Many macrofungus sclerotia are well-known medicinal herbs, health food and nutritional supplements. However, the prevalent adulterant commercial products are major hindrances to their incorporation into mainstream medical use in many countries. The mushroom sclerotia of Lignosus rhinocerotis, Poria cocos, Polyporus umbellatus, Pleurotus tuber-regium and Omphalia lapidescens are commonly used in traditional Chinese medicine. In this study, IR macro-fingerprint method was used in the identification of these sclerotia. The results showed that the spectrum of L. rhinocerotis (LR) was comparable with P. cocos with 94.4% correlation, except that the peak at 1543cm(-1) of LR appeared in lower intensity. The spectrum of P. umbellatus and P. tuber-regium was also correlated (91.5%), as both spectra could be clearly discriminated in that P. umbellatus spectrum has small base peaks located at the range of 1680-1500cm(-1). O. lapidescens was not comparable with all the other sclerotia as its spectrum was totally different. Its base peak was broad and derivated equally along the range. The first IR has revealed the dissimilarity among five mushrooms sclerotia. The second derivative and 2DIR further enhanced the identification in detail.
Studies on fungal taxonomy and fungal diversity are crucial for a better understanding of the interactions between fungi and their habitats. To date, there is no published record on macrofungal diversity in Pulau Bidong, Terengganu. Therefore, this study aimed to identify macrofungi species found with their respective substrates and to determine the macrofungal diversity in the island. The study was conducted at two different occasions and comprised of two trails near Universiti Malaysia Terengganu Research Station in Pulau Bidong, Terengganu. Collectionof fresh macrofungi was made and additional data including host information, distinctive features of each fungal sample and several environmental parameters was also recorded. Fresh specimens were later observed and identified before being dried prior to storage. Overall, 65 macrofungal species with 2 unidentified species belonging to 21 families and 34 genera were recorded. Trail A and trail B recorded diversity index of2.67 and 3.14 for Shannon Index while Simpson index recorded 0.90 and 0.94 respectively. The macrofungal diversity was dominated by family Polyporaceae while rare species discovered were from family of Tricholomataceae, Hericiaceae, Stereaceae, Schizophyllaceae, Sclerodermataceae, Dacrymycetaceae, Tremellaceae, Russulaceae and Clavulinaceae. The most common macrofungal substrates was decayed branches while other macrofungal substrates found were decayed trunks and leaves, soil,termite mounds, and living tree. In conclusion, the macrofungal species were randomlydistributed with high diversity in both trails. Therefore, it is recommended that anincrease in sampling trips, sampling efforts and areas covered be provided to increase the number of macrofungal species discovered and accuracy of diversity studies in the future.
Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut+ transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 μg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 μg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.
Four fungal isolates: Simplicillium chinense (iso 9, accession no. KX425621), Penicillium simplicissimum (iso 10, KP713758), Trichoderma asperellum (iso 11, KP792512), and Coriolopsis sp. (1c3, KM403574) were subjected to a series of induced-tolerance training under high metal concentrations to determine if greater tolerance could be achieved from constant exposure to such conditions. Adaptive tolerance assay (Tolerance Index, TI) and Field-Emission Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX) characterized their metal tolerance. "Untrained" S. chinense, P. simplicissimum and T. asperellum showed tolerance towards 4000-4500ppm Al(III) (TI: 0.64-0.71), 1000ppm Cr(III) (0.52-0.83) and Pb(II) (0.32-0.88). With tolerance training, tolerance towards 2000-6000ppm Al(III), 500-3000ppm Pb(II) and 2000-3000ppm Cr(III) were achieved (TI: 0.01-0.82) compared to untrained cultures (0.00-0.59). In contrast, tolerance training for Coriolopsis sp. and P. simplicissimum was less successful, with TI values similar or lower than untrained cultures. SEM-EDX analysis proposed biosorption and bioaccumulation as mechanisms for metal removal. The latter was demonstrated with the removal of Cr(III) and Pb(II) by S. chinense (12.37 and 11.52mgg-1, respectively) and T. asperellum (10.44 and 7.50mgg-1). Induced-tolerance training may render benefit in the long run, but this delicate approach is suggestively species and metal dependent.
Macrofungi of the order Polyporales are among the most important wood decomposers and caused economic losses by decaying the wood in standing trees, logs and in sawn timber. Diversity and distribution of Polyporales in Peninsular Malaysia was investigated by collecting basidiocarps from trunks, branches, exposed roots and soil from six states (Johor, Kedah, Kelantan, Negeri Sembilan, Pahang and Selangor) in Peninsular Malaysia and Federal Territory Kuala Lumpur. This study showed that the diversity of Polyporales were less diverse than previously reported. The study identified 60 species from five families; Fomitopsidaceae, Ganodermataceae, Meruliaceae, Meripilaceae, and Polyporaceae. The common species of Polyporales collected were Fomitopsis feei, Amauroderma subrugosum, Ganoderma australe, Earliella scabrosa, Lentinus squarrosulus, Microporus xanthopus, Pycnoporus sanguineus and Trametes menziesii.
Neuritin is important in neuritogenesis, neurite arborization, and neurite extension. Lignosus rhinocerotis sclerotia extracts and nerve growth factor (NGF) have been well documented to possess positive neurite stimulatory effects. However, the correlation of neuritin expression with neurite outgrowth of L. rhinocerotis and NGF cotreatment of PC12 cells remains unknown. Thus, the present study investigated neuritin expression in PC12 cells treated with 5 ng/mL of NGF and L. rhinocerotis extracts (20-1280 μg/mL) concurrently for 48 h. The neurite outgrowth score was quantitated, and total protein was harvested for enzyme-linked immunosorbent assay. There was a significant difference (P = 0.051) in neuritin protein abundance in 640 μg/mL of L. rhinocerotis aqueous cotreatment with 5 ng/mL of NGF-treated cells (5 ± 0.39 ng/mL) and 50 ng/mL of NGF-treated PC12 cells (5 ± 0.48 ng/mL) compared to untreated cells (1.9 ± 0.65 ng/ mL), with an average neurite length of 98 ± 3.66, 106 ± 3.00, and 73 ± 4.79 μm, respectively. Expression of microtubule element β3 tubulin was increased in PC12 cells treated with 50 ng/mL of NGF (3.5 ± 0.21-fold) and also cells cotreated with 640 μg/mL of extract and 5 ng/mL of NGF (4.9 ± 0.29-fold) compared to untreated cells. Upregulation of β3 tubulin expression in this study confirmed the elongation of PC12 cell processes. Correlation analysis showed that neuritin protein abundance is positively proportional to the average neurite length in PC12 cells cotreated with L. rhinocerotis extract and 5 ng/mL of NGF. This study highlights that neuritin modulation is involved in neurite outgrowth induced by L. rhinocerotis treatment. To our knowledge, this is the first report to show that tiger milk mushroom extracts induce neuritin expression.
Laccases hold great potential for biotechnological applications, particularly in environmental pollutant remediation. Laccase activity is governed by the solvent environment, and ionic liquids (ILs) emerge as a versatile solvent for activation or stabilization of enzymes. Herein, effects of cholinium-based ILs formulated with carboxylic acids, inorganic acid, and amino acids as anionic species, on the catalytic activity of laccase from Trametes versicolor were investigated by experimental and computational approaches. Experimental results showed that laccase activity was enhanced by 21.39 % in 0.5 M cholinium dihydrogen citrate ([Cho][DHC]), in relation to the laccase activity in phosphate buffer medium. However, cholinium aminoate ILs negatively affected laccase activity, as evidenced by the partial deactivation of laccase in both cholinium glycinate and cholinium phenylalaninate, at concentrations of 0.1 M and 0.5 M, respectively. Molecular dynamics studies revealed that the enhancement of laccase activity in [Cho][DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. Enhanced catalytic performance of laccase in [Cho][DHC] was postulated to be driven by the high accumulation level of dihydrogen citrate anions around laccase's surface. [Cho][DHC] holds great promise as a cosolvent in laccase-catalyzed biochemical reactions.
Lignosus rhinocerus (known locally as 'Tiger Milk mushroom') is the most important medicinal mushroom used by the indigenous communities of Malaysia to treat fever, cough, asthma, cancer, food poisoning and as a general tonic. The sclerotium of the mushroom is the part with medicinal value. Lignosus rhinocerus was hitherto unexploited commercially because of limited supply. Recently, the mushroom was successfully cultivated.
The traditional application of the sclerotium of Lignosus rhinocerotis (tiger's milk mushroom) by the indigenous folks as tonic and remedy to treat a variety of ailments has been documented in Malaysia. Indigenous communities claimed to have consumed the decoction to boost their alertness during hunting. Mental alertness is believed to be related to neuronal health and neuroactivity. In the present study, the cell viability and neuritogenic effects of L. rhinocerotis sclerotium hot aqueous and ethanolic extracts, and crude polysaccharides on rat pheochromocytoma (PC-12) cells were studied. Interestingly, the hot aqueous extract exhibited neuritogenic activity comparable to NGF in PC-12 cells. However, the extracts and crude polysaccharides stimulated neuritogenesis without stimulating the production of NGF in PC-12 cells. The involvements of the TrkA receptor and MEK/ERK1/2 pathway in hot aqueous extract-stimulated neuritogenesis were examined by Trk (K252a) and MEK/ERK1/2 (U0126 and PD98059) inhibitors. There was no significant difference in protein expression in NGF- and hot aqueous extract-treated cells for both total and phosphorylated p44/42 MAPK. The neuritogenic activity in PC-12 cells stimulated by hot aqueous and ethanolic extracts, and crude polysaccharides of L. rhinocerotis sclerotium mimicking NGF activity via the MEK/ERK1/2 signaling pathway is reported for the first time.
Natural compounds found in Lignosus rhinocerus like polysaccharides and polysaccharide-protein complexes have the capabilities to modulate the immune system. It possesses antitumor and anti-inflammatory properties and is commonly used in Southeast Asia and Southern China to alleviate illness. To investigate its immunomodulating properties, composition of polysaccharides and the expression of cytokines/chemokines from L. rhinocerus (TM02®) cultivar treated RAW 264.7 were explored. It was revealed, CWE contains linear polysaccharides with 1,4-linkages and rhinoprolycan fraction (HMW & MMW) possesses 1,4-Glcp and 1,6-Glcp backbone and branched chain (1,3,6-Glcp, 1,4,6-Glcp, 1,3,6-Glcp, 1,2,4,6-Glcp). Cytokines profile showed upregulation from CWE (IL-5: 12.078 ± 1.225), HMW (IL-6: 7.297 ± 0.338; TIMP-1: 3.358 ± 0.200), MMW (IL-5: 15.412 ± 5.823; TIMP-1: 1.747 ± 0.053), and LMW (MIP-2: 3.495 ± 0.416; TIMP-1: 7.573 ± 0.088) and possible involvement of NF-κB and MAPK signaling pathway. Further in vivo studies are needed to fully understand the immunomodulatory effects of TM02®.
The aim of this study was to determine the antiulcer activity of Lignosus rhinocerotis in rats. A total of 48 Sprague-Dawley rats were used in ethanol-induced, aspirin-induced, and water immersion-restraint stress-induced ulcer models. Rats were equally divided into 4 groups for each model and orally administered 5 mL/kg distilled water, 20 mg/kg omeprazole, as well as 250 and 500 mg/kg of L. rhinocerotis powder. L. rhinocerotis powder at both 250 and 500 mg/kg doses demonstrated significant (P < 0.05) protection against gastric ulceration in all the induced ulcer models. Histological studies revealed severe damage and hemorrhage of gastric mucosa in the negative control group for all ulcer-induced models. The study suggests that L. rhinocerotis powder possesses dose-dependent antiulcer activity in the gastric mucosa, as ascertained grossly and histologically, compared with the negative control groups.
In Malaysia and China, the sclerotium of Lignosus rhinocerotis is used by local communities and traditional medicine practitioners as a general tonic and remedy to treat a variety of ailments, including inflammation-associated disorders. In this study, 10 samples from different preparations of L. rhinocerotis sclerotium, including a hot aqueous extract (HAE), an ethanol extract (EE), fractions from the HAE and EE, and crude polysaccharides, were tested for their in vitro cytotoxic and nitric oxide (NO) inhibitory activities in lipopolysaccharide (LPS)--stimulated BV2 microglia. Of the 10 samples tested, HAE was the least cytotoxic toward BV2 microglia, with a half-maximal inhibitory concentration of 176.23 ± 2.64 mg/mL at 24 hours of incubation and 20.01 ± 1.69 mg/ mL at 48 hours of incubation. The inhibition of NO production was explored by pretreatment of BV2 microglia with samples at 2 incubation time points (4 and 24 hours) before the stimulation by LPS for 24 hours. After 24 hours of pretreatment, 8 of the 10 samples inhibited NO production by 50% or more, and cytotoxic effects were not observed. Among the 8 active samples, 500 µg/mL of HAE, 250 µg/mL of an n-butanol fraction of the HAE, and 250 µg/mL of an ethyl acetate fraction of HAE showed maximum inhibition of NO production by 88.95%, 86.50%, and 85.93%, respectively. These results suggest that the L. rhinocerotis sclerotium may contain secondary metabolites that have the potential to inhibit NO production.
Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications.
Lignosus also known as "Tiger Milk Mushroom," is classified in the family Polyporaceae and mainly consumed for its medicinal properties in Southeast Asia and China. The sclerotium is known as the part with medicinal value and often used by the natives to treat a variety of ailments. Lignosus tigris Chon S. Tan, one of the species of the Malaysia Tiger Milk mushroom, has recently been successfully cultivated in laboratory. Earlier studies have demonstrated the L. tigris cultivar E sclerotia exhibited beneficial biomedicinal properties. This study evaluated the potential toxicity of L. tigris E sclerotia in a 28-day sub-acute oral administration in Sprague Dawley (SD) rats. L. tigris E sclerotial powder was administered orally at three different doses of 250, 500, and 1000 mg/kg to the SD rats once daily, consecutively for 28-days. Body weight of the rats was recorded and general behavior, adverse effects, and mortality were observed daily throughout the experimental period. At the end of the experiment, blood hematology and biochemistry, relative organ weights, and histopathological analysis were performed. Results showed that there were no mortality nor signs of toxicity throughout the 28-day sub-acute toxicity study. Oral administration of the L. tigris E sclerotial powder at daily dose up to 1000 mg/kg had no significant effects in body weight, relative organ weight, blood hematological and biochemistry, gross pathology, and histopathology of the organs. L. tigris E sclerotial powder did not cause any treatment-related adverse effect in the rats at different treatment dosages up to 1000 mg/kg. As the lethal dose for the rats is above 1000 mg/kg, the no-observed-adverse-effect level (NOAEL) dose is more than 1000 mg/kg.
Lignosus rhinocerotis has a long history of use by the indigenous community within East Asia to treat a range of health conditions including asthma and chronic cough. To date, there is limited scientific evidence to support its therapeutic effects in relieving these airways conditions. In this study, we examined the effects of the different molecular weight fractions [high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW)] obtained from the cold water sclerotial extract (CWE) of L. rhinocerotis on airways patency using airway segments isolated from Sprague Dawley rat in an organ bath set-up. It is demonstrated that the HMW and MMW fractions exhibited higher efficacy in relaxing the pre-contracted airways when compared to the CWE and LMW fraction. In addition, the HMW fraction markedly supressed carbachol-, 5-hydroxytrptamine-, and calcium-induced airway contractions. CWE demonstrated a lower efficacy than the HMW fraction but it also significantly attenuated carbachol- and calcium-induced airway contractions. Results showed that the bronchorelaxation effect of CWE and fractions is mediated via blockade of extracellular Ca2+ influx. The composition analysis revealed the following parts of carbohydrate and proteins, respectively: HMW fraction: 71 and 4%; MMW fraction: 35 and 1%; and LMW fraction: 22 and 0.3%. Our results strongly suggest that the polysaccharide-protein complex or proteins found in the HMW and MMW fractions is likely to contribute to the bronchorelaxation effect of CWE.
Twenty-eight days subacute toxicity studies performed in rats using sclerotial powder of Lignosus cameronensis cultivar was conducted to assess its safety for consumption prior to other scientific investigations on its medicinal benefits, nutraceutical or pharmaceutical application of the mushroom. The study was conducted at 250, 500, and 1000 mg/kg sclerotial powder of L. cameronensis cultivar (n = 5 for each respective dose, on both male and female groups) while control groups received only distilled water. At the end of the study (29th day), the animals were sacrificed followed by blood and organs collection for analysis. Subacute toxicity studies done shows that sclerotial powder of L. cameronensis cultivar at 250, 500, and 1000 mg/kg did not induce treatment related changes on behavioral patterns, gross physical appearance, growth pattern, body weight gain, values of hematological and clinical biochemical panels as well as histopathological findings on kidney, spleen, heart, lung and liver of the experimental rats. The no-observed-adverse-effect level dose for sclerotial powder of L. cameronensis cultivar in 28-days sub-acute toxicity study is determined to be 1000 mg/kg.
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.
A national treasure mushroom, Lignosus rhinocerus, has been used to treat variety of ailments by local and indigenous communities in Malaysia. The aim of this study was to investigate the potential of the most valuable part of L. rhinocerus, the sclerotium, on neurite outgrowth activity by using PC-12Adh cell line. Differentiated cells with one thin extension at least double the length of the cell diameter were scored positive. Our results showed that aqueous sclerotium L. rhinocerus extract induced neurite outgrowths of 24.4% and 42.1% at 20 μg/mL (w/v) of aqueous extract alone and a combination of 20 μg/mL (w/v) aqueous extract and 30 ng/mL (w/v) of NGF, respectively. Combination of NGF and sclerotium extract had additive effects and enhanced neurite outgrowth. Neuronal differentiation was demonstrated by indirect immunofluorescence of neurofilament protein. Aqueous sclerotium extract contained neuroactive compounds that stimulated neurite outgrowth in vitro. To our knowledge this is the first report on neurite-stimulating activities of L. rhinocerus.
Lignosus rhinocerus, the tiger milk mushroom, is one of the most important medicinal mushrooms used by the indigenous people of Southeast Asia and China. It has been used to treat breast cancer. A cold water extract (LR-CW) prepared from the sclerotia of L. rhinocerus cultivar was found to exhibit antiproliferative activity against human breast carcinoma (MCF-7) and human lung carcinoma (A549), with IC(50) of 96.7 μg/mL and 466.7 μg/mL, respectively. In comparison, LR-CW did not show significant cytotoxicity against the two corresponding human normal cells, 184B5 (human breast cell) and NL 20 (human lung cell). DNA fragmentation studies suggested that the cytotoxic action of LR-CW against cancer cells is mediated by apoptosis. Sephadex G-50 gel filtration fractionation of LR-CW yielded a high-molecular-weight and a low-molecular-weight fraction. The high-molecular-weight fraction contains mainly carbohydrate (68.7%) and small amount of protein (3.6%), whereas the low-molecular-weight fraction contains 31% carbohydrate and was devoid of protein. Only the high-molecular-weight fraction exhibited antiproliferative activity against cancer cells, with IC(50) of 70.0 μg/mL and 76.7 μg/mL, respectively. Thus, the cytotoxic action of the LR-CW is due to the high-molecular-weight fraction, either the proteins or protein-carbohydrate complex.
The sclerotium of the "tiger's milk mushroom" (Lignosus rhinocerotis) is used as tonic and folk medicine for the treatment of cancer, fever, cough and asthma by the local and indigenous communities. It is traditionally prepared by either boiling or maceration-like methods; however, there is no attempt to understand how different processing methods might affect their efficacies as anticancer agents.