Coronary artery calcium and cystatin C for risk stratification of MACCEs and all-cause death in symptomatic patients.

OBJECTIVES: The aim of this study was to examine the independent and joint associations of baseline coronary artery calcium score (CACS) and cystatin C (Cys-C) with the risk of major adverse cardiac and cerebrovascular events (MACCEs) and all-cause death in symptomatic populations. METHODS: The study included 7140 patients with symptom of chest pain who underwent cardiac computerized tomography examinations to measure CACS. All of them had serum Cys-C results. Endpoints were set for MACCEs and all-cause death events. RESULTS: A total of 7140 participants were followed for a median of 1106 days. A total of 305 patients had experienced MACCEs and 191 patients had experienced all-cause death. CACS ≥ 100 and Cys-C ≥ 0.995 mg/L were independently associated with an increased risk of MACCEs (adjusted hazard ratio [HR]: 1.46; 95% confidence interval [CI]: 1.15-1.85; p = .002 and adjusted HR: 1.57; 95% CI: 1.24-2.00; p < .001, respectively). Compared with CACS < 100 and Cys-C < 0.995 mg/L patients, CACS ≥ 100 and Cys-C ≥ 0.995 mg/L patients had the highest risk of MACCEs and all-cause death (adjusted HR: 2.33; 95% CI: 1.64-3.29; p < .001 and adjusted HR: 2.85; 95% CI: 1.79-4.55; p < .001, respectively). Even in patients with CACS < 100, Cys-C ≥ 0.995 mg/L was also associated with a higher risk of MACCEs and all-cause death than Cys-C < 0.995 mg/L (adjusted HR: 1.76; p = .003 and adjusted HR: 2.02; p = .007, respectively). CONCLUSIONS: The combined stratification of CACS and Cys-C showed an incremental risk of MACCEs and all-cause death, reflecting complementary prognostic value. Our results support the combination of the two indicators for risk stratification and event prediction.

The efficacy and safety of self-administered acupressure on respiratory tract infection in chronic kidney disease: a randomized controlled trial.

Background: Respiratory tract infection (RTI) is associated with a higher risk of kidney failure in patients with chronic kidney disease (CKD), without effective precautions. Self-administered acupressure (SAA) has been shown to potentially prevent RTI, but still lack of clinical evidence in CKD. The present randomized controlled trial assessed the efficacy and safety of SAA in preventing RTI recurrence in patients with CKD. Methods: Participants with CKD who had been diagnosed with RTI on more than 2 occasions in the preceding 12 months were enrolled between November 6, 2017, and August, 6, 2018. They were randomly assigned (1:1) to receive daily SAA combined with usual care (intervention) or usual care alone (control) for 24 months. The primary outcome was time to first RTI. Secondary outcomes were RTI rate, kidney function, proteinuria and serum immune indicators, detected by the clinical laboratory in the hospital. The study would be discontinued if the participant met the criteria of stopping the study. Kaplan-Meier method and multivariable Cox proportional hazards regression were used to compare the primary outcome between the two groups. Results: Among the 540 patients screened, 114 participants were randomly assigned to the intervention group (n=57) or the control group (n=57). The median follow-up duration was 24.4 months. Compared with controls, participants in the intervention group did not have a significantly lower risk of RTI according to Kaplan-Meier analysis, but did have a significantly lower risk of RTI according to competing risk analysis (HR 0.65, 95% CI: 0.42-1.00; P=0.05), when considering endpoint (dialysis or death) and loss to follow-up as competing risks, and had a significantly lower rate of RTI [1.65 vs. 2.19 episodes per patient-year, respectively; incidence rate ratio (IRR) 0.75, 95% CI: 0.62-0.92; P=0.006]. Apart from lower study serum IgG levels in the intervention group at 24 months (mean difference 0.68 g/L; 95% CI: 0.07-1.29; P=0.029), all other secondary outcomes and overall adverse events were comparable between the 2 groups. Conclusions: SAA is a promising effective and safe therapy for preventing RTI in patients with CKD. However, the efficacy of SAA in children and adolescents still needs further study. Trial Registration: Chinese Clinical Trials Registry identifier: ChiCTR-IOR-17012654.

Combining ultra-high-performance liquid chromatography quadruple exactive orbitrap mass spectrometry with chemometrics to identify and verify the blood-activating components of hawthorn.

Hawthorn, one of the widely-used Chinese herbal medicines, has been used to treat blood stasis syndrome in the clinic, but its blood-activating components are unclear. This study combined the ultra-high-performance liquid chromatography-quadruple exactive-orbitrap mass spectrometry with chemometrics to identify the blood-activating components of hawthorn. Different polar fractions of hawthorn aqueous extracts were extracted and mixed to prepare 14 samples. The contents of 25 chemical components for 14 samples were determined by the proposed quantitative method which was validated in terms of linearity, precision, stability, repeatability, and recovery, while the blood-activating effect was evaluated by measuring the whole blood viscosity, plasma viscosity, and plasma fibrinogen levels. Then the partial least squares model was established on the spectrum-effect relationship. The result showed that vitexin-2″-O-rhamnoside, rutin, citric acid, malic acid, gallic acid, and fumaric acid could reduce the whole blood viscosity, plasma viscosity, and plasma fibrinogen levels in blood stasis model rats, and these components were the blood-activating components of hawthorn. This study provided a scientific basis for clarifying the blood-activating components of hawthorn, and the spectrum-effect approach proved to be an effective approach to discovering the bioactive components of Chinese herbal medicines.

Developing the liquid chromatography-mass spectrometry method for simultaneously quantifying five components in rat serums after oral administration of hawthorn aqueous extracts and its application to a pharmacokinetic study.

Hawthorn, one of the widely-used traditional Chinese medicines, has been used to treat dyspepsia, hyperlipidemia, and cardiovascular disease in the clinic. Our previous study revealed that gallic acid, neochlorogenic acid, cryptochlorogenic acid, vitexin, and quercetin were active components of hawthorn. In this study, a simple, precise, and reliable liquid chromatography-mass spectrometry method was developed for the simultaneous quantification of five components in rat serums. The separation was achieved on the Hypersil GOLD C18 column, and the mobile phases consisted of 0.1% acetic acid water and methanol at a flow rate of 0.3 mL/min. The mass spectrometry data acquisition was performed on Q-Extractive-Orbitrap mass spectrometry with an electrospray ionization source in negative ion mode. The proposed liquid chromatography-mass spectrometry method was validated in terms of linearity, intra- and inter-precision, accuracy, recoveries, matrix effects, and stability. Then this newly proposed liquid chromatography-mass spectrometry method was successfully applied to a pharmacokinetic study on rats after oral administration of hawthorn aqueous extracts. This study provided relevant information on the pharmacokinetics of active components of hawthorn and explained the underlying mechanism of their bioactivity.

Therapeutic effects of different polar fractions of hawthorn extract on blood stasis model rats revealed by liquid chromatography-mass spectrometry metabolomics.

Hawthorn, a commonly used traditional Chinese medicine, has been suggested to have therapeutic effects on cardiovascular disease. However, effective fractions of hawthorn extract in the treatment of cardiovascular disease, together with possible therapeutic mechanisms, remain unclear. This study aimed to investigate the effects of four different polar fractions of hawthorn extract on blood stasis model rats, and explore the possible metabolic mechanisms by using a liquid chromatography-mass spectrometry metabolomics approach. Evaluation of hemorheology and fibrinogen showed that n-butanol and ethyl acetate fractions of hawthorn extract had significant therapeutic effects on blood stasis model rats. Furthermore, metabolomics analysis showed that n-butanol and ethyl acetate fractions of hawthorn extract could reverse imbalanced biomarkers in plasma and urine of blood stasis model rats. Additionally, metabolic pathway analysis revealed that plasma biomarkers were responsible for several important pathways, including d-glutamine and d-glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, alanine, aspartate, and glutamate metabolism, sphingolipid metabolism, and arginine biosynthesis. Meanwhile, urine biomarkers were responsible for some important pathways, including phenylalanine metabolism, tyrosine metabolism, and lysine degradation. This study demonstrated that n-butanol and ethyl acetate fractions of hawthorn extract had significant therapeutic effects on blood stasis model rats, and the underlying mechanisms involved multiple metabolic pathways.

LC-MS based plasma metabolomics study of the intervention effect of different polar parts of Hawthorn on hyperlipidemia rats.

Hawthorn, a well-known traditional Chinese medicine is used for the treatment of dyspepsia syndrome, cardiovascular disease, and hyperlipidemia. Hawthorn has complex composition. However, the effective fraction and mechanisms of action in alleviating hyperlipidemia are unknown. Therefore, the aim of this study was to evaluate the effects of four different polar components of hawthorn on hyperlipidemia rats, and to explore underlying mechanisms of action through liquid chromatography-mass spectrometry based plasma metabolomics. Hyperlipidemia rat model was established by feeding rats using a high-fat diet. High-fat model rats were then treated with four polar components of hawthorn for 14 consecutive days. Plasma samples were collected and subjected to biochemical and metabolomics analysis. Biochemical analysis showed that hawthorn n-butanol and ethyl acetate extracts had the highest efficacy on hyperlipidemia rats. Water fraction showed a partial effect, whereas petroleum ether extract was not effective against hyperlipidemia rats. Furthermore, liquid chromatography-mass spectrometry metabolomics analysis showed that the most effective fraction of hawthorn reversed the metabolic disorder in plasma of hyperlipidemia rats. Metabolomics analysis showed that hawthorn exerts its activity by modulating lipid metabolism, energy metabolism, oxidative stress, and amino acid metabolism.

Astragalus membranaceus (Huang Qi) as adjunctive therapy for diabetic kidney disease: An updated systematic review and meta-analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Historical literature and pharmacological studies both suggest that Astragalus membranaceus (A. membranaceus) holds potential benefits for diabetic kidney disease (DKD), which is a growing burden with limited proven renal protective options. AIM OF THE STUDY: An updated systematic review was conducted to evaluate the efficacy and safety of A. membranaceus preparations as an adjunctive therapy to conventional therapies for DKD. METHODS: Nine databases and five clinical trial registries were searched for randomized controlled trials (RCTs) of A. membranaceus preparations added to conventional therapies compared with conventional therapies alone for people with DKD. Study screening, data collection, and quality assessment were performed independently by two reviewers. Estimated effects were pooled as mean differences or standardized mean differences with 95% confidence intervals by using a random-effects model. RESULTS: Sixty-six studies, involving 4785 DKD participants, were included. The quality of the included studies was low due to methodological shortfalls. Meta-analysis showed that additional use of A. membranaceus injection reduced more albuminuria (32 RCTs, 2253 participants; SMD: 2.05 [-2.49, -1.61], I2 = 94%), proteinuria (26 RCTs, 1812 participants; SMD: 1.85 [-2.34, -1.37], I2 = 95%), and serum creatinine levels (32 RCTs, 2880 participants; -14.78 µmol/L [-19.22, -10.33], I2 = 97%) than conventional therapies alone did. An anti-albuminuria effect was also observed in the oral A. membranaceus preparation group (four RCTs, 236 participants; SMD: 1.27 [-1.82, -0.73], I2 = 73%). Meta-regression suggested that the treatment effect of A. membranaceus injection was associated with the baseline serum creatinine level. The adverse-events profile was similar between the additional A. membranaceus and control groups. CONCLUSION: The low quality of evidence suggested that adjunctive use of A. membranaceus preparations in addition to conventional therapies may be effective and tolerated for short-term reduction of albuminuria, proteinuria, and serum creatinine in DKD patients. The findings should be considered with caution due to the lack of high-quality RCTs and significant heterogeneity and publication bias. Further RCTs are needed to confirm the long-term efficacy and safety of A. membranaceus preparations, especially of the oral form, in patient-important outcomes.

An automatic approach for constructing a knowledge base of symptoms in Chinese.

BACKGROUND: While a large number of well-known knowledge bases (KBs) in life science have been published as Linked Open Data, there are few KBs in Chinese. However, KBs in Chinese are necessary when we want to automatically process and analyze electronic medical records (EMRs) in Chinese. Of all, the symptom KB in Chinese is the most seriously in need, since symptoms are the starting point of clinical diagnosis. RESULTS: We publish a public KB of symptoms in Chinese, including symptoms, departments, diseases, medicines, and examinations as well as relations between symptoms and the above related entities. To the best of our knowledge, there is no such KB focusing on symptoms in Chinese, and the KB is an important supplement to existing medical resources. Our KB is constructed by fusing data automatically extracted from eight mainstream healthcare websites, three Chinese encyclopedia sites, and symptoms extracted from a larger number of EMRs as supplements. METHODS: Firstly, we design data schema manually by reference to the Unified Medical Language System (UMLS). Secondly, we extract entities from eight mainstream healthcare websites, which are fed as seeds to train a multi-class classifier and classify entities from encyclopedia sites and train a Conditional Random Field (CRF) model to extract symptoms from EMRs. Thirdly, we fuse data to solve the large-scale duplication between different data sources according to entity type alignment, entity mapping, and attribute mapping. Finally, we link our KB to UMLS to investigate similarities and differences between symptoms in Chinese and English. CONCLUSIONS: As a result, the KB has more than 26,000 distinct symptoms in Chinese including 3968 symptoms in traditional Chinese medicine and 1029 synonym pairs for symptoms. The KB also includes concepts such as diseases and medicines as well as relations between symptoms and the above related entities. We also link our KB to the Unified Medical Language System and analyze the differences between symptoms in the two KBs. We released the KB as Linked Open Data and a demo at https://datahub.io/dataset/symptoms-in-chinese .

[Qualitative-Quantitative Analysis of Rice Bran Oil Adulteration Based on Laser Near Infrared Spectroscopy].

The purpose of this study is mainly to have qualitative-quantitative analysis on the adulteration in rice bran oil by near-infrared spectroscopy analytical technology combined with chemo metrics methods. The author configured 189 adulterated oil samples according to the different mass ratios by selecting rice bran oil as base oil and choosing soybean oil, corn oil, colza oil, and waste oil of catering industry as adulterated oil. Then, the spectral data of samples was collected by using near-infrared spectrometer, and it was pre-processed through the following methods, including without processing, Multiplicative Scatter Correction(MSC), Orthogonal Signal Correction(OSC), Standard Normal Variate and Standard Normal Variate transformation DeTrending(SNV_DT). Furthermore, this article extracted characteristic wavelengths of the spectral datum from the pre-processed date by Successive Projections Algorithm(SPA), established qualitatively classified calibration methods of adulterated oil through classification method of Support Vector Machine(SVM), optimized model parameters(C, g) by Mesh Search Algorithm and determined the optimal process condition. In extracting characteristic wavelengths of the spectral datum from pretreatment by Backward interval Partial Least Squares(BiPLS) and SPA, quantitatively classified calibration models of adulterated oil through Partial Least Squares(PLS) and Support Vector Machine Regression(SVR) was established respectively. In the end, the author optimized the combination of model parameters(C, g) by Mesh Search Algorithm and determined the optimal parameter model. According to the analysis, the accuracy of prediction set and calibration set for SVC model reached 95% and 100% respectively. Compared with the prediction of the adulteration oil content of rice bran oil which was established by the PLS model, the SVR model is the better one, although both of them could implement the content prediction. Furthermore, the correlation coefficient R is above 0.99 and the Root Mean Square Error (MSE) is below 5.55 x 10(-4). The results show that the near-infrared spectroscopy technology is effective in qualitative-quantitative analysis on the adulteration of rice bran oil. And the method is applicable to analyze adulteration in other oils.

Hugan Qingzhi Exerts Anti-Inflammatory Effects in a Rat Model of Nonalcoholic Fatty Liver Disease.

Ethnopharmacological Relevance. The Hugan Qingzhi tablet (HQT) is a traditional Chinese medicine used for treating NAFLD (nonalcoholic fatty liver disease). The present study evaluated the anti-inflammatory effects of HQT in rats with NAFLD. Materials and Methods. HQT was administered daily to the NAFLD experimental groups. Biochemical markers, histopathological data, and oxidative stress/antioxidant biomarkers were determined. Proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were detected by enzyme-linked immunoassay. Expressions of silent information regulator 1 (SIRT1) and acetylated-nuclear-factor kappaB-p65 (Ac-NF-κB-p65) were performed by western blotting. Results. At high and moderate doses, HQT was highly effective in decreasing serum alanine aminotransferase (P < 0.01), aspartate aminotransferase (P < 0.01), hepatic total cholesterol (P < 0.01), triglycerides (P < 0.01), and free fatty acid levels (P < 0.01). Moreover, high and moderate doses of HQT reduced hepatic levels of the proinflammatory cytokines TNF-α (P < 0.01), IL-1ß (P < 0.01), and IL-6 (P < 0.01), enhanced SIRT1 expression, and depressed Ac-NF-κB-p65 expression at protein level. Conclusions. In our NAFLD rat model, HQT exerted substantial anti-inflammatory and antioxidant activities, possibly involving the regulation of SIRT1 and Ac-NF-κB-p65 expression.

[Establishment of a L-02 cell model of hepatic steatosis].

OBJECTIVE: To establish an in vitro cell model for investigating hepatic steatosis in non-alcoholic fatty liver disease. METHODS: L-02 cells cultured in 1640 containing 10% fetal bovine serum were divided into control group and model group. At 70%-80% confluency, L-02 cells in the model group were exposed to a long-chain mixture of free fatty acids (FFA, oleate and palmitate ) for 24 h, and cells in control group were treated with fresh medium. Lipid droplets in the cells were observed and total lipid content was determined with Oil Red O staining. The morphology of lipid droplets, trilyceride level, malonaldehyde content and cell apoptosis rate were evaluated to verify the cell model, and the effect of Huganqingzhi tablet on the lipid droplets was observed. RESULTS: A large number of lipid droplets were found in the cell model, which showed markedly increased level of triglyceride without significant changes of malonadehyde content or cell apoptosis rate. Intervention with two doses of Huganqingzhi tablet significantly decreased the number of lipid droplets and trilyceride content in the cell model. CONCLUSION: hepatic steatosis L-02 cell model can be established by long-chain mixture of free fatty acids (oleate:spalmitate=2:1) for therapeutic drug studies.

Hugan Qingzhi medication ameliorates hepatic steatosis by activating AMPK and PPARα pathways in L02 cells and HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hugan Qingzhi tablet (HQT), a lipid- lowering traditional Chinese medicine formula, has been used for the prevention and treatment of nonalcoholic fatty liver (NAFLD). AIM OF THE STUDY: This study was realized to evaluate the effects of HQT-medicated serum on hepatic steatosis using in vitro experiments with cells and explore the relevant mechanisms with method of serum pharmacology. MATERIALS AND METHODS: A model of hepatic steatosis in the L02 and HepG2 cells was induced by free fatty acid (FFA). The components in the HQT-medicated serum were assayed by high-performance liquid chromatography. Intracellular lipid droplets were detected by Oil Red O staining, and their ultrastructure was examined by transmission electron microscope. The biochemical parameters, including triglyceride (TG), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH), were measured with commercial kits. Furthermore, the expression of adiponectin, AMP-activated protein kinase (AMPK) phosphorylation, sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator activated receptor-α (PPARα), carnitine palmitoyltransferase 1 (CPT-1), and acetyl-CoA oxidase 1 (ACOX1) was analyzed by Western blot and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Moderate- and high-dose HQT-medicated serum reduced (P<0.05 or P<0.01) the accumulation of lipid droplets and the cellular TG content in L02 and HepG2 cells. They caused significant reductions (P<0.01) in LDH, AST, ALT and MDA and significant increase (P<0.05 or P<0.01) in T-AOC in the culture medium. They also caused increase (P<0.05 or P<0.01) in GSH level and SOD activity in FFA-induced steatotic L02 and HepG2 cells. Furthermore, moderate- and high-dose HQT-medicated serum enhanced (P<0.01) adiponectin expression in a concentration-dependent manner and increased (P<0.05 or P<0.01) the phosphorylation of AMPK and the expression of PPARα, CPT-1, and ACOX1, and reduced (P<0.05 or P<0.01) the expression of SREBP-1. CONCLUSION: The results suggested that HQT-medicated serum exerts a preventive effect against hepatic steatosis, and the potential mechanism might be activation of AMPK and PPARα pathways.

Effectiveness of Ganoderma lucidum preparation in treating simian acquired immune deficiency syndrome / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effectiveness of the traditional Chinese herbal medicine Lingzhi (Ganoderma lucidum) preparation in treating simian acquired immune deficiency syndrome (SAIDS).</p><p><b>METHODS</b>Five female adult Chinese rhesus monkeys were inoculated rectally with SIVmac239, and were all diagnosed as SAIDS by laboratory and clinical examinations 17 months later. Of these 5 monkeys, 3 (#393, #374, and #381; treatment group) were orally administered with Ganoderma lucidum (2 spores powder capsules plus 2 spores oil capsules on a daily basis), and the remaining other two monkeys (#348 and #361) served as control and did not receive treatment.</p><p><b>RESULTS</b>Animal #393 (treatment group), #361 (control group) and #348 (control group) died of SAIDS (opportunity infection) 3.5 months, 6 months, and 11 months later, respectively. Two animals (#374 and #381) survived. The necropsy revealed depletion and/or exhaustion of their lymphoid tissue. In the monkey #374, the peripheral CD4(+) T lymphocyte increased by 30% in the 6(th) month compared with the baseline level and then fluctuated. The plasma viral load gradually fell and reached about 1 log(10) in the treatment group, but remained stable in the control group. As shown by pathological examinations, the lymph node and spleen of monkeys #374 (treatment group) and #381 (treatment group) showed rehabilitation and reconstruction in the lymphatic tissue, thymus, nerve tissue of gyrus hippocampi, pituitary gland, pineal body, thyroid gland, adrenal gland, and ovary. In the control group, however, animals experienced depletion of lymph nodes, atrophy of spleen, disappearance of thymus, and other disorders in endocrine organs.</p><p><b>CONCLUSION</b>Ganoderma lucidum preparation may have certain protective effect on the immune system, nervous system, and endocrine system of monkeys with SAIDS.</p>

Miniaturization of the structure elucidation of novel natural products--two trace antibacterial acylated caprylic alcohol glycosides from Arctostaphylos pumila.

High-throughput isolation, purification and analysis methods applied to natural products libraries from plants gave rise to the discovery of two novel acylated caprylic alcohol glycosides (1, 2) produced by Arctostaphylos pumila. The NMR spectra were acquired using the CapNMR probe and performed on mass-limited samples, which enabled us to elucidate the structures of 2,6-diacetyl-3,4-diisobutyl-1- O-octylglucopyranoside (1, 200 microg) and 2,6-diacetyl-3,4-dimethylbutyl-1- O-octylglucopyranosid (2, 70 microg). Compounds 1 and 2 exhibited antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus with an MIC of 128 microg/mL and 64 microg/mL, respectively.

A combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats.

Attempts to develop gossypol and steroidal hormones alone as a male contraceptive have been tested for many years; however, both caused undesirable side effects that have prevented their acceptance. In this study, we formulated a regimen of combined gossypol at a low dose of 12 mg/kg or a high dose of 50 mg/kg plus methyltestosterone 20 mg/kg and ethinylestradiol 100 g/kg daily (12 mg G+H and 50 mg G+H) administered for 6 weeks in adult rats. The possible roles of germ cell apoptosis and related genes expression were studied by techniques of TdT-mediated dUTP nick end-labeling (TUNEL), agarose gel electrophoresis of low-molecular-weight DNA, in situ hybridization and reverse transcription-polymerase chain reaction detection. Results showed that germ cell apoptosis and related genes expression were significantly induced after combined drug administration. The apoptosis index increased 3.86- and 9.65-fold in the 12-mg and 50-mg G+H-treated groups, respectively, as compared to the control group. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The apoptosis-related genes fas mRNA expression levels increased 0.44- and 1.39-fold, bax mRNA 0.74- and 2.56-fold, caspase-3 mRNA 0.60- and 1.29-fold, and caspase-9 mRNA 2.50- and 4.08-fold, respectively, in the 12-mg and 50-mg G+H-treated groups vs. the control group. These results indicated that our drug regimen applied as a contraceptive could induce rat germ cell apoptosis. The apoptotic process involved fas system, bax and caspase family genes and the apoptotic extent and cell types were gossypol dose-dependent.

High-throughput method for the production and analysis of large natural product libraries for drug discovery.

High-throughput methods were applied to the production, analysis, and characterization of libraries of natural products in order to accelerate the drug discovery process for high-throughput screening in the pharmaceutical and biotechnology industries. Library production integrates automated flash chromatography, solid-phase extraction, filtration, and high-throughput parallel four-channel preparative high-performance liquid chromatography to obtain the libraries in 96- or 384-well plates. Libraries consist of purified fractions with approximately one to five compounds per well. Libraries are analyzed prior to biological screening by a high-throughput parallel eight-channel liquid chromatography-evaporative light scattering detection-mass spectrometry system to determine the molecular weight, number, and quantity of compounds in a fraction. After biological screening, active fractions are rapidly purified at the microgram level and individual compounds are rescreened for confirmation of activity. Structures of active compounds are elucidated by NMR spectroscopy and mass spectrometry. Utilization of a novel microcoil probe allows NMR data to be gathered on 50 microg. As a demonstration, a library was made from the stem bark of Taxus brevifolia. Biological screening in the National Cancer Institute's in vitro panel of three cancer cell lines demonstrates that the process enables the discovery of active anticancer compounds not detected in the flash fractions from which the library originates.