Extraction, Composition Analysis And Biological Activity Of Volatile Oil From The Root Bark Of Dalbergia Chinensis

As a traditional Chinese herb,cortex moutan is pungent,cold and nontoxic,recorded in“Shen Nong's Herbal Classic”.Paeonia ludlowii,endemic to the Nyingchi,Mainling county of South-East Tibet,belongs to the Paeonia of the family of Paeoniaceae.Local Tibetans as early as a few hundred years ago has been using its root to treat of gynecological diseases,skin diseases,cardiovascular and cerebrovascular diseases.However,the chemical components and pharmacology activities of the root of P.ludlowii have not been reported.The paper expects to extract and obtain essential oil from the root of P.Ludlowii?PEO?,analyze the chemical compositions,study the anti-tumor,anti-fungal,antioxidant activities of PEO and its main component,and find out the possible mechanism.Firstly,extract PEO by using steam distillation method,soaking method and reflux method and analyze extracted PEO by GC-MS.Compare these three different extraction results and determine the best extraction method.Secondly,study on anti-tumor activity of PEO and its main component on human lung cancer A549 cells.The proliferation of A549 cells was determined by CCK-8 assay and colony formation test;the morphology of A549 cells was observed under inverted,fluorescence and transmission electron microscopes.The expression and localization of Tubulin and Cyt c in A549 cells was studied by cell immunofluorescence assay;the formation of ROS was observed by confocal microscope;the activity of Caspase-3 enzyme was analyzed by ELIASA assay;the mRNA expression was detected by RT-PCR.Then,study on anti-fungal effects of PEO and its main component.The inhibitory action was determined by the method of drug sensitive plate on Epidermophytom floccosum,Trichophyton tonsurans and Microsporum gypseum;the morphological changes of these fungi were detected by scanning electron microscopy.Finally,study on antioxidant activity of PEO and its main component.The scavenging rate of DPPH free radical?DPPH?,ABTS radical?ABTS⁺?,hydroxyl radical?·OH?and superoxide radical?·O₂^-?were measured;the total antioxidant capacity was determined by FRAP assay.It was showed that steam distillation method was confirmed as the best extracted method and the extraction rate was 1.99%±0.23%.The results of GC-MS analysis showed there were 55 main components in steam distillation extracted PEO and paeonol occupied 62.07%±3.49%of the total extraction,which is the main component of PEO.The anticancer results showed both PEO and paeonol inhibited proliferation of A549 cells with concentration and time dependence.After 48 h,IC₅₀ value of PEO is2.02±0.19?g/mL and 1.89±0.16?g/mL,and paeonol IC₅₀ value is 12.92±0.23?mol/L and 12.47±0.21?mol/L in CCK-8 and clone formation experiment respectively;cell morphology changes of apoptosis was obviously observed as cell shrinkage,fibroblastsvacuolization,nuclearcondensation,irregularshape,chromatin fragmentation,apoptotic body,etc.;the structure of tubulin was changed to form aggregate and Cyt c was released;mRNA level of cyclin B1 and bax was concentration-dependentlyupregulated,wellsurvivinconcentration-dependently downregulated;ROS was concentration-dependently released;caspase-3 was activated.The antifungal results showed that the PEO and paeonol have significant antifungal effects in all three fungi strains.The minimum inhibitory concentration?MIC?of PEO was 10?g/mL and the minmum fungicidal concentration?MFC?was 20?g/mL;the MIC of paeonol was 12.5?g/mL and the MFC was 25?g/mL.EC₅₀ value of PEO,calculated by the scavenging rate of DPPH·,ABTS⁺,·OH and·O₂^-,is 4.06±0.49 mg/mL,0.72±0.016 mg/mL,3.41±0.06 mg/mL and 5.78±0.20 mg/mL,and EC₅₀ value of paeonol is respectively 55.90±2.64 mg/mL,0.074±0.0017 mg/mL,2.35±0.07 mg/mL and 0.88±0.04 mg/m L.The total antioxidant value of PEO and paeonol was 0.46±0.01 and 19.74±0.31,respectively in the FRAR test.In this paper,it was determined that the optimum method to extract PEO was steam distillation.PEO and paeonol could significantly inhibit proliferation and induced apoptosis of lung cancer A549 cells.Both of them suppress growth in all three kinds of dermatophytes,T.tonsurans,E.Floccosum and M.gypseum,and inhibitory effect of PEO is slightly higher than paeonol.PEO and paeonol had a good scavenging effect on DPPH·?ABTS⁺?·OH and·O₂^-,and total antioxidant capacity put on good antioxidant activity.In summary,P.ludlowii in Tibet have excellent biological activities and has broad application prospects in medicine.