Humulus lupulus L. Extract Prevents Ovariectomy-Induced Osteoporosis in Mice and Regulates Activities of Osteoblasts and Osteoclasts / 中国结合医学杂志

OBJECTIVE@#To systematically evaluate the protective effects of Humulus lupulus L. extract (HLE) on osteoporosis mice.@*METHODS@#In vivo experiment, a total of 35 12-week-old female ICR mice were equally divided into 5 groups: the sham control group (sham); the ovariectomy with vehicle group (OVX); the OVX with estradiol valerate [EV, 0.2 mg/(kg•d)] the OVX with low- or high-dose HLE groups [HLE, 1 g/(kg•d) and 3 g/(kg•d)], 7 in each group. Treatment began 1 week after the ovariectomized surgery and lasted for 12 weeks. Bone mass and trabecular bone mircoarchitecture were evaluated by micro computed tomography, and bone turnover markers in serum were evaluated using enzyme-linked immunosorbent assay (ELISA) kits. In vitro experiment, osteoblasts and osteoclasts were treated with HLE at doses of 0, 4, 20 and 100 µg/mL. Biomarkers for bone formation in osteoblasts and bone resorption in osteoclasts were analyzed.@*RESULTS@#Compared with the OVX group, HLE exerted bone protective effects by the increase of estradiol (P<0.05), the improvement of cancellous bone structure, bone mineral density (P<0.01) and the reduction of serum alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), bone gla-protein, c-terminal telopeptides of type I collagen (CTX-I) and deoxypyridinoline levels (P<0.01 for all). In vitro experiment, compared with the control group, HLE at 20 µg/mL promoted the cell proliferation (P<0.01), and increased the expression of bone morphogenetic protein-2 and osteopontin levels in osteoblasts (both P<0.05). HLE at 100 µg/mL increased the osteoblastic ALP activities, and HLE at all dose enhanced the extracellular matrix mineralization (both P<0.01). Furthermore, compared with the control group, HLE at 20 µg/mL and 100 µg/mL inhibited osteoclastic TRAP activity (P<0.01), and reduced the expression of matrix metalloproteinase-9 and cathepsin K (both P<0.05).@*CONCLUSION@#HLE may protect against bone loss, and have potentials in the treatment of osteoporosis.

Effects of icariin on bone metabolism in iron overload mice / 药学学报

This study was designed to investigate the effects of icariin on bone metabolism in osteoprotic mice induced by iron overload, the model of iron overload mice was established by intraperitoneal injection of iron dextran (100 mg·kg-1). Sixty 2-month-old C57/BL6 male mice were randomly divided into six groups, including normal control group, model group, N-acetyl-L-cysteine (NAC)-treated group, icariin (50, 100 and 200 mg·kg-1)-treated group. Except for the mice in control group, the mice were intraperitoneal injected weekly with iron dextran (100 mg·kg-1) to establish the model of iron overload mice. The NAC and icariin were suspended in 0.5% CMC-Na solution, and administered orally for six times one week according to body weight. The mice in normal group and model group were given the same volume of 0.5% CMC-Na solution. Three months later, the organs, serum and femurs of mice were collected. Serum biochemical parameters were detected with an ELISA kit, the distal femur bone density and trabecular bone microstructure were analyzed by Micro-CT, and the mechanical properties of femur were measured by universal mechanical analyzer. Compared with the normal control group, iron overload decreased the bone mineral density and deteriorated the micro-architecture structure and bone mechanical properties in femur of mice, increased the level of iron, phosphorus and activity of tartrate resistant acid phosphatase-5b (TRACP-5b), reduced the level of osteocalcin (OCN) in serum, decreased the activity of superoxide dismutase (SOD) of liver tissues, increased the content of malondialdehyde (MDA) of liver tissues. Icariin increased bone mineral density, improved the micro-architecture and mechanical properties of bone tissue, reduced the levels of iron and phosphorus, decreased the activity of TRACP-5b and enhanced the levels of OCN in serum, and also decreased the activity of MDA in liver tissue of iron overload mice. These results suggest that icariin is able to reduce bone loss and improve bone microstructure and mechanical properties in iron overload mice through regulation of bone metabolism via anti-oxidation.

Study on exosite inhibitor of cathepsin K from Herba Ecliptae fractions / 药学学报

Cathepsin K (CTSK) is considered a critical pharmaceutical target in the treatment of osteoporosis. CTSK exerts proteolytic activities against regulatory proteins besides its collagenase function, which may account for some of the adverse reactions when blocked by active site-directed inhibitors. Exosite inhibitors that can discriminate between the therapeutic collagenase and other biological activities of CTSK specifically inhibit the collagenase activity of CTSK without interfering with the other proteolytic activities of the protease. Active recombinant CTSK was expressed in Pichia pastoris, and purified by n-butyl sepharose and SP sepharose column chromatography. Herba Ecliptae is a common traditional Chinese medicine in the treatment of bone diseases. Collagenase assay and benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) substrate assay based on CTSK are applied to verify the exosite inhibitors. n-Butanol extract of Herba Ecliptae are the most active fraction and eclalbasaponin IX isolated from n-butanol fraction is the potential exosite inhibitor of CTSK.

Research progress of chemical constituents and pharmacological activities in Humulus lupulus / 中国中药杂志

Hops, the female inflorescences of the hop plant (Humulus lupulus), are widely used in the brewing industry to add bitterness and aroma to beer. Combining with the relevant literature, the chemical composition(resinae, volatile oil, polyphenol and polysaccharide) in hops and their pharmacological effects are reviewed in this paper so as to present some sights for further application research and development.

Estrogenic activity of osthole and imperatorin in MCF-7 cells and their osteoblastic effects in Saos-2 cells / 中国天然药物

There is an increasing interest in phytoestrogens due to their potential medical usage in hormone replacement therapy (HRT). The present study was designed to investigate the in vitro effects of estrogen-like activities of two widespread coumarins, osthole and imperatorin, using the MCF-7 cell proliferation assay and their alkaline phosphatase (ALP) activities in osteoblasts Saos-2 cells. The two compounds were found to strongly stimulate the proliferation of MCF-7 cells. The estrogen receptor-regulated ERα, progesterone receptor (PR) and PS2 mRNA levels were increased by treatment with osthole and imperatorin. All these effects were significantly inhibited by the specific estrogen receptor antagonist ICI182, 780. Cell cycle analysis revealed that their proliferation stimulatory effect was associated with a marked increase in the number of MCF-7 cells in S phase, which was similar to that observed with estradiol. It was also observed that they significantly increased ALP activity, which was reversed by ICI182,780. These results suggested that osthole and imperatorin could stimulate osteoblastic activity by displaying estrogenic properties or through the ER pathway. In conclusion, osthole and imperatorin may represent new pharmacological tools for the treatment of osteoporosis.

Dynamic changes of flavonoids in Actinidia valvata leaves at different growing stages measured by HPLC-MS/MS / 中国天然药物

Flavonoids are a large group of phenolic secondary metabolites havinga wide range of biochemical and pharmacological effects. Quantitative analysis of flavonoid profiles in the genus Actinidia, which has not been intensively conducted, is useful to a better understanding of the pattern and distribution of flavonoids. In the present work, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed to profile the flavonoids, which was then used to determine the dynamic change of 17 biologically active flavonoids in the leaves of Actinidia valvata at the main growing stages, including glucuronides and acylated di- and triglycosides of flavonoids. The contents of flavonoid triglycosides were significantly higher than other flavonoids. The highest concentrations of kaemperol glycosides were observed in June, while other flavonoids showed highest concentrations in October. On the other hand, the contents of four isorhamnetin glycosides were increased sharply in September to October. The flavonoid profiles seem to be related to temperature, UV-B, and water deficit. Further studies are required to examine the functions of flavonoids in the Actinidia valvata and the underlying molecular mechanisms of actions.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method for determination of actinoside E in rat plasma and application to a pharmacokinetic study / 中国天然药物

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of actinoside E in rat plasma. The analytes were extracted by ethyl acetate and an analogue of actinoside F was used as the internal standard. The mobile phase consisted of methanol-water (50: 50, V/V) containing 0.1% formic acid was delivered at a flow rate of 0.3 mL·min(-1) to a Zorbax SB-C18 column (100 mm × 2.1 mm, 3.5 μm). The detection was performed by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatograph run time of 3.0 min. Calibration curves of actinoside E were linear in the range of 0.5-2 500 ng·mL(-1). In this range, intra- and inter-day precision ranged from 1.7% to 7.5% and 2.0% to 8.9%, respectively. The accuracy ranged from 95.7% to 108.6%, and extraction recovery from 83.2% to 85.5%. This method was successfully applied to a pharmacokinetic study of actinoside E in rats after intravenous (5 mg·kg(-1)) and oral (100 mg·kg(-1)) administration, and the results showed that actinoside E was poorly absorbed with an absolute bioavailability being approximately 0.27%.

Total saponin from root of Actinidia valvata Dunn prevents the metastasis of human hepatocellular carcinoma cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To extract the active component from the root of Actinidia valvata Dunn and to investigate the effects on hepatocellular carcinoma (HCC) cells in vitro.</p><p><b>METHODS</b>Total saponin was extracted from the root of A. valvata (TSAVD). HCC cells, such as BEL-7402, HepG2, PLC, SMMC-7721, MHCC-97-H, and MHCC-97-L, were treated with TSAVD in 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay. BEL-7402 and MHCC-97-H cells were also treated respectively with TSAVD at different concentrations for 24 h in wound healing and adhesion assays, and the effects of TSAVD on BEL-7402 and MHCC-97-H cells mobility and adhesion abilities were observed. Meanwhile, the effects of TSAVD on invasion and migration of BEL-7402 and MHCC-97-H cells were also investigated by transwell chamber in invasion and migration assays.</p><p><b>RESULTS</b>TSAVD at 1.5 mg/mL inhibited BEL-7402 cell proliferation with inhibition ratios (IRs) of 61.08%, 74.12%, 84.55% at 24, 48, and 72 h, respectively. Meanwhile, TSAVD inhibited MHCC-97-H proliferation in a concentration-dependent manner from 1.5 to 0.5 mg/mL, with the IR of 36% at 1.5 mg/mL at 24 h. For SMMC-7721, PLC, and HepG2, the IR was lower than 30% at 1.5 mg/mL at 24 h. In the wound healing assay, mobility abilities of BEL-7402 and MHCC-97-H cells in TSAVD treated groups were significantly weaker than those of the control group. After pretreatment for 24 h with TSAVD, adhesion abilities were reduced in both MHCC-97-H and BEL-7402 cells, with IRs of 48.50%±4.86% and 49.85%±5.25% at 200 μg/mL. The IRs of MHCC-97-H and BEL-7402 cells in the migration assay were 49.13%±2.91% and 79.37%±0.09% at 200 μg/mL. In the invasion assay, IRs were 69.78%±4.88% and 82.48%±0.25% at 200 μg/mL.</p><p><b>CONCLUSIONS</b>Of all HCC cells, the highest inhibition by TSAVD was seen for BEL-7402 proliferation. TSAVD could restrain adhesion, invasion, mobility, and migration abilities of BEL-7402 and MHCC-97-H cells in vitro.</p>

A comparison between the root and stalk of Actinidia valvata / 第二军医大学学报

Objective: To make a comparative study on the root and stalk of Actinidia valvata, so as to lay a ground for expanding medicinal part of Actinidia valvata. Methods: A comparative study was made between the root and stalk of Actinidia valvata on the following aspects: macroscopical properties, physical and chemical properties, contents of active constituents, and pharmacological actions. Results: The root and stalk of Actinidia valvata both showed positive reaction for alkaloids and saponin, and both had 280 nm absorption peak in UV-Vis spectrum. The aqueous extraction yield of the root and stalk were 5.35% and 5.68%, respectively; the contents of dihydrodehydrodiconiferyl alcohol were 0.005 01% and 0.006 41%, respectively. The IC50 of root and stalk extracts against HL60 were 62.39 mg and 75.51 mg, respectively; and against K562 were 70.47 mg and 77.46 mg, respectively. Their extracts had similar scavenging activity for free radicals and reductive effect; neither of them had analgesia actions. Conclusion: The root and stalk of Actinidia valvata have similar physical and chemical properties, contents of active constituents and pharmacological actions, suggesting that it is highly possible to substitute the root of Actinidia valvata with stalk for medicinal usage.