Comparison of cuticular wax extraction from pear fruit by different organic solvents

Abstract：【Objective】Establishing a comprehensive extraction protocol is the foundation for studying plant cuticular wax. Currently, organic reagents widely used in the study of extraction of plant cuticular wax, such as chloroform, dichloromethane, etc., are mostly highly toxic and pose certain risks to the physical and mental health of experimental personnel. Therefore, the safety and effectiveness of different organic solvents for extracting pear peel cuticular wax were compared and identified in this study, in order to screen out low-toxic and efficient extraction solvents for pear peel cuticular wax, and replace the chloroform of traditional extraction solvent with higher toxicity, laying a foundation for pear peel cu-ticular wax related research work.【Methods】The fruit of Cuiguan pear at 90 days after flowering was used as test material, and the cuticular wax of peel was extracted using chloroform as control group and seven other organic solvents with relatively low toxicity, including dimethyl carbonate, ether, butyl acetate, acetone, ethyl acetate, methanol, and n- hexane. The components of cuticular wax extracted by these different solvents were detected by gas chromatography-mass spectrometry (GC-MS), and the extraction effect of cuticular wax were also compared from different perspectives such as the number of extracted cuticular wax compounds, content of total cuticular wax, and content of specific compounds. 【Results】The number of cuticular wax compounds extracted from pear peel using two solvents, methanol and ether, is the least, with only 28 and 24, respectively, which have a generally low affection of cuticular wax extraction. Thirty- eight compounds of cuticular wax were extracted using butyl acetate, slightly lower than the number of compounds of cuticular wax extracted using chloroform, and the distribution of the extracted compounds of cuticular wax is similar to that of chloroform. Therefore, the extraction effect of butyl acetate was similar to that of chloroform. In addition, the volatility of butyl acetate was much lower than that of chloroform, even though it was detected with higher extraction effect and much lower toxicity (1/14 toxicity of chloroform). Although n-hexane had the lowest toxicity and good extraction effect on alkanes, accounting for 73.7% of the total wax, its extraction effect on terpenoids was insufficient, only accounting for 6.3% of the total wax, and it also belongs to flammable and explosive hazardous chemicals. Taken together, these four organic solvents are not suitable as improved solvents for extracting cuticular wax. On the contrary, the content of total cuticular wax extracted using acetone was the highest among all groups (0.56 mg· cm-2 ) and was 2.3 times higher than that of chloroform. Thirty-six compounds of cuticular wax were extracted using acetone, slightly lower than the number of cuticular wax extracted by chloroform. The content of terpenoids extracted using acetone was almost three times higher than that of chloroform. Therefore it could be used as an improved solvent for the extraction of terpenoids from pear pericarp. The number of compounds of cuticular wax extracted using ethyl acetate was the highest, up to 41, and the distribution of the extracted compounds of cuticular wax was similar to that of chloroform. Ethyl acetate, which has 1/6 toxicity of chloroform, was found to have a higher efficiency of cuticular wax extraction than chloroform. Thus it could be used as an alternative cuticular wax extraction solvent. In addition, the number of extracted cuticular wax compounds, content of total cuticular wax, and content of specific compound extracted were detected using dimethyl carbonate, which has similar wax extraction effect compared with chloroform but its toxicity was only 1/14 of chloroform. The results indicated that it could be an alternative solvent for extracting pear peel cuticular wax. Fifty-four wax compounds found in eight organic solvent detection were analyzed through principal component analysis (PCA). The eight organic solvent detection could be divided into five groups based on the relationships between organic solvents (scores) and their 54 wax compounds (loadings). Butyl acetate and ethyl acetate formed the first group, which were characterized by high concentrations of nonacosane in the extraction. Group two contained acetone and ether, which were characterized with high concentrations of terpenoids and low concentrations of alkanes. Group three including methanol, which were mainly characterized by low concentrations of terpenoids and alkanes. n-hexane characterized by high concentrations of alkanes and low concentrations of terpenoids in the extract formed group four. The fifth group contained chloroform and dimethyl carbonate with no significant characteristics.【Conclusion】Acetone was the dominant solvent for the extraction of terpenoids from pear fruit peel and can be used as an improved solvent. The extraction effect of cuticular wax using ethyl acetate was equivalent to that of chloroform and dimethyl carbonate, and it also haslower toxicity, which could be used as the optimal solvent to replace chloroform in the cuticular wax extraction progress. Thus, these selected solvents will be helpful for the work of extraction of cuticular wax and related components in plant.