Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry / 中国天然药物

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.

Toxic effects of aqueous extract of crotalariae assamicae semen in rats and possible mechanism in association with liver damage / 中国中药杂志

<p><b>OBJECTIVE</b>To study the toxic effects of aqueous extract of Crotalariae Assamicae Semen (CAS), one of the pyrrolizidine alkaloid-containing Chinese herbal medicines, in rats and the possible mechanism in association with liver damage.</p><p><b>METHOD</b>The aqueous extract of CAS (CASE) was prepared by the conventional water extracting-alcohol precipitating method. The LD50 value of CASE in rats was determined by Kärber method. Rats were randomly divided into four groups in which three groups were orally administered with different doses of the CASE and one group with distilled water as control. Toxic effects were assessed by morphological, biochemical and histopathological changes. Moreover, in vitro metabolism using rat liver microsomes was also conducted and applied for the exploration of the underlying mechanism of liver damage.</p><p><b>RESULT</b>The LD50 value of CASE in Wistar rats was (2.36 +/- 0.26) g x kg(-1). The toxic effects were found in all groups of rats dosed with CASE, in which serum levels of ALT and AST were significantly elevated, and the obvious and dose-dependent damages in liver and lung were observed by histopathological examination. Moreover, the liver tissue-bound pyrroles were detected and generated in a dose-dependent manner, and the pyrrole metabolites observed in the in vitro microsomal metabolism. All the evidences suggested a strong correlation between metabolism and toxicity of CASE in rats.</p><p><b>CONCLUSION</b>CASE could induce the acute toxicity in rats, of which liver and lung were the major targets. Toxic effects were strongly correlated with pyrrolizidine alkaloids in CAS. The possible mechanism for its liver toxicity may be related to the formation of pyrrole metabolites as well as the corresponding tissue-binding products.</p>

[Chemical constituents from rhizome of Daphne papyracea var. crassiuscula].

OBJECTIVE: To study chemical constituents from rhizome of Daphne papyracea var. crassiuscula. METHOD: Ethyl acetate fraction of 75% ethanol extracts from rhizome of D. papyracea var. crassiuscula, and its strucutre was identified by spectral method. RESULT: Nine compounds were separated and identified as daphneticin (1), daphnetin (2), hydrangetin (3), daphnoretin (4), 1-4'-hydroxyphenyl-5-phenyl-2 (E)-en-1-pentanone (5), daphneolon (6), 3beta-O-acetyl-olean-12-en (7), and (+)-usnic acid (8). CONCLUSION: Compounds 1-8 were separated from D. papyracea var. crassiuscula for the first time. Compound 8 was separated from the genus for first time.

Estrogen-like effects of Menoprogen on female ovariectomized rats / 中国中药杂志

<p><b>OBJECTIVE</b>To study the estrogen-like action mechanism of Menoprogen on ovariectomized female rats.</p><p><b>METHOD</b>Ovariectomized rat model (OVX) was established and estradiol (17beta-estradiol, E2) was used as positive control. The uterine coefficient and serum E2 level were determined after administration of Menoprogen for 2 weeks. The uterine vascular endothelial growth factor (VEGF), water channel protein (aquaporin, AQP), estrogen receptor (ER), progesterone receptor (PR) and the expression of proto-oncogenes (c-jun, c-fos) were observed by immunohistochemical method. Yeast two-hybrid assay was applied to detect the existence of components combining with ERalpha or ERbeta in Menoprogen.</p><p><b>RESULT</b>Both Menoprogen and E2 could significantly elevate the uterine coefficient of OVX rats, increase the level of serum E2 and up-regulate the expressions of VEGF, AQP2 as well as AQP5 in uterus. E2, not as E2 Menoprogen couldn't promote the expressions of ERalpha, PR, c-jun and c-fos in OVX rat uterus. And yeast two-hybrid assay showed no components combining with ERalpha or ERbeta in Menoprogen.</p><p><b>CONCLUSION</b>Menoprogen has estrogen-like effect, and can be used to treat menopause syndrome. The risk of estrogen-mediated endometrial cancer is low for this treatment because its mechanism is different from estrogen-like substances.</p>

Pyrrolizidine alkaloids-containing Chinese medicines in the Chinese pharmacopoeia and related safety concerns / 药学学报

It has been well-known that many medicinal plants used in traditional Chinese medicine contain hepatotoxic pyrrolizidine alkaloids (HPAs), and some even have been recorded in many editions of the Chinese Pharmacopoeia (ChP). In order to clarify the current status of these PAs-containing Chinese materia medica and proprietary Chinese formulae, the ChP 2010, the newest version, and the related safety issues were thoroughly investigated and analyzed on the current advances in research. Total nine crude drugs (not including the processed slices) were found to contain HPAs, which may be present in tens of Chinese proprietary drugs prepared with these crude drugs. Because of the lack of the alkaloid limitation in most monographs, their potential threats to human health may be underestimated. For this reason, attention should be drawn to the importance of the issue. The key point is to conduct the basic studies immediately on these PA-containing herbal plants or products, whose possible hazards need to be carefully assessed. Further efforts should also be made to elevate the criteria for quality control and ensure the drugs' safety in clinic for human health.

Pyrrolizidine alkaloids-containing Chinese medicines in the Chinese pharmacopoeia and related safety concerns / 药学学报

It has been well-known that many medicinal plants used in traditional Chinese medicine contain hepatotoxic pyrrolizidine alkaloids (HPAs), and some even have been recorded in many editions of the Chinese Pharmacopoeia (ChP). In order to clarify the current status of these PAs-containing Chinese materia medica and proprietary Chinese formulae, the ChP 2010, the newest version, and the related safety issues were thoroughly investigated and analyzed on the current advances in research. Total nine crude drugs (not including the processed slices) were found to contain HPAs, which may be present in tens of Chinese proprietary drugs prepared with these crude drugs. Because of the lack of the alkaloid limitation in most monographs, their potential threats to human health may be underestimated. For this reason, attention should be drawn to the importance of the issue. The key point is to conduct the basic studies immediately on these PA-containing herbal plants or products, whose possible hazards need to be carefully assessed. Further efforts should also be made to elevate the criteria for quality control and ensure the drugs' safety in clinic for human health.

HCV protease inhibitory, cytotoxic and apoptosis-inducing effects of oleanolic acid derivatives.

PURPOSE: To evaluate oleanolic acid derivatives on liver disease related bioactivities, 29 oleanolic acid derivatives of several series were tested for their inhibitory activity on hepatitis C viral protease and for their cytotoxic effects on Hep G2 cells. RESULTS: The amino derivatives showed potent cytotoxicity, among which, the beta-amino isomer exhibited more distinct cytotoxicity than the alpha-isomer. The cytotoxicity of hemiesters and hemiamides varied as the chain lengths varied. The oxalic and malonic hemiesters showed weaker cytototoxicity than oleanolic acid, while those with longer chain lengths showed higher cytotoxicity. Contrary to the cytotoxic activity, the free amino derivatives showed little inhibitory activity on HCV protease. All the hemiesters and hemiamides showed high activity against HCV protease. CONCLUSION: The findings that addition of amino-group enhanced the cytotoxicity and that introduction of acidic group increased the inhibition on HCV protease may be useful for further design and synthesis of triterpene derivatives as drug candidates for liver diseases.

Study on the metabolism of the precipitation of Xiexin decoction in rats I / 中国中药杂志

<p><b>OBJECTIVE</b>To study the process in vivo of the components in the precipitation of Xiexin decoction.</p><p><b>METHOD</b>The components in the rats' urine samples after oral administration of baicalin, single herb decoction of Radix Scutellariae or the precipitation of Xiexin decoction were analyzed by LC-MS-MS method; and the absorption of baicalin in different samples were calculated by intestinal in situ loop method.</p><p><b>RESULT</b>The urinary excretion amounts of baicalin in three samples were not obviously different, whereas the time reach elimination-peak of baicalin in them had significant difference. The absorption of baicalin in the precipitation was obviously greater than that in the single herb decoction and single baicalin. We found that wogonoside and 7-O-glucuronide chrysin were the metabolites presenting in the rat urine samples after oral administration of baicalin by LC-MS-MS, which had never been reported.</p><p><b>CONCLUSION</b>The resident time in the body of baicalin in the precipitation is prolonged, compared with that in the single herb decoction and single baicalin. The metabolites may be the potential biological components in vivo of baicalin in the precipitation.</p>

Determination of trigonelline by HPLC and study on its pharmacokinetics / 药学学报

<p><b>AIM</b>To develop a sensitive and specific HPLC method for determination of trigonelline in rabbit plasma, and study the pharmacokinetics in rabbit.</p><p><b>METHODS</b>After ig of fenugreek extract and i.v. of trigonelline in rabbit, the biological samples could be well purified after precipitation of protein with methanol and acetonitrile. Asahipak NH2P-50 column was used, the mobile phase consisted of acetonitrile-water (90:10) at a flow-rate of 1.2 mL.min-1, and detection wavelength was set at UV 265 nm. The column temperature is 30 degrees C.</p><p><b>RESULTS</b>The calibration curve was linear in the range from 0.98 mg.L-1 to 31.28 mg.L-1, with r = 0.9986, the detection limit of this method was 50 micrograms.L-1. The concentration-time curves of trigonelline in rabbits after ig and i.v. administration were shown to fit one-compartment and two-compartment open model, respectively. The main parameters after ig of fenugreek extract were as follow: T1/2(Ka) was 0.9 h, T1/2(Ke) was 2.2 h, V was 0.64 L.kg-1, AUC was 1.93 mg.min.L-1. The main parameters after i.v. of trigonelline were as follows: T1/2 alpha was 10.8 min, T1/2 beta was 44.0 min, K21 was 0.044 min-1, K10 was 0.026 min-1, K12 was 0.017 min-1, AUC was 931.0 mg.min.L-1.</p><p><b>CONCLUSION</b>Trigonelline showed a middle rate of absorption and fast rate of elimination in rabbit. Meanwhile, the method is simple, accurate, with a good reproducibility, and it provide a basic method for the investigation of trigonelline and fenugreek pharmacokinetics.</p>

[Determination of trigonelline in Trigonella foenum-graecum by HPLC].

OBJECTIVE: A HPLC method is established to determine the content of trigonelline in Trigonella foenum-graecum. METHOD: The medicinal material was extracted by petholeum ether-ethanol. Asahipak NH2P-50 column was used, mobilephase consisted of acetonitrile-water(75:25) and detection wavelength was set at UV 265 nm. RESULT: The standard curve was linear in the range of 3.68-73.60 micrograms.mL-1 with the correlation coefficient of 0.9999. The average recovery rate and RSD were 97.4% and 1.83% (n = 6) respectively. CONCLUSION: It provides scientific indexes for quality control of T. foenum-graecum.

Studies on the Chemical Constituents of Japanese Buckeye Seed (Aesculus turbinata)Part Ⅱ * Isolation and Identification of Escin IVc and Isoescins Ia, Ib / 中草药

Three triterpenoid saponins were isolated from 90％ ethanolic extract of the seeds of Aes-culus turbinata Bl.. Their structures were identified on the basis of spectral data as escin IVc:22α-O-tigloyl-28-O-acetylprotoaescigenin 33-O-[β-D-glucopyranosyl (1→2)][β-D-glucopyranosyl (1→4)]-β-D-glucopyranosiduronic acid (Ⅰ), isoescin Ia: 21β-O-tigloyl-28-O-acetylprotoaescigenin-3β-O-[β-D-glucopy-ranosyl (1→2)][β-D-glucopyranosyl (1→4)]-β-D-glucopyranosiduronic acid (Ⅳ), and isoescin Ib:21β-O-angeloyl-28-O-acetylprotoaescigenin-3β-O-[β-D-glucopyranosyl (1→2)][β-D-glucopyr anosyl (1→4)]-β-D-glucopyranosiduronic acid (Ⅴ). Escin IVc and isoescins Ia, Ib were obtained for the first time from thisplant. Escins Ia, Ib, IVc and isoescins Ia, Ib had inhibitory effect against recombinant HIV-1 protease atconcentration of 100 μmol/L in vitro.