【作者】 李志文；

【导师】 周宝利；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2009， 博士

【摘要】 本文通过显色法、薄层层析法、分光光度法和高效液相色谱法来综合分析与鉴定茄子中糖苷生物碱的主要成分。以高效液相色谱法作为定量手段,研究不同的提取纯化条件对于茄子中α-茄碱提取率的影响,应用超声波提取技术及高效液相色谱法对茄子体内α-茄碱含量分布的规律进行研究,通过模拟茄子糖苷生物碱的释放过程从而研究其释放途径。采用室内生物测定和室外盆栽相结合的方式,通过糖苷生物碱对病菌生长和致病性、黄瓜和甜瓜幼苗抗性生理以及根际微生物种群数量的影响等方面研究,探讨茄子糖苷生物碱在防御蔬菜病原真菌方面的重要作用及作用机理,并且评价茄子糖苷生物碱对自身和其它作物的化感作用,从而为今后更好的利用茄子中糖苷生物碱进行医疗保健及化学生态活性研究、提高茄子自身营养品质和抗病性提供理论依据。为作物抗病育种及天然食品防腐剂和新型生物源农药的开发与利用提供了潜在资源,为进一步完善不同蔬菜作物间合理的间、混、套作提供理论基础。主要研究结果如下:1.本研究建立了以Waters Nova-pak C₁₈柱为色谱柱;乙腈-0.05 mol·L^-1K₂HPO₄（70:30,H₃PO₄调pH值到4.5）为流动相;0.7 mL·min^-1流速;25℃柱温;205 nm检测波长;10μL进样量的茄子中α-茄碱的HPLC检测方法。并证明,该方法精密度高、准确性好,适用于茄子中糖苷生物碱的定性与定量分析。2.本研究建立了以超声波技术为提取手段,以10倍量的70%甲醇为提取剂,以50℃下提取60 min为提取条件的茄子中α-茄碱的提取方法,并确定了以无水乙醚液-液萃取为纯化手段,提取过程中最佳酸溶pH为2-3,最佳碱沉pH为10-11。该方法α-茄碱平均回收率为98.0%,RSD为1.23%（n=3）,说明该提取方法提取率和精密度高、稳定性好,合理可行。3.明确了茄子中糖苷生物碱的含量分布特征。不同器官中α-茄碱含量大小顺序为:种子＞果实＞叶片＞根系＞侧枝＞主茎:叶片和主茎中α-茄碱含量的最高值出现在5月份左右,而根系则出现在6月份。不同品种类型的茄子果实中α-茄碱含量不同,紫茄中α-茄碱含量显著高于绿茄,门茄果实中α-茄碱含量显著高于对茄、四面斗等其它时期的果实,未成熟的果实中α-茄碱含量显著高于成熟的茄子果实,果肉中α-茄碱含量显著高于果皮。4.阐明了茄子糖苷生物碱释放途径。茄子可通过根系分泌、残体分解和种子萌发的途径向环境中释放糖苷生物碱,其中残体分解和种子萌发是主要途径,根系只能主动分泌极少量的糖苷生物碱,雨水淋溶或浸泡几乎不会使茄子植株中糖苷生物碱释放到环境中。从茄苗现蕾到四面斗果实成熟是根系分泌糖苷生物碱的主要时期,门茄坐果至对茄成熟,茄子根际土壤中α-茄碱含量增加最显著。植株不同部位残茬腐解后释放出的糖苷生物碱量大小顺序为:叶＞根＞茎,随着残株分解时间的延长,根际土壤中茄碱含量显著增高。茄子种子萌发过程释放的生物碱含量与种子萌发密切相关,该生物碱可抑制茄子种子萌发。5.明确了茄子糖苷生物碱对蔬菜病原菌的抗性。茄子糖苷生物碱对黄瓜枯萎菌和甜瓜枯萎菌具有较显著的抑制作用,对于辣椒枯萎菌和番茄灰霉菌抑制作用较弱、而对于茄子黄萎菌未表现抑制作用,对于黄瓜和甜瓜枯萎菌的抑制作用主要表现在抑制菌体生长和致病性两方面。茄子糖苷生物碱在中性至弱碱性条件下抑菌活性最强,在长时间高温条件下失去抑菌活性;无论是抗病还是感病品种,茄子糖苷生物碱对于黄瓜枯萎病和甜瓜枯萎病具有较好的预防效果,但对抗病品种的预防效果要好于感病品种。糖苷生物碱水解后的产物抑菌活性显著下降,说明糖苷生物碱结构与抑菌活性关系密切。6.研究了茄子糖苷生物碱对自身和其它蔬菜的化感作用。茄子糖苷生物碱对于茄科蔬菜作物种子萌发具有显著的抑制作用,而对瓜类和菜豆种子萌发则呈现“低促高抑”的双重浓度效应,糖苷生物碱对于五种蔬菜的化感抑制效应大小顺序为:茄子＞番茄＞菜豆＞黄瓜＞甜瓜。茄子糖苷生物碱对于茄科蔬菜作物种子萌发生理活动和生理代谢具有较显著的抑制作用,而对瓜类和菜豆种子的影响较小。茄子糖苷生物碱对其它蔬菜生长的影响主要体现在种子萌发阶段,对幼苗生长无显著影响,高浓度糖苷生物碱会抑制茄子幼苗生长;茄子糖苷生物碱可通过影响黄瓜和甜瓜幼苗抗性生理来提高自身抗病性。7.明确了茄子糖苷生物碱对黄瓜根际土壤生物活性的影响。茄子糖苷生物碱处理植株同对照相比根际放线菌和细菌数量增加,真菌数量减少,A/F比值和B/F比值均升高,土壤微生物总量增加。茄子糖苷生物碱对黄瓜苗期根际微生物的作用效果显著大于初花期,初花期根际微生物数量显著多于苗期。茄子糖苷生物碱处理使黄瓜根际真菌种群总数和有害种群数量减少,但有益真菌种群数量增加。茄子糖苷生物碱处理可以提高黄瓜苗期根际土壤过氧化氢酶、多酚氧化酶、脲酶和酸性磷酸酶活性,从而提高黄瓜幼苗抗病性。综上所述,本试验通过研究确定了茄子中糖苷生物碱的提取测定方法,从而为大量生产茄碱工艺的开发提供技术参数;分析了糖苷生物碱在不同茄子品种和不同部位中的含量分布特征,从而为寻找高糖苷生物碱含量的种质资源提供理论依据;明确了茄子糖苷生物碱的化学生态学功能,为开发安全、环保的天然防腐剂及生物农药提供潜在资源,为进一步研究、改善茄子栽培生态环境提供理论依据。更多还原

【Abstract】 Different methods which include coloration method,TLC method,spectrophotography method and HPLC method was designed to comprehensive analysis and consultation the composition of glycoalkaloid in eggplant.Ultrasonic wave extraction and HPLC were used to study regulation that theα-solanine content distributes in eggplant.Release pathway of glycoalkaloid from eggplant was studied by imitating release process of glycoalkaloid from eggplant.Adopt the indoor living creature measurement and outdoor potted plant combines together.By the research that influence of glycoalkaloid on germ growth,pathogenicity, resistance physiology of cucumber and melon’s seedling and rhizosphere microorganisms population,we discussed the function and mechanism of glycoalkaloid in eggplant on vegetable germ defense,and evaluated the allelopathy of glycoalkaloid in eggplant on itself and other plant.Thus glycoalkaloid in eggplant can be used in medical care and chemical ecology,and made the nutritional quality and disease resistance of eggplant increased.It also can provide potential resources and theoretical basis to the crop resistance breeding, development and utilization of food preservative,bio-pesticide and intercropping between different vegetable.1.We established a HPLC detection method ofα-solanine in eggplant which used Waters Nova-pak C₁₈ as chromatographic column,acetonitrile-0.05 mol·L^-1K₂HPO₄（70:30,the pH of H₃PO₄ was adapted to 4.5） as mobile phase,velocity of flow was 0.7 mL·min^-1.Column temperature was 25℃.Detection wavelength was 205 um.Injection volume was 10μL.This technique has been proved to be good precision and accuracy and adapt to qualitative and quantitative analysis of glycoalkaloid in eggplant.2.Our study established the extraction method of from eggplant using ultrasonic.Extraction solvent was 10 times amount 70%methanol.The extraction process last 60 min under 50℃. Anhydrous aether extraction was used to be purification ofα-solanine.The best pH during the process thatα-solanine dissolve in acid was 2 to 3.The best pH during the process whichα-solanine deposit in aqueous alkali was 10 to 11.The average recovery ofα-solanine was 98.0%.RSD was 1.23%（n=3）.All of the above proved the ultrasonic extraction has good precision and accuracy.3.The content ofα-solanine in eggplant has been studied in this research.And content order ofα-solanine in different organs is seeds>fruits>leave>roots>lateral branch> main stem.The maximum content ofα-solanine in leave and main stem appeared at May.The roots’ appear at June.There was some difference about content ofα-solanine among different type of eggplant fruits.Content ofα-solanine in purple eggplant was significantly higher than green eggplant. Content ofα-solanine in first fruit was significantly higher than second fruit and third fruit. Content ofα-solanine in immature fruit was significantly higher than mature fruit.And the content ofα-solanine in flesh was significantly higher than the pericarp.4.Release path of glycoalkaloid in eggplant was clarified.The glycoalkaloid in eggplant can be released through root exudation,remnants body decomposition and seed germination. Main path to release glycoalkaloid are remnants body decomposition and seed germination. Only a little glycoalkaloid was released through root exudation.Rainwater eluviate almost couldn’t make glycoalkaloid in eggplant released to environment.The main period of glycoalkaloid release from eggplant root was from seedling alabastrum to the mature of the third eggplant.Content ofα-solanine in the soil around eggplant root significantly increase at the period which from the first fruit to the second fruit.The content order of released glycoalkaloid from remnants body at different part of eggplant was leaf>root>stem.Along with remnants decompose,glycoalkaloid content in soil which around roots significantly increased.The content of glycoalkaloid which released through seed germination closely related to seed germination.That glycoalkaloid can inhibit eggplant seed to germinate.5.The resistance of glycoalkaloid in eggplant on vegetable phytopathogen was studied.There is significant inhibition which glycoalkaloid in eggplant acted on F.omysporum cucumerinum and F.oxysporum melonis,and poor inhibition on F.oxysporum vasinfectum and Botrytis cinerea.There is almost none inhibition which glycoalkaloid in eggplant acted on Verticillium dahliae.The inhibition on F.omysporum cucumerinum and F.oxysporum melonis expressed in hypha growth and pathogenicity.The inhibition of glycoalkaloid in eggplant appeared the strongest at neutrality and alkalescence.But it would lost inhibition at high temperature for long time.Glycoalkaloid in eggplant has better prevention on F.omysporum cucumerinum and F.oxysporum melonis whatever the eggplant was resistant variety or not.But the prevention effect of resistant variety was better than susceptible variety.The inhibition of glycoalkaloid significantly decreased after hydrolysis.So that structure of glycoalkaloid closely related to the inhibition.6.Allelopathy which glycoalkaloid in eggplant acted on itself and other vegetable had been studied.The glycoalkaloid in eggplant had significant inhibition on seed germination of solanaceous vegetable.However,the seed germination of melon and bean were promoted by glycoalkaloid at lower concentration but inhibited at higher concentration.The strength order of allelopathy which glycoalkaloid acted on other five vegetable is eggplant>tomato>bean >cucumber>melon.Physiological activity and metabolism of solanaceous vegetable seed germination was significantly inhibited by glycoalkaloid in eggplant.And the inhibition on melon and bean was lower than solanaceous vegetable.The affection which glycoalkaloid in eggplant acted on other vegetable mainly expressed at seed germination and no significant affection on seedling growth.Higher concentration of glycoalkaloid could inhibit seedling growth of eggplant.Disease resistance of glycoalkaloid increased through affect the resistance physiology of cucumber and melon.7.Affection that glycoalkaloid in eggplant acted on biological activity in rhizosphere soil of cucumber.By the treatment of glycoalkaloid in eggplant,content of actinomyces and bacterium in rhizosphere soil increased,fungi content decreased,ratio of A/F and B/F increased,microorganism content in soil increased.Action of glycoalkaloid in eggplant on rhizosphere microorganism at seedling stage of cucumber was significant higher than initial bloom stage.Microorganism content at initial bloom stage was significantly higher than seedling stage.Fungi and harmful colony population of rhizosphere were decreased by glycoalkaloid in eggplant treatment.And beneficial fungi population increased.Activity of catalase,polyphenoloxidase,urase and phenol at seedling stage were increased by glycoalkaloid in eggplant treatment.So that disease resistance of cucumber seedling increased.In summary,the extracting and determinating method of glycoalkaloid in eggplant were determined in this study,which will provide technical parameters for development of glycoalkaloid production process.The content distribution in different cultivars and positions of eggplant was analyzed,which will provide theoretical basis for searching for germplasms with high glycoalkaloid content.The chemical ecology functions of glycoalkaloid in eggplant were cleared,which will provide potential sources for developing safe and environmental natural preservative and biological agrochemical,which will also provide theoretical basis for further research and improvement of ecological environment of eggplant cultivation.更多还原