Neuroprotection of total steroid saponins from Dioscorea zingiberensis against transient focal cerebral ischemia-reperfusion injury in rats via anti-inflammatory and antiapoptotic effects.

Total steroid saponins isolated from Dioscorea zingiberensis have shown a variety of beneficial bioactivities. However, there are no reports about their neuroprotective effects, until now. Therefore, in the present study, we explored the neuroprotective effects of the total steroid saponins from D. zingiberensis on rats against transient focal cerebral ischemia-reperfusion and their underlying mechanisms. Healthy adult Sprague-Dawley rats were randomly assigned to six groups. After pretreatment with D. zingiberensis total steroid saponins intragastrically for six days, the rats were subjected to an ischemia injury by surgery on the middle cerebral artery occlusion for 90 min. Compared to the ischemia-reperfusion group, the D. zingiberensis total steroid saponin group of rats, especially those given a 30-mg/kg dose, showed not only a marked reduction in neurological deficit scores, cerebral infarct volume, and brain edema, but also an increase in neuron survival (Nissl bodies) in the hippocampal cornuammons 1 and cortex hemisphere of the ipsilateral ischemia. At the same time, the inflammatory cytokines in serum induced by the middle cerebral artery occlusion were significantly decreased by the preadministration of D. zingiberensis total steroid saponins. Furthermore, the increase of caspase-3 was evidently reduced in the hippocampal cornuammons 1 and cortex of the hemisphere injured brain. Finally, the downregulating antiapoptotic Bcl-2 and upregulating proapoptotic Bax proteins were obviously suppressed. Taken together, the current findings suggest that D. zingiberensis total steroid saponins played a potential neuroprotective role against a severe injury induced by transient focal cerebral ischemic reperfusion in a rat experimental model, and this role may be mediated by its anti-inflammatory and antiapoptotic actions.

[Study on HPLC-eLSD fingerprint of total steroid saponins in herbs of Dioscorea zingiberensis].

To establish a HPLC-ELSD fingerprint for total steroid saponins in herbs of Dioscorea zingiberensis. Welchrom C,8 (4. 6 mm x 250 mm,5 microm) chromatographic column was adopted and eluted with the mobile phase of acetonitrile (A)-water (B) at the flow rate of 1.0 mL min-1. The column temperature was room temperature. The ELSD conditions were as follows: the temperature of drift tube was 90.0 degreeC, the flow rate of carrier gas (N2) was 2. 8 L min-1, and the injection volume was 10 microL. After the detection of 10 batches of samples,the common mode of HPLC-ELSD fingerprint for total steroid saponins in herbs of D. zingiberensis was established for the first time,and 25 common peaks were determined. Among them, 10 peaks were identified by comparing with reference substances. The similarities of 10 batches of herbs were evaluated in the common mode. All of them were higher than 0. 80. This method is so accurate, reliable and highly repeatable that it can provide scientific basis for evaluating and controlling the quality of total steroid saponins in herbs of D. zingiberensis.

Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

BACKGROUND: Mushrooms have been used in Asia as traditional foods and medicines for a long time. Ergosta-4,6,8(14),22-tetraen-3-one (ergone) is one of the well-known bioactive steroids, which exists widely in various medicinal fungi such as Polyporus umbellatus, Russula cyanoxantha, and Cordyceps sinensis. Ergone has been demonstrated to possess cytotoxic activity. However, the molecular mechanisms by which ergone exerts its cytotoxic activity are currently unknown. METHODS: In the present study, ergone possessed a remarkable anti-proliferative activity toward human hepatocellular carcinoma HepG2 cells. We assayed the cell cycle by flow cytometry using PI staining; investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V/PI staining; observed the nuclear fragmentation by Hoechst 33258 staining and studied the protein expression of Bax, Bcl-2, p-53, procaspase-3, -8, -9, PARP and cleaved PARP by Western blotting analysis. RESULTS: Cells treated with ergone showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. Furthermore, PARP-cleavage; activation of caspase-3, -8, -9; up-regulation of Bax and down-regulation of Bcl-2 were observed in HepG2 cells treated with ergone, which show that both the intrinsic and extrinsic apoptotic pathways are involved in ergone-induced apoptosis in HepG2 cells. Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in HepG2 cells in a caspase-dependent manner. GENERAL SIGNIFICANCE: In this study, we reported for the first time that ergone-induced apoptosis through activating the caspase. These results would be useful for the further utilization of many medicinal fungi in cancer treatment.

Serum metabonomics study of adenine-induced chronic renal failure in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

An ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC Q-TOF MS) metabonomics approach was employed to study the serum metabolic profiling of adenine-induced chronic renal failure (CRF) rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the CRF and the normal control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass, isotopic pattern and MS/MS fragments information obtained from UPLC Q-TOF MS analysis. Significant differences in the serum level of creatinine, amino acids and LysoPCs were observed, indicating the perturbations of amino acid metabolism and phospholipid metabolism in adenine-induced CRF rats. This research proved that metabonomics is a promising tool for disease research.

Effect of total glycosides from Eucommia ulmoides seed on bone microarchitecture in rats.

Du-Zhong is one of the most important tonic herbs in traditional Chinese medicine for the treatment of bone fractures and other bone diseases. The present study examined whether Du-Zhong seed extract named total glycosides from Eucommia ulmoides seed (TGEUS) could display an increased effect on bone density and bone strength of the femur in rats. Sprague-Dawley rats were used and randomly assigned to the normal group and the TGEUS group (400 mg/kg body weight/day). Daily oral administration of TGEUS was found to increase significantly the biomechanical quality of the femur. The mechanical changes were associated with a bone mineral density (BMD) increase or even with some improvements in microarchitecture. Micro-CT analysis of the distal femur showed that TGEUS significantly increased the bone volume/tissue volume (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N) and trabecular thickness (Tb.Th), and decreased trabecular separation (Tb.Sp) and the structure model index (SMI) in normal rats. To conclude, TGEUS taken orally increased bone density and altered bone histomorphology, suggesting that TGEUS might be a potential alternative medicine for the treatment of postmenopausal osteoporosis.

Cytotoxic steroids from Polyporus umbellatus.

The steroids ergone (1), (22E, 24R)-ergosta-7,22-dien-3ß-ol (2), 5α,8α-epidioxy-(22E,24R) -ergosta-6,22-dien-3ß-ol (3), ergosta-6,22-dien-3ß,5α,6ß-triol (4), and polyporusterone B (5) were isolated from Polyporus umbellatus by bioassay-guided approach. They showed potent anticancer activity against HepG2 cells. Ergone displayed remarkable anticancer activity against HepG2, Hep-2, and Hela cancer cells, of which HepG2 cells were the most sensitive. Furthermore, the cytotoxic effects of ergone on normal human cells (HUVEC) were smaller than on cancer cells. The results showed that ergone had more selective cytotoxic activity against cancer cells than against normal cells.

Quantitative HPLC method and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one, a natural product with diuretic activity from Polyporus umbellatus.

A simple and specific HPLC method with dual wavelength UV detection for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma was developed and proved to be efficient. The method used ergosterol as internal standard (IS). Following a single-step protein precipitation, the analyte and IS were separated on an Inertsil ODS-3 column with a mobile phase containing methanol-water (99:1, v/v) at a flow rate of 1 mL/min. The analytes were detected by using UV detection at wavelength of 350 (ergone) and 283 (IS) nm, respectively. The calibration curve was linear over the range of 0.1-2.0 µg/mL and the lower limit of quantification was 0.1 µg/mL. The intra-day and inter-day precision studies showed good reproducibility with RSD less than 8.5%. The intra-day and inter-day accuracy ranged from 95.6 to 104%. Mean extraction recovery was above 95% at the low, medium and high concentrations. The present HPLC-UV method was simple and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in male SD rats after administration of 20 mg/kg dose of solution of ergone.

Simultaneous determination of eight major steroids from Polyporus umbellatus by high-performance liquid chromatography coupled with mass spectrometry detections.

Polyporus umbellatus is a widely used diuretic herbal medicine. In this study, a high-performance liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometric detection (HPLC-APCI-MS) method was developed for qualitative and quantitative analysis of steroids, as well as for the quality control of Polyporus umbellatus. The selectivity, reproducibility and sensitivity were compared with HPLC with photodiode array detection and evaporative light scattering detection (ELSD). Selective ion monitoring in positive mode was used for qualitative and quantitative analysis of eight major components and beta-ecdysterone was used as the internal standard. Limits of detection and quantification fell in the ranges 7-21 and 18-63 ng/mL for the eight analytes with an injection of 10 microL samples, and all calibration curves showed good linear regression (r(2) > 0.9919) within the test range. The quantitative results demonstrated that samples from different localities showed different qualities. Advantages, in comparison with conventional HPLC-diode array detection and HPLC-ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements along with characteristic retention time, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Polyporus umbellatus matrixes.

[Determination of rosmarinic acid in seeds and its processed products of Perilla frutescens var. typica and Perilla frutescens var. acuta].

OBJECTIVE: To determine the content of rosmarinic acid in seeds and its processed products of Perilla frutescens var. typica (PFT), Perilla frutescens var. acuta (PFA) and Perilla frutescens var. acuta form. discolor (PFAD). METHODS: The content of rosmarinic acid was determined by RP-HPLC with Welch C18 (250 mm x 4.6 mm, 5 microm) column, using methanol-phosphonic acid solution (39:61) as mobile phase at flow rate of 1.0 mL/min. UV detection wavelength was set at 330 nm and column temperature was set at 28 degrees C. RESULTS: The content of rosmarinic acid in roasted seeds of PFT is higher than in its seeds, but that of PFA and PFAD are lower; The content of rosmarinic acid in seeds of PFT, PFA and PFAD decline after the seeds are honey-pocessed and made into frost-like powder, and that of honey-processed one decline by a larger margin. CONCLUSION: The content of rosmarinic acid in seeds of PFT, PFA and PFAD are prone to decline by heat-processing.

Qualitative and quantitative analysis of the diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS.

Polyporus umbellatus is a widely used anti-aldosteronic diuretic in Traditional Chinese medicine (TCM). A new, sensitive and selective high-performance liquid chromatography-fluorescence detector (HPLC-FLD) and high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS/MS) method for quantitative and qualitative determination of ergosta-4,6,8(14),22-tetraen-3-one(ergone), which is the main diuretic component, was provided for quality control of P. umbellatus crude drug. The ergone in the ethanolic extract of P. umbellatus was unambiguously characterized by HPLC-APCI, and further confirmed by comparing with a standard compound. The trace ergone was detected by the sensitive and selective HPLC-FLD. Linearity (r2 > 0.9998) and recoveries of low, medium and high concentration (100.5%, 100.2% and 100.4%) were consistent with the experimental criteria. The limit of detection (LOD) of ergone was around 0.2 microg/mL. Our results indicated that the content of ergone in P. umbellatus varied significantly from habitat to habitat with contents ranging from 2.13 +/- 0.02 to 59.17 +/- 0.05 microg/g. Comparison among HPLC-FLD and HPLC-UV or HPLC-APCI-MS/MS demonstrated that the HPLC-FLD and HPLC-APCI-MS/MS methods gave similar quantitative results for the selected herb samples, the HPLC-UV methods gave lower quantitative results than HPLC-FLD and HPLC-APCI-MS/MS methods. The established new HPLC-FLD method has the advantages of being rapid, simple, selective and sensitive, and could be used for the routine analysis of P. umbellatus crude drug.

Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain.

Gentiopicroside is one of the secoiridoid compound isolated from Gentiana lutea. It exhibits analgesic activities in the mice. The anterior cingulate cortex (ACC) is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission and induce glutamate NMDA NR2B receptor expression in the ACC. But little is known about Gentiopicroside on the persistent inflammatory pain and chronic pain-induced synaptic transmission changes in the ACC. The present study was undertaken to investigate its analgesic activities and central synaptic modulation to the peripheral painful inflammation. Gentiopicroside produced significant analgesic effects against persistent inflammatory pain stimuli in mice. Systemic administration of Gentiopicroside significantly reversed NR2B over-expression during the chronic phases of persistent inflammation caused by hind-paw administration of complete Freunds adjuvant (CFA) in mice. Whole-cell patch clamp recordings revealed that Gentiopicroside significantly reduced NR2B receptors mediated postsynaptic currents in the ACC. Our findings provide strong evidence that analgesic effects of Gentiopicroside involve down-regulation of NR2B receptors in the ACC to persistent inflammatory pain.

[Quantitative analysis of two major secoiridoid glucosides in fruits of Ligustrum lucidum].

OBJECTIVE: To develop an HPLC method for determination of two major secoiridoid glucosides (nuezhenoside and G13) from Fructus Ligustri Lucidi. and determinae nuezhenoside and G13 in samples from different acquisition times and different places. METHOD: The sample was separated on a YMG-C18 (4.6 mm x 250 mm, 10 microm) column. The mobile phase was acetonitrile-water, gradient elution (CH3CN: 0 min, 20%; 15 min, 25%) with the flow rate of 1 mL x min(-1), the detection wavelength was 240 nm, and the column temperature was set at 25 degrees C. RESULT: In HPLC analysis ,the linear ranges of nuezhenoside and G13 were 0.341-34.1 microg and 0.331-33.1 microg, respectively; their average recoveries were 97.5% (RSD 1.35%) and 98.1% (RSD 1.82%), respectively. The results of content determination (nuezhenoside and G13) of samples from different places varied greatly. This may be caused by different species sources and different preparation methods, etc. The experiment led up to the fact that from the beginning to the middle of November the content of nuezhenoside and G13 reached the maximum. CONCLUSION: The established method can be used to determine two major secoiridoid glucosides (nuezhenoside and G13) in Fructus Ligustri Lucidi simultaneously. Meanwhile it can be used for the quality control of Fructus Ligustri Lucidi.

Cinchonidinium chloride monohydrate.

In the title salt, C(19)H(23)N(2)O(+)·Cl(-)·H(2)O, the ions and the water mol-ecule are held together by O-H⋯Cl, N-H⋯Cl, O-H⋯O, O-H⋯N and C-H⋯Cl hydrogen bonds, forming a three-dimensional framework. The vinyl group is disordered over two orientations with refined occupancies of 0.564 (16) and 0.436 (16). The cell parameters of the title compound have been reported previously [Griffiths (1952 ▶). Acta Cryst.5, 290-291].

[Enrichment process of gentiopicroside from the leaves of Gentiana macropylla with macroporous resin].

OBJECTIVE: To select the best type of macroporous resin for enriching gentiopicroside from the leaves of Gentiana macropylla, and study its optimal conditions and parameters. METHODS: Adsorption and desorption characteristics of gentiopieroside were carried out on the resins. HPLC was used to determine the content of gentiopieroside. RESULTS: The better resin D-101 was chosen, and the optimal parameters were obtained. CONCLUSION: The process is feasible to enrich gentiopicroside from the leaves of Gentiana macropylla Pall.

[Study on antioxidant activity of two major secoiridoid glucosides in the fruits of Ligustrum lucidum Ait].

The antioxidant effects of two major secoiridoid glucosides (nuezhenoside and G13, separated in our laboratory) from Fructus Ligustri Lucidi had already been assayed though DPPH radicals, respectively. The results revealed that these ingredients showed significant antioxidant activities. A positive correlation existed between total content and antioxidant activity. G13 had shown higher antioxidant activity than nuezhenoside. It implied that the structure of secoiridoid glucoside was postitive to antioxidant activity. Otherwise, the results could promote the deep research of Furctus Ligustri Lucidi on antioxidant mechanism.

[Progress in regulatory T cells research].

Regulatory T cells (Treg) are functionally mature T cell subpopulations which are key players of maintaining the balance of immunological defense system. Treg can proliferate in vivo or in vitro by antigen specific way or non-antigen specific way, and actively control the properties of other immune cells by suppressing their functional activity and their proliferation as well.

[Content comparison of pinoresinol diglucoside in original and reborn bark of Eucommia ulmoides].

OBJECTIVE: To evaluate the reborn barks quality, antihypertensive effective component of pinoresnol diglucoside (PDG) in three times reborn barks were determined. METHODS: A YWG C18 column (10 microm, 250 x 4.6 mm) was used with a mobile phase of methanol-water (30:75) and flow rate of 1.0 (ml/min). The detective wave-length was set at 277nm and the column temperature at room temperature. RESULTS: PDG in the first reborn barks are slightly higher than the original ones, and in the second reborn barks are similar with the barks before girdling (the fist reborn barks), but in the third reborn barks, PDG are much lower than the barks before girdling (the second reborn barks). CONCLUSION: In order to ensure reborn barks quality, we suggest that the girdling bark regeneration can be made two times, the time between the first and the second girdling is not less than five years. PDG in the third reborn barks should be enhanced.

[Studies on the chemical constituents in root of Gentiana macrophylla from Shaanxi].

OBJECTIVE: To in vestigate the constituents in root of Gentiana macrophylla. METHOD: Various column chromatographic techniques were used for isolation and purification of the principles. The structures were elucidated on the basis of spectral data (UV, IR, MS, 1H-, 13C-NMR etc.) and identified by comparing with standard substance. RESULT: Eight compounds were identified. Four compounds isolated from the chloroform fraction are: 5-carboxyl-3,4-dihydrogen-1H-2-benzopyran-1-one (1), erythrocentauric acid (2), roburic acid (3), oleanolic acid (4). Water fraction gave four known secoiridoid glucosides. They were: gentiopicroside (5), swertiamarine (6), sweroside (7), 6'-O-beta-D-glucosylgentiopicroside (8). CONCLUSION: 1 is a novel compound. It was named as erythrocentauric acid. 2 was isolated from genus Gentiana and 8 was isolated from G. macrophylla for the first time.

[Enhancement by FK506 of triptolide-induced inhibition of expression of COX-2 and iNOS in human rheumatoid arthritis synovial fibroblasts].

To explore the effects of FK506 on the inhibition by triptolide (TP) of cell proliferation and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their inducing products PGE2, NO in human rheumatoid arthritis synovial fibroblasts (RASF), and to study the mechanisms of combination of FK506 and TP in RA therapy, RASF used in the experiments were obtained from synovial tissue of patients with RA and were cultured. RASF were pretreated with FK506(10-1000 nmol/L)for 2 h, then the cells were stimulated with TNF alpha(20 microg/L) in the presence or absence of TP (10 microg/L). The RASF proliferation was determined by [(3)H]-TdR incorporation, and the productions of PGE2 and NO in culture supernatants of RASF were detected with competitive ELISA and enzyme reduction of nitrate. Expression of COX-2 and iNOS mRNA in RASF were analyzed by semi quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western blot method and cellular enzyme immunoassay in synovial fibroblasts. NF-kappa B activity in whole-cell extract of RASF was also measured by an ELISA-based method. Results showed that neither FK506 nor TP at lower concentration (10 microg/L) alone affected TNF alpha-induced COX-2, iNOS expression and production of PGE2, NO in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 microg/L) down-regulated COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was positively correlated with FK506 concentrations (10-1000 nmol/L). NF-kappa B activity in TNF alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 microg/L) treatment than those with TP (10 microg/L) alone. No change was observed in inhibition of proliferation of synovial cells after combined treatment of FK506 and TP. In conclusion, FK506 enhanced TP-mediated down-regulation of COX-2, iNOS and their inducing products PGE2, NO in human RASF by suppressing the activity of NF-kappa B.

[Effects of intrasplenic transplantation of IL-18 gene-modified fetal hepatocytes on mouse immune function]

OBJECTIVE: To investigate the effects of IL-18 gene-modified fetal hepatocytes (AdmIL-18/MNL.CL2) intrasplenic transplantation on mouse immune function. METHODS: Forty mice were evenly divided into 4 groups of 10. Each group received an intrasplenic transplantation one of the following: AdmIL-18/BNL.CL2, Ad-LacZ/BNL.CL2 (virus control), BNL.CL2 (cell control) and PBS (blank control). After two weeks, the mice were sacrificed. Serum cytokine levels, Mpsi and splenic cell culture supernatant and liver tissue extracts supernatants were measured using ELISA. Hepatic cytokines mRNA expression were determined by RT-PCR. THe cytotoxicity of peritoneal Mpsi and NK activity of spienocytes were detected by LDH release assay. The proliferation of splenic lymphocytes was determined by MTT assay. RESULTS: The IL-18, IL-2,IFN-gamma, TNF-alpha levels of serum, Mpsi and splenocyte culture supernatant, liver tissue extracts supernatants in mice transplanted with AdmIL-18/BNL.CL2 were higher and the IL-4, IL-10 levels were lower compared to their levels in other 3 groups. The highest IL-18, IL-2, IFN-gamma, TNF-alpha and the lowest IL-4, IL-10 mRNA expressions in the liver were observed in mice transplanted with AdmIL-18/BNL.CL2. The mice transplanted with AdmIL-18/BNL.Cl2 showed significantly increases cytotoxicity of Mpsi, NK activity and splenic cell proliferation compared with the other 3 groups. CONCLUSION: AdmIL-18 can be effectively transfected into mice fetal heptocytes which subsequently IL-18. Intransplenic transplantation of IL-18 gene-modified fetal hepatocytes may augment mouse immune function and provide an useful basis for targeted gene therapy of liver disease.