本文選題：花椒精油　切入點：超臨界二氧化碳萃取　出處：《合肥工業(yè)大學》2017年碩士論文

【摘要】：本論文主要研究花椒精油的萃取工藝、化學成分分析和抗菌活性。通過單因素試驗和響應(yīng)面設(shè)計分析試驗確定最佳萃取工藝條件;并采用GC-MS分析法來確定花椒精油的主要化學成分。利用體內(nèi)和體外抗菌試驗研究花椒精油對大腸桿菌的抑制作用,并研究體外抑菌機理,以及從分子水平研究花椒精油對大腸桿菌致腸道感染的抑制作用。主要的研究內(nèi)容和結(jié)果如下:1、花椒精油的提取。利用超臨界二氧化碳萃取法萃取花椒精油,通過單因素試驗和響應(yīng)面分析設(shè)計法得到優(yōu)化的提取工藝條件。條件為:萃取壓力為26.3MPa、萃取時間為2.53h、萃取溫度為50℃、萃取CO2流量為21L/h、萃取花椒粉目數(shù)為60目,此時花椒精油得率為11.15%。2、花椒精油的主要化學成分分析。采用氣-質(zhì)聯(lián)用儀GC-MS分析法對花椒精油的主要化學成分進行分析,結(jié)果表明花椒精油主要含有兩大類化合物,分別是烴類含氧有機化合物和萜烯類化合物。其中烯烴類化合物24種,占比41.372%;醇類化合物12種,占比為18.923%;酯類化合物13種,占比為35.870%;酮類化合物5種,占比為2.330%;醛類化合物2種,占比為0.157%;酸類化合物3種,占比為1.348%。其中含量較高的5種化合物分別是:2,6,9,11-十二烯~(-1)-羧酸甲酯,占比為12.428%;乙酸-4-萜烯酯,占比為11.249%;氧化石竹烯,占比為8.768%;D-檸檬烯,占比為7.902%;桉葉油醇,占比為5.935%。3、花椒精油的體外抗大腸桿菌活性及機理研究。通過體外抗菌實驗研究花椒精油對大腸桿菌的抑制作用。采用二倍稀釋法,得到花椒精油抑制大腸桿菌的最低抑菌濃度為24mg.mL~(-1),且在細菌生長的延至期和對數(shù)初期加入花椒精油后,細菌生長明顯受到抑制;采用濾紙片法,得到花椒精油的抑菌圈直徑約為20mm±1.0mm,結(jié)果表明花椒精油具有較強的抑制大腸桿菌作用。通過革蘭氏染色法,掃描電鏡的觀察以及細菌細胞溶出物的測定,初步確定花椒精油的抑菌機理是破壞大腸桿菌細胞的完整結(jié)構(gòu),導致細胞膜破裂,細胞內(nèi)容物滲出,最終致使大腸桿菌死亡。4、花椒精油體內(nèi)抗大腸桿菌致腸道感染活性及分子機理研究。通過腹腔注射大腸桿菌建立小鼠腹瀉模型,研究花椒精油體內(nèi)抗大腸桿菌致腸道感染活性。實驗小鼠分為6組:正常對照組、模型對照組、陽性對照組(黃連素)、花椒精油高劑量組、花椒精油中劑量組、花椒精油低劑量組,連續(xù)給藥6天。通過小鼠癥狀評判、腹瀉率計算、以及小鼠體重、攝食和攝水量分析,研究結(jié)果表明花椒精油對小鼠大腸桿菌感染腹瀉有良好的改善效果,呈劑量依賴性。對小鼠小腸組織病理組織學切片觀察,結(jié)果表明模型對照組小鼠小腸組織明顯受到損傷,小腸絨毛組織結(jié)構(gòu)不完整、萎縮斷裂、脫落明顯、腸腺組織結(jié)構(gòu)模糊,粘膜下層明顯水腫,浸潤大量炎性細胞;花椒精油高劑量組對上述小腸病理組織學改變具有明顯的改善作用,小腸絨毛排列整齊有序,很少腫脹脫落,基本沒有炎性細胞浸潤,小腸上皮組織較完整。結(jié)果表明花椒精油對大腸桿菌感染致小鼠腹瀉有明顯改善作用,對損傷的腸道組織具有明顯的保護作用。在大腸桿菌致小鼠腹瀉的發(fā)病過程中,多種炎性細胞因子發(fā)揮了重要的作用。本文研究了小鼠小腸組織Toll樣受體2(TLR-2)、Toll樣受體4(TLR-4)、腫瘤壞死因子α(TNF-α)、白細胞介素6(IL-6)、白細胞介素8(IL-8)5種炎性細胞因子的基因表達,以及花椒精油的干預作用。結(jié)果表明,大腸桿菌致腸道感染小鼠的小腸組織中TLR-2、TLR-4、TNF-α、IL-6、IL-8基因表達量明顯升高,經(jīng)花椒精油干預后,小腸組織中TLR-2、TLR-4、TNF-α、IL-6、IL-8基因表達量均明顯降低,說明花椒精油抑制大腸桿菌感染致腸道功能紊亂的機理可能與調(diào)節(jié)相關(guān)炎性細胞因子表達有關(guān)。綜上所述,采用超臨界二氧化碳萃取法可以得到較優(yōu)的花椒精油提取工藝,并對花椒精油的主要化學成分進行了分析。花椒精油對大腸桿菌有明顯的體內(nèi)體外抑制作用,對大腸桿菌感染致小鼠腹瀉和腸道組織損傷具有明顯的改善和保護作用。
[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.

【學位授予單位】：合肥工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TQ914.1

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