铁皮石斛种胚原球茎液体悬浮培养研究

目的 建立铁皮石斛原球茎液体悬浮培养体系，为大规模生产石斛奠定基础。方法 对铁皮石斛原球茎悬浮培养条件进行比较选择，并测定生长增殖量。结果液体悬浮培养的原球茎分散性好、整齐均一。悬浮培养的最适培养基为1／4MS＋40mg／LVC，pH5、6，摇床转速为50r／min，15d继代一次，黑暗培养。培养基中添加0、5mg／LABA预培养30d，原球茎的质量更高、同步性更好。结论获得了快速增殖原球茎的液体悬浮培养的最佳条件，为铁皮石斛开发利用创造条件。     

A comparative assessment of the potential of polysaccharide production and intracellular sugar composition within Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (Aphyllophoromycetideae)

Ganoderma lucidum is a well-known medicinal mushroom species in which polysaccharides are one of the major sources of biological activity. The species was considered as a species-complex due to significant variations in morphological, biochemical, and genetic features among populations with a worldwide distribution. This fact was the basis for setting the aim of this research: to study intraspecific diversity in polysaccharide production and intracellular sugar composition among selected G. lucidum strains. The presence ofintraspecific diversity among 10 G. lucidum strains, from different areas worldwide, was noted. Values of produced mycelia biomass and intracellular polysaccharides were found in wide ranges (3.1 - 28.2 g L(-1) and 20.0 - 53.3 mg g(-1), respectively), while differences in extracellular polysaccharide amounts were minor (0.2 - 1.5 mg mL(-1)). The significant quantitative and qualitative differences in intracellular sugar composition were noted. Glucose was the predominant sugar in almost all strains except one (HAI 447), where sucrose was dominant. The potential of polysaccharide production and intracellular sugar composition could be one more taxonomic criterion for strain characterization within G. lucidum. The differences in intracellular sugar composition and proportions could be reflected in features of produced polysaccharides and also in their biological activities.     

Cryopreservation of Digitalis obscura selected genotypes by encapsulation-dehydration.

Shoot-tips from several genotypes of the cardenolide-producing perennial shrub Digitalis obscura L. were successfully cryopreserved using the encapsulation-dehydration technique. Precultivation on MS medium containing 0.5 M sucrose, followed by 2.5 h dehydration (final weight 30 %) induced shoot regrowth in 42 % of cryopreserved shoot-tips. Cold-hardening of the in vitro cultures before sucrose treatment dramatically increased shoot recovery up to 86 %. The optimized cryopreservation protocol was then employed using different shoot cultures from five D. obscura genotypes. Responses to cryopreservation depended mainly on the genotype, best results being obtained when shoot tips from HU3 and LL11 were used. Prolonged subcultures reduced proliferation rates in both control and cryopreserved HU3 shoot-tips, whereas long-term storage in LN did not affect the shoot recovery rate of the genotype. RAPD markers were employed to evaluate possible somaclonal variation occurring in shoots regenerated through successive subcultures and after cryopreservation. The band patterns revealed differences between the original parent plant and the shoots grown in vitro, especially after a prolonged subculture (84.9 % of matches for HU3 after 16 subcultures vs. 93.4 % for AY3 after 2 subcultures). Nevertheless, match percentages were higher (98.6 % to 99.5 %) when band patterns from subcultured shoots were compared to those obtained from their respective control or frozen progenies indicating that cryopreservation ensure genetic stability of selected Digitalis obscura genotypes.     

松杉灵芝菌丝体多糖(FI_0-c)对人组织瘤细胞和人外周血白细胞产生炎症性细胞因子的影响(Ⅱ)(英文)

目的:比较研究水溶性多糖(FI_0-c)及其氯磺酸修饰产物(FI_0-c-S)对人炎症性细胞因子产生的影响.方法:应用氯磺酸修饰法对多糖进行化学修饰.用放射免疫分析法(RIA)及逆转录聚合酶链反应(RT-PCR)测FI_0-c和FI_0-c-S对人组织瘤细胞(THP-1)和人外周血单核细胞(PBMC)分泌各种与炎症有关的细胞因子,白介素-1(IL-1α)和肿瘤坏死因子α(TNFα)的影响及对mRNA表达的影响.结果:FI_0-c和FI_0-c-S(浓度分别为4,40,400 mg/L)显著提高 低剂量组LPS 10 mg/L协同PMA 200 nmol/L诱导的THP-1细胞产生TNFα的量,然而,这些多糖明显地抑制高剂量组LPS 100 mg/L协同PMA诱导的THP-1细胞产生TNFα.在无刺激的条件下FI_0-c能够诱导比较多量的IL-1α产生,但是FI_0-c或FI_0-c-S却都明显抑制高剂量或低剂量LPS和PMA诱导的THP-1细胞产生IL-1α.低浓度FI_0-c 4 mg/L显著抑制高剂量组LPS 100 mg/L协同PMA诱导的THP-1细胞产生IL-1或TNFα mRNA及蛋白质的量.结论:松杉灵芝菌丝体水溶性多糖在不同的刺激条件下具有双向免疫调节作用.化学修饰的多糖可改变原多糖对细胞因子产生的调节方向.     

Contents of some metabolites in the peel and flesh of the medicinal mushroom Wolfiporia cocos (F.A. Wolf) Ryvarden et Gilb. (higher Basidiomycetes)

Wolfiporia cocos (F.A. Wolf) Ryvarden et Gilb., also named Fu-Ling in Chinese, is a traditional Chinese medicine. Its inner parts of the sclerotia have an invigorating activity in addition to diuretic and sedative activities, while the epidermis has only diuretic activity and no invigorating activity. In the present study, samples were collected from three sites (Wuding, Tengchong and Fengqing) of Yunnan province and separated into peel and flesh parts. The contents of metabolites including polysaccharides, total triterpenoids, total flavonoids, total saponins, and mannitol were analyzed. Wuding samples had the highest polysaccharide (6.87 mg/g) and total triterpenoids (10.12 mg/g). Fengqing samples indicated the highest total flavonoids (4.53 mg/g), total saponins (28.66 mg/g), and mannitol (64.20 mg/g). The contents of components in the dry samples were higher than the fresh ones. Total triterpenoids, total flavonoids, and total saponins in the peel of investigated W. cocos were all higher than in the flesh. However, polysaccharide and mannitol in the peel were uniform with the flesh. The different distribution of the metabolites provided the basis for further study of pharmacology.     

Furanocoumarins in Pastinaca sativa L. in vitro culture

Callus cultures of Pastinaca sativa L. (parsnip), Apiaceae, were cultivated on variants of Linsmaier-Skoog's medium, containing varying quantities (0.1-10.0 mg/l) of phytohormones: NAA-BAP and IBA-BAP which allowed to obtain 1.5-3-fold fresh biomass growth during 6-week subcultures. HPLC analyses showed that tissues cultured in vitro produced psoralen, bergapten, xanthotoxin, isopimpinellin and umbelliferone which are well known metabolites in plants growing under natural conditions. Total content of coumarins depended on the nature and quantity of phytohormones present in the medium, and ranged from 115.7 to 408.5 mg/100 g of the dry weight, isopimpinellin being the metabolite which dominated quantitatively (maximum content of 238.9 mg/100 g). Psoralen was also accumulated in callus tissues at considerable amounts (maximum content of 108.8 mg/100 g). This metabolite dominated in vegetative plant parts that have been analysed in our study (leaves, stems, roots) but its contents were lower than in the material from in vitro culture (48.9 mg/100 g 10.6 mg/100 g and 14.9 mg/100 g, respectively). Imperatorin was not detected in callus tissues although it dominated in the analysed fruits of the studied plant (200.0 mg/100 g). The best of the tested media in respect of promoting tissue biosynthetic capabilities was that which contained 3 mg/l NAA and 1 mg/l BAP. The studies showed that in vitro cultures of Pastinaca sativa L. can be a convenient model to study the biosynthesis of furanocoumarins and also a potential rich source of these compounds, particularly isopimpinellin.     

Optimization of fermentation process of Cordyceps militaris and antitumor activities of polysaccharides in vitro

The influence of medium composition and cultural conditions on simultaneous yield of mycelia, intracellular polysaccharide, adenosine, and mannitol by Cordyceps militaris CGMCC 2909 was investigated with desirability functions in this study. An optimization strategy based on the desirability function approach, together with response surface methodology (RSM) has been used to optimize medium composition, and the optimal medium was obtained via the desirability as follows: yeast extract 10.33 g/L, sucrose 27.24 g/L, KH₂PO₄ 5.60 g/L and the optimal culture conditions are initial pH 6, 25°C, rotation speed 150 r/minute, inoculum size 4%(v/v), and medium capacity 40 mL/250 mL. Under these conditions, the yield of mycelia, intracellular polysaccharide, adenosine and mannitol reached 12.19 g/L, 0.6 g/L, 61.84 mg/L, and 1.38 g/L, respectively, and the D value was 0.77. Furthermore, the polysaccharides showed significant antitumor activities against HeLa and HepG2 in vitro in a dose-dependent manner in 72 hours. At a concentration of 1000 mg/mL, the inhibition rate of polysaccharides was 92.38% and 98.79%. The IC50 for HeLa and HepG2 were 70.91 μg/mL and 97.63 μg/mL, respectively.     

Production of biomass, polysaccharides, and ganoderic acid using non-conventional carbon sources under submerged culture of the Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (higher Basidiomycetes)

The effect of different non-conventional carbon sources was studied in the submerged culture of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, for simultaneous production of mycelial biomass, bioactive ganoderic acid, and polysaccharides, in less time, using non-conventional carbon sources to minimize the high costs of current culture media. The optimal medium composition was defined as (g/L): 50 of barley flour, 0.2 of KH2PO4, 0.1 of MgSO4?7H2O, and 1 NH4Cl. Cultivated under this complex culture medium, the mycelial biomass production was 23.49 ± 0.37 g/L; the extracellular polysaccharides production was 2.72 ± 0.11 g/L; the intracellular polysaccharides production was 2.22 ± 0.06 g/L; the ganoderic acids production was 299.67 ± 11.63 mg/L. One liter of culture medium developed in this project was priced at USD $ 0.11 if barley flour is used as carbon source or $ 0.13 with oat flour in order to get a good amount of products of interest.     

四氯化碳气体对初代培养大鼠肝细胞的损害

研究四氯化碳(CCl_4)挥发气体在试管内对大鼠初代培养肝细胞的损害,导致谷丙转氨酶(GPT)外逸;观察了影响CCl_4作用的因素,并初步观察了几种药物在试管内对CCl_4毒性的影响,发现咖啡酸钠盐在10mg/L以上,甘草甜素铵盐在100mg/L可使GPT明显降低,韭叶柴胡多糖在类似浓度则无明显作用。上述结果表明离体的初代培养大鼠肝细胞为研究肝毒剂的毒理作用,护肝药物的作用原理及肝细胞的代谢提供了良好的研究系统。     

鲜铁皮石斛提取物抗Lewis肺癌的机制研究

目的考察鲜铁皮石斛生物碱、多糖提取物对小鼠Lewis肺癌的抑制作用,并探讨其作用机制。方法制备Lewis肺癌实体瘤小鼠模型,将80只实体瘤模型小鼠均分为模型组、环磷酰胺（CTX）组、鲜铁皮石斛生物碱提取物高、中、低剂量（20,10,5g·kg^-1）组、鲜铁皮石斛多糖提取物高、中、低剂量（20,10,5g·kg^-1）组。除CTX组在开始给药后第3,6,9天腹腔注射CTX（40mg·kg^-1）外,其余各组连续给药20d。观察鲜铁皮石斛生物碱、多糖提取物对荷瘤小鼠瘤重、脾脏指数、血清中白细胞介素－2（interleukin－2,IL－2）和肿瘤坏死因子-α（TNF-α）水平的影响。结果鲜铁皮石斛生物碱、多糖各剂量组的瘤体重量均低于模型组,多糖高剂量组,生物碱高、中剂量组抑制瘤重效果显著（P＜0．05）;鲜铁皮石斛生物碱、多糖各剂量组的脾脏重量、脾脏指数均高于模型组,多糖高、中剂量组效果最好（P＜0．05）;不同剂量生物碱和多糖组均能提高TNF-α、IL-2水平,生物碱高剂量组,多糖高、中剂量组提高效果显著（P＜0．05）。结论鲜铁皮石斛生物碱、多糖提取物对Lewis肺癌荷瘤小鼠肿瘤有明显的抑制作用,其作用机制可能与提高脾脏指数,调节血清中TNF-α、IL-2水平有关。     

蜜环菌酒的研制

目的蜜环菌酒富含多糖,具有天麻酒的相似药理活性,可作为天麻酒的替代品。方法将富含多糖的蜜环菌发酵液进行蜜环菌酒生产工艺的优化,制备蜜环菌酒。结果蜜环菌酒的主要成分为酒精16%、多糖0.301mg/mL、蛋白质44.9mg/L、氨基酸3.30g/L、总酸4.8g/L、总酯3.21g/L。结论蜜环菌酒色、香、味、体纯正,含有功能成分。     

用于Hib结合疫苗生产的新型候选菌株的评价

目的 观察b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)760705株在连续传代过程中的稳定性.方法 将Hib 760705株工作种子批菌种连续传代,对第5、第8、第10代Hib培养物进行全面检测,包括培养特性(细菌培养、卫星试验)、染色镜检、生化反应,以检测第5、第8、第10代Hib的生物学特性.同时采用血清凝集试验和聚合酶链反应荚膜分型方法进行b型荚膜多糖稳定性检测.结果 Hib 760705株工作种子批培养物在连续传代过程中具有典型的细菌学特性,能够稳定地产生b型荚膜多糖.结论 Hib 760705株有明确的来源和背景,可以稳定传代,具备作为Hib结合疫苗生产用候选菌株的条件.     

Coumarin compounds in Ammi majus L. callus cultures

Callus cultures of Ammi majus L. (bishop's weed), Apiaceae, were maintained on variants of Linsmaier-Skoog's (L-S) medium differing in the content of the phytohormones, alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) (0.1-10.0 mg/l). The increments of callus tissue fresh weight showed considerable differences (1.4 bis 4.4-fold) during 4-week subcultures on the media tested. HPLC analysis revealed the presence of six compounds in the extracts of callus tissues, which are known metabolites in plants growing under natural conditions: the linear furanocoumarins psoralen, bergapten, xanthotoxin, isopimpinellin, imperatorin and their precursor umbelliferone. The total contents of coumarin compounds under examination showed marked differences dependent on the phytohormone concentrations in the medium. They ranged from 40.95 to 871.05 mg/100 g of dry weight. Imperatorin was the metabolite dominating among the furanocoumarins in calli from almost all tested variants of L-S medium (maximum content of 169.27 mg/100 g). Callus tissue cultured on one of the variants (0.1 mg/l NAA, 0.1 mg/l BAP) yielded in high content of xanthotoxin (145.33 mg/100 g). On the majority of media, umbelliferone was also accumulated at high quantities (maximum content of 536.29 mg/100 g). The medium containing 0.1 mg/l NAA and 0.1 mg/l BAP was the best for the accumulation of analysed coumarins. This medium favoured the formation of embryogenic callus. Xanthotoxin also dominated quantitatively among the furanocoumarins in the tested vegetative organs and fruits of the plant grown under natural conditions (leaves: 26.10 mg, roots: 5.55 mg, fruits: 3010.41 mg/100 g). Maximum contents of this metabolite in in vitro culture were many times higher than those found in vegetative plant organs but manifold lower than in fruits. On the other hand, maximum contents of imperatorin obtained in in vitro culture were many times higher in comparison with those detected in vegetative organs and fruits (leaves: 14.10 mg, roots: 3.30 mg, fruits: 94.70 mg/100 g of dry weight.). The A. majus L. callus culture, established in the course of the present experiments, can be considered a valuable model for studies of the biosynthesis of coumarin compounds, and a potential source of the psoralen 8-alkoxy derivatives imperatorin and xanthotoxin and their precursor umbelliferone.     

用于Hib结合疫苗生产的新型候选菌株的评价

目的 观察b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)760705株在连续传代过程中的稳定性.方法 将Hib 760705株工作种子批菌种连续传代,对第5、第8、第10代Hib培养物进行全面检测,包括培养特性(细菌培养、卫星试验)、染色镜检、生化反应,以检测第5、第8、第10代...     

层析法提纯A、C、Y、W135群脑膜炎球菌荚膜多糖工艺的建立

目的 建立A、C、Y、W135群脑膜炎球菌荚膜多糖层析纯化工艺。方法 采用陶瓷羟基磷灰石和DEAE Sepharose FF层析柱，在PBS体系中纯化A群多糖。以0.5%脱氧胆酸钠预处理C群多糖，用陶瓷羟基磷灰石层析柱在PBS体系中纯化C群多糖。以含有脱氧胆酸钠的平衡缓冲液溶解Y和W135群多糖，再用Capto Adhere和Capto DEAE层析柱串联纯化。纯化的多糖经Sephadex G-25 Medium层析柱脱盐后冻干，按中国药典2015年版三部的要求进行检定。结果  经过层析纯化，A、C、Y、W135群多糖的蛋白质含量分别降至3.7、4.2、5.4和5.3 mg/g，核酸含量分别降至1.2、3.0、1.1和0.8 mg/g，均符合药典要求。此外，磷、唾液酸和O-乙酰基含量等指标亦均符合药典标准。结论 建立了A、C、Y、W135群脑膜炎球菌荚膜多糖的层析纯化工艺。     

Sedum telephium L. polysaccharide content affects MRC5 cell adhesion to laminin and fibronectin

In traditional medicine the fresh leaves and juice of Sedum telephium L. are used as wound-healing promoters. Cell adhesion represents a primary event in wound repair and in tissue homeostasis, and therefore we have investigated the effect of Sedum juice and its main fractions, polysaccharides and flavonols, on human fibroblast (MRC5) adhesion to fibronectin and laminin. Our findings revealed that total Sedum juice strongly inhibited cell adhesion to laminin and fibronectin (EC50 1.03+/-0.12 mg mL(-1)). This anti-adhesive feature was concentrated mainly in the two polysaccharide fractions (EC50 values comprised between 0.09 and 0.44 mg mL(-1)). The flavonol fractions did not seem to contribute to this effect. A first attempt to elucidate the polysaccharide-related anti-adhesive feature of Sedum juice was also performed. The results confirmed that natural polysaccharides, with chemical structures different from heparin, were able to interfere with integrin-mediated cell behaviour and they contributed to the outstanding effects of Sedum juice and to the role of polysaccharides in cell-matrix interaction.     

栉孔扇贝不同部位多糖的提取分离及抗肿瘤活性研究

目的 研究栉孔扇贝不同部位（柱、性腺、肝脏、外套膜、鳃、足）多糖的抗肿瘤活性。方法 以新鲜栉孔扇贝为原材料,经胰蛋白酶水解、醇沉后得到栉孔扇贝不同部位多糖,通过柱前衍生高效液相色谱法进行栉孔扇贝不同部位多糖的单糖组成分析,以人宫颈癌细胞（Hela）和人肝癌细胞（HepG2）为对象对栉孔扇贝不同部位多糖的抗肿瘤活性进行研究。结果 从新鲜栉孔扇贝中提取得到六种多糖,分别为栉孔扇贝柱多糖（CFCP）、栉孔扇贝性腺多糖（CFGOP）、栉孔扇贝肝脏多糖（CFLP）、栉孔扇贝外套膜多糖（CFMP）、栉孔扇贝鳃多糖（CFGIP）、栉孔扇贝足多糖（CFFP）,其中CFCP仅含一种单糖,其余五种多糖均含十种单糖,且单糖摩尔比各不相同。栉孔扇贝六种部位多糖对Hela细胞的抑制率均随多糖浓度的增大而增大,其中CFLP抑制率最强。CFCP、CFGOP、CFMP对HepG2细胞的抑制率均较弱,CFLP、CFGIP、CFFP对HepG2细胞的抑制率均随多糖浓度的增大而增大,其中CFFP抑制率最强。结论 栉孔扇贝不同部位多糖对Hela细胞的抑制率由高到低依次为CFLP>CFGOP>CFMP>CFGIP>CFFP,对HepG2细胞的抑制率由高到低依次为CFFP>CFLP>CFGIP。同时,CFFP、CFMP、CFGIP、CFLP、CFGOP五种多糖对Hela细胞的抑制作用普遍高于HepG2细胞。     

Accumulation of thiophenes by cell cultures of tagetes patula and the release of 5-(4-Hydroxy-1-butynyl)-2,2'-bithiophene into the medium

Calli of TAGETES PATULA, subcultured one or two times, were used to start liquid cultures. Calli of both subcultures contained thiophene-biocides, mainly 5-(but-3-en-1-ynyl)-2,2'-bithiophene (BBT) and 5-(4-acetoxy-1-butynyl)-2,2'-bithiophene (BBTOAc). Secondary calli contained about 3 times higher concentrations of thiophenes than tertiary calli. The liquid batch cultures consisted of coarse cell aggregates (3-8 mm), the volume of which was doubled each 15 days. In contrast to callus cultures grown on solid medium, cell aggregates grown in liquid medium released thiophenes. The release into the medium was at least 2.3 mg.dm (-3) in 90 days by an average amount of 100 g of fresh cell aggregates and occurred mainly as 5-(4-hydroxy-1-butynyl)-2,2'-bithiophene (BBTOH). In callus BBTOH did hardly occur. The amount of thiophenes recovered in the medium corresponded to 0.02% of the cell dry weight. The productivity of the liquid cultures was not related to the productivity of the original calli, neither was the thiophene production related to the growth rate of the biomass in the liquid cultures. The results indicate that not the selection of calli for thiophene content, but rather the selection of calli with the ability to form cell aggregates is of relevance for the production of thiophenes in liquid cultures.     

Effect of Matteuccia struthiopteris polysaccharides on systemic lupus erythematosus-like syndrome induced by Campylobacter jejuni in BALB/c mice

Matteuccia struthiopteris is a nature plant, which contains a lot of potential active components. In the present study, we investigated the effect of polysaccharides extracted from Matteuccia struthiopteris on lupus-like syndrome induced by Campylobacter jejuni CJ-S131 in BALB/c mice. Mice were randomly divided into normal, model control, SLE model (vehicle treated), Matteuccia struthiopteris polysaccharides treated (30 and 15 mg x kg(-1)) groups and prednisone 5 mg x kg(-1) treated groups. The effect of Matteuccia struthiopteris polysaccharides (Ms) on weight and organ index of BALB/c mice was detected. Autoantibodies and total IgG production were measured by enzyme linked immunosorbent assay. Proteinuria was measured and kidneys were examined by light microscopy. Compared with SLE model group, treatment with Matteuccia struthiopteris polysaccharides 30 and 15 mg x kg(-1) reduced weight loss and Matteuccia struthiopteris polysaccharides 15 mg x kg(-1) reduced spleen swelling (P < 0.05). The increased production of autoantibodies and total immunoglobulin G (IgG) were also significantly inhibited. Matteuccia struthiopteris polysaccharides protected kidney against glomerular injury in BALB/c mice with reduced immunoglobulin deposition and lowered proteinuria (P < 0.01). Matteuccia struthiopteris polysaccharides had a protective effect on lupus-like syndrome induced by CJ-S131 in BALB/c mice.