【作者】 邵菊芳；

【导师】 刘炯天；

【作者基本信息】 中国矿业大学 ， 化学工艺， 2013， 博士

【摘要】 银杏是我国的珍贵树种，其特有的活性物质黄酮具有抗氧化、抑菌消炎、抗癌和防治心脑血管疾病等诸多功效，开发前景光明。为满足日益扩大的市场需求，发展银杏深加工产业，利用细胞培养技术大规模生产银杏黄酮为工业生产提供原料是一条有效途径。本文在前人的研究基础上，对银杏愈伤组织的诱导、继代及悬浮细胞系培养条件进行了系统的研究；建立了银杏黄酮糖苷的荧光光谱检测方法；以收获的悬浮培养细胞为对象，优化了加热回流提取及超声波提取工艺参数，推导了显著提取因子对黄酮糖苷得率的模型方程；并探讨了黄酮糖苷提取动力学过程；推测了黄酮糖苷种类；对银杏细胞黄酮体外抗氧化及抑菌活性进行了验证，得到如下结论：1.在细胞诱导试验中，培养基以DCR为好，添加激素BA可以提高诱导率。但在继代培养中MS培养基和激素KT在促进愈伤组织生长方面有积极作用。活性炭作为吸附剂，虽然对细胞的生长表现出一定的抑制作用，但是在黄酮积累上起到促进作用。2.蔗糖在悬浮培养中有利于银杏细胞生长，葡萄糖利于黄酮合成，硝态氮和氨态氮混合能促进细胞的生长，但对黄酮的合成来讲硝态氮更为有利。接种量40g/L、装液量100mL、封口膜封口、添加前体物苯丙氨酸以及24h光照为培养高产黄酮银杏细胞的较优条件，添加柠檬酸能促进细胞生长，而维生素C（Vc）既有利于黄酮的合成，又对细胞的褐化抑制效果较好。3.建立了荧光光谱法测定银杏细胞总黄酮的检测方法，结果表明，以芦丁为标样，纯甲醇溶解，10%Al（NO₃）₃添加4mL，pH3.6的乙酸-乙酸钠2.5mL条件下，荧光强度与芦丁浓度成线性关系，回归方程为y=29.92x+36.49（R²=0.986），线性范围1.8×10^-6-3.2×10^-5mol/L，加标回收率为101.3%。该方法灵敏度高，重现性好，操作简单，成本低，具有良好的应用前景。4.利用响应面法（RSM）优化了银杏悬浮细胞中黄酮糖苷加热回流乙醇提取工艺，得出影响提取得率的显著因素（提取时间、提取温度和乙醇浓度）的二次多项式模型。最佳提取条件为：提取时间3.2h、提取温度76.85℃、乙醇浓度72.05%。此条件下黄酮糖苷提取得率为1.81%，与模型预测值一致，说明利用RSM优化提取条件，建立的模型预测银杏细胞黄酮糖苷准确可靠，重复性好。5.利用二次回归正交旋转组合试验设计，建立了超声波法以提取温度、提取时间、超声功率和乙醇浓度为自变量，黄酮得率为因变量的二次回归方程。最优工艺为以纯乙醇作提取溶剂，在超声功率500W条件下于80℃提取50min。此条件下所得到的黄酮糖苷得率为3.03%，与模型预测值吻合，相对误差较小，说明此二次回归方程可用于实际生产中银杏黄酮糖苷的预测。6.以悬浮培养细胞为原料，在Fick第一定律的基础上，推导了黄酮糖苷乙醇浸提的动力学模型，考察了提取温度对黄酮糖苷质量浓度的影响，计算了不同温度下黄酮糖苷的平衡质量浓度，求得了表观速率常数以及提取过程表观活化能。验证试验表明，黄酮糖苷质量浓度和相对萃余率的理论值和实际值在0-360min范围内基本吻合，说明建立的动力学模型是能够用来描述银杏细胞黄酮糖苷的实际提取过程的。7.利用HPLC-UV-ESI-MSn法在350nm波长下，50-1000m/z正离子模式质谱检测了槲皮素、山萘酚和异鼠李素3种苷元的裂解规律。并在相同的色谱、质谱条件下对培养细胞黄酮提取物中黄酮类化合物进行了鉴定。推测了8种黄酮糖苷，分别为：槲皮素-3-O［2-O,6-O-（α-L-二鼠李糖基）］-β-D-葡萄糖苷；山萘酚-3-O［2-O,6-O-（α-L-二鼠李糖基）］-β-D-葡萄糖苷；杨梅黄酮--3-O［6-O-（α-L-鼠李糖基）］-β-D-葡萄糖苷；槲皮素-3-O［2-O-（α-L-鼠李糖基）］-β-D-葡萄糖苷；异鼠李素-3-O［6-O-（α-L-鼠李糖基）］-β-D-葡萄糖苷；山萘酚-3-O［6-O-（α-L-鼠李糖基）］-β-D-葡萄糖苷、槲皮素-3-O-鼠李糖-2-O-（6-O-对香豆酰）-β-D-葡萄糖苷、山萘素-3-O-鼠李糖-2-O-（6-O-对香豆酰）-β-D-葡萄糖苷。8.银杏细胞黄酮糖苷的体外抗氧化及抑菌试验结果表明：银杏细胞黄酮糖苷对羟自由基和过氧化氢的清除率都显著高于柠檬酸、植酸和Vc；对超氧阴离子的清除率低于Vc，对Fe2+的螯合能力和对植物油脂的抗自氧化能力，受试各浓度下均显著高于阳性对照。体外抑菌试验表明随着黄酮浓度的增加，抑菌效果在增强，受试浓度下抑菌圈直径最大达2.2cm，最小为1.1cm，对大肠杆菌的最低抑菌浓度为0.141mg/mL。2.25mg/mL浓度作用10h时抑菌率达到81%。更多还原

【Abstract】 Ginkgo biloba is precious species in our country for its specific active substancesof flavonoids, which have some sanitarian function as antioxidant, bacteriostat,antiphlogistic, anticarcinogen, preventing cardiovascular and cerebrovascular diseasesand so on. Prospect of exploitation of Ginkgo biloba is perspective.It is an effectiveway to use cell culture technique for large-scale production of Ginkgo bilobaflavonoids to provide raw material for industrial production in attention of the furtherprocessing industry development of Ginkgo biloba. Based on the the studies ofpredecessors, the induction and subculture conditions of callus were investigated inthis article.The suspendsion cultural conditions ofGinkgo biloba cell wereexhaustivllyresearched. Fluorescence spectrum method was established for flavonoidsdetection. Extracting technological parameters offlavonoids glycosideswere optimized,and thereafter the model equation of significantly factors on extraction wasestablished. The kinetic mechnism of flavonoids glycosides extraction was alsoapproached. The identification of flavonoids glycosides was also studiedby UV,HPLC and MS. The anti-oxidation and the inhibition bacteria activity of flavonoidsfrom suspension cultural cell in vitro were verified. The main results from studies areachieved as following:In the callus induction experiment, DCR medium and hormone BA do well thanMS medium and KT hormone. But MS subculture medium and hormone KT has apositive effect in promoting callus growth. Active carbon as adsorbent can inhibit cellgrowth, but play a promoting role in the accumulation of flavonoids.Sucrose was the favourite carbon source for the suspension cultural cell. Andglucose was most suitable for flavonoids synthesis. The mixture of nitrate-nitrogenand ammoniacal nitrogen can promote cell growth, but nitrate-nitrogen was in favourof flavonoids synthesis. The optimal cultural condition was of inoculum40g/L celland phenyl alanine precursor in100mL liquid medium with film sealing in24h lightper day for high yield flavonoids. Citric acid can promote cell growth, while Vitaminc was conducive to flavonoids synthesis. And the inhibition of cell brown wasperformed by Vitamin C.The method of fluorospectrophotometry for total flavonoids detection wasestablished with standard rutin. Results indicated that fluorescence intensity and rutinconcent was linear relationship with regression equation for y=29.92x+36.49, （R2=0.986）. Linear range was1.810^-63.210^-5mol/L. Standard recovery of101.3%was achieved. The analysis results obtained from suspension cultured cell of Ginkgobiloba suggested that the development method is convenient for flavonoids detection.Ethanol extraction condition was optimized by response surface methodology.Quadratic polynomial model about significant factors, extraction time, temperatureand ethanol concent on flavonoids yield was obtained. The optimal extractionconditions was ethanol concent72.05%for3.285h at76.85℃. In this condition, theyield of flavonoids was1.8101%, which was consistent with the predictive value ofthe model. It was demonstrated that the model was accurate and reliable for flavonoidglycosides yield prediction.The regression equation was deduced by quadratic regression orthogonal rotationcombination design experiments.The optimal condition of ultrasonic-assisted methodwas ethanol concent100%and ultrasonic power500W at80℃for50min. The yieldof flavonoids in revification tests was3.0345%, consistent with the predicted value ofthe model. Repeatability and reliability were performed well by the model.The extraction dynamics model for flavonoids from suspension cultured cell wasdeduced on the base of Fick’ first law. This model was adapted to the extraction ofactive components of medicine plants, which belongs to an apparent first order kineticequation. Revification tests showed that the theoretic value of the content offlavonoids and relative raffinate rate basically consistent with experimental value in0-360min. The established dynamic model can be used to describe the actualextraction process of flavonoid glycosides from Ginkgo biloba cell.The fragmentation regularity of quercetin, kaempferol, isorhamnetin wasstudied using HPLC-UV-ESI-MSnmethod in350nm wavelength, at the range of50-1000m/z scanning scope. In the same conditions, flavonoidsfrom the extraction ofcultured cells were identified. Eightflavonoidglucosides were presumed respectivelyfor3-O［2-O,6-O-bis （α-L-rhamnosyl）-β-D-glucosyl］quercetin;3-O［2-O,6-O-bis（α-L-rhamnosyl）-β-D-glucosyl］kaempferol;3-O［6-O-（α-L-rhamnosyl）-β-D-glucosyl］myricetin;3-O［2-O-（α-L-rhamnosyl）-β-D-glucosyl］quercetin;3-O［6-O-（α-L-rhamnosyl）-β-D-glucosyl］isorhamnetin;3-O［6-O-（α-L-rhamnosyl）-β-D-glucosyl］ kaempferol；3-O［2-O-（6-O-p-coumaroyl-β-D-glucosyl）-α-L-rhamnosyl］ quercetin；3-O ［2-O-（6-O-p-coumaroyl-β-D-glucosyl）-α-L-rhamnosyl］kaempferol.The anti-oxidation and bacteriostasis of flavonoids in vitro from suspension cultured cell of Ginkgo biloba were studied. The results showed that the clearance forhydroxy radical and hydrogen peroxide by flavonoids significantly better than citricacid, phytic acid and Vitamin C. But the clearance for superoxide anion by flavonoidswas more below than Vitamin C. The chelation ratio with Fe2+was significantlyhigher than active control. Antioxidative capacity of flavonoids on plant oil was alsostronger than active control in experimental content. The results of bacteriostasisexperiment in vitro showed that bacteriostatic activitywas enhanced with the increaseof flavonoids concentration. The maximum of inhibition zone diameter was2.2cmand minimum for1.1cm in the experimental content. The minimum of bacteriostaticconcentration on Escherichia coli was0.141mg/mL. The bacteriostatic ratio wouldachived81%with the condition for flavonoids concentration2.25mg/mL and actingtime10h.更多还原