Effect of ultrasound assisted H2O2/Vc treatment on the hyperbranched Lignosus rhinocerotis polysaccharide: Structures, hydrophobic microdomains, and antitumor activity.

The effect of ultrasonic intensity (28.14, 70.35, and 112.56 W/cm2) on Lignosus rhinocerotis polysaccharide (LRP) degraded by ultrasound assisted H2O2/Vc system (U-H/V) was investigated. U-H/V broke the molecular chain of LRP and improved the conformational flexibility, decreasing the molecular weight, intrinsic viscosity ([η]) and particle size. The functional groups and hyperbranched structure of LRP were almost stable after U-H/V treatment, however, the triple helix structure of LRP was partially disrupted. With increasing ultrasonic intensity, the critical aggregation concentration increased from 0.59 mg/mL to 1.57 mg/mL, and the hydrophobic microdomains reduced. Furthermore, the LRP treated with U-H/V significantly inhibited HepG2 cell proliferation by inducing apoptosis. The increase in antitumor activity of LRP was closely associated with the reduction of molecular weight, [η], particle size and hydrophobic microdomains. These results revealed that U-H/V treatment facilitates the degradation of LRP and provides a better insight into the structure-antitumor activity relationship of LRP.

Isolation, structural features, rheological properties and bioactivities of polysaccharides from Lignosus rhinocerotis: A review.

L. rhinocerotis, an edible and medicinal mushroom, has long been utilized as folk medicine and nutritional food in Southeast Asia and southern China. Polysaccharides are the main bioactive substances of L. rhinocerotis sclerotia, and they have attracted extensive attention of researchers both at home and abroad. In the past few decades, various methods have been applied to extract polysaccharides from L. rhinocerotis (LRPs) and the structural features of LRPs are closely related to the used methods of extraction and purification. Many studies have confirmed that LRPs possess various remarkable bioactivities, including immunomodulatory, prebiotic, antioxidant, anti-inflammatory and anti-tumor activities and intestinal mucosa protective effect. As a natural polysaccharide, LRP has the potential to be a drug and functional material. This paper systematically reviews the recent studies on structural characteristics, modification, rheological properties and bioactivities of LRPs, and provides a theoretical basis for an in-depth study of the structure-activity relationship, and utilization of LRPs as therapeutic agents and functional foods. Additionally, the further research and development of LRPs are also prospected.

Neuroprotective effects of a novel peptide from Lignosus rhinocerotis against 6-hydroxydopamine-induced apoptosis in PC12 cells by inhibiting NF-κB activation.

According to previous studies, oxidative stress is a leading cause of dopaminergic neuron death and may contribute to the pathogenesis of Parkinson's disease (PD). In the current study, we used chromatography of gel filtration to identify a novel peptide (Lignosus rhinocerotis peptide [LRP]) from the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden. Its neuroprotective effect was evaluated using an in vitro PD model constructed by 6-hydroxydopamine (6-OHDA)-stimulated to apoptosis in PC12 cells. The molecular weight of LRP is determined as 1532 Da and the secondary structure is irregular. The simple amino acid sequence of LRP is Thr-Leu-Ala-Pro-Thr-Phe-Leu-Ser-Ser-Leu-Gly-Pro-Cys-Leu-Leu. Notably, LRP has the ability to significantly boost the viability of PC12 cells after exposure to 6-OHDA, as well as enhance the cellular activity of antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). LRP also lowers the level of malondialdehyde (MDA), decreases the activation performance of Caspase-3, and reduces 6-OHDA-induced apoptosis via inhibition of nuclear factor-kappa B (NF-κB) activation. These data indicate that LRP may have the potential to act as a neuroprotective agent.

Ultrasound-induced changes in rheological behavior and hydrophobic microdomains of Lignosus rhinocerotis polysaccharide.

Ultrasound is increasingly applied to modify the structures and physicochemical properties of polysaccharides. Hence, this work investigated the ultrasound-induced changes in the rheological behavior and hydrophobic microdomains of Lignosus rhinocerotis polysaccharide (LRP). With an increase in ultrasonic time, the apparent viscosity, storage modulus, loss modulus, and the final percentage recovery of LRP/water system increased to reach the maximum after 10 min treatment and then decreased. These results indicated that short-term (10 min) ultrasound could increase the strength of the network structure of LRP/water system, while longer-term ultrasound (30 and 60 min) weakened the network structure. The self-healing properties of LRP/water system was not affected by ultrasound treatment according to repeated strain and time sweep data. The critical aggregation concentration of the LRP/water system decreased from 2.5 to 1.8 mg/mL after 10 min ultrasound and the number of hydrophobic microdomains increased, suggesting that ultrasound promoted the hydrophobic aggregation of LRP.

Rheological properties and critical concentrations of a hyperbranched polysaccharide from Lignosus rhinocerotis sclerotia.

The application of polysaccharides in the food industry mainly depends on their rheological properties and the polysaccharides in different concentration regions exhibit different rheological properties due to the interactions between polymer chains. Hence, this work investigated the concentration-dependent rheological behavior of Lignosus rhinocerotis polysaccharide (LRP) in water and determined the critical concentrations. The intrinsic viscosity of LRP was 378 ± 32 mL/g and the LRP exhibited more apparent shear-thinning behavior with increasing concentration. The LRP critical overlap and aggregation concentration in water was ~ 2.5 mg/mL, implicating the formation of hydrophobic regions may result from the aggregation and overlap between hyperbranched LRP molecules. The LRP/water system showed higher storage modulus than loss modulus with slight frequency dependence at the concentration of 15 mg/mL, exhibiting the structured liquid behavior. When the concentration increased from 10 mg/mL to 30 mg/mL, the compliance recovery percentage value increased from 58.51% to 92.30%, indicating the formation of a strong gel network in the LRP/water system. Furthermore, the micro-rheological test revealed that the LRP/water system exhibited a concentration-dependent increase in elasticity and viscosity and deterioration in fluidity.

A polysaccharide from Lignosus rhinocerotis sclerotia: Self-healing properties and the effect of temperature on its rheological behavior.

This work investigated the self-healing properties of Lignosus rhinocerotis polysaccharide (LRP) and the effect of temperature on its rheological behavior. Dynamic sweep tests (strain sweep, frequency sweep, and time sweep) showed that the LRP/water system possessed self-healing properties due to the entangled network formed by hyperbranched LRP molecular chains. The flow activation energy of LRP solution calculated by Arrhenius equation was shown to decrease with increasing LRP concentration, indicating that LRP solution at higher concentration was less sensitive to temperature. Temperature ramp test exhibited that LRP had a glass transition temperature (Tg) determined as 49.35 °C and the temperature effect was irreversible. Microrheological test revealed that the LRP aqueous solution can form a gel at room temperature with the concentration ≥ 20 mg/mL. This work provided a theoretical basis for the development of LRP-based self-healing materials and facilitated a deep understanding of the temperature effect on rheological behavior of LRP.

Restitution of epithelial cells during intestinal mucosal wound healing: The effect of a polysaccharide from the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerotis (Cooke) Ryvarden cultivar TM02, also known as tiger's milk mushroom, is regarded as important folk medicine in Malaysia, while is used for the treatment of liver cancer, chronic hepatitis, gastric ulcer in traditional Chinese medicine. However, there is no compilation of scientific evidence that its protection for gastric, and no attempts have been made to understand how polysaccharides in Lignosus rhinocerotis might promote intestinal mucosal wound healing. AIM OF THE STUDY: This study aimed to investigate the effect and mechanism of ß-glucan prepared from L. rhinocerotis using an enzymatic method on epithelial restitution during intestinal mucosal damage. MATERIALS AND METHODS: Based on FT-IR, MALDI-TOF-MS, HPSEC-MALLS-RID, and AFM, the structure of polysaccharides from L. rhinocerotis was analysed. In addition, polysaccharides were used to test for wound healing activity in IEC-6 cells by measuring cell migration, proliferation, and expression of cell division control protein 42, Rac-1, RhoA, and Par-3. RESULTS: ß-glucan was extracted using enzyme-assisted extraction, and a yield of approximately 8.5 ± 0.8% was obtained from the dried biomass. The ß-glucan extracted by enzyme-assisted extraction (EAE) of polysaccharides was composed entirely of D-glucose with a total carbohydrate content of 95.5 ± 3.2%. The results of HPLC, FTIR, and MALDI-TOF-MS analyses revealed EAEP to be confirmed as ß-glucan. The molecular weight of prepared ß-glucan was found to be 5.315 × 104 g/mol by HPSEC-MALLS-RID. Furthermore, mucosal wound healing studies showed that the treatment of IEC-6 with a ß-glucan concentration of 200 µg/mL promoted cell migration and proliferation, and it enhanced the protein expression of cell division control protein 42, Rac-1, RhoA, and Par-3. CONCLUSIONS: The present study reveals that the prepared ß-glucan accelerates intestinal epithelial cell proliferation and migration via activation of Rho-dependent pathway. Hence, ß-glucan can be employed as a prospective therapeutic agent for the treatment of diseases associated with gastrointestinal mucosal damage, such as peptic ulcers and inflammatory bowel disease.

Nutritional and 13-Week Subchronic Toxicological Evaluation of Lignosus rhinocerotis Mycelium in Sprague-Dawley Rats.

Lignosus rhinocerotis (Tiger's Milk mushroom) is a novel mushroom with sclerotium belonging to the Polyporaceae family and has been reported widely to possess anti-cancer, anti-cough, antioxidant, gastro-protective, immuno-modulating, and neurite-stimulating properties. As numerous studies have proven the tremendous medicinal values of L. rhinocerotis, it is necessary to understand its nutrition as well as its safety for the recipient. Previous research on L. rhinocerotis has mainly focused on the naturally occurring sclerotium and may have overlooked mushroom mycelia from submerged liquid fermentation, which ensures a high uniform quantitative biomass production as well as a high biological value. Hence, this is the first report on the evaluation of nutrition and 13-week repeated oral toxicity of L. rhinocerotis mycelium (LRM). The LRM powder contained 9.0 ± 4.2% moisture, 1.9 ± 1.3% ash, 1.6 ± 2.2% crude lipid, 8.4 ± 5.3% crude protein, 79.3 ± 4.6% carbohydrate, and 364 kcal/100 g energy. The total free amino acid ranged from 349 to 5636 mg/100 g and the umami index of freeze-dried LRM powder was 0.37. For safety assessment, ninety-six rats were divided into four groups, each consisting of twelve male and twelve female rats. Test articles were administered by oral gavage to rats at 850, 1700, and 3400 mg/kg body weight/day for 13 weeks and reverse osmosis water was used as the control. All animals survived to the end of the study. During the experiment period, no abnormal changes were observed in clinical signs, body weight, or ophthalmological examinations. No adverse or test article-related differences were found in urinalysis, hematology, or serum biochemistry parameters between the treatment and control groups. Necropsy and histopathological examination indicated no treatment-related changes. According to the above results, the no-observed-adverse-effect level (NOAEL) of L. rhinocerotis was identified to be greater than 3400 mg/kg body weight (BW)/day in Sprague-Dawley rats.

Lipid-rich fraction of the sclerotium of Tiger Milk Mushroom Lignosus rhinocerotis (Agaricomycetes) attenuates LPS-induced inflammation in BV2 cells via Nrf2 pathway

Lignosus rhinocerotis (tiger milk mushroom) is widely used by the indigenous people of Malaysia as a traditional remedy. The present study was carried out in order to evaluate the antioxidant, cytotoxic and anti-neuroinflammatory activities of L. rhinocerotis extract on brain microglial cells (BV2). The antioxidant activity was evaluated by 2,2-diphenyl-1-picryhydrazyl (DPPH•), 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) scavenging assays, and ferric reducing antioxidant power (FRAP). The FRAP, DPPH and ABTS•+ scavenging capacities of the TE3 fraction were 420.77 mg FE/g, 58.01%, and 7%, respectively. The cytotoxic activity was determined by MTS assay. The in vitro model of anti-neuroinflammatory property was evaluated by measuring the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced BV2 cells. The TE3 fraction showed a significant NO reduction at 1 to 100 µg/mL. The TE3 fraction down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) genes while it upregulated heme oxygenase (HO-1) and NADPH quinone acceptor oxidoreductase-1 (NQO-1) genes. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription was also activated. The chemical component of the active fraction (TE3) was identified by gas chromatography-mass spectrometry (GCMS). Overall, the BV2 in vitro model anti-neuroinflammatory activity of L. rhinocerotis may be caused by the lipid constituents identified in the fraction

Intranasal administration of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) extract attenuates airway inflammation in murine model of allergic asthma.

Asthma is a chronic inflammatory disorder in the airways that involves the activation of cells and mediators. Lignosus rhinocerotis (Cooke) Ryvardan or Tiger Milk mushroom is a medicinal mushroom that is traditionally used to treat inflammatory diseases including asthma. In this study, the protective effects of intranasal administration of L. rhinocerotis extract (LRE) in ovalbumin (OVA)-induced airway inflammation mouse model were investigated. Mice were sensitized by intraperitoneal (i.p) injection on days 0 and 14, followed by a daily challenge with 1% OVA from days 21 to 27. Following OVA challenge, LRE and dexamethasone were administered via intranasal and i.p. injection respectively. On day 28, the level of serum immunoglobulin (Ig)E, differential cell counts and T-helper (Th) 2 cytokines in bronchoalveolar lavage fluid (BALF) fluid, cell subset population in lung-draining lymph nodes (LNs), leukocytes infiltration and mucus production in the lungs of the animals was measured. Results demonstrated that intranasal administration of LRE significantly suppressed the level of inflammatory cell counts in BALF as well as populations of CD4+ T-cells in lung draining LNs. Apart from that, LRE also significantly reduced the level of Th2 cytokines in BALF and IgE in the serum in OVA-induced asthma. Histological analysis also demonstrated the amelioration of leukocytes infiltration and mucus production in the lungs. Overall, these findings demonstrated the attenuation of airway inflammation in the LRE-treated mice therefore suggesting a promising alternative for the management of allergic airway inflammation.

Antibacterial activity of biosynthesized gold nanoparticles using biomolecules from Lignosus rhinocerotis and chitosan.

A simple, cost-effective, and environmentally friendly method is needed for synthesizing metal nanoparticles, including gold nanoparticles (AuNPs). In this study, AuNPs were synthesized with Lignosus rhinocerotis sclerotial extract (LRE) and chitosan (CS) as reducing and stabilizing agents, respectively. Different LRE concentrations from cold and hot water extraction (CWE and HWE, respectively) were used to reduce chloroauric acid (HAuCl4) to form AuNPs. Positively charged chitosan stabilized AuNPs (CS-AuNPs) mediated by LRE exhibited a surface plasmon resonance (SPR) band at 533 nm. The CS-AuNPs synthesized using CWE had a smaller particle size (49.5 ±â€¯6.7-82.4 ±â€¯28.0 nm) compared to that of the HWE samples (80.3 ±â€¯23.4-125.3 ±â€¯41.5 nm), depending on LRE concentration. FTIR results suggested protein and polysaccharides in LRE were the sources of reducing power, reducing gold ions to AuNPs. CS-AuNPs were mostly spherical with higher LRE concentrations, whereas some triangular, pentagonal, irregular, and rod shaped AuNPs were observed at lower LRE concentrations. CS-AuNPs mediated by LRE displayed effective antibacterial activity against gram-negative (Pseudomonas aeruginosa and Escherichia coli) and gram-positive bacteria (Staphylococcus aureus and Bacillus sp.). Thus, the biosynthesized AuNPs using LRE and chitosan provide opportunities for developing stable and eco-friendly nanoparticles with effective antibacterial properties.

Airway Relaxation Effects of Water-Soluble Sclerotial Extract From Lignosus rhinocerotis.

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.

Data on the Lignosus rhinocerotis water soluble sclerotial extract affecting intracellular calcium level in rat dorsal root ganglion cells.

The data in this article contain supporting evidence for the research manuscript entitled "Bronchodilator effects of Lignosus rhinocerotis extract on rat isolated airways is linked to the blockage of calcium entry" by Lee et al. (2018) [1]. The data were obtained by calcium imaging technique with fluorescent calcium indicator dyes, Fura 2-AM, to visualize calcium ion movement in the rat dorsal ganglion (DRG) cells. The effects of L. rhinocerotis cold water extract (CWE1) on intracellular calcium levels in the DRG cells were presented.

Bronchodilator effects of Lignosus rhinocerotis extract on rat isolated airways is linked to the blockage of calcium entry.

BACKGROUND: Lignosus rhinocerotis (Cooke) Ryvarden is a popular medicinal mushroom used for centuries in Southeast Asia to treat asthma and chronic cough. The present study aimed to investigate the effect of this mushroom on airways patency. MATERIALS AND METHODS: The composition of L. rhinocerotis TM02 cultivar was analyzed. Organ bath experiment was employed to study the bronchodilator effect of Lignosus rhinocerotis cold water extract (CWE) on rat isolated airways. Trachea and bronchus were removed from male Sprague-Dawley rats, cut into rings of 2 mm, pre-contracted with carbachol before adding CWE into the bath in increasing concentrations. To investigate the influence of incubation time, tissues were exposed to intervals of 5, 15 and 30 min between CWE concentrations after pre-contraction with carbachol in subsequent protocol. Next, tissues were pre-incubated with CWE before the addition of different contractile agents, carbachol and 5-hydroxytrptamine (5-HT). The bronchodilator effect of CWE was compared with salmeterol and ipratropium. In order to uncover the mechanism of action of CWE, the role of beta-adrenoceptor, potassium and calcium channels was investigated. RESULTS: Composition analysis of TM02 cultivar revealed the presence of ß-glucans and derivatives of adenosine. The extract fully relaxed the trachea at 3.75 mg/ml (p < 0.0001) and bronchus at 2.5 mg/ml (p < 0.0001). It was observed that lower concentrations of CWE were able to fully relax both trachea and bronchus but at a longer incubation interval between concentrations. CWE pre-incubation significantly reduced the maximum responses of carbachol-induced contractions (in both trachea, p = 0.0012 and bronchus, p = 0.001), and 5-HT-induced contractions (in trachea, p = 0.0048 and bronchus, p = 0.0014). Ipratropium has demonstrated a significant relaxation effect in both trachea (p = 0.0004) and bronchus (p = 0.0031), whereas salmeterol has only affected the bronchus (p = 0.0104). The involvement of ß2-adrenoceptor and potassium channel in CWE-mediated airway relaxation is ruled out, but the bronchodilator effect was unequivocally affected by influx of calcium. CONCLUSIONS: The bronchodilator effect of L. rhinocerotis on airways is mediated by calcium signalling pathway downstream of Gαq-coupled protein receptors. The airway relaxation effect is both concentration- and incubation time-dependent. Our findings provide unequivocal evidence to support its traditional use to relieve asthma and cough.

Effect of ultrasound on size, morphology, stability and antioxidant activity of selenium nanoparticles dispersed by a hyperbranched polysaccharide from Lignosus rhinocerotis.

The differences between ultrasonic and non-ultrasonic approaches in synthesizing Lignosus rhinocerotis polysaccharide-selenium nanoparticles (LRP-SeNPs) were compared in terms of size, morphology, stability and antioxidant activity by UV-VIS, FT-IR, X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) with high resolution TEM. Results indicated that the SeNPs were associated with the LRP macromolecules in a physical adsorption pattern without breaking chemical bonds, and the ultrasonic treatment reduced the size of SeNPs, narrowed the size distribution as well as improved the stability. Due to the LRP compact coil structure loosed under ultrasonic cavitation, the SeNPs could be easily diffused into the LRP internal branches instead of gathering on the LRP surface and were well dispersed and eventually stabilized throughout the extended branches. After ultrasound treatment, the SeNPs had a minimum average diameter of ∼50 nm and the LRP-SeNPs could remain homogeneous and translucent for 16 days within 200 nm size. Furthermore, the ultrasound-treated LRP-SeNPs exhibited higher DPPH and ABTS radical-scavenging abilities than those untreated with ultrasound. This difference may be attributed to the reason that ultrasound can reduce the SeNPs size and increase the specific surface area, which provides sufficient active sites to react with the free radicals and suppress the oxidizing reactions. The integrated results demonstrated that ultrasound played a crucial role in the dispersion, size control, stabilization and antioxidant activity of SeNPs.

Oral reproductive and developmental toxicity of Lignosus rhinocerotis mycelium in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerotis (L. rhinocerotis), also known as the tiger milk mushroom, is widely used as traditional medicine and as soup ingredient in Malaysia and Hong Kong. It is edible and is used traditionally for the treatment of fever, cough, asthma, wounds, chronic hepatitis, gastric ulcers and cancers. In view of its safety profile, little information is found in scientific literature. The objective of this study is to investigate developmental toxicity of L. rhinocerotis in pregnant Sprague-Dawley (SD) rats. MATERIALS AND METHODS: Eighty pregnant SD female rats were used in this study for three treatment groups and a control group, each consisting of 20 pregnant female rats. Three doses of 850mg/kg/day (Low-dose), 1700mg/kg/day (Mid-dose) and 3400mg/kg/day (High-dose) were selected for the study, whereas 10mL/kg distilled water was served as the control. Examinations were conducted on pregnant rats and fetuses respects to mortality, body weight, body weights gains, food consumption and clinical observations. The pregnant females were gross necropsied on G20, followed by maternal and fetus examination, to evaluate the teratogenicity, reproductive and developmental performance of L. rhinocerotis mycelium. RESULTS: Results showed that no L. rhinocerotis mycelium-related animal death and abnormal clinical sign were noted. No statistical differences were noted in maternal mean body weight and maternal mean body weight gains. Some animals in the high-dose group appeared audible respiration due to dosing accident, it resulted in lower food consumption but not relevant to L. rhinocerotis mycelium treatment. In maternal gross necropsy, no L. rhinocerotis mycelium-related gross lesion was noted. In maternal examination, parameters of gravid uterus weight, implantation number, corpora lutea number, litter size, live or dead fetal number, male or female fetus number, resorption number, fetal sex ratio (M/F), pre-implantation loss and post-implantation loss were all within the normal reference ranges and showed no significant difference when compared to the control group. In fetus examination, including external, visceral and skeletal evaluations, there were no significant changes between any of the L. rhinocerotis mycelium treated groups and the control group. CONCLUSIONS: Based on the study results, the no-observable-adverse-effect level (NOAEL) for pregnant female rats under the conditions of this study was 3400mg/kg/day.

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

BACKGROUND: Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. RESULTS: Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, ß-elemene, ß-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 µg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 µg/ml). CONCLUSIONS: This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.

A hyperbranched ß-d-glucan with compact coil conformation from Lignosus rhinocerotis sclerotia.

An alkali-soluble polysaccharide was extracted from Lignosus rhinocerotis sclerotia (LRP). Its structural characteristics were determined by GC-MS, FT-IR, GC, 1D and 2D NMR combined with Smith degradation and methylation analysis. The LRP had a (1→3)-ß-d-Glcp backbone with every three residues bearing a (1→6)-linked and hyperbranched side chain that contained three (1→6)-ß-d-Glcp residues as secondary main chain and two terminal ß-d-Glcp residues linked at O3. The degree of branching was 0.76 from GC-MS analysis, implying a highly branched structure for LRP. The Mw, z1/2, Rh and [η] values of LRP in 0.25M LiCl/DMSO were measured by SEC-MALLS-Vis-RI combination technology to be 2.88×105g/mol, 30.36nm, 22.34nm and 131.50ml/g, respectively. Furthermore, the exponent α of [η]-Mw, ß of z1/2-Mw, the fractal dimension df and molecular parameter ρ were determined to be 0.20, 0.33, 2.50 and 1.36, demonstrating that the LRP was a hyperbranched polysaccharide and adopted a compact coil conformation in LiCl/DMSO.

Structure, molecular conformation, and immunomodulatory activity of four polysaccharide fractions from Lignosus rhinocerotis sclerotia.

Four polysaccharide fractions, LRP-1, LRP-2, LRP-3 and LRP-4 were extracted stepwise from Lignosus rhinocerotis sclerotia with distilled water at 25, 95, 120°C and 1.0M NaOH solution at 4°C. Their structure, molecular size and chain conformation were clarified using SEC-MALLS-RI, GC, FT-IR and UV-vis. Furthermore, their immunomodulatory activities were evaluated by the model of cyclophosphamide (Cy)-induced immunosuppression. The LRP-1 and LRP-2 were polysaccharide-protein complexes (46-68% ß-d-glucan and 27-48% protein), while LRP-3 and LRP-4 were absolutely composed of ß-d-glucose. The LRP-4 with low polydispersity had much higher molecular weight (Mw, 5.86×106g/mol) and intrinsic viscosity ([η], 202.6ml/g) than other LRP fractions. Based on Mw, radius of gyration (z1/2) and [η] data with the exponent ß of z1/2-Mw and its U-shaped curve, all four LRP fractions were highly branched macromolecules and LRP-3 showed a more compact sphere-like conformation than LRP-2 in aqueous solution. Additionally, all four LRP fractions exhibited protective effects against Cy-induced immunosuppression in mice by improving immune organs as well as stimulating the release of major cytokines TNF-α and INF-γ. This work provides a theoretical basis for the application of polysaccharides and their protein complexes from Lignosus rhinocerotis sclerotia in food- or drug-based therapies.

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.