摘要
笃斯越桔(Vaccinium uliginosum L.),广泛分布于黑龙江、内蒙古、大兴安岭以及吉林省长白山地区,天然资源十分丰富。研究表明其具有抗氧化、抗肿瘤、利尿、消炎、解毒之功效,还有抗衰老保健作用等。笃斯越桔是一种药食兼用的植物。果实中有机酸就有十几种,如:柠檬酸、琥珀酸、富马酸、色素类、萜衍生物、矿物元素等,已被制成果酱、果汁、果酒等,经济价值高,在国际市场上有较大的需求。但笃斯越桔枝干和叶片,作为笃斯越桔开发的主要副产物,资源丰富,有资料表明其中含有较多的黄酮类和鞣质,有收敛、抑菌及维生素D样作用,但由于不能直接食用,被当作燃料使用或被废弃,导致其开发利用受到极大限制,研究报道较少。槲皮素也具有独特的保健功能,如抗肿瘤,抗炎,降糖、抗氧化以及血管软化等作用。
从天然产物中草药中提取分离制备有效成分,应用于医药、保健食品及食品添加剂等,符合第三代保健食品的要求,已越来越受到全世界的认同和人们的青睐。有学者预言含中草药的保健食品,特别是植物性中草药功效成分的保健食品,将成为人类研究开发的焦点。
本文以长白山道地中药材笃斯越桔为研究对象,采用高效液相色谱法和紫外可见分光光度法对比研究了笃斯越桔中槲皮素的含量测定方法;在笃斯越桔槲皮素含量测定的基础上,利用二次回归正交旋转组合设计法构建了笃斯越桔乙醇粗提物酸水解制备槲皮素的工艺数学模型;采用有机溶剂萃取和柱层析法对笃斯越桔槲皮素纯化工艺进行了系统研究。制备得到笃斯越桔槲皮素纯化产品后,对其稳定性和其中有机溶剂残留及其体内外抗氧化性进行了研究。结果表明,笃斯越桔槲皮素提取物对光、热以及一般食品添加剂均较稳定,具有抗氧化功能,可以清除体内过多自由基,进而增强机体抗氧化与抗衰老功能。本研究是吉林省科技厅应用基础引导项目(20050567)——长白山道地中药材笃斯越桔有效成分及其功能特性研究课题的一部分。
Vaccinium uliginosum L.,as a kind of food or medicine,is widely distributed in Northeast and Inner Mongolia of China.Vaccinium uliginosum L.contains lots of bioactive compounds,whose fruits extractive can be used to treat diarrhea,dysentery, stomatitis etc.and whose leaves extractive can be used to treat eyes diseases,vascular disorders,Diabetic complication etc.and it has been considered as one of the five human healthy foods by International Food and Agriculture Organization.Quercetin has been found to exhibit lots of biological functions as antioxidation,anti-tumour effect,diuresis,anti-inflammation,anti-angiosclerosis and so on.Therefore,the detection and the technology of the hydrolysis,purification and preparation of the quercetin in the Vaccinium uliginosum L.and its stability and antioxidation were studied in this thesis,which can provide functional ingredient for the functional foods and the rational foundation for the further exploitation of the Vaccinium uliginosum L.
The reversed phase high-performance Liquid chromatography and Ultraviolet-Visible spectrophotometric methods were employed to analysis for the quantitative estimation of quercetin of Vaccinium uliginosum L..The validation of the method was carried out,including precision,stability,reproducibility and the application of sample recovery of the experiments.On the basis of the establishment of standard curve,the regression equation of RP-HPLC and UV methods were as that: Y=2.265977×10~(-7)X+3.554018×10~(-3) and Y=5.78537X+0.00141,with the high linear correlation(coefficient correlation was 0.9999660 and 0.99914,respectively).The condition of the RP-HPLC were that:the column Shimpack VP-ODS (4.6mm×250mm,5μm),mobile phase:methanol and 0.5%of phosphoric acid(50/50), the flowing rate:0.8 mL/min,the column temperature:30℃,the detecting wavelength:360nm and the sample size:10μL.The results were as follow:the precision test RSD=1.13%(n=5),the reproducibility test RSD=0.78%(n=5),the mean value of the application of sample recovery was 99.88%and RSD=0.63%.All the above shows that the precision,reproducibility and the application of sample recovery of the method were good.The content of quercetin in the leaves,trunks, fruits of Vaccinium uLiginosum L.was 141.20mg,89.90mg and 19.98mg per 100g raw material and the RSD value was 0.82%,0.56%and 2.04%,respectively.Based on the complexation recation between the quercetin and the antimony trichloride,it was detected that the contents of quercetin in the leaves,trunks and fruits of the Vaccinium uliginosum L..The results of validation of the method were that:precision test RSD=1.22%(n=5),reproducibility test RSD=1.24%(n=5),stability test of samples RSD=0.46%(n=5),average recovery rate of samples Y=102.779%,RSD=2.03% (n=5).The content of quercetin in the leaves,trunks,fruits of Vaccinium uLiginosum L.was 153.41mg,98.35mg and 22.34mg per 100g raw material and the RSD value was 1.92%,4.77%and 4.07%,respectively.Relatively speaking,UV spectrometry is inexpensive and less time consuming,while the HPLC method has a higher sensitivity, and is more reliable and efficient.Both the proposed methods are precise and accurate for the quantification of quercetin in different parts of Vaccinium uliginosum L..
Mathematical model of quadratic regression orthogonal combination designed were employed to obtain the prepare technical parameters of preparing quercetin in acid hydrolysis experiment of the ethanol extracts of the Vaccinium uliginosum L.. The mathematical regression model of the hydrolysis conditions of quercetin in the Vaccinium uliginosum L.was got as follow:Y=1.90468+0.30484X_1+0.12710X_2+ 0.17333X_3-0.19887X_1~2-0.27633X_2~2-0.12828X_3~2,which can exactly predict the content of the quercetin in the Vaccinium uliginosum L..On the condition of that:the range of sulphuric acid concentration 0～4%,time of hydrolysis 30～90min,the amount of sulphuric acid 40～80 times,the result was that the order of the influence of the three factors on the content of quercetin was as fowllow:sulphuric acid concentration＞time of hydrolysis＞the amount of sulphuric acid.The best hydrolysis conditions were:sulphuric acid concentration:2.729～3.343%,time of hydrolysis: 60.357～69.634min,the amount of sulphuric acid:62.426～70.974 times.In order to get close to the practical manufacture and diminish the energy consumption,the optima condition was settled as follow:the sulphuric acid concentration 3%,the time of hydrolysis 65min and the amount of sulphuric acid 67 times.This technology is applied to industrialized produce eligibly.After the hydrolysis,the content of quercetin in the samples was risen from 153 mg,98mg and 22mg per 100g material to 215 mg,147 mg and 30 mg,respectively and the purity of the quercetin in the hydrolysis product was risen from 437 mg,501 mg and 29mg per 100g crude extract to 1772mg,1972 mg and 1095mg,respectively.
The solvent extraction and column chromatography were employed to purify quercetin after the hydrolysis process.The hydrolysate of the Vaccinium uliginosum L.was first extracted by petroleum ether,then by acetic ether and the quercetin was enriched from 1792mg per 100g sample to 7752mg.After the extract of acetic ether, then the extraction was purified by the AB-8 macroporous resin column chromatography and gradient eluted by ethanol.The purity of quercetin in the eluted sample,which was got at the conditions of 25℃,flow rate of eluting 1.0mL/min and the concentration of ethanol 80%,was 16793 mg per 100g sample detected by RP-HPLC.This eluted sample was further purified by silica gel and gradient eluted by chloroform and methanol.The sample got first by the eluting solvent of chloroform: methanol=10:1,then eluted repeatedly by solvent of chloroform:methanol:water= 10:1:1,in which the contents of quercetin was 31269 mg in 100 mg sample by RP-HPLC.
As a functional ingredient,the stability of quercetin in the Vaccinium uliginosum L.by which the safety and effective are assured were studied in this research.The effect of the light,temperature,pH value,oxidation and reducing agent,metal ions,as well as food additives on the stability of quercetin in the Vaccinium uliginosum L. were studied.The results showed that quercetin are more stable in neutral condition (pH 7-9) compared to alkaline or acid condition;high temperature and sun light can affect the stability of quercetin,It is not so sensitive to mostly metal ions,but the stability of quercetin is reduced when Zn~(2+)、Fe~(2+)、Al~(3+)、Cu~(2+) is respective added to its medium whereas other metal ions have no such effect on its stability,which should be avoided in production.Oxidant has protective effects on quercetin,while reducing agent has destructive effects.Food additives,including carbohydrate,Vc,citric acid and Sodium Chloride have little effect on the stability of quercetin in the Vaccinium uliginosum L.,while the Sodium Benzoate have strongly destructive effect on quercetin.Therefor the volume of the preservative should be strictly controlled in the process of functional foods.
The capillary gas chromatography method coupled with FID detection was developed to simultaneously determine 4 different residual organic volatile solvents which are methanol,ethanol,trichlormethane and acetic ether,in the quercetin extract of the Vaccinium uliginosum L.On the conditions of that:the HP-5 capillary column (30m×0.32mm×0.25μm),the column temperature:firstly the column temperature was 40℃,remaining 1min,then rose to 50℃at the rate of 5℃/min,remaining 2 min,after that it continue to rising to 56℃at the rate of 2℃/min,finally the column temperature should be 80℃,with the rising rate of 20℃/min,introduction port temperature:220℃, detecting room temperature:250℃,hydrogen flow:30mL/min,air flow:300mL/min, constant pressure,carrier gas(N_2) flow:1.0mL/min;split stream sampling,split ratio: 100:1,sample size:1.0μL,the equation of linear regression and the coefficient correlation were obtained as that:y=25516.9676X-2.4698(0.9999),y=36171.949X-3.13896(0.9999),y=32453.1275X-2.5656(0.9999),y=6727.8341X-2.5291(0.9999). The mean value of the application of sample recovery were 98.42%,100.05%, 100.73%,99.64%and RSD were 3.672%,3.650%,2.257%,4.140%(n=5).The developed method was able to determine the residual organic volatile solvents in quercetin extract of Vaccinium uliginosum L.with excellent resolution,precision and recovery.The residual organic volatile solvents were not detected in quercetin extract of the Vaccinium uliginosum L..
The vitro and vivo antioxidation.of quercetin extract of Vaccinium uliginosum L. were studied in this research.In the vitro antioxidation,the inhibition rate of scavenging DPPH free radical was investigated in quercetin purified sample,Vc and BHT and the result shows that its order of inhibition rate was as follow: Vc＞quercetin＞BHT.The reducing power of the samples was investigated and the linear correlation between reducing power and concentration was established.The reducing power of 0.05mg/ml BHT,Vc were equate to quercetin of 0.021mg/ml and 0.0865mg/ml,respectively.In the vivo antioxidation,three indexes for antioxidation were investigated.In the aspect of erythrocuprein(SOD) in blood serum of mouse,the antioxidation capacity of middle dose and the high dose of purified quercetin were significant than blank group.In the aspect of glutathion peroxidase(GSH-Px),the antioxidation capacity of the high dose purified quercetin sample was the highest, besides the VE group.In the aspect of malonaldehyde(MDA),which is the degradation products of lipid peroxidation,the antioxidation capacity of middle dose and the high dose of purified quercetin had the statistical significance.The above-mentioned experiment results show that:after eating the quercetin extract of Vaccinium uliginosum L.,the three indexes for antioxidation in blood serum of mice significantly better than the mice of blank group,which proved that the quercetin of Vaccinium uliginosum L.has anti-oxidation effect,and can enhanced anti-aging of organism and to remove excess free radicals in vivo.
引文
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