Current Bioactive Compounds 2017, 13, 28-40

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RESEARCH ARTICLE

ISSN: 1573-4072 eISSN: 1875-6646

Ganoderma lucidum (Ling-zhi): The Impact of Chemistry on Biological Activity in Cancer

Temitope O. Lawal1, Sheila M. Wicks2 and Gail B. Mahady3,*

1Department of Pharmacy Practice, Clinical Pharmacognosy Laboratories, University of Illinois at Chicago, Chicago, IL 60612, USA and Department of Pharmaceutical Microbiology, University of Ibadan, Ibadan, Nigeria; 2Department of Clinical Anatomy, City Colleges of Chicago and Rush University, Chicago, IL 60612, USA; 3Department of Phar- macy Practice, Clinical Pharmacognosy Laboratories, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA

Abstract: Background: Ganoderma (Ganodermataceae), a genus of medicinal mushrooms, has been employed as an herbal medicine in Traditional Chinese medicine (TCM) for over 2000 years. The fruiting bodies have been used historically in TCM, while the mycelia, and spores are now also used as a tonic, to stimulate the immune system and treat many diseases, including cancers. Objective: To review the anti-cancer research on G. lucidum (GL) from 2005 up to May 2016 and correlate these data with analysis of the active chemical constituents. Methods: Literature searches were performed from 2005 to May 2016 in various databases such as Pub- A R T I C L E H I S T O R Y Med, SciFinder, Napralert, and Google Scholar for peer-reviewed research literature pertaining to Gano- Received: January 23, 2016 Revised: June 2, 2016 derma lucidum and cancer. Accepted: June 9, 2016 Results: Of the >200 known Ganoderma species, only two species, G. lucidum (W. Curtis: Fr.) P. Karst DOI: 10.2174/15734072126661606140748 and G. sinense Zhao, Xu et Zhang, are officially recognized as “Ling-zhi” in the Chinese Pharmacopoeia. 01 Both species are thought to have similar biological effects. “Ling-zhi” is purported to have anticancer effects, and is also used in the promotion of health and longevity. Numerous polysaccharides and triterpenes have been identified in G. lucidum and have anticancer effects in both cell culture and animal models. Conclusion: The current published literature suggests that G. lucidum extracts have excellent potential for use in cancer therapy, as both the triterpenes and polysaccharides appear to have anti-cancer effects both in vitro and in vivo. However, many problems still exist including significant quality issues including taxonomy and identification of species, parts used, and the production of extracts with active constituents. Keywords: Ganoderma lucidum, Ling-zhi, Ganodermataceae, cancer, polysaccharides, triterpenes.

INTRODUCTION Enzymes contained within the mushroom help to promote the breakdown the wood constituents including lignins Ganoderma (Ganodermataceae), a genus of medicinal and cellulose in rotting trees [6]. In terms of distribution, mushrooms has been employed as an herbal medicine in while Ganoderma are primarily used in Asia as medicines, Traditional Chinese medicine (TCM) for over 2000 years [1- these mushrooms are also found in many countries of the 5]. The genus “Ganoderma” was named by Karsten in 1881, world, including Malaysia where they grow on rubber trees. and means "brightness or shiny", and “shiny skin” in Greek To facilitate the identification of the genus and species, [6, 7]. Ganoderma mushrooms are characterized by their many physical and geographical characteristics are used, large basidiocarps [8]. They are leather-like, and may or may including shape and color of the fruiting body, host specific- not have a stem [8]. The fruiting bodies (carpophore, myo- ity as well as place of origin [2]. carp) are generally found growing in a characteristic fan-like shape on the trunks of both live and rotting trees [6]. USE IN TRADITIONAL CHINESE MEDICINE (TCM) The medicinal use of Ganoderma, also commonly re- *Address correspondence to this author at the Director, Clinical Pharma- ferred to as “Reishi” (Japan) or “Ling-zhi” (Chinese) dates cognosy Laboratories, Department of Pharmacy Practice, College of Phar- back more than 2000 years in TCM [9]. The fruiting bodies macy, University of Illinois at Chicago, 833 South Wood St., MC 877, were used in TCM to increase energy, stimulate the immune Chicago, IL 60612, USA; Tel: 312-996-1669; Fax: 312-413-5894; E-mail: [email protected] system and to treat many diseases, including arthritis, asthma and bronchitis, heart disease, hepatitis, hypertension, nephri-

1875-6646/17 $58.00+.00 © 2017 Bentham Science Publishers Current Bioactive Compounds Anti-Cancer Activities of Ganoderma lucidum (Ling-zhi) Current Bioactive Compounds 2017, Vol. 13, No. 1 29 tis, and cancers [2-11]. The term “Ling-zhi” in China indi- ents from Ling-zhi act either additively or synergistically to cates spiritual strength and longevity, and thus Ling-zhi is produce its overall pharmacologic effects. Interestingly, the considered a powerful herbal medicine that enhances well- quantity of triterpenes present in both Ganoderma species ness and long life [2]. known as “Ling-zhi” is significantly different, and it is reported that G. sinense has a much lower percentage of triter- Of the >200 known Ganoderma species, only two spe- penes than G. lucidum [12-15]. cies, G. lucidum (W. Curtis: Fr.) P. Karst and G. sinense Zhao, Xu et Zhang, are officially recognized as “Ling-zhi” In the study by Da et al., [15], the chemistry of the two in the Chinese Pharmacopoeia, and research suggests that official species of Ling-zhi in the Chinese Pharmacopoeia they may have similar pharmacological effects [12]. Ling-zhi was investigated to compare the types of chemical constitu- was also mentioned in the “Classic of the Materia Medica” ents present in both G. sinense and G. lucidum. Thirty-two (502-536 AD) and a book entitled the “Ben Cao Gang Mu”, batches of commercial Ling-zhi samples were tested using which was thought to be one of the initial Chinese pharma- multiple-optimized chromatographic and spectroscopic copoeias (1590 AD) [2]. Ganoderma mushrooms are primar- methods, to determine the chemical variances between these ily cultivated in Sichuan, Zhejiang, Jiangxi and Hunan prov- species. Results from this study showed definite differences inces, although wild collection is also still performed [1]. between G. sinense and G. lucidum in terms of the types of chemical constituents present and their quantity [15]. The In TCM, Ling-zhi is a medicinal herb used for nourishing most striking difference in the chemistry was the lack of the and tonifying chi of zang organs, as well as nourishing chi most common triterpenes in G. sinense, while analysis of G. and blood [1]. Ling-zhi has also been used to treat stress, lucidum (GL), showed hundreds of triterpenes, including the weakness, as well as mental fatigue. Ling-zhi may be em- ganoderic acids (G) A, B, C2, D, F, G, H and K and gan- ployed alone, or in combination with other herbs to increase oderenic acids (GE) A-D (Fig. 1) many of which possessed its therapeutic efficacy [1]. Ling-zhi has also been used for antitumor activities. A difference in the types of polysaccha- the treatment of anxiety, heart palpitations, lack of sleep and rides contained in G. lucidum and G. sinense was not directly poor sleep quality [1]. Thus, the therapeutic effects of Ling- observed, however in the principle component analysis, a zhi include tonifying effects, energy-enhancing activities, difference in the polysaccharide content was also shown for improving heart activity, improving memory, as well as re- the two species [15]. ducing the ageing process [2]. In 2010, excerpts from the Chinese Pharmacopoeial monograph for Ling-zhi state that it In a 2012 investigation, Xie et al., [12] found similarities “acts to replenish Qi, ease the mind, and relieve cough and in the polysaccharide content of the two species of Ling-zhi asthma, and it is recommended for dizziness, insomnia, pal- that were obtained from various areas in China. Using high pitations, and shortness of breath” [2]. performance size exclusion chromatography-evaporative light scattering detection (HPSEC-ELSD) and HPSEC- Among cultivated mushrooms, Ling-zhi is unique in that MALLS-RI (multi-angle laser light scattering analyses), this it is used more as a medicinal agent rather than a food. In group showed that both experimentally generated profiles, terms of commercial production, G. lucidum (GL) products and the molecular weights of isolated polysaccharides were are available in many forms including crude drug, dried comparable. Analysis of digested polysaccharides (using the preparations, phytomedicines, and teas. Such products are enzymes pectinase and dextranase) also showed similarity in manufactured from the mycelium, and fruiting bodies of the the structures of the monosaccharides [15]. However, the mushroom. Reported therapeutic effects of Ling-zhi include polysaccharide content may not be as important as the struc- a reduction in blood sugar levels for the treatment of diabe- ture of the polysaccharides present. Polysaccharides contain tes, enhancement of immune function, liver protection, an- large numbers of single sugars (monosaccharides) that are timicrobial, as well as possible adjunct therapy for cancer. joined through O-glycoside linkages [16]. Polysaccharides The mushroom is commonly used to make soup in southern exist in two forms: homo-polysaccharides are repeating units China, and it is marketed as a botanical dietary supplement of one monosaccharide, while hetero-polysaccharides are worldwide [13]. It is purported to have anticancer effects and composed of more than two types of monosaccharides. In is also used to increase overall well being as well as increase medicinal mushrooms, the primary polysaccharides include life span. The goal of this review was to review the in vitro the glucans that contain varying kinds of glycosidic linkages, and in vivo effects of GL on cancer from 2005 up to 2016, including (1-3)-, (1-6)- glucans or (1-3)--glucans. In addi- and to correlate these data with chemical analysis of the action, heteroglycans may also occur in medicinal mushrooms. tive constituents. The anticancer effects of GL were previ- Mushroom-derived polysaccharides are known to have anti- ously reviewed up to 2005 [13]. cancer and antitumor effects. Furthermore, there has been a considerable amount of research performed which indicates CHEMISTRY that -D-glucan derivatives impact the immune system by Chemical analysis of Ling-zhi fruiting bodies, mycelium increasing the levels or activities of the cellular and humoral and spores shows the presence of a wide range of chemical immune responses, increasing the effects of macrophages, constituents including phenols, sterols, amino acids, lignins, mononuclear cells, and neutrophils [16]. polysaccharides and terpenes [13, 14]. While the polysaccha- Polysaccharides isolated from Ling-zhi have shown a rides, peptidoglycans, and triterpenes are thought to be the variety of pharmacological effects including a reduction in primary biologically active constituents of Ling-zhi, hun- inflammation, blood sugar, and ulcers, as well as having an- dreds of compounds have been isolated and identified [15]. Since many of these chemical compounds have some bio- titumor, and immune stimulating activities [2, 17-21]. The polysaccharides are generally extracted from Ling-zhi using logical activity, it is most likely that the chemical constitu- 30 Current Bioactive Compounds 2017, Vol. 13, No. 1 Lawal et al.

R4 O

O OH

R1 R2 H

Compound R1 R2 R3 R4 Double Bond

GEA =O -OH -OH H 20,22

GEB -OH -OH =O H 20,22

GEC -OH -OH -OH H 20,22

GED =O -OH =O H 20,22

GA =O -OH -OH H ---

GB -OH -OH =O H ---

GC2 -OH -OH -OH H ---

GD =O -OH =O H ---

GF =O =O =O H ---

GG -OH -OH =O -OAc ---

GH -OH =O =O -OAc ---

GK -OH -OH =O -OAc ---

Fig. (1). Triterpenes constituents present in G. lucidum, namely ganoderenic acids A (GEA), B (GEB), C (GEC), D (GED), and ganoderic acids A (GA), B (GB), C2 (GC2), D (GD), F (GF), G (GG), H (GH), K and (GK). Modified from Da et al., [15]. hot aqueous extraction techniques, and then they are precipi- sponse in animal models, and these doses are similar to the tated using alcohols. Chemical structure analyses of the iso- commonly used TCM dose of Ling-zhi used in clinic [1]. lated polysaccharides suggest that glucose is the primary Numerous GL polysaccharide preparations are currently sugar substituent [17, 22]. For example, Ganoderan, a poly- marketed as herbal medicinal products in the treatment of saccharide with a molecular weight of 20 kDa is a -glucan chronic diseases, such as diabetes, cancer and liver diseases [16]. This compound isolated from G. lucidum has been [1, 23]. shown to have antitumor effects in a rodent model of cancer [16]. The composition of ganoderan is mainly glucose with In 2015, Sun et al., [25] investigated the in vitro antican- 4% protein. However, polysaccharides may also contain xy- cer activities of GLPS. This group assessed the effects of lose, mannose, galactose, or fucose/fructose [23, 24]. GLPS on the levels of cytokines in mononuclear lymphocytes. Mouse splenic mononuclear lymphocytes were incu- In 2014, Meng et al. [1] investigated the effects of semi- bated with GLPS and B16F10 cell culture supernatant and purified polysaccharides obtained from hot water extracts then activated by phytohemagglutinin. Treatment with from G. lucidum (GLPS) and G. sinense (GSPS) on the con- GLPS, fully or partly suppressed the production of the cyto- centration of the cytokines interleukin-1, interleukin-6, kines at both the mRNA and protein levels, and these data interleukin-10, and tumor necrosis factor- in RAW 264.7 suggest that GLPS may play a role in reducing cancer via macrophages. Results of this investigation demonstrated that this mechanism [25]. polysaccharides from both species significantly increased the levels of all cytokines tested, but the GLPSs were more po- In addition to polysaccharides (PSs), peptidoglycans tent than GSPSs. The overall results of this study demon- (PGs) are also known to be present in Ling-zhi, as well as strated that GLPSs and GSPSs enhanced macrophage func- proteoglycans with antiviral effects [26-30]. Along with the tion, by increasing phagocytosis, and by enhancing the pro- polysaccharides and peptidoglycans, the other naturally oc- duction of nitric oxide and cytokines [1]. Such data suggests curring constituents of G. lucidum (GL) that have anti-tumor that while GLPS and GSPS can both stimulate macrophage activities are the terpenes, a class of compounds with a car- activities, the GLPSs appear to be more effective. Further- bon skeleton made up of one or more isoprene C5 units [13]. more, the results demonstrated that concentrations of GLPSs The most common terpenes in GL are the triterpenes, a sub- in the range of 19–300 mg/ml stimulate the immune re- class that contains a C30 carbon skeleton and molecular Anti-Cancer Activities of Ganoderma lucidum (Ling-zhi) Current Bioactive Compounds 2017, Vol. 13, No. 1 31 weights in the range of 400 to 600 kDa, and a basic la- reduce the proliferation and metastases of breast cancer cells nostane skeleton (Fig. 2). These compounds are highly com- [43]. In one study, a GLE (in concentrations of 250-1000 plex and often oxidized [31, 32]. The primary triterpenes in g/mL) reduced the migration of MCF-7 cells caused by GL are based on a lanostane skeleton (Fig. 2) [33]. Isolation oxidation, through a mechanism that involved the down- of the triterpenes from G. lucidum is achieved by the use of regulation of the MAPK pathway. The extract reduced organic solvents, including the alcohols, as well as organic Erk1/2 phosphorylation, down-regulated c-FOS expression solvents such as acetone, dichloromethane, petroleum ether and inhibited AP-1 and NF-B. This extract also reduced IL- or combinations of solvents. Ganoderic acids A and B (Fig. 8 levels in MCF-7 cells [43]. Data generated from a 2014 1) were some of the initial ganoderic acids isolated, but since study also support these data [44]. These investigators stud- that time over 100 triterpenes have been isolated and identi- ied the effects of the same Ganoderma extract (GLE) on the fied from G. lucidum. While the ganoderic and lucidenic progression of tumors and metastatic disease in a rodent acids predominate the triterpenes, many other compounds model of cancer. In this study, MDA-MB-231 cancer cells including the ganoderals, ganoderiols, and ganoderic acids were implanted into nude mice and GLE in a dose of 100 have been isolated and identified from G. lucidum (Fig. 3) mg/kg was orally administered to the animals for 4 weeks. [15, 32, 34-42]. Some of the G. lucidum triterpenes with an- Although treatment of the mice with GLE reduced the size of titumor activities include ganoderic acid A, B, D, F, H, and the largest tumors, the results were not statistically signifi- K, and ganoderic acid DM (Fig. 1) [15]. cant. However, GLE treatments did significantly reduce MDA-MB-231 breast cancer cell metastases, and reduced The quality control/quality assurance (QA/QC) of Ling- expression of genes responsible for invasion [44]. zhi is difficult due to the various species and parts used. For example, there are six triterpenes constituents of G. lucidum In another recent study, the same extract, GLE, inhibited fruiting bodies that give a bitter taste, thus for products pre- inflammatory breast cancer cell (IBC) proliferation [45]. IBC pared with the fruiting bodies alone, these specific triterpe- is an aggressive and lethal disease that manifests without a nes may be used to determine the QC/QA [15]. However, typical tumor mass [45]. Current treatment involves the use since the bitter taste is not present in the mycelium, this of non-targeted systemic chemotherapy, surgery, and radia- method would not be useful for quality control for other tion. One investigation showed that the treatment of SUM- Ganoderma products. In fact, quality control for these prod- 149 IBC cells with GLE reduced the expression of the phos- ucts is difficult, as many chemical constituents have been phoinositide-3-kinase (PI3K)/AKT/mammalian target of shown to have some biological activity, including the triter- rapamycin (mTOR) pathway that regulates cell proliferation. penes. However, while many biologically active triterpenes In vivo, severe combined immunodeficient (SCID) mice that have been isolated and identified, to date there are few ex- were inoculated with IBC cells and then administered GLE amples of biologically active triterpenes being used in clinic (orally, 28 mg/kg) for 13 weeks showed a 50% reduction in as successful therapeutic agents [15]. Thus, in terms of using both tumor growth and weight. Tumors from the GLE Ling-zhi in clinical trials it is critical that these products are treated animals exhibited a suppression of E-cadherin, prepared the traditional way, according to the Chinese Phar- mTOR, eIF4G, and p70S6K, as well as a reduction of macopoeia, or they are chemically and biologically standard- ERK1/2 signaling [45]. ized based on experimental data. In terms of active anti-breast cancer constituents of G. lucidum (GL), the ganoderic acids (GA-A, GA-F and GA-H; see Fig. 1) were tested against MDA-MB-231, an invasive mammary cancer cell line [46]. The proliferation of MDA- MB-231 cells was determined after treatment with GA-A, GA-F and GA-H (0.10, 0.25 and 0.50 mM) for up to 72 h. A significant inhibition of growth was observed with GA-A and GA-H and an IC50 of ~ 0.5 mM was determined. The

HO results also showed that both GA-A and GA-H inhibited the growth of the cells by reducing the transcription factors AP- 1 and NF-B, down-regulating the expression of Cdk4 and uPA. However, similar concentrations of GA-F had no effect Lanosterol on cell proliferation [46]. In another study by Wu et al., Fig. (2). The primary triterpenes in Ganoderma lucidum are based (2013), ganoderiol A (GA; Fig. 3), dihydrogenated GA and on a lanostane skeleton. Lanosterol is an example of this skeletal GA isomers, inhibited both the adhesive capabilities and type. migratory properties of MDA-MB-231 cells in a dose range of 5-20 g/ml [47]. The GL triterpenes also reduced the ac- BREAST CANCER tivity of focal adhesion kinase (FAK), as well as reduced the Breast cancer is the primary cancer that is feared by most communication between FAK and SRC, leading to paxillin women and many times can progress to metastatic disease, deactivation. Application of GL triterpenes to MDA-MB- which is invasive and resistant to most therapies, with a high 231 cells led to a reduction in the expression of RhoA, Rac1, mortality [43]. Oxidative stress is a factor that initiates can- and Cdc42, as well as Wiskott-Aldrich Syndrome protein cer and its progression via reactive oxygen species (ROS) (N-WASP) and Cdc42. This study demonstrated that GL increasing the risk of angiogenesis and metastatic disease. triterpenoids reduced the metastatic process via the FAK- Ganoderma lucidum extracts (GLE) have been reported to SRC-paxillin signaling pathway [47]. 32 Current Bioactive Compounds 2017, Vol. 13, No. 1 Lawal et al.

OAc OH COOH O COOH

OAc

O AcO O OH H

Ganodermanotriol Lucidenic Acid B Fig. (4). Chemical structure of acetylated ganoderic acid (3,15- biacetoxy-22-hydroxy-5-lanosta-7,9(11),24-trien-26-oic acid (GA- T1) that induced programmed cell death in Hela cervical cancer cells as described by Liu et al. [48]. Acetylation increased the observed cytotoxicity.

CH2OH COLORECTAL CANCER HO OH Colorectal cancer is common in many countries, and is now one of the top three cancers diagnosed in the United

O States [49]. The association between dietary factors and the risk of colon cancer and death is well documented. Interest- HO ingly, there appears to be an inverse relationship between the ingestion of mushrooms and the risk of gastric cancers [49]. GANODERIOL A Of these medicinal mushrooms, GL is well recognized as Fig. (3). Structural examples of the lucidenic acids and ganoderiols being effective for reducing colon cancer risk in animal stud- from G. lucidum. ies. Extracts of GL have been shown to inhibit colon cancer in numerous in vivo models, and in vitro GL inhibits cell CERVICAL CANCER proliferation and induced programmed cell death in human colon cancer cells [50-52]. In a 2015 study, Liu et al. reported that ganoderic acids (GAs) isolated from G. lucidum possess anticancer activities In a 2006 study, Xie et al. [53] tested different fractions via a mechanism that involves the formation of reactive oxy- of a GL extract (GLE) against the SW 480 human colorectal gen species (ROS) [48]. This investigation determined the cancer cell line. In this study, two GLE fractions, one effects of four structurally related GAs, i.e. GA-T, GA-Mk, polysaccharide and triterpenoid, containing fraction (GLE- and two deacetylated derivatives of GA-T (Fig. 4), 3,15- 1), and the other triterpenoid-containing fraction minus biacetoxy-22-hydroxy-5-lanosta-7,9(11),24-trien-26-oic polysaccharides (GLE-2) were investigated. The results of acid (GA-T1) and 3-acetoxy-15,22-bihydroxy-5-lanosta- this study demonstrated that GLE-1 and GLE-2 were able to 7,9(11),24-trien-26-oic acid (GA-T2) on the antioxidant de- reduce the growth of SW 480 cells, but the GLE-2 fraction, fense system and the induction of programmed cell death in at a concentration of 50 g/ml, was more active than the HeLa cervical cancer cells. The approximate IC50 to HeLa GLE-1 at a concentration of 250 g/ml, however GLE1 also and ranking order of the four GAs – T2 (with two hydrox- had antioxidant effects and reduced DNA synthesis, thus yls), T1 (with one hydroxyl), Mk (with one hydroxyl) and T suggesting that the fraction containing both the (no hydroxyl) were 82 > 31 30 > 11 M [48]. The results polysaccharides and triterpenes had better cytotoxic effects indicated that the tested GAs enhanced apoptosis by decreas- than the polysaccharide extract alone [53]. ing the mitochondrial membrane potential and by activating In a 2008, and 2009 study, Chen and co-workers demon- both caspase-9 and caspase-3 [48]. They further reported that strated that purified ganoderic acid T (GA) isolated from GL GAs enhanced the production of reactive oxygen species by inhibited the growth of the colon cancer cell line HCT-116 reducing glutathione (GSH) level, superoxide dismutase [54, 55]. GA enhanced cell aggregation and prevented HCT- (SOD) and glutathione peroxidase (GPX) activities. Adding 116 cells from adhering to the extracellular matrix [54, 55]. antioxidants such as N-acetyl cysteine, catalase and diphen- GA also inhibited NF-B, suggesting that this compound has yleneiodonium chloride inhibited the above effects. The re- anti-inflammatory effects. In the Lewis Lung Carcinoma sults also showed that there was a decrease in the mitochon- (LLC) model, GA reduced tumor proliferation and the de- drial membrane potential (m) in HeLa cells treated with velopment of metastatic disease, as well as reduced the ex- up to 40 M of GAs for 24 h. The relative ranking order of pression of the matrix metalloproteinase (MMP)-2 and these GAs for their induced m disruption was: GA-T2 < MMP-9 mRNA [54], further suggesting that the triterpenes GA-T1 GA-Mk < GA-T. The results of this study indicate isolated from GL have cytotoxic effects. Another GL triter- that GAs induce HeLa apoptosis through increasing oxida- pene named ganodermanontriol (GNDT; Fig. 5) inhibited the tive stress and by reducing antioxidant responses. Acetyla- growth of HCT-116 and HT-29 colon cancer cells (~10- 80 tion of hydroxyl groups in GAs may contribute to their pro- M, each) by impacting cell cycle through a reduction of oxidant activities and cytotoxicity [48]. cyclin D1 [56]. A reduction in Cdk-4 and PCNA expression Anti-Cancer Activities of Ganoderma lucidum (Ling-zhi) Current Bioactive Compounds 2017, Vol. 13, No. 1 33

24 R 25 4 R7 11

9 R6 8

7 R3

R1 R2 H

Compound R1 R2 R3 R4 R5 R6 R7 Double Bond

Ganolucidic acid E O H OH O H COOH H 8,9

Lucidumol A O O H H OH OH CH3 8,9

Ganodermanontriol O H H H OH OH CH2OH 7,8;9,11 7-oxo-ganoderic acid Z OH O H H H COOH H 8,9;24,25

15-hydroxy-ganoderic acid S O H OH H H COOH H 8,9;24,25 Ganoderic acid DM O O H H H COOH H 8,9;24,25

Fig. (5). Various triterpenes isolated by from G. lucidum. Ganodermanontriol reduces the growth of both HCT-116 and HT-29 colon cancer cells [58]. was observed, but Cdk-2, p21 and cyclin E expression was GLPs promoted the expression of Fas and caspase-3 pro- not seen. GNDT increased the levels of E-cadherin and ß- teins, whilst reducing the expression of cleaved poly catenin proteins in the HT-29 cells. Furthermore, in a HT-29 (ADP-ribose) polymerase. Their data indicated that GLPs xenograph model, GNDT inhibited tumor growth in nude demonstrated potential antitumor activity in human colon mice by reducing cyclin D1 protein expression in the tumors. cancer cells, predominantly by the inhibition of migration No adverse effects were observed [56]. and an increase in programmed cell death. Furthermore, induction of the Fas/caspase-dependent apoptosis pathway is A triterpene extract derived from GL (GLT, 1-50 g/ml) involved in the cytotoxicity of GLPs [60]. reduced the growth of the human colon cancer cell line HT-29 and inhibited HT-29 tumor growth in rodents [57]. The triter- Furthermore, a 2010 clinical study involving patients pene extract arrested the cell cycle at the G0/G1 stage and in- with colorectal adenomas investigated the effects of GL [61]. duced autophagy in the HT-29 cancer cells by increasing the An aqueous extract from a cultured medium of GL mycelia development of autophagic vacuoles. In the HT-29 cells, GLT (GLM) was tested for its potential chemopreventative effects increased the Beclin-1 and LC-3 expression. These are two in humans [61]. In this clinical study, Japanese patients who proteins that are well known to be involved in the induction of were diagnosed with colorectal adenomas were selected to autophagy. The GLT also reduced the activity of p38 mitogen- participate. The GLM group was administered 1.5 g/day of activated protein kinase. In a rodent xerograph model, GLT the extract orally for one year. A colonoscopy was scheduled also inhibited the colon cancer tumor growth by inducing 12 months after the initiation of therapy and information autophagy, indicating a novel mechanism of action [57]. about all adenomas was recorded. 96 patients were enrolled in the GLM group and 102 eligible patients in the no- In a 2014 study, the effects of GL polysaccharides (GLP) treatment control group. The number of adenomas in the were investigated in HCT-116 cells [59]. HCT-116 cells that control no-treatment group was increased at 12 months by were treated with varying amounts of GLP (0.625-5 mg/ml), 0.66 +/- 0.10, however in the GLM group, treatment de- showed a significant reduction in cell proliferation (P< 0.01). creased the number of adenomas by -0.42 +/- 0.10 (p < The cytotoxic effects of GLP were due to inhibition of cell 0.01). In the control patients, the size of the adenomas in- migration, changes in cell morphology, elevations in cellular creased to 1.73 +/- 0.28 mm, but in the GLM treated pa- calcium levels and release of LDH. Western blotting analysis tients, the adenomas significantly decreased to -1.40 +/- 0.64 showed that the expression of the proteins Fas and caspase-3 mm (p < 0.01) [61]. were increased after treatment with GLP. GLP appears to increase apoptosis in HCT-116 cells by increasing calcium GASTRIC CANCER release and apoptosis [59]. In a 2015 study, Liang et al. [60] evaluated the anticancer effects and underlying molecular In 2015, Reis et al. [62] examined the effects of a metha- mechanisms of GLPS isolated from GL on LoVo human nol extract of G. lucidum on the gastric cancer cell line colon cancer cells. The experimental results demonstrated (AGS). Treatment of the AGS cells with the methanol ex- that the GLPS-mediated anticancer effect in LoVo cells was tract induced autophagy by increasing the production of characterized by cytotoxicity, migration inhibition, enhanced autophagosomes. In addition, the LC3-II levels in the cells DNA fragmentation, morphological alterations and increased were also enhanced, and there was also a reduction in the lactate dehydrogenase release. The authors further reported amount of p62, indicating that the GL methanol extract in- that the activation of caspases-3, -8 and -9 was involved in duced autophagy. An increase in the levels of both LC3-II GLP-stimulated apoptosis. Additionally, treatment with and p62 was also observed when the AGS cells were treated 34 Current Bioactive Compounds 2017, Vol. 13, No. 1 Lawal et al. with both the methanol extract and lysosome protease inhibi- tors. Results from these experiments showed that the GL methanol extract induced autophagy and did not alter the rate of autophagy flux [62]. In another 2015 study by Ruan et al.,

[58] six triterpenoids namely ganolucidic acid E, lucidumol O A, ganodermanontriol (Fig. 5), 7-oxo-ganoderic acid Z, 15- hydroxy-ganoderic acid S, and ganoderic acid (GA) were O isolated from a GL extract and identified through chromatographic means. The compounds were tested in three human HO cancer cell lines, namely Caco-2, HepG2, and HeLa cells. Each of the compounds inhibited the growth of these cell 9,11-dehydroergosterol peroxide lines via apoptosis in a concentration dependent manner, with IC50s of 20.87 – 84.36 mM [58]. In the HeLa cell line, the six compounds increased the sub-G1 accumulations by 22% and in the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay, 43.03% of the cells showed apoptosis, which was also confirmed using the an- O nexin-V assay. The 7-oxo-ganoderic acid Z and ganoderic acid DM also induced apoptotic changes in Caco-2 cells. No O compound was effective against HepG2 cells [58]. HO LIVER CANCER Ergosterol peroxide Since liver cancer is an important cause of mortality in Asian countries, G. lucidium (GL) is often used as a func- Fig. (6). Two active peroxide compounds, 9, 11-dehydroergosterol tional food in China as adjunct therapy for this disease [55]. peroxide [9(11)-DHEP] and ergosterol peroxide (EP) isolated from One in vitro study investigated the cytotoxicity of an ethanol GLE. The median inhibitory concentrations of 9(11)-DHEP and EP extract of GL mycelia (GLE) on Hep 3B liver cancer cells on the growth of Hep 3B were 16.7 and 19.4 g/ml, respectively that were grown on a black soybean based media that also [55]. contained Astragalus membranaceus, a traditional herb from China [55]. Treatment of the Hep 3B cells with the GLE cells was determined in vitro [64]. Cultured PG cells were extract resulted in a concentration and time-dependent reduc- treated with GLPP in varying concentrations, and proliferation in cell growth. The median inhibitory concentration tion, cell migration, adhesion, zymography and RT-PCR assays were performed. The GLPP did not directly inhibit (IC50) of the GLE was 156.8, 89.9 and 70.1 g/mL, after treatment for 24, 48, and 72 h, respectively. Two identified PG cell proliferation, however when pretreated with GLPP, peroxides, namely 9,11-dehydroergosterol peroxide [9(11)- PG cell motility and adhesion were significantly inhibited. DHEP] and ergosterol peroxide (EP) (Fig. 6), appeared to be GLPP also reduced the activity of matrix metallopeptidase 9 the actives and had an IC50 in the Hep 3B cells of 16.7 and and the expression of its mRNA. The observed effects were 19.4 g/mL, respectively [55]. concentration dependent. A 41.53% inhibition was observed at 100 mg/L GLPP [64]. In a 2015 study, Li et al. [63] examined the therapeutic effects of GL polysaccharides (GLPS) on hepatocellular car- OVARIAN CANCER cinoma (HCC) in mice. This investigation measured the effects of GLPS on the ratio of regulatory T cells (TR) and Ovarian cancer (OC) is a serious gynecological cancer, effector T cells (TE) in a rodent model of liver cancer [63]. with a high mortality rate generally due to late diagnosis, as The concentrations of microRNAs (miRNAs) and mRNA well as a lack of safe and effective treatments for late stage were determined using qPCR and a microRNA-125b (miR- disease [65]. The majority of patients will experience cis- 125b) inhibitor was employed to reduce the levels of miR- platin resistance, thus new strategies to treat the disease are 125b. The results of this study showed that GLPS reduced urgently needed. One in vitro study tested the cytotoxicity tumor growth and this was due to an increase in the balance and anti-migration effects of a semi-purified polysaccharide of TEs to TRs. In vitro, GLPS treatment of the T cells re- extract of GL on the ovarian cancer cells IOSE-398, EOC, duced the expression of Notch1 and FoxP3 by increasing the OV2008, C13, A2780s and A2780-cp [65]. The results of expression of miR-125b. In vivo the miR-125b inhibitor also this study showed that GLPS (at 1 mg/ml) inhibited the inhibited the antitumor effects of GLPS on tumor load in the growth of the ovarian cancer lines by apoptosis and the mice. The results of this investigation showed that GLPS down-regulation of anti-apoptotic proteins. A semi-purified may have a very interesting antitumor mechanism in that GLPS extract reduced the resistance of ovarian cancer cells suppresses the growth of HCC via miR-125b-inhibition of to cisplatin, through a mechanism that involved an increase the increase of TRs and also suppresses its function [63]. in the expression of p53 and a reduction in the expression of Akt [65]. Since ovarian cancer is extremely difficult to treat, LUNG CANCER and cisplatin resistance is common, the adjunct use of GLPS The effects of a GL polysaccharide peptide (GLPP) on to reverse chemoresistance in these patients may be a very the invasion potential of the human lung carcinoma (PG) exciting and clinically significant advancement.

Anti-Cancer Activities of Ganoderma lucidum (Ling-zhi) Current Bioactive Compounds 2017, Vol. 13, No. 1 35

In a 2011 study, Hsieh and Woo [66] investigated the animals, the tumor cells in the three GLPS groups all showed antitumor activities of a water and ethanol extract of GL in a significant reduction in RNA and DNA content, at an oral human ovarian OVCAR-3 cells. Both GL extracts inhibited dose of 200 mg/kg (P = 0.000). A significantly reduced cell growth after 72 hrs of treatment, but the ethanol extract RNA/DNA ratio was also observed in all three GLPS groups (GLE) was more effective than the aqueous extract (GLA), treated with 400, 200, or 100 mg/kg, (P = 0.003, 0.000, with an IC50 of 10 g/ml. In addition, the GLE inhibited col- 0.008), respectively. All doses of GLPS raised the percent- ony formation in the OVCAR-3 cells and was more effective age of G2/G2 phase cells. GLPS also decreased the percent- than the GLA [66]. The GLE disrupted the cell cycle of age of G2/M phase tumor cells, while CTX did not (P = OVCAR-3 through a mechanism that involved a reduction in 0.000). The study results indicated that GLPS treatments the expression of cyclin D1. These authors also showed that inhibit DNA and RNA synthesis in the tumor cells, and may the GLE and GLA induced the activities of specific enzymes enhance host immune function thereby interfering with the such as SOD and catalase, and specific phase II detoxifica- normal cell cycles [68]. tion enzymes, namely NAD(P)H:quinone oxidoreductase 1 The antitumor effects of sulfated and carboxymethylated- and glutathione S-transferase PT [66]. Since the aqueous modified polysaccharides isolated from G. lucidum (S-GL extract would have contained primarily polysaccharides, it is and CM-GL) were investigated in S-180 cells [69]. Both likely that the both the polysaccharides and the triterpenes polysaccharide S-GL and CM-GL reduced the growth of S- played a role in the activity. 180 cells in a concentration-dependent manner, having an Dai et al. [67] investigated potential mechanism of action IC50 of 26 and 38 g/mL, respectively. The polysaccharides for GL in the human ovarian cancer cell line HO8910 also exhibited antitumor effects in S180 solid tumors in (HOCC) and HPOC. Both the cell lines were treated with a BALB/c mice. No toxicity was observed after administration commercial extract of GL, and the effects of GL treatment of the polysaccharide preparations. Both S-GL and CM-GL on cell viability was determined using an MTT assay. Also polysaccharide reduced cell growth by inhibiting the cell assessed in this study was the expression of vascular endo- cycle at the G2/M phase. In terms of a mechanism of action thelial growth factor (VEGF) and connexin 43 (Cx43). Cx43 the pro-apoptotic protein Bax was up-regulated while the expression in HOCC was reduced using small interference expression of the anti-apoptotic protein Bcl-2 was reduced. RNAs. These authors reported that GL treatment concentra- The chemical modification of these two polysaccharides was tion-dependently (10 g/ml) reduced the growth of HOCC, shown to be an important aspect of their antitumor effects VEGF expression and an increase in Cx43 expression in the [69]. cancer cells [67]. Furthermore, the effects of GL on the viability of HOCC cells were reduced when Cx43 expression Another in vivo study demonstrated the anti-tumor effects of a purified polysaccharide peptide isolated from GL was reduced but the effect of GL on VEGF expression was (GLPP) [70]. GLPP displayed an average MW of 6,600. In not altered. The study concluded that extracts of GL reduced this study, GLPP was tested for its antitumor effects and to the growth of ovarian cell lines through a mechanism that determine a potential impact on the immune response. The involved a reduction of VEGF expression, and an increase in study also determined the possible effects of GLPP on the the expression of Cx43, by down-regulating the expression of VEGF and up-regulating the downstream Cx43 expres- reduction of adverse effects in mice caused by cyclophos- phamide (CTX) treatment and Cobalt-60 (Co) radiation. sion. The study concluded that extracts of GL were poten- GLPP was administered to the mice at a dose of 100 and 300 tially promising treatments for ovarian cancer [67]. mg/kg body weight per day. These treatments reduced the ASCITIC TUMORS AND IMMUNE FUNCTION growth of S180, Heps, and EAC tumor cells that had been implanted in mice. In addition, GLPP treatments increased Mouse sarcoma S180 is an aggressive form of cancer that phagocytosis in a dose-dependent manner suggesting an ef- is useful in studying rodent models as after they are injected fect on the immune system. In addition, at a dose of 300 into the peritoneum they grow very quickly and have a gen- mg/kg body weight per day, the GLPP treatment was as ef- eration time between twelve to eighteen hours [68]. As these fective as PSP (1000 mg/kg) in reducing adverse effects ob- cells grow in the peritoneum they begin to block the flow of served after CTX and Co radiation treatments [70]. The lymphatic fluids, causing a backup of these fluids into the study suggests that GLPP may be of potential benefit to use peritoneal cavity causing ascites. The S180 cell line is also as an adjuvant treatment in combination with standard of capable of causing metastatic disease of the primary organs care cancer treatments to reduce adverse effects and enhance such as the lung, liver, kidneys, pancreas, intestine and the immune response. stomach in a period of weeks, leading to mortality within three to four weeks. The ascites caused by the growth of In 2010, Zhang et al., isolated and identified a novel S180 is a highly aggressive cancer in rodent models. Li and proteoglycan, named GLIS that was obtained from a GL co-workers [68] investigated the effects of GLPS on RNA extract and was shown to directly stimulate the activation of and DNA, and the cell cycle of S180 cells in rodents. GLPS B-lymphocytes in vitro, as well as in BALB/c mice inocu- was administered orally at doses of 100-400 mg/kg to mice lated with S180 sarcoma [71]. In this study, S180 tumor- bearing mice were treated with this proteoglycan and the - with S180 ascitic tumors. Controls included mice treated with normal saline or subcutaneous injection of cyclophos- lymphocytes were isolated from the spleen. These - phamide (CTX) at 25 mg/kg. Treatments were administered lymphocytes were activated after GLIS treatment, and GLIS once daily for 9 days and then ascitic tumor cells were har- administration also enhanced the production of immuno- vested and the RNA and DNA levels, as well as the cell cy- globulin. In the bone marrow, GLIS also increased the activ- cle alterations were measured. Compared with saline treated ity of macrophages and enhanced the production of sub- 36 Current Bioactive Compounds 2017, Vol. 13, No. 1 Lawal et al. stances that regulate the immune response, including inter- R3 leukin-1, tumor necrosis factor- and nitric oxide. The phagocytic capacity of macrophages was also increased as COOH well as their cytotoxicity actions against tumor cells. GLIS treatments suppressed the proliferation of the S180 tumor in BALB/c mice by as much as 60%, indicating the potent effects that GLIS had on stimulating the immune system that R2 played an important role in its antitumor activity [71]. AcO R1

MISCELLANEOUS CANCER AND TUMOR CELL 1. R1 = OH, H; R2 = H2; R3 = OAc LINES 3. R1 = OAc, R2 = OH, R3 = OAc R3 Two extracts of GL were assessed for their potential to treat B16 mouse melanoma and sarcoma tumors in mice and rats [72]. A semi-purified extract containing primarily triter- COOH penes (GLT), as well as an extract containing acidic terpenoids (GLAT), were prepared. B16 mouse melanoma cells were injected under the skin of C57BL/6 mice. Administra- 2. R1 = OAc, H; R2 = OH, H; R3 = OAc R2 4. R1 = OH, H; R2 = H2; R3 = OAc tion of either GLT or GLAT suppressed the growth of the 5. R1 = OAc, H; R2 = OH, H; R3 = H2 R melanoma cells after they were inoculated subcutaneously 1 into syngeneic C57BL/6 mice. In cell culture, GLT exerted a strong effect and reduced the proliferation of the B16 cells. Fig. (7). A new ganoderic acid (GA), 3,22-diacetoxy-7- In the cancer cell lines, L929-mouse fibrosarcoma and C6-rat hydroxyl-5-lanost-8,24E-dien-26-oic acid (1), together with four astrocytoma, GLT reduced cell viability, and induced apop- known compounds GA-Mk (2), -Mc (3), -S (4) and -Mf (5), were tosis via a mechanism that involved the caspases and an in- isolated from G. lucidum mycelia. The structure of compound 1 crease in the expression of p53. The up-regulation of Bcl-2 was elucidated using HRMS, IR, UV, 1D and 2D NMR. When tested in vitro for their cytotoxic activities against 95D and HeLa was also observed. Interestingly, GLT appeared to increase tumor cell lines, these compounds had an IC50 concentrations rang- reactive oxygen species (ROS) production, and treatment of ing from 14.7 to 38.5 M [73]. the cells with N-acetyl cysteine reduced the effect of GLT- induced apoptosis, indicating that the promotion of ROS by GLT was part of the mechanism of action [72]. extract reduced prostate cancer cell proliferation in a concen- In a 2013 study, a fraction from a GL extract containing tration dependent manner. The GLT also induced apoptosis, triterpenes, including a novel compound named “ethyl 7- as well as arrested the cell cycle in the G1 phase. Study of hydroxy-4,4,14-trimethyl-3,11,15-trioxo-5-chol-8-en-24- caspase-3 and caspase-9 indicated that GLT reduced cell oate”, and the cytotoxic effects of this new compound were viability through an apoptotic mechanism by increasing the determined in the cancer lines K562, HL-60, CA46, HepG2, activity of these two caspases. Furthermore, GLT arrested SW480 and SMMC-7221 [73]. The results showed that this the cell cycle in these cancer cell lines at G1 by increasing new compound exhibited good cytotoxicity against the leu- the expression of p21 and by reducing the expression of cy- kemia cell lines HL-60 and CA46 with an IC50 of 25.98 and clin-dependent kinase 4 and E2F1. The results of this study 20.42g/ml, respectively [73]. In another 2013 study, Li et indicate that the GLT fraction reduced the growth of prostate al. [74] reported the isolation of a novel ganoderic acid cells by inducing apoptosis and G1 cell cycle arrest [75]. named 3,22-diacetoxy-7-hydroxyl-5-lanost-8,24E-dien- 26-oic acid from a GL mycelium extract. This group also COMBINATION OF G. LUCIDUM WITH STANDARD isolated and identified four previously identified compounds CHEMOTHERAPY namely GA-Mk, GA-Mc, GA-S and GA-Mf (Fig. 7) [74]. A combination study investigated the effects of a poly- The compounds were identified using standard structure elu- saccharide isolated from G. lucidum named LZP-F3 and the cidation methods including high-resolution mass spec, and chemotherapeutic agents, cisplatin and arsenic trioxide (AT), 2D-nuclear magnetic resonance. All of the isolated com- in human urothelial cancer cell lines [76]. The cell lines in- pounds, including the new structure were tested in cell cul- cluded: NTUB1, a cisplatin-resistant cell line (N/P14), as ture to determine their effects on 95D and HeLa cancer cells. well as an arsenic-resistant cell line (N/As0.5) [76]. Using The results showed that all compounds had activity and the the MTT assay and a median-effect analysis the authors IC50 concentration range was 14.7 to 38.5M [74]. showed that LZP-F3 reduced the chemoresistance of N/P14 In 2015, Wang et al. [75] investigated the effect of a total and N/As0.5 to cisplatin and arsenic. The mechanism of ac- triterpenoid fraction isolated from G. lucidum (GLT) on a tion appears to involve an increase in the activity p38 and a number of prostate cell lines, including LNCaP, 22Rv1, PC- reduction in the expression of Akt. In a concentration of 10 3, and DU-145. They also tested the extract against an im- g/mL, LZP-F3 impacted the cell cycle at G1, and the cell mortalized line of benign prostate hyperplasia cells (BPH) cycle arrest appeared to be due to an increase in of p21. [75]. Flow cytometry was used to assess the effects of the When LZP-F3 and AT were combined the effect was syner- GLT extract on cell proliferation, apoptosis, and the cell cy- gistic and inhibited the growth of all cancer cells. The cle. The results of this study demonstrated that the GLT mechanism by which cell growth was inhibited included

Anti-Cancer Activities of Ganoderma lucidum (Ling-zhi) Current Bioactive Compounds 2017, Vol. 13, No. 1 37 increase of FAS, enhancement of caspase 3 and 8, an in- this cell line to DOX. Treatment of the HepG2/ADM cells crease in the expression of Bax and Bad, as well as a reduc- with GAB increased rhodamine-123 accumulation by reduction in Bcl-2 and Bcl-xL [76]. ing its efflux. Using silencing RNAs for ABCB1, it was further discovered that the ABCB1 was necessary for this ef- In a study by Yue et al. [77], the interaction of a group of fect; however neither ABCB1 expression, nor the alteration triterpenes isolated from G. lucidum with doxorubicin was of the ABCB1 ATPase activity appeared to be involved in assessed in HeLa cells [77]. The triterpenes tested included: the mechanism of action [48]. 3,7,15-trihydroxy-4,4,14-trimethyl-11-oxochol-8-en-24-oic acid, 3,7-dihydroxy-4,4,14-trimethyl-11,15-dioxochol-8-en- Investigation of the molecular docking of GAB demon- 24-oic acid (LCN); 3-hydroxy-4,4,14-trimethyl-7,11,15- strated that GAB bound to different regions of ABCB1 than trioxochol-8-en-24-oic acid; 12-acetoxy-3-hydroxy-4,4,14- verapamil. Further results indicated that GAB overcame trimethyl-7,11,15-trioxochol-8-en-24-oic acid (lucidenic acid chemoresistance of MDR cancer cells to chemotherapy by E); and (5) 7,15-dihydroxy-4,4,14-trimethyl-3,11-dioxochol- inhibiting the ABCB1 transport function of ABCB1, thereby 8-en-24-oic acid (77; Fig. 8). When combined with doxoru- increasing the chemotherapeutic effect. These results again bicin (DOX), GLT or LCN enhanced the effect of DOX and suggest that GAB has significant effects against MDR cancer the effect was found to be synergistic in HeLa cells. Treating cells and may be a novel ABCB1-mediated MDR reversal the cells with GLT increased the expression of numerous agent [48]. proteins that were involved in cell growth, cell cycle, programmed cell death, and oxidation. Treatment of the cells CONCLUSION increased the production of ROS by DOX. The study con- Products prepared from the fruiting bodies of Ling-zhi cluded that there is a synergistic effect produced when DOX have been used in TCM for thousands of years. More re- and GLT are used together, and that the mechanism by cently, the mycelia and spores of have also been used in the which GLT sensitizes the cells to DOX appears to involve an manufacture of these products. Specific therapeutic effects of increase in oxidation, and programmed cell death [77]. These Ling-zhi include the treatment of diabetes, as an immune results correlate well with the previous studies and suggest stimulant, liver protection, antimicrobial, and as an adjunct that GL extracts and pure compounds can reduce or reverse therapy for cancer. The results of many cell culture and ani- chemoresistance to standard chemotherapeutic drugs. mal studies suggest that various extracts produced from G. lucidum that contain polysaccharides and/or triterpenes have cytotoxic and anti-tumor effects. Analysis of the chemistry shows that hundreds of compounds have been identified, although the polysaccharides, peptidoglycans, and triterpenes are thought to be the primary biologically active constituents. Biological assessment of the extracts containing polysaccharides show an enhanced immune response by increasing macrophage function, increasing phagocytosis, and enhancing nitric oxide production, as well as increasing the interleukins (IL-1, IL-6, and IL-10). However, the polysaccharides have also been shown to have cytotoxic effects on colon and lung cancer in vitro, and one small human study suggested that the polysaccharides also reduced colorectal adenomas. In vivo, oral administration of the GL derived Fig. (8). The structures of the GL triterpenes: (1),7,15-trihydroxy- polysaccharides reduced liver and sarcoma tumors in rodent 4,4,14-trimethyl-11-oxochol-8-en-24-oic acid; (2) 3,7-dihydroxy- models. In addition, GL-derived triterpenes have shown 4,4,14-trimethyl-11,15-dioxochol-8-en-24-oic acid (LCN); (3) 3- good activities in breast, cervical, colon, gastric, liver, and hydroxy-4,4,14-trimethyl-7,11,15-trioxochol-8-en-24-oic acid; (4) ovarian cancer cells. Furthermore, some studies showed sig- 12-acetoxy-3-hydroxy-4,4,14-trimethyl-7,11,15-trioxochol-8-en- nificant anti-cancer effects using GL extracts containing both 24-oic acid (lucidenic acid E); and (5) 7,15-dihydroxy-4,4,14- the polysaccharides and triterpenes. Finally, combination trimethyl-3,11-dioxochol-8-en-24-oic acid [77]. Compound LCN studies of GL polysaccharides or triterpenes with standard (2) was synergistic with doxorubicin in HeLa cells. chemotherapeutic drugs also show synergistic anti-cancer effects, indicating a potential to use G. lucidum in combination with chemotherapy as an adjunct treatment. Thus, over- The effects of ganoderenic acid B (GAB, Fig. 1), a la- all Ling-zhi shows significant potential for the development nostane-like triterpene isolated from a GL extract, were de- as adjunct therapy for cancer. termined on ATP-binding cassette (ABCB1) mediated multidrug resistance (MDR) [48]. ABCB1 is a glycoprotein However, to date few in vivo xenograph studies (consid- that has been well investigated as it is thought to cause ered the gold standard) have been performed to validate multidrug resistance in cancer cells, and is a product of the these results, and additional clinical trials are needed to as- MDR1 gene. In the study by Liu et al. [48] HepG2/ADM sess the effectiveness of using GL as an adjunct to chemo- cells were treated with GAB and DOX, vincristine or pacli- therapy. Since both the triterpenes and polysaccharides ap- taxel. The results showed that GAB could reverse the effects pear to have activity against a wide range of cancer and tu- of ABCB1-induced MDR. In addition, in ABCB1/MCF- mor cells in cell culture and animal models, it is possible that 7/ADR breast cancer cells, GAB reversed the resistance of these chemical constituents may act either additively or syn- 38 Current Bioactive Compounds 2017, Vol. 13, No. 1 Lawal et al. ergistically to produce the overall effects. Further studies [11] Liu, R.M.; Li, Y.B.; Zhong, J.J. Cytotoxic and pro-apoptotic effects looking specifically at synergistic and additive effects be- of novel ganoderic acid derivatives on human cervical cancer cells in vitro. Eur. J. Pharmacol., 2012, 681(1-3), 23-33. tween GL extracts and chemotherapeutic agents are also [12] Xie, J.; Zhao, J.; Hu, D.J.; Duan, J.A.; Tang, Y.P.; Li, S.P. Com- needed. parison of Polysaccharides from Two Species of Ganoderma. Molecules, 2012, 17, 740-752. Some of the most significant problems with Ling-zhi [13] Yuen, J.W.M.; Gohel, M.D.I. Anticancer effects of Ganoderma research are the issues of sourcing, identification and authen- lucidum: A review of scientific evidence. Nutr. Cancer, 2005, 53, tication of the materials and overall quality control, all which 11-17. need to be addressed for potential future animal or human [14] Guo, X.Y.; Han, J.; Ye, M.; Ma, X.C.; Shen, X.; Xue, B.B.; Che, Q.M. Identification of major compounds in rat bile after oral studies. In recent years, there has been much taxonomic con- administration of total triterpenoids of Ganoderma lucidum by fusion associated with GL and other species, and the identi- high-performance liquid chromatography with electrospray fication of Ling-zhi has come into question [78-80], and ionization tandem mass spectrometry. J. Pharm. Biomed. Anal., these issues need to be addressed and resolved. A recent 2012, 63, 29-39. study by Zhou et al. [79], analyzed 32 collections for their [15] Da, J.; Wu, W.Y.; Hou, J.J.; Long, H.L.; Yao, S.; Yang, Z.; Cai, L.Y.; Yang, M.; Jiang, B.H.; Liu, X.; Cheng, C.R.; Li, Y.F.; Guo, morphological and molecular phylogenetic perspectives D.A. Comparison of two official Chinese pharmacopoeia species of from Asia, Europe and North America and have discussed Ganoderma based on chemical research with multiple technologies the issues and taxonomic positions of these species. It is and chemometrics analysis. J. Chromatogr. A, 2012, 1222, 59-70. critical the Ling-zhi products used in future clinical trials are [16] Ren, L.; Perera, C.; Hemar, Y. Antitumor activity of mushroom polysaccharides: a review. Food Funct., 2012, 3, 1118-1130. well defined and analyzed, to ensure that these studies can be [17] Bao, X.; Fang, J.; Li, X. Structural characterization and immuno- repeated and duplicated. modulating activity of a complex glucan from spores of Gano- derma lucidum. Biosci. Biotech. Biochem., 2001, 65(11), 2384- CONFLICT OF INTEREST 2391. [18] Xie, J.T.; Wang, C.Z.; Wicks, S.; Yin, J.J.; Kong, J.; Li, J.; Li, The authors confirm that this article content has no con- Y.C.; Yuan, C.S. Ganoderma lucidum extract inhibits proliferation flict of interest. of SW 480 human colorectal cancer cells. Exp. Oncol., 2006, 28(1), 25-29. [19] Hikino, H.; Konno, C.; Mirin, Y.; Hayashi, T. Isolation and hypo- ACKNOWLEDGEMENTS glycemic activity of ganoderans A and B, glycans of Ganoderma We would like to express our deep appreciation to the lucidum fruit bodies. Planta Med., 1985, 51, 339-340. [20] Miyazaki, T.; Nishijima, M. Structural examination of a water- Schlumberger Foundation for their fellowship support of this soluble, antitumor polysaccharide of Ganoderma lucidum. Chem. work. The contents are solely the responsibility of the Pharm. Bull., 1981, 29, 3611-3616. authors and do not necessarily represent the official views of [21] Tomoda, M.; Gonda, R.; Kasahara, Y.; Hikino, H. Glycan struc- the funding agency. tures of Ganoderans B and C, hypoglycemic glycans of Ganoderma lucidum fruit bodies. Phytochemistry, 1986, 25, 2817-2820. [22] Wang, Y.Y.; Khoo, K.H.; Chen, S.T.; Lin, C.C.; Wong, C.H.; Lin, REFERENCES C.H. 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