Study On The Extraction,Purification And Biological Activities Of Lignans From Cinnamanum Camphoral Leaf

Cinnamoum camphoratrees are planted in the south of China.But they are very popular in the north of China as its cold resistance and protect environment.The leaves can absorb toxic gases such as S,F,prevent sand,keep from bacterial and kill insect.The leaves and stems of Cinnamomum camphora tree can dispel cold,relieving pain,help rescue various pains.Many references have shown that lignans exist in Cinnamomum camphora leaves.We used various methods to extract lignans from Cinnamomum camphora leaves,such as refluxing method,microwave technology,ultrasonic wave technology.Then macroporous resin(AB-8),silica gel(100-200 mesh),ODS were used to purify extractions,respectively.At last,the HPLC was used to analysis the purity of extractions.Then,we researched the antioxidant,antibacterial and anticancer activities of them.The results as shown:(1)The ethanol refluxing method was used to extract lignans from Cinnamanum camphoral leaves,and orthogonal test was used to determine the optimum conditions.When ethanol concentration was 100%,ration of material to liquid was 1:4(g/mL),refluxing time was 2 h,the yield of lignans was best(2)The experiment is about lignans from Cinnamanum camphoral leaves with microwave extraction technology.Some factors which influenced yield of lignans were researched with response surface methodology.Finally,the maximum yield was 42.69 mg/g with microwave time 5 min,ethanol concentration 80%,ration of material to liquid 1:26(g/mL)and extraction temperature 60 ?.(3)The experiment is about lignans from Cinnamanum camphoral leaves with ultrasonic extraction technology.Some factors which influenced yield of lignans were researched with response surface methodology.Finally,the maximum yield was 45.31 mg/g When ultrasonic time was 3 min,ethanol concentration was 60%,ration of material to liquid was 1:26(g/mL)and extraction time was 78 min.(4)According to the cleance rate of DPPH·,·OH,O2-· and reducing power,we could gain the results that the antioxidant activity increased with the concentration increased.The ICso values of cleance rate of DPPH· for Vc,BHT,lignans were 0.2 mg/mL,0.157 mg/mL,0.569 mg/mL,respectively.The IC50 values of cleance rate of ·OH for Vc,BHT,lignans were 0.516 mg/mL,0.796 mg/mL,0.767 mg/mL,respectively.The IC50 values of cleance rate of O2-· for Vc,BHT,lignans were 2.45 mg/mL,2.58 mg/mL,2.13 mg/mL,respectively.And the reducing power is better with the concentration of Vc,BHT,lignans increased.(5)To isolate and identify the lignans extracts from the leaves of Cinnamomum camphora and research the antibacterial activity of lignans.The compounds were purified by AB-8 macroporous resin,silica gel,Sephadex LH-20,ODS column chromatography.The structures of the compounds were identified on the basis of chemical methods(Ecgrine and Label reaction)and spectral methods(UV,IR,MS,H-NMR,C-NMR).Three compounds are identified as(+)pinoresinol-4-O-glucoside(1);pinoresinol(2),sesamin(3).Compounds 1,2,3 are reported in the leaves of this species for the first time.The purity of pinoresinol-4-O-glucoside,pinoresinol,sesamin were evaluated by HPLC.(6)The agar disc diffusion method and dilution broth method were used to determine the antibacterial activities of(+)pinoresinol-4-O-glucoside,pinoresinol,sesamin.Pinoresinol exhibiting antimicrobial properties and unharmful for human health provide promising antimicrobials for food safety and preservation application,but their antibacterial mechanisms are not yet fully understood.Herein,pinoresinol from Cinnamomum camphora used as a model natural compound was extracted and isolated.Results indicated that 62.5 ?g/mL pinoresinol completely inhibit the growth of five bacteria.Much effort was focused on elucidating the mechanism of antibacterial action of pinoresinol against P.aeruginosa and B.subtilis by determination of cell permeability and observing the changes of cell microstructure using scanning electron microscope(SEM).The leakage of the soluble saccharides and proteins were detected and up to 13.89 and 21.76 ?g/mL,respectively for P.aeruginosa and 11.08 and 19.05 ?g/mL,respectively for B.subtilis.Measurements of the release of the soluble saccharides and proteins confirmed the disruptive action of pinoresinol on cytoplasmic membrane.The damages of the pinoresinol on cell wall were confirmed using Gram staining and scanning electron microscopy(SEM).We concluded that the antibacterial action mechanism of the pinoresinol is attributed to the physiological and morphological changes in bacterial cells.(7)Pinoresinol was extracted from Cinnamomum camphora leaves and evaluated for its cytotoxicity against human cervical cancer cell lines(HeLa)and Human Umbilical Vein Endothelial Cells(HUVEC)cells by the MTS metabolism assay,displaying IC50 values at 200 ?g/mL for HeLa cells.The cytotoxicity of pinoresinol against HeLa was corroborated with flow cytometry,Hoechst staining and nuclear morphology assessment as apoptosis inducer.The induced inhibition of proliferation of HeLa by pinoresinol was associated with S phase arrest.(8)An efficient electrochemical approach has constructed on multi-walled carbon nanotubes(MWCNTS)and Au NPs,which were used to modify the glassy carbon electrode(GCE)to increase the specific surface area and enhance the conductivity.The modified materials were characterized with scanning electron microscopy(SEM),Transmission electron microscope(TEM),FT-IR,X-ray diffractometry(XRD)to evaluate its morphology,crystallinity,composition.Cyclic voltammetry(CV)was used to optimize sensing condition.Under optimized condition,the HeLa cells dropped on the surface of working electrode could be detected with CV and electrochemical impedance spectroscopy(EIS)in a huge range from 102-106 cells/mL.CV and EIS were used to evaluate the effect of pinoresinol against HeLa cells.The results showed electron transfer resistance(Ret)will decrease and current will increase once HeLa cells treated with pinoresinol.These results showed that the pinoresinol is strong inducers of apoptisis and arrest cell cycle in HeLa cells and the new electrochemical method could be applied to detect pinoresinol against cancer cells.