A dual mechanism with H2S inhibition and membrane damage of morusin from Morus alba Linn. against MDR-MRSA.

It's urgent to discover new antibiotics along with the increasing emergence and dissemination of multidrug resistant (MDR) bacterial pathogens. In the present investigation, morusin exhibited rapid bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) by targeting the phospholipid of bacterial inner membrane, increasing membrane rigidity and disrupting bacterial homeostasis together with the membrane permeability, which caused fundamental metabolic disorders. Furthermore, morusin can also accumulate ROS, suppress H2S production, and aggravate oxidative damage in bacteria. Importantly, morusin also inhibited the spread of wounds and reduced the bacterial burden in the mouse model of skin infection caused by MRSA. It's a chance to meet the challenge of existing antibiotic resistance and avoid the development of bacterial resistance, given the multiple targets of morusin.

Morusin shows potent antitumor activity for melanoma through apoptosis induction and proliferation inhibition.

BACKGROUND: The discovery of new anti-melanoma drugs with low side effect is urgently required in the clinic. Recent studies showed that morusin, a flavonoid compound isolated from the root bark of Morus Alba, has the potential to treat multiple types of cancers, including breast cancer, gastric cancer, and prostate cancer. However, the anti-cancer effect of morusin on melanoma cells has not been investigated. METHODS: We analyzed the effects of morusin on the proliferation, cell cycle, apoptosis, cell migration and invasion ability of melanoma cells A375 and MV3, and further explored the effects of morusin on tumor formation of melanoma cell. Finally, the effects of morusin on the proliferation, cycle, apoptosis, migration and invasion of A375 cells after knockdown of p53 were detected. RESULTS: Morusin effectively inhibits the proliferation of melanoma cells and induces cell cycle arrest in the G2/M phase. Consistently, CyclinB1 and CDK1 that involved in the G2/M phase transition were down-regulated upon morusin treatment, which may be caused by the up-regulation of p53 and p21. In addition, morusin induces cell apoptosis and inhibits migration of melanoma cells, which correlated with the changes in the expression of the associated molecules including PARP, Caspase3, E-Cadherin and Vimentin. Moreover, morusin inhibits tumor growth in vivo with little side effect on the tumor-burden mice. Finally, p53 knockdown partially reversed morusin-mediated cell proliferation inhibition, cell cycle arrest, apoptosis, and metastasis. CONCLUSION: Collectively, our study expanded the spectrum of the anti-cancer activity of morusin and guaranteed the clinical use of the drug for melanoma treatment.

Apoptotic and anti-Warburg effect of Morusin via ROS mediated inhibition of FOXM1/c-Myc signaling in prostate cancer cells.

Though Morusin is known to induce apoptotic, antiprolifertaive, and autophagic effects through several signaling pathways, the underlying molecular mechanisms of Morusin still remain unclear until now. To elucidate antitumor mechanism of Morusin, cytotoxicity assay, cell cycle analysis, Western blotting, TUNEL assay, RNA interference, immunofluorescense, immunoprecipitation, reactive oxygen species (ROS) measurement, and inhibitor study were applied in this study. Morusin enhanced cytotoxicity, increased the number of TUNEL positive cells, sub-G1 population and induced the cleavages of PARP and caspase3, attenuated the expression of HK2, PKM2, LDH, c-Myc, and Forkhead Box M1 (FOXM1) along with the reduction of glucose, lactate, and ATP in DU145 and PC3 cells. Furthermore, Morusin disrupted the binding of c-Myc and FOXM1 in PC-3 cells, which was supported by String and cBioportal database. Notably, Morusin induced c-Myc degradation mediated by FBW7 and suppressed c-Myc stability in PC3 cells exposed to MG132 and cycloheximide. Also, Morusin generated ROS, while NAC disrupted the capacity of Morusin to reduce the expression of FOXM1, c-Myc, pro-PARP, and pro-caspase3 in PC-3 cells. Taken together, these findings provide scientific evidence that ROS mediated inhibition of FOXM1/c-Myc signaling axis plays a critical role in Morusin induced apoptotic and anti-Warburg effect in prostate cancer cells. Our findings support scientific evidence that ROS mediated inhibition of FOXM1/c-Myc signaling axis is critically involved in apoptotic and anti-Warburg effect of Morusin in prostate cancer cells.

Morusin Protected Ruminal Epithelial Cells against Lipopolysaccharide-Induced Inflammation through Inhibiting EGFR-AKT/NF-κB Signaling and Improving Barrier Functions.

Using phytogenic extracts for preventing or treating rumen epithelial inflammatory injury is a potential alternative to antibiotic use due to their residue-free characteristics. In this study, the efficacy of Morus root bark extract Morusin on ruminal epithelial cells (RECs) against pathogenic stimulus was investigated for the first time. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative real-time polymerase chain reaction (qPCR) results showed that the Morusin did not affect the cell viability of RECs and exerted anti-inflammatory effects in a concentration-dependent manner. Transcriptome analysis further revealed that the Morusin significantly downregulated the inflammatory-response-related cell signaling, while it upregulated the cell-proliferation-inhibition- and barrier-function-related processes in RECs upon lipopolysaccharide (LPS) stimulation. The epidermal growth factor receptor (EGFR) blocking and immunoblotting analysis further confirmed that the Morusin suppressed LPS-induced inflammation in RECs by downregulating the phosphorylation of protein kinase B (AKT) and nuclear factor-kappaB (NF-κB) p65 protein via inhibiting the EGFR signaling. These findings demonstrate the protective roles of Morusin in LPS-induced inflammation in RECs.

Antitumor Effect of Morusin via G1 Arrest and Antiglycolysis by AMPK Activation in Hepatocellular Cancer.

Though Morusin isolated from the root of Morus alba was known to have antioxidant, anti-inflammatory, antiangiogenic, antimigratory, and apoptotic effects, the underlying antitumor effect of Morusin is not fully understood on the glycolysis of liver cancers. Hence, in the current study, the antitumor mechanism of Morusin was explored in Hep3B and Huh7 hepatocellular carcninomas (HCC) in association with glycolysis and G1 arrest. Herein, Morusin significantly reduced the viability and the number of colonies in Hep3B and Huh7 cells. Moreover, Morusin significantly increased G1 arrest, attenuated the expression of cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase 6 (CDK6) and upregulated p21 and p27 in Hep3B and Huh7 cells. Interestingly, Morusin significantly activated phosphorylation of the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) but attenuated the expression of the p-mammalian target of protein kinase B (AKT), rapamycin (mTOR), c-Myc, hexokinase 2(HK2), pyruvate kinases type M2 (PKM2), and lactate dehydrogenase (LDH) in Hep3B and Huh7 cells. Consistently, Morusin suppressed lactate, glucose, and adenosine triphosphate (ATP) in Hep3B and Huh7 cells. Conversely, the AMPK inhibitor compound C reduced the ability of Morusin to activate AMPK and attenuate the expression of p-mTOR, HK2, PKM2, and LDH-A and suppressed G1 arrest induced by Morusin in Hep3B cells. Overall, these findings suggest that Morusin exerts an antitumor effect in HCCs via AMPK mediated G1 arrest and antiglycolysis as a potent dietary anticancer candidate.

The Beneficial Effects of Morusin, an Isoprene Flavonoid Isolated from the Root Bark of Morus.

The root bark of Morus has long been appreciated as an antiphlogistic, diuretic and expectorant drug in Chinese herbal medicine, albeit with barely known targets and mechanisms of action. In the 1970s, the development of analytic chemistry allowed for the discovery of morusin as one of 7 different isoprene flavonoid derivatives in the root bark of Morus. However, the remarkable antioxidant capacity of morusin with the unexpected potential for health benefits over the other flavonoid derivatives has recently sparked scientific interest in the biochemical identification of target proteins and signaling pathways and further clinical relevance. In this review, we discuss recent advances in the understanding of the functional roles of morusin in multiple biological processes such as inflammation, apoptosis, metabolism and autophagy. We also highlight recent in vivo and in vitro evidence on the clinical potential of morusin treatment for multiple human pathologies including inflammatory diseases, neurological disorders, diabetes, cancer and the underlying mechanisms.

Stress Granule Formation Attenuates RACK1-Mediated Apoptotic Cell Death Induced by Morusin.

Stress granules are membraneless organelles composed of numerous components including ribonucleoproteins. The stress granules are characterized by a dynamic complex assembly in response to various environmental stressors, which has been implicated in the coordinated regulation of diverse biological pathways, to exert a protective role against stress-induced cell death. Here, we show that stress granule formation is induced by morusin, a novel phytochemical displaying antitumor capacity through barely known mechanisms. Morusin-mediated induction of stress granules requires activation of protein kinase R (PKR) and subsequent eIF2α phosphorylation. Notably, genetic inactivation of stress granule formation mediated by G3BP1 knockout sensitized cancer cells to morusin treatment. This protective function against morusin-mediated cell death can be attributed at least in part to the sequestration of receptors for activated C kinase-1 (RACK1) within the stress granules, which reduces caspase-3 activation. Collectively, our study provides biochemical evidence for the role of stress granules in suppressing the antitumor capacity of morusin, proposing that morusin treatment, together with pharmacological inhibition of stress granules, could be an efficient strategy for targeting cancer.

Morusin Suppresses Cancer Cell Invasion and MMP-2 Expression through ERK Signaling in Human Nasopharyngeal Carcinoma.

The most important cause of treatment failure of nasopharyngeal carcinoma (NPC) patients is metastasis, including regional lymph nodes or distant metastasis, resulting in a poor prognosis and challenges for treatment. In the present study, we investigated the in vitro anti- tumoral properties of morusin on human nasopharyngeal carcinoma HONE-1, NPC-39, and NPC-BM cells. Our study revealed that morusin suppressed the migration and invasion abilities of the three NPC cells. Gelatin zymography assay and Western blotting demonstrated that the enzyme activity and the level of matrix metalloproteinases-2 (MMP-2) protein were downregulated by the treatment of morusin. Mitogen-activated protein kinase proteins were examined to identify the signaling pathway, which showed that phosphorylation of ERK1/2 was inhibited after the treatment of morusin. In summary, our data showed that morusin inhibited the migration and invasion of NPC cells by suppressing the expression of MMP-2 by downregulating the ERK1/2 signaling pathway, suggesting that morusin may be a potential candidate for chemoprevention or adjuvant therapy of NPC.

Prenylated phenolics as promising candidates for combination antibacterial therapy: Morusin and kuwanon G.

Combination of antibiotics with natural products is a promising strategy for potentiating antibiotic activity and overcoming antibiotic resistance. The purpose of the present study was to investigate whether morusin and kuwanon G, prenylated phenolics in Morus species, have the ability to enhance antibiotic activity and reverse antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. Commonly used antibiotics (oxacillin, erythromycin, gentamicin, ciprofloxacin, tetracycline, clindamycin) were selected for the combination studies. Checkerboard and time-kill assays were used to investigate potential bacteriostatic and bactericidal synergistic interactions, respectively between morusin or kuwanon G and antibiotics. According to both fractional inhibitory concentration index and response surface models, twenty combinations (14 morusin-antibiotic combinations, six kuwanon G-antibiotic combinations) displaying bacteriostatic synergy were identified, with 4-512-fold reduction in the minimum inhibitory concentration values of antibiotics in combination. Both morusin and kuwanon G reversed oxacillin resistance of methicillin-resistant Staphylococcus aureus. In addition, morusin reversed tetracycline resistance of Staphylococcus epidermidis. At half of the minimum inhibitory concentrations, combinations of morusin with oxacillin or gentamicin showed bactericidal synergy against methicillin-resistant Staphylococcus aureus. Fluorescence and differential interference contrast microscopy and scanning electron microscopy showed an increase in the membrane permeability and massive leakage of cellular content in methicillin-resistant Staphylococcus aureus exposed to morusin or kuwanon G. Overall, our findings strongly indicate that both prenylated compounds are good candidates for the development of novel antibacterial combination therapies.

[The Effect of Morusin on Stemness Phenotype of Laryngeal Cancer Stem Cell].

OBJECTIVE: To investigate the regulation effect of Morusin on stemness phenotype of laryngeal cancer stem cells. METHODS: Separation and detection the proportion of CD133 + laryngeal cancer stem cells through flow cytometry; evaluation the self-renewal ability of CD133 + laryngeal cancer stem cells by tumor sphere formation assay; exploring the migration ability of CD133 + laryngeal cancer stem cells by Transwell assay; analyzing the cytotoxicity of chemotherapy drugs on CD133 + laryngeal cancer stem cells by modified MTT assay; detection of the expression levels of stemness associated markers by immunofluorescence staining, RT-qPCR and Western blot. After treatment with different concentrations of Morusin, cells were performed the above experiments for detection the self-renewal ability, migration ability, cytotoxicity resistance and expression of stemness associated markers. RESULTS: Flow cytometry analysis showed that the proportion of CD133 + laryngeal cancer stem cells was (3.50±0.34)%, while after enrichment, the proportion increased to (93.20±5.23)%. CD133 + laryngeal cancer stem cells exhibited better self-renewal ability (P<0.001) and migratory ability (P<0.05); they were resistant to the cytotoxicity of chemotherapy drug (P<0.05), and highly expressed of stemness associated markers. After being treated with Morusin, the self-renewal and migratory abilities of CD133 + laryngeal cancer stem cells were reduced (P<0.05). In addition, after treated with Morusin, CD133 +laryngeal cancer stem cells were more sensitive to chemotherapy drugs; moreover, the expression levels of stemness associated markers were decreased. CONCLUSION: CD133 + laryngeal cancer stem cells possessed stemness phenotypic characteristics. Morusin attenuated stemness phenotype of laryngeal cancer stem cells, which may be related to its down-regulation effect on stemness associated markers.

A UPLC-MS/MS method for comparative pharmacokinetics study of morusin and morin in normal and diabetic rats.

The aim of this study was to establish and validate a rapid, selective and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for simultaneous quantitations of morin and morusin, and to investigate their pharmacokinetics difference between normal and diabetic rats after oral administration. Plasma samples were pretreated via protein precipitation with acetonitrile. Genkwanin was used as internal standard (IS). Analytes and IS were separated on a Thermo Hypersil Gold C18 column (50 × 4.6 mm, 3 µm) using gradient elution. The mobile phase consisted of acetonitrile and 0.1% formic acid in water at a flow rate of 0.5 mL/min. Mass spectrometry detection was carried out by means of negative electrospray ionization source and multipe-reaction monitoring mode. The transitions of m/z 300.9 → 151.2 for morin, m/z 419.2 → 297.1 for morusin and m/z 283.1 → 268.2 for IS were chosen for quantification. Calibration curves were linear in the range of 1.01-504.2 ng/mL (r2 ≥ 0.99) for morin and 1.02-522.3 ng/mL (r2 ≥ 0.99) for morusin. The lower limit of quantification was 1.02 ng/mL for morin and 1.05 ng/mL for morusin. The extraction recovery was >85.1% for each analyte. No obvious matrix effect was observed under the present UPLC-MS/MS conditions during all of the bioanalysis. The stability study demonstrated that morin and morusin remained stable during the whole analytical procedure. The method was successfully applied to support the pharmacokinetic comparisons of morin and morusin between normal and diabetic rats.

Anti-Allergic and Anti-Inflammatory Effects of Kuwanon G and Morusin on MC/9 Mast Cells and HaCaT Keratinocytes.

Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disease. The use of immunomodulatory corticosteroids in AD treatment causes adverse side effects. Therefore, novel natural anti-inflammatory therapeutics are needed. The aim of the present study was to investigate the anti-allergic and anti-inflammatory activities of kuwanon G and morusin. To investigate the effect of kuwanon G and morusin on skin inflammation, enzyme-linked immunosorbent assays (ELISA) to quantitate secreted (RANTES/CCL5), thymus- and activation-regulated chemokine (TARC/CCL17), and macrophage-derived chemokine (MDC/CCL22) were performed, followed by Western blotting to measure the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and nuclear transcription factor-κB (NF-κB) p65 in tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-stimulated HaCaT keratinocytes. In order to evaluate the anti-allergic effects, ELISA to quantify histamine and leukotriene C4 (LTC4) production and Western blotting to measure 5-lipoxygenase (5-LO) activation were performed using PMA and A23187-stimulated MC/9 mast cells. Kuwanon G reduced the release of RANTES/CCL5, TARC/CCL17, and MDC/CCL22 via down-regulation of STAT1 and NF-κB p65 signaling in TNF-α and IFN-γ-stimulated HaCaT keratinocytes. Kuwanon G also inhibited histamine production and 5-LO activation in PMA and A23187-stimulated MC/9 mast cells. Morusin inhibited RANTES/CCL5 and TARC/CCL17 secretion via the suppression of STAT1 and NF-κB p65 phosphorylation in TNF-α and IFN-γ-stimulated HaCaT keratinocytes, and the release of histamine and LTC4 by suppressing 5-LO activation in PMA and A23187-stimulated MC/9 mast cells. Kuwanon G and morusin are potential anti-inflammatory mediators for the treatment of allergic and inflammatory skin diseases such as AD.

Protective Effects of Morus Root Extract (MRE) Against Lipopolysaccharide-Activated RAW264.7 Cells and CCl4-Induced Mouse Hepatic Damage.

BACKGROUND/AIMS: Inflammation is one of the main contributors to chronic diseases such as cancer. It is of great value to identify the potential activity of various medicinal plants for regulating or blocking uncontrolled chronic inflammation. We investigated whether the root extract of Morus australis possesses antiinflammatory and antioxidative stress potential and hepatic protective activity. METHODS: The microwave-assisted extractionwere was used to prepare the ethanol extract from the dried root of Morus australis (MRE), including polyphenolic and flavonoid contents. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells was examined the anti-inflammatory and anti-oxidative potential of MRE. CCl4-induced mouse hepatic damage were performed to detect the hepatic protective potential in vivo. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. RESULTS: MRE contained approximately 23% phenolic compounds and 3% flavonoids. The major flavonoid component of MRE was morusin. MRE and morusin inhibited lipopolysaccharide-induced production of nitrite and prostaglandin E2 in RAW264.7 cells. MRE and morusin also suppressed the formation of intracellular reactive oxygen species and the expression of iNOS and COX-2. In an in vivo study, a thiobarbituric acid reactive substances assay showed that MRE inhibited CCl4-induced oxidative stress and expression of nitrotyrosine. MRE also decreased CCl4-induced hepatic iNOS and COX-2 expression, as well as CCl4-induced hepatic inflammation and necrosis in mice. CONCLUSION: MRE exhibited antiinflammatory and hepatic protective activity.

Induction of apoptosis by morusin in human non-small cell lung cancer cells by suppression of EGFR/STAT3 activation.

This study was designed to validate the anticancer effects of morusin in human non-small cell lung cancer (NSCLC) cells. Morusin suppressed the cell growth and colony formation in a concentration-dependent manner in H1299, H460 and H292 cells. These anticancer activities were related with apoptosis induction proved by the accumulation of chromatin condensation, PARP cleavage, increase of sub-G1 phage and annexin V-positive cell population. Interestingly, signal transducer and activator of transcription 3 (STAT3) was dephosphorylated by morusin. Morusin suppressed the transcriptional activity of STAT3 and down-regulated the expression of STAT3 target genes. In addition, morusin inhibited the phosphorylation of epithelial growth factor receptor (EGFR), an upstream regulator of STAT3. The docking study showed that morusin directly binds to the tyrosine kinase domain of EGFR. Furthermore, the anticancer effects of morusin were consistently observed in erlotinib-resistant H1975 cells expressing L858R and T790 M mutant EGFR, suggesting that morusin can be used for the advanced NSCLC with acquired resistance to EGFR TKI. Taken together, our results demonstrate that morusin induced apoptosis in human NSCLC cells regardless of EGFR mutation status through inhibition of EGFR/STAT3 activation.

Morusin Functions as a Lipogenesis Inhibitor as Well as a Lipolysis Stimulator in Differentiated 3T3-L1 and Primary Adipocytes.

Conflicting results for morusin activity during adipogenic differentiation are reported in 3T3-L1 adipocytes and cancer cells. To elucidate the influence of morusin on fat metabolism, their anti-obesity effects and molecular mechanism were investigated in 3T3-L1 cells and primary adipocytes. Morusin at a dose of less than 20 µM does not induce any significant change in the viability of 3T3-L1 adipocytes. The accumulation of intracellular lipid droplets in 3T3-L1 adipocytes stimulated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, 10 µg/mL insulin in DMEM containing 10% FBS (MDI)-significantly reduces in a dose-dependent manner after morusin treatment. The phosphorylation level of members in the MAP kinase signaling pathway under the insulin receptor downstream also decrease significantly in the MDI + morusin-treated group compared to MDI + vehicle-treated group. Also, the expression of adipogenic transcription factors (PPARγ and C/EBPα) and lipogenic proteins (aP2 and FAS) are significantly attenuated by exposure to the compound in MDI-stimulated 3T3-L1 adipocytes. Furthermore, the decrease in the G0/G1 arrest of cell cycle after culturing in MDI medium was dramatically recovered after co-culturing in MDI + 20 µM morusin. Moreover, morusin treatment induces glycerol release in the primary adipocytes of SD rats and enhances lipolytic protein expression (HSL, ATGL, and perilipin) in differentiated 3T3-L1 adipocytes. Overall, the results of the present study provide strong evidence that morusin inhibits adipogenesis by regulating the insulin receptor signaling, cell cycle and adipogenic protein expression as well as stimulating lipolysis by enhancing glycerol release and lipolytic proteins expression.

Rapid, microwave-accelerated synthesis and anti-osteoporosis activities evaluation of Morusin scaffolds and Morusignin L scaffolds.

Described herein is a facile and efficient methodology toward the synthesis of Morusin scaffolds and Morusignin L scaffolds 4-9 and 12via a novel three-step approach (Michael addition or prenylation, cyclization and cyclization) and use a rapid, microwave-accelerated cyclization as the key step. Furthermore, their biological activities have been preliminarily demonstrated by in vitro evaluation for anti-osteoporosis activity. These Morusin, Morusignin L and newly synthesized compounds 5b, 6a, 8e, 8f greatly exhibited the highest potency, especially at the 10-5mol/L (P<0.01), and had good in vitro anti-osteoporosis activities using the commercially available standard drug Ipriflavone as a positive control. The mechanisms associated with anti-osteoporosis effects of these compounds may be through the inhibition of TRAP enzyme activity and bone resorption in osteoclasts, and promotion effect of osteoblast proliferation in vitro. The results indicated that Morusin scaffolds and Morusignin L scaffolds may be useful leads for further anti-osteoporosis activity screenings.

Quantification of morusin using LC-MS in rat plasma: Application to a pharmacokinetic study.

A sensitive LC-MS method was developed for the quantification of morusin in rat plasma using praeruptorin C as internal standard. After extraction with diethyl ether, post-treatment samples were chromatographed on a Hypersil C18 column. An isocratic mobile phase consisting of methanol-water (70:30, v/v) was applied at a flow rate of 0.4 mL/min. Detection was performed via electrospray ionization source with positive ion mode using selected ion monitoring mode at m/z 443.1 for morusin and m/z 451.0 for IS. Acceptable linearity (r2 ≥ 0.99) was observed over the concentration range of 1.5-800 ng/mL. This method was successfully applied in the pharmacokinetics study of morusin in rats.

Morusin inhibits glioblastoma stem cell growth in vitro and in vivo through stemness attenuation, adipocyte transdifferentiation, and apoptosis induction.

Glioblastoma multiforme (GBM) cancer stem cells (GSCs) are responsible for the progression and recurrence of GBM after conventional therapy. Morusin possesses anti-cancer activity in vitro. The purpose of this study is to confirm the growth inhibition effect of morusin on human GSCs growth in vitro and in vivo and to explore the possible mechanism of its activity. Human GSCs were enriched under nonadhesive culture system, and characterized through neurosphere formation, toluidine blue staining, immunofluorescence staining, Western blotting analysis of stemness markers of CD133, nestin, Sox2 and Oct4, and tumorigenecity in vivo; the growth inhibition effect of morusin on human GSCs in vitro and in vivo were tested by cell cytotoxicity, neurosphere formation inhibition, adipogenic differentiation, apoptosis induction, and tumor growth inhibition in vivo assays. The potential molecular mechanisms underlying the growth inhibition effect of morusin on GSCs in vitro and in vivo were investigated with Western blotting evaluation of stemness, adipogenic, and apoptotic proteins in morusin treated GSCs and tumor tissues. GSCs enriched under nonadhesive culture system possess stemness characterstics; Morusin inhibited GSCs growth in vitro and in vivo, it reduced stemness of GSCs, induced them adipocyte-like transdifferention and apoptosis. Morusin has the potential to inhibit human GSCs growth in vitro and in vivo through stemness attenuation, adipocyte transdifferentiation, and apoptosis induction.

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes.

1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes. 2. 100 µM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 µM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 µM, respectively. 3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 µM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively. 4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

Bioguided isolation, identification and bioactivity evaluation of anti-fatigue constituents from Schizophyllum commune.

This study aims to clarify the specific anti-fatigue components of Schizophyllum commune (S.commune) and analyze its potential anti-fatigue mechanism. The main anti-fatigue active ingredient of S.commune was locked in n-butanol extract (SPE-n) by activity evaluation. Twelve compounds were identified by high performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS). The anti-fatigue effect of morusin is the most predominant among these 12 ingredients. The determination of biochemical indices showed that morusin could increase liver glycogen reserves, improve the activity of antioxidant enzymes in liver, and reduce reactive oxygen species (ROS) content in muscle tissue, thereby reducing myocyte damage. Further studies revealed that morusin could reduce the level of oxidative stress by activating Nrf2/HO-1 pathway, thus alleviating the fatigue of mice caused by exhaustive exercise. The current findings provide a theoretical basis for the development of natural anti-fatigue functional food.