Extraction of pharmaceutical components from Ginkgo biloba leaves using supercritical carbon dioxide

Ginkgo biloba extract (GBE) has many remarkable pharmacological and clinical effects, and it is the most frequently used product as a phytomedicine in many countries. The combination of primary extraction with 70% ethanol followed by extraction using supercritical carbon dioxide provides an efficient and economical means for obtaining flavonoids and terpenoids from Ginkgo biloba leaves. The supercritical fluid extraction (SFE) is affected by pressure, temperature, and the concentration of modifier in the extractant. At the most favorable experimental conditions of 300 MPa, 60 degrees C, and carbon dioxide containing 5% ethanol as modifier, the yield of GBE powder is 2.1% (based on the air-dry weight of Ginkgo biloba leaves) compared to a yield of only 1.8% by conventional solvent extraction. The contents of flavonoids and terpenoids in SFE products are 35.9% and 7.3%, respectively, which are significantly higher than the general standards of 24% and 6%, respectively.     

Biologically active secondary metabolites from Ginkgo biloba

Three new compounds, (7E)-2beta,3alpha-dihydroxy-megastigm-7-en-9-one (1), 3-[5,7-dihydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-yl]-4-methoxybenzoic acid (2), and 4'-O-methyl myricetin 3-O-(6-O-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside (3), were isolated from Ginkgo biloba, together with 27 known compounds. The structures of the new compounds were determined primarily from 1D- and 2D-NMR analysis. The 4-O-methylbenzoic acid structural feature at C-8 in 2 is encountered for the first time. The antioxidant activities of 29 compounds isolated from Ginkgo biloba were evaluated on intracellular reactive oxygen species in HL-60 cells. It was found that quercetin, kampferol, and tamarixetin had antioxidant activity that was approximately 3-fold greater than that of their respective glycosides and also approximately 3-fold greater than that of a standard ascorbic acid with an IC(50) at maximum effectiveness.     

Chromatographic profiles and identification of new phenolic components of Ginkgo biloba leaves and selected products

Ginkgo biloba leaves and their extracts are one of the most widely used herbal products and/or dietary supplements in the world. A systematic study of the phenolic compounds is necessary to establish quality parameters. A modified LC-DAD-ESI/MS method was used to obtain chromatographic profiles for the flavonoids and terpene lactones of Ginkgo biloba leaves. The method was used to identify 45 glycosylated flavonols and flavones, 3 flavonol aglycones, catechin, 10 biflavones, a dihydroxybenzoic acid, and 4 terpene lactones in an aqueous methanol extract of the leaves. The extracted G. biloba leaf products contained the same flavonoids as the raw leaves except for the lack of biflavones. The detected glycosylated flavonol contents were equal to or more than 0.0008% of the dry plant material. This is the first report of the presence of more than 20 of these flavonoids in G. biloba.     

银杏花粉黄酮组分分离纯化及其清除DPPH自由基能力

为了分离纯化银杏花粉中具有潜在抗氧化活性的黄酮组分及研究其潜在抗氧化活性,该文对银杏花粉的总黄酮进行提取和分步萃取,采用1,1-diphenyl-2-picrylhydrazyl(DPPH)自由基清除率检测和组分分析锁定待纯化萃取相。通过DPPH-HPLC-PAD技术筛选目标清除自由基组分,并采用制备型液相色谱技术分离纯化这些组分,然后采用紫外可见吸收光谱、质谱、核磁共振氢谱、碳谱等技术进行结构鉴定,最后通过测定DPPH自由基清除率初步研究其潜在抗氧化活性。结果显示,银杏花粉粗提物具有清除DPPH自由基活性,对DPPH的半抑制率(IC50值)为1.32 mg/m L,乙酸乙酯相和正丁醇相的IC50值为0.46、0.84 mg/m L,分别是粗提物的34.85%和63.64%。可见,乙酸乙酯相的清除DPPH活性最高。进一步研究显示,乙酸乙酯相富集了6种主要银杏花粉黄酮,而且均具有清除DPPH活性。经纯化和结构鉴定分别为山奈酚-3,4’-双-O-β-D-葡萄糖苷(1)、山奈酚-3-O-β-D-葡萄糖基-7-O-α-L-鼠李糖苷(2)、山奈酚-3-O-β-D-葡萄糖苷(3)、山奈酚-3-O-α-L-鼠李糖苷(4)、柚皮素(5)和山奈酚(6)。其清除DPPH自由基的活性由高到低依次为:化合物6化合物2化合物3化合物4化合物1化合物5。其中山奈酚清除DPPH的IC50值为0.017 mg/m L,与维生素C及芦丁相比没有显著性差异(P0.05)。上述研究为银杏花粉功能成分的深入研究奠定了基础。     

银杏磷素营养及其营养诊断方法的研究初报

通过对施磷量和银杏植株不同叶位叶片全磷量及单叶重的测定 ,并进行回归分析。结果表明 :银杏上位叶的全磷含量比下位叶的高 ;单叶重则相反 ,下位叶的比上位叶的高 ;施磷量对叶片全磷量和单叶重有着明显的影响 ,供磷偏低或过高 ,都使叶片全磷含量和单叶重下降 ;长枝条 8、 12位叶的全磷量与施磷量的回归关系达极显著者或显著水平 ,其叶片全磷量能反映供磷水平 ;8位叶的单叶重与施磷量间的回归关系达显著水平 ,其单叶重能反映供磷水平 ;长枝条 8、12位叶可以作磷素营养诊断部位 ,而 8位叶则是最佳部位 ,其叶片全磷量和单叶重可作为磷素营养诊断的指标     

银杏内生真菌刺盘孢遗传多样性的初步分析

为了研究银杏内生真菌的刺盘孢菌株的遗传分化,选用了分离自江苏5个县市的14个菌株,利用RAPD技术进行遗传多样性分析。从20个随机引物中筛选出5个引物,扩增出62条DNA带,在此基础上分析并建构了遗传相关聚类图。结果表明,不同地区的刺盘孢菌株其遗传差异性不同,14个菌株可聚为两大类,其中启东、邳州的6株聚为一类,南京、南通和泰兴的8株又聚为一类。所有的14个菌株的相似性系数范围在46%~89%。     

银杏内生真菌多样性与产黄酮类物质真菌的分离和鉴定

采用传统组织培养法从银杏根、叶片和茎中分离内生真菌,以相对频率作为指标分析其多样性,通过显色反应、薄层层析(TLC)、高效液相色谱(HPLC)等方法对内生真菌的发酵产物进行初步分析,筛选能够产黄酮类物质的内生真菌,并通过分光光度法测定总黄酮产量;同时根据菌株的形态学特征,结合真菌r DNA的居间序列(internal transcribed spacer,ITS)的系统进化分析进行菌株鉴定。结果表明:共分离得到217株内生真菌,分属于5纲、8目、10科、13属;经初步筛选,2株分离自根组织的内生真菌GF111和GF521具有产黄酮类化合物的能力,发酵液总黄酮产量分别为(14.50±1.30)mg/L和(21.10±1.30)mg/L;初步鉴定2株产黄酮内生真菌GF111和GF521分别为卷枝毛霉Mucor circinelloides和尖镰孢菌Fusarium oxysporum。可见,内生真菌在银杏不同部位在数量、种群及其组成上存在着较大差异,并且菌株GF111和GF521产黄酮能力较高,可作为生物发酵生产黄酮类物质的备选菌株。本研究为更好地开发和保护银杏资源提供了依据。     

Anti-inflammatory effects of isoflavone powder produced from soybean cake

Soybean cake, a byproduct obtained during the processing of soybean oil, has been shown to be a rich source of isoflavones. The objectives of this study were to use soybean cake as raw material for processing into powder and to evaluate the anti-inflammatory activity. Eleven treatments, including powders of malonylglucoside, glucoside, acetylglucoside, aglycone, ISO-1, and ISO-2, as well as genistein standard, gamma-PGA, control, normal, and PDTC, were used for evaluation. A total of 77 mice were each provided daily with tube feeding for 4 weeks at a dose of 0.3 mL of aqueous solution from each treatment, and inflammation was induced with intraperitoneal injection of 1 mg/kg of body weight lipopolysaccharide (LPS). Results showed that all of the isoflavone powders and genistein standard were effective in inhibiting LPS-induced inflammation, lowering leukocyte number in mice blood and reducing production of IL-1beta, IL-6, NO, and PGE2 in both peritoneal exudate cell supernatant and peritoneal exudate fluid. All of the isoflavone treatments failed to retard T cell proliferation; however, both ISO-1 and ISO-2 could inhibit B cell proliferation. The difference in anti-inflammatory activity was minor between any of the isoflavone treatments.     

银杏果仁抗菌蛋白的分离纯化及其基因克隆和原核表达初步研究

通过40%及80%(NH4)2>SO4>分级盐析,CM Sephorose F.F.离子交换层析和Superdex 75凝胶过滤层析;从银杏(Ginkgo biloba)种仁中分离纯化到一种表观分子量约为12 KD的抗菌蛋白.经过MALDI-TOF-MSPMF鉴定表明,该蛋白与一种新型抗菌蛋白Gnk2(ginkbilobin-2)具较高匹配率(P<0.05);根据ESI-MS/MS内肽氨基酸测序结果设计简并引物,扩增该蛋白基因一段,经比对发现与Gnk2基因序列相似性也达99%.按照Gnk2基因序列设计同源引物,利用RT-PCR克隆该抗菌蛋白全长基因,提交GeneBank(Accession No.FJ865399),并以此构建原核表达载体pGEX-GK2-1转化大肠杆菌(Escherichia coli)BL21(DE3),SDS-PAGE和Western blot分析表明,融合蛋白在大肠杆菌中能够高效表达.     

Anti-inflammatory effects of phenolic compounds isolated from the fruits of Artocarpus heterophyllus

Artocarpus heterophyllus Lam is a large evergreen tree cultivated throughout Southeast Asia for its fruits. Its leaves and roots have been used for medicinal purposes. The aim of this work was to study the in vitro anti-inflammatory effects of phenolic compounds isolated from the ethyl acetate extracts of the fruits of Artocarpus heterophyllus. Three phenolic compounds were characterized as artocarpesin [5,7,2',4'-tetrahydroxy-6-(3-methylbut-3-enyl) flavone] ( 1), norartocarpetin (5,7,2',4'-tetrahydroxyflavone) ( 2), and oxyresveratrol [ trans-2,4,3',5'-tetrahydroxystilbene] ( 3) by spectroscopic methods and through comparison with data reported in the literatures. The anti-inflammatory effects of the isolated compounds ( 1- 3) were evaluated by determining their inhibitory effects on the production of proinflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. These three compounds exhibited potent anti-inflammatory activity. The results indicated that artocarpesin ( 1) suppressed the LPS-induced production of nitric oxide (NO) and prostaglandin E 2 (PGE 2) through the down-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions. Thus, artocarpesin ( 1) may provide a potential therapeutic approach for inflammation-associated disorders.     

Selective responses of three Ginkgo biloba leaf-derived constituents on human intestinal bacteria

The selective responses of Ginkgo biloba leaf-derived materials against six intestinal bacteria was examined using an impregnated paper disk method and compared with that of bilobalide, ginkgolides A and B, kaempferol, and quercetin. The components of G. biloba leaves were characterized as kaempferol 3-O-alpha-(6' "-p-coumaroylglucosyl-beta-1,4-rhamnoside), kaempferol 3-O-(2' '-O-beta-D-glucopyranosyl)-alpha-L-rhamnopyranoside, and quercetin 3-O-alpha-(6' "-p-coumaroylglucosyl-beta-1,4-rhamnoside) by spectroscopic analysis. The growth responses varied with each bacterial strain tested. At 2 mg/disk, kaempferol 3-O-alpha-(6' "-p-coumaroylglucosyl-beta-1,4-rhamnoside) and quercetin 3-O-alpha-(6' "-p-coumaroylglucosyl-beta-1,4-rhamnoside) revealed potent inhibition against Clostridium perfringens, and kaempferol 3-O-(2' '-O-beta-D-glucopyranosyl)-alpha-L-rhamnopyranoside showed a clear inhibitory effect on Escherichia coli. At 0.5 mg/disk, quercetin 3-O-alpha-(6' "-p-coumaroylglucosyl-beta-1,4-rhamnoside) showed a strong activity against C. perfringens, but weak activity was exhibited by kaempferol 3-O-alpha-(6' "-p-coumaroylglucosyl-beta-1,4-rhamnoside) against C. perfringens and kaempferol 3-O-(2' '-O-beta-D-glucopyranosyl)-alpha-L-rhamnopyranoside against E. coli. No inhibition was observed from treatments conducted with bilobalide, ginkgolides A and B, kaempferol, or quercetin. Furthermore, these isolated compounds did not inhibit Bifidobacterium bifidum, B. longum, B. adolescentis, or Lactobacillus acidophilus.     

银杏氮素营养诊断技术研究

应用果园调查、土壤植株化验分析与大田氮肥施用量试验相结合的方法 ,对银杏氮素营养诊断技术进行了研究。结果表明 ,土壤氮素的最佳诊断形态为土壤有效氮 ,其诊断指标 (mg/kg)为 :高 115.2 ,中 78.8～ 115.2 ;低78.8。银杏植株氮素营养诊断最佳诊断时期为 7月 ,最佳诊断部位为长枝条 8位叶 ,诊断形态为全氮 ,其诊断指标 (N % )为 :高 2.04、中 1.80～ 2.04、低 1.80。     

不同介质条件对银杏淀粉糊流变特性的影响

淀粉糊流变特性是影响淀粉类食品加工品质的主要因素。该文采用布拉班德尔黏度计,考察不同介质条件如浓度、pH值、蔗糖和食盐对银杏淀粉糊黏度特性的影响。银杏淀粉糊的布拉班德尔黏度测定结果表明：随着银杏淀粉糊浓度增加,其起糊温度降低,峰值黏度增高,热稳定性、冷稳定性下降,凝沉性增强;60 g/kg银杏淀粉糊黏度性质受酸碱介质的影响较大,在pH值中性7左右,其淀粉糊黏度热稳定性最好,凝沉性最强,但冷稳定性最差;食品介质蔗糖对60 g/kg银杏淀粉糊黏度曲线影响较大,其淀粉起糊温度、峰值黏度随蔗糖浓度增加而增加,淀粉糊黏度的冷稳定性、热稳定性均降低,凝沉性增强;而食盐影响较小。该结果对银杏淀粉类食品深加工开发具有较大的指导意义。     

采前激素处理对银杏种子的脱皮与保鲜效果的研究

研究了乙烯利20、100、500 mg/L;891植物促长素20、100、500 mg/L;脱落酸20、100、500 mg/L等9种不同采前激素催熟处理方法对银杏种子采后脱皮及贮藏保鲜的影响。其中,乙烯利500 mg/L处理,采后4 d呼吸高峰到来,第6 d果肉软化,容易去皮,且贮藏90 d,除霉变率稍高外,浮水率、失水率、硬化率都较低,还原糖、蛋白质、脂肪、淀粉、蛋白质含量保持较高水平,适宜于以食用为目的的贮藏。891植物促长素500 mg/L处理发芽率高,β-淀粉酶、过氧化氢酶(CAT)、脂肪酶、多     

银杏叶药渣用于制备颗粒燃料的工艺能耗试验

为降低银杏叶药渣制备颗粒燃料的能耗,对干燥、粉碎和制粒成型等主要加工环节的单位能耗进行了试验研究.首先,将银杏叶药渣在60～120℃的热风温度下分别恒温干燥至含水率20％,研究干燥温度对干燥能耗的影响;通过正交试验,研究含水率、筛网孔径和主轴转速对粉碎能耗的影响,分析在不同含水率和颗粒度条件下制粒成型的能耗情况.然后根...     

银杏磷素营养诊断技术的研究

应用果园调查与磷肥试验相结合的方法，对银杏磷素营养诊断技术进行了研究。结果表明：银杏土壤磷素营养诊断可以土壤速效磷作为诊断形态，7月份诊断指标（mg/kg^-1)为：高＞14.0，中9.8-14.0，低＜9.8。银杏植株磷素营养诊断最佳诊断时期为7月,最佳诊断部位为长枝条的倒8位叶，以银杏叶片全磷作为诊断形态，其诊断指标（％）为：高＞0．329，中0．265－0．329，低＜0．265。     

银杏钾素营养诊断技术的研究

应用果园调查、土壤植株分析与大田钾肥施用量试验相结合的方法 ,对银杏钾素营养诊断技术进行了研究。结果表明 :土壤钾素以土壤速效钾作为诊断形态 ,其诊断指标 (mg/kg)为 :高 >75 .0 ,中 5 0 .1～ 75 .0 ,低 <5 0 . 1。银杏植株钾素营养诊断最佳诊断时期为 7月 ,最佳诊断部位为长枝条倒 8位叶。诊断形态为叶片全钾 ,其诊断指标 (% )为 :高 >0 .95 8,中 0 .81 8～ 0 .95 8,低 <0 .81 8。     

机械采收作业中银杏树频谱特性与振动响应关系研究

机械采收是林果收获最有效的方法,在受迫振动下果实的掉落不仅受到激振振幅、频率和持续时间的影响,还与果树自身的生长形态和固有频谱特性有关.为了研究银杏树的频谱特性与振动响应之间的关系以及振动响应在不同方向上的差异性,该文在室内冲击激振下测试了一棵Y型银杏树不同方向的频谱特性.然后,通过频谱曲线中峰值点和谷值点所在的频率对果树树干进行简谐激振获得空间加速度响应.结果显示频谱特性与振动响应之间存在一定的对应关系,基频以及10.00Hz以下的激振频率无法激发起很大的振动响应.共振频率能够引起极大的振动响应,但是加速度幅值在低频下较小并且随着激振频率的增加而增大.当频率高于25.00Hz时并不能再次引起较强的加速度响应,样品树的最佳激振频率为23.75Hz.在同一个测试位置,3个方向的振动响应呈现出相似的特性但振幅不同.随着测试位置逐渐远离激振点,沿着果树生长方向的加速度幅值显著增加,并且该方向是振动响应传递过程的主导方向.结果表明在机械采收林果时,可以首先测试果树的频谱来获得共振频率,然后在特定的共振频率下对果树进行激振来获得较强的振动响应,更高的激振频率并不一定引起更强烈的振动响应.同一个测试位置不同方向之间存在差异性,不同位置的果实可以被不同方向的惯性力移除.     

Anti-inflammatory and antifibrotic effects of naringenin in diabetic mice

Renal protective effects of naringenin at 0.5, 1, and 2% of the diet in diabetic mice were examined. Naringenin supplemented at 1 and 2% increased its deposit in liver and kidney of diabetic mice. Compared with the diabetic control group, naringenin treatments at 1 and 2% lowered plasma levels of glucose and blood urea nitrogen, as well as increased insulin level and creatinine clearance (P < 0.05). Naringenin treatments dose-dependently reduced renal tumor necrosis factor-α level and expression (P < 0.05) but only at 1 and 2% significantly decreased production and expression of interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1 (P < 0.05). Naringenin intake at 2% decreased renal formation and expression of type IV collagen, fibronectin, and transforming growth factor-β1 (P < 0.05). This compound at 1 and 2% lowered protein kinase C activity and suppressed nuclear factor κB (NF-κB) p65 activity, mRNA expression, and protein production in kidney. However, this agent only at 2% diminished NF-κB p50 activity, mRNA expression, and protein production (P < 0.05). These results indicate that naringenin could attenuate diabetic nephropathy via its anti-inflammatory and antifibrotic activities.     

Anti-inflammatory effects of resveratrol and oligostilbenes from Vitis thunbergii var. taiwaniana against lipopolysaccharide-induced arthritis

Vitis thunbergii Sieb. and Zucc. var. taiwaniana Lu is an endemic plant in Taiwan used as a dietary supplement for bone health. In this study, human chondrocytes were induced to produce COX-2, MMP-3, -13, and PGE(2) by LPS. An (18)F-FDG microPET imaging system was used to evaluate the anti-inflammatory arthritic effects in vivo. Six stilbenes, resveratrol (1), (+)-ε-viniferin (2), ampelopsin C (3), ampelopsin A (4), (-)-vitisin B (5), and (+)-vitisin A (6), were isolated from the stem part of V. thunbergii, which displayed the strongest PGE(2) inhibition. Among these compounds, 1 significantly decreased COX-2 activity, PGE(2), MMP-3, and -13 production in vitro, and (18)F-FDG uptake and serum PGE(2) in rabbits in vivo. Anti-inflammatory effects were enhanced through the combined usage of 1 and other oligostilbenes. Taken together, the synergistic effects of 1 and oligostilbenes resulted in stem part extracts with lower 1 content displaying the better anti-inflammatory arthritis effects.