| Camphor tree is an evergreen tree belonging to the genus Cinnamomum of Lauraceae.It is subdivided into Cinnamomum camphora and Borneol Cinnamomum camphora,etc.It is widespread in China including south area of the Yangtze River,southwest provinces,and in southern Taiwan.Camphor tree is not only widely used in wood utilization,but also its bark,roots,stems,leaves and fruit can be used to extract essential oil.Essential oils of camphor trees have been widely used in flavors and fragrances,pharmaceutical synthesis,fine chemical synthesis,daily-use chemicals and other fields.The related research,development and utilization have a long history.In this paper,we study leaves and twigs of Borneol Cinnamomum camphora to overcome the current status of the independent research on essential oil and residue that are commonly found in the existing research.In view of the actual process of preparing essential oil with the steam distillation method,the three major residues produced during the preparation of essential oil(condensed water containing essential oil components,water extracts containing water-soluble constituents,and deoiled leaves and twigs containing active ingredients)were subjected to compositional analysis and biological activity studies to eliminate or reduce waste discharge,to explore the green process of essential oil production.In the process of extracting essential oil by steam distillation of leaves and twigs from Borneol Cinnamomum camphora,a large amount of residues are produced: condensed water,water extracts and deoiled leaves and twigs.For condensed water,its physical and chemical properties(color,relative density,refractive index,turbidity,oil content,chemical composition,etc.)and antibacterial activity were studied;for water extracts,its physical and chemical properties(color,solid content,chemical ingredients,etc.),decolorization methods,and antibacterial activity were studied.Deoiled leaves and twigs are large and difficult to handle in the actual production process.Therefore,it is the focus of this study: to propose an efficient extraction method(enzymolysis-ultrasonic-assisted extraction)to preparethe crude ethanol extract from deoiled leaves and twigs.The crude extract was assayed for its total polyphenol content,antibacterial activity,and antioxidant activity;then,crude ethanol extract was fractionally extracted to obtain extraction phases such as n-hexane,methylene chloride,ethyl acetate,n-butanol,and water.In consideration of literature reports and the extraction rate of each extraction phase,ethyl acetatephase is used for further separation,purification and antibacterial activity research.GC and GC-MS techniques were used to analyze the composition and chemical identification of steam condensed water.The results showed that the main components of condensed water were 1,8-Cineole,camphor,borneol,α-terpineol and other oxygenated compounds.The components of the water extract were analyzed by conventional chemical qualitative methods(such as color reaction and precipitation reaction).The results showed that organic acids,amino acids,proteins,peptides,reducing sugars,polysaccharides,glycosides,saponins,phenols,anthraquinones are present in water extract.In addition,the decolorization effect of three adsorbents(diatomite,kaolin,activated carbon)on the water extract was compared and analyzed.It was found that adding 5 % of activated carbon(200 mesh)gave the best decolorization effect.The enzymatic hydrolysis-ultrasonic-assisted extraction process of total polyphenols from deoiled leaves and twigs was studied by Box-Behnken Design-Response Surface Method;The ability to remove DPPH free radicals and hydroxyl radicals was used as an evaluation index of antioxidant activity of the total polyphenol extracts.The results showed that the optimal conditions were as follows: enzyme dosage 1.9 %,ethanol concentration 54 %,liquid-solid ratio 34 m L/g,enzymolysis temperature 50 ℃,enzymolysis time 1.5 h,ultrasonic time 30 min,ultrasonic frequency 45 k Hz,ultrasonic power 360 W,ultrasonic temperature 70 ℃;The average extraction rate of total polyphenols was 22.08 mg/g.The total polyphenol extracts showed a dose-effect relationship for the scavenging activities of DPPH radicals and hydroxyl radicals.The IC50 values were 0.36 and 0.15 mg/m L,respectively,indicating that the total polyphenol extracts have strong antioxidant activity ability.The crude extract was fractionated by solvent extraction to obtain the n-hexane extraction phase,the dichloromethane extraction phase,the ethyl acetate extraction phase,the n-butanol extraction phase and the water extraction phase;The ethyl acetate extract was then separated and purified by silica gel column chromatography,gradient elution and thin layer chromatography.The results showed that the extraction rate of ethyl acetate was the highest,which was 44.93 %;Chloroform-methanol(20:1,V/V)was the ideal thin-layer chromatographic developing agent;the gradient elution rate of ethyl acetate extraction phase was 95.65 %.The components are concentrated on the elution sites of 2,4,5,9,and 11,and each elution site contains 2-4 spots,and the separation effect is good.The plate double dilution method was used toevaluate antimicrobial activity,by determing the minimum inhibitory concentration,with Escherichia coli,Staphylococcus aureus,Bacillus subtilis and other several kinds of bacteria as experimental bacteria.The results showed that,in the experimental concentration range,the crude ethanol extractfrom deoiled leaves and twigs had a strong inhibitory effectagainst the Streptococcus pneumoniae,Bacillus subtilis,and Staphylococcus epidermidis.Fractions No.2,No.4,and No.9 obtained by silica gel column chromatography from ethyl acetate with high yield had certain inhibitory effect on Bacillus subtilis;water extracts had stronger inhibition on Klebsiella pneumonia and Bacillus subtilis;Condensed water has a strong inhibitory effect on Escherichia coli,Bacillus subtilis,Staphylococcus aureus,Candida albicans,and Aspergillus niger. |
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