An alternative and simplified approach to identification and test for minimum content of TCM herbal drugs.

Following a decision of the European Pharmacopoeia (Ph. Eur.) Commission, the Traditional Chinese Medicines (TCM) Working Party started a pilot phase to examine the suitability of a high-performance thin-layer chromatography (HPTLC) minimum content test as an alternative to the classical assay in TCM monographs. This approach was evaluated with two TCM herbal drugs: Fritillaria thunbergii bulbs (FTB) and Corydalis rhizome (CYR). Firstly, the existing HPTLC methods were optimised for both drugs. The new methods were applied to the evaluation of multiple samples, and acceptance criteria for the identification, following Ph. Eur. chapter 2.8.25. High-performance thin-layer chromatography of herbal drugs and herbal drug preparations, were set. The HPTLC test for minimum content of markers was then developed and validated. In this test, the intensity of the marker zone in the fingerprint of the sample is compared to the corresponding zone in the reference solution, which has a concentration giving an intensity equivalent to the acceptance criterion. This test gives a pass or fail result rather than a content and can be performed visually (on the images) or by software (using peak profiles from images; PPI). Reproducibility of the HPTLC methods was evaluated in a collaborative trial including six laboratories. In summary, results for FTB from five laboratories were in agreement. The remaining laboratory did not pass the identification of the samples. For CYR, all laboratories presented the same results for identification. In the test for minimum content, one borderline sample passed in four laboratories and failed in two. All laboratories reached similar conclusions for the other seven samples. The HPTLC methods proposed offer a simplified approach to evaluating identity and minimum content of TCM drugs in a single analysis.

Tripterygium glycoside fraction n2: Alleviation of DSS-induced colitis by modulating immune homeostasis in mice.

BACKGROUND: The Tripterygium glycosides (TG) is the main active extractive of Tripterygium wilfordii Hook F and is widely used in clinical practice to treat inflammatory diseases (including inflammatory bowel disease). However, due to its severe toxicity, TG is restricted to the treatment of many diseases. Therefore, it is necessary to study a new method to obtain the attenuated and synergistic extracts from TG. PURPOSE: Tripterygium glycosides-n2 (TG-n2) was obtained from TG by a new preparation method. In this study, we aimed to investigate the difference in the chemical compositions between TG and TG-n2, further explored its toxicity and therapeutic effects on DSS-induced colitis in mice. METHODS: The major chemical compositions of TG and TG-n2 were analyzed by ultra-performance liquid chromatography (UPLC). Subsequently, acute toxicity test was applied to evaluate the toxicity difference between TG and TG-n2. Dextran sulfate sodium (DSS)-induced acute colitis model was used to explore the therapeutic effect of TG and TG-n2 and their potential mechanisms of action. RESULTS: We found that the chemical compositions of TG-n2 is different from TG. The main difference is the ratio of triptriolide (T11) / triptolide (T9). Acute toxicity test proved that TG-n2 was less toxic than TG. Base on this, further studies showed that TG-n2 has a similar therapeutic effect as compared to TG on attenuating the symptoms of colitis, such as diarrhea, bloody stools, body weight loss, colonic atrophy, histopathological changes, inhibiting cytokines secretion and reducing absolute lymph number. In addition, TG and TG-n2 can increase the apoptosis of T lymphocyte in vivo. Further investigated showed that TG and TG-n2 could increase the expressions of Bax and p62 on CD3-positive T cells. CONCLUSION: This study showed that oral administration of TG-n2 is safer than TG. Moreover, the attenuated TG-n2 has the similar therapeutic effect on treating experimental colitis in mice when compared to TG. Its mechanism may be related to activating the expression of Bax in T cells and inducing T cells autophagy to regulate the survival of T lymphocytes in colitis mice, thus reducing inflammation in colon.

Mechanism and structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase from Saccharomyces cerevisiae.

The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)-adenosyl-L-homocysteine, a series of 35 sterol substrates as acceptor molecules and a series of 10 substrate and high energy intermediate (HEI) sterol analogues as inhibitors of biomethylation. The SMT was found to be selective for sterol, both regio- and stereochemically. The presence of an unhindered 24,25-bond, an equatorially-oriented polar group at C-3 (which must act as a proton acceptor) attached to a planar nucleus and a freely rotating side chain were obligatory structural features for sterol binding/catalysis; no essential requirement or significant harmful effects could be found for the introduction of an 8(9)-bond, 14 alpha-methyl or 9 beta,19-cyclopropyl group. Alternatively, methyl groups at C-4 prevented productive sterol binding to the SMT. Initial velocity, product inhibition, and dead-end experiments demonstrated a rapid-equilibrium random bi bi mechanism. Deuterium isotope effects developed from SMT assays containing mixtures of [3-3H]zymosterol with AdoMet or [methyl-2H3]AdoMet confirmed the operation of a random mechanism, kappa H/kappa D = 1.3. From this combination of results, the spatial relationship of the sterol substrate to AdoMet could be approximated and the topology of the sterol binding to the SMT thereby formulated.

Antifungal sterol biosynthesis inhibitors.

During the course of the last decade, the development of SBIs, and particularly sterol biomethylation inhibitors, has been based on the rational design approach. Successful though this approach has been in elucidating sterol biomethylation enzymology, its limitations are becoming apparent from the findings that: (i) 24,25-double bond metabolism gives rise to cholesterol and ergosterol in a mechanistically similar manner, (ii) 25-azasterols are harmful to human physiology, and (iii) side-chain modified sterols designed to inhibit the SMT enzyme in S. cerevisiae may be ineffective or operate by another kinetic mechanism in a related organism, rendering it therapeutically nonuseful. Nevertheless, it may be possible to ultimately capitalize on the unique aspects of sterol biomethylation chemistry and enzymology to design taxa-specific inhibitors. With increased understanding of the structure and function of SMT enzymes in different fungi, it should be possible to prepare novel mechanism-based inactivators to control SMT activity uniquely and with high specific activity.

Developmental regulation of sterol biosynthesis in Zea mays.

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods from Zea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-(14)C]mevalonic acid, [2-(14)C]acetate, and [2-(3)H]acetate), and stable isotopes (D2O), we discovered that hydroxymethylgutaryl CoA reductase is not "the" rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24 alpha-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an active de novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3 beta-ol. Examination of the mass spectrum and 1H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed.

Sterol specificity of the Saccharomyces cerevisiae ERG6 gene product expressed in Escherichia coli.

The ERG6 gene from Saccharomyces cerevisiae has been functionally expressed in Escherichia coli, for the first time, yielding a protein that catalyzes the bisubstrate transfer reaction whereby the reactive methyl group from (S)-adenosyl-L-methionine is transferred stereoselectively to C-24 of the sterol side chain. The structural requirements of sterol in binding and catalysis were similar to the native protein from S. cerevisiae. Inhibition of biomethylation was observed with fecosterol and ergosterol which suggests that ergosterol may function in wild-type yeast as feedback regulator of sterol biosynthesis.

Dynamic CT scan of intracranial tumors.

Of ninety-eight patients with intracranial tumors examined by dynamic CT scanning, 66 were confirmed operatively and pathologically. Our study showed that: 1) Dynamic CT can be performed safely and easily in outpatients; 2) Acoustic neurinoma, meningioma, pituitary chromophobe adenoma, astrocytoma and metastatic tumor have varying levels of time-density curves; 3) The tissue-blood ratio (TBR) at peak time (TBRp) is a useful indicator of the vascularity of tumoral tissues, and analysis of the time-density curve combining with TBRp is helpful to the differential diagnosis of tumors; 4) Differential diagnosis between tumors and vascular abnormalities such as aneurysm and arteriovenous malformation could be made easily with dynamic CT.

[Phytoecdysteroids of Rhaponticum uniflorum root].

Three phytoecdysteroids I, II and III were isolated from the root of Rhaponticum uniflorum (L.) DC. and their structures were elucidated by chemical and spectroscopic methods (UV, IR, EI-MS, FAB-MS, 1HNMR, 13CNMR, 1H-1H COSY, 1H-1H NOESY, 1H-13CCOSY and CD). Compound II is new and named rhapontisterone, its structure was confirmed as (20R, 22R, 24S)-2 beta, 3 beta, 11 alpha, 14 alpha, 20, 22, 24-heptahydroxy-5 beta cholest-7-en-6-one. The other two, I and III, were identified as ecdysterone and turkesterone, respectively, both are known compounds, but turkesterone was isolated for the first time from the title plant.

[Studies on liposoluble constituents from the aerial parts of Siegesbeckia orientalis L].

Eight compounds were isolated from the aerial parts of Siegesbeckia orientalis L. and their structures were elucidated by spectroscopic methods(IR, EI-MS, 13C-NMR, 1H-NMR, 1H-1H COSY, 1H-1H NOESY and 1H-13C COSY). Compounds I and II are new natural products and named siegesesteric acid(I) and siegesetheric acid(II), their structures were confirmed as ent-17-acetoxy-18-isobutyryloxy-16(alpha)-kauran-19-oic acid(I) and ent-17-ethoxy-16(alpha)-(-)-kauran-19-oic acid(II). The others were identified as known compounds: ent-16 beta, 17-dihydroxy-kauran-19-oic acid (III), kirenol(IV), beta-sitosteryl glucoside(V), heneicosanol(VI), methyl arachidate(VII) and beta-sitosterol(VIII). These compounds, except kirenol and beta-sitosterol, were isolated for the first time from the title plant.

[Antifungal effect of three natural products on the genetic substance of Saccharomyces cerevisiae GL7 and Prototheca wickerhamii].

AIM: To investigate the antifungal effect of three natural products on the genetic substance of Saccharomyces cerevisiae GL7 and Prototheca wickerhamii. METHODS: The normal and treated cells were observed by confocal laser scanning microscope (CLSM) and image analysis to quantitatively described the cell morphology, area, DNA and RNA content. RESULTS: The morphology, area, DNA and RNA contents were changed greatly in the treated cells. CONCLUSION: Solasodine, 4'-hydroxy-3, 5-dimethoxystilbene and dictamnine directly or indirectly interfered the synthesis and function of genetic substance in S. cerevisiae and P. wickerhamii.

[Chemical components of volatile oil from Echinops grijisii Hance].

The volatile oil obtained from Echinops grijisii roots was analysed by GC-MS method. Twenty four constituents have been identified from the oil, among which cis-beta-farnesene and 5-(3-buten-1-ynyl)bithiophene are the main ones.

[Non-anthraquinone constituents from Rheum sublanceolatum C. Y. Cheng et Kao].

OBJECTIVE: Study on the non-anthraquinone constituents from rhizoma and radix of Rheum sublanceolatum. METHOD: The constituents were isolated through column chromatography and identified on the basis of their physiochemical and spectral data. RESULT: Six non-anthraquinone constituents were isolated and identified as n-octacosanic acid, sitosterol, daucosterol, 2-methyl-5-carboxymethyl-7-hydroxychromone, piceatannol and 6-hydroxymusizin-8-O-beta-D-glucopyranoside. CONCLUSION: All these compounds were firstly isolated from R. sublanceolatum.

Effects of triterpenes from Ganoderma lucidum on protein expression profile of HeLa cells.

To elucidate the cytotoxicity mechanism of Ganoderma triterpenes, a chemoproteomic study using five purified ganoderic acids, ganoderic acid F (GAF), ganoderic acid K (GAK), ganoderic B (GAB), ganoderic acid D (GAD) and ganoderic acid AM1 (GAAM1) was conducted. GAF, GAK, GAB, GAD and GAAM1 treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with IC(50) values of 19.5+/-0.6 microM, 15.1+/-0.5 microM, 20.3+/-0.4 microM, 17.3+/-0.3 microM, 19.8+/-0.7 microM, respectively. The protein expression profiles of HeLa cells treated with each ganoderic acid at dose of 15 microM for 48 h were checked using two-dimensional electrophoresis (2-DE). The possible target-related proteins of ganoderic acids, i.e. proteins with same change tendency in all five ganoderic acids-treated groups compared with control, were identified using MALDI-TOF MS/MS. Twelve proteins including human interleukin-17E, eukaryotic translation initiation factor 5A (eIF5A), peroxiredoxin 2, ubiquilin 2, Cu/Zn-superoxide dismutase, 14-3-3 beta/alpha, TPM4-ALK fusion oncoprotein type 2, PP2A subunit A PR65-alpha isoform, nucleobindin-1, heterogeneous nuclear ribonucleoprotein K, reticulocalbin 1 and chain A of DJ-1 protein were identified. Ganoderic acids might exert their cytotoxicity by altering proteins involved in cell proliferation and/or cell death, carcinogenosis, oxidative stress, calcium signaling and ER stress.

Microbial transformation of dehydrocostuslactone by Mucor polymorphosporus.

Biotransformation of dehydrocostuslactone (1) by Mucor polymorphosporus yielded four compounds, and their structures were identified as 11alpha,13-dihydrodehydrocostuslactone (2), 3alpha-hydroxy-11alpha,13-dihydrodehydrocostuslactone (3), 3beta-hydroxy-4beta,15,11alpha,13-tetrahydrodehydrocostuslactone (4) and 2beta-hydroxyl-11alpha,13-dihydrodehydrocostuslactone (5), respectively, on the basis of their spectral data. Among them, compound 5 is a new compound.

A triterpenoid saponin from Albizia julibrissin.

A triterpenoid saponin (1) was obtained from the stem barks of Albizia julibrissin Durazz. Its structure was elucidated as 3-O-[beta-D-xylopyranosyl-(1 --> 2)-alpha-L-arabinopyranosyl-(1 --> 6)-beta-D-glucopyranosyl]-21-O-[(6S)-2-trans-2-hydroxymethyl-6-methyl-6-O-beta-D-quinovopyranosyl-2, 7-octadienoyl]-16-deoxy-acacic acid 28-O-beta-D-glucopyranosyl-(1 --> 3)-[alpha-L-arabinofuranosyl-(1 --> 4)]-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl ester (1), named as Julibroside J26, based on the chemical and spectral methods.

Biotransformation of 24 alpha-methylcholesterol and 24 beta-methylcholesterol by yeast mutant GL7.

Incubation of 24 alpha- and 24 beta-methylcholesterols with yeast mutant GL7 afforded their corresponding C-22-desaturated products under the catalysis of sterol delta 22(23)-desaturase. The metabolites were identified to be 22-dehydro-24 alpha-methylcholesterol (2% yield from 24 alpha-methylcholesterol) and 22-dehydro-24 beta-methylcholesterol (51% yield from 24 beta-methylcholesterol) respectively on the basis of their chromatographic and spectral properties. It was concluded that the sterol delta 22(23)-desaturase prefers the 24 beta-methyl sterols and is highly stereospecific.

Biotransformation of podophyllotoxin to picropodophyllin by microbes.

Biotransformation of podophyllotoxin (PT) by several microbial species has been investigated. Among the fungi tested, it was found that Penicillium strains can isomerize PT to picropodophyllin (PPT) in 8% yield and other strains also transform the substrate into the same product but with lower yield.

A new phenolic glycoside and a new trans-clerodane diterpene from Conyza blinii.

A new phenolic glycoside, 4-propionyl-2,6-dimethoxyphenyl beta-D-glucopyranoside (1) and a new trans-clerodane diterpene named 19-deacetylconyzalactone (2), were isolated from the aerial parts of Conyza blinii.

Antiproliferative activity of the extract of Gleditsia sinensis fruit on human solid tumour cell lines.

BACKGROUND: The fruit extract of Gleditsia sinensis Lam. (GSE) is a traditional herbal medicine that is saponin-rich. However, its activity on solid tumour cell lines has never been demonstrated. METHODS: The activity of GSE was demonstrated in four cancer cell lines (breast cancer MCF-7, MDA-MB231, hepatoblastoma HepG2 and oesophageal squamous carcinoma cell line SLMT-1) using MTT assay, anchorage-independent clonogenicity assay, DNA laddering and in situ cell death detection. RESULTS: The mean MTT(50) (the mean concentration of GSE to reduce MTT activity by 50%) ranged from 16 to 20 microg/ml of GSE. An anchorage-independent clonogenicity assay showed that all of the four solid tumour cell lines gradually lost their regeneration potential after treatment with GSE, DNA fragmentation and TUNEL analysis demonstrated that the action of GSE is both dose- and time course-dependent. CONCLUSIONS: Our results suggest that GSE has a cytotoxic activity and can induce apoptosis in human solid tumour cell lines.