优良性状蚕蛹虫草的筛选及高产虫草素液态发酵条件优化

刘朋肖,马婕馨,刘警鞠,赵国柱,何湘伟

菌物学报 ›› 2021, Vol. 40 ›› Issue (11) : 3046-3057.

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菌物学报 ›› 2021, Vol. 40 ›› Issue (11) : 3046-3057. DOI: 10.13346/j.mycosystema.210114 CSTR: 32115.14.j.mycosystema.210114
研究论文

优良性状蚕蛹虫草的筛选及高产虫草素液态发酵条件优化

作者信息 +

Screening of Cordyceps militaris with excellent traits and optimization of liquid fermentation conditions for highly yielding cordycepin

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文章历史 +

摘要

蛹虫草是重要的食药用真菌,虫草素为其主要活性成分,在抗肿瘤、抗菌、降血糖等方面具有较为突出的功效。蛹虫草菌株间的形态及环境条件差异,对菌株次级代谢产物虫草素产生影响显著。本研究对不同来源的6株蛹虫草菌株（YCC-B、YCC-C、YCC-H、YCC-W、YCC-Y、CGMCC 3.4655）,从蚕蛹体培养子实体性状,液体发酵条件（培养天数、培养方式、外源金属离子等）和传代稳定性等方面筛选优良性状菌株,提高其发酵合成虫草素的能力及稳定性。结果表明,蛹虫草菌株YCC-W在蚕蛹子实体出草及菌体液体发酵产虫草素上综合表现优良,传代稳定;液体发酵培养基中添加外源金属离子Mn2+作为酶的辅基,可以促进虫草素合成;采用振荡-静置相结合的混合发酵培养方式,可以避免单纯振荡培养溶氧量大、菌丝体生长旺盛,而虫草素产生不佳的问题。先振荡培养3d后静置培养至25d时,菌株YCC-W合成虫草素含量最高,可达(874.13±24.25)μg/mL,且稳定性良好。为进一步开发菌种及扩大规模生产提供参考。

Abstract

Cordyceps militaris is an important edible and medicinal fungus, having active ingredient with outstanding effects in anti-tumor, anti-bacteria, reducing blood sugar and other functions. The differences of morphological characters and growth environment of C. militaris strains significantly influence the production of secondary metabolites. In this study, six strains of C. militaris (YCC-B, YCC-C, YCC-H, YCC-W, YCC-Y and CGMCC 3.4655) from different sources were selected based on properties of stromata on tussah pupae, liquid fermentation conditions (culture duration, culture methods, exogenous metal ions, etc.) and reproduction stability for screening the most excellent strain to improve ability and stability of fermentation and synthesis of cordycepin. The results showed that C. militaris YCC-W fruiting body properties performed well on tussah pupae and cordycepin production was promoted in liquid fermentation, showing stable reproduction in subculture. The synthesis of cordycepin was greatly improved by adding the metal ion Mn2+ to the liquid fermentation medium as a coenzyme. The mixed culture method (shaking + standing) can avoid the poor production of cordycepin caused by the strong growth of mycelium with large dissolved oxygen in the simple shaking culture. On the conditions of 3d shake culture proceeded with 22d static culture, production of cordycepin of strain YCC-W reached the highest yield of (874.13±24.25)μg/mL. The results provide reference for further development of the strains and expanding cordycepin production.

关键词

金属离子 / 活性产物 / 腺苷 / 传代培养

Key words

metal ions / active product / adenosine / subculture

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刘朋肖, 马婕馨, 刘警鞠, 赵国柱, 何湘伟. 优良性状蚕蛹虫草的筛选及高产虫草素液态发酵条件优化[J]. 菌物学报, 2021, 40(11): 3046-3057 https://doi.org/10.13346/j.mycosystema.210114
LIU Peng-Xiao, MA Jie-Xin, LIU Jing-Ju, ZHAO Guo-Zhu, HE Xiang-Wei. Screening of Cordyceps militaris with excellent traits and optimization of liquid fermentation conditions for highly yielding cordycepin[J]. Mycosystema, 2021, 40(11): 3046-3057 https://doi.org/10.13346/j.mycosystema.210114
灵芝Ganoderma spp.是担子菌门Basidiomycota、伞菌纲Agaricomycetes、多孔菌目Polyporales、灵芝科Ganodermataceae、灵芝属Ganoderma 真菌(邢佳慧 2019)。萜类化合物是灵芝中一类重要的活性物质,近年来对这类物质的药理活性特别是对抗肿瘤活性的研究,是当代灵芝研究的一个热点(林志彬 2015;陈若芸和康洁 2016)。
灵芝属的不同种中,含有不同类型的萜类化合物。除了多环杂萜、萜烯等类型的萜类化合物外,羊毛甾烷型四环三萜是一类在灵芝属各个种中普遍存在的活性物质(Lin & Yang 2019)。此外,灵芝有子实体、菌丝体和孢子粉3种形态,不同形态的灵芝也会含有不同的萜类物质。赤芝Ganoderma lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai 中的萜类大多为羊毛甾烷型四环三萜,其中以其子实体中三萜种类最多且含量较高,菌丝体中三萜种类少且不同于子实体,孢子粉中三萜种类和含量最少(Ma et al. 2011;于华峥等 2016;崔宝凯和吴声华 2020;戴玉成等 2021)。
前期研究中,虽然发现赤芝子实体的中性三萜组分和菌丝体三萜抗肿瘤活性较好(唐庆九等 2010;Liu et al. 2012;岳亚文等 2020),也有报道将灵芝子实体中26个三萜化合物做了体外活性比较并预测了其作用靶点(杜国华等 2017),但这些研究并不是在同一实验条件下进行,缺乏系统的比较,其结论仍无法对灵芝三萜的抗肿瘤能力和构效关系作出确切说明。
通过计算机辅助发现药物的作用靶点,对确定化合物的靶点以及构效关系的研究具有提示作用。其中,分子对接是研究分子间(如配体和受体)相互作用的一种理论模拟方法(Ferreira et al. 2015),当今很多学者通过分子对接技术筛选抗肿瘤靶点和小分子抑制剂(顾勇亮等 2015;王樟根 2017;Chen et al. 2017)。鉴于目前灵芝三萜类化合物的构效关系研究中所选化合物过少、类型不全面的现状,本研究在前期制备了赤芝Ganoderma lingzhi子实体、菌丝体和层迭灵芝Ganoderma lobatum (Cooke) G.F. Atk.子实体中多种类型的羊毛甾烷型三萜化合物的基础上(岳亚文等 2020;彭小芳等 2021),对灵芝酸、烯酸、酮、醛、醇以及环氧酸类三萜进行了体外抗肿瘤活性的比较,总结具有抗肿瘤活性潜力的羊毛甾烷型三萜的结构特征并进一步通过计算机分子对接技术探索其活性位点,为高活性灵芝三萜化合物的寻找和结构改造提供参考。

1 材料与方法

1.1 材料、试剂和仪器

1.1.1 实验材料

45个羊毛甾烷型三萜化合物购自国家标准物质中心或自制,纯度≥98%。L1210：小鼠白血病细胞株,购自中国科学院上海生命科学研究院细胞资源中心。

1.1.2 主要试剂

胎牛血清(fetal bovine serum,FBS)、RPMI1640培养基、1%双抗(青霉素和链霉素)、磷酸缓冲盐溶液(phosphate buffered saline, PBS) (Gibco公司);5-氟尿嘧啶[3H)-pyrimidinedione,5FU]、二甲基亚砜(dimethyl sulfoxide,DMSO) (Sigma公司);Alamar Blue试剂(Biosource公司)。

1.1.3 主要仪器

生物安全柜、二氧化碳培养箱、超低温冰箱(Thermo公司);高压灭菌锅(TOMY公司);恒温水浴锅(上海一恒科技有限公司);多功能倒置荧光显微镜(Olympus公司);多功能酶标仪(Bio-Tek公司);细胞计数仪(Beckman-Coulter公司);台式高速大容量离心机(Eppendorf公司);漩涡混合器(上海精科实业有限公司)。

1.2 方法

1.2.1 样品溶液的制备

将三萜化合物用DMSO配制成20 mg/mL的母液,再用DMSO逐级稀释为1、4和10 mg/mL的样品工作液。将5FU用DMSO配制成10 mg/mL的溶液,作为阳性对照备用。

1.2.2 抗肿瘤活性测定

将L1210细胞置于RPMI 1640 (含1%双抗和10% FBS,下同)中,于5% CO2、37 ℃培养箱内培养传代。取对数生长期的L1210细胞,用RPMI 1640培养基稀释成2×104个/mL细胞悬液,按200 μL/孔接种于96孔板中,再分别加入1 μL样品溶液、阴性对照(DMSO)和阳性对照(5FU),每组设置3个生物重复。将96孔板置于二氧化碳培养箱内培养72 h取出,用多功能酶标仪在570 nm、600 nm处测定其吸光度(A)值作为初始值,然后加入30 μL 0.1 mg/mL的Alamar Blue试剂,再放入培养箱培养4 h左右,取出再次测定其吸光度(A)值。参照冯娜等(2010)的方法,依据增殖抑制率计算公式计算各样品对肿瘤细胞L1210的增殖抑制率：
细胞抑制率(%)= 1−[117 216×A570(sample)−80 586×A600(sample)][117 216×A570(control)−80 586×A600(control)]

1.2.3 小分子配体的准备

应用画图软件ChemDraw画出三萜化合物的结构,导入软件Discovery Studio 2016 (DS2016)中。考虑到配体数目、对映异构体和pH等因素,采用DS2016中的Prepare Ligand处理该体系,通过此操作给小分子配体进行加氢,产生三维结构和异构体。导入的12个三萜化合物经处理后产生76个用于对接的配体分子。

1.2.4 蛋白受体的准备

研究所用受体蛋白及功能见表1,其晶体结构均源自RCSB PDB蛋白质数据库(https://www. rcsb.org/)。对导出的蛋白晶体进行前处理,删除水分、配体分子,通过Clean Protein模块去除蛋白多构象、补充非完整的氨基酸残基、为蛋白添加极性氢、给蛋白施加 CHARMm力场进行能量优化,最后选择From receptor cavities定义活性位点,将准备好的蛋白保存备用。
表1 分子对接所使用的受体

Table 1 Receptors used for molecular docking

PDB ID 缩写
Abbreviation		 名称
Name		 功能
Function
2XOW p53 p53蛋白
p53 protein		 防止癌变,修复缺陷
Prevent cancer and repair defects
IYSI Bcl-xl 抗凋亡蛋白
Anti-apoptotic proteins		 阻止凋亡
Prevent apoptosis
5HG5 EGFR 表皮生长因子受体
Epidermal growth factor receptor		 加速促进细胞异常生长和分裂,最终导致肿瘤诞生
Accelerate the abnormal growth and division of cells, and eventually lead to the birth of tumors
1M48 IL-2 白细胞介素-2
Interleukin-2		 促进淋巴细胞生长、增殖、分化,能诱导和增强细胞毒活性
Promote the growth, proliferation and differentiation of lymphocyte, induce and enhance cytotoxic activity
1Y6B VEGFR2 血管内皮细胞生长因子受体2
Vascular endothelial growth factor receptor 2		 调节淋巴管内皮细胞和血管内皮细胞,促进淋巴管和血管的生成,还有调节淋巴细胞的迁移等作用
Regulate lymphatic endothelial cells and vascular endothelial cells, promote the production of lymphatic vessels and blood vessels, and regulate the migration of lymphocytes, etc.

1.2.5 分子对接

以三萜化合物为对接配体,以p53、Bcl-xl、EGFR、IL-2和VEGFR2蛋白为对接受体,利用DS2016软件的LibDock模块进行基于结构的配 体-蛋白分子对接,修改对接参数为Conformation Preferences：User Specified,其他参数均为默认值。对接结果以Libdock Score打分函数列出。Libdock Score越高,预测配体与受体蛋白结合的活性越高。另外通过DS2016软件分析对接结果中的配体与受体蛋白之间相互作用类别,找到配体与氨基酸残基对接的主要活性位点。

2 结果与分析

2.1 三萜化合物的抗肿瘤活性

45个三萜化合物的结构通式见图1。根据化合物的来源不同,可分为赤芝子实体酸性三萜、赤芝子实体中性三萜、赤芝菌丝体三萜和层迭灵芝子实体三萜。这些化合物的结构及对小鼠白血病细胞L1210增殖的半数抑制浓度结果分别见表2-表5。阳性对照5FU的增殖抑制率是90.83%。结果表明,在赤芝子实体酸性三萜化合物中,ganoderic acid I、ganoderic acid D的活性较强,其增殖抑制的半数抑制浓度(IC50)值分别为39.54 μmol/L和3.67 μmol/L;其他酸性三萜化合物对L1210的IC50均在50 μmol/L以上;ganoderic acid ε、ganoderenic acid C、ganoderic acid N和ganoderic acid Y在受试浓度范围内甚至未表现出抑制L1210增殖的作用。灵芝子实体中性三萜中,ganoderiol A和ganoderal A的活性较好,其IC50分别为30.30 μmol/L和28.44 μmol/L。灵芝菌丝体中的8个三萜化合物均表现出较强的抑制L1210细胞增殖的能力,IC50均在50 μmol/L以下,其中ganoderic acid T的活性最强,其对L1210的IC50仅为1.92 μmol/L。层迭灵芝中6个树舌环氧酸类三萜化合物对L1210细胞增殖抑制的能力则普遍较差,除applanoxidic acid E外,其他树舌环氧酸类三萜对L1210细胞株的IC50均在150 μmol/L以上。该结论与有关文献报道的结果相吻合(刘如明 2012;唐庆九等 2010;Shao et al. 2020;岳亚文等2020)。
图1 不同类型羊毛甾烷型三萜化合物的结构通式

Fig. 1 General structural formula of lanostane triterpenes with different types.

Full size|PPT slide

表2 灵芝子实体酸性三萜化合物对L1210细胞增殖的抑制作用

Table 2 Inhibition of acidic triterpenes from fruiting bodies of Ganoderma lingzhi to L1210 cell proliferation

灵芝子实体酸性三萜
Acidic triterpenes from fruiting
bodies of G. lingzhi		 母环
Female ring		 R1 R2 R3 R4 R5 R6 R7 IC50
(μmol/L)
Ganoderic acid I A β-OH β-OH =O -H =O β-OH =O 39.54
Ganoderic acid ε A β-OH β-OH =O -H =O -H β-OH -
Ganoderenic acid C B β-OH β-OH =O -H α-OH =O - -
Ganoderic acid C2 A β-OH β-OH =O -H α-OH -H =O 520.54
Ganoderic acid C6 A β-OH =O =O β-OH =O -H =O 2 793.27
Ganoderic acid G A β-OH β-OH =O β-OH =O -H =O 58.26
Ganoderic acid B A β-OH β-OH =O -H =O -H =O 77.32
Ganoderenic acid B B β-OH β-OH =O -H =O =O - 58.81
Ganoderenic acid A B =O β-OH =O -H α-OH =O - 351.85
Ganoderic acid A A =O β-OH =O -H α-OH -H =O 104.19
Ganoderic acid K A β-OH β-OH =O β-OAc =O -H =O 116.97
Ganoderenic acid E B =O β-OH =O β-OH =O =O 135.46
Ganoderic acid H A β-OH =O =O β-OH =O -H =O 417.07
Ganoderenic acid H B β-OH =O =O -H =O =O - 108.75
Lucidenic acid A - - - - - - - - 103.41
Ganoderic acid N A =O β-OH =O -H =O -H =O -
Ganoderic acid D A =O β-OH =O -H =O -H =O 3.67
Ganoderenic acid D B =O β-OH =O -H =O =O 27 094.48
Ganoderic acid Z C β-OH =O =O -H -H 2 289.44
Ganoderic acid F A =O =O =O β-OAc =O -H =O 265.35
Ganoderenic acid F B =O =O =O -H =O =O - 72.91
Ganoderic acid DM C =O =O -H -H -H - - 75.48
Ganoderic acid Y D β-OH -H - - - - - -
Ganoderic acid TN D β-OH β-OAc - - - - - 57.75
注：“-”表示在受试浓度下化合物对L1210无作用,下同
Note: “-” indicates that the compound has no effect on L1210 at the tested concentration, the same below.
表3 灵芝子实体中性三萜化合物对L1210细胞增殖的抑制作用

Table 3 Inhibition of neutral triterpenes from fruiting bodies of Ganoderma lingzhi to L1210 cell proliferation

灵芝子实体中性三萜
Neutral triterpenes from fruiting bodies of G. lingzhi		 母环
Female ring		 R1 R2 R3 R4 IC50
(μmol/L)
Ganodermanontriol F =O α-OH -CH2OH β-OH 51.05
Ganoderiol A F β-OH -OH -CH2OH -OH 30.30
Ganodermanondiol F =O α-OH -CH3 -OH 137.38
Ganoderiol F E =O -CH2OH -CH2OH - 147.92
Ganoderol A E =O -CH3 -CH2OH - 65.21
Ganoderal A E =O -CH3 -CHO - 28.44
Ganoderol B E β-OH -CH3 -CH2OH - 54.28
表4 灵芝菌丝体三萜化合物对L1210细胞增殖的抑制作用

Table 4 Inhibition of triterpenes from mycelia of Ganoderma lingzhi to L1210 cell proliferation

灵芝菌丝体三萜
Mycelial triterpenes		 母环
Female ring		 R1 R2 R3 IC50
(μmol/L)
Ganoderic acid T G α-OAc α-OAc β-OAc 1.92
Ganoderic acid S G α-OH -H β-OAc 19.33
Ganoderic acid P G α-OH α-OAc β-OAc 26.66
Ganoderic acid T1 G α-OAc α-OAc β-OH 21.12
Ganoderic acid Mk G α-OAc α-OH β-OAc 16.71
Ganoderic acid Me G α-OAc α-OAc -H 9.66
Lanosta-7,9(11),24-trien-3α-hydroxy-26-oic acid G α-OH -H -H 29.97
Ganoderic acid R G α-OAc -H β-OAc 31.69
表5 层迭树舌子实体三萜化合物对L1210细胞增殖的抑制作用

Table 5 Inhibition of triterpenes from fruiting bodies of Ganoderma lobatum to L1210 cell proliferation

树舌环氧酸三萜
Applanoxidic acids		 母环
Female ring		 R1 R2 R3 R4 IC50
(μmol/L)
Applanoxidic acid H H β-OH α-OH =O -OH 10 083.46
Applanoxidic acid A I =O =O α-OH - 272.52
Applanoxidic acid G H =O =O β-OH -OH 3 477.67
Applanoxidic acid C H =O =O =O -OH 1 507.38
Applanoxidic acid E I =O =O β-OH - 84.64
Applanoxidic acid F I =O =O =O - 166.01

2.2 三萜化合物与蛋白对接

p53、Bcl-xl、EGFR、IL-2和VEGFR2均是与抗肿瘤作用相关的靶点蛋白,选择这些蛋白作为受体,与实验结果中IC50值小于50 μmol/L的12个三萜化合物产生的76个配体进行分子对接。对接Poses以及LibDock打分结果见 表6。由对接结果可知,除ganoderic acid Mk、ganoderic acid_I、ganoderic acid D不能与靶蛋白EGFR对接外,其他三萜化合物均可以与5种靶点蛋白对接,说明灵芝三萜抑制肿瘤的过程中存在多靶点作用。在这些化合物的对接模拟中,灵芝菌丝体来源的三萜均表现优异。其中,ganoderic acid T的表现最好,其与靶点蛋白p53和Bcl-xl对接位点的数目及对接得分均排在第一位,表明ganoderic acid T与抗肿瘤作用靶点有较强的结合能力。为进一步观察配体分子与靶蛋白发生的相互作用,将每个靶蛋白对应打分最高的配体的对接结果以二维平面图展示出来做进一步分析。
表6 配体与靶蛋白对接

Table 6 Docking of ligand to target protein

配体
Ligand		 p53 Bcl-xl EGFR IL-2 VEGFR2
Poses Score Poses Score Poses Score Poses Score Poses Score
Ganoderic acid P 10 115.52 10 142.42 5 123.16 5 118.39 10 133.25
Ganoderma acid T1 3 106.89 10 140.52 22 111.43 1 102.88 10 135.49
Ganoderic acid S 6 113.64 10 131.19 3 119.55 10 145.95 10 123.33
Ganoderic acid T 10 119.76 10 152.44 4 132.70 2 116.23 10 126.43
Ganoderic acid Me 10 112.97 10 141.92 3 105.81 10 115.93 10 131.55
Ganoderic acid R 10 112.53 10 142.54 2 121.23 10 142.28 10 125.44
Ganoderic acid Mk 2 104.29 10 136.51 - - 9 127.94 10 127.44
Lanosta-7,9(11),24-trien-3α-hydroxy-26-oic acid 2 108.04 10 129.37 2 114.60 10 131.04 10 118.92
Ganoderic acid I 2 113.87 10 134.51 - - 9 119.06 5 105.76
Ganoderic acid D 2 106.48 10 140.06 - - 10 123.40 10 111.06
Ganoderiol A 1 101.96 10 141.78 2 122.61 10 122.90 10 116.80
Ganodera A 1 110.40 10 129.12 3 104.15 10 129.88 10 107.94
注：化合物的众多对接Poses中只给出LibDock打分最高值
Note: Only the highest LibDock score is given among many poses.
在对接模拟研究中,靶蛋白与配体小分子之间存在氢键、烷基化和盐桥等相互作用时有利于提高对接活性(金海晓 2006)。从本研究的对接结果(图2)可知,ganoderic acid T、ganoderic acid S和ganoderic acid T1结构上的乙酰基、羟基、末端羧基与多个氨基酸残基存在氢键相互作用,结构中的烷基存在烷基化作用,这些相互作用对于这些三萜化合物具有很强的抗肿瘤活性可能起到了决定性的影响。
Ganoderic acid T与Bcl-xl的对接打分最高(表6),分析其结果(图2B)发现,靶点蛋白Bcl-xl上的氨基酸残基与ganoderic acid T的乙酰基和末端羧基存在氢键相互作用,包括ALA93与C-3位上的羰基氧原子,PHE101、ARG104与C-15位上的羰基氧原子,TYR105与C-22位上羰基氧原子,GLY142与C-22位上酯基氧原子,ARG143与末端羟基氧原子、羰基氧原子。氨基酸残基ALA97、PH195、VAL145、ALA146还与ganoderic acid T之间存在多个π键-烷基化和烷基化作用。此外,氨基酸残基ARG143还与该化合物侧链末端羧基形成盐桥相互作用。由于众多氨基酸残基与ganoderic acid T的相互作用,使得ganoderic acid T配体与受体蛋白的结合活性最强。
图2 灵芝羊毛甾烷型三萜与靶蛋白分子对接

A：Ganoderic acid T与p53蛋白对接;B：Ganoderic acid T与Bcl-xl蛋白对接;C：Ganoderic acid T与EGFR蛋白对接;D：Ganoderic acid S与IL-2蛋白对接;E：Ganoderic acid T1与VEGFR2蛋白对接

Fig. 2 Molecular docking of lanostane triterpenes from Ganoderma spp. to target proteins.

A: Docking of ganoderic acid T to p53 protein; B: Docking of ganoderic acid T to Bcl-xl protein; C: Docking of ganoderic acid T to EGFR protein; D: Docking of ganoderic acid S to IL-2 protein; E: Docking of ganoderic acid T1 to VEGFR2 protein.

Full size|PPT slide

3 讨论

刘如明(2012)通过研究ganoderic acid T、Mk、T1和T2 4种灵芝酸对Hela细胞的细胞毒性及诱导细胞凋亡的能力发现,乙酰化程度越高的灵芝酸,其细胞毒性及诱导肿瘤细胞凋亡的能力越强,并且ganoderic acid T可以通过靶向p53蛋白抑制癌细胞侵袭、增殖(Chen & Zhong 2011;唐文等 2015)。本研究体外活性实验结果表明,具有3个乙酰氧基的ganoderic acid T对L1210细胞毒性最强,具有2个乙酰氧基的ganoderic acid P次之,只有1个乙酰氧基的ganoderic acid S较弱,这一结果与此前的研究结论一致。本研究进一步通过计算机虚拟对接的方式,将对L1210细胞增殖抑制能力较强的12个灵芝三萜化合物与p53、Bcl-xl、EGFR、IL-2和VEGFR2 (表达异常时会促进肿瘤增殖、迁移)抗肿瘤靶蛋白进行分子对接,结果显示灵芝羊毛甾烷型三萜化合物结构中的乙酰基、羟基、羧基可以与受体蛋白活性口袋里的氨基酸残基之间形成氢键、烷基化(疏水作用)等相互作用,证明了乙酰基、羟基以及末端羧基是该类三萜化合物发挥药效的重要官能团。本研究在定义对接活性位点时选择了From receptor cavities这一选项,因此推测当灵芝三萜化合物与受体腔中的氨基酸残基存在相互作用时,这些三萜化合物对靶蛋白的抑制类型是竞争性抑制。此外,分子对接的结果显示,三萜化合物与氨基酸残基主要通过氢键、具有疏水作用的烷基化等非共价键来发生作用,由此,推测这些三萜化合物对靶蛋白的抑制作用是可逆的。
有研究认为,ganoderic acid A 和ganoderic acid H通过抑制转录因子AP-1和NF-κB从而抑制乳腺癌细胞MDA-MB-231的生长与侵袭行为,推测这一活性与三萜羊毛甾烷结构中C-3、C-7和C-15位上的羟基有关(Jiang et al. 2008)。本研究的体外实验结果表明,在以上3个位点有羟基的化合物,例如ganoderic acid ε、ganoderenic acid C和ganoderic acid N,对小鼠白血病细胞L1210增殖抑制的能力并不显著。因此,关于C-3、C-7和C-15位上羟基的构效关系值得继续探讨。
本研究通过细胞增殖抑制率测定实验对比研究发现,子实体中的三萜醇ganodermanontriol、ganoderal A和ganoderiol A等同菌丝体中的三萜酸ganoderic acid T、ganoderic acid T1和ganoderic acid P一样,母环上都具有△7,8、△9,11共轭双键且抗肿瘤活性突出,因此母环上共轭双键的存在可能有助于提高化合物的抗肿瘤活性。Cheng et al. (2010)研究结果也表明,双键位于7位和9位的化合物比具有7-酮-8-烯结构的化合物表现出更高的活性。此外,这些活性突出的三萜醇类化合物,支链末端的羟基是否与其活性有关,值得进一步探讨。

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析
[1]
Chen LB, Wu GX, Cheng W, Fan XZ, Shi DF, Shi M, Gao H, 2016. Extraction, purification, and antitumor activity of cordycepin from Cordyceps militaris residue medium. Journal of Food Science and Technology, 34(4):73-79 (in Chinese)
[2]
Cui L, Dong MS, Chen XH, Jiang M, Lv X, Yan GJ, 2008. A novel fibrinolytic enzyme from Cordyceps militaris, a Chinese traditional medicinal mushroom. World Journal of Microbiology and Biotechnology, 24(4):483-489
https://doi.org/10.1007/s11274-007-9497-1
http://link.springer.com/10.1007/s11274-007-9497-1
本文引用 [1]
[3]
Cunningham KG, Manson W, Spring FS, Hutchinson SA, 1950. Cordycepin, a metabolic product isolated from cultures of Cordyceps militaris. Nature, 166(4231):949-949
本文引用 [1]
[4]
Das SK, Masuda M, Sakurai A, Kakibara MS, 2010. Medicinal uses of the mushroom Cordyceps militaris: current state and prospects. Fitoterapia, 81(8):961-968
https://doi.org/10.1016/j.fitote.2010.07.010
https://linkinghub.elsevier.com/retrieve/pii/S0367326X10001863
本文引用 [2]
[5]
Dong HH, Li JJ, Zhou X, Jiang YX, 2016. Research progress on extraction and pharmacological activity of cordycepin from Cordyceps militaris. Journal of Jilin Medical University, 37(6):465-467 (in Chinese)
[6]
Fan DD, Wang W, Zhang JJ, 2012. Enhancement of cordycepin production in submerged cultures of Cordyceps militaris by addition of ferrous sulfate. Biochemical Engineering Journal, 60:30-35
https://doi.org/10.1016/j.bej.2011.09.014
https://linkinghub.elsevier.com/retrieve/pii/S1369703X11002592
本文引用 [1]
[7]
Hung YP, Wang JJ, Wei BL, Lee CL, 2015. Effect of the salts of deep ocean water on the production of cordycepin and adenosine of Cordyceps militaris-fermented product. AMB Express, 5(1):53-62
https://doi.org/10.1186/s13568-015-0140-5
http://www.amb-express.com/content/5/1/53
本文引用 [1]
[8]
Lennon MB, Suhadolnik RJ, 1976. Biosynthesis of 3’-deoxyadenosine by Cordyceps militaris: mechanism of reduction. Biochimica et Biophysica Acta, 425:532-536
https://www.ncbi.nlm.nih.gov/pubmed/1083247
本文引用 [1] 摘要
The biosynthesis of 3'-deoxyadenosine (cordycepin) by Cordyceps militaris has been investigated using [U-14C]adenosine and [3-3H]ribose. Crystallization of the resulting radioactive 3'-deoxyadenosine to a constant specific activity showed incorporation of both labeled compounds. A control showed that the 3H:14C ratio of the AMP isolated from the RNA was the same as the 3H:14C ratio in the 3'-deoxyadenosine. The 14C ratio in the adenine: ribose of the [U-14C]adenosine added to the 3'-deoxyadenosine producing cultures of C. militaris and of the isolated 3'-deoxyadenosine was the same, e.g. 50:50. These data provide strong evidence that adenosine in converted to 3'-deoxyadenosine without hydrolysis of the N-riboside bond. Degradation of the 3-deoxyribose from 3'-deoxyadenosine showed that the 3H was retained on carbon-3. These results suggest that the formation of 3'-deoxyadenosine may proceed by a reductive mechanism similar to that for the formation of 2'-deoxynucleotides.
[9]
Li B, 2016. Extraction and separation of cordycepin and adenosine from cultured Cordyceps militaris and inhibitory effect on oxidative stress in kidneys of diabetic mice. Master Thesis, Hefei University of Technology, Hefei. 1-73 (in Chinese)
[10]
Li H, Sun JY, Liao KH, Wang Z, Shan AY, 2016. Anti-inflammatory effect and mechanism study of cordycepin. Tianjin Journal of Traditional Chinese Medicine, 33(5):303-306 (in Chinese)
[11]
Liu XW, Tang JP, 2019, Process optimization of semisolid fermentation of Cordyceps militaris with loofah for cordycepin production. Science and Technology of Food Industry, 42(6):118-124 (in Chinese)
[12]
Luo W, Liu DB, Wu ZW, Xia ZL, Xie HQ, 2011. Study on the active components accumulation in Cordyceps militaris through fermentation process. Food and Fermentation Industries, 37(10):96-99 (in Chinese)
[13]
Ma JX, 2020. Purification and antibacterial and antitumor activity analysis of liquid fermentation cordycepin from Cordyceps militaris. Master Thesis, Beijing Forestry University, Beijing. 1-91 (in Chinese)
[14]
Masuda M, Das SK, Hatashita M, Fujihara S, Sakurai A, 2014. Efficient production of cordycepin by the Cordyceps militaris mutant G81-3 for practical use. Process Biochemistry, 49(2):181-187
https://doi.org/10.1016/j.procbio.2013.10.017
https://linkinghub.elsevier.com/retrieve/pii/S1359511313006193
本文引用 [1]
[15]
Meng XL, Li CX, 2017. Detection method and influencing factors of strain degradation of Cordyceps militaris. Journal of Anhui Agricultural Sciences, 45(13):133-134 (in Chinese)
[16]
Ning BY, Xue SS, Liu H, Liu K, Hao JW, 2020. Study on methods of regeneration of Cordyceps militaris strains. Edible Fungi, 42(3): 24-26+28 (in Chinese)
[17]
Raethong N, Wang H, Nielsen J, Vongsangnak W, 2020. Optimizing cultivation of Cordyceps militaris for fast growth and cordycepin overproduction using rational design of synthetic media. Computational and Structural Biotechnology Journal, 18:1-8
https://doi.org/10.1016/j.csbj.2019.11.003
https://www.ncbi.nlm.nih.gov/pubmed/31890138
本文引用 [1] 摘要
is an entomopathogenic fungus which is often used in Asia as a traditional medicine developed from age-old wisdom. Presently, cordycepin from is a great interest in medicinal applications. However, cellular growth of and the association with cordycepin production remain poorly understood. To explore the metabolism of as potential cell factories in medical and biotechnology applications, this study developed a high-quality genome-scale metabolic model of, NR1329, based on its genomic content and physiological data. The model included a total of 1329 genes, 1821 biochemical reactions, and 1171 metabolites among 4 different cellular compartments. Its growth simulation results agreed well with experimental data on different carbon sources. NR1329 was further used for optimizing the growth and cordycepin overproduction using a novel approach, POPCORN, for rational design of synthetic media. In addition to the high-quality GEM NR1329, the presented POPCORN approach was successfully used to rationally design an optimal synthetic medium with C:N ratio of 8:1 for enhancing 3.5-fold increase in cordycepin production. This study thus provides a novel insight into physiology and highlights a potential GEM-driven method for synthetic media design and metabolic engineering application. The NR1329 and the POPCORN approach are available at the GitHub repository: https://github.com/sysbiomics/-GEM.© 2019 The Authors.
[18]
Shih IL, Tsai KL, Hsieh C, 2007. Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. Biochemical Engineering Journal, 33(3):193-201
https://doi.org/10.1016/j.bej.2006.10.019
https://linkinghub.elsevier.com/retrieve/pii/S1369703X06003160
本文引用 [1]
[19]
Suhadolnik RJ, Weinbaum G, Meloche HP, 1964. The biosynthesis of cordycepin. Journal of the American Chemical Society, 86:948-949
https://doi.org/10.1021/ja01059a057
https://pubs.acs.org/doi/abs/10.1021/ja01059a057
本文引用 [1]
[20]
Sung GH, Hywel-Jones NL, Sung JM, Luangsa-Ard JJ, Spatafora JW, 2007. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology, 57(1):5-59
https://doi.org/10.3114/sim.2007.57.01
https://linkinghub.elsevier.com/retrieve/pii/S0166061614601305
本文引用 [1]
[21]
Suparmin A, Kato T, Dohra H, Park EY, 2017. Insight into cordycepin biosynthesis of Cordyceps militaris: comparison between a liquid surface culture and a submerged culture through transcriptomic analysis. PLoS One, 12(11):e0187052
https://doi.org/10.1371/journal.pone.0187052
https://dx.plos.org/10.1371/journal.pone.0187052
本文引用 [2]
[22]
Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, Reet NV, Amin DN, Oberg B, Andersson B, Kristensson K, Wigzell H, Rottenberg ME, 2009. Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin. PLoS Neglected Tropical Diseases, 3(8):e495
https://doi.org/10.1371/journal.pntd.0000495
https://dx.plos.org/10.1371/journal.pntd.0000495
本文引用 [1]
[23]
Wang CW, Wang Z, Wang JF, Wu HX, 2019. Research progress of cordycepin in Cordyceps militaris. Fujian Agricultural Science and Technology, 50(2):66-70 (in Chinese)
[24]
Wang YM, 2013. The research of genome shuffling breeding and in vitro antioxidant activity of Cordyceps kyushuensis. Master Thesis, Shandong University, Jinan. 1-101 (in Chinese)
[25]
Wang ZS, Gu YX, Yuan QS, 2006. Effect of nutrition factors on the synthesis of superoxide dismutase, catalase, and membrane lipid peroxide levels in Cordyceps militaris mycelium. Current Microbiology, 52(1):74-79
https://doi.org/10.1007/s00284-005-0193-9
http://link.springer.com/10.1007/s00284-005-0193-9
本文引用 [1]
[26]
Wen ZX, Du XF, Meng N, Li YJ, Mi R, Sun YX, Ma SH, Li SY, 2019. Tussah silkmoth pupae improve anti-tumor properties of Cordyceps militaris (L.) Link by increasing the levels of major metabolite cordycepin. RSC Advances, 9(10):5480-5491
https://doi.org/10.1039/C8RA09491H
http://xlink.rsc.org/?DOI=C8RA09491H
本文引用 [1]
[27]
Wongsa B, Raethong N, Chumnanpuen P, Wong-ekkabut J, Laoteng K, Vongsangnak W, 2020. Alternative metabolic routes in channeling xylose to cordycepin production of Cordyceps militaris identified by comparative transcriptome analysis. Genomics, 112(1):629-636
https://doi.org/10.1016/j.ygeno.2019.04.015
https://linkinghub.elsevier.com/retrieve/pii/S0888754319300321
本文引用 [2]
[28]
Wu F, Zhou LW, Yang ZL, Bau T, Li TH, Dai YC, 2019. Resource diversity of Chinese macrofungi: edible, medicinal and poisonous species. Fungal Diversity, 98:1-76
https://doi.org/10.1007/s13225-019-00432-7
https://doi.org/10.1007/s13225-019-00432-7
本文引用 [1]
[29]
Xiao Y, Xing GL, Rui X, Li W, Chen XH, Jiang M, Dong MS, 2014. Enhancement of the antioxidant capacity of chickpeas by solid state fermentation with Cordyceps militaris SN-18. Journal of Functional Foods, 10:210-222
https://doi.org/10.1016/j.jff.2014.06.008
https://linkinghub.elsevier.com/retrieve/pii/S1756464614001996
本文引用 [1]
[30]
Xia YL, Luo FF, Shang YF, Chen PL, Lu YZ, Wang CS, 2017. Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin. Cell Chemical Biology, 24(12):1479-1489
https://doi.org/10.1016/j.chembiol.2017.09.001
https://linkinghub.elsevier.com/retrieve/pii/S2451945617303276
本文引用 [3]
[31]
陈丽冰, 吴光旭, 程薇, 范秀芝, 史德芳, 石猛, 高虹, 2016. 北虫草培养残基中虫草素的提取纯化及抗肿瘤活性. 食品科学技术学报, 34(4):73-79
本文引用 [1]
[32]
董宏鸿, 李建军, 周行, 姜艳霞, 2016. 蛹虫草中虫草素的提取及药理活性研究进展. 吉林医药学院学报, 37(6):465-467
本文引用 [1]
[33]
李兵, 2016. 蛹虫草中虫草素和腺苷的提取分离及其对糖尿病小鼠肾脏氧化应激的影响. 合肥工业大学硕士论文,合肥. 1-73
本文引用 [1]
[34]
李晖, 孙佳瑜, 廖康汉, 王喆, 单爱云, 2016. 虫草素抗炎作用及机制研究. 天津中医药, 33(5):303-306
本文引用 [1]
[35]
罗巍, 刘东波, 吴郑武, 夏志兰, 谢红旗, 2011. 蛹虫草液态发酵过程中有效成分的动态积累变化. 食品与发酵工业, 37(10):96-99
本文引用 [1]
[36]
刘希文, 汤佳鹏, 2021. 蛹虫草丝瓜络半固态发酵产虫草素的工艺优化. 食品工业科技, 42(6):118-124
本文引用 [1]
[37]
马婕馨, 2020. 蛹虫草液体发酵虫草素纯化及抗菌抑癌活性分析. 北京林业大学硕士论文,北京. 91
本文引用 [1]
[38]
孟小丽, 李翠新, 2017. 蛹虫草菌种退化的检测方法和影响因素. 安徽农业科学, 45(13): 133-134+156
本文引用 [1]
[39]
宁宝云, 薛姗姗, 刘慧, 刘康, 郝继伟, 2020. 蛹虫草菌种复壮方法探讨. 食用菌, 42(3): 24-26+28
本文引用 [1]
[40]
王长文, 王忠, 王锦锋, 吴鸿雪, 2019. 蛹虫草虫草素研究进展. 福建农业科技, 50(2):66-70
本文引用 [1]
[41]
王延明, 2013. 九州虫草基因组改组育种与体外抗氧化活性研究. 山东大学硕士论文,济南. 1-101
本文引用 [1]

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