Study On Preparation And Pharmacodynamics Of Oregano Oil Submicron Emulsion For Injection Of Traditional Chinese Medicine Antibiotics

Objective:In recent years,the long-term abuse of antibiotics in clinical has led to the some problems such as“widespread resistance”?“super bacteria”,which caused serious harm to people's lives,masses of people eager to pHarmaceutical workers can look for alternatives to traditional medicine antibiotic products,so the development of new green plant antibiotic is a task which no delay.Oregano oil is a kind of volatile oil,which extracted from origanum,is a pale yellow oily liquid,and taken as raw material for people medicine and health products in worldwide.For a long time,Europe and the United States take it as food antimicrobial preservative,which is one of the commonly used Chinese medicine and animal feed additives in China folk,has the characteristics of safe and efficient,green and no incompatibility etc.This topic according to the characteristics and requirements of modern traditional Chinese medicine to develop a new type of green Chinese medicine antibiotics,which can control caused by bacterial gastrointestinal diseases?such as diarrhea,dysentery,acute enteritis,etc.?,injection of oregano oil submicron emulsion.Compared with the related agents on the market at present,it has better stability and to the digestive tract,work quickly,high bioavailability,the formulation of development not only filled the gap of oregano oil injection in clinical medicine,and wide raw material sources,non-toxic,green environmental protection,is a kind of ideal alternative medicine antibiotics,it highlights the advantages and characteristics of TCM,explore the development direction of natural antibiotics.Methods:?1?To“level of single factor test and orthogonal test method”examines the oregano essential oil steam distillation extraction process,and to combine“Box-Benhnken response surface method”decompression extraction process of oregano essential oil was optimized,and then comparison differences of chemical composition of two kinds of volatile oil extraction process,at the same time the antibacterial activity of volatile oil from 2 kinds of process is studied by“the paper diffusion method”;?2?To“GC-MS method”conducts a comprehensive comparison and analysis to the type of chemical composition of volatile oil from the different parts of oregano herbs?leaf-flower,stem,root?,and combining with“tube plate method”,the DPPH free radical clearance experiment,potassium ferricyanide total reducing power"examines the antimicrobial properties of volatile oil from different parts of and oxidation resistance,for oregano oil biological activity research provides the experimental basis;?3?By“the GC-MS”investigated 7 different origin quality of differences of volatile of Origanum vulgare L.,3 components?carvacrol,thymol and p-cymene?content were determined,combined with the“internal standard method”;?4?The first use of“single factor test and comparative analysis method”examined the pHysical parameters of oregano oil?solubility,relative density,refractive index,conductivity,pH,viscosity,zeta potential,etc.?and by“orthogonal experimental design Box-Benhnken and response surface method”was investigated in the prescription of injection oil,soybean lecithin,Poloxamer188 ratio and amount,optimize the oregano oil submicron emulsion for injection prescription,finally determined the injection of oregano Oil submicron emulsion preparation and sterilization process by orthogonal design combined with the index evaluation method;?5?Using“the HPLC”investigated content determination method of 3 components?carvacrol,thymol and p-cymene?in the injection of oregano oil submicron emulsion,and to determine the content of 3 components;?6?By using transmission electron microscopy?sem?,particle size and particle size distribution measurement method,dextran gel method,platinum plate method and the pHysical parameters of the finished product emulsions?appearance,particle size,coating,surface tension,conductivity,PH value,zeta potential,etc.?,and then through the stability test,hemolysis test,blood vessels irritant experiment,determination of“LD₅₀0 value”examines the finished product of emulsion stability,security,and acute toxicity;?7?Using ELISA reagent to change content in rat serum IL-6,IL-10,TNF-alpHa,CRP for comment Price index.The effects of the injection of oregano oil submicron emulsion against pneumonia,while using the MTT assay,NK cell activity in mice and mononuclear phagocytic function in mice carbon clearance test and peritoneal macropHage phagocytosis experiment of chicken red blood cell to evaluate the injection of oregano oil submicron emulsion for high immunity;?8?By“the method of drug concentration in blood”examines the injection of oregano oil submicron emulsion in the healthy SD rats in vivo pHarmacokinetic process,and combined with“DAS 2.0 pHarmacokinetic analysis software”identified the main pHarmacokinetic parameters.Results:?1?The optimal process of steam distillation essential oil as the medicinal material crushing into powders,adding 10 times amount of water,soaking 2h,extraction 8h;optimum vacuum extraction essential oil as the solid-liquid ratio is 10:1,pressure 558 mbar,refluxing 4.23h;a comparative study of the two methods:the volatile constituents of the steam distillation and vacuum extraction showed some differences?steam distillation extraction were identified25 kinds of components,vacuum extraction method identified 23 components?,at the same time,bacteriostatic compositions of thymol and carvacrol vary with the extraction pressure change to some extent,thus affecting the antibacterial effect of oregano essential oil.?2?37 compounds were identified in the leaves-flowers oils,accounting for 98.78%of the total,11 compounds were identified in the stems oils,accounting for 99.51%of the total;29 compounds were identified in the roots oils,accounting for 98.97%of the total.There are 59 compounds in the essential oils from three different parts,including 3 compounds?Carvacrol,Thymol,Caryophyllin?,which is shared by each extraction parts;different parts of the Origanum essential oils have antibacterial activity,leaf-flower oil antibacterial activity than the root oil and stem oil;scavenging effect of DPPH of oregano oil in different parts:leaf-flower oil>root oil>stem oil,with the increase of concentration,reducing energy also increased gradually.When the concentration is less than 1.25 mg/mL,unable to determine what kind of oil reduction ability is strong,but when the concentration is greater than the1.25mg/mL,extract the reduction site of essential oils appear obvious difference,reducing capacity for:leaf-flower oil>root oil>stem oil.?3?Through the comprehensive comparison in 7 regions of Origanum vulgare L.analysis found that the three regions of Hubei Tuanfeng,Jiujiang,Jiangxi,Shaanxi Xi'an Origanum vulgare better quality,three components of?carvacrol,thymol,p-cymene?total more than 50%;by GC-MS analysis and“internal standard method”was finalized in Hubei oregano essential oil each 1 ml contains carvacrol 4.026 mg,thymol 1.389 mg,p-cymene 0.424 mg,the three components of total content close to 6 mg/ml?4?The effects of the parameters and pHysicochemical properties of bovine oil of oregano oil to find highly soluble,non soluble polar ethers,ketones,oil and ethanol;relative density at room temperature is 0.9511,with a certain viscosity?3.311±0.018 m Pa·s?;its the rate of conductivity and zeta potential at room temperature almost 0;pH value of weak acid,its refractive index will gradually increase with the increase of temperature;the preparation technology optimization,determination of oregano oil submicron emulsion for injection prescription:oregano oil 1g,soy lecithin 1.9g,Poloxamer 188 2.4 g,injection of soybean oil?LCT:MCT=1:1?22 g,glycerol2 g,BHT 0.2 g oleic acid 0.34 g,and the injection water is added to 100 mL;the preparation process for the oil and water two-pHase mixture at 60?,and stir,then collect the solution,using a high-speed shearing machine,adjust to F files(19000 r min^-1),shear a 30min get colostrum;collect colostrum and transferred to the high pressure homogenizer.Under the pressure of 10 bar,homogeneous after10 times,final milk,that is,the final product;injection of oregano oil submicron emulsion sterilization process ultimately determine the medium pH value adjusted to 6 to 8,and 105?for 45 min,moist heat sterilization is adopted.?5?HPLC method for the determination of oregano oil submicron emulsion of three components in the content and chromatograpHic conditions:analysis of chromatograpHic column:Kromasil 100-5 C₁₈8 column?250mm x 4.6mm,5um?column,mobile pHase:in acetonitrile water?54%:46%?in the isocratic elution;the flow rate was 1mL/min;column temperature 30?.The detection wavelength was 274 nm.?6?Properties,pHysical parameters,the oregano oil submicron emulsion stability and safety inspection found that the finished emulsion appearance of spherical particles,the particle size is consistent,uniform particle size distribution and average particle size?Z-ave?was 135.44±2.66 nm,particle size distribution?PDI?was 0.124,the zeta potential was-36.10±+1.15 mV,room temperature viscosity is 12.14 mPa·s,PH=6.43,osmotic pressure=283 osmkg^-1,the material parameters in accordance with the relevant requirements;the stability test results show that the high speed centrifugal,cold hot alternating long-term test cycle and 6 months of pHysical examination there is no obvious change in parameters;safety test showed that the finished emulsion has no obvious hemolysis and vascular irritation,acute toxicity test showed that:t the median lethal dose(LD₅₀)of oregano oil submicron emulsion for injection is1.3475 mg/g.?7?Study in efficacy trials showed that oregano oil submicron emulsion of low dose to medicine group,IL-6,IL-10,TNF-and CRP content is lower than that of model group,the symptoms of pneumonia are improved slightly,but with the model group is not significant.Oregano oil submicron emulsion,high dose group IL-6,IL-10,TNF-and CRP in were significantly lower than those in the model group,that oregano oil submicron emulsion can effectively reduce the secretion of various cytokines,which can effectively improve the symptoms of pneumonia;oil of oregano and improve immunity test results show that oil of oregano has significantly increased macropHage pHagocytosis and lympHocyte transformation of important,and mice spleen and thymus index increased significantly,indicating that oil of oregano has strong ability in promoting animal growth.?8?Oregano oil submicron emulsion in rats in vivo pharmacokinetic parameters:to thymol indices AUC_?0-t?=170266.1ng/L*min,C_max=7695.175 ng/L,T_max=5 min,t_1/2z=60.013 min CL_z=1.184L/min/kg,V_z=106.973 L,MRT_?0-??=43.541 min;to carvacrol indices AUC_?0-t?=72690.93 ng/L*min,C_max=3132.741 ng/L,T_max=5 min,t_1/2z=55.101 min,CL_z=1.499 L/min/kg,V_z=129.186 L,MRT_?0-??=39.877 min,that oregano oil submicron emulsion in vivo using high and rapidly eliminated,suitable for intravenous administration.Conclusion:?1?Using the different extraction process,chemical constituents and antibacterial activity of oregano oil was significantly different,compared to the extraction of volatile oil extracted from the atmospHeric antibacterial more pressure,and the change in content of thymol and carvacrol will change with pressure and occurrence regularity,that method different extraction may directly affect the antimicrobial properties of oregano oil;?2?Different parts of Origanum vulgare?leaf-flower,stem and root?of the essential oil,antibacterial and antioxidant activities,including the“underground part”?root?and“aerial part”?flower-leaf and stem?contain similar antibacterial ingredients,and oregano root also contain a large amounts of fatty acids,the compounds in high root content?oregano relative content greater than 60%?,their presence may have important significance for improving the overall effectiveness and explore the the new effect of essential oil from Origanum,has some medicinal value,it should be retained or development;?3?Prescription,preparation,sterilization of oregano oil submicron emulsion confirmed by this research are proved its utility strong,simple operation,high reproducibility,the finished emulsion particle size,particle size distribution,surface tension is greatly reduced,the zeta potential is less than-36 mV,the encapsulation rate was over 85%,strong stability,can be applied in actual production;?4?The research first establishedraw materials-oregano oil 3 ingredients of GC-MS measurement method,and establish oregano submicron emulsion of 3 composition of HPLC content detection method,results show that the method has strong specificity,high reproducibility,good precision,can be used for quality control of Oregano oil submicron emulsion;?5?The first by pHarmacodynamical experiments verify the oregano oil has good anti-inflammatory and enhance the role of immunity,this and previous reports is consistent,which discovery of anti-inflammatory effect of open up more for a wide range of biological activity of Oregano oil,as well as it provides certain reference and basis for research and development of oregano oil.