| Peroxisome proliferators activated receptors (PPARs) are closely related to humanchronic disease, such as diabetes mellitus and the other metabolic diseases. There aretwo kinds of agonists for PPARs, natural agonists and synthetic agonists. Searching forthe PPARs pan agonists with lower adverse effect and efficient is becoming a focus, dueto the synthetic agonists showed continuously a number of serious of toxicity and sideeffect. However, the natural medicines have played an important role in treating andpreventing human disease for many years, in particular the Traditional ChineseMedicines, which can be administered for a long duration. So, screening new PPARspan agonists with low toxicity and efficient from natural medicines has become aresearch focus on developing the treatment of diabetes mellitus and its complication.In the present study， the PPARα, PPARβ and PPARγ active fractions andcompounds form natural medicines with potential antidiabetic properties were screenedand evaluated on cell model. The results will help to supply and illustrate themechanism of those medicines, and establish the foundation for modern development ofthose natural medicines and the development of new drug for the treatment of diabetes.Firstly, cell-based high-throughput PPARs (PPARα/β/γ) model was developed forthe screening of PPARs agonists from natural medicines. HeLa cells were transfectedwith the re-constructed plasmid pBIND-PPAR (α, β, or γ)–LBD and reporter genepGL4.35, and the known PPARs agonists were used as the positive control (fenofibratefor PPARα, L165041for PPARβ, and rosiglitazone for PPARγ). The screeningconditions were evaluated by analyzing the expression value of luciferase. Meanwhiles,the Z′-factor was firstly introduced as evaluating indicator for the conforimation andevaluation of the quality of the PPARα, PPARβ, and PPARγ high-throughput screeningassay (HTS) model. The results suggested that one kind of simple, stable, specific andmulti-target HTS assay method was constructed for screening of PPARs pan, dual, orsingle agonists on HeLa cell.Secondly, the natural medicines’ extracts were prepared.12different kinds ofmedicines with potential antidiabetic properties {Coptis Chinensis Franch, Ligustrumlucidum Ait., Fructus Gleditsiae Abnormalis, Crataegus pinnatifida Bge L.,Dendrobium, Folium Mori, Salvia Miltiorrhiza Bunge., Rhizoma alismatis, Malusdoumeri (Bois) Chev. leaves, the flower of Edgeworthia gardneri, Pterocephalus hookeri (C. B. Clarke) Hoeck, Huidouba}, were circumfluence extracted in turn byhexane,70%EtOH, EtOAc and H2O. Finally,58different kinds of extracts wereobtained by this extract process.For identifying the PPARs active fractions from those12natural medicines, the58extracts were analyzed on the developed HTS model. The results showed that all the12natural medicines have the ability of activation for PPARγ and PPARβ, except RA.There are26extracts in58extracts can activate PPARγ and PPARβ simultaneously. Theflower of EG-EtOAc extract (EB1) and n-hexane eatract (EA), PH-EA extractdisplayed the most remarkable activity for PPARγ and PPARβ within the testedconcentration range, even higher the positive controls in the same bath. Additional,there still have23extracts with activation for PPARγ and PPARβ, they are LL-EA;FG-EA,-EB1and depositing in alcohol extract (ED); CP-EA; FM-EA and EB3;CC-EB1; DE-EA,-EB1and the supernatant of H2O extract (EC); SM-EC; EG n-BAIextract (EB2) and-EB3; PH-EB1,-EB3and–EC; MM-EA,-EB1and–EC; HU-EA,EB1and EB3.According to the above results of activity assay, in order to seek out the PPARs panagonists,10different kinds of extracts with high activation for PPARγ/β were picked up,and continued to carry out the activity experiment for PPARα. The results demonstratedthat all of those10extracts can be activated on PPARα, the sequence of activationability is (relative to positive controls in the same bath): PH-EA＞EG-EB1＞FG-EB1＞EG-EA＞MM-EC＞PH-EB3＞PH-EB1＞MM-EB1＞PH-EC＞CC-EB1. Thses5natural medicines might through activating the PPARs to play the function of reduceblood glucose and lipid, and anti-flammatory. In this study, some the maximus polarityextract of natural medicines such as MM-EC and EG-EC were found to displaysignificant activation on PPARβ/α and PPARγ/α for the first time, respectively, evenhigher than that of positive controls in the same bath.Afterward, some secondary fractions and pure compounds were carried out theactive test for identifying the high–activity extracts of PPARs contain the PPARs panagonists or not. It was found firstly that:(1)11secondary fractions from EG-EB1canactive PPARγ and PPARβ;(2) among of the9pure compounds from EG-EB1,umbelliferone and pentadecanoic showed the activation on PPARγ and PPARβ at thesame time, the maximum fold activation were1.78-and1.92-fold, and1.74-and5.91-fold, respectively.(3) Berberine was confirmed as one of the PPARs pan against atthe cellular level. At last, the cytotoxicity of th0se10extracts,11secondary fractions from EG-EB1and3pure compounds with activation for PPARs were investigated, and the datum ofMTT experiments showed that5extracts (EG-EA and EB1, PH-EB3, MM-EC andFG-EB1) and11secondary fractions and pure compounds (umbelliferone andpentadecanoic) from EG-EB1didn’t affect on the proliferation of to ECV-304andRAW264.7at the concentration with highest fold activation for PPARs. In addition, theinhibition rates of the other5activate extracts were lower30%for these two cellsproliferation, except for PH-EA and CC-EB1. The inhibition rates of berberine were21.9%and31.3%for ECV-304and RAW264.7cell, respectively.In a word, a multi-receptor model for PPARs (three subtypes, PPARα, PPARβ andPPARγ) was constructed on HeLa cell in the present study, which can be used to screenand evaluate the active fractions and pure compounds for PPARs from natural medicinewith the complex components, effectively. That will offer the theories and experimentalfoundation for searching the novel, safe and high-efficient natural medicines with thetreatment of diabetes mellitus and its complication. |
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