Lentinula edodes, known as "shiitake" is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC(50) values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI(comit)/SI(mit) ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.

Aspergillus niger is an opportunistic human pathogen and a strong air pollutant. A study was conducted with 75 different essential oils for the inhibition of hyphal growth and spore formation in Aspergillus niger. Cinnamomum zeylanicum (bark), Cinnamomum zeylanicum (leaf), Cinnamomum cassia, Syzygium aromaticum and Cymbopogon citratus were the top five essential oils which demonstrated marked inhibitory effect against hyphal growth and spore formation of A. niger. The chemical composition of these five most active essential oils was investigated by gas chromatography-mass spectra (GC-MS). Most of the other essential oils were found challenging to combat A. niger, suggesting their use as strong aroma therapeutic agents.

Despite considerable recent work to reveal different features of mushrooms species, the few studies of antiviral activities are inadequate and therefore further studies are required. Morchella conica, M. esculenta, Terfezia boudieri, Pleurotus ostreatus, Tricholoma anatolicum, Fomes fomentarius, Laetiporus sulphureus, Phellinus igniarius, Porodaedalea pini, and Pyrofomes demidoffii from Turkey were investigated to reveal their in vitro cytotoxic and anti-herpes simplex virus 1 (HSV-1) activities. The crude methanol extracts (MEs) and aqueous extracts (AEs) of fungal species and acyclovir (ACV) were used. Various dilutions were used to assess the cytotoxic effects of fungal species (50-0.10 mg/mL) and ACV (500-0.98μg/mL) on uninfected Vero cells. Maximum nontoxic concentrations were determined for all extracts and ACV by comparing the optical densities of their cell controls. The concentration providing 50% protection against the cytopathic effect caused by the virus, extracts, and ACV (EC50) was calculated,and the half-maximal cytotoxic concentration (CC50) and the selectivity index (SI) were determined, the latter as the ratio of CC50 to EC50. While the AEs of F. fomentarius (EC50, 11.22 mg/mL; SI>4.46), Ph. igniarius (EC50, 9.71 mg/mL; SI>5.15), and P. pini (EC50, 7.16 mg/mL; SI>6.98) showed considerable antiherpetic activity, MEs and AEs of the other fungal species did not showed any effects. The EC50 and SI of ACV were determined as 0.20μg/mL and 3085, respectively. The results demonstrate that F. fomentarius, Ph. igniarius, and P. pini have important anti-HSV-1 activity.

BACKGROUND:Since oxidative stress and inflammation are two linked factors in the pathogenesis of several human diseases. Thus identification of effective treatment is of great importance. Edible mushroom and microalgae are rich in the effective antioxidant phytochemicals. Hence, their beneficial effects on oxidative stress-associated inflammation are extremely required to be investigated.

METHODS:This study evaluated the functional constituents, antioxidant and anti-inflammatory activities of Malaysian Ganoderma lucidum aqueous extract (GLE) and Egyptian Chlorella vulgaris ethanolic extract (CVE). Also, the synergistic, addictive or antagonistic activities of the combination between the two extracts (GLE-CVE) were studied. Expression of inducible nitric oxide synthase, cyclooxygenase-2, and nuclear factor-kappa B, as well as levels of nitric oxide, tumor necrosis factor (TNF)-α, lipid peroxidation, reduced glutathione and antioxidant enzymes were determined using in vitro model of lipopolysaccharide-stimulated white blood cells.

BACKGROUND:A search for new anti-coronaviral drugs to treat coronaviral infections was motivated by an outbreak of severe acute respiratory syndrome (SARS).

OBJECTIVES:In order to find drugs that treat coronavirus infections, including SARS, we screened traditional medicinal herbal extracts and evaluated their antiviral activities on coronavirus replication.

STUDY DESIGN:We employed a plaque assay to evaluate the effect of 22 medicinal herbal extracts on virus replication. We determined the 50% effective concentration (EC50) of each extract that was necessary to inhibit the replication of mouse hepatitis virus A59 (MHV-A59); we also determined 50% cytotoxic concentrations (CC50) for each extract. Northern and Western blot analyzes were performed to investigate antiviral activity in MHV-infected DBT cells, including virus entry, viral RNA and protein expression, and virus release. Coronavirus specific inhibition was also demonstrated using porcine epidemic diarrhea virus (PEDV).

RESULTS:Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, Phellodendron cortex and Sophora subprostrata radix decreased the MHV production and the intracellular viral RNA and protein expression with EC50 values ranging from 2.0 to 27.5 microg/ml. These extracts also significantly decreased PEDV production and less dramatically decreased vesicular stomatitis virus (VSV) production in vitro.

CONCLUSIONS:The extracts selected strongly inhibited MHV replication and could be potential candidates for new anti-coronavirus drugs.

BACKGROUND:A search for new anti-coronaviral drugs to treat coronaviral infections was motivated by an outbreak of severe acute respiratory syndrome (SARS).

OBJECTIVES:In order to find drugs that treat coronavirus infections, including SARS, we screened traditional medicinal herbal extracts and evaluated their antiviral activities on coronavirus replication.

STUDY DESIGN:We employed a plaque assay to evaluate the effect of 22 medicinal herbal extracts on virus replication. We determined the 50% effective concentration (EC50) of each extract that was necessary to inhibit the replication of mouse hepatitis virus A59 (MHV-A59); we also determined 50% cytotoxic concentrations (CC50) for each extract. Northern and Western blot analyzes were performed to investigate antiviral activity in MHV-infected DBT cells, including virus entry, viral RNA and protein expression, and virus release. Coronavirus specific inhibition was also demonstrated using porcine epidemic diarrhea virus (PEDV).

RESULTS:Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, Phellodendron cortex and Sophora subprostrata radix decreased the MHV production and the intracellular viral RNA and protein expression with EC50 values ranging from 2.0 to 27.5 microg/ml. These extracts also significantly decreased PEDV production and less dramatically decreased vesicular stomatitis virus (VSV) production in vitro.

CONCLUSIONS:The extracts selected strongly inhibited MHV replication and could be potential candidates for new anti-coronavirus drugs.

BACKGROUND:Ebola virus (EBOV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have been identified as potential threats to blood safety. This study investigated the efficacy of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate EBOV and MERS-CoV in platelet concentrates (PCs) and plasma, respectively.

STUDY DESIGN AND METHODS:PCs and plasma were spiked with high titers of cell culture-derived EBOV and MERS-CoV, treated with various light doses of ultraviolet C (UVC; THERAFLEX UV-Platelets) or methylene blue (MB) plus visible light (MB/light; THERAFLEX MB-Plasma), and assessed for residual viral infectivity.

RESULTS:UVC reduced EBOV (≥4.5 log) and MERS-CoV (≥3.7 log) infectivity in PCs to the limit of detection, and MB/light decreased EBOV (≥4.6 log) and MERS-CoV (≥3.3 log) titers in plasma to nondetectable levels.

CONCLUSIONS:Both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce EBOV and MERS-CoV infectivity in platelets and plasma, respectively.

BACKGROUND:Ebola virus (EBOV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have been identified as potential threats to blood safety. This study investigated the efficacy of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate EBOV and MERS-CoV in platelet concentrates (PCs) and plasma, respectively.

STUDY DESIGN AND METHODS:PCs and plasma were spiked with high titers of cell culture-derived EBOV and MERS-CoV, treated with various light doses of ultraviolet C (UVC; THERAFLEX UV-Platelets) or methylene blue (MB) plus visible light (MB/light; THERAFLEX MB-Plasma), and assessed for residual viral infectivity.

RESULTS:UVC reduced EBOV (≥4.5 log) and MERS-CoV (≥3.7 log) infectivity in PCs to the limit of detection, and MB/light decreased EBOV (≥4.6 log) and MERS-CoV (≥3.3 log) titers in plasma to nondetectable levels.

CONCLUSIONS:Both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce EBOV and MERS-CoV infectivity in platelets and plasma, respectively.

BACKGROUND:Middle East respiratory syndrome coronavirus (MERS-CoV) has been identified as a potential threat to the safety of blood products. The Mirasol Pathogen Reduction Technology System uses riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against MERS-CoV when tested in human plasma.

STUDY DESIGN AND METHODS:MERS-CoV (EMC strain) was used to inoculate plasma units that then underwent treatment with riboflavin and UV light. The infectious titers of MERS-CoV in the samples before and after treatment were determined by plaque assay on Vero cells. The treatments were initially performed in triplicate using pooled plasma (n = 3) and then repeated using individual plasma units (n = 6).

RESULTS:In both studies, riboflavin and UV light reduced the infectious titer of MERS-CoV below the limit of detection. The mean log reductions in the viral titers were≥4.07 and ≥4.42 for the pooled and individual donor plasma, respectively.

CONCLUSION:Riboflavin and UV light effectively reduced the titer of MERS-CoV in human plasma products to below the limit of detection, suggesting that the treatment process may reduce the risk of transfusion transmission of MERS-CoV.

Poria cocos Wolf., which belongs to the Polyporaceae family, has been widely used as an Oriental traditional herbal medicine for centuries. Its sclerotium has been reported to possess a wide spectrum of pharmacological activities, including free-radical scavenging, anti-viral, anti-microbial, anti-inflammatory and anticancer activities. However, the cellular and molecular mechanisms of apoptosis induction by P. cocos in human cancer cells are poorly understood. In the present study, we investigated the pro-apoptotic potential of an ethanol extract of P. cocos sclerotium (EEPC) in human leukemiaU937 cells in vitro. We found that EEPC induced anti-proliferative effects in U937 cells in a concentration- and time-dependent manner, which was due to apoptotic induction, as evident from morphological changes and flow cytometric assays. EEPC-induced apoptosis of U937 cells was associated with an increase in the Bax:Bcl-2 ratio, the release of cytochrome c to the cytosol, and a decrease in the expression of an inhibitor of the apoptosis family of proteins. The events were accompanied by activation of caspase-8, -9 and -3, and cleaved poly(ADP-ribose) polymerase, suggesting the involvement of both the intrinsic and extrinsic apoptotic cascades. In addition, the overexpression of Bcl-2 caused a significant attenuation of EEPC-induced caspase activation, degradation of PARP, and the collapse of mitochondrial membrane potential, and thereby reversed EEPC-induced cell apoptosis and growth inhibition. Collectively, these data provide insights into the molecular mechanisms underlying EEPC-induced apoptosis in U937 cells, suggesting that EEPC may be a new therapeutic option for the treatment of leukemia.

AIM:We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases.

MATERIALS AND METHODS:The effects of cannabidiol (CBD) and the synthetic cannabinoid WIN-55,212 (WIN) on LNCaP (prostate) and SW480 (colon) cancer cell proliferation were determined by cell counting; apoptosis was determined by cleavage of poly(ADP)ribose polymerase (PARP) and caspase-3 (Western blots); and phosphatase mRNAs were determined by real-time PCR. The role of phosphatases and cannabinoid receptors in mediating CBD- and WIN-induced apoptosis was determined by inhibition and receptor knockdown.

RESULTS:CBD and WIN inhibited LNCaP and SW480 cell growth and induced mRNA expression of several phosphatases, and the phosphatase inhibitor sodium orthovanadate significantly inhibited cannabinoid-induced PARP cleavage in both cell lines, whereas only CBD-induced apoptosis was CB1 and CB2 receptor-dependent.

CONCLUSION:Cannabinoid receptor agonists induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines; however, the role of the CB receptor in mediating this response is ligand-dependent.

Cordyceps militaris is a traditional herbal ingredient frequently used for tonic and medicinal purposes in eastern Asia. The hot water extract of its cultivated fruiting bodies demonstrated a potent cytotoxic effect against the proliferation of the human premyelocytic leukemia cell HL-60, with an IC50 of 0.8 mg/ml for a 12-h treatment. It induced the characteristic apoptotic symptoms in the HL-60 cells, including DNA fragmentation and chromatin condensation, occurring within 12-16 h of treatment at a dose of 1 mg/ml. The activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase were detected during the course of apoptosis induction. These results indicate that the hot water extract of Cordyceps militaris fruiting bodies inhibited cancer cell proliferation by inducing cell apoptosis through the activation of caspase-3, and that the Cordyceps militaris extract may therefore have therapeutic potential against human leukemia.

Khz (fusion of Ganoderma lucidum and Polyporus umbellatus), isolated from the mycelia of G. lucidum and P. umbellatus, exerts anti‑proliferative effects against malignant cells; however, its activity against human breast cancer cells remains to be elucidated. In the present study, cell proliferation was assessed using a 3-(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, and poptosis was examined using annexin V‑propidium iodide staining and flow cytometry. The activation of caspases 7, 8 and 9 were detected in the Khz‑treated cells using western blotting. The results demonstrated that Khz increased the intracellular calcium concentration and induced the production of reactive oxygen species in MCF‑7 breast cancer cells, as determined using flow cytometry. The results also demonstrated that Khz inhibited cell proliferation and induced apoptosis in the MCF‑7 cells. In addition, the mechanism by which Khz induces apoptosis in cancer cells was investigated. Khz induced apoptosis preferentially in transformed cells, with a minimal effect on non‑transformed cells, suggesting its potential as an anticancer therapeutic agent. Oxidative stress is associated with apoptotic and non‑apoptotic cell death, although pro‑oxidative conditions are not a pre‑requisite for apoptosis. Assessment of the activation status of caspases 7, 8 and 9 revealed that the levels of cleaved caspases were significantly increased in the cells treated with Khz. It is widely accepted that calcium signaling is important in apoptosis, and the present study observed an increase in [Ca2+]i in response to Khz treatment. The anti‑proliferative and pro‑apoptotic effects of Khz suggest that this extract may be developed as a potential anticancer agent.

Activation of the cell death program (apoptosis) is a strategy for the treatment of human cancer, and unfortunately a large number of drugs identified as cell cycle-specific agents for killing cancer cells are also toxic to normal cells. The present study demonstrates that the polysaccharide peptide (PSP) extracted from the Chinese medicinal mushroom, Coriolus versicolor, used in combination therapy in China, has the ability to lower the cytotoxicity of certain anti-leukemic drugs via their interaction with cell cycle-dependent and apoptotic pathways. Flow cytometry analysis demonstrated that pre-treatment of PSP (25-100 microg/ml) dose-dependently enhanced the cell cycle perturbation and apoptotic activity of doxorubicin (Doxo) and etoposide (VP-16), but not cytarabine (Ara-C) in human promyelocytic leukemia HL-60 cells. The antagonistic result from combined treatment with Ara-C and PSP may be caused by the removal of HL-60 cells in the G1-S boundary by PSP before exposure to Ara-C. A negative correlation between the increase in apoptotic cell population (pre-G1 peak) with the S-phase cell population expression (R2=0.998), the expression of cyclin E expression (R2=0.872) and caspase 3 activity (R2=0.997) suggests that PSP enhanced the apoptotic machinery of Doxo and VP-16 in a cell cycle-dependent manner and is mediated, at least in part, by the PSP-mediated modulation of the regulatory checkpoint cyclin E and caspase 3. This study is the first to describe the cell cycle mechanistic action of PSP and its interaction with other anticancer agents. Our data support the potential development of PSP as an adjuvant for leukemia treatment, but also imply the importance of understanding its interaction with individual anticancer agents.

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800μg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800μg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.

Cannabinoids possess a number of characteristics that make them putative anticancer drugs, and their value as such is currently being explored in a number of clinical studies. To further understand the roles that cannabinoids may have, we performed gene expression profiling in glioma cell lines cultured with cannabidiol (CBD) and/orΔ9-tetrahydrocannabinol (THC), and pursued targets identified by this screening. Results showed that a large number of genes belonging to the heat shock protein (HSP) super-family were up-regulated following treatment, specifically with CBD. Increases were observed both at the gene and protein levels and arose as a consequence of increased generation of ROS by CBD, and correlated with an increase in a number of HSP client proteins. Furthermore, increases impeded the cytotoxic effect of CBD; an effect that was improved by co-culture with pharmacalogical inhibitors of HSPs. Similarly, culturingglioma cells with CBD and HSP inhibitors increased radiosensitivity when compared to CBD-alone. Taken together, these data indicate that the cytotoxic effects of CBD can be diminished by HSPs that indirectly rise as a result of CBD use, and that the inclusion of HSP inhibitors in CBD treatment regimens can enhance the overall effect.

Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 microM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 microM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 microM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.

Aldose reductase (ALR2) is a key enzyme involved in diabetic complications and the search for new aldose reductase inhibitors (ARIs) is currently very important. The synthetic ARIs are often associated with deleterious side effects and medicinal and edible plants, containing compounds with aldose reductase inhibitory activity, could be useful for prevention and therapy of diabetic complications. Non-psychotropic phytocannabinoids exert multiple pharmacological effects with therapeutic potential in many diseases such as inflammation, cancer, diabetes. Here, we have investigated the inhibitory effects of extracts and their fractions from two Cannabis sativa L. chemotypes with high content of cannabidiol (CBD)/cannabidiolic acid (CBDA) and cannabigerol (CBG)/cannabigerolic acid (CBGA), respectively, on human recombinant and pig kidney aldose reductase activity in vitro. A molecular docking study was performed to evaluate the interaction of these cannabinoids with the active site of ALR2 compared to known ARIs. The extracts showed significant dose-dependent aldose reductase inhibitory activity (>70%) and higher than fractions. The inhibitory activity of the fractions was greater for acidic cannabinoid-rich fractions. Comparative molecular docking results have shown a higher stability of the ALR2-cannabinoid acids complex than the other inhibitors. The extracts of Cannabis with high content of non-psychotropic cannabinoids CBD/CBDA or CBG/CBGA significantly inhibit aldose reductase activity. These results may have some relevance for the possible use of C. sativa chemotypes based preparations as aldose reductase inhibitors.