Study On The Extraction Technology And Biological Activity Of Cannabidiol From Cannabis Sativa L.

Hemp(Cannabis sativa L.)is an annual herbaceous plant.Cannabinoid is the main active component in kenaf.At present,there are more than 140 kinds of natural cannabinoid isolated from hemp plants,among which tetrahydrocannabinol(THC)and cannabidiol(CBD)are the two main active components of cannabinoid in hemp.Studies have shown that CBD has sedative,anti-cancer,anti-epileptic and vasodilation activities.In this study,the HPLC method was used to determine the content of CBD in the ultrasonic-assisted extraction of Hemp leaves,and the effects of different factors on the extraction content were explored.The response surface methodology was used to optimize the conditions for ultrasonic-assisted extraction of CBD and the activity of CBD was studied to provide a scientific basis for the further development and utilization of CBD in hemp.The main results of this paper are summarized as follows:1.An HPLC method was established for the detection of CBD using Agilent HC-C18column(250×4.6 mm,5μm),detection wavelength of 220 nm,flow rate of 1.0 mL/min,column temperature of 28℃,and isocratic elution of mobile phase:methanol-water=90∶10.Under these conditions,the separation effect and peak type were good,and the results were accurate,reliable and stable.The single factor experiment was used to determine the effects of ultrasonic extraction power,extraction temperature,extraction time and solid to liquid ratio on the extraction of CBD content in Hemp leaves.The Box-Benhnken experimental design was performed using Design-expert software to determine and verify the optimal extraction conditions of CBD,and to detect the CBD content in different parts of hemp in Daqing,Heilongjiang Province.The results showed that using methanol as the extraction solvent,the optimal extraction conditions were as follows:extraction power 320 W,extraction temperature81℃,extraction time 26 min,and solid-to-liquid ratio 1∶22 g/mL.Under these conditions,the average content of CBD was 79.53±0.26 mg/g.In Daqing hemps,the CBD content in the tender leaves was the highest,followed by the whole leaf,old leaf,seed and stem,and the CBD content in the root was the lowest.2.Compared with VC,BHA and BHT,the total antioxidant capacity of FRAP,its iron ion reducing ability,DPPH radical and ABTS radical scavenging ability were evaluated.The results of four different antioxidant capacity experiments showed that CBD had significant antioxidant activity.When the concentration of CBD liquid medicine was 0.5 mg/mL,the FRAP value was 132.7,the DPPH free radical scavenging rate was close to 80%,and the ABTS free radical scavenging rate was more than 50%.In addition,the antioxidant capacity of CBD was stronger than that of BHA,lower than that of VC and similar to that of BHT in the reducing force determination experiment.3.With levofloxacin as the positive control,the in vitro inhibition experiments of CBD on S.aureus,A.baumannii,B.subtilis,E.coli,P.aeruginosa and C.albicans were conducted by using filter paper method and double dilution method.The diameter of inhibition zone was measured,and the MIC and MBC were measured to evaluate the inhibition effect.The results showed that,according to the filter paper method,CBD had antibacterial effect on the six selected common bacteria,and was highly sensitive to C.albicans with fungal morphological characteristics.When the concentration of CBD was 5μmol/mL,the diameter of the inhibition zone could reach 10.1 mm Under this concentration,the antibacterial effects of CBD on the other bacteria were as follows:B.subtilis>S.aureus>A.baumannii>P.aeruginosa>E.coli.4.A.baumannii was used as the pathogenic factor to establish the mouse pneumonia model by gavage,and the model mice were divided into the treatment group and the evaluation group.By comparing the evaluation criteria established by the evaluation group,we observed the therapeutic effects of CBD on A.baumannii pneumonia in different development stages,and observed the pathological sections of lung after hematoxylin-eosin staining.The painstaking blood of mice was cultured using TSA colony culture method to compare the plate colony growth of the model group and the treatment group.The results showed that after modeling,CBD could help the recovery of lung tissue in mice and reduce the pathological damage of lung caused by pneumonia.And within 0-6 h,it was the optimal medication time.The amount of heart blood borne bacteria in the model group was>10~5 CFU/mL 12 h after intraperitoneal injection,and that in the treatment group was(2.3±0.53)×10~4 CFU/mL.After24 h and 48 h of drug administration,the amount of heart blood borne bacteria in the treatment group was lower than that in the control group.