Studies On The Anti-inflammatory Effects And Quality Control Of Periploca Forrestii Schltr.

Periploca forrestii Schltr.(P.forrestii)is a well-known Miao’s Medicinal plant,which is found in southwest China.It has been widely used to treat many diseases,including soft tissue injury,rheumatism,arthralgia and abnormal menstruation,with considerable therapeutic efficacy.A number of P.forrestii-based drugs(e.g.,Heiguteng Zhuifeng Huoluo Capsule,Fenghe Chubi Tincture,Compound Xianling Fengshi Vina and Shenglong Qufeng Vina)have been approved by the Chinese Food and Drug Administration.They are prescribed for patients with various inflammatory diseases and get a better therapeutic effect.The constituents of P.forrestii include pregnane glycosides,cardiac glycosides,oligosaccharides,coumarins,flavonoids and triterpenoids.Pharmacological studies have indicated that this plant possesses anti-tumor,anti-inflammatory,analgesic,immunosuppressant and anti-oxidative activities,as well as cardiac effects.However,the mechanisms underlying the anti-inflammatory effect of P.forrestii remain to be elucidated.Meanwhile,the research on the quality standards of the plant is almost empty.In this paper,the anti-inflammatory effects(in vivo and in vitro),effect mechanism,chemical composition analysis and quality standard were investigated comprehensively,which would support its application as a preventive or therapeutic agent for disease.1.To evaluate the active fraction of anti-inflammation effect from P.forrestii.the vitro RAW264.7 cells inflammatory model was established by simulating with LPS.The survival rate was determined by MTT assay.The quantity of nitric oxide(NO)was assayed by Griess reagent.The production of tumor necrosis factor α(TNF-α)was determined by ELISA.Compared with the LPS group,EFPF significantly inhibited the production of NO and TNF-α in LPS-induced RAW 264.7 cells(P<0.001).The result demonstrated that EFPF was the main active fraction of anti-inflammatory effects from P.forrestii.2.The chemical components of EFPF were analyzed by UHPLC-Q-TOF-MS.UHPLC-Q-TOF-MS methods provide retention time,calculated,formulas,UV absorption wavelength,and MS2 fragmentation ions for main peaks of EFPF.Chemical analysis showed that EFPF contain a variety of caffeoylquinic acids such as 1-O-caffeoylquinic acid(1-CQA),3-O-caffeoylquinic acid(3-CQA),5-O-caffeoylquinic acid.The result provided the important evidences for pharmacodynamic material basis.3.The anti-inflammatory effects of EFPF were evaluated using the acetic acid-induced celiac capillary permeability,the xylene-induced mouse ear edema and carrageenan-induced rat paw edema models in vivo.Compared with the control group,EFPF significantly reduced the increase of peritoneal capillary permeability,mouse ear edema and rat paw edema rate.The result indicated that EFPF has a significant anti-inflammatory activity in vivo.4.To investigate the potential mechanisms of EFPF,We further investigated the inflammatory mediators,cytokines and the protein expression of related regulatory proteins in LPS-stimulated RAW264.7 cells,which would support its application as a preventive or therapeutic agent for disease.The morphological features of apoptotic cells were determined by AO/EB staining in LPS-stimulated RAW 264.7 Cells.The apoptosis rate,reactive oxygen species(ROS)content and mitochondrial membrane potentials(MMP)were detected by flow cytometry.The supernatant was analyzed for NO using the Griess reagent,and the levels of inflammatory mediators and cytokines were determined using enzyme-linked immunosorbent assays for prostaglandin E2(PGE2),TNF-α,interleukin 6(IL-6),and interleukin-10(IL-10).The protein expression of NO synthase(iNOS),cyclooxygenase-2(COX-2),nuclear factor κB(NF-κB),and mitogen-activated protein kinases(MAPKs)including extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and p38 MAPK were examined by western blot.Compared with the LPS group,EFPF significantly increased the survival rate,ROS content and MMP(P<0.05,P<0.005,P<0.001).EFPF significantly inhibited the LPS-stimulated production of inflammatory factors such as NO,PGE2,TNF-α,and IL-6 in a concentration-dependent manner(P<0.05),and increased the IL-10 production(P<0.05).EFPF significantly inhibited the mRNA expressions of TNF-a、IL-6 and increased the mRNA expressions of IL-10 in LPSstimulated RAW 264.7 cells(P<0.005,P<0.001).And also significantly inhibited LPS-induced expression of iNOS and COX-2 proteins,suppressed the phosphorylation and degradation of IκBα,decreased the level of p65 and inhibited the phosphorylation of p38,ERK1/2 and JNK(P<0.05,P<0.005).These results suggested EFPF exerted anti-inflammatory effect by inhibiting the apoptosis,reducing the protein expressions of iNOS and COX-2 and the production of the inflammation factors,including TNF-α,IL-6,NO and PGE2,mainly by inhibiting LPS-mediated stimulation of NF-κB and MAPK signaling pathways.5.To establish a method for determination of P.forrestii,The content of total caffeic acid was determined by ultraviolet spectrophotmetry,the content of multiple index components were determined by HPLC,and moisture content,total ash,acid insoluble ash and organic chlorine pesticides were determined.The method of quality control for P.forrestii was established,and the quality standards have been improved,which provide the scientific basis and theory foundation for the development and application of P.forrestii.