Extraction Of Tobacco Flavor Compounds From Discarded Tobacco Leaves And Their Application In Cigarettes And In Vitro Bioactivities

Tobacco is a very important economic crop and is one of the most commonly studied plants. In China, tobacco cultivation and production are among the highest in the world. Every year, more than25%of the tobacco leaves obtained are discarded during cigarette processing. The discarded tobacco leaves are not used for other purposes and pollute the environment despite containing an abundance of potentially useful compounds, such as aromatic compounds, alkaloids, solanesol, proteins, and polysaccharides. With the problem "smoking and health" have attracted more and more concern, to reduce the tar content in cigarette is imperative. How to keep the aroma style when tar content reducing at the same time becomes one of the most important problems in cigarette enterprise. Therefore, it is important to study and utilize the tobacco leaves discarded during processing.In this study, the aromatic compounds of discarded tobacco leaves were analyzed by hydrodistillation, simultaneous distillation extraction and GC-MS, the extraction methods of tobacco essential oil were established. In vitro antioxidant and antimicrobial activities of tobacco essential oils and extracts were also studied. Then the extraction, purification, and in vitro bioactivities of tobacco polyphenol were studied and evaluated. Results were summarized as follows:1. The aromatic compounds were analyzed by hydrodistillation, simultaneous distillation extraction and GC-MS, a total of55compounds were identified by hydrodistillation representing about81.97%of the total weight.72compounds were identified by simultaneous distillation extraction. The results showed that the major aroma components of discarded tobacco leaves were Solanone, Damascone, Geranyl acetone, Megastigmatrienone,β-Ionone, Benzaldehyde, Benzyl Alcohol, Safranal, and Dihydroactinidiolide, which belong to a group of key tobacco flavor compounds. Thus. discarded tobacco leaves could be a good resource for tobacco essence used in cigarette products.2. Extraction of essential oil from discarded tobacco leaves using a combination of solvent extraction and steam distillation method was explored. According to the RSM results, the optimal extraction conditions to obtain the highest yield were determined as follows:a particle size of40mesh, a temperature of41℃, a liquid-solid ratio of6.27mL/g, a time of5h, an extraction number of3. The predicted extraction yield value of tobacco essential oil was3.44%. A verification experiment was performed using the derived optimum extraction conditions, and the yields of the resulting products were determined. The experimental values (means of5measurements) was3.38±0.03%. No significant differences (p>0.05) between the actual and predicted values were found. These data proved the model designed in this study was valid.3. Extraction of essential oil from discarded tobacco leaves using a short path molecular distillation was explored. Because of the requirement of molecular distillation, petroleum ether (30-60℃) was selected as the best extraction solvent. The optimal extraction conditions to obtain the highest yield were determined as follows:a vacuum of0.lmbar, a scraped film speed of400r/min, a sample velocity of2.5mL/min, a distillation temperature of90℃. The extraction yield of tobacco essential oil was6.38%under optimum extraction condition.4. Application of tobacco essential oil and extracts in cigarette flavoring were studied. Based on the results of smoking sensory evaluation, after adding in cigarettes, tobacco essential oils extracted from discarded tobacco leaves by different methods can improve the smooth and increase the abundance of aroma during smoking when added in cigarettes, they can also improve temperament fragrant, rich incense smoke. All the results showed that tobacco essential oils extracted in this study were good tobacco essence.5. In vitro antioxidant activity of tobacco essential oil and solvent extracts were evaluated by different methods. Results indicated that samples tested in the study had different antioxidant activities under different test method. For example, water extract TE-3had higher antioxidant activity than the synthetic antioxidant BHA in superoxide anion radical scavenging activity assay, while in β-carotene-linoleic acid model system assay, ethyl acetate extract and tobacco essential oil had the highest antioxidant activity. Based on the results of bioassay-guided method, the antioxidant activity of tobacco essential oil and extracts were resulted from high content of tobacco polyphenol. In vitro antimicrobial activities were determined by disc diffusion method, results indicated that tobacco essential oil TEO-1extracted by SE-SD had better antimicrobial activity against three strains of tested microorganisms.6. Ultrasonic assisted extraction of crude tobacco polyphenol from discarded tobacco leaves was explored. The optimal extraction parameters were as follows:the concentration of ethanol was55%, the ratio of solid to liquid was1:25, the extraction temperature was50℃, the extraction time was20min, the ultrasonic power was90W, the extraction number was3. Under optimum conditions, the extraction yield of crude tobacco polyphenol was65.91mg CAE/g. Absorption and desorption experiments were performed to screen the suitable resin from6types of macroporous absorbent resins for purification of tobacco polyphenol from discarded tobacco leaves. Results indicate that macroporous resin D101possesses the strongest absorption and desorption capacity. The optimum absorption and desorption conditions of macroporous resin D101are concentration of feeding solution2.5mg/mL, pH value4.0, feeding rate2.0mL/min, elute ethanol concentration80%, elution rate2.0mL/min. The dynamic recovery of tobacco polyphenol was70.37%under the optimum condition. Identified by RP-HPLC, the major contents of tobacco polyphenol purified by macroporous resin D101were chlorogenic acid (6.2±0.015%), scopoletin (0.8±0.002%), rutin (11.4±0.055%), quercetin (1.08±0.023%), representing about20%of the total weight.7. In vitro antioxidant activity of tobacco polyphenol purified by macroporous resin D101were determined by DPPH radical scavenging assay, β-carotene-linoleic acid model system assay and reducing power assay. In vitro antimicrobial activity of tobacco polyphenol was determined by disc diffusion method. All the results showed that the purified tobacco polyphenol had strong antioxidant and antimicrobial activity compared with positive control.