| Tobacco is an important economical crop,the contents of fragrant precursors,such as the pigment,polyphenols and nonvolatile organic acid,play an important role in the aroma quality of cigarettes.Ninety percent of acetone and fifty percent of methanol is used to extract the pigments and polyphenols of tobacco leaf in the tobacco industry standard method,YC/T 382-2010 and YC/T 202-2006.The nonvolatile organic acid of tobacco leaf is commonly extracted with methanol.As all of the three methods extracted the tabacco leaf only 1 time,thus the pigment,polyphenols and nonvolatile organic acid may not be extracted from the tobacco leaf completely.In this paper,the increased solvent polar gradient method was used to extract the secondary metabolites in tobacco leaf and the main pigment,polyphenols and organic acid were determined by liquid chromatography.A new method is provided for the extraction of main pigment,polyphenols and organic acid from tobacco leaf,simultaneously and completely to give some reference for the evaluation of tobacco quality.The results are as follows:(1)The fragrant precursor of tobacco was extracted by increased solvent polar gradient method with n-hexane,chloroform,ethyl acetate,acetone,ethanol and water.More pigment and polyphenols were extracted as compared with that of the industry standard extraction methods,and more nonvolatile organic acid was extracted than the commonly used methanol method.Take the Yunyan 105 as example,the total lutein and β-carotene extracted by increased solvent polar gradient methods is 24.6% and 455.5% higher than that of the industry standard method,respectively.The total chlorogenic acid and rutin extracted by the increased solvent polar gradient methods is 10.6% and 15.9% higher than that of the industry standard method,respectively.While the total oxalate,malic acid and citric acid extracted by increased solvent polar gradient method is 25.0%,30.0% and 28.6% larger than that of the commonly used extraction method of nonvolatile organic acid.This indicats that the increased solvent polar gradient method is more suitable for the extraction of the main pigment,polyphenols and nonvolatile organic acid from tobacco leaf than that of the tobacco industry standard methods of pigment,polyphenols and commonly used method for nonvolatile organic acid extraction.(2)No lutein and β-carotene were detected in the extraction of ethyl acetate,acetone,ethanol and water extractions of Yunyan 105,G80 and Yunyan 87 after ectracted by n-hexane and chloroform.Thus,the pigments of tobacco leaf can be extracted by n-hexane and chloroform completely.The recovery of lutein and β-carotene is 96.3% to 102.6% and 95.8% to 104.4%,respectively,with the ralative standard deviation less than 5%.This indicates that the increased solvent polar gradient method is accurate and reproducibile for the quantitative analysis of the main pigments in tobacco leaf.(3)Chlorogenic acid and rutin can be detected in all of the six extractions.However,the chlorogenic acid and rutinin in the n-hexane extractions of Yunyan 105,G80 and Yunyan 87 is only 0.10%,0.19% and 0.28% of the total chlorogenic acid,and 0.08%,0.08% and 0.31% of the total rutin extracted with the increased solvent polar gradient method,respectively.As the chlorogenic acid and rutin in the n-hexane extraction is very low,the extraction of chlorogenic acid and rutin in tobacco leaf can be finished by chloroform,ethyl acetate,acetone,ethanol and water.The recovery of chlorogenic acid and rutin is 97.1% to 115.0% and 92.7% to 105.2%,respectively,with the ralative standard deviation less than 10%.This indicates that the increased solvent polar gradient method is accurate and reproducibile for quantitative analysis of the main polyphenols in tobacco leaf.(4)The oxalate extracted by n-hexane,chloroform,ethyl acetate,acetone,ethanol and water is 0.79%,29.82%,7.93%,13.84%,20.64% and 27.31% of the total oxalate in the leaf of Yunyan 105 C3 F.The malic acid in chloroform,ethyl acetate and ethanol extraction is 15.43%,59.10% and 25.12% of the total malic acid extracted by the increased six solvents polar gradient method,respectively,while only 0.35% of malic acid was extracted by the left three solvent,n-hexane,acetone,ethanol and water.The total citric acid extracted by acetone,ethanol and water is 20.19%,56.14% and 23.42% of the total citric acid extraxted by the increased six solvents polar gradient method,respectively.As the oxalate,malic acid and citric acid in the n-hexane extraction is very low,the extraction of oxalate,malic acid and citric acid in tobacco leaf can be finished by chloroform,ethyl acetate,acetone,ethyl alcohol and water,also.The recovery of organic acid is 92.5% to 108.7% with a ralative standard deviation of less than 5.2%.This indicates that the increased solvent polar gradient method is accurate and reproducibile for quantitative analysis of the main organic acid in tobacco leaf. |
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