MAJOR BIOACTIVE TRITERPENOIDS FROM GANODERMA SPECIES AND THEIR THERAPEUTIC ACTIVITY: A REVIEW

Ganoderma a traditional Chinese medicine popularly used for complementary cancer therapy and longevity for centuries. The vast amount of study has been performed on the medicinal properties of Ganoderma lucidum. G. lucidum contains various compounds with a high grade of biological activity, which increase the immunity. Several of these substances belong to the triterpenoids and polysaccharides. Proteins, sterols, phenols, lipids, etc., are also present. Ganoderma triterpenes are important secondary metabolites of G. lucidum. Ganoderma triterpenes are limestone-tetracyclic terpenes which have been reported to possess antioxidant, antitumor, anti-human immunodeficiency virus, anticancer, anti-inflammation, cytotoxic, hepatoprotective, and neuroprotective activities. This review deals with most important triterpenes isolated from Ganoderma and their therapeutic effects.


INTRODUCTION
Ganoderma is a wood-rotting mushroom with hard fruiting body and grows on decaying tree stumps or logs. Ganoderma is known by various popular names as "Reishi" in Japan, "Lingzhi" in China, and "Yeongji" in Korea. The taxonomical studies have reported about 300 species which belong to genus Ganoderma and the majority of which are distributed in tropical regions. Species of Ganoderma are corky, tough, and thick. Ganoderma does not have the fleshy texture and thus do not qualify to be considered as edible mushrooms [1,2]. Some of the important species of Ganoderma on which most of the research work on medical aspects have been carried out are as follows: Ganoderma lucidum, G. sinensis, G. theaecolum, G. zonatum, G. applanatum, G. pfeifferi, G. tsugae, G. resinaceum, G. amboinense, G. colossum, G. formosanum, G. australe, and G. atrium. G. lucidum is the most commonly characterized medicinal mushroom of the genus Ganoderma [3][4][5][6][7][8]. The more weight of Ganoderma mushroom is due to its high water content up to 90%, which makes extracts of mushroom dehydrated powder and residual 10% of its mass consist of protein (10-40%), carbohydrate (3-28%), fiber (3-32%), fat (28%), and ash (8-10%). Besides, various other compounds such as provitamin D2 [9], C19 fatty acids [10], and essential nutrients such as copper and zinc [11] have also been found to be present. With the minerals potassium, calcium, phosphorous, magnesium, selenium, iron, zinc, and copper represent most of the mineral content [12,13]. Rex, 2014 [14], reported that G. lucidum contains about 72 μg Se/g of dry weight and thus can act as a good source of essential micronutrients like selenium. G. lucidum is found across the world and is considered as an effective supplement for the prevention and treatment of many diseases since ages. Triterpenes and polysaccharides from G. lucidum have been found to possess anti-inflammatory and antioxidant activity. The polysaccharide and the water extract from G. lucidum have shown to possess immune modulator and antitumor activities. In addition, G. lucidum have a wide variety of bioactive compounds such as terpenoids mostly triterpenoids, carbohydrates including polysaccharides and glycoproteins, steroids, phenolic compounds, nucleotides, and their derivatives. The proteins of Ganoderma mushroom contain different essential amino acids. Lysine and leucine represent the highest percentages. G. lucidum, also contains a large share of polyunsaturated fatty acids as compared to the total fatty acids, which are the highest contributors for the best human health [6,12].
Ganoderma has been generally admitted as nutritional supplement across the world due to its long-term safety and tolerance. G. lucidum possess a vast array of medicinal properties. The extremely important G. lucidum in oriental traditional medicine has been used as remedy against various chronic diseases such as antitumor [15,16], antioxidant [17], immunoregulation [18,19], hepatoprotection [20], hypoglycemic effect [21,22], antibacterial activity [23], reduction of blood cholesterol [24,25], inhibition of angiogenesis [26,27], antifibrotic activity [28], anti-human immunodeficiency virus (HIV) activity [29], and reduction of lower urinary tract symptoms [30]. The above bioactivities of Ganoderma have been found due to the important bioactive substances such as polysaccharides and triterpenoids. Despite the vast array of reported medicinal attributes of Ganoderma; however, the pathways and mechanisms of action of these bioactive substances from Ganoderma remain poorly defined. With further advancement in modern research technologies, clear and detailed insights into these pathways and mechanisms of action are becoming increasingly possible in which G. lucidum can influence the observed health benefits. Understanding these mechanisms could lead to more robust use of Ganoderma as an anticarcinogenic agent. With improvement in techniques, better separation and purification methods have proved very beneficial for the isolation and identification of bioactive substances from G. lucidum. However, modern researchers have primarily focused more on two active components, namely triterpenes and polysaccharides. In the foregoing account, emphasis has been given on the research work carried out by different scientists on major bioactive triterpenoids found in G. lucidum and other species of Ganoderma.

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have molecular weights ranging from 400 to 600 KDa. Triterpenes isolated from Ganoderma species show remarkable therapeutic and pharmacological properties on a number of human diseases including cancer pharmacological properties [1,16,18,20,31,33]. The triterpene extracts of G. lucidum are known to induce apoptosis of multiple human cancer cell lines [16]. However, the cytotoxic activity of triterpenes varied significantly across different subtypes of triterpenes [16]. Most triterpenoids extracted and identified from Ganoderma have shown robust biological activities ( Table 1). The GAs isolated from Ganoderma have shown antiviral, anticancer, antioxidant, hepatoprotective, cytotoxic, antiplatelet aggregation, and inhibition of histamine release and hypocholesterolemic activities [7,[34][35][36][37][38][39][40]. The most abundant triterpenic acid from G. lucidum is GA T which shows significant anticancer activity both in vivo and in vitro experiments [41,42]. GA has been found to inhibit tumor invasion by inhibiting matrix metalloproteinase (MMP)-9 expressions [42]. Another triterpenic acid GA D has been shown to directly bind to 14-3-3ζ protein [43] and this binding may contribute to the facilitation of apoptosis observed in human epithelial cell line (HeLa) cell [43]. Ganoderiol F (GA-F) a tetracyclic triterpene found in Ganoderma lucidum [36,44] [36,45]. GA-F has also been demonstrated in vivo in rats with LLC tumor cells [46]. The other forms of the isolated triterpenes from Ganoderma lucidum have been reported to show cytotoxic activity in the p338, HeLa, human hepatoma cell line (BEL-7402), and human gastric cancer cell line (SGC-7901) [47,48].
Recently, Hsu et al., 2018 [49], tested a new atheroprotective effect of G. lucidum, an arterial condition which is associated with chronic oxidative stress and inflammation, using a carotid artery ligation mouse model. In this study, the ligation of the artery generated disturbed blood flow, a critical atherogenic factor with no cure currently. These authors studied that G. lucidum protected arteries from disturbed flow-induced atherogenesis and the triterpenoid fraction is the critical constituents for these effects. Ganoderma triterpenoids alleviated oxidative stress and inflammation, thereby preventing neointimal hyperplasia in the ligated arteries through daily oral dosage after 2 weeks. Specific triterpenes or a mixture of triterpenes have been isolated and identified from G. lucidum and other species of Ganoderma with various health benefits, the results of which have been published. The various health benefits of Ganoderma triterpenes are as follows.

Anticancer activities
The triterpene extracts identified from G. lucidum and other Ganoderma species have shown anticancer property under in vivo conditions [12,33,[50][51][52]. The carcinogenic effects shown by various types of extracts from G. lucidum include various cancer cell lines (breast, colon, lung, pancreas, prostate, and skin) [12,52]. The known mechanisms through which the extracts of G. lucidum exhibit anticancer activities include direct inhibition of cell proliferation through cancerspecific cell cycle arrest and apoptosis [41,[53][54][55]. G. lucidum extracts, in addition, can lead to downregulation of cell cycle-associated proteins, resulting in cell cycle arrest [54,56,57]. Studies of the triterpene extracts from G. lucidum have shown that these extracts can arrest the cell cycle at the G1 phase [54,55]. The mechanism for this inhibition of cell cycle at G1 phase is by the downregulation of cyclin D1 through the modulation of the β-catenin pathways [58]. Cyclin D1 is the key regulator of cyclin-dependent kinase which is very important for the transition of G1/S phase of the cell cycle [59]. About 30% of colon cancer has an overexpression of cyclin D1, due to the abnormal β-catenin signaling pathway [60]. The triterpene from G. lucidum, ganodermanontriol has been found to inhibit the proliferation of human colorectal carcinoma cell lines (HCT116 and HT-29) by inhibiting the expression of β-catenin, thus controlled levels of cyclin D1 is expressed [54]. The triterpene extracts from Ganoderma can also cause inhibition of G2/M transition, apart from inhibiting G1 phase of cell cycle [38]. It has been studied that the triterpene extract of Ganoderma can suppress the activity of protein kinase C (PKC), leading to a prolonged G2 phase, by treatment with the triterpene-enriched ethanol soluble fractions (WEES-G6). PKC is selectively activated during G2 phase of the cell cycle and belongs to the class of serine-threonine protein kinases [61]. During the G2 phase of the cell cycle, PKC has been found to be involved in the regulation of nuclear disassembly [62]. Various studies have reported that the use of PKC inhibitors can arrest the G2 phase of the cell cycle [63,64]. In addition, the level of cycling B, a kinase, which is responsible for the transition from G2 to M phase, is reduced by WEES-G6 [38]. Due to the activity of WEES-G6, the c-Jun N-terminal kinase (JNK) and p38 kinase, both of which are mitogen-activated protein kinase which responded to cellular stress are activated [38]. JNK is considered very critical regulator of transcription which can activate tumor suppressors such as p53 [65][66][67]. Johnson and Lapadat [67] observed cell cycle arrest in triterpene-treated human hepatoma (HuH-7) carcinoma, but no effect has been seen in a normal human liver cell line, which further supports the use of triterpenes as therapeutic anticancer agent. Jiang Similarly, Chen et al., 2010 [42], revealed that GA T from G. lucidum inhibits the tumor growth through inhibition of MMP expression.
A recent study by researchers reported that gold nanoparticles (Au-NPs) synthesized from G. lucidum and then conjugated with drug doxorubicin show robust and significant anticancer drug accumulation and cytotoxic activity against MCF-7-doxbreast cancer cell line. Au-NPs efficiently inhibited the growth of MCF-7-doxbreast cancer cell line at higher concentration (400 μM/ml) by 97%. mRNA expression of ABCB1 gene and CDNA synthesized from human breast cancer cell line (MCF-7) showed reduced expression. It is important to conclude that the pharmacological activity of G. lucidum exhibits the anticancer activity of newly synthesized Au-NPs conjugated with drug doxorubicin. However, further research is required under in vivo conditions to report toxicity if any, due to newly synthesized Au-NPs. Au-NPs synthesized from G. lucidum conjugated with drug doxorubicin could prove as possible and strong source of drug delivery for anticancer inducing drug preparation which can benefit treatment of breast cancers [69].

Cytotoxic activities
The triterpene extracts identified from G. lucidum have been to show cytotoxic effects under in vitro conditions (on cancer cell lines) [12]. Various cytotoxic compounds from G. lucidum have been found to trigger apoptosis, leading to programmed cell death [52,53]. The triterpenes from G. lucidum also observe to cause apoptosis of various cancer cell lines, and this has been found to be due to the increase of proapoptotic proteins and decrease of antiapoptotic proteins [41,53]. The structure-activity relationship of GA-DM was investigated and it was shown to inhibit the proliferation of the aggressive human prostate cancer cell line PC3 [70].
The mechanisms by which triterpenes from G. lucidum induce apoptosis in human cancer cell lines include mitochondria-dependent pathway followed by activation of caspase cascade [70,71]. The mitochondrialdependent apoptotic pathway also known as intrinsic apoptotic pathway involves the decrease in mitochondrial potential followed by the release of cytochrome c from the mitochondria [72,73]. The cytochrome c which is released from the mitochondria into the cytosol is known to trigger the caspase cascade which leads to apoptosis. This caspase cascade involves caspase 9 and caspase 3 which have been studied to have higher expressions in different human cancer cell lines when treated with the triterpenes extract from G. lucidum [41,71,74].
The release of cytochrome c depends on the ratio of Bax/Bcl-2 balance [75]. It has also been observed that when the ratio of Bax/Bcl-2 is increased, apoptosis is triggered. The Bcl-2 family proteins can be either proapoptotic or antiapoptotic. Bcl-2 associated X protein (Bax) and Bcl-2 associated death promoter (Bad) are proapoptotic while as Bcl-2 is antiapoptotic. Various studies have revealed that during the treatment of different human cancer cell lines with the triterpenes of G. lucidum, the ratio of Bax/Bcl-2 is increased which, therefore, increases Bax expression while downregulating Bcl-2 expression [71]. Liu et al., 2012 [70], observed cytotoxic and proapoptotic effect of GA derivatives on human cervical cancer cells under in vitro conditions.

Antioxidant activity
The major contributor to increased cancer risk is known to be the oxidative stress. Reactive oxygen species (ROS) and free radicals are produced as by-products of metabolic processes involving redox enzymes and electron transfer during bioenergetics, as well as due to exposure to some exogenous chemicals [76]. Free radicals and ROS can damage cells and tissues by the process of oxidation and long-term accumulation of such damage due to free radicals and ROS causes aging and various age-associated diseases [77]. ROS and free radicals have the potential to cause damage to proteins and DNA within cells, leading to oxidative stress, which can be countered by antioxidative enzymes and repair mechanisms. However, it has been observed that excessive oxidative stress can override the innate protective system, leading to a variety of physiological disorders including cancer [78]. These cancer

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cells further contribute to cancer progression by generating increased levels of free radicals relative to normal cells [78]. Various studies have suggested that this cancer-causing damage might be reduced or prevented with the help of antioxidants from the extracts of Ganoderma species [78,79].
Various other studies have also shown that the triterpene extracts of G. lucidum have antioxidant activity and have the potential to reduce oxidative damage by directly scavenging free radicals generated in the cell due to the increase in the activity of superoxide dismutase and catalase which are enzymes involved in removing harmful free radicals and ROS [80,81]. Smina et al., 2011 [82,83], revealed in mice that triterpenes from G. lucidum showed antioxidant activity which may be due to increased activity of antioxidant enzymes and they further observed that total terpenes from G. lucidum prevent radiation-induced DNA damage and apoptosis in splenic lymphocytes of mice under in vitro conditions. In a recent study by Smina et al., 2016 [84], total triterpenes from G. lucidum were highly effective in reducing the levels of lipid peroxidation and protein oxidation to near normal values in both liver and brain tissues in Swiss albino mice under in vivo conditions. Total triterpenes, when administered under vivo conditions, were also found to be successful in restoring the antioxidant enzyme activities and glutathione level in liver and brain of irradiated mice. Administration of total triterpenes, before radiation exposure, significantly decreased the DNA strand breaks.

Anti-HIV activity
HIV, which induces a lethal and incurable condition known as acquired immunodeficiency syndrome (AIDS), is a highly infectious virus affecting an estimated 35 million people all over the world [85]. The treatment strategies for HIV, which are currently in use, involve delaying the progression of disease into AIDS [85]. Various compounds that exhibit inhibitory effects against AIDS have been identified from G. lucidum, and related species of Ganoderma such as triterpenes have shown anti-HIV-1 protease activity [22,86]. Mizushina et al., 1999 [87], observed the inhibition of HIV Type 1 transcriptase due to lucidinic acid and lucidinic lactones isolated from G. lucidum. el-Mekkawy et al., 1998 [29], have assayed 13 compounds for anti-HIV activity isolated from G. lucidum. El Dine et al., 2008 [88], observed anti-HIV-1 protease activity of triterpenoids from G. colosseum. The inhibitory activity of triterpenoids isolated from Ganoderma species against HIV has also been reported by Cassels and Asencio, 2011 [89]. Various compounds out of these have shown anti-HIV-1 activity, which includes GA A which showed robust activity against HIV proteases. However, muchextended research is to be carried out to ascertain a mechanistic basis for G. lucidum extracts and other species of Ganoderma as anti-HIV agents. In addition, determination of the structure-activity relationship between triterpenes from G. lucidum and HIV proteases must be performed as well.

Antimetastatic potential
Cancer metastasis is very complex phenomenon in which cancer cells split from the primary tumor cells and invade other tissues, thereby leading to the formation of secondary tumors. Cancer metastasis dramatically reduces the rate of survival and cure, when left untreated [90]. Several key proteins which are involved in metastasis of cancer may be regulated by triterpenes of G. lucidum and other species of Ganoderma [64,91]. MMP is a family of proteins which cause degradation of extracellular matrix and thereby promote cancer metastasis [32,92,93]. The triterpenoid GA-Me extracted from G. lucidum suppressed the invasion of 95-D, LLC, and HCT-116 metastatic cancer cell lines through inhibition of MMP-9 expression [75,94]. Chen et al. [95] revealed that GA T extracted from G. lucidum inhibits the tumor invasion through inhibition of MMP expression. Interleukin (IL-8) and various angiogenic factors such as vascular endothelial growth factor (VEGF) caused induction of angiogenesis and resulted in the promotion of metastasis [96]. It is further suggested that the expression of IL-8 is upregulated during oxidative stress, and therefore, overexpression of IL-8 is involved in the metastasis of breast cancer cell lines [97,98]. Studies have reported that oxidative-induced IL-8 expression was reduced in breast cancer cell lines after treatment with triterpenoid extracts of G. lucidum [99].

Hepatoprotective activity
It has been studied that GA B isolated from Ganoderma species showed significant hepatoprotection property [100]. However, it was observed that when the doses of GA B were increased 10 times than the normal, it did not further reduce glutamic oxaloacetic transaminase and glutamic pyruvic transaminase levels in the serum of the mice [101]. Chen and Yu, 1993 [102], have reported that ganosporic acid A has shown significant activity of lowering the levels of GPT in mice with liver injury by carbon tetrachloride (CCL 4 ) and exhibits hepatoprotective effect. Lin et al., 2003 [38], and Liu et al., 2014 [103], observed that triterpenoids such as GA XL, XL2, and ganoderic in from the extracts of G. lucidum and G. theaecolum have good hepatoprotective properties suppress the growth of hepatoma cells. Wu et al., 2016 [104], observed the hepatoprotective effects and mechanism of the action of triterpenoids from G. lucidum on α-amanitin-induced liver injury in mice. Wu et al., 2016 [105], studied the hepatoprotective effect of Ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic cells. GAs, namely, GAs R and S, from the cultured mycelium of G. lucidum have shown strong hepatoprotective activity in galactosamine-induced cytotoxicity in cultured rat hepatocytes. The triterpenoid extracts from Ganoderma can prevent liver damage induced by CCL 4 and galactosamine in rats [106]. The triterpenoids from G. lucidum have shown significant protection against immunological liver damage in mice in vitro and in vivo.

Anti-inflammatory potential
About 20% of the cancers are considered to be the result of inflammation [115,116]. The carcinogenesis is promoted due to the chronic overexpression of inflammatory cytokines such as IL-6, VEGF, and tumor necrosis factor-α [117,118]. The administration of a triterpene extract of G. lucidum is known to suppress the inflammatory cytokine secretion in macrophage cells, therefore, reducing the level of inflammation [119].

CONCLUSION
The beneficial health attributes of Ganoderma species are due to the presence of various bioactive compounds. Ganoderma genus, in general, and G. lucidum, in particular, can be considered as a natural pharmacy store besides being natural therapeutic machinery. There are two main groups of bioactive substances triterpenes and polysaccharides that have been studied in detail. Triterpenoids have been reported as having cytotoxic, hepatoprotective, anti-inflammatory, anti-HIV, neurotrophic, along with antitumor, anticancer, and antioxidant activities. In addition, because the various bioactive compounds isolated from G. lucidum

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did not show any toxic side effects, the demand for this mushroom as health fortifying food, a natural remedy, and dietary food is increasing day by day and attracting the interests of the scientific community and industrial community as well. However, due to the lack of results, intense investigation needs to be performed in the field (e.g., human clinical trials). Till now, the available data suggest that G. lucidum has a high potential to be accepted as a good health food supplement for patients experiencing cancer therapy. This available knowledge and further investigation would facilitate the development of new nutraceuticals and pharmacological formulations.

CONFLICTS OF INTEREST
It is hereby stated that the above article is consented for publication by all authors in this journal and, therefore, declares no conflicts of interest.