Hyperxylones A and B, two polycyclic polyprenylated acylphloroglucinols with a benzoyl substituted bicyclo[3.2.1]octane core from Hypericum beanii.

Two new polycyclic polyprenylated acylphloroglucinols (PPAPs) possessing a rare benzoyl substituted bicyclo[3.2.1]octane core, hyperxylones A (1) and B (2), along with three new dearomatized isoprenylated acylphloroglucinols (DIAPs), hyperxylones C - E (3-5), were isolated from the roots of Hypericum beanii. The structures of 1-5 were determined by high-resolution electrospray ionization mass spectroscopy (HRESIMS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses, gauge-independent atomic orbital (GIAO) NMR calculations, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 were biomimetically semi-synthesized starting from 5 and 4, respectively, enabling the correct stereochemical assignment of 5 and 4. Moreover, compounds 1 and 2 showed anti-nonalcoholic steatohepatitis (NASH) activity by inhibiting lipid deposition in L02 cells; compounds 3 and 5 exhibited nitric oxide (NO) inhibitory activity in lipopolysaccharides (LPS)-induced RAW264.7 cells.

Diverse polycyclic polyprenylated acylphloroglucinols with anti-neuroinflammatory activity from Hypericum beanii.

A phytochemical investigation on the roots of Hypericum beanii resulted in the isolation of six new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperberlones A-F, along with fourteen known analogues. The structural characterization of these compounds was carried out by analyzing the HRESIMS data, 1D and 2D NMR spectroscopic data, electronic circular dichroism (ECD) calculations, and gauge-independent atomic orbital (GIAO) NMR calculations. Hyperberlone A (1) was a caged PPAP with a rare tricyclo[4.3.1.03,8]decane carbon skeleton. It was deduced to be biosynthetically generated from hyperbeanol C (8) through key Paternò-Büchi reaction, radical cascade cyclizations, and retro-aldol reaction. Compounds 4, 6, 7, 9, 14, and 16 exhibited significant nitric oxide (NO) production inhibitory effects in lipopolysaccharide (LPS)-induced BV-2 microglial cells with IC50 values of 6.11-25.28 µM. Moreover, compound 4 significantly decreased the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-induced BV-2 microglia, as well as the phosphorylation of JNK.

[Study on drug-target binding kinetics profiles of flavonoids in Chrysanthemum morifolium and xanthine oxidase].

Based on the target occupancy mathematical model, the binding kinetic process of potential active ingredients of lowering uric acid in Chrysanthemum morifolium with xanthine oxidase(XOD) was evaluated. The potential active ingredients of lowering uric acid in Ch. morifolium were screened by UPLC-Q-Exactivems MS technology, reference substance identification and in vitro enzymatic kinetics experiments. The binding kinetic parameters of xanthine oxidase and potential inhibitor in Ch. morifolium were determined by surface plasma resonance(SPR). The verified mathematical model of the XOD target occupancy evaluated the kinetic binding process of inhibitors and xanthine oxidase in vivo. According to UPLC-Q-Exactive MS and reference substance identification, 39 potential uric acid-lowering active ingredients in Ch. morifolium extracts were identified and the inhibitory activities of 23 compounds were determined. Three potential xanthine oxidase inhibitors were screened, namely genistein, luteolin, and apigenin. whose IC_(50 )were 1.23, 1.47 and 1.59 µmol·L~(-1), respectively. And the binding rate constants(K_(on)) were 1.26×10~6, 5.23×10~5 and 6.36×10~5 mol·L~(-1)·s~(-1), respectively. The dissociation rate constants(K_(off)) were 10.93×10~(-2), 1.59×10~(-2), and 5.3×10~(-2 )s~(-1), respectively. After evaluation by different administration methods, the three selected compounds can perform rapid and sustained inhibition of xanthine oxidase in vivo under combined administration. This study comprehensively evaluated the target occupancy process of three effective components in different ways of administration in vivo by UPLC-MS, concentration-response method, SPR technology and xanthine oxidase target occupancy model, which would provide a new research idea and method for screening active ingredients in traditional Chinese medicine.

Pulsed electromagnetic fields prevented the decrease of bone formation in hindlimb-suspended rats by activating sAC/cAMP/PKA/CREB signaling pathway.

Microgravity is one of the main threats to the health of astronauts. Pulsed electromagnetic fields (PEMFs) have been considered as one of the potential countermeasures for bone loss induced by space flight. However, the optimal therapeutic parameters of PEMFs have not been obtained and the action mechanism is still largely unknown. In this study, a set of optimal therapeutic parameters for PEMFs (50 Hz, 0.6 mT 50% duty cycle and 90 min/day) selected based on high-throughput screening with cultured osteoblasts was used to prevent bone loss in rats induced by hindlimb suspension, a commonly accepted animal model to simulate the space environment. It was found that hindlimb suspension for 4 weeks led to significant decreases in femoral and vertebral bone mineral density (BMD) and their maximal loads, severe deterioration in bone micro-structure, and decreases in levels of bone formation markers and increases in bone resorption markers. PEMF treatment prevented about 50% of the decreased BMD and maximal loads, preserved the microstructure of cancellous bone and thickness of cortical bone, and inhibited decreases in bone formation markers. Histological analyses revealed that PEMFs significantly alleviated the reduction in osteoblast number and inhibited the increase in adipocyte number in the bone marrow. PEMFs also blocked decreases in serum levels of parathyroid hormone and its downstream signal molecule cAMP, and maintained the phosphorylation levels of protein kinase A (PKA) and cAMP response element-binding protein (CREB). The expression level of soluble adenylyl cyclases (sAC) was also maintained. It therefore can be concluded that PEMFs partially prevented the bone loss induced by weightless environment by maintaining bone formation through signaling of the sAC/cAMP/PKA/CREB pathway. Bioelectromagnetics. 39:569-584, 2018. © 2018 Wiley Periodicals, Inc.

A selective knockout method for discovery of minor active components from plant extracts: Feasibility and challenges as illustrated by an application to Salvia miltiorrhiza.

Natural products have been recognized to play an invaluable role in drug discovery. However, efficient discovery of minor active constituents from natural sources is challenging due to the low abundance and complex matrices. In this study, we developed a selective knockout method to discover minor bioactive components from complex phytochemical mixtures, using a Chinese medicine as an example. Based on the chromatographic fingerprint, six major components in the ethyl acetate extract of the root of Salvia miltiorrhiza (EASM) were selectively knocked out via high-resolution peak fraction (HRPF) approach. The remaining extract was automatically enriched and fractionated to generate a chemical library consisting of 62 minor components with contents less than 3‰. Simultaneously, a parallel mass-spectrometry (MS) analysis was performed to ensure purity and to characterize the structure of the compound in each fraction. Via an antioxidant response element (ARE)-driven luciferase reporter system, 33 minor components were screened out as nuclear factor erythroid 2-related factor 2 (Nrf2) activators and 30 components were identified. Here, the Nrf2 activation activities of 21 components have been reported for the first time. Different from the existing methods for discovery of active compounds from natural products, in the developed method of this manuscript, the major components are selectively removed, and the fractions of the minor components are prepared after several times of preparative HPLC enrichment by high-resolution peak fraction approach. It improves the prospective discovery of minor active components from complex medicinal herbs.

[Effect of flavonoids from hedysari radix on pulmonary functions of pulmonary fibrosis rat].

OBJECTIVE: To study the effect of flavonoids from Hedysari Radix on pulmonary functions of pulmonary fibrosis rat and its mechanism. METHODS: 72 Wistar rats were randomly divided into 6 groups: blank control group, model group, prednisone group, Hedysari Radix flavonoids low, medium and high dosage group. The rat model was established by propelling bleomycin into bronchial tree through endotracheal intubation with laryngoscope. The pulmonary fanctions were measured. RESULTS: Hedysari Radix flavonoids could normalize the pulmonary functions of rats with bleomycin-induced pulmonary fibrosis. CONCLUSION: Hedysari Radix flavonoids can inhibit the process of pulmonary fibrosis.

[Comparative study of Hedysari Radix and Astragali Radix alternative classic tonification prescriptions on humoral immunity in immunosuppressed mice].

OBJECTIVE: To compare the regulating effects of Hedysari Radix and Astragali Radix alternative classic tonification prescriptions on humoral immunity in immunosuppressed mice. METHODS: The immunosuppressed mouse model was induced by cyclophosphamide. The mice were administered intragastically with same dose of Hedysari Radix and Astragali Radix alternative Buzhong Yiqi Yiqi Yangxue,Yupingfeng oral liquid and Fuqi Zhihan granules for antagonistic experiments in vivo. And spleen index, HC50, CD19+B lymphocyte subgroup and content of serum IL-4 were determined after treatment. RESULTS: Both groups of Hedyseri Radix and Astragali Radix could antagonize immunosuppressive action caused by cyclophosphamide. They both could significantly raise spleen index, HC50, CD19+ B lymphocyte subgroup and content of serum IL4 in different degree. And Yupingfeng aqueous extract of Hedysari Radix substitute Astragali Radix was better than Yupingfeng oral liquid in raising spleen index. There were no significant differences among the rest Hedysari Radix and Astragali Radix alternative groups. CONCLUSION: Hedysari Radix compatibility with other drugs compared with original prescription has similar role in humoral immunity regulation.

[Comparative study of Radix Hedyseri as sulstitute for Radix Astragali of yupingfeng oral liquid on cellular immunity in immunosuppressed mice].

OBJECTIVE: To study on Radix Hedyseri as substitute for Radix Astragali of Yupingfeng oral liquid on cellular immunity in immunosuppressed mice. METHODS: The model of immunosuppression mice were induced by Cyclophosphamide. And the same dose of Radix Hedyseri and Radix Astragali alternative Yupingfeng oral liquid was intragastric administrated into mice; Antagonistic experiments were observed in vivo. Determined the thymus gland index, spleen index, phagocytosis of the macrophage, proliferation index of T lymphocyte, kill and wound activity, T lymphocyte subgroup, and content of IL-13 of serum. RESULTS: Yupingfeng oral liquid and Yuping-feng aqueous extract of Radix Hedyseri substitute Radix Astragali both could significant raise thymus gland index and spleen index, and clearly increase the phagocytosis of the macrophage. They both could antagonize immunosuppressive action caused by Cyclophosphamide, which could promote T lymphocyte proliferation, kill and wound activity, quantity of T lymphocyte subgroup, and production of IL-1beta with different degree. And Yupingfeng aqueous extract of Radix Hedyseri substitute Radix Astragali increased spleen index and T lymphocyte proliferation was better than those of Yupingfeng oral liquid. CONCLUSION: Radix Hedyseri and Radix Saposhnikoviae, compatible with Rhizoma Atractylodis Macrocephalae compared with Yupingfeng oral liquid in cell immunity regulation has a similar role, and better in the recovery of spleen weight and T cell proliferation.

Emodin protects rat liver from CCl(4)-induced fibrogenesis via inhibition of hepatic stellate cells activation.

AIM: To investigate the role of emodin in protecting the liver against fibrogenesis caused by carbon tetrachloride (CCl(4)) in rats and to further explore the underlying mechanisms. METHODS: Rat models of experimental hepatic fibrosis were established by injection with CCl(4); the treated rats received emodin via oral administration at a dosage of 20 mg/kg twice a week at the same time. Rats injected with olive oil served as a normal group. Histopathological changes were observed by hematoxylin and eosin staining. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and hepatic hydroxyproline content were assayed by biochemical analyses. The mRNA and protein relevant to hepatic stellate cell (HSC) activation in the liver were assessed using real-time reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, western blotting and enzyme-linked immunosorbent assay. RESULTS: The degree of hepatic fibrosis increased markedly in the CCl(4) group compared to the normal group (P < 0.01), and decreased markedly in the emodin group compared to the CCl(4) group according to METAVIR scale (P < 0.01) compared with those in the normal control group (51.02 +/- 10.64 IU/L and 132.28 +/- 18.14 IU/L). The activities of serum ALT and AST were significantly higher in rats injected with CCl(4) (289.25 +/- 68.84 IU/L and 423.89 +/- 35.67 IU/L, both P < 0.05). The activities of serum ALT and AST were significantly reduced by administration of emodin (176.34 +/- 47.29 IU/L and 226.1 +/- 44.52 IU/L, both P < 0.05). Compared with the normal controls (54.53 +/- 13.46 mg/g), hepatic hydroxyproline content was significantly higher in rats injected with CCl(4) (120.27 +/- 28.47 mg/g, P < 0.05). Hepatic hydroxyproline content was significantly reduced in the rats treated with emodin at 20 mg/kg (71.25 +/- 17.02 mg/g, P < 0.05). Emodin significantly protected the liver from injury by reducing serum AST and ALT activities and reducing hepatic hydroxyproline content. The mRNA levels of transforming growth factor-beta1 (TGF-beta1), Smad4 and alpha-SMA in liver tissues were significantly down-regulated in SD rats that received emodin treatment. Furthermore, significant down-regulation of serum TGF-beta1 protein levels and protein expression of Smad4 and alpha-SMA in liver tissues was also observed in the rats. Emodin inhibited HSC activation by reducing the abundance of TGF-beta1 and Smad4. CONCLUSION: Emodin protects the rat liver from CCl(4)-induced fibrogenesis by inhibiting HSC activation. Emodin might be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.