Studies On The Volatile Oil, Bioactivities And Germplasm Innovation Technique Of Mosla Chinensis Maxim In Hunan

Mosla chinensis Maxim is Chinese traditional herb and belongs to Labiatae and Molsa,which is annual herbage,specially found in China.From The Tang Dynasty, Mosla chinensis Maxim was used as traditional Chinese medicine or civilian medicine. Recently years,the resources of wild Mosla chinensis Maxim reduced rapidly.The studies,conservations,utilizations of this unique resources were urgent extremely.In this paper,hunan wild Mosla chinensis Maxim was choosen as material,It was studied that volatile oil extraction technique,bioactivities,secretory structures,and the induction technique of autotetraploid.The main results and conclusions were as follows:1,Chemical compositions analysis and volatile oil extraction technique of Mosla chinensis MaximThe chemistry system detection methods were used to analysis Mosla chinensis Maxim,the results showed the material contained polysaccharide,amino acids,protein, tannins,flavonoides,coumarins,esters,phenols,sterides.Sterides,coumarins were first found in Mosla chinensis Maxim.It was optimized that the petroleum benzin extraction technique and enzyme-aided petroleum benzin extraction technique.Five extraction methods were compared which were super critical fluids CO₂ extraction,enzyme-aided petroleum benzin extraction, ultrasonic-assisted petroleum benzin extraction,petroleum benzin extraction, hydrodistillation.Petroleum benzin extraction was raised as the feasible extraction method after considered the equipments,costs and extraction rate.It was established that GC analytical method and fluorescence detection method of thymol by the experiments of liner correlation,accuracy and recovery rate.Gas chromatogram analytical method:the linear equation was y=86954x-7998.6 and correlation coefficients was 0.9963(n=6)in 0～2.85mg/mL for standard thymol,the RSD=1.88(n=5),the average recovery was 100.33%,RSD=1.69%(n=5).Fluorescence spectrophotometer method:under Ex=275nm and Em=297nm,the linear equation of the fluorescence intensity(â… )and the concentration(C,μg/mL)of thymol wasâ… =3716.5C+371.43 and correlation coefficients was r=0.9958(n=7)in 0～1.84μg/mL for thymol.RSD=0.40(n=5),the average recovery was 94.71%,RSD=1.22%(n=5). GC-MS analysis results suggested volatile oil of Mosla chinensis Maxim in Hunan contained phenols,alkenes,esters,fatty acids,alkanes,alcohols etc.The MCMVO was extracted by supercritical CO₂ contained thymol,carvacrol,P-cycmene,humulene etc. The thymol content accounted for 56.25%,and the content of carvacrol was 19.21%. The content of thymol and carvacrol of MCMVO extracted by organic solvent was 58.33%and 22.54%.10 compositions were first extracted from Mosla chinensis Maxim.2,Acute toxicity test and subchronic toxicity testThe results of acute toxicity test suggested:LD₅₀of MCMVO to mice was 1.303±0.116 mL/kg·bw,95%interval range was 1.419～1.197mL/ kg·bw.The results of subchronic toxicity test demonstrated:The MCMVO could not obviously promote the mice grow,and had no obvious influence on the hemoglobin(HB),red blood cell(RBC), white blood cell(WBC)and WBC counter;MCMVO had the effect on reversible reduction the blood total cholesterol(TCHO)and blood urea nitrogen(BUN)in 8 weeks.3,Bioactivities of Mosla chinensis Maxim volatile oilMCMVO had certain anti-bacterium and anti-fungi effects,and the inhibitory effects on SA and AFL was most powerfully among the total experimented bacterium and fungi.The inhibitory effects on SA was confirmed to transform permeability of cellular membrane,depress utilization ability of SA to nitrogen source,interfere with the function of cell organ and further suppress cell metabolism.The action mechanism of MCMVO on AFL was MCMVO can destroy cell wall and plasmalemma of AFL, active suppress AFL sporular germination.0.79 g/L and 0.39 g/L dose group of MCMVO had the stronger effects on anti-NDV than 1.0 g/L dose group of virazole.The MCMVO could improve carbon clearance index and phagocytic index of mice, The MCMVO made DTH action of skin strengthen which was caused by DNFB;The MCMVO could increase weight of chest gland and spleen of normal mice.The MCMVO had dual regulating effects to the movement of intestines and stomach.Low concentration of which promoted the movement of intestines and stomach,and high concentration of which restrained the intestines and stomach from moving. MCMVO had scavenging and reduction capacity of radical oxygen such as O₂^-,H₂O₂,·OH,and could significantly recover bioactivity of GSH-Px,SOD and lessened the content of MDA.4.Studies on the relationship between structure and activity of thymol and carvacrol in MCMVOThymol and carvacrol were main compounds in MCMVO,their structures were similar,but the activities were different evidently.In this paper,thymol and carvacrol geometry molecular conformations were optimized by semi-empirical AM1,ab initio calculation RHF/STO-3G,RHF/3-21G,RHF/6-31G,density functional theory B3LYP/STO-3G,B3LYP/3-21G,B3LYP/6-31G.Bond length,bond angle,dihedral angle,IR spectrum characteristic absorption peak,nuclear magnetic resonance ¹H-NMR, ¹³C-NMR were calculated and were compared with experimentally observed value with statistical method.The results displayed:density functional theory B3LYP/6-31G, B3LYP/3-21G were the most accurate.Considering the optimizing time and accurate degree,B3LYP/3-21G were adopted as molecular configuration optimizing method. After the B3LYP/3-21G geometry optimizing,it was studied that the effects of substituent group on bond length of O-H,O,H difference of electric charge, disassociation energy of O-H,highest occupied molecular orbital energy,lowest unoccpied molecular orbital energy etc.Analysis results indicated:the antioxygen activities depended on bond length of O-H,O,H difference of electric charge, disassociation energy of O-H,highest occupied molecular orbital energy,lowest unoccpied molecular orbital energy,and methyl and isopropyl appeared position effect on these parameters,the influence rule of methyl on parameters were ortho->meta->paratope-,the influence rule of isopropyl on parameters were paratope->meta->ortho-. After the B3LYP/3-21G geometry optimizing,it was stuied that the correlation between 17 sturcture parameters and phenols bioactivity.The results exhibited:lowest unoccpied molecular orbital energy,TE,logP,S,V structure parameters appeared significant correlativity.The composition analysis on the front liner orbital showed:electro-negativity activity center of phenols were O and 1C.Along with small substituent groups pulled into,electronegativity activity centers were unchanged,but contribution of O reduced,contribution of C raised.The distribution of front liner orbital were more uniformizatin.Electropositivity activity centers of phenols were 6C,3C,2C,5C,and along with small substituent groups pulled into,electropositivity activity center changed slightly,but all along located meta- of-OH group.5,Studies on the secretory structures and development of peltate glandular hairsPeltate glandular hairs were found on the leaves and stems surface of Mosla chinensis Maxim.Peltate glandular hairs consisted of one basel cell,one stalk cell and eight secretory cell.It was researched that the development of peltate glandular hairs on the leaves of Mosla chinensis Maxim.The results showed:the initial cells,originated from the protodermal cells,desintegrated a basal cell,a stalk cell and an apical cell through two successive periclinal divisions,the apical cell would continue to divide anticlinally up to form 8 secretory cells of peltate glandular hairs.MCMVO were confirmed to reside in secretory cells by histochemical method.6,Clonal technique system establishment of Mosla chinensis Maxim somatic cell and the induction of Mosla chinensis Maxim autotetraploidIt was established that clonal technique system of Mosla chinensis Maxim somatic cell.Shoot stem were taken as explants,generation mode existed two styles.One was the explants of stem-segment with bud propagated by regenerating directly tussock plantlets from the explants,the other was the explants of stem-segment with bud propagated by forming callus on the explants and differentiated bud from the callus. Clump shoot regeneration medium:MS+6BA0.5～1.0mg/L+IBA0.5～1.0 mg/L;Clump shoot regeneration medium from axillary buds:MS+6BA0.5～1.0 mg/L+NAA0.5～1.0 mg/L;Efficiency seedling induction medium:MS+6BA0.5mg/L+IBA0.1 mg/L+GA0.5～1.0mg/L,Rooting medium:1/2MS+IBA2.0 mg/L.The autotetraploid of Mosla chinensis Maxim were first breeded from various materials.the chromosome numbers of diploid were 18(2n=2x=18,x=9),and variation plantlet chromosome numbers were 36(2n=2x=36,x=9),they were autotetraploid.