Xiaolin Yang

Antiosteoporotic activity of echinacoside in ovariectomized rats.

PURPOSE Echinacoside (ECH), isolated from Cistanche tubulosa (Schrenk) R. Wight stems, has been reported to enhance bone regeneration in MC3T3-E1 cells in vitro. The objectives of this study were to investigate the antiosteoporotic effect of ECH on bone metabolism in the ovariectomized (OVX) rat model of osteoporosis in vivo. METHODS Fifty-six aged 6 months female Sprague-Dawley rats were randomly assigned into sham-operated group (SHAM) and six OVX subgroups (n=8 each). The OVX rats were then subdivided into six groups treated with vehicle (OVX), Xian-ling-gu-bao (XLGB, 0.5 g/kg body weight/day, orally), 17β-estradiol (E2, 50 μg/kg body weight/day, orally) or ECH (30, 90, and 270 mg/kg body weight, daily, orally) for 12 weeks respectively. We evaluated the pharmacological effects of E2, XLGB and ECH against osteoporosis by evaluating the body weight, uterus wet weight, serum and urine biochemical parameters, bone mineral density (BMD), bone biomechanical properties, bone microarchitecture, bone histomorphology and uterus immunohistochemistry. RESULTS In OVX rats, the increases of body weight, serum hydroxyproline (HOP) levels, and the decreases of uterus wet weight and BMD were significantly reversed by ECH treatment. Moreover, three dosages of ECH completely corrected the increased urine concentration of calcium (Ca), inorganic phosphorus (P), and HOP observed in OVX rats. Furthermore, Micro-CT analysis results of distal femur showed that all ECH-treated groups notably enhanced bone quality compared to OVX group (p<0.05). Consistent with this finding, total femur BMD and biomechanical strength of tibia were significantly improved (p<0.05) after 12 weeks ECH administration. Histological results also showed the protective activity of ECH through promotion of bone formation and suppression of bone resorption. In addition, the ECH administration also significantly enhanced the expression of ER in the uteri according to immunohistochemical evaluation (p<0.05). Those findings, based on the serum and urine biochemical, BMD, Micro-CT, biomechanical test, histopathological and immunohistochemical parameters, showed that ECH has a notable antiosteoporotic effect, similar to estrogen, especially effective for prevention osteoporosis induced by estrogen deficiency. CONCLUSION These results suggest that ECH, as a new class of phytoestrogen, has a remarkable antiosteoporotic activity, and may be a promising candidate for treatment of postmenopausal osteoporosis induced by estrogen deficiency in a natural way through herbal resources.

Anti-rheumatoid arthritic activity of flavonoids from Daphne genkwa

The aim of the study was to investigate the anti-rheumatoid arthritic activity of four flavonoids from Daphne genkwa (FFD) in vivo and in vitro. Flavonoids of D. genkwa were extracted by refluxing with ethanol and purified by polyamide resin. An in vivo carrageenan-induced paw edema model, tampon-granuloma model and Freunds complete adjuvant (FCA)-induced arthritis mouse model were used to evaluate the anti-rheumatoid arthritic activities of FFD. Moreover, nitric oxide (NO) release and neutral red uptake (NRU) in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells were used to evaluate the anti-inflammatory effect in vitro. In addition, antioxidant effect of FFD was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A high dose of FFD significantly reduced the degree of acute inflammatory paw edema in mice as a response to carrageenan administration (p<0.01). FFD displayed a dose-dependent inhibition of granuloma formation in mice (p<0.05). FFD also inhibited chronic inflammation in adjuvant-induced arthritis rats when administered orally at the dose of 50mg/kg/day (p<0.001). In addition, FFD suppressed the production of NO and exhibited immunoregulatory function in LPS-activated RAW264.7 cells in a dose-related manner. Simultaneously, FFD revealed conspicuous antioxidant activity with IC50 values of 18.20μg/ml. FFD possesses significant anti-inflammatory and antioxidant activity, which could be a potential therapeutic agent for chronic inflammatory disorders such as rheumatoid arthritis.

Protective effects of timosaponin B-II on high glucose-induced apoptosis in human umbilical vein endothelial cells

This study was designed to investigate the action of timosaponin B-II, a main bioactive compound in Anemarrhena asphodeloides Bunge, on the prevention from high glucose-induced cytotoxicity and apoptosis in human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved. The results showed that compared with the normal control group, exposure of HUVECs to high glucose media for 72 h resulted in a significant increase in lactates dehydrogenise release, reactive oxygen species production, Caspase-3 activity and the percentage of apoptotic cells (p<0.01). However, pretreatment with timosaponin B-II significantly increased the viability of HUVECs and decreased lactates dehydrogenise release, Caspase-3 activity and the apoptosis rate in a concentration-dependent manner (p<0.05). In addition, timosaponin B-II notably decreased the amount of reactive oxygen species and malondialdehyde, as well as promoted glutathione peroxidase activity, endothelial nitric oxide synthase activity and nitric oxide release (p<0.05). These results suggest that timosaponin B-II has the antiapoptotic effect in endothelial cells through inhibition of high glucose-induced oxidative stress and has the potential for preventing diabetic cardiovascular complications.

Antioxidant effects of Genkwa flos flavonoids on Freund׳s adjuvant-induced rheumatoid arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE Genkwa flos (Daphne genkwa Sieb. et Zucc.), a Chinese herbal medicine, has been traditionally used for over two thousand years in China for inflammation related symptoms, including joint pain. To evaluate the antioxidative effects of flavonoid aglycones (FA) isolated from Genkwa flos on adjuvant arthritis in rats and to identify the relationship between antioxidant potential and whole blood viscosity (WBV). MATERIALS AND METHODS FA compounds were identified using LC-MS and the content was assayed by HPLC. Arthritis was induced by an intradermal injection of Freund׳s complete adjuvant in the footpad. The effects of FA on paw volumes, secondary arthritis scores, histopathology of joints, and body and organ weights were measured. The antioxidant effects of FA and WBV were determined. RESULTS LC-MS analysis showed that the FA contained four major compounds: luteolin, apigenin, hydroxygenkwanin and genkwanin. FA significantly decreased paw edema, arthritis scores, and weight loss. These observations were consistent with the reduction of oxidative stress and the improvement of the WBV. CONCLUSION FA significantly decreased arthritis in a rat model through antioxidant and hemorheological modulatory mechanisms. The Genkwa flos flavonoids may have clinical potential for the treatment of rheumatoid arthritis.

Efficacy and Safety of Echinacoside in a Rat Osteopenia Model

This study aimed to investigate the efficacy and safety of echinacoside (ECH) using an osteopenia rat model. Forty-eight 6-month-old female Sprague-Dawley rats were randomly divided into one sham-operated group (SHAM) and five OVX (ovariectomized) subgroups: SHAM with vehicle 0.5% carboxymethylcellulose sodium (0.5% CMC-Na) and OVX with vehicle (OVX), OVX with 17β-estradiol (E2), and OVX with ECH of graded doses (ECH-L, ECH-M, and ECH-H). The effects of ECH and E2 on serum biochemical parameters, bone mineral density (BMD), bone biomechanical properties, bone microarchitecture, and immunohistochemistry were examined, and safety assessments were also evaluated. The results showed that ECH treatments improved total femur BMD, bone microarchitecture, and biomechanical properties and decreased serum marker levels in comparison to OVX group. Moreover, ECH administration significantly increased osteoprotegerin (OPG) level, and decreased receptor activator of nuclear factor-κB ligand (RANKL) level in serum, as well as in proximal femur. Importantly, ECH treatment ameliorated the lipid parameters without the overall incidences of adverse events of uterus and mammary gland compared to OVX and SHAM groups. This study demonstrated that administration of ECH for 12 weeks can effectively and safely prevent OVX-induced osteoporosis in rats via increasing the OPG/RANKL ratio.

Neuroprotective effects of polygalacic acid on scopolamine-induced memory deficits in mice.

BACKGROUND Polygala tenuifolia Willd is a Traditional Chinese Medicine used for the treatment of learning and memory deficits. Triterpenoid saponins, the main bioactive compounds of Polygala tenuifolia Willd, are easily hydrolyzed to polygalacic acid (PA). PURPOSE The present study was undertaken to investigate the neuroprotective effects of PA on scopolamine-induced cognitive dysfunction and to elucidate its underlying mechanisms of action. METHODS PA (3, 6, and 12 mg/kg) was administered orally to mice for fourteen days, and scopolamine (1 mg/kg) was injected intraperitoneally for fourteen days to induce memory impairment. Memory-related behaviors were evaluated using the Morris water maze. Cholinergic and neuroinflammatory activities were measured in brain tissue. Superoxide dismutase activities, malondialdehyde and reduced glutathione contents were also measured in the brains. RESULTS Treatment with scopolamine significantly increased the escape latency time, decreased the number of crossings, and shortened the time spent in the target quadrant, while PA reversed these scopolamine-induced effects. PA significantly improved cholinergic system reactivity, as indicated by decreased acetylcholinesterase (AChE) activity, increased choline acetyltransferase (ChAT) activity, and elevated levels of acetylcholine (ACh) in the hippocampus and frontal cortex. PA also significantly ameliorated neuroinflammation and oxidative stress in mice. CONCLUSION These results suggest that PA might exert a significant neuroprotective effect on cognitive impairment, driven in part by the modulation of cholinergic activity and neuroinflammation.

enhancement of absorption and bioavailability of echinacoside by verapamil or clove oil

Purpose This present study investigated the absorption kinetics of echinacoside (ECH) in situ and in vitro and its oral bioavailability in rats. Additional aim was to find an agent(s) to promote ECH absorption and oral bioavailability among two efflux proteins and three absorption promoters. Methods ECH absorption behaviors were investigated by everted gut sac model in vitro and single-pass intestinal perfusion model in situ. Pharmacokinetics study was performed to investigate the influences of verapamil and clove oil on ECH bioavailability in vivo. All samples were measured at different time intervals by high performance liquid chromatography. Results The results showed that the effective permeability coefficient (Peff) and apparent permeability coefficient of ECH were 0.83×10−6–3.23×10−6 cm/s and 2.99×10−6–9.86×10−6 cm/s, respectively. The Peff among duodenum, jejunum, and ileum were not statistically different, but they were higher than colon (P<0.01), which demonstrated that intestinal ECH absorption was poor and site dependent. Additionally, verapamil and clove oil significantly increased the jejunal Peff of ECH both in situ and in vitro. Moreover, the bioavailability of ECH in combination with verapamil and clove oil were increased by 1.37-fold (P<0.05) and 2.36-fold (P<0.001), respectively, when compared to ECH group. Overall, verapamil and clove oil facilitated ECH absorption and oral bioavailability. Conclusion The absorption and bioavailability of ECH were enhanced by verapamil and clove oil, respectively, both in vitro and in vivo. Consequently, the combination of verapamil and clove oil with ECH will be a promising and effective approach to promote intestinal absorption and oral bioavailability of ECH.

Absorption characteristics of the total alkaloids from Mahonia bealei in an in situ single-pass intestinal perfusion assay.

AIM To investigate the absorption characteristics of the total alkaloids from Mahoniae Caulis (TAMC) through the administration of monterpene absorption enhancers or protein inhibitors. METHOD The absorption behavior was investigated in an in situ single-pass intestinal perfusion (SPIP) assay in rats. RESULTS The intestinal absorption of TAMC was much more than that of a single compound or a mixture of compounds (jatrorrhizine, palmatine, and berberine). Promotion of absorption by the bicyclic monoterpenoids (borneol or camphor) was higher than by the monocyclic monoterpenes (menthol or menthone), and promotion by compounds with a hydroxyl group (borneol or menthol) was higher than those with a carbonyl group (camphor or menthone). The apparent permeability coefficient (Papp) of TAMC was increased to 1.8-fold by verapamil, while it was reduced to one half by thiamine. The absorption rate constant (Ka) and Papp of TAMC were unchanged by probenecid and pantoprazole. CONCLUSION The intestinal absorption characteristics of TAMC might be passive transport, and the intestinum tenue was the best absorptive site. In addition, TAMC might be likely a substrate of P-glycoprotein (P-gp) and organic cation transporters (OCT), rather than multidrug resistance protein (MRP) and breast cancer resistance protein (BCRP). Compared with a single compound and a mixture of compounds, TAMC was able to be absorbed in the blood circulation effectively.

Simultaneous quantification of Akebia saponin D and its five metabolites in human intestinal bacteria using ultra-performance liquid chromatography triple quadrupole mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ/MS) method was developed for simultaneous quantification of Akebia saponin D (ASD) and its five metabolites in intestinal mixtures of bacteria from human feces. After protein precipitation, the analytes and internal standard (IS), glycyrrhetinic acid, were determined in selected ion recording (SIR) mode with negative ion ESI source. Chromatographic separation was carried out on an ACQUITY UPLC™ BEH C18 column (100mm×2.1mm, 1.7μm) using gradient elution. The mobile phase consisted of solvents A (acetonitrile) and B (0.1% aqueous formic acid) at the flow rate of 0.4mL/min. Each sample was chromatographed within 10.5min including equilibration time. The linearity ranged from 0.1 to 100μg/mL for ASD, and 2-1000ng/mL for five metabolites, Dipsacus saponin A (M1), HN-saponin F (M2), hederagenin-28-O-β-d-glucopyranoside (M3), Akebia saponin PA (M4), hederagenin (M5). The limits of detection (LOD) were 0.41, 0.59, 0.61, 0.55, 0.52 and 0.31ng/mL for ASD, M1, M2, M3, M4 and M5, respectively. The intra- and inter-day precision was all within 11.1% and accuracy ranged from -8.33% to 12.47%. The conversion rate of five metabolites was 41.21% in 24h. The method was validated and successfully applied to quantification of ASD and its five metabolites in human intestinal bacteria.

Determination of chikusetsusaponin V and chikusetsusaponin IV in rat plasma by liquid chromatography–mass spectrometry and its application to a preliminary pharmacokinetic study

A sensitive liquid chromatography-electrospray ionization-mass spectrometry method has been developed and validated for determination of two major bioactive saponins in rat plasma after oral administration of saponins extracted from Rhizoma Panacis Japonici, including chikusetsusaponin V and chikusetsusaponin IV for the first time. Akebia saponin D was used as the internal standard (IS). Plasma samples were prepared by protein precipitation with methanol. A Phenomenex C18 column (150 × 4.6 mm, 4 µm) was used as the analytical column with a mobile phase of acetonitrile and 0.05% aqueous formic acid. Mass spectrometric detection was achieved by single quadrupole mass spectrometer equipped with an electrospray ionization interface operating in negative ionization mode. Calibration curves showed good linearity over the concentration range of 5-500 ng/mL for the two analytes in rat plasma. The lower limit of quantification was 5 ng/mL. The intra- and inter-batch precisions were within 10.3% and accuracy ranged from -3.9 to 5.4%. The method was validated and successfully applied to the preliminary pharmacokinetic study of chikusetsusaponin V and chikusetsusaponin IV in rat plasma after oral administration of saponins extracted from Rhizoma Panacis Japonici.