Cordyceps fungi: natural products,pharmacological functions and developmental products

Objectives Parasitic Cordyceps fungi, such as Cordyceps sinensis, is a parasitic complex of fungus and caterpillar, which has been used for medicinal purposes for centuries particularly in China, Japan and other Asian countries. This article gives a general idea of the latest developments in C. sinensis research, with regard to the active chemical components, the pharmacological effects and the research and development of products in recent years. Key findings The common names for preparations include DongChongXiaCao in Chinese, winter worm summer grass in English. It has many bioactive components, such as 3′‐deoxyadenosine, cordycepic acid and Cordyceps polysaccharides. It is commonly used to replenish the kidney and soothe the lung, and for the treatment of fatigue. It also can be used to treat conditions such as night sweating, hyposexuality, hyperglycaemia, hyperlipidaemia, asthenia after severe illness, respiratory disease, renal dysfunction, renal failure, arrhythmias and other heart disease and liver disease. Because of its rarity and outstanding curative effects, several mycelia strains have been isolated from natural Cordyceps and manufactured by fermentation technology, and are commonly sold as health food products. In addition, some substitutes such as C. militaris and adulterants also have been used; therefore, quality control of C. sinensis and its products is very important to ensure their safety and efficacy. Summary Recent research advances in the study of Cordyceps, including Cordyceps mushrooms, chemical components, pharmacological functions and developmental products, has been reviewed and discussed. Developing trends in the field have also been appraised.     

冬虫夏草繁育品质量控制和药理活性研究进展

冬虫夏草为传统的名贵中药材,在中国有悠久的药用历史,具有补肾、益肺、止血、化痰的功效。近年来,冬虫夏草繁育品的研究获得突破,实现了冬虫夏草的大规模产业化繁育。冬虫夏草繁育品基原符合中国药典规定,与野生冬虫夏草相比,冬虫夏草繁育品不仅具有相似的化学成分和药理活性,还具有较低的重金属含量,安全性更容易控制。     

发酵虫草菌粉指纹图谱研究

目的通过对发酵虫草菌粉指纹图谱的深入研究,为发酵虫草菌粉类产品的质量提高提供思路。方法液质联用确定发酵虫草菌粉类产品(百令胶囊)指纹图谱中6个主色谱峰的化学成分;采用中药色谱指纹图谱相似度评价系统,比较天然虫草和不同厂家发酵虫草菌粉指纹图谱相似度及不同干燥方式对指纹图谱的影响。结果百令胶囊指纹图谱中的6个主色谱峰成分为尿苷5-单磷酸、鸟苷酸、5’-腺嘌呤核苷酸、尿苷、鸟苷、腺苷。发酵虫草菌粉的指纹图谱与天然虫草均存在差异,其中百令胶囊的指纹图谱与天然虫草最接近。干燥方式对指纹图谱影响较大,采用沸腾干燥方式对应的指纹图谱主色谱峰的面积比最接近天然虫草晒干方式。结论指纹图谱可准确反映发酵虫草菌粉质量及质量控制方式的有效性。     

Analysis of sterols and fatty acids in natural and cultured Cordyceps by one-step derivatization followed with gas chromatography–mass spectrometry

Ten free fatty acids namely lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, docosanoic acid and lignoceric acid and four free sterols including ergosterol, cholesterol, campesterol and β-sitosterol in natural (wild) Cordyceps sinensis, Cordyceps liangshanensis and Cordyceps gunnii, as well as cultured C. sinensis and Cordyceps militaris were first determined using pressurized liquid extraction (PLE), trimethylsilyl (TMS) derivatization and GC–MS analysis. The conditions such as the amount of reagent, temperature and time for TMS derivatization of analytes were optimized. Under the optimum conditions, all calibration curves showed good linearity within the tested ranges. The intra- and inter-day variations for 14 investigated compounds were less than 3.4% and 5.2%, respectively. The results showed that palmitic acid, linoleic acid, oleic acid, stearic acid and ergosterol are main components in natural and cultured Cordyceps which could be discriminated by hierarchical clustering analysis based on the contents of 14 investigated compounds or the 4 fatty acids, where the contents of palmitic acid and oleic acid in natural Cordyceps are significantly higher than those in the cultured ones.     

Distinction of water-soluble constituents between some Paecilomyces (= Isaria) and Cordyceps fungi by capillary electrophoresis

In order to reveal the differences between Cordyceps, Paecilomyces (= Isaria) and Nomuraea, we collected seven entomogenous fungi grown in natural field and analyzed the profiles of water-soluble constituents derived from some different sources of Cordyceps, Paecilomyces and Nomuraea by determination using capillary electrophoresis. C. sinensis and C. kyushuensis showed similar peak clusters of protein migrated at 5–7, 8–9, and 12–20 min. The peak clusters obtained from N. atypicola was similar to those of C. sinensis and C. kyushuensis. The water-soluble constituent clusters of C. militaris migrated at 5–9 and 10–15 min were partly different from those of other Cordyceps. It was also revealed that the P. tenuipes and P. cicadae showed lesser peak clusters rather than Cordyceps. These results indicated that the profiles of protein of these entomogenous fungi by capillary electrophoretic analysis could serve as fingerprints for classification and medicinal quality control of Cordyceps.     

冬虫夏草人工繁育品、野生品及亚香棒虫草中氨基酸比较

目的：比较冬虫夏草人工繁育品、野生品与亚香棒虫草中水解和游离氨基酸的含量。方法：采用氨基酸分析仪测定冬虫夏草人工繁育品、野生品以及亚香棒虫草中17种氨基酸的含量。结果：冬虫夏草人工繁育品的水解氨基酸含量为16.776%~19.080%,野生冬虫夏草为14.857%~21.959%,亚香棒虫草为13.043%~14.933%。冬虫夏草人工繁育品的游离氨基酸含量为1.767%~2.373%,野生冬虫夏草为1.753%~2.521%,亚香棒虫草为2.856%~3.197%。结论：冬虫夏草人工繁育品和野生品中氨基酸含量基本一致,和亚香棒虫草有显著性差异。本研究为冬虫夏草的鉴别及人工繁育品的进一步开发利用提供科学依据。     

Quality control of Cordyceps sinensis, a valued traditional Chinese medicine

Cordyceps sinensis, a well-known and valued traditional Chinese medicine, is also called DongChongXiaCao (winter worm summer grass) in Chinese. It is commonly used to replenish the kidney and soothe the lung for the treatment of fatigue, night sweating, hyposexualities, hyperglycemia, hyperlipidemia, asthemia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease, and liver disease. As the rarity and upstanding curative effects of natural Cordyceps, several mycelial strains have been isolated from natural Cordyceps and manufactured in large quantities by fermentation technology, and they are commonly sold as health food products in Asia. In addition, some substitutes such as Cordyceps militaris also have been used and adulterants also confused the market. Therefore, quality control of C. sinensis and its products is very important to ensure their safety and efficacy. Herein, markers and analytical methods for quality control of Cordyceps were reviewed and discussed.     

基于网络药理学和分子对接探讨冬虫夏草抗肿瘤的作用机制

目的 基于网络药理学方法和分子对接技术探究冬虫夏草抗肿瘤的作用机制。方法 利用TCMSP、CNKI、PubMed、Drugbank、Stitch和Swiss target prediction等平台检索冬虫夏草的化学成分和作用靶点;通过GeneCards、OMIM等数据库筛选肿瘤相关基因,运用Cytoscape 3.7.2构建冬虫夏草活性成分-靶点网络,通过String数据库对关键靶点构建网络互作(PPI)网络,并进行基因本体(GO)基因和京都基因和基因组百科全书(KEGG)通路富集分析,最后利用AutoDock Vina软件和Pymol软件对药物有效活性成分和关键靶点进行分子对接验证。结果 共得到冬虫夏草22个化合物,86个抗肿瘤共同靶点,主要包括环加氧酶(PTGS)2、丝裂原活化蛋白激酶3(MAPK3)、过氧化物酶体增生激活受体γ(PPARG)、胱天蛋白酶3(CASP3)、JUN基因等关键靶点。GO分析与KEGG通路结果显示,冬虫夏草抗肿瘤涉及到多种生物学过程以及PPAR、花生四烯代谢、5-羟色胺信号通路等多种信号通路。将关键化合物和靶点进行分子对接,提示冬虫夏草抗肿瘤可能的前5个主要活性成分11,14-二十碳二烯酸、花生四烯酸、黄豆黄素、胆甾醇和豆甾醇与关键靶点PTGS2、PTGS1、PGR、HMGCR和CNR1均能自发结合。结论 初步探讨了冬虫夏草抗肿瘤的主要活性成分、相关靶点及相关通路,发现冬虫夏草可以通过多成分、多靶点、多通路抗肿瘤,为后期实验验证提供了参考依据。     

冬虫夏草抗肿瘤及免疫调节作用的研究进展

冬虫夏草为我国传统名贵中药,具有抗肿瘤、降血脂、免疫调节、祛痰、平喘、抗心律失常、保护肝肾功能等多种药理学作用。随着对冬虫夏草免疫机制的深入研究,为其作为抗肿瘤药物提供了有力证据。综述了冬虫夏草及其活性成分对黑色素瘤、甲状腺癌、胃癌、肺癌、乳腺癌等多种肿瘤的抑制作用,主要从吞噬细胞、自然杀伤细胞、树突状细胞、B/T淋巴细胞以及促进细胞因子的分泌等几个方面探讨冬虫夏草对其免疫活性的影响。其免疫调节的作用机制主要通过T淋巴细胞介导的细胞免疫应答和B/T淋巴细胞介导的体液免疫应答。     

Morphological and microscopic identification studies of Cordyceps and its counterfeits

Macroscopic and microscopic studies were applied to distinguish Cordyceps sinensis (Berk.) Sacc. and its 5 common counterfeits. Transverse sections of stroma and larvae and surface sections of stroma of C. sinensis, Cordyceps gunnii, Cordyceps barnesii, Cordyceps gracilis, Cordyceps liangshanensis and Cordyceps militaris were examined and their morphological and microscopic features photographed. The main morphological and microscopic features of the 6 species of Cordyceps were basically similar except for certain diagnostic differences. These included macroscopic differences from C. sinensis as follows: the stroma of C. gunnii is stout and rough with sterile bulgy or branched apex; the larvae of C. barnesii has a pair of teeth on the head; the stroma of C. liangshanensi is thread-like; C. gracilis is without stroma; and C. militaris is without larvae. There were also microscopic differences: from C. sinensis as follows: the stroma of C. barnesii is without perithecia; C. gunnii, C. liangshanensis and C. gracilis are without bristles on the larva body. These differences allow C. sinensis and its counterfeits to be easily distinguished.     

基于代谢组学技术的虫草鉴别研究

目的 采用超高效液相色谱-飞行时间质谱（UPLC-Q-TOF-MS）对不同产地虫草化学成分进行鉴定与比较分析。方法 采用超高效液相-飞行时间质谱（UPLC-Q-TOF-MS）及多元统计分析技术对实验结果进行分析,并对差异化合物进行鉴定。结果 对不同产地的虫草进行了成分鉴定,核苷类物质含量高。不同产地虫草药材在组分种类和相对量上存在着一定的差异。湖南、湖北、丽江的虫草含有其他成分,丽江虫草含有大量的虫草素,湖南虫草含有一些黄酮类物质。采用主成分分析（PCA）将不同产地药材分别聚成不同组别,其中西藏（那曲）、四川、青海之间聚集紧密,而凉山、湖北、湖南及丽江虫草散布在四周,差别非常显著。结论 UPLC-Q-TOF-MS结合多元统计分析方法可为虫草药材的质量评价提供依据。     

何首乌炮制与肝毒性研究进展

何首乌及其炮制品为临床常用中药,对人体有多种有益效果。临床服用何首乌引起肝中毒问题时有报道,但由于何首乌导致肝毒性的机制尚未完全明确,炮制后的何首乌仅被认为是相对安全,目前仍缺乏有效控制何首乌炮制品的科学方法。本文从何首乌的炮制工艺、化学成分、肝毒性、药代动力学等角度汇总分析何首乌研究进展,以探索何首乌质量控制方法和下一步研究思路。     

Enhancement of Neuromuscular Activity by Natural Specimens and Cultured Mycelia of Cordyceps sinensis in Mice

The present study was aimed to evaluate the effect of natural specimen and laboratory cultured mycelia of Cordyceps sinensis on neuromuscular activity in mice. The powder of natural specimen and laboratory cultured Cordyceps sinensis was orally administered at the dose rate of 100, 300 and 500 mg/kg for 30 days. Natural specimen and in vitro propagated Cordyceps sinensis showed significant (P<0.05) enhancement in neuromuscular endurance and antidepressant activity at 300 and 500 mg/kg as compared to the control group. However, the fungus did not proved to be as effective as fluoxetine in exhibiting antidepressant action. Muscular endurance was determined on a Rota rod apparatus while antidepressant (mood elevating) activity was measured on a photoactometer in Swiss albino mice. The effects produced by both natural specimens and laboratory cultured Cordyceps sinensis were comparable and showed almost equal potency.     

Immunomodulatory effect of polysaccharides from submerged cultured Cordyceps gunnii

Context: The genus Cordyceps (Clavicipitaceae) is a group of entomopathogenic fungi that is widely used as tonic food or invigorant with broad-spectrum medicinal properties in China. Cordyceps gunnii (Berk.)Berk (C. gunnii), is also well known as the Chinese rare caterpillar fungus and has similar pharmacological activities with Cordyceps sinensis (C. sinensis). Polysaccharides (PS) from various Cordyceps species have demonstrated many interesting biological activities, including antitumor, immunopotentiation, hypoglycemic, and hypocholesterolemic activities.

Objective: To investigate the effect of C. gunnii PS on the immunostimulatory antitumor function and expression of immune related cytokines in normal, immuno-suppressive, and H22-bearing mice, respectively. Methods: C. gunnii PS were extracted with hot water at 80°C for 2 h. Normal, immuno-suppressive, and H22-bearing mice were treated with PS respectively. By detecting the value of macrophage phagocytic index, proliferation of lymphocytes, natural killer (NK) cell activity and expression of related cytokines, interleukin (IL-4), tumor necrosis factor-α (TNF-a) and interferon-γ (IFN-γ), and tumor inhibition index in H22-bearing mice additionally, the effect of PS on immunostimulatory antitumor function and its mechanism were studied.

Results: The total sugar content of the PS was determined to be 95% after purification. PS markedly increased the thymus and spleen indexes, the macrophage phagocytosis, the proliferation of splenic cells, and the level of IFN-γ and TNF-α. In tumor growth inhibition test, PS showed remarkable inhibition effects.

Conclusion: PS from the C. gunnii could enhance nonspecific immunological function, humoral immunity, cellular immunity in mice, and inhibit tumor growth.     

桑枝的化学成分及其药理活性研究进展

桑枝作为应用广泛的传统中药,其化合物类型丰富,主要含有黄酮类、生物碱、香豆素类、芪类和三萜类等成分,还含有氨基酸、有机酸、挥发油及多种维生素等。现代药理学研究表明桑枝具有镇痛、抗炎、降血糖、抗氧化等多种药理作用。笔者归纳总结了近年来相关文献,对桑枝中的化学成分和药理作用进行简要综述,并为其进一步的质量控制及开发利用提供参考。     

蛹虫草菌丝体多糖与子实体多糖体外免疫调节活性的研究

目的 分析蛹虫草菌丝体多糖（mycelia polysaccharides of Cordyceps militaris,MPS）与蛹虫草子实体多糖（polysaccharides from fruiting body of Cordyceps militaris,FPS）的体外免疫活性。方法 以RAW264.7细胞株为受试对象,从细胞增殖、中性红吞噬能力、NO释放水平角度系统考察不同浓度的MPS与FPS的免疫调节活性,并分析其量化关系。结果 MPS与FPS均可明显促进RAW264.7细胞株的增殖、对中性红的吞噬能力和NO释放水平,且均呈现出浓度依赖性的特征。二者对RAW264.7细胞株增殖的促进作用无显著性差异,但在免疫调节最佳作用浓度方面却存在一定差异,MPS在100μg·mL^-1时表现出最好的细胞吞噬能力,而FPS在200μg·mL^-1时表现出最高的细胞吞噬能力。结论 MPS与FPS均具有显著的体外免疫活性。     

冬虫夏草菌丝体水提醇沉物体外抗肿瘤活性研究

目的 研究冬虫夏草菌丝体水提醇沉物的体外抗肿瘤活性及其机制。方法 采用热水浸提-醇沉法得到水提物,高效液相色谱仪测定水提醇沉物中腺苷的量;采用MTT法测定其抗肿瘤活性,利用流式细胞仪结合碘化丙锭染色法检测其对细胞周期的抑制。结果 实验表明水提醇沉物能抑制人肝癌HepG2细胞株及大细胞肺癌NCI-H460细胞株的增殖,并呈浓度相关性;半数抑制浓度（IC₅₀）值分别为（1.49±0.19）和（1.67±0.27）mg/mL。细胞周期分析表明,水提醇沉物分别阻滞HepG2及NCI-H460细胞周期于G₂/M期、S期,并可诱导上述两种细胞发生凋亡。结论 冬虫夏草水提醇沉物通过阻滞HepG2及NCI-H460细胞周期循环,诱导其凋亡,从而表现出良好的增殖抑制活性。为深入研究冬虫夏草菌丝体水提醇沉物抗肿瘤的机制提供了实验证据。     

冬虫夏草繁育品中重金属及有害元素分布特征研究及风险评估

目的：对冬虫夏草繁育品干品和鲜品中重金属及有害元素分布特征进行研究，以期为无公害冬虫夏草的繁育提供技术支持。方法：采用微波消解法对干品和鲜品样品进行有机破坏处理，以ICP-MS法为检测方法，对干品和鲜品共30批次的样品中铅(Pb)、镉(Cd)、砷(As)、汞(Hg)、铜(Cu)5种重金属及有害元素进行测定，结合化学计量学对测定结果的含量特征进行分析；结合课题组前期建立的风险评估方法(HI或MOE法)从食用及药用两方面对分析结果进行风险评估。结果：分析过程中各元素在线性范围内线性关系良好，进样精密度RSD在0.34%～0.79%之间，各元素的回收率在93.0%～103.0%之间。30批次样品中，Cu、Cd全部检出，含量范围分别是2.1～8.98 mg·kg^-1和0.02～0.25 mg·kg^-1;As有26批检出，含量范围是0.10～0.48 mg·kg^-1;Pb有16批检出，含量范围是0.10～0.50 mg·kg^-1;Hg仅有4批检出，含量范围是0.02～0.05 mg·kg^-1     

Comprehensive quality evaluation and comparison of Angelica sinensis radix and Angelica acutiloba radix by integrated metabolomics and glycomics

Angelica radix (Danggui in Chinese) used in China and Japan is derived from two species of Angelica, namely Angelica sinensis and Angelica acutiloba, respectively. The differences in quality between A. sinensis radix (ASR) and A. acutiloba radix (AAR) should be therefore investigated to guide the medicinal and dietary applications of these two species. Secondary metabolites and carbohydrates have been demonstrated to be the two major kinds of bioactive components of Danggui. However, previously, quality comparison between ASR and AAR intensively concerned secondary metabolites but largely overlooked carbohydrates, thus failing to include or take into consideration an important aspect of the holistic quality of Danggui. In this study, untargeted/targeted metabolomics and glycomics were integrated by multiple chromatography-based analytical techniques for qualitative and quantitative characterization of secondary metabolites and carbohydrates in Danggui so as to comprehensively evaluate and compare the quality of ASR and AAR. The results revealed that not only secondary metabolites but also carbohydrates in ASR and AAR were different in type and amount, which should collectively contribute to their quality difference. By providing more comprehensive chemical information, the research results highlighted the need to assess characteristics of both carbohydrates and secondary metabolites for overall quality evaluation and comparison of ASR and AAR.     

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.