不同灵芝菌丝体中三萜与多糖含量的比较

根据十九种不同来源的灵芝菌株的生长速度及长势，从中选出了菌丝体阶段生长性能良好的10个菌株进行液体培养，并测定了菌丝中三萜和多糖的含量。结果表明，不同灵芝菌株在三萜和多糖含量上存在着一定的差异，其中多糖含量以汉城2号，日本灵芝，南韩灵芝为高；三萜含量却以韩国灵芝、灵芝0772、灵芝0771为高，并且二者的含量不存在必然的联系，这为灵芝优良品种的筛选提供了理论依据。     

灵芝三萜的生物活性及潜在应用研究

灵芝作为重要的药用真菌,其主要生物活性成分包括多糖、糖肽及三萜类化合物等,其中,灵芝三萜类化合物系高度氧化的羊毛甾烷衍生物,表现出强大且独特的生物活性及药理价值,目前灵芝三萜含量已成为《美国药典（United States Pharmacopoeia）》对灵芝质量的评价标准。综述目前关于灵芝三萜生物活性的研究成果,包括细胞毒性和抗肿瘤活性、抗氧化和保肝活性、神经保护作用、助眠特性、抗糖尿病活性等;概述灵芝三萜在功能性食品和保健品开发中的潜在应用价值,并探讨其未来的研究方向。     

灵芝三萜结构和生物活性及高产三萜菌株的研究进展

灵芝（Ganoderma spp.）是著名的药用真菌,含有多种活性化合物,灵芝三萜是其主要的活性代谢成分之一,具有抗肿瘤、免疫调节、降血糖等功能。笔者总结了灵芝三萜的分离纯化、结构、生物活性及高产三萜育种工作的研究进展,并对今后灵芝的研究工作进行展望,以期为灵芝三萜的开发利用提供参考。     

灵芝三萜和多糖提取工艺研究

采用醇提法、水提法分别提取灵芝三萜和多糖,并与超声波辅助提取效果进行比较。结果,灵芝三萜的最佳提取条件为：采用10倍无水乙醇,在60℃提取60 min,提取2次,三萜提取率为0.76%;灵芝多糖的最佳提取条件为：用90℃热水提取90 min,料（g）液（mL）比1∶20,提取2次,多糖提取率为2.67%。超声波辅助提取,灵芝三萜和多糖的提取率可分别提高20.5%和13.2%。     

利用大孔树脂制备灵芝活性三萜的研究

用D101大孔树脂作为填料,不同浓度乙醇梯度洗脱灵芝(Ganoderma lucidum)子实体提取物,利用HPLC对得到的不同洗脱物进行了分析对比,并利用体外抗肿瘤模型对洗脱物进行了活性研究.结果表明,90％和95％乙醇洗脱获得了灵芝中极性较弱的三萜组分,得率共计为6.7％.该三萜组分具有较强的抗肿瘤活性,其中,以95％乙醇洗脱获得的三萜组分活性最强,对肿瘤细胞K562、LNCaP增殖抑制的IC50值分别达到14.67 μg/mL和0.87 μg/mL.     

灵芝三萜提取工艺优化

以灵芝T0菌株的菌丝体为原料,采用超声辅助提取三萜,在单因素试验结果的基础上,通过正交试验得到优化最佳工艺条件.结果表明,在乙醇浓度85％、提取时间30 min、料液比1:30的条件下,灵芝菌丝体三萜的平均得率达到了3.50％.该结果为优化灵芝菌丝体三萜的提取工艺提供了有利参考.     

不同日常食用加工方法对灵芝中活性成分利用的比较

为阐明日常的食用加工方法是否能充分利用灵芝(Ganoderma lingzhi)中的活性成分,用苯酚硫酸法和HPLC法分别检测龙芝二号子实体片经水煮、焖烧、水泡、酒浸四种加工处理的提取液中灵芝多糖和灵芝三萜含量,结果水煮、焖烧、水泡40min后多糖基本全部溶出,三萜溶出率分别为85.5%、83.1%、51.1%;50%的白酒浸泡7d后三萜溶出率为95.0%,而多糖溶出率仅为0.31%;用这些方法比较龙芝二号与菌草灵芝的处理结果,结果多糖和三萜溶出效果基本相同;通过比较,焖烧40min能使灵芝片中有效成分大部分溶出。     

超临界CO2萃取灵芝子实体三萜工艺优化及其与醇提法比较研究

采用超临界CO2 (SFE-CO2)技术萃取灵芝(Ganoderma lucidum)子实体中的三萜成分,通过单因素试验和正交试验确定最佳条件为:子实体粒度10目、夹带剂95％乙醇、压力35 Mpa、温度40℃、时间2.5h、CO2流量35 g/min、夹带剂体积:子实体重6:1(mL/g),萃取物的得率为2.42％,总三萜的得率为0.98％,萃取物中三萜的含量为40.5％.与传统的醇提法相比,SFE-CO2法中粗提物和总三萜的萃取率略低,但粗提物中三萜的含量较高,且HPLC研究表明,SFE-CO2的粗提物中三萜的种类较多.     

不同粉碎程度对灵芝多糖与三萜提取得率的影响

以3个品种的灵芝(Ganoderma lucidum)子实体为材料,比较了不同粉碎程度对灵芝多糖与三萜提取效果的影响,结果表明,超细粉碎工艺能够有效地破碎灵芝子实体中的菌丝细胞,显著提高灵芝多糖的提取率,但对三萜的提取结果没有影响.     

白肉灵芝子实体和菌丝体活性成分的比较

对四川和西藏栽培的白肉灵芝(Ganoderma leucocontextum)子实体(编号分别为GLFS、GLFT)及液体发酵菌丝体(编号为GLM)中多糖、三萜、糖醇、核苷含量及多糖分子量分布特征进行比较.结果表明:GLFS和GLFT中多糖含量相对较低,分别为0.87％和0.79％,GLM多糖含量相对较高,为1.54％...     

灵芝孢子对肿瘤细胞生长的抑制效果研究

应用3种肿瘤细胞模型对未破壁灵芝孢子、机械破壁灵芝孢子和全灵芝剥壁孢子进行抗肿瘤试验研究。结果表明,未破壁及不同破壁方式的灵芝孢子对人恶性乳腺癌细胞(MT-1)、人恶性淋巴癌细胞株(Jurkat)、人恶性脑肿瘤细胞(U343)均有不同程度的抑制作用,抑制效果强弱为全灵芝剥壁孢子机械破壁灵芝孢子未破壁灵芝孢子,表明破壁的确能提高灵芝孢子对肿瘤细胞的抑制作用,而且选择破壁的方法也很重要,生物酶法破壁能使灵芝孢子的生物活性成分得到充分释放,所以大大提高灵芝孢子的功效。     

Hypoxic preconditioning alleviates oxygen-glucose deprivation in PC12 cells:the involvement of autophagy

AIM: To examined the effects of hypoxic preconditioning(HPC) on oxygen-glucose deprivation(OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy.METHODS: Cultured PC12 cells were randomly divided into control group, HPC group, 3-methyladenine(3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group. CCK-8 assay was used to detect the cell viability. The caspase-3 activity was also tested. TUNEL staining and flow cytometry were used to detect the cell apoptosis. The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot.RESULTS: Compared with control group, the viability of PC12 cells was significantly reduced, and the activity of caspase-3 was significantly increased in OGD group. Compared with 3-MA+ HPC+OGD group and OGD group, the viability of PC12 cells was significantly increased, and the activity of caspase-3 was significantly reduced in HPC+OGD group(P<0.05). The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate(P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in HPC+OGD group(P<0.05). Compared with control group, the protein level of cleaved caspase-3 was significantly increased in OGD group(P<0.05). Compared with OGD group, the protein level of cleaved caspase-3 was significantly decreased, and the levels of LC3-2 and beclin-1 were significantly increased in HPC+OGD group(P<0.05).CONCLUSION: OGD decreases cell survival and induces apoptosis.Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC12 cells from OGD induced injury.     

EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by mTOR regulation

AIM:Hypoxia (evoked by CoCl2)-induced apoptosis and autophagy are emerging as crucial events in the etiopathology of many neurodegenerative diseases. Epigallocatechin gallate (EGCG) is the active ingredient in tea polyphenols with abilities of anti-apoptosis and anti-autophagy, but the mechanism has not been fully elucidated. In recent years, studies have reported that the mammalian target of rapamycin (mTOR) involved in the regulation of a variety of neurological like differentiation and maturation of nerve cells, anti-oxidative stress, etc. Therefore, we investigate that whether EGCG protects PC12 from hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. METHODS:The expression of mTOR and beclin-1 were detected by Western blotting. The expression of caspase-3 was measured by ELISA. The cell viability was detected by CCK-8 assay. The LC-3 expression in nucelus was observed by immunofluorescence. RESULTS:Hypoxia induced apoptosis and autophagy in PC12 cells. EGCG antagonized hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. Blocking the pathway of mTOR reversed the protective effect of EGCG on PC12 cells. CONCLUSION: EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by controlling mTOR regulation.     

Relationship between morphological changes of autophagy and apoptosis in PC12 cells induced by oxygen-glucose deprivation and reoxygenation

AIM: To investigate the relationship between morphological changes of autophagy and apoptosis in the PC12 cells induced by oxygen-glucose deprivation and reoxygenation. METHODS: The PC12 cells were randomly divided into normal control group, oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group. The cells in oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group were exposed to reoxygenation (12 h) after 3 h of oxygen-glucose deprivation, and autophagy inhibitor 3-methyladenine and autophagy activator rapamycin were added into the cells at the same time. Using transmission electron microscope and monodansylcadaverine fluorescence staining, the morphological changes of autophagosome were observed. The apoptosis of the PC12 cells were analyzed by flow cytometry with Annexin V-FITC/PI staining and TUNEL method. RESULTS: Compared with normal control group, the numbers of autophagosomes and the apoptotic rates increased in oxygen-glucose deprivation and reoxygenation group (P<0.05). Compared with oxygen-glucose deprivation and reoxygenation group, the numbers of autophagosomes decreased obviously (P<0.05) and the apoptotic rates increased markedly in autophagy inhibitor group (P<0.05). The numbers of autophagosomes increased obviously (P<0.05), the apoptotic rates decreased markedly (P<0.05), the autophagosomes became bigger in size, and autolysosomes was also found in autophagy activator group. CONCLUSION: Oxygen-glucose deprivation and reoxygenation induce autophagy in PC12 cells, and autophagy inhibits cell apoptosis to play a protective role.     

Protective effect of procyanidins on PC12 cells exposed to Aβ25-35

AIM: To investigate the protective effect of procyanidins on the PC12 cells exposed to Aβ_25-35 and the mechanisms.METHODS: Aβ_25-35 at 25 μmol/L was used to treat the PC12 cells for 48 h, and the PC12 cells were pretreated with procyanidins at 25, 50 and 100 mg/L for 24 h. The cell vitality was measured by MTT assay. The content of reactive oxygen species (ROS) was detected by DCFH-DA staining. The change of mitochondrial membrane potential was examined by JC-10 staining. The apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of activated caspase-3 was determined by Western blot.RESULTS: Under the exposure of the PC12 cells to Aβ_25-35, procyanidins increased the cell viability, reduced intracellular ROS level, prevented mitochondrial membrane potential decline, attenuated the caspase-3 activation and inhibited the apoptosis of PC12 cells (P<0.05 or P<0.01).CONCLUSION: Procyanidins have a significant protective effect on the PC12 cells exposed to Aβ_25-35. Its mechanism may be related to removing intracellular ROS induced by Aβ_25-35, relieving the damage to the mitochondrial membrane, and thereby inhibiting cell apoptosis.     

Effects of Mage-H1 expression on PC12 cell differentiation and cell cycle

AIM: To explore the effects of melanoma-associated antigen H1 (Mage-H1) on cell proliferation and differentiation. METHODS: A phase contrast microscope was used to observe the morphological changes of PC12 cells treated with or without nerve growth factor (NGF). The expression of Mage-H1 in pre-and post-differentiated PC12 cells was detected by RT-PCR and Western blotting, and its potential effects on the cell cycle were analyzed by flow cytometry. RESULTS: After induced by NGF for 8 days, over 92% of PC12 cells were differentiated. The relative levels of Mage-H1 mRNA and protein in the differentiated PC12 cells were 4.6 times and 2.6 times higher than those in control cells,respectively. Moreover, the PC12 cells transiently expressed Mage-H1 were significantly arrested in G₀-G₁ phase as compared to the cells transfected with an empty vector.CONCLUSION: Mage-H1 inhibits the proliferation of PC12 cells and promotes the differentiation of the cells.     

激素对菊花愈伤组织诱导和丛生芽分化的影响

利用菊花的叶片为外植体,在附加不同激素浓度的MS培养基上诱导愈伤组织。通过对6-BA和NAA在菊花愈伤组织诱导和丛生芽作用的研究,筛选合适的激素配比。结果表明:愈伤组织诱导的最适培养基为MS+6-BA 2.0 mg/L+NAA 1.0 mg/L,丛生芽诱导的最适培养基为MS+6-BA 2.0~3.0 mg/L+NAA 0.1~0.5 mg/L,可获得较高的分化率。丛生芽继代培养基为MS+6-BA 2 mg/L+NAA 0.1 mg/L;试管苗在1/2MS+NAA 0.1 mg/L+20 g/L蔗糖的生根培养基上均可生根。     

不同抗性辣椒品种根系分泌物对疫霉菌的影响

为分析辣椒根系分泌物与抗疫病的相关性,测定了4 个不同抗性辣椒品种的根系分泌物对辣椒疫霉菌的影响。结果表明：对辣椒疫病表现抗病的PRMND 和DNP56 的根系分泌物对疫霉菌的菌丝生长、游动孢子囊形成、游动孢子释放和休止孢子萌发均有显著的抑制作用,而对辣椒疫病表现高度感病的科星6 号和新机遇的根系分泌物对疫霉菌的菌丝生长没有明显影响,科星6 号根系分泌物对游动孢子囊的形成没有明显的影响,而新机遇根系分泌物对游动孢子囊形成有明显抑制作用,科星6 号和新机遇的根系分泌物对游动孢子释放和处理8 h 后对休止孢子萌发具有明显的抑制作用,但其抑制效果显著低于抗病品种。     

不同培养基及糖浓度对丰花月季叶愈伤组织诱导的影响

以丰花月季叶片为材料,在MS,B5,White,WPM4种基本培养基和在合不同糖浓度为0、30、60、90g/L的MS培养基上,进行愈伤组织的诱导.结果表明:4种培养基中,MS培养基的诱导率最高,4种糖浓度中,以糖浓度为30g/L的诱导率最高.最佳培养基为MS+6-BA0.1 mg/L+NAA 0.1 m/L+糖30g/L+琼脂7.5 g/L.