Jinjun Hou

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 dynamic multiple reaction monitoring method for the multiple components quantification of complex traditional Chinese medicine preparations: Niuhuang Shangqing pill as an example

It is a challenging task to simultaneously and quantitatively analyze multiple components in DFF [Da-Fu-Fang, namely, complex traditional Chinese medicine (TCM) preparations containing more than ten TCMs] due to their numerous and extreme complex chemical compositions possessing a wide variety of chemical and physical features, and their very low content. Rather than using a conventional mass spectrometry (MS) method with multiple reaction monitoring (MRM), in the current study, this challenge was addressed by using dynamic multiple reaction monitoring (DMRM). Using a DFF, Niuhuang Shangqing pill, which is composed of 19 TCMs, as a model, a rapid (one run in 20min), sensitive [lower limit of detection (LOD) and limit of quantitation (LOQ) were achieved comparable with MRM] and accessible (a standard HPLC/MS/MS instrumentation was employed) MS method was successfully developed for the simultaneous quantification of 41 bioactive components which represented 15 of the 19 medicinal plants. A comparison of LOD and LOQ using MRM and DMRM was made to quantitatively reveal that the latter demonstrated advantages over the former. Meanwhile, a standard operating procedure concerning the development of a new DMRM method was recommended. The MS data were obtained in the positive ion mode with electrospray ionization as the ion source, acetonitrile and water as mobile phase and a Kinetex C18 core-shell column (100mm×2.10mm, 2.6μm, Phenomenex Inc.) as the analytical column. This method was then applied to 32 batches of samples. It transpired, through principal component analysis and orthogonal partial least squares discriminant analysis, that the consistency of the products was relatively good within one company, but poor among different companies among the 32 samples; one failed to qualify in terms of the Chinese Pharmacopeia. This work illustrated that the proposed DMRM method was particularly suitable for quantifying the trace components in DFF and capable of ensuring the quality of DFF.

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.

HPLC/qTOF-MS-oriented characteristic components data set and chemometric analysis for the holistic quality control of complex TCM preparations: Niuhuang Shangqing pill as an example

The quality control of Da-Fu-Fang (DFF), referring to the traditional Chinese medicine (TCM) preparations comprising more than 10 TCMs, is challenging due to their extreme chemical complexity. In this study, a strategy is proposed for the holistic quality control of DFFs based on HPLC/qTOF-MS-oriented characteristic components data set (CCDS) and chemometric analysis. Niuhuang Shangqing pill (NHSQP), composed of 19 TCMs, is used to illustrate this strategy. The fingerprint profiling of NHSQP by HPLC/qTOF-MS resulted in the characterization of 190 compounds, comprising 47 unambiguously identified by reference standard comparison. A CCDS containing 60 characteristic components was constructed by analyzing the MS spectral differentiation of the crude drugs, a laboratory-made NHSQP powder, and negative control preparations. With the established CCDS, it was possible to simultaneously monitor 16 out of the 19 drugs involved in NHSQP. Subsequently, 26 NHSQP samples from different vendors were evaluated by the qualitative and semi-quantitative analyses of their LC/MS fingerprint data. The 60 characteristic components were detected in all of the NHSQP samples, which demonstrated their authenticity. When compared with the standard sample No. 3, however, 15 of the NHSQP samples exhibited inferior quality. Samples No. 21 and No. 13 differed significantly based on a PCA score plot, and the components responsible for the differentiation were confirmed to originate from different TCMs. This strategy is a powerful and easy method to implement and provides a potential approach to establishing the holistic quality control of complex TCM preparations.

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.

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

HIGHLIGHTSSFC was used to separate and isolate two pairs of 7‐epimers of SOAs.Acetonitrile stabilized two pairs of epimers and was used as modifier in SFC.Two achiral UPC2 methods were established on the Torus 1‐AA and Diol columns.Preparative SFC enabled isolation of four SOA compounds with the purity >95%.SFC provides a solution to preparation of high‐purity reference standards. ABSTRACT 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.

Selective and comprehensive characterization of the quinochalcone C-glycoside homologs in Carthamus tinctorius L. by offline comprehensive two-dimensional liquid chromatography/LTQ-Orbitrap MS coupled with versatile data mining strategies

Quinochalcone C-glycosides (QCGs) are a series of pharmacologically bioactive components chemotaxonomic for Carthamus tinctorius L. The low abundance and ubiquitous interference from flavonoid O-glycosides (FOGs) frequently hinder the systematic exposure and characterization of these QCG homologs. We herein present an offline comprehensive two-dimensional liquid chromatography/linear ion-trap quadrupole/Orbitrap mass spectrometry (2D LC/LTQ-Orbitrap MS) approach coupled with versatile data mining strategies, to systematically characterize the QCGs from C. tinctorius. Initially, an offline 2D LC system, with an orthogonality of 71% and a theoretical peak capacity of 7654, was established by combining an Acchrom XAmide column and a BEH Shield RP-18 column. Subsequently, the water extract of C. tinctorius was separated by first dimensional hydrophilic interaction liquid chromatography (HILIC) yielding twelve fractions, which were further analyzed by reversed-phase ultra-high performance liquid chromatography/LTQ-Orbitrap MS using high energy C-trap dissociation (HCD) and collision-induced dissociation (CID) in the negative ion mode. The characteristic product ion filtering of m/z 119.05 (C8H7O−) in the HCD spectra, ring double bond equivalent (RDB 10–30), characteristic UV absorption around 405 nm, preferred 0,2X0 cleavage for C-glycosides, and diagnostic product ions analysis, were simultaneously employed for the structural elucidation of QCGs. Ultimately, 163 QCQ homologs were putatively characterized, and 149 are potential new ones. Particularly, nine dimers of QCG-FOG have not been previously reported. The obtained results have greatly expanded the knowledge on the structural diversity of QCGs, demonstrating the potency of the offline comprehensive 2D LC/LTQ-Orbitrap MS approach in separation and characterization of minor herbal components.

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.

Venenum Bufonis induces rat neuroinflammation by activiating NF-κB pathway and attenuation of BDNF

ETHNOPHARMACOLOGICAL RELEVANCE Venenum Bufonis (VB), also called toad venom, has been widely used in clinic as a cardiotonic, anohyne and antineoplastic agents both in China and other Asian countries. However, its neurotoxicity and cardiotoxicity limit its wide clinical application. Compared with extensive attention attracted with cardiotoxicity, the toxic effect of VB on Central Nervous System (CNS) is much less studied. AIM OF THE RESEARCH This study was performed to examine the neurotoxicity caused by VB on Sprague Dawley (SD) rats, then to clarify the mechanism in vivo by investigating its action on the neuroinflammation which possibly attributed to the activation of nuclear factor κB (NF-κB) pathway and the attenuation of brain-derived neurotrophic factor (BDNF). MATERIALS AND METHODS Rats administrated with 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution and VB (100mg/kg, 200mg/kg and 400mg/kg) were sacrificed at 2h, 4h, 6h, 8h, 24h and 48h. The brain level of neurotransmitters and their corresponding receptors, pro-inflammatory cytokines, BDNF/TrkB and NF-κB pathway-related proteins were examined, respectively. RESULTS VB administration induced severe neurologic damage and neuroinflammation, as indicated by the disordered 5-hydroxytryptamine (5-HT), dopamine (DA) and their corresponding receptors, together with the over production of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). VB also notably promoted the expression of p-NF-κBp65, p-IκBα, p-IKKα and p-IKKβ and down-regulated the expression of BDNF and TrkB. CONCLUSION This study demonstrates that VB triggers neurotoxicity which probably is induced by neuroinflammation via activating of NF-κB pathway and attenuating the expression of BDNF.