Two new triterpenoids from the leaves of Cyclocarya paliurus (Batalin) Iljinskaja.

Two previously undescribed triterpenoids (1-2), along with thirteen known compounds (3-15) were isolated from a CHCl3-soluble extract of the leaves of Cyclocarya paliurus. Their structures were established on the basis of chemical and spectroscopic approaches. These compounds were assessed for their therapeutic effects on diabetic nephropathy (DN)-evoked fibrosis through High-Glucose and transforming growth factor-ß1 (TGF-ß1) challenged HK-2 cells. Among them, compounds 3, 5 and 8 could remarkedly decrease the level of fibronectin to relieve DN with 27.66 ± 2.77%, 6.09 ± 0.57% and 17.74 ± 5.83% inhibition rate at 10 µM, 10 µM and 1 µM, respectively.

Engineering a Reliable and Convenient SARS-CoV-2 Replicon System for Analysis of Viral RNA Synthesis and Screening of Antiviral Inhibitors.

The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.

Pyrazolo[1,5- a]pyridine Inhibitor of the Respiratory Cytochrome bcc Complex for the Treatment of Drug-Resistant Tuberculosis.

Respiration is a promising target for the development of new antimycobacterial agents, with a growing number of compounds in clinical development entering this target space. However, more candidate inhibitors are needed to expand the therapeutic options available for drug-resistant Mycobacterium tuberculosis infection. Here, we characterize a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5- a]pyridine-3-carboxamide. TB47 is active (MIC between 0.016 and 0.500 µg/mL) against a panel of 56 M. tuberculosis clinical isolates, including 37 multi-drug-resistant and two extensively drug-resistant strains. Pharmacokinetic and toxicity studies showed promising profiles, including negligible CYP450 interactions, cytotoxicity, and hERG channel inhibition. Consistent with other reported QcrB inhibitors, TB47 inhibits oxygen consumption only when the alternative oxidase, cytochrome bd, is deleted. A point mutation in the qcrB cd2-loop (H190Y, M. smegmatis numbering) rescues the inhibitory effects of TB47. Metabolomic profiling of TB47-treated M. tuberculosis H37Rv cultures revealed accumulation of steps in the TCA cycle and pentose phosphate pathway that are linked to reducing equivalents, suggesting that TB47 causes metabolic redox stress. In mouse infection models, a TB47 monotherapy was not bactericidal. However, TB47 was strongly synergistic with pyrazinamide and rifampicin, suggesting a promising role in combination therapies. We propose that TB47 is an effective lead compound for the development of novel tuberculosis chemotherapies.

[Chemical Constituents from Thalictrum fortune].

Objective: To investigate the chemical constituents of Thalictrum fortunei. Methods: Compounds were separated and purified by chromatographic methods and the structures were identified by their physicochemical properties and spectroscopic data. Results: Ten compounds were isolated and identified as bergenin( 1),1-( 4-hydroxy-3-methoxy)-phenyl-2-[4-( 1,2,3-trihydroxypropyl)-2-methoxy]-phenoxym-1,3-propandiol( 2) 、4-( 2-hydroxyethyl)-2-methoxyphenyl-O-ß-D-glucopyranoside( 3),meliasendanin D( 4),2-( 4-hydroxy-3-methoxyphenyl)-ethyl-O-ß-D-glucopyranoside( 5),kizutasaponin C( 6),2-( 3-hydroxy-4-methoxyphenyl)-ethyl-O-ß-Dglucopyranoside( 7),ß-sitosterol( 8),3-O-ß-D-glucopyranosyl( 1→6)-ß-D-glucopyranosyl( 22 S,24Z)-cycloart-24-en-3ß,22,30-tetraol-26-O-ß-D-glucopyranoside( 9) and 3-O-ß-D-quinovopyranosyl( 1→6)-ß-D-glucopyranosyl( 1→4)-ß-D-fucopyranosyl( 22 S,24Z)-cycloart-24-en-3ß,22,26-triaol-26-O-ß-D-glucopyranoside( 10). Conclusion: Compounds 1 ~ 6 are isolated form this plant for the first time.

Transport of ginkgolides with different lipophilicities based on an hCMEC/D3 cell monolayer as a blood-brain barrier cell model.

AIMS: In this report, the transport of ginkgolides with different lipophilicities was investigated using an hCMEC/D3 cell monolayer as a blood-brain barrier (BBB) cell model in vitro in an attempt to explain ginkgolide transport path mediated by lipophilicity. MAIN METHODS: The log P values of ginkgolides were determined by measuring the distribution of the molecule between oil and water. Additionally, the cytotoxicity of ginkgolides on hCMEC/D3 cells was assayed with the MTT method. Ginkgolide contents were determined with an ultra performance liquid chromatograph equipped with an evaporative light scattering detector (ULPC-ELSD) method. Apparent permeability coefficients (Papp) and efflux ratios (PappBL→AP/PappAP→BL) were then calculated to describe the transport characteristics of ginkgolide. KEY FINDINGS: The transport of ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J across the hCMEC/D3 cell monolayer was non-directional. Additionally, ginkgolide C transport on the cell monolayer was time- and concentration-dependent in the paracellular pathway controlled by cytochalasin D (a tight junction modulator). The transport of ginkgolide N, ginkgolide L, and ginkgolide K across the cell monolayer displayed clear directionality at low ginkgolide concentrations. This behavior indicated that the transport of ginkgolide N, ginkgolide L, and ginkgolide K was influenced by the transcellular pathway containing an efflux protein accompanied by the paracellular pathway for passive diffusion. Additionally, the transport of ginkgolide K was increased significantly by co-culturing with a P-gp inhibitor. SIGNIFICANCE: These findings provide important information for elucidating ginkgolide transport pathways and may be beneficial for the design of ginkgolide molecules with high neuroprotective effects.

Neuroprotective effect of Ginkgolide K against H2O2-induced PC12 cell cytotoxicity by ameliorating mitochondrial dysfunction and oxidative stress.

Mitochondria and oxidative stress play important roles in neuronal cell death associated with cerebral ischemia. Elevated level of reactive oxygen species (ROS) and mitochondrial dysfunction are thought to be responsible for cerebral ischemia injury along with neural cells death through several apoptotic mechanisms. In this study, exposure of rat pheochromocytoma (PC12) cells to hydrogen peroxide (H2O2) at the concentration of 0.3 mM for 24 h caused significant loss of cell viability, lactate dehydrogenase (LDH) release from cells, ascent of ROS level and mitochondrial membrane potential (MMP) decrease. Moreover, the activities of caspase-9, caspase-8 and caspase-3 all were increased in H2O2-induced PC12 cells. However, pretreatment with ginkgolide K (GK) solutions of different concentrations (10, 50, 100 µM) for 24 h prior to exposuring to H2O2 significantly increased cells viability, suppressed LDH release, attenuated ROS level, prevented cytochrome c release from mitochondria and boosted MMP expression. In addition, ginkgolide K notably inhibited the caspase-3 and caspase-9 but not caspase-8 activities in exogenous H2O2-treated PC12 cells. These results demonstrated that ginkgolide K protected PC12 cells from H2O2-induced apoptosis by restoring MMP expression, ameliorating oxidative stress and subsequently leading to inhibit the activity of caspase-3 protein. Therefore, the present study supported that ginkgolide K may be a promising neuroprotective compound for cerebral ischemia treatment.

α-Glucosidase inhibitory effect and simultaneous quantification of three major flavonoid glycosides in Microctis folium.

Microctis Folium, the leaves of Microcos paniculata L., is a commonly used herbal tea material. The methanol extract of Microctis Folium and its principle compounds vitexin (1), isovitexin (2) and isorhamnetin 3-O-ß-D-rutinoside (3) were investigated for their α-glucosidase inhibitory effects. The extract showed strong α-glucosidase inhibitory effect (IC50 = 61.30 µg/mL) and the three flavonoid glycosides 1-3 exerted satisfactory α-glucosidase inhibitory effects, with IC50 values of 244.0 µM, 266.2 µM and 275.4 µM, respectively. A simple and reliable HPLC-DAD method was developed for the quantification of the three flavonoid glycosides in Microctis Folium and applied successfully to determine contents of these components in samples collected from different locations. This study suggested that Microctis Folium may be a promising candidate for development of herbal antidiabetes drugs, and vitexin, isovitexin and isorhamnetin 3-O-ß-D-rutinoside can be the biomarkers and chemical markers for this plant substance.

New cycloartane glycosides from the aerial part of Thalictrum fortunei.

Four new cycloartane glycosides, 3-O-ß-D-xylopyranosyl-(1 → 6)-ß-D-glucopyranosyl-(1 → 4)-ß-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3ß,22,26-triol 26-O-(6-O-acetyl)-ß-D-glucopyranoside (1), 3-O-α-L-arabinopyranosyl-(1 → 6)-ß-D-glucopyranosyl-(1 → 4)-ß-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3ß,22,26-triol 26-O-(6-O-acetyl)-ß-D-glucopyranoside (2), 3-O-ß-D-glucopyranosyl (24S)-cycloartane-3ß,16ß,24,25,30-pentaol 25-O-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranoside (3) and 3-O-ß-D-glucopyranosyl (24S)-cycloartane-3ß,16ß,24,25,30-pentaol 25-O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranoside (4), were isolated from the aerial parts of Thalictrum fortunei. Their structures were established on the basis of extensive NMR and HR-ESI-MS analyses, along with acid hydrolysis.

[Research progress of Ypsilandra thibetica, a medicinal plant of Liliaceae].

Ypsilandra thibetica belongs to the family Liliaceae. Its whole plant has the medicinal functions of heat-clearing and detoxifying, relieving congestion and other effects, and is used as the folk medicine to cure scrofula, dysuria embolism and other symptoms. Previous chemical studies revealed that its major and active ingredient is steroidal saponin. Up to now, more than fifty steroidal saponins, mainly composed of spirostan and furostanol types, have been described. Pharmacological and clinical studies have demonstrated that Y. thibetica has anti-tumor, uterine contractions, hemostatic and antibacterial activities, in particular for the treatment of a variety of gynecological hemorrhagic diseases. In an effort to provide references for the advanced research and development of this species, this paper summarized the research progress on its pharmacognosy, including botany and authentication, its isolated secondary metabolites, biological activities and pharmacological applications. In addition, some advantages of this species which could be potentially used as a substitute for Paridis Rhizoma, one of ingredients of the well-known drug "Yunnan Baiyao", together with the future prospect are also briefly included.

[Study on pharmacokinetics of ginkgolide B injection in Beagle dogs].

OBJECTIVE: To study the pharmacokinetics of ginkgolide B injection in Beagle dogs. METHODS: Determined the serum concentration of ginkgolide B by LC-MS and calculated its parameter of pharmacokinetics via DAS 2.0 software. RESULTS: After intravenous drips of 0.62, 2.07 and 10.35 mg/kg ginkgolide B, parameters of pharmacokinetics of ginkgolide B were as follows: Tmax were 0.444, 1, 1 h; Cmax were 0.764, 3.024, 11.013 mg/L; AUC(0-1) were 1.007, 3.644, 16.646 mg x h/Lo. CONCLUSION: Ginkgolide B has two compartment model in Beagle dogs.

Two new saponins from Thalictrum fortunei.

Two new cycloartane glycosides were isolated from the aerial parts of Thalictrum fortunei (Ranunculaceae). The chemical structures of these compounds were elucidated as 3-O-ß-D-glucopyranosyl (1 → 4)-ß-d-fucopyranosyl-(22S,24Z)-cycloart-24-en-3ß,22,26,30-tetraol 26-O-ß-D-glucopyranoside and 3-O-ß-D-glucopyranosyl (1 → 4)-ß-D-fucopyranosyl-(22S,24Z)-cycloart-24-en-3ß,22,26,29-tetraol 26-O-ß-D-glucopyranoside by extensive 1D and 2D NMR methods, HR-ESI-MS, and hydrolysis. Their cytotoxic activities toward human hepatoma Bel-7402 cells, human colon carcinoma LoVo cells, and human non-small-cell lung cancer NCIH-460 cells were evaluated by MTT assay, respectively.

Anti-tumour and immunomodulating activities of diosgenin, a naturally occurring steroidal saponin.

Diosgenin is a naturally occurring steroidal saponin abundantly present in many medical plants. In this study, diosgenin could significantly inhibit the growth of sarcoma-180 tumour cells in vivo, and remarkably increase thymus and spleen weights of S-180-bearing mice and upgrade the secretion level of TNF-α in serum. Moreover, diosgenin could stimulate lymphocyte transformation and enhance phagocytic capability of macrophages in vitro, and remarkably promoted the secretion of NO and TNF-α in macrophages. These results suggested that diosgenin could improve both specific and non-specific cellular immune responses, and the anti-tumour effects of diosgenin were achieved by immunostimulating properties instead of direct cytotoxicity.

Neuroprotective effect of ginkgolide K against acute ischemic stroke on middle cerebral ischemia occlusion in rats.

Ginkgolide K, a natural platelet-activating factor receptor antagonist, was isolated from the leaves of Ginkgo biloba. However, little is known about its neuroprotective effect in ischemia-reperfusion (I/R)-induced cerebral injury. Hence, the present study was carried out to investigate the effect of ginkgolide K on neuroprotection and the potential mechanisms in the rat I/R model induced by middle cerebral artery occlusion (MCAO). The rats were pretreated with ginkgolide K 2, 4 and 8 mg/kg (i.v.) once a day for 5 days before MCAO. Neurological deficit score (NDS), brain water content, 2,3,5-triphenyltetrazolium chloride (TTC) staining and pathology of brain tissue, as well as indexes of oxidative stress [superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) and nitric oxide synthase (NOS)] were measured at 24 h after ischemia. The results indicated that pretreatment with ginkgolide K significantly diminished the volume of infarction and brain water content, and improved NDS. Moreover, ginkgolide K markedly reversed the level of MDA, NO, NOS and SOD to their normal state in serum or cerebral ischemic section. In addition, hematoxylin and eosin staining showed the neuronal injury was significantly improved after being pretreated with ginkgolide K. These findings demonstrate that ginkgolide K exhibits neuroprotective properties through its antioxidative action in MCAO rats.

A triterpenoid from Thalictrum fortunei induces apoptosis in BEL-7402 cells through the P53-induced apoptosis pathway.

Thalictrum fortunei S. Moore, a perennial plant distributed in the southeastern part of China, has been used in Traditional Chinese Medicine for thousands of years for its antitumor, antibacterial and immunoregulatory effects. In order to investigate the active components and the mechanism of the anti-tumor effects of Thalictrum fortunei, the growth inhibitory effects of eight triterpenoids isolated from the aerial parts of the plant on tumor cell lines were examined by 3-(4,5)-dimethylthiazoy1-3,5-diphenyltetrazolium bromide (MTT) assay. The MTT-assay results showed that the inhibitory activity of 3-O-ß-D-glucopyranosyl-(1→4)-ß-D-fucopyranosyl(22S,24Z)-cycloart-24-en-3ß,22,26-triol 26-O-ß-D-glucopyranoside (1) was stronger than that of the other seven tested triterpenoids on human hepatoma Bel-7402 cell line (Bel-7402), human colon lovo cells (LoVo), human non-small cells lung cancer NCIH-460 cells (NCIH-460) and human gastric carcinoma SGC-7901 cells (SGC-7901) after 48 h treatment in vitro, with the IC(50) values of 66.4, 84.8, 73.5, 89.6 µM, respectively. Moreover, the antitumor mechanism of compound 1 on Bel-7402 cell was explored through nucleus dyeing, fluorescence assay, flow cytometry and western blot. The flow cytometric analysis results revealed that compound 1 caused apoptosis and mitochondrial membrane potential (MMP) loss in Bel-7402 cells. A fluorescence assay indicated that intracellular reactive oxygen species (ROS) were markedly provoked by compound 1 treatment compared to control cells. Immunoblot results showed that compound 1 significantly increased the expression levels of cleaved caspase-3, P53 and Bax protein, and decreased the expression level of Bcl-2 protein. These findings indicate that compound 1 inhibits the growth activity of tumor cells, probably through the P53 protein-induced apoptosis pathway.

[Determination of geniposide, crocin and crocetin in different processing products of fructus gardeniae by HPLC-ELSD].

OBJECTIVE: To develop a HPLC- ELSD method for determination the contents of geniposide, crocin and crocetin in different processing products of Fructus Gardeniae. METHODS: The separation was performed in the HyperClone ODS C18 column (250 mm x 4. 6 mm, 5 microm) with linear gradient elution using methanol-water and 0.05% phosphoric acid in water, the flowing rate was 0.8 mL/min, the column temperature was 30 degrees C, and the ELSD parameter was as follow: 70 degrees C as atomization temperature and 2.0 L/min as the gas flowing rate. RESULTS: The contents of geniposide and crocin in raw, yellowish, carbocoal and scorched Fructus Gardeniae decreased with the deepening of processing degree. However, the content of crocetin in carbocoal and scorched Fructus Gardeniae increased comparing with the raw one. CONCLUSION: This is a simple and credible quality control method, and can be used for the quality control and comprehensive evaluation for different processed products of Fructus Gardeniae.

A new decalin derivative from red yeast rice.

A new decalin derivative, monascusic acid A (1), together with a new natural product (2), was isolated from the ethanol extract of red yeast rice. Their structures were elucidated by spectroscopic methods.

Four new cycloartane glycosides from Thalictrum fortunei.

Four new cycloartane glycosides were isolated from the aerial parts of Thalictrum fortunei (Ranunculaceae). The chemical structures of these new glycosides were elucidated as 3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3beta,22,26-triol 26-O-beta-D-glucopyranoside, 3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3beta,22,26-triol 26-O-beta-D-quinovopyranosyl-(1-->6)-beta-D-glucopyranoside, 3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3beta,22,26-triol 26-O-beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranoside, and 3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3beta,22,26-triol 26-O-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside by extensive NMR methods, HR-ESI-MS, and hydrolysis. This is the first report of (22S,24Z)-3beta,22,26-trihydroxycycloartan-24-ene (thelictogenin A, 5) being glycosylated at C-26.

[Chemical constituents from rhizome of Pulsatilla dahurica].

OBJECTIVE: To study the chemical constituents from rhizome of Pulsatilla dahurica. METHOD: The constituents were isolated and purified by various chromatographic methods. AR compounds were identified on the basis of spectral analysis and physico-chemical characters. RESULT: Six compounds were isolated from the 70% alcohol extract of the rhizome identified as hederagenin ( I ), hederagenin 3-O-alpha-L-arabinopyranoside (II), hederagenin 3-O-beta-D-glucopyranosyl(1-->2)-alpha-L-arabinopyranoside (III), hederagenin 3-O-beta-D-glucopyranosyl(1 -->2) [beta-D-glucopyranosyl(1-->4)]-alpha-L-arabinopyranoside (IV), beta-sitosterol (V) and daucosterol (VI), respectively. CONCLUSION: Compounds I approximately VI were isolated from this plant for the first time.