Study On Resource Chemistry Of Sophora Alopecuroides L.

This dissertation was divided into five chapters.In Chapter 1,a detailed review was given about the latest research progress of resource chemistry of Sophora alopecuroides.Then,the research idea and experimental program of this paper were proposed on the basis of this review.In Chapter 2,chemical constituents from the root of S.alopecuroides were studied.The compounds were isolated by silica gel and polyamide column chromatography,and their structures were identified on the basis of spectral analysis and physicochemical data.As a result,thirteen compounds were obtained from the ethyl acetate extract and butanol extract,and their structures were elucidated as oxysophocarpine(1),oxymatrine(2),matrine(3),sophocarpine(4),sophoramine(5),sophoridine(6),cytisine(7),alopecurone A(8),alopecurone B(9),sophoraflavanone G(10),leachianone A(11),isokurarinone(12),kushenol F(13).In Chapter 3,the resource chemistry evaluation on the S.alopecuroides were performed,and the results were summarized in six sections.Section 1:resource chemistry evaluation of the alkaloids.1.To determine the distribution and content of quinolizidine alkaloids in different seed organs(seed coat and cotyledon),a reliable method has been established using ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry(UPLC-MS/MS).The results show there were remarkable differences in their contents in seed coat and cotyledon.The total contents of 7 alkaloids in cotyledons(mean value of two batches:38.52 mg/g)are much higher than those in seed coat(mean value of two batches:6.40 mg/g).With respect to the content of each analyte,oxymatrine,oxysophocarpine and cytisine are the most abundant in the cotyledon,and are 8-12 times higher than in the seed coat.higher than those in the cotyledons.These results suggested that the alkaloids in the seeds of S.alopecuroides are mainly distributed in the cotyledons.In addition,the percentage contents of each alkaloid in the cotyledons and the seed coat are quite different.For example,the oxymatrine takes the largest percentage in the cotyledons(32.6%),whereas the sophoridine takes the largest percentage in the seed coat(33.4%).2.HILIC-UPLC-TQ-MS/MS method was established to study the distribution and dynamic changes of seven alkaloids and lysine in different parts and different phenological growth stage of S.alopecuroides.Based on the results,the means of total contents of the seven alkaloids in different parts in June to October ranked as follows,seed>leaf>root>stem>legume,and the total contents of the seven alkoids in stem,leaf,and leguem in June to October gradually decreased,and in seed gradually increased until September.As for the individual compounds determined in S.alopecuroides,oxymatrine and oxysophocarpine were found to be the most abundant alkaloids in different parts and different phenological growth stage of this plant.In addition,some mathematical models were established for the relationship between the content of quinolizidine alkaloids and lysine in the dynamic changes.Section 2:resource chemistry evaluation of the amino acids.With the application of HILIC-UPLC-TQ-MS,a rapid,sensitive and non-derivative method for analysis of amino acids has been established.The validated method was successfully applied for the simultaneous characterization and quantitation of 20 amino acids in different parts and different phenological growth stage of S.alopecuroides.The results indicated that S.alopecuroides were rich in the free amino acids.In the samples from different phonological phases,the total contents of the amino acids were obviously various,even for the same organ.In sample 4(root,20110925)the content reached as high as 22.45 mg/g,whereas it was only 3.30 mg/g in sample 15(legume,20110925).In the stem,the total content of the amino acids was different across the growing stages.The highest peak achieved in June 48.55 mg/g and decreased through the growing season,reaching the lowest concentration 4.83 mg/g in October.In addition to the protein amino acids,three nonprotein amino acids including GABA,Hpro,and Cit were found in S.alopecuroides samples.In addition,relative quantitative determination of amino acids with several amino acids selected for the best accuracy was investigated.The accuracies of relative quantitative method for amino acids determinations suggest that it is feasible to quantify amino acids by the proposed relative quantitative determination method.Section 3:resource chemistry evaluation of the nucleosides and nucleobases.To explore the profiles of the nucleosides and nucleobases in different parts and different phenological growth stage of S.alopecuroides,an HILIC-UPLC-TQ-MS method was established and validated.The results showed that almost all the analytes were accompanied with the whole period of S.alopecuroides growth and development,but the two nucleotide composition was not detected;the compounds showed certain regularity in plant growth process of S.alopecuroides.During June to October growth period of root and stem of S.alopecuroides,the contents of guanine and cytosine decreased gradually.During June to August growth period of leaf,the contents of guanine and cytosine increased gradually,while the contents of inosine decreased gradually.During July to October growth period of legume,the contents of guanine decreased gradually;During July to October growth period of seed,the contents of xanthine reached the maximum in September,while the contents of most of these components were the most in August,which is different from the dynamic change regularity of alkaloid.Section 4:resources chemistry evaluation of flavonoidsA comparative study on the total flavonoids distribution and dynamic changes in S.alopecuroides was carried out using UV-Vis spectrophotometry.Based on the results,the means of total contents of flavonoids in different parts in June to October ranked as follows root(2.27%)>leaf(2.01%)>stem(0.69%)>seed(0.59%)>legume(0.31%).The total contents of flavonoids in root,stem,and leaf of S.alopecuroides increased gradually with consistent trends during the growth period,while the total contents of flavonoids in legume gradually decreased during growth period,with opposite trend compared with other organs,and the total amount of flavonoids in seed changed slightly,with a stable trend.Section 5:resource chemistry evaluation of the polysaccharides.The total contents of neutral and acidic polysaccharides in different parts and different phonological growth stage of S.alopecuroides were determined using UV-Vis spectrophotometry.Based on the results,the means of total contents of acidic polysaccharides in different parts in June to October ranked as follows leaf(16.75%)>stem(13.03%)>root(12.91%)>legume(11.94%)>seed(7.58%).The means of total contents of neutral polysaccharides in different parts in June to October ranked as follows leaf(13.68%)>root(12.91%)>seed(11.74%)>stem(10.09%)>legume(9.61%).Section 6:resources chemistry evaluation of the inorganic elementsA comparative study on the 29 inorganic elements in different parts and different phonological growth stage of S.alopecuroides was carried out by using ICP-AES.The results showed that the inorganic elements can be enriched in certain organs,such as Ca?Cu?Mn are mainly distributed in the leaves,the mean of the content of Ca in leaf is 59.6%.Na is mainly distributed in the stem(58.3%),Zn,P,K,are mainly distributed in seeds,the mean of the content of Zn in seed is 66.3%.The content of these elements show certain regularity in different phenological periods such as the content of Ca gradually increased with the growth in the legume and the content of Zn gradually decreased with the growth in the leaves.In Chapter 4,Ultrasonic assisted extraction method and ultrasound bath-assisted enzymatic hydrolysis method for the extraction of alkaloids were compared.The results were summarized in two sections.Section 1:optimization of ethanol and acidic water ultrasound extraction of quinolizidine alkaloids from the seed of S alopecuroides using response surface methodology1.Box-Behnken Design(BBD),a widely used form of response surface methodology(RSM),was used to investigate the effect of process variables on the ultrasound assisted extraction method.Three independent variables including the proportion of ethanol(%),extraction time(min)and solvent-to-material ratio(mL/g)were studied.The results showed that the optimal extraction condition was obtained with the proportion of ethanol of 62.22%,extraction time of 70.00 min and solvent-to-material ratio of 125.52 mL/g for combination of 7 kinds of alkaloids.The results suggested that the ultrasonic assisted extraction under optimal extraction conditions gave the best yield of 7 kinds of alkaloids.That were sophocarpine(387.32?g/g),oxysophocarpine(9731.39?g/g),oxymatrine(9400.16?g/g),matrine(421.58?g/g),sophoramine(315.73?g/g),sophoridine(7103.59?/g)and cytisine(7751.33?g/g)respectively.2.Box-Behnken Design(BBD),a widely used form of response surface methodology(RSM),was used to investigate the effect of process variables on the ultrasound assisted extraction method.Three independent variables including pH,extraction time(min)and solvent-to-material ratio(mL/g)were studied.The results showed that the optimal extraction condition was obtained with the pH of 2.3,extraction time of 66.95 min and solvent-to-material ratio of 115.70 mL/g for combination of 7 kinds of alkaloids.The results suggested that the ultrasonic assisted extraction under optimal extraction conditions gave the best yield of 7 kinds of alkaloids.That were sophocarpine(395.32?g/g),oxysophocarpine(12383.53?g/g),oxymatrine(12256.97?g/g),matrine(419.52?g/g),sophoramine(316.69?g/g),sophoridine(8173.94?/g)and cytisine(8363.42?g/g/g)respectively.Section 2:Optimization of ultrasound bath-assisted enzymatic hydrolysis extraction of quinolizidine alkaloids from the seed of S alopecuroides using response surface methodologyBox-Behnken Design(BBD),a widely used form of response surface methodology(RSM),was used to investigate the effect of process variables on the ultrasound assisted extraction method.Three independent variables including pH,extraction temperature(?),extraction time(min)and solvent-to-material ratio(mL/g)were studied.The results showed that the optimal extraction condition was obtained with the pH of 5.12,extraction temperature of 53.96?,extraction time of 59.74 min and solvent-to-material ratio of 112.01 mL/g for combination of 7 kinds of alkaloids.The results suggested that the ultrasonic assisted extraction under optimal extraction conditions gave the best yield of 7 kinds of alkaloids.That were sophocarpine(410.46?g/g),oxysophocarpine(15377.73?g/g),oxymatrine(14635.47?g/g),matrine(422.18?g/g),sophoramine(325.36?g/g),sophoridine(9864.06?/g)and cytisine(9222.39?g/g)respectively.In chapter 5,the utilization of S alopecuroides was studied and the results were summarized as two sections.Section 1:Study on potential hypoglycemic effects of the resources ingredients from S alopecuroides in alloxan induced diabetic mice.Choose alloxan diabetic mice,and take blood glucose,glucose tolerance and the content of TC,TQ ALT,AST,BUN,and SCR in serum as the evaluation indexes,to research the effects of the total flavonoids,total alkaloids and polysaccharides in S alopecuroides on diabetic mice and diabetic complications.The results showed that:compared with the model group,total polysaccharides in S alopecuroides can significantly decrease blood glucose in mice,improve the glucose intolerance of diabetic mice,significantly reduce TC,TG level in serum of diabetic mice,significantly decrease liver and kidney indexes and serum AST,ALT,BUN,SCR indexes,and relieve renal lesion in liver and kidney of diabetic mice.High doses of total flavonoids can significantly reduce blood glucose in model mice,and can reduce the level of serum TC and TG in model mice,significantly decrease the liver index,and serum AST,ALT indexes,and relieve liver pathological changes in diabetic of mice.Although the total alkaloids of S alopecuroides in this experiment showed no hypoglycemic effect,it was revealed that alkaloids can improve blood lipids levels in diabetic mice.The above results showed that total polysaccharid and total flavonoids in S alopecuroides have obvious hypoglycemic effect,and has certain improvement on diabetic kidney and liver complications,which is a proof of effective development and utilization of the resources components in S.alopecuroides.Section 2:Study on potential hypolipidemic effects of the resources ingredients from S alopecuroides.High fat diet caused hyperlipidemia model mice were selected,the content of mouse serum TC,TG,LDL-C,HDL-C,ALT,AST,BUN,SCR,MDA was taken as evaluation indexes,and total flavonoids,total polysaccharides,and total protein antilipemic function in S.alopecuroides were studied.The results showed that:compared with the model group,total flavonoids and total polysaccharide in S alopecuroides can significantly reduce model mice serum TG and TC,significantly increased HDL-C content,reduce the content of MDA,significantly decrease the liver index and serum AST and ALT index,and relieve hyperlipemia mice liver damage.Total protein in S.alopecuroides cannot reduce the content of TC in serum,but have the same effects as total flavonoids and total polysaccharide as for the other indexes.The above results showed that total polysaccharide,total flavonoids,and total protein in S.alopecuroides have significant hypolipidemic effect,and have positive significance for the prevention and treatment of hyperlipidemia and fatty liver.