花椒精油的提取工艺、化学成分分析与抗菌活性研究

发布时间：2018-04-02 20:29

本文选题：花椒精油　切入点：超临界二氧化碳萃取　出处：《合肥工业大学》2017年硕士论文

【摘要】：本论文主要研究花椒精油的萃取工艺、化学成分分析和抗菌活性。通过单因素试验和响应面设计分析试验确定最佳萃取工艺条件;并采用GC-MS分析法来确定花椒精油的主要化学成分。利用体内和体外抗菌试验研究花椒精油对大肠杆菌的抑制作用,并研究体外抑菌机理,以及从分子水平研究花椒精油对大肠杆菌致肠道感染的抑制作用。主要的研究内容和结果如下:1、花椒精油的提取。利用超临界二氧化碳萃取法萃取花椒精油,通过单因素试验和响应面分析设计法得到优化的提取工艺条件。条件为:萃取压力为26.3MPa、萃取时间为2.53h、萃取温度为50℃、萃取CO2流量为21L/h、萃取花椒粉目数为60目,此时花椒精油得率为11.15%。2、花椒精油的主要化学成分分析。采用气-质联用仪GC-MS分析法对花椒精油的主要化学成分进行分析,结果表明花椒精油主要含有两大类化合物,分别是烃类含氧有机化合物和萜烯类化合物。其中烯烃类化合物24种,占比41.372%;醇类化合物12种,占比为18.923%;酯类化合物13种,占比为35.870%;酮类化合物5种,占比为2.330%;醛类化合物2种,占比为0.157%;酸类化合物3种,占比为1.348%。其中含量较高的5种化合物分别是:2,6,9,11-十二烯~(-1)-羧酸甲酯,占比为12.428%;乙酸-4-萜烯酯,占比为11.249%;氧化石竹烯,占比为8.768%;D-柠檬烯,占比为7.902%;桉叶油醇,占比为5.935%。3、花椒精油的体外抗大肠杆菌活性及机理研究。通过体外抗菌实验研究花椒精油对大肠杆菌的抑制作用。采用二倍稀释法,得到花椒精油抑制大肠杆菌的最低抑菌浓度为24mg.mL~(-1),且在细菌生长的延至期和对数初期加入花椒精油后,细菌生长明显受到抑制;采用滤纸片法,得到花椒精油的抑菌圈直径约为20mm±1.0mm,结果表明花椒精油具有较强的抑制大肠杆菌作用。通过革兰氏染色法,扫描电镜的观察以及细菌细胞溶出物的测定,初步确定花椒精油的抑菌机理是破坏大肠杆菌细胞的完整结构,导致细胞膜破裂,细胞内容物渗出,最终致使大肠杆菌死亡。4、花椒精油体内抗大肠杆菌致肠道感染活性及分子机理研究。通过腹腔注射大肠杆菌建立小鼠腹泻模型,研究花椒精油体内抗大肠杆菌致肠道感染活性。实验小鼠分为6组:正常对照组、模型对照组、阳性对照组(黄连素)、花椒精油高剂量组、花椒精油中剂量组、花椒精油低剂量组,连续给药6天。通过小鼠症状评判、腹泻率计算、以及小鼠体重、摄食和摄水量分析,研究结果表明花椒精油对小鼠大肠杆菌感染腹泻有良好的改善效果,呈剂量依赖性。对小鼠小肠组织病理组织学切片观察,结果表明模型对照组小鼠小肠组织明显受到损伤,小肠绒毛组织结构不完整、萎缩断裂、脱落明显、肠腺组织结构模糊,粘膜下层明显水肿,浸润大量炎性细胞;花椒精油高剂量组对上述小肠病理组织学改变具有明显的改善作用,小肠绒毛排列整齐有序,很少肿胀脱落,基本没有炎性细胞浸润,小肠上皮组织较完整。结果表明花椒精油对大肠杆菌感染致小鼠腹泻有明显改善作用,对损伤的肠道组织具有明显的保护作用。在大肠杆菌致小鼠腹泻的发病过程中,多种炎性细胞因子发挥了重要的作用。本文研究了小鼠小肠组织Toll样受体2(TLR-2)、Toll样受体4(TLR-4)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素8(IL-8)5种炎性细胞因子的基因表达,以及花椒精油的干预作用。结果表明,大肠杆菌致肠道感染小鼠的小肠组织中TLR-2、TLR-4、TNF-α、IL-6、IL-8基因表达量明显升高,经花椒精油干预后,小肠组织中TLR-2、TLR-4、TNF-α、IL-6、IL-8基因表达量均明显降低,说明花椒精油抑制大肠杆菌感染致肠道功能紊乱的机理可能与调节相关炎性细胞因子表达有关。综上所述,采用超临界二氧化碳萃取法可以得到较优的花椒精油提取工艺,并对花椒精油的主要化学成分进行了分析。花椒精油对大肠杆菌有明显的体内体外抑制作用,对大肠杆菌感染致小鼠腹泻和肠道组织损伤具有明显的改善和保护作用。
[Abstract]:This paper mainly studies the extraction of essential oil, chemical composition analysis and antibacterial activity. By single factor test and response surface analysis design test to determine the best extraction conditions; and using GC-MS analysis method to determine the main chemical components of essential oil. The use of in vivo and in vitro antibacterial test of Xanthoxylin inhibition on Escherichia coli. Study on the antibacterial mechanism and in vitro, and to study the molecular Xanthoxylin induced inhibition of intestinal infection of Escherichia coli. The main research contents and results are as follows: 1. Extraction of essential oil by supercritical carbon dioxide extraction of essential oil, by single factor experiment and response surface analysis and design method to optimize the extraction condition.. conditions for extraction pressure 26.3MPa, extraction time 2.53h, extraction temperature of 50 DEG CO2, extraction flow is 21L/h, extraction of pepper powder 60 mesh At this time, essential oil yield of 11.15%.2, analysis of main chemical components of essential oil. By gas chromatography-mass spectrometry GC-MS method for analysis of main chemical components of essential oil were analyzed. The results show that Xanthoxylin mainly contains two kinds of compounds are hydrocarbon oxygenated organic compounds and terpene compounds including olefin. A compound of 24, accounting for 41.372%; 12 alcohol compounds, accounted for 18.923%; 13 kinds of ester compounds, accounting for 35.870%; 5 kinds of ketone compounds, accounting for 2.330%; 2 kinds of aldehydes, accounted for 0.157%; acid compound 3, accounting for 1.348%. 5 of them were higher were 2,6,9,11- twelve ene ~ (-1) - carboxylic acid methyl ester, accounted for 12.428%; acetic acid -4- ester terpene, accounted for 11.249%; caryophyllene oxide, accounted for 8.768%; D- limonene, accounted for 7.902%; eudesmol, accounted for 5.935%.3, the body of Xanthoxylin Study on activity and mechanism of Escherichia coli resistant. Inhibition by in vitro antibacterial experiment of zanthoxylum oil on Escherichia coli. By two times dilution method, get the minimum inhibitory concentration of Xanthoxylin inhibited Escherichia coli 24mg.mL~ (-1), and the addition of essential oil in bacterial growth until the early logarithmic period and after bacterial growth significantly inhibited; filter paper method was used to get the bacteriostatic ring Xanthoxylin diameter 20mm + 1.0mm. The results show that Xanthoxylin has a strong role in inhibiting Escherichia coli by Gram staining. The determination of dissolution, scanning electron microscope and bacterial cells, preliminarily determined the antibacterial mechanism of Xanthoxylin is complete destruction of E. coli cells, leading to cell membrane rupture, cell content leakage, ultimately resulting in the death of Escherichia coli.4, intestinal infection and the activity of molecules induced by anti Escherichia coli Xanthoxylin in vivo Study on the mechanism of mice. The diarrhea model by intraperitoneal injection of Escherichia coli, intestinal infection caused by Xanthoxylin in vivo study activity against Escherichia coli. The experimental mice were divided into 6 groups: normal control group, model control group, positive control group (berberine), Xanthoxylin high dose group, dose group of pepper oil, pepper oil low dose group administered continuously for 6 days. The mice diarrhea symptoms evaluation, rate, and body weight, food intake and water intake analysis, the results show that Xanthoxylin has good improving effect on diarrhea in mice infected with Escherichia coli in a dose-dependent manner. The observation of mouse small intestine pathological slices, the results show that the model control group of small intestine the mice were damaged, villus tissue structure is not complete, atrophic fracture, shedding significantly, fuzzy structure of intestinal gland tissue, submucosal edema, infiltration of inflammatory cells; pepper The essential oil of high dose group has obvious effect on the intestinal pathological change of intestinal villi, neat and orderly, rarely swelling off, no infiltration of inflammatory cells and the intestinal epithelial tissue integrity. The results showed that the essential oil of induced diarrhea has obvious improvement effect on Escherichia coli infection, has obvious protective effect on the injury of intestinal tissue. The pathogenesis in Escherichia coli induced diarrhea in a variety of inflammatory cytokines play an important role. This paper studies the Toll like receptor 2 (TLR-2) in small intestine of mice, Toll like receptor 4 (TLR-4), tumor necrosis factor alpha (TNF- alpha), interleukin 6 (IL-6). Interleukin 8 (IL-8 5) expression of inflammatory cytokine genes, and the intervention effect of zanthoxylum oil. The results showed that TLR-2, intestinal infection in mice induced by Escherichia coli in small intestine of TLR-4, TNF- alpha, IL-6, IL-8 gene expression Was increased by Xanthoxylin intervention, TLR-2, intestinal TLR-4, TNF- alpha, IL-6, IL-8 gene expression was significantly decreased, indicating the mechanism of Xanthoxylin inhibit Escherichia coli infection caused by intestinal dysfunction may be associated with regulation of inflammatory cytokine expression. In summary, using supercritical carbon dioxide extraction method to get the better of the essential oil extraction process, and the main chemical components of the essential oil were analyzed. Zanthoxylum oil has inhibitory effect on Escherichia coli in vitro and in vivo significantly, on mice diarrhea and intestinal tissue injury has improved and protected the infection of Escherichia coli.

【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ914.1

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