| 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. |
