Three novel dalbergiphenol hybrids from the heartwood of Dalbergia cochinchinensis.

A novel discovery of two hybrid benzodioxepin-dalbergiphenol epimers, named cochindalbergiphenols A-B (1-2), and a benzofuran-dalbergiphenol hybrid, named cochindalbergiphenol C (3), were isolated and identified from the heartwood of Dalbergia cochinchinensis. The structures of all the isolated compounds were identified through NMR and HRESIMS techniques, while the absolute configurations were determined by comparing the experimental and calculated ECD spectra. Compounds 1-3 exhibited potential protective effects against hypoxia/reoxygenation (H/R) induced injury in H9c2 cells.

[UPLC-Q-TOF-MS metabolomic study on improvement of acute myocardial ischemia in rats by Dalbergia cochinchinensis heartwood].

This paper aimed to study the effect of Dalbergia cochinchinensis heartwood on plasma endogenous metabolites in rats with ligation of the left anterior descending coronary artery, and to analyze the mechanism of D. cochinchinensis heartwood in improving acute myocardial ischemic injury. The stability and consistency of the components in the D. cochinchinensis heartwood were verified by the establishment of fingerprint, and 30 male SD rats were randomly divided into a sham group, a model group, and a D. cochinchinensis heartwood(6 g·kg~(-1)) group, with 10 rats in each group. The sham group only opened the chest without ligation, while the other groups established the model of ligation. Ten days after administration, the hearts were taken for hematoxylin-eosin(HE) staining, and the content of heart injury indexes in the plasma creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH), energy metabolism-related index glucose(Glu) content, and vascular endothelial function index nitric oxide(NO) was determined. The endogenous metabolites were detected by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS). The results showed that the D. cochinchinensis heartwood reduced the content of CK-MB and LDH in the plasma of rats to relieve myocardial injury, reduced the content of Glu in the plasma, improved myocardial energy metabolism, increased the content of NO, cured the vascular endothelial injury, and promoted vasodilation. D. cochinchinensis heartwood improved the increase of intercellular space, myocardial inflammatory cell infiltration, and myofilament rupture caused by ligation of the left anterior descending coronary artery. The metabolomic study showed that the content of 26 metabolites in the plasma of rats in the model group increased significantly, while the content of 27 metabolites decreased significantly. Twenty metabolites were significantly adjusted after the administration of D. cochinchinensis heartwood. D. cochinchinensis heartwood can significantly adjust the metabolic abnormality in rats with ligation of the left anterior descending coronary artery, and its mechanism may be related to the regulation of cardiac energy metabolism, NO production, and inflammation. The results provide a corresponding basis for further explaining the effect of D. cochinchinensis on the acute myocardial injury.

[Quality evaluation of Dalbergiae Odoriferae Lignum by HPLC fingerprint and multi-component quantitative analysis].

The present study detected the component content in Dalbergiae Odoriferae Lignum by HPLC fingerprint and the multi-component determination method. HPLC analysis was performed on the Agilent ZORBAX SB-C_(18) column(4.6 mm×250 mm, 5 µm). Acetonitrile-0.5% phosphoric acid aqueous solution with gradient elution was employed as the mobile phase. The flow rate was 1.0 mL·min~(-1) and the column temperature was maintained at 30 ℃. The detection wavelength was 210 nm and the sample volume was 10 µL. The similarity of 18 batches of Dalbergiae Odoriferae Lignum was 0.343-0.779, indicating that there were great differences between different batches of Dalbergiae Odoriferae Lignum. Eighteen common peaks were identified, including eight flavonoids such as liquiritigenin and latifolin. The mass fractions of liquiritigenin, luteolin, naringenin, isoliquiritigenin, formononetin, dalbergin, latifolin, and pinocembrin were in the ranges of 0.134 1%-0.495 2%, 0.028 2%-0.167 0%, 0.016 3%-0.591 3%, 0.053 5%-0.188 0%, 0.142 4%-0.640 1%, 0.068 0%-0.590 7%, 0.003 2%-1.980 7%, and 0.009 6%-0.740 2%, respectively. Eighteen batches of Dalbergiae Odoriferae Lignum were divided into three categories by cluster analysis and eight differential components in Dalbergiae Odoriferae Lignum were marked by partial least-squares discriminant analysis(PLS-DA). The cumulative variance contribution rate was 90.5%. The HPLC fingerprint combined with the multi-component determination method for Dalbergiae Odoriferae Lignum is easy in operation and accurate in results, with good repeatability and reliability. The quality of Dalbergiae Odoriferae Lignum can be evaluated and analyzed by the PLS-DA model. This study is expected to provide a reference for the quality control and clinical application of Dalbergiae Odoriferae Lignum.

[Molecular mechanism of Dalbergia cochinchinensis heartwood in regulation of energy metabolism to treat myocardial ischemia based on network pharmacology and experimental validation].

Dalbergia cochinchinensis(DC) is chemically similar to the valuable and scarce Chinese herb Dalbergiae Odoriferae Lignum, and both of them belong to the Dalbergia Leguminosae. DC is used for treating cardiovascular diseases and cancer. However, its potent active ingredient groups and molecular mechanisms in anti-myocardial ischemia are not fully clarified. In this study, the active ingredient groups, targets, and signaling pathways of DC heartwood for the treatment of myocardial ischemia were screened out based on network pharmacology and molecular docking technology, and the effects were verified by the rat model of acute myocardial ischemia induced by isoprenaline(ISO). The molecular mechanism of DC heartwood was elucidated based on the target of multi-ingredient and multi-target pathways. The crossing targets of DC heartwood for the treatment of myocardial ischemia were identified through the screening of active ingredients in DC heartwood and the prediction of targets. The Kyoto Encyclopedia of Genomes(KEGG) pathway enrichment and Gene Ontology(GO) functional annotation were performed. AutoDock was used to bind the active ingredient groups to the pathway targets. Finally, the molecular mechanism of myocardial ischemia treatment by DC heartwood extracts in the treatment of myocardial ischemia was revealed through the rat model of ISO-induced acute myocardial ischemia by performing electrocardiogram(ECG), hemodynamic, cardiac enzymes, hematoxylin-eosin(HE) staining, high-energy phosphate compounds, reverse transcription polymerase chain reaction(RT-PCR), and Western blot pharmacodynamic experiments, based on the multi-ingredient and multi-target action of active ingredient groups and pathway targets. The network pharmacology showed that the 18 ingredients of DC heartwood corresponded to 510 targets, 629 myocardial ischemia-related targets, and 101 cross-targets. GO and KEGG enrichment analyses showed that DC heartwood was involved in the hypoxic response, vasoconstriction, and nitric oxide biosynthesis, and had effects on the molecular functions of hemoglobin binding, protein binding, and adenosine triphosphate(ATP) binding. It regulated the signaling pathways such as hypoxia-inducible factor 1(HIF-1), vascular endothelial growth factor(VEGF), and phosphatidylinositol-3-kinase/protein kinase B(PI3 K/AKT) to act on myocardial ischemia. Experimental studies showed that DC heartwood slowed down the heart rate and ST segment change(ΔST), and increased systolic blood pressure(SBP), diastolic blood pressure(DBP), and mean arterial pressure(MBP) in rats with ISO-induced acute myocardial ischemia. It also reduced plasma lactate dehydrogenase(LDH), creatine kinase isoenzyme MB(CK-MB), and glutamate transaminase(AST) levels, relieved myocardial fiber disorders and inflammatory cell infiltration, and increased ATP and cellular energy(EC) levels. DC heartwood increased the mRNA expressions of calmodulin-dependent protein kinase kinase(CAMKK) in the myocardial tissue, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3), mammalian target of rapamycin(mTOR), PI3 K, VEGF, endothelial nitric oxide synthase(eNOS), HIF-1α in the myocardial tissue. It decreased the mRNA expression of pyruvate dehydrogenase(PDH), and increased the protein expressions of PFKFB3, VEGFA, and eNOS. Molecular docking showed that liquiritigenin, stigmasterol, isodalbergin, latifolin, 4-methoxydalbergione, dibutyl terephthalate, 2,4-dihydroxy-5-methoxybenzophenone in DC heartwood produced bio-binding activities with epidermal growth factor receptor(EGFR), HIF-1α, CAMKK, PI3 K, mTOR, and PDH, respectively. Therefore, the active ingredient groups of DC heartwood act on the HIF-1 signaling pathway, regulate cardiomyocyte energy metabolism, and increase ATP energy charge in a multi-ingredient and multi-target manner, improving cardiac function and histopathological changes to protect rats with acute myocardial ischemia induced by ISO.

[Study on effect of anemoside B4 in improving COPD rats by regulating IL-12/STAT4 and IL-4/STAT6 signaling pathways].

To study the effect of anemoside B4 on rats with chronic obstructive pulmonary disease (COPD).Seventy-two SD male rats were randomly divided into blank group and model group.The method of exposure to cigarette smoke and combined with lipopolysaccharide (LPS) was used to replicate the rat model of COPD.After the model was maintained for 5 weeks,the rats were randomly divided into model group,dexamethasone group (0.81 mg·kg~(-1)) and anemoside B4 low,medium and high (2,4,8 mg·kg~(-1)) dose groups,a group of 12 animals were administered,and then the administration was started.The administration was maintained until the28th day,and the pulmonary function parameters of rats were measured by an animal pulmonary function instrument.After testing the rat lung function parameters,immediately draw rat alveolar lavage fluid (BALF),and use high-throughput protein chip technology to determined the expression levels of inflammatory cytokines in rat BALF.HE staining was used to observe the general pathological changes of rat lung and tracheal tissue.Masson staining was used to observe the collagen deposition in rat lung tissue.Real-time q PCR method was used to determine the mRNA expression level of related genes in rat lung tissue.Western blot method was used to determine the expression levels of related proteins in rat lung tissues.According to the findings,compared with the model group,the dexamethasone group and the anemoside B4 drug groups had different degrees of increase in the lung function parameters of rats (P<0.01,P<0.05),improved the expression level of inflammatory cytokines in the BALF of rats to varying degrees (P<0.01,P<0.05),and improved the pathological structure of rat lung tissue to varying degrees.Relative mRNA expressions of matrix metalloproteinase 2 (MMP-2),matrix metalloproteinase 12 (MMP-12),matrix metalloproteinase inhibitor 1 (TIMP-1),interleukin-6 (IL-6),and transforming growth factor-ß1 (TGF-ß1) were significantly reduced (P<0.01);whereas relative mRNA expressions of matrix metalloproteinase 9(MMP-9) and matrix metalloproteinase inhibitor 2 (TIMP-2) were increased significantly (P<0.01).The mRNA and protein expression levels of T-box transcription factor (T-bet),interleukin-12 (IL-12) and signal transducer and activator of transcription 4(STAT4) reduced to varying degrees (P<0.01,P<0.05).The mRNA of transcription factor GATA3 (binding protein-3),interleukin-4 (IL-4) and signal transducer and activator of transcription 6 (STAT6) in rat lung tissues and the protein expression levels of IL-4 and STAT6 were increased to varying degrees (P<0.01,P<0.05).In conclusion,anemoside B4 has a certain protective effect on COPD rats caused by cigarette smoke exposure and combined with LPS.The mechanism of action may be related to the regulation of IL-12/STAT4 and IL-4/STAT6 signaling pathways.

Chemical-activity-based quality marker screening strategy for Viscum articulatum.

Viscum articulatum Burm. f. is a parasitic plant rich in flavonoids, triterpenoids, and catechins and has a high nutritional value. It has been reported that consuming V. articulatum can prevent cardiac diseases. In this study, six bioactive compounds, including catechins, triterpenoids, and phenylpropanoid glycosides, were determined in alcohol extracts of the plant using HPLC. The anti-inflammatory and antioxidant activities of three catechins, two triterpenoids, and three combination drugs were measured in cardiomyocytes, and the results showed that the anti-inflammatory activity was significantly enhanced while retaining strong antioxidant activity when epicatechin and ursolic acid were used in combination. The main quality markers epicatechin and ursolic acid were screened based on the specificity of the genuine herb and a potent synergistic effect, and the lowest limitation contents of V. articulatum which could discriminate it from some other taxonomically similar materials were accordingly determined. This self-built lowest limitation content of the two screened quality markers could quickly and accurately reflect the efficacy in terms of chemical composition and reverse the disorderly market use of nongenuine herbs or confusing species for adulteration. This study is of some significance for market regulation, drug development, and clinical medication.

Effects of Eucommia ulmoides extract against renal injury caused by long-term high purine diets in rats.

Diets of overloaded purine-rich foods for a long time are one of the important reasons to cause renal lesions. Eucommia ulmoides is one of the traditional Chinese medicine herbs, which has been used to recover functions of the kidney. However, its mechanism remains unclear. The aim of this study was to explore the effects and protective mechanism of Eucommia ulmoides extract on renal injury caused by long-term high purine diets in rats. SD rats underwent an intragastric adenine (200 mg kg-1 d-1) administration for 9 weeks and were treated for 15 weeks. The results demonstrated that Eucommia ulmoides extract significantly reduced serum Cre and BUN levels in rats. H&E and Masson's trichrome stains showed notable lowering of the infiltration of inflammatory cells, the formation of fibrous tissues and collagen fibers, and improvement in the pathological morphology of kidneys. It also suppressed the protein and mRNA expressions of TGF-ß1 and α-SMA and enhanced E-cadherin expression. Meanwhile, Eucommia ulmoides extract prominently inhibited the mRNA expression of Col I, Col III, Col IV, TIMP-1, and TIMP-2 and promoted expressions of MMP-1, MMP-2 and MMP-9. Through our study, it is the first time to prove that Eucommia ulmoides extract could ameliorate renal interstitial fibrosis and may involve in the regulation of the extracellular matrix (ECM) degradation enzyme (MMPs/TIMPs) system, promotion of the expression of E-cadherin, and suppression of expressions of TGF-ß1 and α-SMA. The results provide a significant implication for the utilization of Eunomia Ulmoides extract as functional foods to enhance renal functions and improve renal injury caused by high purine diets.

Latifolin protects against myocardial infarction by alleviating myocardial inflammatory via the HIF-1α/NF-κB/IL-6 pathway.

CONTEXT: The Traditional Chinese herb medicine Dalbergia odorifera T. Chen (Fabaceae), exerted a protective effect on myocardial ischaemia. Latifolin is a neoflavonoid extracted from Dalbergia odorifera. It has been reported to have the effects of anti-inflammation and cardiomyocyte protection. OBJECTIVE: To investigate whether latifolin can improve myocardial infarction (MI) through attenuating myocardial inflammatory and to explore its possible mechanisms. MATERIALS AND METHODS: Left coronary artery was ligated to induce a rat model of MI, and the rats were treated with sodium carboxymethyl cellulose (CMC-Na) or different doses of latifolin (25, 50, 100 mg/kg/d) by oral gavage for 28 days. Serum contents of myocardial enzyme were measured at seven and fourteen days after treatment. Cardiac function, infarct size, histopathological changes and inflammatory cells infiltration was assessed at 28 days after treatment. Western blotting was used to investigate the underlying mechanisms. RESULTS: Latifolin treatment markedly decreased the contents of myocardial enzymes, and increased left ventricular ejection fraction (85.27% vs. 59.11%) and left ventricular fractional shortening (62.71% vs. 45.53%). Latifolin was found to significantly reduced infarction size (27.78% vs. 39.07%), myocardial fibrosis and the numbers of macrophage infiltration (436 cells/mm2 vs. 690 cells/mm2). In addition, latifolin down-regulated the expression levels of hypoxia-inducible factor-1α (0.95-fold), phospho-nuclear factor-κB (0.2-fold) and interleukin-6 (1.11-fold). DISCUSSION AND CONCLUSIONS: Latifolin can protect against myocardial infarction by improving myocardial inflammation through the HIF-1α/NF-κB/IL-6 signalling pathway. Accordingly, latifolin may be a promising drug for pharmacological treatment of ischaemic cardiovascular disease.

[Study on neoflavonoids from heartwood of Dalbergia latifolia].

Dalbergiae Odoriferae Lignum is derived from heartwood of Dalbergia odorifera,which is national Ⅱ level of rare and endangered protective plants in China. Its resources are scarce and its price is high. In order to find substitutes of D. odorifera,the chemical constituents of 70% ethanol extract of heartwood of D. catifolia were systematically studied by using silica gel,Sephadex LH-20 column chromatography,and semi-preparative HPLC. Sixteen neoflavanoids were isolated and identified as eight dalbergiphenols( 1-8),three dalbergiones( 9-11),two dalbergins( 12,13),two benzophenones( 14,15) and one other type neoflavanoids( 16) based on spectroscopic data analyses and/or comparing the spectroscopic data with those in literature. Among them,compounds 3,7 and 11 were isolated from the genus Dalbergia for the first time,and compounds 2,4-6,8,14 and 15 were isolated from the D. latifolia for the first time. Ten neoflavonoids were both discovered from D. latifolia and D. odorifera.

[Effect of neoflavonoid latifolin isolated from Dalbergia odorifera on acute myocardial ischemia in rats and its mechanism of Nrf2 signaling pathway].

The present study was designed to evaluate the cardioprotective effect of latifolin on pituitrin(Pit) or isoproterenol(ISO)-induced myocardial injury in rats, and further investigate its underlying mechanisms. Rats were administrated sublingually with pituitrin or subcutaneously with isoproterenol to induce acute myocardial ischemia in rats, and lead II electrocardiograph was recorded. In rats with isoproterenol, ELISA assay or colorimetric method was used to detect the content or activity of myocardial injury markers in serum, and the SOD activity and MDA content in myocardium were detected by colorimetric assay; histopathological examination was conducted by HE staining; the frozen section of myocardial tissues was used for DCFH-DA fluorescent staining to detect the content of ROS in myocardium; Western blot was used to detect the protein expression levels of Nrf2, Keap1, HO-1 and NQO1 in myocardium. Results showed that latifolin significantly inhibited ST-segment changes induced by pituitrin or isoproterenol, and increased heart rate. Further mechanism study showed that latifolin reduced cardiac troponin I(cTnI) level, aspartate transaminase(AST) and lactate dehydrogenase(LDH) activities in serum, increased myocardial superoxide dismutase(SOD) activity and reduced myocardial malondialdehyde(MDA) level, and protected myocardium with less necrosis, infiltration of inflammatory cells and fracture of myocardial fibers. Furthermore, latifolin obviously reduced ROS level in myocardium, inhibited the expression of Kelch-like ECH-associated protein-1(Keap1), increased the nuclear translocation of nuclear factor erythroid 2 related factor 2(Nrf2), and promoted the expression of Heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) in myocardial tissues. Our data suggest that latifolin has a potent protective effect against pituitrin or isoproterenol-induced myocardial injury, which may be related to inhibition of oxidative stress by activating Nrf2 signaling pathway.

[Neoflavonoids and their pharmacological activities in Dalbergia genus].

Neoflavonoids are a kind of characteristic components in the Dalbergia genus. Based on the previous researches, 59 neoflavonoids have been obtained from the Dalbergia genus. According to their molecular skeleton, the neoflavonoids can be divided intodalbergiphenols, dalbergiones, dalbergins, benzophenones and other types. Modern research shows that neoflavonoids displayed a variety of pharmacological activities, such as anti-osteoporosis, anti-inflammatory, antitumor, anti-androgen, anti-allergic, antioxidation etc. This paper reviewed neoflavonoids and their pharmacological functions, which could provide the valuable reference for comprehensive utilization and new drug development in the Dalbergia genus.

[Protective Effects of Different Extracts of Imperatae Rhizoma in Rats with Adriamycin Nephrosis and Influence on Expression of TGF-ß1, and NF-κB p65].

OBJECTIVE: To study the protective effect of Imperatae Rhizoma extract on renal tissues in rats with Adriamycin nephrosis, and to explore the possible mechanism. METHODS: The nephrosis model was induced by adriamycin 6.5 mg/kg intravenously in rats. The rats were randomly divided into seven groups, including normal group, model group, predisone group, petroleum ether groups, ethyl acetate group ,alcohol group, and water parts group, and treated for eight weeks. The protein content of 24 hours urine excretion was tested respectively by automatic biochemistry analyzer each week. After eight weeks, BUN, CRE, TCHO, TG, TP and ALB in serum were examined by automatic biochemistry analyzer. The TNF-α in serum was measured by ELISA. The expression of TGF-ß1, and NF-κB p65 in renal tissues were detected by immunohistochemistry respectively. The pathological changes in the renal were observed by light microscope. RESULTS: Compared with the model group ,the proteinuria of the rats in ethyl acetate group obviously reduced at the 6th, 7th, 8th week, the content of TNF-α in serum and the expression of TGF-ß1, and NF-κB p65 in renal tissues significantly reduced, but the content of TP and ALB in serum were increased obviously. In the alcohol and water parts groups ,the level of TG in serum and the protein content of 24 hours urine excretion of the 6th and 7th week were significantly decreased. The ethyl acetate, alcohol and water parts groups ameliorated the changes of pathology in renal. CONCLUSION: The different extracts of Imperatae Rhizoma had different protective effect in rats with adriamycin nephrosis, and the effect of ethyl acetate group was more stronger than that of other groups. The mechanism may be related to reducing the expression of NF-κB p65 and TGF-ß1, and the content of TNF-α inrenal tissues of rats.

[Phenylpropanoids constituents of Eucommia ulmoides leaves].

OBJECTIVE: To investigate phenylpropanoids constituents from Eucommia ulmoides leaves. METHODS: Various column chromatographic methods were used in isolation and purification. Physiochemical constant determination and spectral analysis were adopted to determine the chemical structures of phenylpropanoids. RESULTS: Nine phenylpropanoids were isolated and identified as caffeic acid (1), chlorogenic acid methylester (2), syringin (3), guaiacylglycerol (4), 5-methoxy-guaiacylglycerol (5), 5,9-dimethoxy-guaiacylglycerol (6), 9-n-butyl-guaiacylglycerol (7), 9-n-butyl-isoguaiacylglycerol (8), 8'-methoxy-olivil (9). CONCLUSION: Compounds 5 - 9 are isolated from this plant for the first time.

[Study on chemical constituents of iridoids from eucommiae folium].

OBJECTIVE: To investigate the chemical constituents of iridoids from Eucommiae Folium. METHODS: The compounds were isolated and purified by various column chromatography, the structures were identified on the basis of physiochemical properties and spectral analysis. RESULTS: Nine iridoids were isolated and identified as asperuloside (1), daphylloside (2), scandoside methyl ester (3), loganin (4), 8-epi-loganin (5), 7-epi-loganin (6), deacetyl asperulosidic acid methyl ester (7), geniposide (8) and geniposidic acid (9). CONCLUSION: Compounds 2 - 7 are isolated from this plant for the first time, compounds 4 - 6 are isolated from this genus for the first time.

Chemical composition, antimicrobial property and microencapsulation of Mustard (Sinapis alba) seed essential oil by complex coacervation.

In this study, the essential oil from mustard seed was isolated by simultaneous steam distillation and extraction (SDE) and analyzed by gas chromatography-mass spectrometry (GC-MS). Fourteen components were identified in the mustard seed essential oil with allyl isothiocyanate being the main component (71.06%). The essential oil has a broad-spectrum antimicrobial activity with inhibition zones and MIC values in the range of 9.68-15.57 mm and 128-512 µg/mL respectively. The essential oil was subsequently encapsulated in complex coacervation microcapsules with genipin, a natural water-soluble cross-linker. The optimum parameters for the hardening effectiveness of the genipin-hardened essential oil microcapsules were 8h at 40°C and pH 10.0 with a genipin concentration of 0.075 g/g gelatin. The genipin-hardened microcapsules had a particle size of mainly 5-10 µm and strong chemistry stability which is potential for its application in food preservation.

[Study on chemical constituents of Eucommia ulmoides leaves].

OBJECTIVE: To investigate the chemical constituents of Eucommia ulmoides leaves. METHODS: Various column chromatography were used in the isolation and purification, physiochemical constant determination and spectral analysis were adopted to determine the chemical structures. RESULTS: Ten compounds were isolated and identified as borreriagenin (1), n-butyl-O-ß-D-fructopyranoside (2), α-D-glucopyranosyl-(1-->1')-3'-amino-3'-deoxy-ß-D-glucopyranoside (3), ß-D-fructofuranosyl-α-D-galactopyranoside (4), ß-D-fructose (5), diisobutyl phthalate (6), 5-hydroxy-9-isopropylether-guaiacylglycerol (7), 4-hydroxyphenylethanol-8-O-ß-D-apiofuranosyl(1-->6)-ß-D-glucopyranoside (8), lariciresinol (9), and (3S,5R,6R,9S)-tetrahydroxy-7-ene-megastigmane (10). CONCLUSION: All compounds are isolated from this genus for the first time.

Antitumor activity of Pulsatilla chinensis (Bunge) Regel saponins in human liver tumor 7402 cells in vitro and in vivo.

Pulsatilla chinensis (Bunge) Regel is a Chinese medicinal herb for "blood-cooling" and detoxification. Now it is used for the treatment of malignant tumor, but the antitumor mechanisms and toxic side effects of P. chinensis are unclear. The present study was undertaken to investigate if P. chinensis saponins (PRS) possesses anticancer effects and toxic side effects in human liver tumor 7402 cells in vitro and vivo. 7402 cells were treated with different concentrations of PRS for 24h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was assessed by flow cytometry. The in vivo effect of PRS on 7402 tumor cells transplanted in athymic nude mice was investigated. 15 saponins were isolated and identified from PRS. PRS inhibited the proliferation of human liver tumor 7402 cells in vitro by apoptosis. 19 days after administration of PRS (100, 200mg/kg), the weight of tumor mass was markly decreased in nude mice. The anti-tumor effect of PRS in vivo was associated with a significant increase in the 7402 apoptosis rate. Although PRS inhibited the weight of mice, it showed almost no effect on leukocyte number, liver and spleen weight index. Light microscopic histopathological examination showed that PRS had no specific lesion in organ. These results suggested that P. chinensis saponins exert potential anticancer activity in treating tumors in nude mice and no toxic side effects.

[Chemical constituents of rhizoma imperatae and their anti-complementary activity].

OBJECTIVE: To study the chemical constituents of Rhizoma Imperatae and their anti-complementary activity. METHODS: By the hemolysis test, the petroleum extraction, ethyl acetate extraction, n-butanol extraction and the water extraction was tested for anti-complementary activity. Compounds were isolated and purified by silica gel column chromatography, Sephadex LH-20 and reversed-phase column chromatography. The structures were identified by the various spectroscopic data of ESI-MS, 1H-NMR, 13C-NMR. The compounds were evaluated for anti-complementary activity in vitro. RESULTS: The petroleum extraction, ethyl acetate extraction showed significant anti-complementary activity. Ten compounds were isolated from the petroleum and EtOAc soluble fractions and identified as cylindrin (1), arundoin (2), friedelin (3), beta-sitosterol (4), siderin (5), ethyl p-hydroxybenzoate (6), 5-methoxyflavone (7), vanillic acid (8), trans-p-coumaric acid (9), 5-hydroxymethylfurfural (10). CONCLUSION: Compounds 6, 7, 8, and 10 are isolated from the genus for the first time, and compounds 3, 8 and 9 inhibited the complement system towards the classical pathway.

[Relationship between HPLC fingerprint chromatogram and inhibitory effect on respiratory burst of rat PMN of leaves of crataegus].

OBJECTIVE: To study the relationship between HPLC fingerprint chromatogram and inhibitory effect on respiratory burst of rat PMN of leaves of crataegus L. METHOD: HPLC fingerprint peaks of different species of hawthorn leaves were isolated and used for the effective experiment on the respiratory burst of rat PMN. The mathematic models of the relationship between the area and the effect of fingerprint peaks were established. According to the mathematic models, the HPLC fingerprint were change into bioactive fingerprint (include effective fingerprint and potency fingerprint) with the helps of mathematics, chemometrics, computer program simulation and etc. RESULT: The chromatogram-effect relationship of leaves of crataegus. on respiratory burst of rat PMN was established. According to this relationship, the activities of fourteen samples of leaves of crataegus. were forecasted. It was positive correlation between the expected value and the practical value. And the correlation coefficients was 0.968 (P < 0.01). CONCLUSION: An all-around evaluative system, which includes not only chemical identification but also effective evaluation for traditional Chinese medicine was established. It will provide a new idea for study on fingerprint chromatogram of traditional Chinese medicine.

Screening the proteins that interact with calpain in a human heart cDNA library using a yeast two-hybrid system.

Calpain, a cytosolic cysteine protease, requires calcium ions for activity. It has been reported that calpain is involved in the degradation of myofibrillar and neurofilament proteins, and the activation of phosphorylase b kinase and protein kinase C. More recently, calpain was shown to participate in apoptosis. In order to understand the calpain-related signal transduction pathway and its changes during hypertrophy, and especially in hypertension, we screened a human heart cDNA library to find proteins that interact with calpain. 1) Using PCR we amplified the full-length, domain II, domain III and domain IV cDNA of calpain (calcium-activated neutral protease, CANP) I large subunit respectively. 2) Then the fragments were cloned into pGBKT7 vector, resulting in 4 bait expression constructs (pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III, and pG BKT7-CANP IV). 3) After 4 bait vectors were transformed into AH109 by the lithium acetate-mediated method, AH109/pGBKT7-CANP, AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III, and AH109/pGBKT7-CANP IV were obtained, respectively. 4) After the human heart cDNA library was sequentially transformed into AH109/ pGBKT7-CANP, 1000-1200 positive clones were grown on SD/Trp-Leu-Ade-His-. Only 150 positive clones were obtained through a colony-lift filter assay to detect beta-galactosidase activity. 5) Total 105 clones among above 150 positive clones were eliminated through that the duplicate, pseudopositive and autoactive detection, respectively. 6) Finally, sequencing eliminated clones with a wrong open reading frame (ORF). Eight clones were cancelled with wrong ORF. The remaining 37 positive clones were analyzed using BLAST software available on the Internet and classified as follows: 1. enzymes or proteins related to signal transduction in the cell; 2. contraction proteins 3. matrix proteins 4. unknown proteins. 7) In order to determine which domain of the calpain I large subunit was involved in the interaction with these real clones, the 37 clones were transformed into AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III or AH109/pGBKT7-CANP IV. Among these 37 clones, 29 clones could interact with domain II, 5 clones could interact with domain III and 6 clones could interact with domain IV. Thus, we successfully constructed 4 bait expression vectors, pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III and pGBKT7-CANP IV, and obtained 37 real positive clones that interacted with the calpain I large subunit by screening a human heart cDNA library using pGBKT7-CANP as bait. Among them, 29 clones could interact with domain II of the calpain I large subunit, where the active site of calpain is located. Additional studies will be needed to clarify the calpain-related signal transduction pathway in greater detail.