罗汉果醇抗肿瘤活性及其作用机制研究

罗汉果醇是罗汉果皂苷的苷元,有研究报道罗汉果皂苷V具有防癌抑癌作用。该研究采用噻唑蓝实验( MTT法)检测罗汉果醇对不同肿瘤细胞增殖的抑制情况,以及不同浓度的罗汉果醇对CNE1细胞的增殖抑制率;应用细胞克隆形成实验进一步验证罗汉果醇对CNE1细胞增殖的抑制作用;采用Annexin V/PI 双染法检测罗汉果醇对CNE1细胞凋亡的影响;以实时定量PCR技术检测罗汉果醇对CNE1细胞中Caspase-3、Sur-vivin、Bax和Bcl-2基因的mRNA 表达水平的影响。结果表明：罗汉果醇能显著抑制DU145、HepG2、A549、CNE1、CNE2细胞的增殖,其中对CNE1细胞增殖的抑制作用最为显著,并呈剂量依赖性,其半数抑制浓度IC50为(81.48±4.73)μmol·L-1;通过对CNE1细胞进一步的克隆形成实验,也验证了这一点;Annexin V/PI 双染法可见随着浓度的增加,凋亡比例增加;实时定量PCR技术检测显示罗汉果醇处理后,促凋亡基因Caspase-3、Bax的表达增加,抗凋亡基因Survivin、Bcl-2的表达减少。因此,罗汉果醇可能是通过促进Caspase-3、Bax等促凋亡基因和抑制Survivin、Bcl-2等抗凋亡基因的表达,来诱导肿瘤细胞凋亡,进而发挥抗肿瘤活性。     

亚肉座菌天然产物的分离鉴定及抗肿瘤活性

亚肉座菌能侵染粉虱和介壳虫,具有悠久的生物防治历史,但关于其天然产物的种类及抗肿瘤活性研究尚不深入。本研究借助硅胶柱、HPLC等色谱技术从亚肉座菌发酵液中分离纯化到6个化合物。基于核磁共振和质谱数据解析,化合物1–6依次鉴定为:5α,8α-表二氧-24(R)-甲基胆甾-6,22-二烯-3β-醇5α,8α-epidioxy-24(R)-methylcholesta-6,22-diene-3β-ol(1),杜斯塔宁dustanin(2),3β-乙酸基-15α,22-二羟基何伯烷3β-acetoxy-15α,22-dihydroxyhopane(3),麦角固醇ergosterol(4),7β,15α,22-三羟基何伯烷7β,15α,22-trihydroxyhopane(5)和5-(羟甲基)呋喃-3-羧酸flufuran(6),化合物1和6为首次从虫生真菌中获得。对化合物进行抗肿瘤活性测试发现,化合物1和5对人肝癌细胞(BEL-7404)展示显著的抗肿瘤活性,IC50值分别为28μmol/L和12.4μmol/L,而它们对人正常肝脏细胞(HL-7702)和肾脏上皮细胞(HEK-293T)均未呈现出明显活性。该研究对阐明亚肉座菌代谢产物及其活性提供了重要数据,为开发抗肿瘤新药提供了理论参考。     

白柄肉齿菌Sarcodon leucopus多酚生物碱提取物的体外抗氧化及降血糖活性

为探讨西藏白柄肉齿菌Sarcodon leucopus子实体多酚生物碱提取物(PAE)的抗氧化和降糖活性,本研究考察了PAE对DPPH自由基清除能力、细胞内抗氧化能力(CAA)和糖尿病关键酶(大鼠小肠来源麦芽糖酶、蔗糖酶和α-淀粉酶)活性的抑制能力.结果表明,PAE具有一定的抗氧化活性,对DPPH自由基的半数抑制浓度(...     

重组人RGD-sTRAIL的表达、纯化及抗肿瘤活性研究

利用PCR技术构建RGD短肽与人肿瘤坏死因子凋亡配体(胞外区114-281)的融合基因,将该DNA片段克隆到原核表达载体pET-11a中。重组质粒转化大肠杆菌BL21(DE3),IPTG诱导后可表达相对分子质量约为20000的目的蛋白,占菌体蛋白的20％左右,且大多数重组蛋白以不溶的包涵体形式存在。Western印迹表明目的蛋白具有人sTRAIL的抗原性。表达产物经变性、复性、离子交换层析和分子筛等步骤,可以得到纯度大干95％的RGD-sTRAIL重组蛋白。肿瘤细胞体外实验发现,纯化后的RGD-sTRAIL重组蛋白能明显抑制人肺癌细胞A549生长,并呈剂量依赖性。研究结果表明,通过复性,包涵体中的RGD-sTRAIL蛋白得到正确的折叠,并在体外具有杀伤肿瘤细胞的活性,从而为进一步研究体内靶向性杀伤肿瘤细胞奠定了基础。     

斑褐孔菌石油醚提取物体外抗肿瘤活性研究

探讨斑褐孔菌石油醚提取物(PEFP)体外抗肿瘤活性及其机制。采用磺酰罗丹明染色法(SRB法)考察PEFP对人肝癌细胞SMMC-7721、人胃癌细胞SGC-7901、人喉癌上皮细胞Hep-2增殖的影响。倒置相差显微镜、透射电镜观察PEFP作用SMMC-7721后的细胞形态及超微结构的变化;流式细胞术检测PEFP作用SMMC-7721后细胞周期的改变情况。实验结果显示PEFP对上述三种人恶性肿瘤细胞均表现出明显的抑制效果,且呈剂量效应关系,其半数抑制浓度(IC50)分别为52.72、69.18、58.88μg/mL。取1/2 IC50浓度的PEFP作用于SMMC-7721 48 h后,细胞形态结构及细胞周期发生改变,G0/G1期细胞增多,S期细胞明显减少。PEFP可能通过阻滞SMMC-7721细胞周期于G0/G1期来抑制肿瘤细胞的增殖,具有较明显体外抗肿瘤活性。     

中国的齿状真菌研究3.肉齿菌属

对中国肉齿菌属的3个种进行了总结,其中花状肉齿菌为中国新记录种.该种采集自海南省儋州市热带植物园,主要特征:子实体花朵状、覆瓦状叠生,囊状体棒状或纺锤形,担孢子广椭圆形.根据采集的标本对该种进行了详细的描述及显微结构绘图,对该种与其它种类的联系和区别进行了讨论,并列出了附有每种简要描述的检索表.     

阔叶鳞盖蕨和粗毛鳞盖蕨(碗蕨科)配子体发育的研究

以腐殖土为基质,对阔叶鳞盖蕨Microlepia patyphylla（D．Don）J．Sm．和粗毛鳞盖蕨M.strigosa（Thunb．）Presl进行了孢子繁殖;利用光学显微镜观察和记录了它们的孢子萌发和配子体发育过程。结果表明,两者的孢子及配子体性状极为相似：孢子同型,三裂缝,萌发慢;孢子萌发需要光,萌发方式为书带蕨型Vittaria-type;配子体生长慢,发育类型为铁线蕨型Adiantum—type;原叶体心形或其他形状,无毛状体,多数为雌雄异株。性器类型为薄囊蕨型Leptosporangiate-type。它们既具有大量的原始性状,也具有少数进化性状。与阔叶鳞盖蕨相比,粗毛鳞盖蕨的配子体发育特征更为原始。说明粗己鳞盖蕨的系统位置更加低下。首次观察到阔叶鳞盖蕨中细胞自然死亡时的叶绿体聚集现象。粗毛鳞盖蕨原叶体老化时叶绿体呈现规则的、相互镶嵌的多边形的形状以及在正常光照下粗毛鳞盖蕨某些细胞中的部分叶绿体成链珠状排列等现象。     

紫陀螺菌化学成分及其抑制肿瘤细胞增殖活性的研究

以紫陀螺菌为对象,研究其子实体的化学成分及其抑制肿瘤细胞增殖活性。采用溶剂提取、柱层析和高效液相色谱等方法分离纯化化学成分,通过核磁共振和质谱技术鉴定单体化合物结构,运用结晶紫法评价单体化合物抑制肿瘤细胞增殖活性。从乙酸乙酯提取物中共分离鉴定6个单体化合物,分别为(22E,24R)-麦角甾-5,7,22-三烯-3β-醇(1)、3β,5α-二羟基-(22E,24R)-麦角甾-7,22-二烯-6-酮(2)、(22E,24R)-麦角甾-7,22-二烯-3β,5α,6β-三醇(3)、吲哚-3-甲酸甲酯(4)、4,4-二甲基-1,7-庚二酸(5)和(8E,10E)-12羰基十八碳-8,10-二烯酸(6),其中化合物1为主要成分,相对含量为23.8%。活性测试结果表明3对人乳腺癌细胞株MCF-7 细胞、人胰腺癌细胞株PANC-1细胞和人乳腺癌细胞株MDA-MB-231细胞具有微弱的细胞增殖抑制活性。本研究首次报道了紫陀螺菌化学成分,对深入挖掘其在健康领域中的开发价值具有重要意义。     

阔叶鳞盖蕨配子体发育及卵发生的研究

采用显微镜和透射电镜对阔叶鳞盖蕨(Microlepia platyphylla)的配子体发育和卵发生过程进行了观察,以阐明其卵发生的细胞学机制,探讨其演化地位。阔叶鳞盖蕨孢子褐色,四面体形,具三裂缝,接种5~10d后孢子萌发,经丝状体和片状体阶段发育为心形原叶体,原叶体发育是铁线蕨型,通常为雌雄异株,精子器产生于不规则配子体的表面,颈卵器产生于心形原叶体生长点的下方,性器官是薄囊蕨型。卵发生研究表明,阔叶鳞盖蕨颈卵器产生于生长点下方表面细胞,经两次分裂形成了顶细胞、初生细胞和基细胞。其中初生细胞再经两次不等分裂产生卵细胞、腹沟细胞和颈沟细胞,此3个细胞通过胞间连丝紧密相连,随发育,腹沟细胞与卵细胞间形成了分离腔,但在孔区处始终通过胞间连丝相连,成熟卵细胞上形成了卵膜和受精孔,卵核表面产生了核外突,通过比较表明阔叶鳞盖蕨卵发生与蕨（Pteridium aquilinum）卵发生相似。     

Anti-inflammatory Cyathane Diterpenoids from Sarcodon scabrosus

Four novel diterpenoids were isolated from the fruiting bodies of Sarcodon scabrosus (Fr.) Karst. (Boraginaceae) together with neosarcodonin A. One of the novel compounds was elucidated to be a cyathane diterpenoid, namely neosarcodonin O, by its spectral data. The others were characterized as 19-O-linoleoyl, 19-O-oleoyl and 19-O-stearoyl derivatives of sarcodonin A, after comparison with the authentic samples synthetically prepared from sarcodonin A. These compounds, together with the five 19-O-acyl derivatives synthesized from sarcodonin A, each exhibited inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears by topical application.     

Anti-inflammatory cyathane diterpenoids from Sarcodon scabrosus

Four novel diterpenoids were isolated from the fruiting bodies of Sarcodon scabrosus (Fr.) Karst. (Boraginaceae) together with neosarcodonin A. One of the novel compounds was elucidated to be a cyathane diterpenoid, namely neosarcodonin O, by its spectral data. The others were characterized as 19-O-linoleoyl, 19-O-oleoyl and 19-O-stearoyl derivatives of sarcodonin A, after comparison with the authentic samples synthetically prepared from sarcodonin A. These compounds, together with the five 19-O-acyl derivatives synthesized from sarcodonin A, each exhibited inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears by topical application.     

New Antibacterial Diterpenoids from the Sarcodon scabrosus Fungus

The structures of new antibacterial diterpenoids that had been isolated from Sarcodon scabrosus were established by chemical and spectral means to be sarcodonin L (2) and M (3), both having the cyathane skeleton. Other antibacterial compounds were identified to be allocyathin B₂ (1), sarcodonin G (4) and sarcodonin A (5) by comparing their spectral data with those of authentic samples.     

Anti-inflammatory compounds from the bitter mushroom, Sarcodon scabrosus

A bioassay-guided purification procedure from the methanol extract of Sarcodon scabrosus led to the isolation of several anti-inflammatory compounds: sarcodonin A (1) and G (2), and related compounds (3, 4 and 5). We named these related compounds neosarcodonin A (3), B (4) and C (5) and elucidated their structures on the basis of spectral data. Topical application of each of these compounds to mouse ears suppressed TPA-induced inflammation. Neosarcodonin C (5) exhibited the highest activity and inhibited the TPA-induced edema on mouse ears by up to 87% with a 200-microg application.     

粗糙肉齿菌子实体化学成分研究

利用硅胶柱层析、RP-8层析及Sephadex LH-20分离的方法,从高等真菌粗糙肉齿菌(Sarcodon scabrosus)干燥子实体的甲醇提取物中分离并鉴定了8个化合物,经现代光谱技术鉴定分别为苯甲酸(1)、麦角甾-5,7,22-三烯-3β-醇(2)、2',3'-二乙酰基-3,4,5',6',4″-五羟基-对联三苯(3)和1-乙基-β-D-吡喃葡萄糖苷(4)、对羟基苯甲酸(5)、丁二酸(6)、腺嘌呤(7)、腺嘌呤核苷(8).以上化合物均为首次从粗糙肉齿菌中获得.     

Secoscabronine M, a novel diterpenoid from the Chinese bitter mushroom Sarcodon scabrosus

Secoscabronine M (1) is a hemiacetal cyathane diterpenoid that was isolated from the fruiting bodies of the basidiomycete Sarcodon scabrosus (Fr.) Karst. Compound 1 possesses a novel structure with a bond cleavage between C-3 and C-4. The structure of the new compound was elucidated by means of spectroscopic methods, including two-dimensional nuclear magnetic resonance experiments. The absolute configuration of 1 was established by analysis of circular dichroism spectroscopy and also by employing time-dependent density functional theory calculations. In addition, compound 1 was confirmed to be an equilibrium mixture of two epimers (15S and 15R) at position C-15 in polar solvents by one-dimensional nuclear magnetic resonance analysis.     

Scabronine M, a novel inhibitor of NGF-induced neurite outgrowth from PC12 cells from the fungus Sarcodon scabrosus

A novel cyathane diterpenoid, designated scabronine M (1), was isolated from the fruiting bodies of the mushroom Sarcodon scabrosus together with 10 known compounds. The structure of the new compound was elucidated on the basis of extensive spectroscopic analysis including 2D-NMR. Among these compounds, only scabronine M (1) significantly inhibited dose-dependently NGF-induced neurite outgrowth in PC12 cells without cytotoxicity, possibly through suppressing the phosphorylation of the receptor Trk A and the extracellular signal regulated kinases (ERK). This is the first report of novel neurite outgrowth-inhibiting activity in PC12 cells of this group of diterpenoids.     

Synthesis of Some Di- and Tridepsipeptides Containing Hydroxy-methylbutyric Acid Involved in the Structure of a Depsipeptide Isolated from the Mushroom Sarcodon scabrosus

Some di- and tridepsipeptides containing hydroxymethylbutyric acid were synthesized. A comparison of their NMR spectra with those of the degradation products from the Sarcodon scarbrosus depsipeptide enabled assigning the natural compound preferentially to be cyclo-Hmb-Ile-Hmb-Ala-Thr-. The configuration of the residue of Hmb-Ile is postulated to be L-L, although it could possibly be D–D.     

薄芝糖肽的抗肿瘤作用及其作用机制研究

目的研究薄芝糖肽的抗肿瘤作用,并探讨其作用机制.方法采用体内移植性肿瘤实验与体外细胞培养相结合的方法,对肿瘤细胞增值程度、细胞凋亡情况及肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ及其mRNA的表达情况等进行研究.结果(1)薄芝糖肽在50、100、200μg/ml剂量下显著抑制移植性肉瘤S180的生长.(2)薄芝糖肽不能直接抑制人急性早幼粒白血病细胞(HL-60)的体外培养,不能诱导其凋亡.(3)薄芝糖肽与小鼠腹腔巨噬细胞和脾淋巴细胞共同培养显著抑制HL-60细胞生长并诱导其凋亡,TNF-α、IFN-γ及其mRNA的表达水平显著升高.结论薄芝糖肽可通过促进TNF-α和IFN-γ的mRNA的表达,增加TNF-α、IFN-γ的分泌而抑制肿瘤的生长.