Effects of Total Saponins from Dioscorea Nipponica Makino on Monosodium Urate-Induced M1-Polarized Macrophages through Arachidonic Acid Signaling Pathway: An in vitro Study / 中国结合医学杂志

OBJECTIVE@#To investigate and reveal the underlying mechanism of the effect of total saponins from Dioscoreae nipponica Makino (TSDN) on the arachidonic acid pathway in monosodium urate (MSU) crystal-induced M1-polarized macrophages.@*METHODS@#M1 polarization of RAW264.7 cells were induced by 1 µ g/mL lipopolysaccharide (LPS). The methylthiazolyldiphenyl-tetrazolium bromide method was then used to screen the concentration of TSDN. MSU (500 µ g/mL) was used to induce the gouty arthritis model. Afterwards, 10 µ g/L TSDN and 8 µ mol/L celecoxib, which was used as a positive control, were added to the above LPS and MSU-induced cells for 24 h. The mRNA and protein expressions of cyclooxygenase (COX) 2, 5-lipoxygenase (5-LOX), microsomal prostaglandin E synthase derived eicosanoids (mPGES)-1, leukotriene B (LTB)4, cytochrome P450 (CYP) 4A, and prostaglandin E2 (PGE2) were tested by real-time polymerase chain reaction and Western blotting, respectively. The enzyme-linked immunosorbent assay was used to test the contents of M1 markers, including inducible nitric oxid synthase (NOS) 2, CD80, and CD86.@*RESULTS@#TSDN inhibited the proliferation of M1 macrophages and decreased both the mRNA and protein expressions of COX2, 5-LOX, CYP4A, LTB4, and PGE2 (P<0.01) while increased the mRNA and protein expression of mPGES-1 (P<0.05 or P<0.01). TSDN could also significantly decrease the contents of NOS2, CD80, and CD86 (P<0.01).@*CONCLUSION@#TSDN has an anti-inflammation effect on gouty arthritis in an in vitro model by regulating arachidonic acid signaling pathway.

Total Saponin Fraction of Dioscorea Nipponica Makino Improves Gouty Arthritis Symptoms in Rats via M1/M2 Polarization of Monocytes and Macrophages Mediated by Arachidonic Acid Signaling / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of effects of total saponin fraction from Dioscorea Nipponica Makino (TSDN) on M1/M2 polarization of monocytes/macrophages and arachidonic acid (AA) pathway in rats with gouty arthritis (GA).@*METHODS@#Seventy-two Sprague Dawley rats were randomly divided into 4 groups (n=18 in each): normal, model, TSDN at 160 mg/kg, and celecoxib at 43.3 mg/kg. Monosodium urate crystal (MSU) was injected into the rats' ankle joints to induce an experimental GA model. Blood and tissue samples were collected on the 3rd, 5th, and 8th days of drug administration. Histopathological changes in the synovium of joints were observed via hematoxylin and eosin (HE) staining. The expression levels of arachidonic acid (AA) signaling pathway were assessed via real-time polymerase chain reaction (qPCR) and Western blot. Flow cytometry was used to determine the proportion of M1 and M2 macrophages in the peripheral blood. An enzyme-linked immunosorbent assay (ELISA) was used to detect interleukine (IL)-1 β, tumor necrosis factor-alpha (TNF-α), IL-4, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4).@*RESULTS@#HE staining showed that TSDN improved the synovial tissue. qPCR and Western blot showed that on the 3rd, 5th and 8th days of drug administration, TSDN reduced the mRNA and protein expressions of cyclooxygenase (COX)2, microsomal prostaglandin E synthase-1 derived eicosanoids (mPGES-1), 5-lipoxygenase (5-LOX), recombinant human mothers against decapentaplegic homolog 3 (Smad3), nucleotide-binding oligomerization domain-like receptor protein 3 (NALP3), and inducible nitric oxide synthase (iNOS) in rats' ankle synovial tissues (P<0.01). TSDN decreased COX1 mRNA and protein expression on 3rd and 5th day of drug administration and raised it on the 8th day (both P<0.01). It lowered CD68 protein expression on days 3 (P<0.01), as well as mRNA and protein expression on days 5 and 8 (P<0.01). On the 3rd, 5th, and 8th days of drug administration, TSDN elevated the mRNA and protein expression of Arg1 and CD163 (P<0.01). Flow cytometry results showed that TSDN decreased the percentage of M1 macrophages while increasing the percentage of M2 in peripheral blood (P<0.05 or P<0.01). ELISA results showed that on the 3rd, 5th, and 8th days of drug administration, TSDN decreased serum levels of IL-1 β, TNF-α, and LTB4 (P<0.01), as well as PGE2 levels on days 3rd and 8th days (P<0.05 or P<0.01); on day 8 of administration, TSDN increased IL-4 serum levels and enhanced IL-10 contents on days 5 and 8 (P<0.05 or P<0.01).@*CONCLUSION@#The anti-inflammatory effect of TSDN on rats with GA may be achieved by influencing M1/M2 polarization through AA signaling pathway.

In Vivo Intestinal Absorption Characteristics of Dioscin from Dioscorea nipponica Makino Extract in Rat / 医药导报

Objective To study absorption characteristic of dioscin from Dioscorea nipponica Makino extract in rat intestine.Methods Single-pass intestinal perfusion (SPIP) model was used for rat in situ and HPLC was used to determine the concentrations of dioscin.The effects of different intestinal segments,drug concentration and P-glycoprotein (P-gp) inhibitor on intestinal absorption were investigated.Results Dioscin could be absorbed in the whole intestine,the absorption rate constant (Ka) and the apparent coefficient (Papp) of dioscin decreased following the sequence of ileum > duodenum =jejunum > colon.Absorption parameters of dioscin had no significant difference at different concentrations (40,80,120 mg·L-1).There were significant differences in Ka and Papp values between P-gp inhibitor group and no P-gp inhibitor group(P<0.05).Conclusion The saturate phenomena was not observed under the test range of drug concentration,and the absorption mechanism may be passive diffusion transport.Dioscin in Dioscorea nipponica Makino extract may be the substrate of P-gp.

Isolation, identification and antimicrobial activity of endophytic fungi from Dioscorea nipponica / 药物评价研究

Objective To isolate endophytic fungi from the fresh leaves of Dioscorea nipponica Makino,and to determine their antimicrobial activities.Methods Endophytic fungi from the fresh leaves of Dioscorea nipponica Makino were isolated by tissue isolation method,and the preliminary identification was carried out by slide plate culture,tissue culture,and plug culture.After isolating the endophytic fungi,the antimicrobial activity from the isolated endophytic fungi was detected using filter paper method with Staphylococcus aureus,Staphylococcus albus,Staphylococcus citreu,Micrococcus tetragenus,Escherichia coli,Salmonella typhi,Pseudomonas aeruginosa,Bacterium termo,Bacillus endocarditidiscapsulatus,Shigella flexneri,Bacterium paratyphosum B indicator microorganisms.Results A total of 52 strains of the endophytic fungi were isolated.According to morphological characteristics,10 endophytic fungi producing spores were identified as two orders,three families,and nine genera.The extracts of fungal mycelium had the antibiotic activity of strains of C37,C30,and C50.With the identification by morphology and molecular biology,strain C37 was determined to belong to the genus Gibberella,and C30,C50 to Alternaria.Conclusion The antibacterial activity of these endophytes in the leaves ofDioscorea nipponica Makino could be exploited for the development of new antibacterial biological agents.

Dioscorea Extract (DA-9801) Modulates Markers of Peripheral Neuropathy in Type 2 Diabetic db/db Mice

The purpose of this study was to investigate the therapeutic effects of DA-9801, an optimized extract of Dioscorea species, on diabetic peripheral neuropathy in a type 2 diabetic animal model. In this study, db/db mice were treated with DA-9801 (30 and 100 mg/kg, daily, p.o.) for 12 weeks. DA-9801 reduced the blood glucose levels and increased the withdrawal latencies in hot plate tests. Moreover, it prevented nerve damage based on increased nerve conduction velocity and ultrastructural changes. Decrease of nerve growth factor (NGF) may have a detrimental effect on diabetic neuropathy. We previously reported NGF regulatory properties of the Dioscorea genus. In this study, DA-9801 induced NGF production in rat primary astrocytes. In addition, it increased NGF levels in the sciatic nerve and the plasma of type 2 diabetic animals. DA-9801 also increased neurite outgrowth and mRNA expression of Tieg1/Klf10, an NGF target gene, in PC12 cells. These results demonstrated the attenuation of diabetic peripheral neuropathy by oral treatment with DA-9801 via NGF regulation. DA-9801 is currently being evaluated in a phase II clinical study.

Effects of different treatments on seed germination of Dioscorea nipponica / 中草药

Objective: To study the effects of different temperature conditions (10, 15, 20, 25, and 30 °C) and different concentration of gibberellin (GA3) and naphthalene acetic acid (NAA) on the germination responses of Dioscorea nipponica seeds. Methods: The seed germination rate and germination vigor were recorded and analyzed statistically, through setting conditions including cold stratification and soaking treatment with different concentration of GA3/NAA in culture dishes. Results: After sand stratification at 4 °C, the germination percentage and germination vigor of D. nipponica seeds were the highest of 80.00% and 53.33% at 25 °C, respectively. After soaking in 100 mg/L GA3 for 24 h without stratification, the germination percentage and vigor of D. nipponica seeds were 66.65% and 48.35%, respectively; After soaking in 1 mg/L NAA for 12 h, the germination percentage and germination vigor were 58.35% and 3.35%, respectively. The germination process treated with low temperature sand stratification was the fastest. The seeds began to germinate at the day 6, and germinated completely within 12 d. Conclusion: The seeds should germinate at 25 °C after sand stratification at 4 °C for 60 d, or the seeds without stratification would germinate after being treated with 100 mg/L GA3.

Physicochemical properties of starch obtained from Dioscorea nipponica / 中草药

Objective To study the medicinal plants of Dioscorea nipponica, the physicochemical pro-perties of starch in D. nipponica were investigated by means of various analytical methods. Methods D. nipponica starch was characterized by scanning electron microscope (SEM), X-ray diffraction, granule size analysis, DSC, and Brabender Viscograph system. Results Compared with tapioca and potato starch, the morphology of D. nipponica starch showed smaller particles, oval shaped, and dissimilar granules in size. The crystal type of D. nipponica starch was C-type pattern. The amylose content in D. nipponica starch was 26.3%. The starch separated from D. nipponica showed the highest transition temperature and intermediate enthalpy of gelatinization between potato and tapioca. According to the viscosity measurement with Brabender viscograph, D. nipponica starch exhibited lower peak viscosity, higher setback and lower breakdown viscosity. Conclusion D. nipponica starch is obtained from D. nipponica. Significant differences from D. nipponica and other tuber starches in physicochemical properties are obtained due to their geographical origin.