【作者】 刘世尧；

【导师】 李加纳； 白志川；

【作者基本信息】 西南大学 ， 作物学， 2012， 博士

【摘要】 皱皮木瓜(Chaenomeles speciosa (Sweet) Nakai)是蔷薇科(Rosaceae)苹果亚科(Maloideae)木瓜属(Chaenomeles)多年生落叶小乔木,原产于亚洲东部,世界各地均有分布,是我国南北各地广范栽培的重要果树资源。皱皮木瓜果实营养丰富、气味芳香,富含超氧化物歧化酶、齐墩果酸、熊果酸、黄酮类、皂甙类、有机酸类等多种活性成分,是一种药食兼用的水果类型。然而皱皮木瓜果肉维管系统发达、石细胞丰富、有机酸含量极高,质地坚硬、口感酸涩,除药用外,难以作为鲜食水果供人们食用,由此造成大量的野生皱皮木瓜资源浪费。随着近年来国内外植物提取物市场需求量的激增,以皱皮木瓜果实为原料进行活性成分的规模化生产已成为我国皱皮木瓜果树资源产品深度开发的新方向；同时作为蔷薇科典型高酸果树品种,对皱皮木瓜果实高酸积累机理的探索研究,可以为苹果亚科近缘果树果实有机酸代谢及调控研究提供基础数据。但国内外对皱皮木瓜果实活性成分的提取、分离纯化工艺研究及果实有机酸代谢均在不同程度上受到研究方法陈旧和技术手段落后等瓶颈问题制约,尚处于起步阶段。代谢组学(Metabonomics/Metabolomics)研究技术是对某一生物或细胞在特定生理时期内所有低分子量代谢产物同时进行定性和定量分析的一门新学科,它是以组群指标分析为基础,以高通量检测和数据处理为手段,以信息建模与系统整合为目标的系统生物学分支,适用于生物体复杂组分代谢机理的研究。低温超声萃取技术(Low-temprature Ultrasound Extracting Technology)是近年来发展起来的一种新型分离技术,它是在低温条件下通过空化效应实现对萃取的强化作用,在超声场中空化效应产生的微小气泡瞬间被激活,一系列动力学过程伴随超声空化产生的微射流、冲击波等机械效应,加快两相间的传质速度,从而促进细胞内有效成分的溶出。离心分配色谱(Centrifugal Partition Chromatography, CPC)属于现代液液逆流分配色谱的一种,高效离心分配色谱技术(High Performance Centrifugal Partition Chromatography, HPCPC)是由离心分配色谱发展来的液相色谱新技术,具有很强的分离纯化性能,已被广泛应用于天然产物和中药有效成分的分离纯化。但以上技术迄今均鲜见于皱皮木瓜果实开发方面的研究报道。本论文以我国特色果树资源皱皮木瓜为试材,利用基于RP-HPLC-PDA检测平台与GC-MS检测平台的植物代谢组学研究技术对皱皮木瓜果实中的水溶性和脂溶性有机酸的组成、含量波动范围以及果实全发育期主要有机酸代谢规律进行探索研究,获得了代表样品有机酸组分定性定量检测数据、全国十个道地产区12批样品有机酸组成和含量差异数据及重庆皱皮木瓜全发育期果实有机酸代谢规律数据,分析了皱皮木瓜果实高酸积累机制；同时利用现代低温超声萃取技术、高效离心分配色谱分离技术、高效液相色谱以及气相-质谱联用检测技术,对其果实超氧化物歧化酶、齐墩果酸熊果酸、挥发油等3大类活性成分进行了提取、分离纯化和鉴定研究,并进行皱皮木瓜果实SOD酶学性质检测、齐墩果酸熊果酸提取工艺优化和纯度鉴定以及挥发油组成和含量检测,完善相关工艺并获得相应数据。有关研究结果如下：1.不同产区皱皮木瓜有机酸组成检测及全发育期果实有机酸代谢规律采用HPLC-PDA法共检出皱皮木瓜水溶性有机酸13种,采用GC-MS共检出其脂溶性有机酸43种,方法稳定可靠。国内十个道地产区12批样品水溶性有机酸总含量范围为：119.841-276.046mg·g-1,四川仪陇最高而重庆綦江三角最低；样品脂溶性有机酸总含量以山东沂州最高而陕西白河最低；聚类分析筛选出水溶性有机酸和脂溶性有机酸高、中、低酸典型品种各一组,且其组成各不相同。采用代谢组学方法研究重庆皱皮木瓜果实不同生长期水溶性与脂溶性有机酸的动态变化,结果表明：在果实发育前期两类有机酸的种类和数量随时间的增高而提高,7月中下旬达高最高水平,果实成熟期时随生长时间迅速减少；全发育期苹果酸、富马酸与水溶性总有机酸积累过程呈显著正相关,而α-酮戊二酸与之呈负相关；苹果酸、柠檬酸、奎尼酸等9种与果实脂溶性有机酸积累过程呈显著正相关,而戊酸与之呈负相关,说明苹果酸是皱皮木瓜果实高酸积累的关键因子。本节建立了皱皮果实有机酸代谢组学基本研究方法,揭示了重庆皱皮木瓜全发育期果实有机酸代谢规律。2.皱皮木瓜超氧化物歧化酶提取工艺优化与酶学性质检测皱皮木瓜果实超氧化物歧化酶最佳提取工艺为：以pH=9.32的0.05mol·L-1磷酸氢二钠溶液作为提取溶剂,料液比1：3,以聚乙烯聚吡咯烷酮作为果实酚类物质吸附剂,采用低温(0℃)超声(800W)萃取处理20mmin,目标酶蛋白提取率达到最大值。皱皮木瓜果实超氧化物歧化酶蛋白分子量约为38.5kD,在347nm波长处具有特征性紫外吸收峰,且双氧水和氯仿-乙醇(3：5)对目标酶蛋白活性具有明显抑制作用而氰化物作用不明显,因此初步皱皮木瓜超氧化物歧化酶为铁型SOD；同时酶液在60℃,恒温水浴中酶活保持时间约为10-15min,在pH=6.0-9.0酸度范围内目标酶蛋白活性较稳定。本研究有效消除了多酚与高酸对皱皮木瓜超氧化物歧化酶溶出率的抑制,明确了皱皮木瓜超氧化物歧化酶的酶学性质。3.皱皮木瓜齐墩果酸和熊果酸提取纯化与高效离心分配色谱分离皱皮木瓜齐墩果酸和熊果酸最佳提取工艺为：以95%乙醇作提取溶剂,料液比1：30,250W60℃超声处理20min,目标化合物提取率达到最大；总浸提物经过碱溶酸沉与氯仿萃取处理可使目标提取物得到进一步纯化；以正己烷-乙酸乙酯-甲醇-水(7：3：5：1,V/V)作为两相系统,下相作为固定相,上相作为流动相,采用上行模式洗脱,在流动相流速1.5mL·min-1、转速1000rpm条件下,齐墩果酸和熊果酸混合物在180min内得到有效分离,馏份重结晶后高效液相色谱仪纯度鉴定结果分别为96.9%与94.3%。本研究解决了皱皮木瓜三萜类成分同分异构体分离的难题。4.皱皮木瓜挥发油提取与分离鉴定采用水蒸汽蒸馏法提取皱皮木瓜鲜果挥发油,提取率约为0.016%；气相-质谱联用仪分离鉴定出样品挥发油组分106种,其中酯类物质(52种)占80.95%、醇类物质(18种)占6.16%、羧酸类(12种)占4.80%、烷烃和烯烃类(14种)占4.29%、酮类(3种)占1.62%,及少量萘类、吡喃类、醛类成分。皱皮木瓜果实挥发油提取率低于光皮木瓜(0.022%),皱皮木瓜果实极性挥发油组成与光皮木瓜相似但含量低于光皮木瓜,弱极性挥发油组分数量高于光皮木瓜。二者均含有多个应用价值较高的挥发油组分。本研究解决了重庆皱皮木瓜果实挥发油组成不明的问题。综上所述本研究结果不仅对于阐明皱皮木瓜果实有机酸组成及全发育期果实有机酸代谢机理,丰富蔷薇科果树果实有机酸代谢理论,而且对我国皱皮木瓜资源果实超氧化物歧化酶、齐墩果酸熊果酸、挥发油等3大类活性成分深度开发提供基础数据。研究结果具有重要的理论意义和实践价值。更多还原

【Abstract】 Chaenomeles speciosa (Sweet) Nakai is Dicotyledoneae Rosaceae Maloideae Chaenomeles Lindl. defoliate perennial small tree. C. speciosa is native to eastern Asia, widely natively distributed in the world, and is one of the most important fruit tree resources in China. The fruit of C. speciosa has abundant nutrients and aromatic smell, and it contains many biological active components including superoxide dismutase (SOD), oleanolic acid(OA) and ursolic acid (UA), flavonoids, saponins and organic acids, etc. and is an edible fruit and can be used as medicine. However, there are strengthed vascular bundles, rich stone cells and extremly high organic acids content in its fruit flesh. Excepting for being used for medicine, the fruit has hard texture and tastes sour and puckery, and it is difficult to directly supply for people as fresh fruit. Which caused great wastes of C. speciosa fruits. With the significant increasing demand of plant extracts in domestic and foreign markets in recent years, scale Production of its biological active components extracts regarding C. speciosa fruit as materials has become a new way for developping of C. speciosa. At the same time, C. speciosa fruit organic acids metabolism studying is especially necesarry as a high organic acids contents fruit tree type. Which could provide the basic data for metabolism researching and regulating of organic acids in fruit of the fruit tree which is closely related to Maloideae. But researching on the extracting, seperating and purifying technology of the main biological active components and studying of the fruit organic acids metabolizing are conditioned by the old studying methods and backward technological means in different degree, that is still in its beginning stage.Metabonomics is a new subject that qualitatively and quantitatively analyze metabolites within a certain period in some living thing or cells. Metabonomics is branch of systems biology that is on the basis of cluster-parameters testing, and by the means of high-throughput detecting, to have information modeling and system integrating as a purpose. So it is suitable to study on the metabolism of the complicated metabolites in organism. Ultrasound extracting technology is a new extracting technology developed in recent years. This way realized strengthening effect to the active component extracting by ultrasonic cavitation. The micro bubbles were created in ultrasonic treatment field and cultivated for a moment. At the same time a series of dynamics process following along with its mechanical effects including micro-jet and shock-wave etc. produced by ultrasounic cavitation field accelerates the mass spectra transfer velocity between two phases. Which gives a boost to dissolution rate of the effective components. Centrifugal partition chromatography(CPC) technology is one of the modern liquid-liquid counter current chromatography technologies. High performance centrifugal partition chromatography (HPCPC) is a new technology of liquid chromatography derived from centrifugal partition chromatography(CPC). And HPCPC has a very strong seperating and purifying ability. Nowaway it has been widely used to seperating and purifying of the natural products and effective components of traditional Chinese drugs(TCD). But up to the present time, these technologies are rarely used to exploitation studying of Chaenomeles speciosa fruit product.Regarding Chaenomeles spesiosa which is pensable resources in China as experimential material, Metabonomics technology basing on RP-HPLC-PDA and GC-MS was utilized for its fruit organic acids studying including compositon, concentration range of fruit water-soluble and fat-soluble organic acids of12samples from10main producing areas of China. In addition,organic acids metabolizing law was explored during the whole developping period of C. speciosa produced in Qijiang,Chongqing and its high-acids mechanism was analyzed. At the same time, this paper has studied on extraction,seperation and purification technologies of the3main functional components including superoxide dismutase (SOD), oleanolic acid(OA) and ursolic acid (UA), essential oil etc. in its fruit. Its’SOD enzyme properties was detected, extraction routes of OA and UA was optimized and their products purity was determined, and the composition and relative content of fruit essential oil were measured, all the data was obtained. The results of this study are as follows:1. Determination of organic acids in C. speciosa fruits samples from defferent producing area in China and studying on metabolizing raw of organic acids during the whole developping period of C.speciosa fruit produced in ChongqingThe study detected13water-soluble organic acids from C. speciosa fruit by HPLC-PDA and43fat-soluble organic acids by GC-MS.The steady and reliable detecting method were set up. The total water-soluble organic acids content is from119.841to276.046mg·g-1, the highest sample is from Yilong,Sichuan and the lowest is from Qijiang,Chongqing. The highest fat-soluble total organic acids sample is from Qizhong,Shandong and the lowest one is from Baihe,Shanxi. SPSS13.0clustering analysis found the high, middle and low acids strains of each water-soluble organic acids and fat-soluble organic acids. The dynamic expressing law of organic acids accumulating of C. speciosa fruit were detected and analyzed by Metabonomics technology basing on principal components analysis (PCA) of HPLC-PDA and GC-MS dectectong data. The results showed that the species and quantities of two types of organic acids increased in growing time during the earlier-developing stage, but decreased in the later-developing stage. Their main parameters reached the maximum value in July. The accumulation process of malate and fumaric acid were significantly positive with total water-organic acids but a-ketoglutarate was negative with them during the whole developing period of C. speciosa fruit.9kinds of fat-soluble acids including Malate, citrate and quinic acid etc. were significantly positive with total fat-organic acids but pentanoic acid was negative with total fat-organic acids during the whole developing period of C. speciosa fruit. The results set up the basic Metabonomics studying method of fruits organic acids,showed that malate is one of the key factors for C.speciosa fruit high-acids accumulation and explored the organic acids metabolizing law during the whole developing period of C. speciosa fruit produced in Qijiang, Chongqing.2. Extraction of SOD and determination of the enzyme properties in C. speciosa fruitThe best process conditions of SOD extraction in C. speciosa fruit:extraction solvent is pH=9.32,0.05mol·L-1Na2HOP4solution, the ratio of gardenia to liquor was1:3, phenolic constituents adsorptive material is PVPP, and in0℃low temperature with800W ultrasonic assisting extration for20min. Now target SOD Enzyme extracting rate reached maximum. The molecular weight of C. speciosa fruit SOD is38.5kD and its characteristic absorption peak is at347nm. H2O2and chloroform-alcohol(3:5) significantly inhibited its SOD enzyme acitivity, but KCN didn’t. So C. speciosa fruit SOD is Fe-SOD. At the same time, the enzyme activity was stable within pH6.0-9.0and within10-15min at60℃. The studying results removed the inhibit of high acid and phenolic Constituents to the dissolution efficiency of fruit SOD and SOD enzyme properties were clear.3. Extraction and purification of OA and UA of C. speciosa fruit and their seperation by HPCPCThe best process conditions of OA and UA extraction in C. speciosa fruit:extraction solvent is alcohol(95%), the ratio of gardenia to liquor is1:30, at60℃and with250W ultrasonic assisting extration for20min. When OA and UA extracting rate reach maximum. The rude extracts were further purified by means of aqueous alkali and acid precipitation and chloroform extraction. Hexane-ethyl Acetoacetate-methanol-water(7:3:5:1. V|V) was used as two phases system, the lower phase was used as stationary phase and the upper phase was used as mobile phase, increased model eluting, and with1.SmL·min-1flowing rate and at1000rpm rotating speed, OA and UA were effectively seperated within180min by HPCPC. The two fractions were further purified by recrystallization. The two samples purity seperately is96.9%and94.3%determined by HPLC. The studying result disposed of the seperating problem of OA and UA isomers.4. Extraction, seperation and identification of essential oil from C.speciosaThe C. speciosa fresh fruit essential oil was extracted by steam distilling method and its extracting rate is about0.016%. GC-MS seperated and identified106components of C. speciosa fruit essential oil including80.95%ester matter,6.16%alcohols,4.80%organic acids,4.29%alkanes and alkenes,1.62%ketones, and some naphthalenes, pyrans, aldehydes,etc. Its extracting rate was lower than C. sinensis (0.022%). The polar fractions of its essential oil components composition is similar with C. sinensis but their content was a bit lower lower than C. sinensis but there are more non-polar fractions constituents in C. speciosa than C. sinensis. Both of them contain several valuable active components. The studying result discovered the composition of essential oil of C. speciosa and C. sinensis produced in Chongqing.In conclusion, this paper not only discovered the composition of organic acids in C. speciosa fruit from defferent producing area in China and metabolizing law of its fruit during its developing period from Qijiang,Chongqing, but also provided the basic theoretical data for mai functionall active components including SOD.OA and UA, essential oil of its" fruit.What we have studied above, there were an important theory meaning and practical value direction for enriching fruit organic acids matabolizing studying theory of Rosaceae fruit.更多还原