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菌物学报, 2022, 41(9): 1341-1353 doi: 10.13346/j.mycosystema.220040

综述

灵芝菌丝体三萜及其药理活性的研究进展

冯娜,1,*, 岳亚文2, 程池露3, 杨梅2, 汪旵4, 张劲松,1,*

1.上海市农业科学院食用菌研究所 国家食用菌工程技术研究中心 农业部南方食用菌资源利用重点实验室 上海市农业遗传育种重点开放实验室,上海 201403

2.华东理工大学药学院制药工程与过程化学教育部工程研究中心 上海市新药设计重点实验室,上海 200237

3.上海海洋大学食品学院,上海 201306

4.华东理工大学化工学院,上海 200237

Research progress of triterpenes from mycelia of Ganoderma lingzhi and its pharmacological effects

FENG Na,1,*, YUE Yawen2, CHENG Chilu3, YANG Mei2, WANG Chan4, ZHANG Jingsong,1,*

1. Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungal Resources and Utilization (South), Ministry of Agriculture of P.R. China, Key Laboratory of Agricultural Genetics and Breeding of Shanghai, Shanghai 201403, China

2. Engineering Research Centre of Pharmaceutical Process Chemistry, Ministry of Education, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

3. School of Food Science, Shanghai Ocean University, Shanghai 201306, China

4. School of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China

收稿日期: 2022-01-24   接受日期: 2022-02-25  

基金资助: 上海市科技兴农重点攻关项目(沪农科创字(2018)第1-1号)

Corresponding authors: fengna006@163.com, syja16@saas.sh.cn

Received: 2022-01-24   Accepted: 2022-02-25  

摘要

菌丝体作为灵芝生长发育过程中的一个特定生理阶段,会产生不同于子实体和孢子的特定结构的灵芝酸类三萜化合物。本文总结了灵芝菌丝体中已发现和鉴定的三萜化合物结构、生物活性及其构效关系的研究进展,以期为灵芝菌丝体三萜在生物合成、代谢调控等的基础研究及相关产品的开发应用提供科学参考。

关键词: 灵芝; 菌丝体; 四环三萜; 药理活性

Abstract

During vegetative growth and development of Ganoderma lingzhi (or Ganoderma lucidum), mycelia produce triterpenes with specific structures, which are different from the tritepenes in fruiting bodies and basidiospore. This paper summarized the progress of researches on structures, bioactivities and structure-activity relationship of triterpenes isolated and identified from mycelia of G. lingzhi in order to provide scientific reference for the basic research in biosynthesis, metabolic regulation and application of triterpenes and their related products.

Keywords: Ganoderma lingzhi; mycelia; tetracyclic triterpenes; pharmacological activity

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本文引用格式

冯娜, 岳亚文, 程池露, 杨梅, 汪旵, 张劲松. 灵芝菌丝体三萜及其药理活性的研究进展[J]. 菌物学报, 2022, 41(9): 1341-1353 doi:10.13346/j.mycosystema.220040

FENG Na, YUE Yawen, CHENG Chilu, YANG Mei, WANG Chan, ZHANG Jingsong. Research progress of triterpenes from mycelia of Ganoderma lingzhi and its pharmacological effects[J]. Mycosystema, 2022, 41(9): 1341-1353 doi:10.13346/j.mycosystema.220040

灵芝Ganoderma lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai是担子菌门Basidiomycota、伞菌纲Agaricomycetes、多孔菌目Polyporales、多孔菌科Polyporaceae、灵芝属Ganoderma真菌,俗称赤芝。长久以来,东亚地区的灵芝一直被认为与欧洲灵芝G. lucidum是同一种。直到1994年,Moncalvo et al. (1994,1995)对来自不同地域的所谓的“Ganoderma lucidum”进行了分子学研究,证实了欧洲、亚洲和美洲的灵芝实际上可被分类为2个物种。经过系统的研究和比较,分类学家们将分布在东亚地区(包括中国、日本等)的灵芝命名为G. lingzhi (Cao et al. 2012;戴玉成等 2021)。目前,在中国广泛人工栽培的灵芝,大多数属于G. lingzhi。野外生境下,除了灵芝G. lingzhi外,欧洲灵芝G. lucidum (Curtis) P. Karst.在中国也有分布(戴玉成等 2013;Si & Dai 2016)。

20世纪80年代初,包括欧洲、亚洲在内的很多学者对野生和人工栽培的灵芝进行了化学成分的研究,三萜作为其中一类重要的活性物质,一直是植化学家们分离和研究的对象。由于当时认知水平的限制,很多研究对象都被归属为欧洲灵芝G. lucidum。Nishitoba et al. (1986)发现采集自日本Hasuta和Ageo地区的2个灵芝菌株的子实体分别产生了C30灵芝酸和C27赤芝酸2种不同类型的三萜化合物,这是最早从化学成分方面发现2个灵芝菌株之间存在差异的报道。

灵芝菌丝体中的三萜,除了少数几个三萜醇类化合物在子实体和孢子中也有发现外,作为主要成分的灵芝酸类三萜化合物,几乎都是菌丝体所特有的。灵芝菌丝体中这些灵芝酸类化合物,在抗肿瘤活性上有着优异的表现。虽然过去诸多研究中的灵芝种学名为G. lucidum,但研究的对象实际是目前的灵芝或东亚灵芝G. lingzhi,故本文所整理归纳的灵芝G. lingzhi菌丝体中的三萜化合物,实际也包括了过去相关文献中学名为G. lucidum的文献,并总结了其生物活性和构效关系研究进展,以期为灵芝菌丝体三萜的生物合成、代谢调控方面的研究和抗肿瘤先导化合物的开发提供帮助。

1 灵芝菌丝体三萜的来源与发酵方式

Toth et al. (1983)首次从采自法国Gif-sur- Yvette地区的灵芝菌丝体中分离得到羊毛甾烷型三萜化合物,但未具体说明菌种来源、培养基配方和发酵过程。Tseng et al. (1984)将来自中国台湾的灵芝菌株NTU G001进行了液体发酵条件的优化,在(28±2) ℃全光照的条件下将菌丝体静置14 d,然后比较6种合成培养基(synthetic media)和7种完全培养基(complete media)对菌丝体生物量的影响,结果发现该菌在完全培养基上生长最佳,尤其是PDB (马铃薯250 mg/mL,葡萄糖20 mg/mL)和PCSB (马铃薯125 mg/mL,玉米125 mg/mL,蔗糖20 mg/mL),生物量分别达到了(451±19) mg/100 mL和(467±41) mg/100 mL。随后,几位学者对灵芝菌丝体的化学成分展开了研究,并发表了一系列化合物的结构。但在这些研究中,研究的对象逐渐从TP-1 (中国台湾种)、AT-4 (ATCC 32471)、AT-5 (未说明来源)和CJ-3 (未说明来源) 4个菌株,集中在中国台湾采集的菌株TP-1上,发酵过程调整为(28±1.5) ℃的温度下,菌丝体静置培养30 d。培养基的配方为:葡萄糖30 mg/mL,麦芽提取物20 mg/mL,蛋白胨1 mg/mL,pH 6.5 (Lin & Shiao 1987;Shiao et al. 1987;Lin et al. 1988a,1988b;Shiao et al. 1988a,1988b)。

Yang et al. (2002)对中国台湾糖业公司研究所保有的灵芝菌丝体进行分离鉴定,发现得到的三萜化合物中有3个是3位被酮基取代的新化合物,但在研究中,菌种的来源和发酵的过程未有叙述。

几乎在中国台湾学者开始研究的同时,日本2个研究团队也开展了灵芝菌丝体中次生代谢产物的分离工作。Hirotani et al. (1986,1987)对灵芝菌丝体也进行了液体静置发酵。其发酵的条件为:25 ℃黑暗条件下,静置培养6周。培养基配方为:葡萄糖15.00 mg/mL,大豆蛋白胨1.00 mg/mL,酵母提取物0.50 mg/mL,KH2PO4 0.50 mg/mL,NaCl 0.10 mg/mL,MgSO4·7H2O 0.05 mg/mL,CaCl2 0.11 mg/mL,pH 5.5 (1 mol/L HCI调节)。该研究中,用于发酵的灵芝菌种来源不明。Nishitoba et al. (1987a,1987b)对采集自日本Hasuta地区的灵芝菌株进行固体发酵,从菌丝体中分离得到了一系列以往没有报道的灵芝酸类三萜。值得注意的是,此研究采用的菌种在子实体生长过程中,其菌柄会形成3个分叉,且产生了C27类型的赤芝酸(Nishitoba et al. 1986)。

国内对灵芝菌丝体中化学成分的研究起步较晚。毋艳(2004)对山东四维制药股份有限公司提供的泰山灵芝菌丝体进行了分离纯化,获得了4个新的三萜化合物和5个已知的甾醇化合物。但该研究中,灵芝菌种的来源和菌丝体的发酵过程并没有提及。

钟建江教授带领的团队对灵芝菌种CGMCC5.616 (中国普通微生物菌种保藏管理中心)进行了发酵条件的优化,创建了两阶段发酵方法,即第一阶段为液体振荡培养,第二阶段培养时,菌丝体先液体振荡培养4 d后再转入静置培养12 d。此培养模式下,灵芝酸的含量可以由对照的1.36 mg/100 mg (干重)上升到3.19 mg/100 mg (Fang & Zhong 2002)。从该菌种中,汤亚杰、李英波等人先后分离得到10个三萜化合物(Li et al. 2009;Wang et al. 2010;李英波2013;Li et al. 2013)。

岳亚文等(2020)采用先液体发酵再固体发酵的方法,对浙江一种无孢灵芝(即一种孢子产量较低的灵芝品种龙芝2号)进行了菌丝体的发酵并分离鉴定了其中的化合物,得到了10个已知三萜化合物和1个首次发现的天然灵芝酸化合物。该实验中,液体发酵的种子培养基为:1 000.0 mL水中加入豆饼粉20.0 g,葡萄糖25.0 g,MgSO4∙H2O 1.5 g,KH2PO4 3.0 g,自然pH。固体培养基为:大米98%,1% CaSO4,1%蔗糖。

2 灵芝菌丝体三萜的结构类型

从以上灵芝的发酵菌丝体中,共分离得到了70种三萜化合物(表1),均为羊毛甾烷型四环三萜(图1,图2)。由于研究工作和文章发表的时间比较集中,出现了同一化合物有不同名称的现象,例如:ganoderic acid Q、ganoderic acid Mk和ganodermic acid P1实际上为同一化合物。虽然ganoderic acid Mk的C22位上连接的乙酰氧基(-OAc)构型未定,但从化合物的化学位移数据可以判断,其构型同ganoderic acid Q和ganodermic acid P1相同,都是α型(Hirotani et al. 1987;Nishitoba et al. 1987b;Shiao et al. 1988a)。此外,也有作者没有仔细核对文献,误将ganoderic acid P当成一个新的化合物从而用其化学名(22S,24E)-3α-hydroxy-15α, 22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid发表(Yang et al. 2002)。菌丝体中的这些化合物大多数为三萜酸,在C26位上存在一个羧基,且C24、C25位上存在一个双键结构,仅个别化合物在C26位上为羟基取代,属于三萜醇类物质。在三萜酸的母环上,存在有2种双键结构,一类是△7(8)、△9(11)共轭双键,另一类则是△8(9)双键,这决定了2类三萜酸的紫外吸收不一致。母环上有共轭双键的灵芝三萜酸化合物,例如ganodermic acid N、ganodermic acid O、ganodermic acid Q、ganodermic acid P1、ganodermic acid P2、ganoderic acid Ja、ganoderic acid Jb、ganoderic acid TN、ganoderic acid TO、ganoderic acid TQ等,其最大紫外吸收多集中在235、243和252 nm。存在△8(9)一个双键,又在C11位上有酮基的灵芝三萜化合物,例如3-O-acetylganoderic acid B、ethyl 3-O-acetylganoderate B、ethyl ganoderate J、methyl O-acetyl ganoderate C、methyl 3,7,11,15,23-pentaoxolanost-8-en-26-oate等,其最大紫外吸收在253 nm左右。而仅存在△8(9)双键且C11位上没有任何取代的三萜酸类化合物,例如ganoderic acid Ma、ganoderic acid Mb、ganoderic acid Mc、ganoderic acid Md、ganoderic acid Mg、ganoderic acid Mh、ganoderic acid Mi、ganoderic acid Mj、ganoderic acid DH和7-O-ethyl ganoderic acid O,其最大紫外吸收在210 nm左右。这一类化合物中,ganoderic acid Mc、ganoderic acid DH和7-O-ethyl ganoderic acid O均在C-7位上有不同官能团的取代,在质子性溶剂(例如甲醇)中表现极不稳定,很容易生成其他三萜化合物(Wang et al. 2011;李英波 2013)。

表1   灵芝菌丝体中的三萜化合物

Table 1  Triterpenes in mycelia of Ganoderma lingzhi

编号
No.
化合物名称
Compound names
分子式
Formulas
分子量
Molecular
weights
参考文献
References
1Ganoderic acid UC30H48O4472Toth et al. 1983
2Ganoderic acid VC32H48O6528Toth et al. 1983
3Ganoderic acid WC34H52O7572Toth et al. 1983
4Ganoderic acid XC32H48O5512Toth et al. 1983
5Ganoderic acid YC30H46O3454Toth et al. 1983
6Ganoderic acid ZC30H48O3456Toth et al. 1983
7Ganoderic acid RC34H50O6554Hirotani et al. 1986
8Ganoderic acid SC32H48O5512Hirotani et al. 1986
9Ganoderic acid TC36H52O8612Hirotani et al. 1986
10Ganoderic acid OC36H54O9630Hirotani et al. 1987
11Ganoderic acid PC34H50O7570Hirotani et al. 1987
12Ganoderic acid Q
Ganoderic acid Mk
Ganodermic acid P1
C34H50O7570Hirotani et al. 1987
Nishitoba et al. 1987b
Shiao et al. 1988a
137-O-methyl ganoderic acid OC37H56O9644Hirotani et al. 1987
14Ganoderic acid MaC34H52O7572Nishitoba et al. 1987a
15Ganoderic acid MbC36H54O9630Nishitoba et al. 1987a
16Ganoderic acid McC36H54O9630Nishitoba et al. 1987a
17Ganoderic acid MdC35H54O7586Nishitoba et al. 1987a
18Ganoderic acid MeC34H50O6554Nishitoba et al. 1987a
19Ganoderic acid MfC32H48O5512Nishitoba et al. 1987a
20Ganoderic acid MgC35H54O8602Nishitoba et al. 1987b
21Ganoderic acid MhC34H52O8588Nishitoba et al. 1987b
22Ganoderic acid MiC33H52O6544Nishitoba et al. 1987b
23Ganoderic acid MjC35H54O7586Nishitoba et al. 1987b
24Ganodermic acid SC34H50O6554Shiao et al. 1987
25Ganodermic acid RC34H50O6554Shiao et al. 1987
26Lanosta-7,9(11),24-trien-3α-ol-26-oic acidC30H46O3454Lin & Shiao 1987
27Lanosta-7,9(11),24-trien-3α-acetoxy-26-oic acidC32H48O4496Lin & Shiao 1987;
Zhu et al. 2020
2822β-acetoxy-3α,15α-dihydroxylanosta-7,9(11),24-trien-26-oic acidC32H48O6528Lin et al. 1988a
2922β-acetoxy-3β,15α-dihydroxylanosta-7,9(11),24-trien-26-oic acidC32H48O6528Lin et al. 1988a
303α,15α-diacetoxy-22α-hydroxylanosta-7,9(11),24-trien-26-oic acidC34H50O7570Lin et al. 1988a
313α,15α,22α-trihydroxylanosta-7,9(11),24-trien-26-oic acidC30H46O5486Lin et al. 1988a
323β, 15α,22β-trihydroxylanosta-7,9(11),24-trien-26-oic acidC30H46O5486Lin et al. 1988a
333β,15α-diacetoxy-22α-hydroxylanosta-7,9(11),24-trien-26-oic acidC34H50O7570Lin et al. 1988a
343β,15α-diacetoxylanosta-8,24-dien-26-oic acidC34H52O6556Lin et al. 1988a
35Ganodermic acid NC32H48O5512Shiao et al. 1988a
36Ganodermic acid OC32H48O5512Shiao et al. 1988a
37Ganodermic acid QC32H46O5510Shiao et al. 1988a
38Ganodermic acid P2C34H50O7570Shiao et al. 1988a
39Ganodermic acid JaC30H46O4470Shiao et al. 1988a
40Ganodermic acid JbC30H46O4470Shiao et al. 1988a
41Ganodermic acid T-NC32H48O5512Lin et al. 1988b
42Ganodermic acid T-OC32H48O5512Lin et al. 1988b
43Ganodermic acid T-QC32H46O5510Lin et al. 1988b
44Lanosta-7,9(11),24-trien-15α-acetoxy-3α-hydroxy-23-oxo-26-oic acidC32H46O6526Shiao et al. 1988b
45Lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acidC36H52O8612Shiao et al. 1988b
46Lanosta-7,9(11),24-trien-3α,15α-diacetoxy-23-oxo-26-oic acidC34H48O7568Shiao et al. 1988b
47Lanosta-7,9(11),24-trien-3α-acetoxy-15α,22β-dihydroxy-26-oic acidC32H48O6528Shiao et al. 1988b
48Lanosta-7,9(11),24-trien-3α-acetoxy-15α-hydroxy-23-oxo-26-oic acidC32H46O6526Shiao et al. 1988b
49Lanosta-7,9(11),24-trien-3α-hydroxy-26-oic acidC30H46O3454Lin & Shiao 1989
50(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acidC30H44O5484Yang et al. 2002
51(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acidC32H46O6526Yang et al. 2002
52(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acidC34H48O7568Yang et al. 2002
533,22-dihydroxy-7,9,24-triene-26-oicC30H46O4470Wu 2004
54Ganoderic acid S2C32H48O5512Wu 2004
55Ganoderic acid S3C30H44O4468Wu 2004
56Ganoderic acid S4C30H46O4470Wu 2004
573-O-acetylganoderic acid BC32H44O8556Li et al. 2009
583-O-acetylganoderic acid KC32H44O8556Li et al. 2009
598β,9α-dihydroganoderic acid CC30H38O7510Li et al. 2009
60Ethyl 3-O-acetylganoderate BC34H48O8584Li et al. 2009
61Ethyl ganoderate JC32H44O7540Li et al. 2009
62Methyl O-acetylganoderate CC35H46O10626Li et al. 2009
63Methyl 3,7,11,15,23-pentaoxolanost-8-en-26-oateC31H40O7524Li et al. 2009
647-O-ethyl ganoderic acid OC38H58O9658Wang et al. 2010
653α,22β-diacetoxy-7α-hydroxyl-5α-lanost-8,24E-dien-26-oic acidC34H50O8586Li et al. 2013
66Ganoderic acid DHC34H52O7572Li 2013
67Ganoderol BC30H48O2440Feng et al. 2015
68Ganoderic acid T1C34H50O7570Yue et al. 2020
69GanodermenonolC30H46O2438Yue et al. 2020
70GanodermanondiolC30H48O3456Yue et al. 2020

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图1

图1   灵芝菌丝体三萜化合物结构类型

Fig. 1   Structural types of triterpenes in mycelia of Ganoderma lingzhi.


图2

图2   灵芝菌丝体三萜化合物的结构

Fig. 2   Structures of triterpenes isolated from mycelia of Ganoderma lingzhi.


3 灵芝菌丝体三萜化合物的生物活性

早期的研究中,由于分离得到的三萜化合物的量很少,较少有人进行活性的验证和作用机制的研究。Toth et al. (1983)从灵芝菌丝体中得到ganoderic acid U、ganoderic acid V、ganoderic acid W、ganoderic acid X、ganoderic acid Y、ganoderic acid Z时,首次报道了这些化合物具有体外抑制肝癌细胞增殖的活性,这为后人对菌丝体三萜化合物的抗肿瘤研究提供了方向。

Hirotani et al. (1986)发现ganoderic acid R和ganoderic acid S可以减轻半乳糖胺诱导产生的肝中毒,该研究也没有进一步更深入的探讨。

Wang et al. (1990)认为,ganodermic acid S的化学结构类似于脱氧胆酸盐,而脱氧胆酸可以结合到血小板膜中导致血小板功能的抑制。因此,研究者对ganodermic acid S开展了一系列的对血小板凝集作用的研究。结果发现,在浓度超过20 μmol/L时,ganodermic acid S会引起血小板聚集,且细胞聚集程度与化合物浓度呈线性关系。Ganodermic acid S浓度在20-50 μmol/L之间时,会造成磷酸肌苷与花生四烯酸释放的相互转化。扫描电子显微镜显示,在聚集阈值即20 μmol/L以下时,血小板形态为针状盘状;在阈值以上,血小板细胞向上聚集成不规则的穗状。为解释ganodermic acid S如何影响血小板对胶原蛋白的反应,进一步对作用通路进行了研究,结果发现,ganodermic acid S主要通过抑制TXA2依赖性信号的Gq-PLCβ1受体通路而非G1受体通路来抑制胶原反应,而胶原反应中酪氨酸激酶依赖通路在聚集中起主要作用(Su et al. 1999a,1999b)。

此外,ganodermic acid S与前列腺素(PG)E1联用,以浓度依赖的方式增强了血小板的环磷酸腺苷(adenosine monophosphate,AMP)合成。在7.5 μmol/L剂量下,ganodermic acid S可将(PG)E1单独诱发的环AMP合成的水平提高1.8倍,并通过肌球蛋白轻链和普列克底物蛋白的磷酸化、α-颗粒的分泌、细胞聚集和蛋白质酪氨酸磷酸化等途径增强了(PG)E1对血小板胶原反应的抑制作用。Ganodermic acid S与(PG)E1联用后,还基本消除了致密颗粒的分泌和血栓素(TX) B2的形成(Su et al. 2000)。

刘如明(2012)采用MTT法检测了ganoderic acid T、ganoderic acid Mk、ganoderic acid Me、ganoderic acid S、ganoderic acid Mf和7-O-ethyl ganoderic acid O对4种肿瘤细胞(肺癌细胞95D、卵巢癌细胞HO-8910、胰腺癌细胞SW1990和宫颈癌细胞HeLa)和一种正常的成纤维细胞(HF)增殖的抑制能力,结果证明6种化合物均具有一定的细胞毒性。

Ganoderic acid Me可以抑制C57BL/6小鼠Lewis肺癌的肿瘤生长和肺转移。腹腔注射ganoderic acid Me (28 mg/kg)可显著增强NK细胞活性,白细胞介素-2 (IL-2)和干扰素-γ (IFN-γ)的表达增加,核因子-κB (NF-κB)的表达上调,这提示ganoderic acid Me可以通过提高免疫功能有效抑制肿瘤生长和肺转移(Wang et al. 2007)。Chen et al. (2008)发现ganoderic acid Me通过下调基质金属蛋白酶2/9 (MMP2/9)基因表达抑制95-D肺癌细胞的浸润转移,还可抑制Lewis肺癌细胞的生长和扩散,且具有显著增强免疫功能作用。Ganoderic acid Me也可诱导野生型人P53肿瘤细胞凋亡。吲哚胺2,3-双加氧酶(IDO-)介导的微环境在肿瘤免疫逃逸中起着重要作用,通过建立稳定的IDO-过度表达2LL细胞克隆,检测到ganoderic acid Me可以通过增强小鼠lewis肺癌细胞中IDO-的表达和激活,诱导T细胞凋亡,抑制CD8+T细胞活化和增强Treg介导的免疫抑制,有助于在肺肿瘤中创造耐受性环境(Que et al. 2014)。此外,ganoderic acid Me可使多药耐药癌细胞KB-A-1/Dox中阿霉素和罗丹明123的含量均增加100%,推测其可能通过影响膜蛋白P-gp的活性来提高多药耐药细胞对阿霉素的敏感性(唐文 2013a)。

作为迄今为止结构式上有最多乙酰氧基的灵芝三萜,ganoderic acid T表现出较强的抗肿瘤活性,也是至今研究比较详细的一个化合物。Ganoderic acid T以剂量依赖性方式对多种人类癌细胞系(肺腺癌细胞A549、PC9、PC9-IR、H1650和H1975,肺癌细胞95-D,结肠癌细胞HCT-116,肺癌Lewis)产生细胞毒性(Lai et al. 2009)。

Ganoderic acid T可能通过抑制MMP-2和MMP-9的表达,从而以剂量和时间依赖性方式抑制高转移性人类肺癌细胞95-D的转移。细胞聚集和粘附实验结果表明,ganoderic acid T能促进细胞聚集,同时抑制细胞与细胞外基质(ECM)的粘附(Xu et al. 2010)。Ganoderic acid T还通过诱导细胞凋亡和G1期细胞周期阻滞显著抑制95-D的增殖。在诱导凋亡过程中,ganoderic acid T通过与线粒体功能障碍和p53表达相关的内在途径诱导转移性肺肿瘤细胞凋亡(Tang et al. 2006)。

在对人结肠癌细胞HCT-116的实验中,ganoderic acid T同样表现出对细胞增殖的抑制能力,且以剂量依赖性方式抑制HCT-116细胞的迁移、促进细胞同型聚集以及抑制HCT-116细胞与细胞外基质的粘附。此外,通过比较ganoderic acid T作用于HCT-116的p53+/+和p53-/- 两种类型细胞后的结果,可以发现ganoderic acid T引起的细胞毒性依赖于p53基因,p53基因可能是ganoderic acid T抑制人癌细胞抗侵袭的重要靶点(Chen & Zhong 2008;Chen &Zhong 2011)。

体内实验证实,ganoderic acid T在体内能够抑制Lewis肺癌肿瘤的生长和转移。将ganoderic acid T (28 mg/kg)皮下注射到C57BL/6小鼠体内后,其对Lewis肺癌肿瘤生长和肺转移的抑制率分别达到63.35% (P<0.001)和78.33% (P<0.001)。从肿瘤组织中提取mRNA进行qRT-PCR分析后,发现ganoderic acid T通过下调MMP-2和MMP-9 mRNA在体内的表达来抑制肿瘤的转移(Chen et al. 2010)。Lai et al. (2009)通过给药后小鼠的体重、肿瘤的大小、小鼠的存活率、肿瘤的重量以及小鼠的肝脏和肺的转移性肿瘤菌落几个方面,验证了ganoderic acid T能有效抑制A549肺腺癌肿瘤的生长与癌细胞的转移。

用不同浓度的ganoderic acid T处理后,再用γ射线照射HeLa宫颈癌细胞,研究其对辐射敏感性的影响。结果证实ganoderic acid T增加了肿瘤细胞的DNA损伤,干扰了DNA修复,阻断细胞周期在G1期,增加ROS生成,并降低线粒体膜电位。Ganoderic acid T在辐射条件下还可诱导ATP耗竭,从而在促进细胞凋亡的同时增加细胞坏死(Shao et al. 2021)。

Ganoderic acid T处理后,使得多药耐药KB-A-1/Dox细胞中阿霉素和罗丹明123的含量分别增加了40%和20%,表明ganoderic acid T同ganoderic acid Me类似,也可以提高多药耐药KB-A-1/Dox细胞对阿霉素的敏感性(唐文 2013b)。

4 灵芝菌丝体三萜化合物的构效关系研究

由于灵芝菌丝体三萜具有相似的结构特征,多个化合物仅存在取代基(羟基或乙酰氧基)个数的不同、取代位点的不同、α/β立体构型的不同,通过实验可以比较和总结这些取代基、取代位点或构型对化合物活性的影响,因此灵芝菌丝体三萜是研究构效关系的绝佳对象。有关的研究取得了一定的进展。

8个三萜化合物:ganodermic acid S、ganodermic acid R、ganoderic acid N、ganoderic acid X、ganoderic acid O、ganoderic acid Mf、ganoderic acid Jb和ganoderic acid Ja,其中有4对是C-3α/β立体异构体,2对是C-3/C-15位置异构体,所有化合物均含有一个共同的C-26羧基,其构效关系通过激活血小板磷脂酶C (PLC)的试验进行了阐明。试验结果表明,这些化合物的激活能力强弱与其取代基相关的关系为:2个乙酰氧基取代基>一个乙酰氧基取代基> 2个羟基取代基。此外,含有C-3β取代基的三萜类化合物比C-3α类化合物更有效。Ganoderic acid Ja是一种具有C-3α和C-15α羟基取代基的三萜化合物,实验结果证实,即使在较高浓度下,其活性也不足以诱导血小板聚集(Wang et al. 1994)。

作为同分异构体,ganoderic acid Mf和ganoderic acid S均通过线粒体介导的途径诱导人HeLa细胞凋亡,但它们具有不同的细胞周期阻滞特异性。Ganoderic acid Mf将肿瘤细胞阻滞于G1期,而GAS将肿瘤细胞阻滞于S期(Liu & Zhong 2011)。

刘如明(2012)以宫颈癌细胞HeLa为模型,考察了ganoderic acid T及其羧基端衍生物(TLTO-A、TLTO-Me、TLTO-Ee、TLTO-Pe)的抗肿瘤活性和作用机制。结果表明,酰胺衍生物TLTO-A的细胞毒性最强,3种酯化衍生物TLTO-Me、TLTO-Ee、TLTO-Pe的细胞毒性与GA T相似,其毒性顺序为:TLTO-A>GA T≈ TLTO-Me≈TLTO-Ee≈TLTO-Pe。5种化合物均将HeLa细胞周期阻滞于G1期,且都可以通过线粒体途径诱导肿瘤细胞发生凋亡,其中以TLTO-A的促凋亡活性最强。从结构上来看,5种化合物的区别仅在C-26位的取代基不同,因此,推测ganoderic acid T的C-26羧基是一个活性修饰位点。

5 展望

本文所述灵芝菌丝体是指处于营养生长阶段的次级菌丝,该生物体不同于灵芝生殖生长过程中的三级菌丝(即子实体),在灵芝的生长过程中执行不同的生理生化功能,因此产生了独特结构的三萜代谢产物。灵芝子实体中的三萜酸结构中,母环结构上多数为C8、9双键与C11位酮基。而在灵芝菌丝体中,半数以上的三萜酸在母环上有共轭双键。这种母环上有共轭双键且具有多个乙酰氧基取代的三萜酸化合物,有着优异的抗肿瘤活性,是抗肿瘤药物先导化合物发现的一个重要来源。灵芝菌丝体中还有很多三萜化合物的活性和作用机制研究尚存在空白,有必要对这些结构类型不同的化合物进行活性的筛选和比较,探讨其构效关系,为高效低毒抗肿瘤药物的开发提供更充分的研究基础。

与灵芝子实体相比,灵芝发酵的生产周期短,条件精准可控,且三萜化合物产量高,灵芝菌丝体因此被用作研究三萜生物合成和代谢调控的对象。很多学者采用了外源物添加、增强/敲除某个基因、或导入外源基因等多种方式,来探讨三萜合成转化以及高产的可能性(Xu & Zhong 2011;Xu et al. 2013;Li et al. 2016;Xu et al. 2019)。在这些研究中,迫切需要建立灵芝菌丝体特有的三萜化合物的定量分析方法,通过化合物的有无或含量变化,来解释说明其生物合成过程中的上下游关系,从而精准获得影响三萜生物合成的关键基因和代谢通路。

以往灵芝命名的混淆造成了灵芝菌丝体中这些三萜化合物来源的混乱,有研究发现,Ganoderma lingzhi Ganoderma lucidum的子实体不仅存在分子生物学和形态学上的差异,在三萜的组成和含量上也有很大不同,Ganoderma lucidum中的三萜含量明显低于Ganoderma lingzhi (Hennicke et al. 2016)。因此,有必要在分别制备出2个灵芝种的菌丝体化合物后,进行系统的分析和比较,从化学分类的角度进一步补充说明2个物种的区别。

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