A Status Review of the Bioactive Activities of Tiger Milk Mushroom Lignosus rhinocerotis (Cooke) Ryvarden

Edible and medicinal mushrooms are regularly used in natural medicines and home remedies since antiquity for ailments like fever, inflammation, and respiratory disorders. Lignosus rhinocerotis (Cooke) Ryvarden is a polypore found in Malaysia and other regions in South East Asia. It can be located on a spot where a tigress drips milk while feeding, hence the name “tiger's milk mushroom.” The sclerotium of L. rhinocerotis is highly sought after by the native communities in Malaysia to stave off hunger, relieve cough and asthma, and provide stamina. The genomic features of L. rhinocerotis have been described. The pharmacological and toxicity effects, if any, of L. rhinocerotis sclerotium have been scientifically verified in recent years. In this review, the validated investigations including the cognitive function, neuroprotection, immune modulation, anti-asthmatic, anti-coagulation, anti-inflammatory, anti-microbial/ anti-viral, anti-obesity, anti-cancer/ anti-tumor, and antioxidant properties are highlighted. These findings suggest that L. rhinocerotis can be considered as an alternative and natural medicine in the management of non-communicable diseases. However, there is a paucity of validation studies including human clinical trials of the mycochemicals of L. rhinocerotis.


INTRODUCTION
Medicinal mushrooms have been valued and used since ancient times by the Chinese, Korean, Japanese, Egyptians, and European communities. They are valued not only for the culinary purposes but also for their nutritional and medicinal values (Manzi et al., 1999). The greatest attribute of mushrooms, besides their taste, is their peculiar healing properties. Recently, ethnomycological knowledge of medicinal mushrooms for their curative properties is being tapped. Lignosus rhinocerotis (Cooke) Ryvarden, belonging to the Polyporaceae family is regarded as a rare and valuable traditional medicine and it can only be located in a certain geographic regions encompassing South China, Thailand, Malaysia, Indonesia, Philippines, Papua New Guinea, New Zealand, and Australia ; Figure 1).
Lignosus rhinocerotis, however, was only collected from the wild. In the wild, the tiger milk mushroom grows solitary and makes the collection process time and energy consuming (Abdullah et al., 2013). Since the 2000s, large-scale cultivation of L. rhinocerotis in a controlled environment was made successful in Malaysia, overcoming the cost and supply problem (Lau et al., 2011(Lau et al., , 2013a(Lau et al., , 2015. Commercialization of this mushroom was then made possible and this opened the opportunity to investigate the potential pharmacological and nutraceutical properties of this mushroom for functional food or dietary supplements.

DESCRIPTION OF L. RHINOCEROTIS
This mushroom consists of the pileus (cap), stipe (stem), and sclerotium (tuber) (Figure 2). Its morphology is unusual for a polypore as the fruiting body (cap and stem) raises from the tuber under the ground, rather than from woody substrate. The cap and stem are woody while the sclerotium is a compacted mass of fungal mycelium containing food reserves. The sclerotium is white and gives a milk-like solution; and it even tastes like milk (Tan et al., 2010). As the irregular shaped sclerotium remains underground, the collection of the mushroom is challenging. Thus, due to lack of samples, limited study is done on this national treasure.
In Malaysia and Indonesia, this mushroom is known as "cendawan susu rimau" which literally means "tiger milk mushroom" (Burkill, 1930). Early documentation of this mushroom were given by Ridley and Corner (Ridley, 1900;Corner, 1989). In the early twentieth century, the taxonomy of L. rhinocerotis depended primarily on morphological observations. However, in recent years, specimens of "susu rimau" collected in these regions were confirmed as L. rhinocerotis on the basis of both micro-and macromorphological characteristics, as well as molecular approaches. To date, L. rhinocerotis is noted to be the "most commonly occurring member of Lignosus in Malaysia" (Lai et al., 2009;Tan et al., 2010;Choong et al., 2014). Figure 2 shows the taxonomic classification of L. rhinocerotis.
Lignosus rhinocerotis was often mistaken for Pleurotus tuberregium or Lentinus tuber-regium. Thus, the use of molecular markers in identifying L. rhinocerotis has proven to overcome the drawback of classic taxonomy methods (Sotome et al., 2008;Cui et al., 2011). There are even genetic markers and DNA barcode markers being developed to identify the Lignosus spp.
Recently, Tan et al. (2015) reported successful cultivation of the mushroom, which would overcome the supply problem and make possibilities for more investigation to be done on L. rhinocerotis. Other researchers also cultivated this mushroom using mycelium in submerged culture techniques. Further, the optimization of substrate formulation was done for the cultivation of L. rhinocerotis in conditions that mimic their natural environment. Based on the optimized formulation, the pilot cultivation was conducted. Both sclerotia and sporophores were successfully produced (Abdullah et al., 2013).

Ethnomycological Aspects of L. rhinocerotis
According to folklore, it is believed that the mushroom emerges on the spot where the milk of a tigress had accidentally dribbled during lactation. The sclerotium of the mushroom resembles the "congealed white mass of milk" (Corner, 1989;Chang, 2015). Different tribal communities in Malaysia have different referral names, such as "betes kismas" by the Semai (Chang and Lee, 2010), "tish am ong" by the Kensiu (Mohammad et al., 2012), and "Pěti' Aa" by the Besisi (Skeat, 1896). The Batak people from Indonesia denoted L. rhinocerotis as "Ndurabi" (Karo) (Hilton and Dhitaphichit, 1978). Lignosus rhinocerotis found in China were called "how gui kou or hurulingzhi" (in Chinese), which means "tiger milk Ganoderma" (Huang, 1999;Yokota, 2011). In Japan, it is known as "hijiritake" (Lee and Chang, 2007).
Besides the traditional beliefs that L. rhinocerotis was derived from the tiger's milk, there are many other folklore beliefs about this mushroom. The Semai (indigenous people of Malaysia) believes that L. rhinocerotis could reinstate the spirit of a crop and guarantee a lavish harvest. The sclerotia are habitually used during paddy farming and prayer ritual for a bountiful crop yields (Haji Taha, 2006). Alternatively, some crops, for instance, paddy is positioned in a flower-filled container, and suspended over the mushroom (Skeat and Ottoblagoen, 1906).
The Besisi (or MahMeri) declares that the mushroom is easier to be spotted after the full moon (Hartland, 1909). Lignosus rhinocerotis were also associated with childbirth customs. The Semang (or Negrito) people believe that L. rhinocerotis hold "the soul of an unborn tiger cub" and that "the soul is conveyed when the tiger eats it" (Cumming, 1903;Robert, 1986).
Lignosus rhinocerotis is mentioned in various stories from different cultural background, and in most cases, are linked to their medicinal properties. For example, in the story of "Indra Bangsawan", "tigress' milk" is the remedy for a princess who became infected with an eye disease and lost her vision (Beveridge, 1909). A Mughal emperor, Jahangir (1568-1616) also wrote that the "milk of a tigress was of great use for brightening eyes" (Green, 2006;Lee et al., 2009).
Based on ethnobotanical uses, various tribes of the Orang Asli in Malaysia (the Semai, Temuan, and Jakun) use L. rhinocerotis to relief asthma, cough, food poisoning, swollen breasts, joint  pain, liver illness, swollen body parts, and as a general tonic (Lee and Chang, 2007;Ismail, 2010). It is also used to nurse women after childbirth (during postpartum period) and also to stave off hunger. The L. rhinocerotis is not only popular among the indigenous people but also the urban population in Malaysia (Hattori et al., 2007). The sclerotia of L. rhinocerotis are occasionally sold in Traditional Chinese Medicine (TCM) store in Malaysia. They are used by the TCM practitioner to revitalize the body of the patients. According to Sabaratnam et al. (2013), infusions of L. rhinocerotis are said to improve the overall wellness of the individual by enhancing the vitality, energy, and alertness.
There are many ways this mushroom is prepared and consumed to treat illness. In earlier years, the sclerotium was pounded and the juice was infused with water and drunk as a tonic. The mushrooms are grounded or sliced, then boiled with water for drinking or soaked into Chinese wine for external applications (Chang and Lee, 2004). The sclerotium is also eaten raw and with betel leaves to relieve a cough and sore throat. The preparation methods of decoction and/or topical medicine vary among tribes.

Genomic and Proteomic Studies of L. rhinocerotis
Genome sequencing of L. rhinocerotis was carried out . A comparative genomics and phylogenetic analysis were performed. The L. rhinocerotis genome is widely composed of sesquiterpenoid biosynthesis genes. Moreover, the genome of L. rhinocerotis appears to code for 1,3-β-and 1,6-β-glucans, as well as lectin, laccase, and other fungal immune-modulatory proteins (FIPs).
Subsequently, proteomic profiling of L. rhinocerotis sclerotial proteins were carried out using various spectrometers. Table 1 shows the genomic and proteomic studies of L. rhinocerotis. While some of the proteins were unknown, a majority of the proteins were detected as lectin (39.13%) which were speculated for defense mechanisms in L. rhinocerotis. The other proteins identified from the study were of pharmacological interest, for example, FIPs (2.52%), antioxidant proteins i.e., manganese superoxide dismutase (Mn-SOD, 0.91%) and glutathione S-transferase (GST, 0.54%). There is a paucity of studies of the myconutrients in this mushroom. Furthermore, it is also necessary to study the molecular and genetic basis of the identified components, and the medicinal and/or nutraceutical properties of L. rhinocerotis.

Nutritional Composition of L. rhinocerotis
Analysis of the chemical compositions of L. rhinocerotis was carried out by Yap et al. (2013). The wild strain (WT Rhino) and the commercial strain (TM02) sclerotial powder were analyzed based on the Association of Official Analytical Chemists (AOAC) procedures. In general, the nutrient composition of the sclerotium of cultivated strain was higher when compared to the wild strain.
The major constituents of L. rhinocerotis sclerotia were carbohydrates (monosaccharides and disaccharides) while the fat content was <1%. As reported by Lau et al. (2013a), the beta (β)-glucans represented the dominant glucans in the aqueous extracts of L. rhinocerotis, which was 82-93% of total glucan (w/w). The protein content of the sclerotium of TM02 was 3.6times higher than that of the wild-type. The essential amino acid content (g/kg dry weight) of the commercial strain was 4times higher than that in the wild strain. The minerals (calcium, potassium, sodium, and magnesium) were also higher in the cultivated strain. Table 2 shows the chemical compositions of L. rhinocerotis.
Therapeutic Values of L. rhinocerotis Anti-asthmatic Activity The efficacy of L. rhinocerotis in treating asthmatic symptoms has been validated in several in vitro and in vivo studies. The effects of L. rhinocerotis extracts on ovalbumin-induced allergic asthma in Sprague-Dawley rats were investigated (Malagobadan et al., 2014). The treatment with L. rhinocerotis extracts significantly reduced asthmatic parameters, for instance, the total immunoglobulin E (IgE) in serum, and T-helper type 2 (Th2) cytokine levels  in bronchoalveolar lavage fluid (BALF). It also inhibited the number of eosinophil in BAFL and diminished the infiltration of eosinophil in the lungs.
A study on airway inflammation in an asthmatic model was reported (Johnathan et al., 2016). The hot aqueous extract of L. rhinocerotis (500 mg/kg) was effective in reducing asthmarelated parameters. The extract significantly ameliorated the increase of the total IgE (14.9 ng/ml) in serum, as well as IL-4 (16.2 pg/ml), IL-5 (38.8 ng/ml), and IL-13 (80.5 pg/ml) levels in BALF. It also effectively decreased eosinophils numbers in BALF while attenuating eosinophil infiltrations in the lungs. Sequential extraction using five solvents, i.e., petroleum ether, diethyl ether, hexane, ethyl acetate, and methanol, was conducted prior to gas chromatography-mass spectrometry (GC-MS) analysis (Johnathan et al., 2016). GC-MS examination uncovered five major groups (alkane, fatty acids, benzene, phenol, and dicarboxylic acid) with a total of 18 constituents in L. rhinocerotis. Linoleic acid, octadecane, and 2,3-dihydroxypropyl elaidate were present in abundance in the L. rhinocerotis extract (Johnathan et al., 2016).

Anti-coagulant and Fibrinolytic Activities
Cardiovascular diseases can be caused by thrombosis due to fibrin aggregation in the blood (Lee et al., 2005). Natural anti-coagulant and fibrinolytic agents have been used to treat thrombolytic conditions, especially edible mushrooms. Recently, the edible mushroom "wood ear fungus, " Auricularia polytricha (Mont.) Sacc was found to be able to produce protease-like fibrinolytic enzymes (Mohamed Ali et al., 2014).
The anti-coagulant activity of the crude aqueous extract of L. rhinocerotis was also reported. Kho (2014) demonstrated that the crude extracts of L. rhinocerotis caused a 1.2 cm-lytic zone in a fibrin plate assay. Subsequently, aqueous two phase system (ATPS) was employed to partition, purify, and concentrate the protein fraction of the mushroom extract. As a result, a fibrinolytic enzyme with a specific activity of 151.61 U/mg was  isolated and the molecular size was estimated to be between 55 kDa and 60 kDa. The anti-platelet activity of the aqueous extract of L. rhinocerotis was also reported using fresh human blood (Teo, 2014). The crude extract, after subjected to ATPS, yielded a partially purified protease-like enzyme with size ranging from 50 to 55 kDa.
Sidek Ahmad et al. (2014) tested six different wild sclerotia (LR1 to LR6) of L. rhinocerotis obtained from Perak, Malaysia, for their fibrinolytic activities. Out of the six sclerotia used, the LR1 sclerotium exhibited proteolytic activity with a clear zone of 1.31 cm diameter in skim milk agar plates while giving a clear zone of 0.97 cm when tested with fibrin plates. It is presumed that the fibrinolytic activity of L. rhinocerotis is attributed to the presence of a protease.

Anti-inflammatory Activity
The anti-inflammatory activity of the sclerotium of L. rhinocerotis was previously reported with its hot aqueous, cold aqueous, and methanol extracts (Lee et al., 2012b(Lee et al., , 2013b. Lee et al. (2014) reported that the three extracts of L. rhinocerotis exhibited antiinflammatory properties as shown by the carrageenan-induced paw edema test using Sprague-Dawley rats. The cold aqueous extract, the most potent extract, was subjected to separation by Sephadex G50 gel filtration chromatography. The resulting high-molecular-weight protein fraction was further assessed for anti-inflammatory activity in lipopolysaccharide (LPS)induced RAW 264.7 macrophage cells. The protein fraction was shown to inhibit tumor necrosis factor alpha (TNF-α) production.
The anti-inflammatory effect of L. rhinocerotis hot aqueous and ethanol extracts on RAW 264.7 cells was further tested (Baskaran et al., 2012;Baskaran, 2015). The ethanol extract showed significant decrease (48.3-88.5%) of nitric oxide (NO) production from 0.01 to 100 µg/mL dose-dependently but the aqueous extract did not show a significant reduction. The ethanol extract was able to activate signal transducer and activator of transcription 3 (STAT3) pathway by reducing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions while increasing the interleukin 10 (IL-10) expression. Nallathamby et al. (2016) analyzed the ethyl acetate fraction from the ethanol extract of L. rhinocerotis. The fraction significantly reduced the NO production in microglial (BV2) cells by 12 to 70% at 10 and 100 µg/mL; respectively. The major compounds of the ethyl acetate fraction were revealed as linoleic acid, oleic acid, and ethyl linoleate. The identified compounds were further tested individually for their anti-inflammatory activities. Treatment with linoleic acid significantly suppressed iNOS and COX2 expression by 1.2-fold as compared to the control. In another study, LPS-induced BV2 cells pretreated with hot aqueous extract (500 µg/mL), n-butanol fraction of hot aqueous extract (250 µg/mL), and ethyl acetate fraction of hot aqueous extract (250 µg/mL), showed maximum inhibition of NO production by 88.95, 86.50, and 85.93%, respectively (Seow et al., 2017). These studies represent the first evidence of antiinflammatory properties of L. rhinocerotis using brain microglial BV2 cells.

Anti-microbial Activity
Four extracts of the wild L. rhinocerotis sclerotium, i.e., petroleum ether, chloroform, methanol, and aqueous extracts, were screened for their anti-microbial properties (Mohanarji et al., 2012). The four extracts were tested against 15 pathogenic bacteria, including Staphylococcus, Corynebacterium, Bacillus, Streptococcus, Klebsiella, Salmonella, Pseudomonas, Escherichia, and Micrococcus spp.; as well as four fungi species including Candida spp and Mucor sp. Antifungal and antibacterial activities of the extracts were evaluated by measuring the inhibition zone using disc diffusion assay. The methanol and aqueous extracts (30 mg/mL) showed significant inhibition against the tested microbes except for Streptococcus pyogenes and Serratia marcescens. A qualitative phytochemical analysis showed the presence of alkaloids, protein, gums and mucilage, and flavonoids in the extracts of L. rhinocerotis (Mohanarji et al., 2012).

Anti-obesity and Hepatoprotective Activities
A study by Hoe (2014) provided evidence that the aqueous extract of L. rhinocerotis mitigated non-alcoholic fatty liver disease in high-fat-diet induced obese hamsters. Further, the extract inhibited weight gain in high-fat-diet fed hamsters. The L. rhinocerotis extract did not show any side effects in the experimented animals with regards to the weight of organs i.e., brain, heart, thymus, liver, kidney, adrenal glands, spleen, testicles, and epididymis.
The L. rhinocerotis at low dose demonstrated the highest effect in reducing the weights of liver and adipose tissues with no histological abnormalities found in the organs and tissues. The serum biochemical, as well as the liver and kidney function parameters, were also measured. The results showed that the extract reversed the serum levels of several biochemical parameters in the hamsters. The L. rhinocerotis aqueous extract was also shown to inhibit reactive oxygen species (ROS) production and reduced cluster of differentiation 68 (CD68) and COX2 expressions in the liver. The extract also downregulated mRNA expressions of different genes, namely TNF-α, IL-1β, IL-6, transforming growth factor beta (TGF-β), and collagen-type 1 (Colla1)-related inflammation and collagen-type 1 (Colla1)related inflammation and fibrosis caused by obesity.

Antioxidant Properties
The cold aqueous, hot aqueous, and methanol extracts of wild and cultivated sclerotium of L. rhinocerotis were assessed for their antioxidant capacities (Yap et al., 2013). The total phenolic content (TPC) of the extracts were calculated in terms of gallic acid equivalents (mg GAE/g). The TPC of the extracts ranged from 19.3 to 29.4 mg GAE/g extract. The ability of the extracts to reduce ferric ions and scavenge free radicals was also measured by using Ferric reducing antioxidant power (FRAP) assay and DPPH• (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay; respectively. The FRAP value was between 0.006 to 0.016 mmol/min g extract. The DPPH radical scavenging values were 0.05-0.2 mmol Trolox equivalents (TE)/g. The extracts from wild sclerotium of L. rhinocerotis had higher antioxidant capacity compared to the cultivated sclerotium. The methanol extract displayed higher activity compared to the aqueous extracts. It is noteworthy to mention that both strains exhibited significantly higher superoxide anion radical scavenging activity when compared to rutin.
Similar analyses were done using aqueous extract of L. rhinocerotis (Hoe, 2014). The extract showed half maximal effective concentration (EC 50 ) values of DPPH scavenging and ferrous ion chelating activity at 135.16 ± 2.47 mg/mL and 503.34 ± 10.44 mg/mL; respectively. The TPC and flavonoid contents were 366.23 ± 5.06 mg GAE and 28.67 ± 2.5 mg rutin equivalent/g; respectively. In a study by Suziana Zaila (2013), the ferric reducing capacity of pressurized methanol extract was higher than the aqueous extract.
The free radical scavenging abilities, reducing properties, metal chelating activities, and inhibitory effects of lipid peroxidation by L. rhinocerotis extracts of mycelium from submerged cultivated and the culture broth were reported by Lau et al. (2014). The aqueous methanol extracts from the mycelium and culture broths showed comparable antioxidant effects with the aqueous methanol extracts of sclerotium. The results suggested that the mycelia of L. rhinocerotis obtained in submerged cultivation can also be a valuable source of antioxidant compounds.

Anti-tumor/Anti-cancer Activities
The traditional claims that L. rhinocerotis had anti-tumor/anticancer properties were investigated in several cancer and solid tumor cell lines. The first study on the anti-tumor properties of L. rhinocerotis sclerotium specified that the water-soluble polysaccharide-protein complex (PR-HW) and alkali-soluble βglucan (PR-CA) possessed notable host-mediated anti-tumor activity on the Sarcoma 180 implanted male BALB/c mice (Lai, 2005). The PR-HW exhibited apoptotic effect on human acute promyelocytic leukemia cells (HL-60), human chronic myelogenous leukemia cells (K562), and human acute monocytic leukemia cells (THP-1) with IC 50 of 100 mg/ml, 400 mg/ml, and >400 mg/ml; respectively after 72 h of incubation (Lai, 2005;Lai et al., 2008). Besides, flow cytometric analysis revealed that the onset of apoptosis could be linked to the cell cycle arrest at Gap1 (G1) phase. The cold-water extract (LR-CW) prepared from the sclerotium of L. rhinocerotis exhibited antiproliferative activity in human breast carcinoma cells (MCF-7) and human lung carcinoma cells (A549), with IC 50 of 96.7 and 466.7 µg/mL, respectively. In contrast, the LR-CW did not show significant cytotoxicity toward the two corresponding normal human cells, i.e., human breast cells (184B5) and human lung cells (NL 20). From the results of DNA fragmentation assay, the cell death was attributed to apoptosis. The high-molecular-weight fraction of the cold water extract exhibited cytotoxicity toward MCF7 and A549 cancer cells, with IC 50 of 70.0 and 76.7 µg/mL, respectively (Lee et al., 2012a).
The mycelial extract of L. rhinocerotis cultured in a stirred tank reactor was also reported to exhibit cytotoxic effect on cervical cancer cells (Ca Ski) (Abdullah et al., 2010). The ammonium sulfate precipitate obtained from the mycelial protein fraction showed a growth inhibition of 72-82% against Ca Ski cells. The aqueous methanol extract of mycelial and culture broth of L. rhinocerotis from liquid fermentation did not exert cytotoxicity toward the normal cell lines (Lau et al., 2014).
In conclusion, there is a consistency among the reported studies, that the alcoholic extracts were not cytotoxic toward various cancer cell lines (Yap et al., 2013;Lau et al., 2014). However, the selectivity of the extracts on non-tumorigenic cells lines is not consistent (Lau et al., 2013a;Lee et al., 2014). The cultivation, processing, and preparation of extract may affect the cytotoxicity toward different cell lines. The chemical compositions of the extract may also influence the degree of cytotoxic level toward the cell lines. According to the reported literature, the primary identified bioactive components are more to high-molecular-weight hydrophilic components (Lai et al., 2008;Lau et al., 2014), protein-carbohydrates complex , and protein/peptides (Lau et al., 2013b).

Anti-viral Activity
The mycelial protein fractions of L. rhinocerotis were previously reported to inhibit human papilloma virus (HPV) activity (Abdullah et al., 2010). The anti-dengue activity tested by plaque reduction assay against dengue virus type-2 (DENV-2) strain was detected in hot aqueous extract of L. rhinocerotis at IC 50 of 520 µg/mL (Ellan et al., 2013). Hot aqueous extract of L. rhinocerotis also inhibited dengue viral RNA synthesis by 99.68%. The extract showed anti-dengue activity in viral penetration assay but did not have a significant effect on DENV-2 virucidal activity and viral attachment. The antidengue activity was correlated with the carbohydrate content in the hot aqueous extract of L. rhinocerotis (2.0 mg/mL) (Ellan et al., 2014). This suggested that the L. rhinocerotis has antidengue activity attributed to the polysaccharide content in the sclerotium.

Immunomodulatory Actions
Immune modulatory activity can lead to anti-inflammation and anti-tumor effects. The immune responses are mediated by various immune cells and their secondary secretory components (Wong and Cheung, 2009). Guo et al. (2011) investigated the effect of aqueous extract of L. rhinocerotis sclerotium on RAW 264.7 and primary macrophages isolated from BALB/c mice. The mushroom treatment promoted a significant up-regulation of pinocytosis, leading to an increase in the production of ROS, NO, and TNF-α production. The iNOS expression in both RAW 264.7 cells and primary macrophages was also increased. The expression of Dectin-1 + cells on the cell surface was shown to decrease. On the other hand, the complement receptor (CR3 + ) and toll-like receptor (TLR2 + ) were increased in response to aqueous extract-treated primary macrophage. The aqueous extract also increased the phosphorylation of I-kappa-B alpha (IKBα), which then triggered the nuclear factor kappa B (NF-κB) signaling pathway. Therefore, the immunomodulatory effect of L. rhinocerotis could be intervened by macrophage activation via the NF-κB signal pathway.
The in vitro results from the previous studies were further tested in in vivo studies using healthy BALB/c mice and athymic nude mice. Their serum cytokine profile, splenocytes, and peritoneal exudate cells (PECs) were analyzed . The hot water extract and sonication-assisted cold alkalisoluble polysaccharides (PRS) of L. rhinocerotis increased the spleen weight of both healthy BALB/c mice and athymic nude mice. The hot water extract activated the neutrophil production whereas the PRS stimulated other innate immune cells.
The combined effect of L. rhinocerotis supplementation and resistance training on immune functions/parameters such as CD3 + and CD4 + T lymphocytes; and CD4 + B lymphocytes were measured in young males between 19 to 25 years . The results indicated that the combination of extracts and resistance training did not affect the immune functions of the subjects.
In another study, a water-soluble polysaccharide-protein complex (PRW1) was isolated from the sclerotium of Polyporus rhinocerus Cooke (synonym of L. rhinocerotis) and further purified by membrane ultrafiltration (Liu et al., 2016). It showed a significant increase of NO production and enhanced the release of a variety of cytokines, for example, granulocyte colony stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF). Accompanying the release of stimulating factors were the IL-6, IL12p40/70, monocyte chemoattractant protein-1 (MCP-1), MCP-5, macrophage inflammatory protein (MIP-1-α), MIP-2, regulated on activation normal T cell expressed and secreted (RANTES), soluble tumor necrosis factor receptor I (sTNFRI), and TNF-α. L. rhinocerotis also triggered extracellular signal-regulated kinase (ERK) phosphorylation and increased the iNOS expression. It was the first report that provided the molecular mechanism of the immunostimulatory properties of Polyporus-derived protein complex on RAW 264.7cells. Polyporus rhinocerus sclerotium may have a potential application for cancer immunotherapy.
It is noteworthy to clarify that while the myconutrients of L. rhinocerotis can improve immune system by activating several immune effector cells (pro-inflammatory), they too, can suppress the immune system by inhibiting certain inflammation markers (anti-inflammation). This "immunomodulating properties" is very common among fungal metabolites, especially the mushroom polysaccharides.

Neuritogenic Properties
The sclerotium of L. rhinocerotis was investigated for neurite outgrowth in rat pheochromocytoma adherent (PC12 adh) cells (Eik et al., 2011). The aqueous extract increased the percentage of neurite bearing cells by 9.8 to 23.6% in PC12 cells. The aqueous and ethanol extracts stimulated maximal neurite outgrowth of 23.6% and 18.5%; respectively at 20 µg/mL. It is also shown that the L. rhinocerotis aqueous extract showed better neurite outgrowth compared to other medicinal mushrooms tested. Furthermore, the combination of 20 µg/mL aqueous extract and 30 ng/mL of nerve growth factor (NGF) enhanced the neurite outgrowth by 42.1% compared to either aqueous extract (24.4%) or NGF (24.6%) alone (Eik et al., 2012).
The aqueous extract of the L. rhinocerotis mycelium also had high neurite outgrowth activity of 21.1% at 20 µg/mL in PC12 cells (John et al., 2011). The synergistic effects of L. rhinocerotis mycelium extract combined with other natural products were further tested on PC12 cells. The treatments with the combination of aqueous extract (20 µg/mL) and Gingko biloba (30 µg/mL) resulted in 39.89% of neurite outgrowth activity when compared to G. biloba alone (31.15%) (John et al., 2012). The increase of neurite outgrowth activity was significant although the synergistic effects are not remarkably high. The effects of L. rhinocerotis mycelium extract (20 µg/mL) bio-augmented with 1 µg/mL of curcumin were subsequently tested (John et al., 2013). The combination has enhanced neurite outgrowth by 27.2% when compared to curcumin alone. Phan et al. (2013) reported neurite outgrowth of aqueous extract of L. rhinocerotis sclerotium and mycelium in mouse neuroblastoma (N2a) cells. At 20 µg/mL, the aqueous extract of sclerotium resulted in 38.1% of neurite bearing cells, which was approximately twice the number of NGF-treated neurite bearing cells. However, the aqueous extract of the mycelium did not cause a significant increase in neurite outgrowth when compared to NGF treatment. In all the studies, neuronal differentiation in the cell lines treated by L. rhinocerotis extracts were further demonstrated by indirect immunofluorescence staining of neurofilament protein.
On the other hand, the ethyl acetate and n-butanol fractions of L. rhinocerotis were found to mimic the neuritogenic activity of NGF by targeting the TrkA receptor and activated the mammalian target of rapamycin (mTOR) signaling pathway with the phosphorylation of the transcription factors, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), and leading to the increased expressions of neuritogenesis biomarker, i.e., the growth associated protein 43 (GAP43), tubulin alpha 4A (TUBA4A), and tubulin beta 1 (TUBB1) in PC12 cells (Seow, 2016).
In a different study, aqueous extract of L. rhinocerotis sclerotium was investigated for stimulation of neurite outgrowth in dissociated cells of the brain, spinal cord, and retina of chick embryo (Samberkar et al., 2015). After 48 h incubation, the aqueous extract at 50 µg/mL induced maximum neurite outgrowth of 20.8 and 24.7% in brain and spinal cord. On the other hand, 20.8% of neurite outgrowth was achieved in retinal cells at 25 µg/mL. The neuronal differentiation by L. rhinocerotis aqueous extract-treated cells was then confirmed by immunofluorescence staining.

Toxicology Evaluation
It is crucial that the toxicity assessment is done both in in vitro and in vivo assays in order to develop L. rhinocerotis as health supplements. The teratogenicity effects of L. rhinocerotis was carried out by Lee et al. (2013a). The freeze-dried sclerotium of L. rhinocerotis was orally administrated to both male and female mice for 28 days and then the animals were allowed to mate for 10 days. The female rats were continuously fed with the sclerotium powder until the pups were delivered (around 7-8 weeks after mating period). The pups were assessed for antifertility and teratogenic effects, if any. The 100 mg/kg sclerotium of L. rhinocerotis did not affect the fertility of the rats. Further, the dosage tested did not trigger teratogenic effects or aberrations in the litter.
The genotoxic effects of the sclerotium of the L. rhinocerotis or its possibility to cause gene mutations were evaluated in the plate incorporation and pre-incubation tests (Lee et al., 2013a). The bacteria strains were Salmonella typhimurium and Escherichia coli strains. There was no genotoxic effects or mutation in all the five strains (TA98, TA100, TA1535, TA1537, and WP2 uvrA) tested.
The genotoxic effects of L. rhinocerotis sclerotia were further evaluated by the bacterial reverse mutation or Ames test, the in-vitro chromosome aberration and in vivo mammalian erythrocyte micronucleus assays (Chen et al., 2013). At the concentration of the mycelium of L. rhinocerotis at 100 mg/mL (5 mg/plate) dose no mutagenic activity in the presence and absence of S9 metabolic activation system was observed in all the five Salmonella strains. The number of structural aberrations in Chinese hamster ovary (CHO-K1) cells was comparable to the negative control. The cells were unaltered and frequencies across the treatment had insignificant damage. The treatment with L. rhinocerotis displayed no adverse effect on the natural micronucleus frequency in both male and female mice. Further polychromatic erythrocytes (PCE) percentage and micronucleus frequency in 2,000 mg/kg L. rhinocerotis orally administrated animals had no significant difference when compared to the control animals.
The effect on cellular viability of L. rhinocerotis extracts of non-tumorigenic cell lines was investigated. The aqueous extract  of L. rhinocerotis sclerotium was not cytotoxic to normal human breast (184B5) and normal lung (NL20) cells (Lee et al., 2012a). The methanol extract of L. rhinocerotis sclerotium also was not cytotoxic to normal human colon (CCD-18co), kidney (HEK-293), nasopharyngeal (NP69), oral (OKF6), rat kidney (NRK-52E), and Vero cell lines (Lau et al., 2013a;Suziana Zaila, 2013). Eik et al. (2012) and Seow et al. (2015) reported that the aqueous and ethanol extracts of the L. rhinocerotis sclerotium were non-cytotoxic to PC12 cells after 48 h incubation. Phan et al. (2013) also reported that the aqueous extracts of the mycelium and sclerotium of L. rhinocerotis were not cytotoxic with IC 50 values of 1.75-5.93 mg/mL to mouse embryonic fibroblasts (BALB/3T3) and N2a cells after 24 h incubation. Baskaran (2015) reported that the aqueous and ethanol extracts were non-cytotoxic to RAW264.7 macrophages cells. Nallathamby et al.(2013Nallathamby et al.( , 2016, too found that the ethanol, hexane, and ethyl acetate fractions obtained from the sclerotium was non-cytotoxic to BV2 microglial cells. The crude polysaccharides, hot aqueous and ethanol extracts of the sclerotium of L. rhinocerotis were shown to be non-cytotoxic to BV2 microglial cells. Further, the hexane, ethyl acetate, n-butanol and aqueous fractions from hot aqueous and ethanol extracts were not cytotoxic to BV2 microglial cells (Nallathamby et al., 2013(Nallathamby et al., , 2016. In a sub-acute toxicity in vivo study using Sprague Dawley rats, the sclerotium powder of cultivated and wild L. rhinocerotis were orally administrated at 250, 500, and 1,000 mg/kg doses (Shien et al., 2011). Neither the cultivated nor the wild L. rhinocerotis sclerotium had adversative effects on the growth rate, blood, and clinical biochemical parameters. There were also no pathological changes in the vital organs like liver, kidneys, spleen, and lungs.
Findings obtained from the chronic toxicity study on L. rhinocerotis TM02 cultivar were also similar (Lee et al., 2013a). The oral administration of the sclerotium powder at the highest dose of 1,000 mg/kg did not cause adverse effects. Therefore, the no-observed-adverse-effectlevel (NOAEL) dose of the sclerotium powder was higher than 1,000 mg/kg. Based on both in vitro and in vivo toxicity assessments reported, indicate that cultivated L. rhinocerotis, the mycelium or sclerotium, were safe for consumption. Table 3 summarizes the medicinal properties of this mushroom. In order to expand the application of this mushroom, certain aspects of research should be further studied in detail.

Future Perspectives
First, since there is a high demand for this mushroom and the current lack of supply, more studies could be conducted on the domestication of L. rhinocerotis by optimizing the cultivation conditions to produce more consistent yield and bioactive components in extracts. Second, in most studies conducted so far, the medicinal properties of L. rhinocerotis were demonstrated using crude mushroom extracts or mixture containing different constituents. It is therefore necessary to identify the active bio-molecules for a better grasp of the medicinal properties and the mechanistic pathways of the novel compounds. Figure 3 provides an overview of the recent findings of the medicinal properties of L. rhinocerotis and the proposed mechanistic pathways of some of the activities.
Third, many findings were based on in vitro results. Thus, these studies should be further investigated in in vivo and human/ clinical studies to validate the uses scientifically. Besides, more applications for L. rhinocerotis extracts/ bioactive compounds should be ventured such as nanotechnology and genomic identifications. Finally, more surveys should be conducted in cooperation with the indigenous people to discover more traditional medicine applications. This will enhance the understanding of the medicinal properties based on traditional knowledge and applications for evidential documentation.

CONCLUSION
The L. rhinocerotis sclerotium is well-known for its ethnomedicinal uses in curing many ailments. The efforts to research, compile, and validate the information scientifically is a continuous process in the development of medicinally relevant products. This review showed that the sclerotium of L. rhinocerotis possessed several potential therapeutic properties that would be useful to human's health. Further research is warranted to identify and isolate chemical components/bioactive components and their mode of actions.

AUTHOR CONTRIBUTIONS
NN, C-WP, SL-SS, AB, and VS have written the first draft of the manuscript. HL and SNAM revised and improved the first draft. All authors have seen and agreed on the finally submitted version of the manuscript.