Comparative quality of the forms of decoction pieces evaluated by multidimensional chemical analysis and chemometrics: Poria cocos, a pilot study.

Many Chinese medicinal materials (CMMs) are parts of plants or fungi that have been processed into different physical forms, termed decoction pieces, that are typically boiled in water for consumption. One CMM may have several decoction pieces forms, e.g., slices, small cubes (dice), or grains. The specifications that have different morphological parameters (shape, size and thickness) for these various decoction pieces have been developed over, in some cases, centuries of practice. Nevertheless, whether and how the form of decoction pieces affects the extraction (decoction) dynamics, and quality stability during storage has not been studied. Here, we investigated Poria cocos (PC) as a pilot study; we explore how the form of PC decoction pieces affects its chemistry using multidimensional chemical evaluation such as ultra-performance liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry (UHPLC-PDA-QTOF-MS/MS), ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) and high performance gel permeation chromatography coupled with charged aerosol detector (HPGPC-CAD), combined with analysis of variance (ANOVA), principal component analysis (PCA), factor analysis (FA) and hierarchical cluster analysis (HCA). The results indicated that different specifications had significant differences, and these specifications could be divided into four groups. The comprehensive results of the chemical analyses undertaken here indicate that the highest potentially available quality of PC decoction pieces was in the forms of curl, ultra-small grains and small grains, followed by thin slices. This information not only is conducive to promoting the standardization of the specification/form of PC decoction pieces and maximizing the benefits from its utilization, but also provide a promising strategy for assessing other CMM decoction pieces in different forms.

Correlation between Quality and Geographical Origins of Poria cocos Revealed by Qualitative Fingerprint Profiling and Quantitative Determination of Triterpenoid Acids.

Poria cocos (Schw.) Wolf (PC) is a well-known saprophytic fungus, and its sclerotium without the epidermis (PCS) is widely used in traditional Chinese medicine and as a functional food in many countries. PCS is normally collected from multiple geographical regions, but whether and how the quality of PCS correlates with where it grows have not been determined. This correlation could be significant both for quality control and optimum utilization of PCS as a natural resource. In this study, a qualitative fingerprint profiling method performed by ultra-performance liquid chromatography (UHPLC) with diode array detection (DAD) combining quadrupole time-of-flight-mass spectrometry (QTOF-MS/MS) and a quantitative UHPLC coupled with triple quadrupole mass spectrometry (QqQ-MS/MS) approach were established to investigate whether and how the quality of PCS correlates with its collection location. A standard fingerprint of PCS was generated by median simulation of 25 tested samples collected from four main producing areas of China, and similarity analysis was applied to evaluate the similarities between the fingerprints of samples and the standard fingerprint. Twenty three common peaks occurring in the fingerprint were unequivocally or tentatively identified by UHPLC-QTOF-MS/MS. Meanwhile, principal component analysis (PCA), supervised orthogonal partial least squares-discriminate analysis (OPLS-DA) and hierarchical cluster analysis (HCA) were employed to classify 25 batches of PCS samples into four groups, which were highly consistent with the four geographical regions. Ten compounds were screened out as potential markers to distinguish the quality of PCS. Nine triterpene acids, including five compounds that played important roles in the clusters between different samples collected from the four collection locations, were simultaneously quantified by using the multiple reaction monitoring (MRM) mode of UHPLC-QqQ-MS/MS. The current strategy not only clearly expounded the correlation between quality and geographical origins of PCS, but also provided a fast, accurate and comprehensive qualitative and quantitative method for assessing the quality of PCS.

Tissue-Specific Analysis of Secondary Metabolites Creates a Reliable Morphological Criterion for Quality Grading of Polygoni Multiflori Radix.

In commercial herbal markets, Polygoni Multiflori Radix (PMR, the tuberous roots of Polygonum multiflorum Thunb.), a commonly-used Chinese medicinal material, is divided into different grades based on morphological features of size and weight. While more weight and larger size command a higher price, there is no scientific data confirming that the more expensive roots are in fact of better quality. To assess the inherent quality of various grades and of various tissues in PMR and to find reliable morphological indicators of quality, a method combining laser microdissection (LMD) and ultra-performance liquid chromatography triple-quadrupole mass spectrometry (UPLC-QqQ-MS/MS) was applied. Twelve major chemical components were quantitatively determined in both whole material and different tissues of PMR. Determination of the whole material revealed that traditional commercial grades based on size and weight of PRM did not correspond to any significant differences in chemical content. Instead, tissue-specific analysis indicated that the morphological features could be linked with quality in a new way. That is, PMR with broader cork and phloem, as seen in a transverse section, were typically of better quality as these parts are where the bioactive components accumulate. The tissue-specific analysis of secondary metabolites creates a reliable morphological criterion for quality grading of PMR.

Aberrant alterations of the expressions and S-nitrosylation of calmodulin and the downstream factors in the brains of the rodents during scrapie infection.

The aberrant alterations of calmodulin (CaM) and its downstream substrates have been reported in some neurodegenerative diseases, but rarely described in prion disease. In this study, the potential changes of Ca2+/CaM and its associated agents in the brains of scrapie agent 263K-infected hamsters and the prion infected cell line SMB-S15 were evaluated by various methodologies. We found that the level of CaM in the brains of 263K-infected hamsters started to increase at early stage and maintained at high level till terminal stage. The increased CaM mainly accumulated in the regions of cortex, thalamus and cerebellum of 263K-infected hamsters and well localization of CaM with NeuN positive cells. However, the related kinases such as total and phosphorylated forms of CaMKII and CaMKIV, as well as the downstream proteins such as CREB and BDNF in the brain of 263K-infected hamsters were decreased. Further analysis showed a remarkable increase of S-nitrosylated (SNO) form of CaM in the brains of 263K-infected hamsters. Dynamic analysis of S-nitrosylated CaM showed the SNO form of CaM abnormally increases in a time-dependent manner during prion infection. Compared with that of the normal partner cell line SMB-PS, the CaM level in SMB-S15 cells was increased, meanwhile, the downstream proteins, such as CaMKII, p-CaMKII, CREB, as well as BDNF, were also increased, especially in the nucleic fraction. No SNO-CaM was detected in the cell lines SMB-S15 and SMB-PS. Our data indicate an aberrant increase of CaM during prion infection in vivo and in vitro.

Remarkable increases of α1-antichymotrypsin in brain tissues of rodents during prion infection.

α1-Antichymotrypsin (α1-ACT) belongs to a kind of acute-phase inflammatory protein. Recently, such protein has been proved exist in the amyloid deposits which is the hallmark of Alzheimer's disease, but limitedly reported in prion disease. To estimate the change of α1-ACT during prion infection, the levels of α1-ACT in the brain tissues of scrapie agents 263K-, 139A- and ME7-infected rodents were analyzed, respectively. Results shown that α1-ACT levels were significantly increased in the brain tissues of the three kinds of scrapie-infected rodents, displaying a time-dependent manner during prion infection. Immunohistochemistry assays revealed the increased α1-ACT mainly accumulated in some cerebral regions of rodents infected with prion, such as cortex, thalamus and cerebellum. Immunofluorescent assays illustrated ubiquitously localization of α1-ACT with GFAP positive astrocytes, Iba1-positive microglia and NeuN-positive neurons. Moreover, double-stained immunofluorescent assays and immunohistochemistry assays using series of brain slices demonstrated close morphological colocalization of α1-ACT signals with that of PrP and PrPSc in the brain slices of 263K-infected hamster. However, co-immunoprecipitation does not identify any detectable molecular interaction between the endogenous α1-ACT and PrP either in the brain homogenates of 263K-infected hamsters or in the lysates of prion-infected cultured cells. Our data here imply that brain α1-ACT is increased abnormally in various scrapie-infected rodent models. Direct molecular interaction between α1-ACT and PrP seems not to be essential for the morphological colocalization of those two proteins in the brain tissues of prion infection.

Urine metabolomics study on the liver injury in rats induced by raw and processed Polygonum multiflorum integrated with pattern recognition and pathways analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum L. is a famous traditional Chinese medicine that has always been perceived to be safe. Recently, the increasing case reports on hepatotoxicity induced by Raw P. multiflorum (RP) have attracted particular attention. However, the diagnosis and identification of RP-induced hepatotoxicity are still very difficult for its unknown mechanism and the lack of specific biomarkers. AIM OF THE STUDY: To further explore the toxicity and metabolic mechanisms involved in the hepatotoxicity induced by RP. MATERIALS AND METHODS: The hepatotoxicity induced by RP and its processed products (PP) (dosed at 20g/kg for 4 weeks) on rats were investigated using conventional approaches including the biochemical analysis and histopathological observations. Further, a urinary metabolomic approach was developed to study the metabolic disturbances caused by RP and PP, followed by the pattern recognition approach and pathways analysis. RESULTS: RP showed obvious hepatotoxity whereas PP did not. 16 potential biomarkers (pyridoxamine, 4-pyridoxic acid, citrate et al.) differentially expressed in RP group were identified compared with the control and PP-treated groups. The pathways analysis showed that vitamin B6 metabolism, tryptophan metabolism and citrate cycle might be the major enriched pathways involved in the hepatotoxicity of the herb. CONCLUSION: 16 differentially expressed metabolites were identified to be involved in the RP-induced hepatotoxicity. Vitamin B6 metabolism might be mostly related to the hepatotoxicity induced by RP. This finding may provide a potential therapeutic target or option to treat hepatotoxicity induced by RP.

Untargeted Metabolomics Reveals Dose-Response Characteristics for Effect of Rhubarb in a Rat Model of Cholestasis.

Cholestasis is a serious manifestation of liver diseases with limited therapies. Rhubarb, a widely used herbal medicine, has been frequently used at a relatively large dose for treating cholestasis. However, whether large doses are optimal and the therapeutic mechanism remain unclear. To explore these questions, the anti-cholestatic effect of five doses of rhubarb (0.21, 0.66, 2.10, 6.60, and 21.0 g/kg) in an alpha-naphthylisothiocyanate (ANIT)-induced rat model of cholestasis was examined by histopathology and serum biochemistry. A dose-dependent anti-cholestatic effect of rhubarb (0.21-6.6 g/kg) was observed, and an overdose of 21.0 g/kg showed a poor effect. LC-MS-based untargeted metabolomics together with pathway analysis were further applied to characterize the metabolic alterations induced by the different rhubarb doses. Altogether, 13 biomarkers were identified. The dose-response curve based on nine important biomarkers indicated that doses in the 0.42-6.61 g/kg range (EC20-EC80 range, corresponding to 4.00-62.95 g in the clinic) were effective for cholestasis treatment. The pathway analysis showed that bile acid metabolism and excretion, inflammation and amino acid metabolism were altered by rhubarb in a dose-dependent manner and might be involved in the dose-response relationship and therapeutic mechanism of rhubarb for cholestasis treatment.

[Studies on chemical constituents of Valeriana plants and their biological activities].

The recent progresses on chemical components and pharmacological activities of the genus Valerianawere summarized.Besides-essential oil, the chemical composition of Valerianais mainly focused on monoterpenoids,sesquiterpenoids,lignans, flavonoids, alkaloids, etc. Iridoids are the main chemical components ofmonoterpenoids. There are two types ofiridoidson the basis of the cyclopentane open or not. The Valerianahas been drawmuch attention for their significant sedation,spasmolysis,antidepression,antitumor, against adenosine A1 receptors and cytotoxicityactivity,and had certain function for cardiovascular disease treatment. Given to the fact of the lack of systematic review and summary of studies on the Valeriana, we summarized and analyze the study literatures on the pharmacological activity of Valerianain recent years, and providedsome basisfor further study.

[Uniformity of Coptidis Rhizoma decoction pieces dispensing based on effective constituent equivalence].

Traditional Chinese medicine (TCM) dispensing is the final step of TCM used for clinical treatment, the stability of TCM dispensing is the guarantee of good clinical effect. Establishment of effect-constituent equivalence for Chinese herbal pieces based on clinical efficacy, can not only guarantee the stability of TCM dispensing, but also relate to the precision of clinical effect. This study chose Coptidis Rhizoma as the model, established effect-constituent equivalence of Coptidis Rhizoma, based on the effect-constituent index already established by our research group, and taking into consideration of homogeneity of clinical dosage and compliance of decoction, the uniformity of dispensing for different specification of Coptidis Rhizoma decoction pieces was studied. This research model was then applied to guide the specification-optimization of Coptidis Rhizoma and its clinical dispensing. The result indicated, effective constituent equivalence could reflect the fluctuation of specification, dosage and decoction to the fluctuation of efficacy; Optimized Coptidis Rhizoma decoction pieces had the characteristic of high homogeneity as for clinical dispensing, good compliance as for decoction, and high effective constituent equivalence. In conclusion, effective constituent equivalence could improve relevance of methods of TCM dispensing control to clinical effect. Preparated Superior-standard Decoction Pieces based on effective constituent equivalence was featured by good quality and a good practice of adjustable dosage, which could promote the development of TCM decoction pieces toward precision medicine.

Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on HepG2 cells using the xCELLigence system.

AIM: To investigate the cytotoxic effects of the six protoberberine alkaloids (PAs) from Rhizoma Coptidis on HepG2 cells. METHOD: A systematic screening was conducted to investigate the dynamic response of HepG2 cells to the PAs using the impedance-based xCELLigence system. Cisplatin was selected as the positive control. The real time, concentration-response curves and the 50% inhibitory concentrations (IC50) were acquired to evaluate the anticancer activity of the PAs. RESULTS: All of the six PAs inhibited cell growth and induce death in HepG2 cells in a time- and concentration-dependent manner. The IC50 values of cisplatin, berberine, columbamine, coptisine, epiberberine, jatrorrhizine, and palmatine were 5.13, 42.33, 226.54, 36.90, 302.72, 383.54, and 456.96 µg·mL(-1), respectively. The results obtained using the xCELLigence system corresponded well with those of the conventional methods. CONCLUSION: The xCELLigence system is a reliable and efficient tool for real-time screening of the cytotoxic effect of compounds in cell-based in vitro assays. Coptisine and berberine, with methylenedioxy group at C2 and C3 on the phenyl ring showed stronger effect.than the other four PAs. However, compared with cisplatin, the six PAs didn't show obvious cytotoxic effect on HepG2 cells. These results provided some useful data for the evaluation of the anticancer compounds, and the clinical application of traditional Chinese medicine.

Comparison of reversed-phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis.

Radix isatidis is a famous anti-influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti-viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint-efficacy relationships between the fingerprints and the anti-viral activity were further investigated with principal component regression analysis. The results showed that the anti-viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.

A strategy for trade monitoring and substitution of the organs of threatened animals.

The use of threatened animals as a source of traditional medicines is accelerating the extinction of such species and imposes great challenges to animal conservation. In this study, we propose a feasible strategy for the conservation of threatened medicinal animals that combines trade monitoring and the search for substitutes. First, DNA barcoding provides a powerful technique for monitoring the trade of animal species, which helps in restricting the excessive use and illegal trade of such species. Second, pharmacological tests have been adopted to evaluate the biological equivalence of threatened and domestic animals; based on such testing, potential substitutes are recommended. Based on a review of threatened animal species and their substitutes, we find that the search for substitutes deserves special attention; however, this work is far from complete. These results may be of great value for the conservation of threatened animals and maintaining the heritage of traditional medicine.

[Chemical constituents from leaves of Rhododendron rubiginosum var rubiginosum].

Thirteen compounds were isolated from the leaves of Rhododendron rubiginosum var. rubiginosum by various chromatographic techniques. On the basis of spectroscopic data, their structures were elucidated as 3,9-dihydroxy-megastigma-5-ene (1), 3 beta-hydroxy-5alpha ,6 alpha-epoxy-7-megastigmen-9-one (2), loliolide (3), ursolic acid(4), 2 alpha, 3 beta-dihydroxy-urs-12-en-28-oic acid (5), 2 alpha, 3 beta,23-trihydroxy-urs-12-en-28-oic acid (6), 7,9-dimethoxyrhododendrol (7), 7-methoxyrhododendrol (8), zingerone (9), isofraxidin (10), scopoletin (11), (+)-pinoresinol (12) and 3'-O-demethylepipinorisenol (13). All compounds were isolated from this plant for the first time, and compounds 1-3, 7-9, and 11-13 were isolated from the genus Rhododendron for the first time.

Comparative analysis of gene expression profiles between cortex and thalamus in Chinese fatal familial insomnia patients.

Fatal familial insomnia (FFI) is a special subtype of genetic human prion diseases that is caused by the D178N mutation of the prion protein gene (PRNP). According to the surveillance data from 2006, FFI accounts for about half of all genetic prion disease cases in China. In this study, global expression patterns of the thalamus and parietal cortex from three patients with FFI were analyzed by Affymetrix Human Genome U133+ 2.0 chip. A total of 1,314 genes in the thalamus and 332 ones in the parietal lobe were determined to be differentially expressed genes (DEGs). The percentage of upregulated DEGs is much less in the thalamus (19.3 %) than that in the parietal lobe (42.8 %). Moreover, 255 of those DEGs showed the same altering tendencies in both tested regions, including 99 upregulated and 156 downregulated ones. The reliability of the results was confirmed by the real-time RT-PCR assays. There were 1,152 and 531 biological processes affected in the thalamus and the parietal lobe, respectively, as well as 391 overlapping ones in both regions. The most significantly changed molecular functions included transcription and DNA-dependent regulation of transcription, RNA splicing, mitochondrial electron transport, etc. The changed functions in the thalamus contained more numbers of DEGs than parietal lobe. According to KEGG classification, there were 167 and 115 different pathways changed in the thalamus and the parietal lobe, respectively, while 102 were changed in both. Interestingly, the top three changed pathways in the three groups mentioned above were Parkinson's disease, Alzheimer's disease, and oxidative phosphorylation. These results demonstrate the greater damage in the thalamus than in the parietal lobe during FFI pathogenesis, which is consistent with previous pathological observations. This study aims to describe the global expression profiles in various brain regions of FFI while proposing useful clues for understanding the pathogenesis of FFI and selecting potential biomarkers for diagnostic and therapeutic tools.

Effects of borneol on the pharmacokinetics of geniposide in cortex, hippocampus, hypothalamus and striatum of conscious rat by simultaneous brain microdialysis coupled with UPLC-MS.

It has been verified that borneol could promote the accumulation of other drugs in the whole brain. In this study, a microdialysis sampling system coupled with UPLC-MS was developed to evaluate the delivery of geniposide to four brain regions (cortex, hippocampus, hypothalamus and striatum) of conscious rats in the absence/presence of borneol: rats were administrated with geniposide alone (300mg/kg, iv) or administrated with both geniposide and borneol (0.2g/kg, ig). The dialysate collected from specific brain area was analyzed by a UPLC-MS system: separated on a BEH C18 column (50mm×2.1mm id, 1.7µm) within 1.5min, and detected in positive ion electrospray mode. The calibration curve was in good linearity over the concentration range of 0.009-90µg/mL. The inter- and intra-day accuracies were within ±10%, and the precisions were within 9.13%. The established method was applied to study the brain pharmacokinetics of geniposide and the results demonstrated that borneol markedly facilitated the delivery of geniposide to hippocampus and hypothalamus, but slightly hampered its delivery in cortex.

Comparison of the pathologic and pathogenic features in six different regions of postmortem brains of three patients with fatal familial insomnia.

Fatal familial insomnia (FFI) is an autosomal dominant prion disease clinically characterized by rapidly progressive insomnia, prominent autonomic alterations and behavioral disturbance. The D178N mutation of the prion protein gene (PRNP) on chromosome 20 in conjunction with methionine at codon 129 is a molecular feature. Although the neuropathological characteristics of FFI are well documented, the neuropathologic and pathogenic features of FFI patients remain poorly understood. Six brain regions of postmortem brains from 3 FFI patients were examined using immunohistochemistry, western blot analyses and quantitative real-time PCR. In all 3 brain specimens, reactive astrogliosis was found to be more severe in the thalamus than in the cortex regions. Western blot analyses showed that all three brains expressed PrP, but only 2 were associated with significantly weak proteinase K (PK) resistance. However, the conformational stabilities of PrPSc in the 3 FFI brains were significantly weaker than those presented in a G114V genetic Creutzfeldt-Jakob disease (gCJD) case. Immunohistochemistry and western blot analyses showed comparable amounts of neuron-specific enolase (NSE)-positive stained cells and NSE protein among the different regions in the three brains. In addition, the transcriptional levels of glial fibrillary acidic protein (GFAP) and NSE-specific mRNAs were coincident with the expression of these proteins. In conclusion, in the present study, we described the detailed regional neuropathology of FFI cases.

Terpenoids from Incarvillea arguta.

One new monoterpenoid, (+)-argutoid A (1), three new iridoids, ( - )-incarvoid A (2), (+)-incarvoid B (3), and incarvoid C (4), and five known compounds were isolated from Incarvillea arguta. Their structures were characterized by means of spectroscopic methods.

Sesquiterpenoids from Incarvillea arguta: absolute configuration and biological evaluation.

Diincarvilones A-D (1-4), incarvilone A (5), and a known compound, argutosine B (6), were isolated from Incarvillea arguta. The structures, including the absolute configurations of the new compounds, were determined by NMR spectroscopy, X-ray diffraction analysis, CD spectroscopy, and a variety of computational methods. The biological properties of these substances, including effects on intracellular Ca(2+) influx, nitric oxide (NO) production, and human cancer cells, were evaluated. The results showed that at the concentration of 10 µM (in HBSS buffer) diincarvilones A and B cause a persistent increase in cytoplasmic calcium levels in A549 cells.

Brachystemols A-C, three new furan derivatives from Brachystemma calycinum.

Three new furan derivatives, brachystemols A-C (1-3), and 13 known compounds (4-15) were isolated from the EtOH extract of Brachystemma calycinum. Their structures were identified by means of spectroscopic methods. Compounds 4-13 were isolated from this plant for the first time.

Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromatography-diode array detector-electrospray mass spectrometry.

An on-line high performance liquid chromatography (HPLC)-diode array detector (DAD)-electrospray ionization mass spectrometry (ESI-MS) method has been developed to quantify simultaneously eight bioactive chemical components in Houttuynia cordata Thunb and related Saururaceae medicinal plants. Simultaneous separation of these eight compounds was achieved on a C(18) analytical column with gradient elution of acetonitrile and 0.2% acetic acid (v/v) at a flow rate of 0.6 mL/min and being detected at 280 nm. These eight compounds were completely separated within 90 min. Good linear regression relationship (r(2)>0.9978) within test ranges was shown in all calibration curves. Good repeatabilty for the quantification of these eight compounds in H.cordata was also demonstrated in this method, with intra- and inter-day variations less than 3.0%. The method established was successfully applied to quantify eight bioactive compounds in closely related species of H.cordata, which provides a new basis for quality assessment of H.cordata.