Purification and characterization of an acid deoxyribonuclease from the cultured mycelia of Cordyceps sinensis

A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by (NH(4))(2)SO(4) precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single- stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: MgCl(2) above 150 mM, MnCl(2) above 200 mM, ZnCl(2) above 150 mM, CaCl(2) above 200 mM, NaCl above 300 mM, and KCl above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other well-characterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.     

Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia.

Cordyceps, one of the well-known traditional Chinese medicines, consists of the dried fungus Cordyceps sinensis growing on the larva of the caterpillar. It is commonly used for the replenishment of body health. One of the known pharmacological effects is its anti-oxidation activity. However, there is a great variation of the quality in different sources of Cordyceps. Here, the water extracts of various sources of natural C. sinensis and cultured Cordyceps mycelia were analyzed for their anti-oxidation activity by using three different assay methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. The results showed that Cordyceps, in general, possesses a strong anti-oxidation activity in all assays tested. However, both natural and cultured Cordyceps showed the lowest inhibition in the lipid peroxidation when compared with the other two assay methods. The cultured Cordyceps mycelia had equally strong anti-oxidation activity as compared to the natural Cordyceps. Besides, the anti-oxidation activities were increased to 10-30 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from Cordyceps polysaccharides.     

Antitumor sterols from the mycelia of Cordyceps sinensis.

Activity guided fractionations led to the isolation of two antitumor compounds 5 alpha,8 alpha-epidioxy-24(R)-methylcholesta-6,22-dien-3 beta-D-glucopyranoside and 5,6-epoxy-24(R)-methylcholesta-7,22-dien-3 beta-ol from the methanol extract of Cordyceps sinensis. Two previously known compounds, ergosteryl-3-O-beta-D-glucopyranoside and 22-dihydroergosteryl-3-O-beta-D-glucopyranoside were also isolated. The structures of hitherto unknown sterols were established by 1D and 2D NMR spectroscopic techniques with the former synthesized in order to confirm the identity of the sugar moiety by chemical correlation. The glycosylated form of ergosterol peroxide was found to be a greater inhibitor to the proliferation of K562, Jurkat, WM-1341, HL-60 and RPMI-8226 tumor cell lines by 10 to 40% at 10 micrograms/ml than its previously identified aglycone, 5 alpha,8 alpha-epidioxy-24(R)-methylcholesta-6,22-dien-3 beta-ol.     

Biotransformation of gentiopicroside by asexual mycelia of Cordyceps sinensis

The biotransformation of gentiopicroside by asexual mycelia of Cordyceps sinensis yielded two products, one of which was proved to be a new pyridine monoterpene alkaloid. The possible mechanisms were discussed.     

Activation of peritoneal macrophages by orally administered ether analogues of lysophospholipids.

Oral administration of dodecylglycerol, inflammatory product of cancerous tissues, and the alkyl lysophospholipid derivative, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3-choline), greatly activated mouse peritoneal macrophages. The activation was dose related and was assessed as increased Fc-mediated ingestion of red blood cells, superoxide production, chemiluminescence activity, and incorporation of radioactive thymidine and leucine. Furthermore, the data show that dodecylglycerol or ET-18-OCH3-choline was capable of inducing equally high levels of macrophage activation and cytotoxic action on tumor cells, just as occurs with intraperitoneal administration. Dodecylglycerol appeared to activate the macrophages at a relatively lower dose (5 micrograms/mouse) than ET-18-OCH3-choline (15 micrograms/mouse). The optimal oral doses required to activate macrophages for ingestion and cytotoxic activities were relatively higher than previously observed when these agents were administered intraperitoneally. Thus, the dose difference provided crucial information for correlating oral dosages with in vivo concentration of these agents as bioassayed by macrophage activation. These observations have extended and further support our earlier findings that these agents are effective immunopotentiators and thus could therapeutically be used to activate macrophages for cytotoxic effects on tumor cells via the oral route.     

Production of mycelia and exo-biopolymer from molasses by Cordyceps sinensis 16 in submerged culture

The molecular weight of exo-biopolymer obtained from a submerged culture of Cordyceps sinensis 16 consisted of a main unit and a subunit of 126 and 68 kDa, respectively. The optimal medium for the production of mycelia and exo-biopolymer was determined to be molasses containing 2% sucrose, 0.9% yeast extract, 0.3% K2HPO4, and 0.4% CaCl2. Using optimized medium, maximum productions of mycelia and exo-biopolymer in shake-flask culture were 54.0 g/L and 28.4 g/L, respectively. This study suggests that large-scale production of mycelia and exo-biopolymer by C. sinensis 16 is possible in submerged culture.     

冬虫夏草菌丝体γ-谷氨酰转肽酶的探测和部分性质研究

本研究首次发现冬虫夏草发酵菌丝体含有较高活力的γ-谷氨酰转肽酶（简称CSGT）,并且通过硫酸铵分级沉淀、疏水层析、凝胶过滤层析、阴离子交换层析和制备电泳的提取纯化程序,将CSGT纯化了2300倍,然后对CSGT的基本酶学性质进行了研究。CSGT的稳定pH范围和温度范围分别为pH8-11和0-20℃, 当pH 9-10 、30℃并且以L-谷氨酸-对-硝基苯胺（简称GpNA）和双甘肽为底物时CSGT的活力达到最大值。几种还原剂均能激活CSGT,说明其活性中心含有巯基。Zn2+, Cu2+, Hg2+ , Mn2+ 等金属离子均强烈抑制CSGT活性,而K+, Ca2+, Mg2+ 和Na+等对CSGT活性没有影响。     

Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters

Broiler chicks were orally dosed with a hot-water extract of mycelia from Cordyceps sinensis (CS-HW) to assess possible substitution of Avilamycin as an antibiotic growth promoter (AGP). The growth performance (body weight gain and survivability) and the health index (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the CS-HW (600 mg/kg diet) and the Avilamycin (20 mg/kg diet) fed group in comparison with the control group (p<0.05). The Avilamycin-fed group and the CS-HW-fed group had similar growth performances but the latter gave a better microbial flora in the small intestines. These results indicate that CS-HW enhances the physiological activity in chicks and can be used as a substitute for AGPs.     

The effect of Zn on the Zn accumulation and biosynthesis of amino acids in mycelia of Cordyceps sinensis

Zinc is an essential trace element in the human body and it participates in various pathways of metabolism. Cordyceps sinensis is a wellknown traditional Chinese medicine that contains cordycepin, cordycepic polysaccharides, proteins, vitamins, trace elements, and many other biological active materials. In this study, we cultured C. sinensis in liquid medium containing Zn ions and then analyzed the biomass, the ratios of total Zn accumulation, and the organic Zn content in the mycelia. The results showed that when the media contain 150 mg/L Zn, the biomass of mycelia of C. sinensis reached 10.7 g/L and the Zn content present in the mycelia reached 4875.1 microg/g. The percentage of Zn accumulated in the mycelia was 34.05% of the total Zn in the media, of which the organic Zn accounted for 76.33%. The results also revealed that the content of total amino acid (TAA) and essential amino acid (EAA) in the mycelia were increased substantially TAA and EAA in the Zn-treated mycelia were 11.1% and 15.2% higher, respectively, than that in the mycelia without Zn treatment. These results will be useful for elucidation of the physiological mechanism of Zn effects on the biological metabolism in the mycelia of C. sinensis.     

The effect of Zn on the Zn accumulation and biosynthesis of amino acids in mycelia of Cordyceps sinensis

Zinc is an essential trace element in the human body and it participates in various pathways of metabolism. Cordyceps sinensis is a well-known traditional Chinese medicine that contains cordycepin, cordycepic polysaccharides, proteins, vitamins, trace elements, and many other biological active materials. In this study, we cultured C. sinensis in liquid medium containing Zn ions and then analyzed the biomass, the ratios of total Zn accumulation, and the organic Zn content in the mycelia. The results showed that when the media contain 150 mg/L Zn, the biomass of mycelia of C. sinensis reached 10.7 g/L and the Zn content present in the mycelia reached 4875.1 μg/g. The percentage of Zn accumulated in the mycelia was 34.05% of the total Zn in the media, of which the organic Zn accounted for 76.33%. The results also revealed that the content of total amino acid (TAA) and essential amino acid (EAA) in the mycelia were increased substantially TAA and EAA in the Zn-treated mycelia were 11.1% and 15.2% higher, respectively, than that in the mycelia without Zn treatment. These results will be useful for elucidation of the physiological mechanism of Zn effects on the biological metabolism in the mycelia of C. sinensis.     

Activation of the mammalian immune system by siRNAs

Inhibition of gene expression through RNA interference (RNAi) is emerging as a powerful experimental tool for gene function and target validation studies. The potential uses of this technology seem unlimited, extending to the prevention and therapy of human diseases. However, recent work demonstrating that there are unanticipated, different nonspecific effects associated with the use of small interfering RNAs in mammals has raised concerns about the safe use of RNAi in vivo. These nonspecific effects include activation of the immune system, potentially harming the individual. The application of screening assays for nonspecific activation of both innate and acquired immunity will be necessary for further development of RNAi as a therapeutic tool.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Protection against radiation-induced bone marrow and intestinal injuries by Cordyceps sinensis, a Chinese herbal medicine

Bone marrow and intestinal damage limits the efficacy of radiotherapy for cancer and can result in death if the whole body is exposed to too high a dose, as might be the case in a nuclear accident or terrorist incident. Identification of an effective nontoxic biological radioprotector is therefore a matter of some urgency. In this study, we show that an orally administered hot-water extract from a Chinese herbal medicine, Cordyceps sinensis (CS), protects mice from bone marrow and intestinal injuries after total-body irradiation (TBI). CS increased the median time to death from 13 to 20 days after 8 Gy TBI and from 9 to 18 days after 10 Gy TBI. Although CS-treated mice receiving 10 Gy TBI survived intestinal injury, most died from bone marrow failure, as shown by severe marrow hypoplasia in mice dying between 18 and 24 days. At lower TBI doses of 5.5 and 6.5 Gy, CS protected against bone marrow death, an effect that was confirmed by the finding that white blood cell counts recovered more rapidly. In vitro, CS reduced the levels of free radical species (ROS) within cells, and this is one likely mechanism for the radioprotective effects of CS, although probably not the only one.     

N6-(2-hydroxyethyl)adenosine,a biologically active compound from cultured mycelia of Cordyceps and Isaria species

The water-ethanol extracts of the cultured mycelia of 17 species of Cordyceps and six species of Isaria were each examined for the presence of     

冬虫夏草固态培养菌丝中纤溶酶的纯化和酶学性质

【背景】血栓性疾病发病率逐年递增,发病人群呈现低龄化趋势,严重地影响着人们的身心健康。因此研发高效、安全、特异性强的溶栓药物具有重要的意义,是血栓性疾病预防与治疗研究领域的热点。【目的】对冬虫夏草菌丝固态培养过程中产生的纤溶酶进行分离纯化,并对纯化的纤溶酶进行酶学性质分析。【方法】通过硫酸铵盐析、阳离子交换色谱和Superdex 75凝胶过滤色谱分离纯化虫草纤溶酶。采用Bradford法测定样品中总蛋白质浓度,纤维蛋白平板法测定纤溶酶活性,Native-PAGE检测纯度,SDS-PAGE测定相对分子量。【结果】在固态培养中冬虫夏草菌丝可以产生至少两种纤溶酶,分别命名为OSP-1和OSP-2。纯化后OSP-1比活力达到4186.25U/mg,纯化倍数为41.69倍。OSP-1由两个亚基构成,相对分子量分别为27.60k D和23.83k D,是一种丝氨蛋白酶。酶学性质分析表明,该酶最适作用温度为40°C,最适pH为4.0。Cu2+可促进OSP-1酶活,而Zn2+会抑制酶活。除了具有较高的纤溶酶活性,OSP-1还可发挥激活纤维蛋白酶原的作用。在水解纤维蛋白原的过程中,该酶可依次降解γ、Aα、Bβ链。【结论】研究发现的OSP-1具有开发成新型溶栓药物的潜力。     

蝉虫草子实体与发酵菌丝体胞内多糖成分分析

比较蝉虫草子实体、孢梗束、虫体以及蝉虫草发酵菌丝体的胞内多糖含量、单糖组成和分子量差异,结果表明,蝉虫草子实体与发酵菌丝中胞内多糖的含量和组成均存在差异。葡萄糖、半乳糖和甘露糖是蝉虫草发酵菌丝体和子实体粗多糖中主要的3种单糖,阿拉伯糖和木糖是子实体粗多糖中的特征性单糖,而果糖属于菌丝体的特征性单糖。子实体粗多糖中高分子量组分（>1×10 ⁶Da）的相对含量明显高于菌丝体粗多糖。本研究对提升蝉虫草发酵多糖产品品质、促进蝉虫草开发应用具有参考价值。     

蛹虫草菌丝体水溶性黄色素组分分析及其稳定性研究

本研究以蛹虫草菌丝体为供试材料,采用高效液相色谱对提取的蛹虫草菌丝体水溶性黄色素的组分进行了分离纯化与测定分析,并以其主要组分YP-4为检测指标,采用温度、光照、pH、金属离子、常见食品添加剂等条件对蛹虫草菌丝体水溶性黄色素稳定性的影响进行了较为系统的研究。结果表明,蛹虫草菌丝体水溶性黄色素由5种结构相似的类胡萝卜素组分组成。其中,主要组分YP-4占到WSYPs总量的57.43%,YP-4的MS data [M-H]为521.2657,紫外特征吸收峰为：209nm、239nm、421nm、445nm、474nm,在445nm处有最大吸收峰。以YP-4为检测指标,对提取蛹虫草菌丝体黄色素的稳定性测试结果表明,该类黄色素具有良好的加工稳定性,但在加工过程中应尽量缩减紫外光照射时间,注意酸性添加剂和Fe ³⁺的用量。这一研究结果将为蛹虫草菌丝体水溶性黄色素乃至其他优质天然黄色素的开发利用提供参考依据。     

Blazeispirane and protoblazeispirane derivatives from the cultured mycelia of the fungus Agaricus blazei

Two blazeispirane derivatives including blazeispirols G and I were isolated from the cultured mycelia of the fungus Agaricus blazei Murill and were established to be (20S, 22S, 23R, 24S)-14 beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-triene-11 alpha,23-diol and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-23,28-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A. Furthermore, four blazeispirol derivatives blazeispirols, U, V, V(1) and Z(1) were isolated form the same source described above. Their structures were determined to be (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxyergosta-4,6,8,11-tetraen-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 alpha,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 beta,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxy-4,5-seco-ergosta-6,8-diene-3,5-dione by extensive 1 D and 2D NMR spectral data.