| Objective:Flavonoids are the secondary metabolites of much plants and have 15-carbon skeleton containing two phenyl rings and a heterocyclic ring.Pharmacological studies of flavonoids have shown a variety of interesting biological activities,including anti-inflammatory,immunomodulatory,antiatherogenic4 and antihyperglycemic activities,especially antioxidantive.Many biological and pharmacological properties of many flavonoids are associated with free radical-scavenging actions.According to the documented research,free radical has high reactivity and oxidizability.Free radicals and other reactive species are considered to be important causative factors in the development of various diseases.Gynostemma pentaphyllum(Cucurbitaceae family),also called "jiao-gu-lan"in Chinese or "Gocaekmbaw" in Zhuang language,is a climbing,perennial herbaceous vine plant.Most reports as concerns G.pentaphyllum are major in gypenoside with bioactivity.Small part of documents has focused on flavonoid ingredient.In this study,we examined the basic composition and content of G.pentaphyllum flavonoids(GPf).The study also evaluated antioxidantive effects of GPf by using H2O2-stimulated A549 cell,RAW264.7 cell model and DPPH free radical scavenging model preliminary revealed the possible antioxidantion mechanism of GPf.The acute lung injury model was established based on lipopolysaccharide induced rats to detect the effects of GPf in vivo and explore its possible protective mechanism.Method:1.The G.pentaphyllum tea(50.0 kg)was exhaustively extracted with 70%EtOH-H2O by ultrasonic(power 500W,frequency 40 kHz)for about 30 min at room temperature.Macroporous resin SP825L、silica gel column、Sephadex LH-20、semipreparative HPLC and so on were used to isolate and purify the total flavonoids.The chemical structure were identified with UV,MS and NMR data.2.The contents of the main flavoboids were analyzed by UPLC.The isolated flavonoids were selected as standards.The analytical method was evaluated by linear calibration curves,stability,accuracy and precision.3.DPPH assay method provides an easy and rapid way to evaluate antioxidants of flavonoids from G.pentaphyllum in vitro.The oxidative damage model was established using different concentrations H2O2 to induce 549 cell、RAW264.7 cell for different hours,and then treated with the flavonoids for 10 h.The effects of flavonoids from G.pentaphyllum on cell viability of A549 and RAW264.7 cell damaged by H2O2 were detected by MTT assay.The contents of ROS were detected by DCFH-DA fluorescent probe method via flow cytometer.The contents of MDA,SOD and GSH were detected by TBA,NBT and DTNB-linked colorimetry assay,respectively.Expressions levels of Nrf2,NQO1 and HO-1 in A549 cells were evaluated by Western blot.4.The SD rats were randomly divided into normal group,model group,the positive control group,the model in the placebo group(hereinafter referred to as analog security group),GPf low dose group group,GPf middle dose group group,GPf high dose group group.In addition to the normal group,the remaining rats were injured by lipopolysaccharide(LPS)tracheal instillation method to establish acute lung injury model.Protein and inflammatory cells in bronchoalveolar lavage fluid(BALF)were measured by BCA method and Wright-Giemsa staining,respectively.HE staining of lung tissue slices were used to observe the pathological changes.TNF-a,IL-10,IL-6 and TGF-β1 in serum and lung issue of rats were measured by ELISA,the level of O2-,MDA,SOD and GSH protein in serum and lung tissue was detected by NO2-,TBA,NBT and DTNB-linked colorimetry assay.Metabolin of rats given GPf by gavage was analyzed by LCMS-IT-TOF to explore the GPf absorption in vivo.Result:1.Twelve flavonoids from G.pentaphyllum were obtained as light yellow,amorphous powder.According to the compounds on high resolution IT-TOF-MS values and 13C NMR spectral data,combining with the reported papers,the chemical structures were identified as rutin(1),4’-O-methyl-kaempferol-3-O-rutinoside(2),ombuoside(3),kaempferol-3-β-D-O-rutinoside(4),isorhamnetin-3-O-β-D-rutinoside(5),quercetin-3-O-β-D-glucoside(6),isorhamnetin(7),kaempferol(8),quercetin(9),respectively.Compound 10,11,12 were speculated as quercetin-3-O-(4"’-O-acetyl)-D-ruti nosi de,methylkaempferol,dimethyl-quercetin,respectivly.Besides,4’-O-methyl-kaempferol-3-O-rutinoside(2)was reported for the first time from this family.2.Senve flavonoids,compound 1,2,3,4,5,6,10 from G.pentaphyllum were quantitatively analyzed by UPLC-MS and narigin was selected as an internal standard.Linear calibration curves with correlation coefficients(r)greater than 0.99 were obtained for all analytes in different concentration.The limit of detection(LOD)and limit of quantification(LOQ)were collected at the several concentrations by the UPLC-MS.Intra-and inter-day stability were determined by assaying mixed standard solution during a single day and on three different days,respectively.The overall inter-day RSD(n=6)were 1.63%,4.01%,3.34%,1.56%,2.15%,3.85%and 2.02%,less than 5%for compound 1,2,3,4,5,6,10,respectively.And the overall intra-day RSD(n=6)were 1.88%,2.98%,1.89%,4.50%,3.12%,1.28%and 2.60%,less than 5%for 1,2,3,4,5,6,10,respectively.The results of quantitative determination on main flavonoids in G.pentaphyllum were 12907.74,341.68,538.56,43.85,451.27,57.11,317.97 pg/g for 1,2,3,4,5,6,10,respectively.3.Flavonoids from G.pentaphyllum showed a degree of DPPH scavenging activity,especially 1,6,8,9,with IC50 values of 7.97±0.18,5.74±0.08,6.56±0.22,3.11± 0.07 μg/mL,respectively.The vitamin C,as a control agent,exhibited high scavenging effect upon DPPH free and IC50 value is 5.16 ± 0.58 μgg/mL.Whereas 2,3,4 and 5 showed low activity,with IC50 values more than 100 μg/mL.The A549 cell、RAW264.7 cell viability were 60.4%,66.2%after treated with 500 μmol/L,300 μmol/L H2O2 for 10 hours,respectively.So they were chosen to be as an oxidant stress model.Compared with H2O2 group,GPf treated cells showed a dose-dependent increase in cell viability,especially rutin(1),4’-O-methyl-kaempferol-3-O-rutinoside(2),isorhamnetin-3-O-β-D-rutinoside(5),quercetin-3-O-β-D-glucoside(6),and quercetin(9)at concentration of 50 μg/mL showed significant high viabilities(p<0.05).They were chosen as candidates for post-study.Compared with normal group,the contents of SOD,GSH and HO-1 expressions in A549 cells were lower after damaged with H2O2.On the contrary,the contents of ROS and MDA expressions were increased.Compared with model group,the contents of SOD,GSH and the expressions of Nrf2,NQO1 and HO-1 were increased after treated with GPf.The contents of ROS and MDA in RAW264.7 cell rose up,as the level of SOD and GSH declined after injured by H2O2(p<0.05).Pretreatment of GPf significantly decreased H2O2-induced ROS and MDA contents,incressed SOD activity and GSH level in RAW264.7 cells.In addition,GPf markedly improved the levels of Nrf2,NQO1 and HO-1 in RAW264.7 cells to the normal level.4.The results showed that damaged lung organization structure,a large amount of inflammatory cells infiltration and accompanied by thickening of alveolar walls were significantly induced by LPS.These results suggest that acute lung injury in rats successfully established by LPS.Pretreatment of GPf significantly decreased LPS-induced lung tissue damage,and inflammatory cells infiltration in HE section.In addition,GPf markedly reduced the levels of protein and inflammatory cells in BALF.Furthermore,results of colorimetry assay showed that GPf inhibited the produce of O2-and MDA in serum and lung tissue of LPS-induced rats,especially MDA decresed significantly compared with control group(p<0.05).The contents of MDA in rat serum,there were significant difference between GPf group and model group(p<0.01).About the contents of GSH,there were statistical difference between control group and model group(p<0.05).There were also significant statistical differences between GPf low-dose group and control group(p<0.01).The levels of GSH in lung tissue of middle-dose group and high-dose group has increased more or less.However,there was no significant statistical difference between GPf group and control group.Compared to control group,IL-6,IL-10,TNF-a and TGF-β1 levels in serum and lung tissue of the model group were significantly increased(p<0.01)and these elevated expression were inhibited by dexamethasone(p<0.01).Compared with the model group,the levels of TGF-β1、IL-10 and IL-6 in serum and lung tissue of the GPf high-dose groups were significantly decreased(p<0.05).Conclusion:1.In this study,twelve flavonoids were isolated from the ethanol extract of G.pentaphyllum,a Zhuang medicine,nine of them were identified by IT-TOF and 13C-NMR.Besides,compound 2 was found from Gynostemma genus for the first time.The main flavonoids were determined by UPLC-MS rapidly and exactly.Furthmore,the established method with good precision and stability is acceptable and reproducible.2.The flavonoids from G.pentaphyllum showed various degrees of of DPPH free radical scavenging activity.According to the results,hydroxyl at 3,3’,4’position is important on DPPH free radical scavenging activity.Otherwise,the results of western blotting showed that GPf significantly improve the expressions of Nrf3,NQO1 and HO-1 in A549 cell and RAW264.7 cell.The determination of ROS,MDA,SOD and GSH demonstrated that GPf can attenuate the effect of H2O2-induced oxidative stress on A549 cell by resisting oxidation and its potential mechanism is associated with Nrf2 signaling pathways.3.Compared to the modle group,the high expression of O2-,MDA and IL-6,IL-10,TNF-α,TGF-β1 inflammatory factor were inhibited by GPf.The expression of Keap1,NQO 1 and HO-1 leveles were regulated to resist oxidative stress response.Otherwise,the level of SOD and GSH increased after deal with GPf.In conlusion,it can be found that GPf protected LPS-induced acute lung injury may be through its antioxidative property.The finding may provide a biological evidence for the application of the G.pentaphyllum to fight the oxidative stress related diseases. |
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