance HILIC/QTOF MS(E) and Progenesis QI.

Animal-derived medicines have been a vital component for traditional Chinese medicine. However, their quality control remains challenging due to the large polarity of the contained endogenous small molecules (ESMs) that are difficult to separate by reversed-phase HPLC. Herein, an intelligentized strategy by ultra-high performance hydrophilic interaction chromatography/quadrupole time-of-flight mass spectrometry (HILIC/QTOF-MS(E)) is presented, and used for the ESMs characterization and differentiation of two geographic origins of earthworm (Guang Di-long, GD; Hu Di-long, HD) as a case study. Chromatographic separation was performed on a BEH Amide column (2.1 × 100 mm, 1.7 µm). The MS(E) data in both negative and positive ion modes were acquired to record the high-accuracy MS and MS/MS data of all precursor ions. Automatic data processing was enabled by use of Progenesis QI software. As a consequence, 926 metabolites among 4705 features and 761 among 3418 features were characterized in the negative and positive modes, respectively, by searching the human metabolome database (HMDB). To reduce the false positive identifications, structural confirmation was conducted by comparison with the reference standards (tR and MS, MS/MS data) or matching with theoretical data or commercial library. Principal component analysis (PCA) of the GD and HD samples showed distinct classifications. Further orthogonal partial least squares discriminant analysis (OPLS-DA) and variable importance in projection (VIP) plot revealed the potential discriminatory markers between GD and HD. The present study provides a powerful and practical strategy that facilitates the primary metabolites characterization and quality evaluation of animal-derived medicines more efficiently. Graphical Abstract A general flowchart illustrating the application of ultra-high performance HILIC/QTOFMS(E) coupled with data processing by Progenesis QI to characterization of the endogenous small molecules and discriminatory analysis of animal-derived traditional medicine: earthworm as a case study.

Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl).

ETHNOPHARMACOLOGICAL RELEVANCE: Pearl and nacre are valuable traditional medicines to treat palpitations, convulsions or epilepsy in China for thousands of years. However, the active ingredients are not clear till now. AIM OF THE STUDY: The main purpose of the current investigation was to assess the anticonvulsant and sedative-hypnotic activity of pearl powder and nacre powder, including their corresponding 6 protein extracts. MATERIAL AND METHODS: Determination of the amino acid composition of the obtained protein was carried out by ultra-performance liquid chromatography (UPLC) combined with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatisation. The influence of the tested drugs on locomotor activity and convulsions latency was recorded. The contents of 5-Hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in brain were detected by enzyme-linked immunesorbent assay (ELISA) kits. In addition, immunohistochemistry was carried out to evaluate the changes of 5-HT3 and GABAB. In parallel, the expressions of them were demonstrated by western blot. RESULTS: The obtained data suggested that pearl original powder (1.1g/kg), pearl water-soluble protein (0.2g/kg), pearl acid-soluble protein (0.275g/kg), pearl conchiolin protein (1.1g/kg), nacre original powder (1.1g/kg), nacre water-soluble protein (0.2g/kg), nacre acid-soluble protein (0.7g/kg) and nacre conchiolin protein (1.1g/kg) could down-regulate the expression of 5-HT3 and up-regulate the level of GABAB to varying degrees compared with the control group. Besides, drug administration also reduced the locomotor activity and increased convulsions latency with a certain mortality. CONCLUSIONS: These findings correlated with the traditional use of pearl and nacre as sedation and tranquilization agents, thus making them interesting sources for further drug development and also providing critical important evidence for the selection of quality control markers.

An efficient and target-oriented sample enrichment method for preparative separation of minor alkaloids by pH-zone-refining counter-current chromatography.

An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and (1)H NMR.

Discriminatory components retracing strategy for monitoring the preparation procedure of Chinese patent medicines by fingerprint and chemometric analysis.

Chinese patent medicines (CPM), generally prepared from several traditional Chinese medicines (TCMs) in accordance with specific process, are the typical delivery form of TCMs in Asia. To date, quality control of CPMs has typically focused on the evaluation of the final products using fingerprint technique and multi-components quantification, but rarely on monitoring the whole preparation process, which was considered to be more important to ensure the quality of CPMs. In this study, a novel and effective strategy labeling "retracing" way based on HPLC fingerprint and chemometric analysis was proposed with Shenkang injection (SKI) serving as an example to achieve the quality control of the whole preparation process. The chemical fingerprints were established initially and then analyzed by similarity, principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to evaluate the quality and to explore discriminatory components. As a result, the holistic inconsistencies of ninety-three batches of SKIs were identified and five discriminatory components including emodic acid, gallic acid, caffeic acid, chrysophanol-O-glucoside, and p-coumaroyl-O-galloyl-glucose were labeled as the representative targets to explain the retracing strategy. Through analysis of the targets variation in the corresponding semi-products (ninety-three batches), intermediates (thirty-three batches), and the raw materials, successively, the origins of the discriminatory components were determined and some crucial influencing factors were proposed including the raw materials, the coextraction temperature, the sterilizing conditions, and so on. Meanwhile, a reference fingerprint was established and subsequently applied to the guidance of manufacturing. It was suggested that the production process should be standardized by taking the concentration of the discriminatory components as the diagnostic marker to ensure the stable and consistent quality for multi-batches of products. It is believed that the effective and practical strategy would play a critical role in the guidance of manufacturing and help improve the safety of the final products.

Ultra-performance liquid chromatography of amino acids for the quality assessment of pearl powder.

Pearls have been widely used as a traditional medicine, in cosmetics, and as a health food supplement in China since ancient times. However, the identification and quality assessment of pearl powder have been challenging tasks because of the similar morphological features and chemical composition of its common adulterants, especially conch powders. In this study, ultra-performance liquid chromatography was combined with pre-column derivatization to rapidly quantify 14 amino acids in pearl powder and its analogues. Based upon the quantification results, a quality criterion of a total amino acid content of not less than 1.10% was proposed for pearl powder. Principal component analysis indicated that leucine and phenylalanine were the amino acids characteristic for distinguishing between pearls and nacres. The area ratio of leucine to phenylalanine was demonstrated to be an effective diagnostic marker to discriminate freshwater cultured pearls, natural seawater pearls, and nacres. The proposed method, involving both the qualitative and quantitative aspects, was subsequently applied to quality assessment of pearl powders purchased commercially in various parts of China; eight out of 18 batches were deemed authentic and unadulterated. In the future, this analytical process should play a significant role in standardizing and providing quality control to the pearl powder market.

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.

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.

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.

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.