Relative Research Of Hepatoprotection And Lung Injury Protection Effects With Active Components Of Meconopsis

Objectives:The flower from several species of Meconopsis belong to Papaveraceae are valuable plants used as traditional Tibetan medicine(TTM) named oubei, and widely used in Tibetan area.According to statistics, oubei was used in more than 120 Tibetan medicine preparations. For Tibetan doctor, the regularity of herb medicine followed by medicinal literature, only flower was used as oubei, however, the low production of flower could not guarantee the supply. Another, only purple or blue flower Meconopsis was used as medicine to treat hepatitis and pneumonia, the other color flower was not used or used less, and over collecting and low usage resulted in many species of Meconopsis was endangered. Moreover, Meconopsis distributed in the fragile ecological environment of alpine meadow,alpine brushes and alpine screes, wild plant resources are rare,so many plants would be wasted if only flower was used. Therefore,resource studies of Meconopsis to demonstrate the rationality of different flower color Meconopsis exhibited similar pharmacological effect, then to alleviate the resource scarce of purple or blue flower Meconopsis. These researches have important implications for Meconopsis resources protection for its reasonable exploitation and utilization.Meconopsis integrifolia(Maxim.)Franch, M. punicea Maxim and M.henrici Bur et Franch were the original of TTM recorded in Tibetan ancient medicinal literature(Yue Wang Yao Zhen, Si Bu Yi Dian and Jing Zhu Ben Cao). The project was based on the distinction effect of hepatoprotection, pneumonia protection, inflammatory cells and antioxidant activity of Meconopsis plant and flower, then screening out M. integrifolia which exhibited strong antioxidant activity,hepatoprotective effect and well inhibitory effect on inflammatory cells to further study. Ulraperformance liquid chromatography coupled with tandem mass spectrometry and high-speed countercurrent chromatography were used for study the active components. Spectrum-effect relationship of M. integrifolia between antioxidant activity and UPLC fingerprints was analyze to elucidate effective substance.Methods and Results:1. Pharmacognostical study on Tibetan Medical Species of MeconopsisTo identify the common Tibetan medical species of Meconopsis.The plant morphology and crude drug were observed; microscopic characteristics of root, scape, leaf and flower were observed by optical microscopy; The pollen was observed by scanning electron microscopy.(1) Original plant and crude drug characteristics could be identified by their flower color.(2)Microscopic characteristics could be distinguished by the level of lignifications of root, and difference of vascular.(3)Powder characteristics could be identified by the difference of endothecium.(4) The pollen of M. integrifolia and M. punicea was spherical, nonaperturate, the difference of them was that the spines of M. punicea was more sharper than that of others and the surface of it was much smoother than that of M. integrifolia. The pollen of M. henrici was tricolpate,and the surface with speculate glyphs. And the distinction of microscopic characteristics of three species of Meconopsis was also obvious. The differences of microscopic characteristics of common Tibetan medical species were obvious, which provided basis for their quality evaluation.2. Enrichment and purification of total flavonoids from Meconopsis integrifolia by macroporous adsorption resinsA method for enrichment and purification of total flavonoids from M. integrifolia by macroporous adsorption resins. Four macroporous resins AB-8, D101, HPD450, HPD600 were chosen with static and dynamic adsorption and desorption experiments to optimize the technical parameters. D101 macroporous resin was good for enrichment and purification of total flavonoids. The optimal condition was sample concentration 0.30 g/m L, elution flow rate 3BV/h, adsorption capacity 51.2 m L/g, sample was first eluted with4 BV of water, then with 3 BV 50% ethanol. The purity of total flavonoids was increase from 2.47% to 33.83%. This method that D101 macroporous resin enrichment and purification of total flavonoids from M. integrifolia was simple and stabile for total flavonoids from M. integrifolia.3. Antioxidant activity, hepatoprotection and lung injury protection effects of flowers and plants from 3 species of Meconopsis3.1 In vitro antioxidant activity profiling of aqueous ethanol extract of Meconopsis flower and total flavonoids of Meconopsis plantThe aqueous ethanol extract of Meconopsis flower and total flavonoids of Meconopsis plant, were comparative study for their in vitro antioxidant. Total phenolics and flavonoids were determined by spectrophotometric techniques. In vitro antioxidant and radical scavenging profiling was determined through: Total antioxidant capacity, radical scavenging effects by DPPH and ABTS methods and super oxide radical scavenging effects. M. integrifolia had the highest contents in phenolics and flavonoids and the highest antioxidant activity. M. henrici had the highest super oxide radical scavenging effects. Aqueous ethanol extract of 3 species of Meconopsis flower could promote producing super oxide radical.The flower and plant of M. integrifolia exhibited excellent in vitro antioxidant activity.3.2 Anti-inflammation of aqueous ethanol extract from Meconopsis flower in lipopolysaccharide stimulated RAW264.7 macrophagesComparative study the difference between aqueous ethanol extract from 3 species of Meconopsis flower. Inflammatory RAW264.7macrophages was stimulated by lipopolysaccharide(LPS), CC-K assay was performed to measure the cell viability, griess assay was used to measure the released nitric oxide(NO), ELISA was applied to detect TNF-α, IL-6 and IL-10, the change of Caspase 3/7 was observed by fluorescence microplate, DCFH-DA was applied to detect ROS. Aqueous ethanol extract from 3 species of Meconopsis flower showed no cytotoxicity on RAW264.7 macrophages at the concentrations of 5, 20, 50 μg/m L(P>0.5). Aqueous ethanol extract from Meconopsis flower exhibited non inhibitory effects of NO. In high concentration level(50, 200 μg/m L), Aqueous ethanol extract from Meconopsis flower showed inhibitory effect on TNF-α. The secretion of IL-6 statistically inhibited by 20, 50 μg/m L extract from M. integrifolia and 50, 200 μg/m L extract from M. henrici.In the concentration of 20 μg/m L of aqueous ethanol extract from Meconopsis flower could increase the secretion of IL-10. Aqueous ethanol extract from Meconopsis flower promoted proliferation of RAW264.7 macrophages stimulated by LPS. Aqueous ethanol extract from Meconopsis flower showed excellent antioxidant activity that reduced the production of ROS in RAW264.7 macrophages induced by LPS. Aqueous ethanol extract from 3 species of Meconopsis flower exhibited anti-inflammation effects, and extract from M.integrifolia flower showed the best effect of reducing the ROS production.3.3 In vivo hepatoprotective effect of total flavonoids of Meconopsis plantThis study aims to compare the hepatoprotective effects of total flavonoids of 3 species of Meconopsis plant in vivo. Mice with Con A-induced liver injury were used to assess the hepatoprotective effect in vivo. The level or activity of glutamate pyruvate transaminase(ALT), aspartate aminotransferase(AST), lactic dehydrogenase(LDH) in the blood serum and superoxide dismutase(SOD), glutathione(GSH), catalase(CAT) and malonaldehyde(MDA)in the liver of the mice were assayed. The pathological changes of liver tissue were examined by HE. In the mice with Con A-induced liver injury, the groups treated with total flavonoids of 3 pecies of Meconopsis plant showed lower levels of ALT, AST, LDH and MDA,and increase SOD. Total flavonoids of 3 species of Meconopsis plant exhibited excellent hepatoprotective effect in vivo, M. henrici showed the best hepatoprotective effect in 3 species of Meconopsis,M. integrifolia had the better effect to increase SOD.3.4 The effect of aqueous ethanol extract from Meconopsis on lung injury induced by lipopolysaccharideTo investigate the effects of aqueous ethanol extract from 3species of Meconopsis in LPS-induced acute lung injury in mice.Acute lung injury mice model were developed by LPS intratracheal instillation. The pathological changes of lung tissue were examined by HE. Ratio of wet/dry lung weight(W/D) was used to evaluate edema degrees of lung tissue. ELISA was applied to detecte TNF-α, IL-1β,IL-6, superoxide dismutase(SOD), malonaldehyde(MDA) and myeloperoxidase(MPO) in lung tissue of the mice were assayed.Pretreatment with M. punicea extract could attenuate histopathologic changes induced by LPS. In LPS group, W/D ratio were obviously increased(P<0.05), but there was not significant differences among model groups and sample groups. Aqueous ethanol extract from 3 species of Meconopsis could reduce the levels of TNF-α and increase the levels of SOD. M. punicea extract showed protective effect on lung injury in mice.4. Separation and purification of four flavonol diglucosides from the flower of Meconopsis integrifolia by high-speed countercurrent chromatographyFlavonoids are the main components of M. integrifolia, which is a traditional Tibetan medicine. However, traditional chromatography separation requires large quantity of raw M.integrifolia and it is very time-consuming. Herein, we applied high-speed counter-current chromatography(HSCCC) in the separation and purification of flavonoids from ethanol extract of M. integrifolia flower. Et OAc/n-Bu OH/H2O(2∶3∶5,v/v/v) was selected as the optimum solvent system to purify the flavonoids from 600 mg crud extract. Bath temperature was35 ℃, detection wavelength was 254 nm, the apparatus was rotated at 900 r/min, at a flow rate of 2.0 m L/min. Four components were separation and purification, namely quercetin-3-O-β-D-glucopyrannosy-(1â†'6)-β-D-glucopyranoside(compound 1, 60 mg), quercetin-3-O-[2’ ’-O-acetyl-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranoside](compound 2, 40 mg), quercetin-3-O-[3’ ’-O-acetyl-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranoside](compound 3, 11 mg) and quercetin-3-O-[6’ ’-O-acetyl-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranoside](compound 4, 16 mg).Among the four compounds, compound 3 and 4 were new discovered acetylated flavonol diglucosides. After the HSCCC separation, the purities of the four flavonol diglucosides were 98%, 95%, 90% and92%, respectively. The structures of them were identified by MS and NMR spectra.5. Study of spectrum-effect relationship and quality evaluation of M. integrifolia5.1 Spectrum-effect relationship on antioxidant activity effects of M. integrifoliaThe spectrum-effect relationship on antioxidant activity effect of M. integrifolia was established based on ulra-performance liquid chromatography, aims to reveal the material basis of M. integrifolia. The fingerprints of total flavonoids of M. integrifolia were established by UPLC, and the antioxidant activity effects were evaluated by the scavenging of DPPH, ABTS, total antioxidant capacity and super oxide radical scavenging effect. The relationship of characteristic absorption band of fingerprints with the pharmacological action was calculated by principle component analysis(PCA) and grey relation analysis(GRA). Twenty night common peaks in the fingerprints were obtained.Based on the result of PCA, DPPH radical scavenging and super oxide radical scavenging activity were elected to related with twenty night common peaks, while correlation bigger than 0.7, we concluded that they were the antioxidant activity components of M.integrifolia. Antioxidant activity effect of M. integrifolia was related to synergistic effect of many components.Analysis by UPLC-MS/MS, we knew that quercetin glycoside were the main components of total flavonoids of M. integrifolia, and also with little alkaloid.5.2 UPLC determination of active components in flower of M.integrifoliaTo determine the quercetin-3-O-β-D-glucopyrannosy-(1â†'6)-β-D-glucopyranoside(RS1), quercetin3-O-[2’ ’-O-acetyl-β-D-glucopyranosyl-(1â†'6)-(1â†'6)-β-D-glucopyranoside](RS2) and isoquercitrin(RS3) in flower of M. integrifolia by UPLC. The chromatographic process was according to the method had established before. Waters ACQUITY UPLC HSS T3 column(100×2.1 mm, 1.8 μm)was used. The mobile phase was consisted of A(acetonitrile) and B(0.1% acetic acid solution), which was used as the mobile phase in isocratic elution mode as follow: 0–10 min, 16% A. The flow rate was set at 300 μL/min, the column temperature was 35 C, and the sample injection volume was 2 μL. The spectra from 210 nm to 400 nm were recorded. The standard curves of three active constituents showed a good linearity in 0.02504–0.5008 mg/m L(R2=1.0000),0.02502–0.5004 mg/m L(R2=0.9999), 0.006275–0.2008 mg/m L(R2=0.9998). The average recoveries were 103.39%, 102.13%, 90.36%,RSD<2.83%. The assay demonstrated that this method is simple and has adequate accuracy to measure active components in flower of M.integrifolia.Conclusion:1. M. integrifolia, M. punicea and M. henrici could identify by their flower color, plant size, the difference of root, the difference of endothecium and pollen.2. M. henrici plant exhibited excellent hepatoprotective effect,so it is to suggest that M. henrici could be use as oubei equivalent with its flower. M. integrifolia showed the best effect of antioxidant activity, an also exhibited good hepatoprotective effect, so it is to suggest that M. integrifolia could be use as oubei equivalent with M. henrici plant and its flower.3. HSCCC was applied to separation and purification of flavonoids from the ethanol extract of M. integrifolia flower.Among the four compounds, all of them were found in M. integrifolia for the first time, and compound 3 and 4 were new discovered acetylated flavonol diglucosides.4. The spectrum-effect relationship on antioxidant activity effect of M. integrifolia based on ulra-performance liquid chromatography was study, we found that quercetin glycoside were the main components of total flavonoids of M. integrifolia, M.integrifolia plant has similar components with its flower.5. Ultra performance liquid chromatography was used to determine the main compounds of M. integrifolia flower, the results showed that M. integrifolia distributed in high altitude in Xiaojin and Maerkang county had better quality.