Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high fatality of piglets, influencing the swine industry. Japanese horse chestnut (seed of Aesculus turbinata) contains many saponin mixtures, called escins, and has been used for a long time as a traditional medicinal plant. Structure-activity relationship (SAR) studies on escins have revealed that acylations at C-21 and C-22 with angeloyl or tigloyl groups were important for their cytotoxic effects. However, the strong cytotoxicity of escins makes them hard to utilize for other diseases and to develop as nutraceuticals. In this research, we investigated whether escin derivatives 1-7 (including new compounds 2, 3, 5 and 6), without the angeloyl or tigloyl groups and with modified glycosidic linkages by hydrolysis, have PEDV inhibitory effects with less cytotoxicity. Compounds 1-7 had no cytotoxicity at 20μM on VERO cells, while compounds 8-10 showed strong cytotoxicity at similar concentrations on PEDV. Our results suggest that escin derivatives showed strong inhibitory activities on PEDV replication with lowered cytotoxicity. These studies propose a method to utilize Japanese horse chestnut for treating PEDV and to increase the diversity of its bioactive compounds.

Rhodatin (1), a meroterpenoid featuring a unique pentacyclic scaffold with both spiro and spiroketal centers, and five unusual acorane-type sesquiterpenoids, named rhodocoranes A-E (2-6, respectively), are the first natural products isolated from the basidiomycete Rhodotus palmatus. Their structures were elucidated by two-dimensional NMR experiments and HRESIMS, while the absolute configuration of the substance family was determined by Mosher's method utilizing 2. Rhodatin strongly inhibited hepatitis C virus, whereas 4 displayed cytotoxicity and selective antifungal activity.

One hundred methanolic plant extracts were screened for antiviral activity against seven viruses. Twelve extracts were found to have antiviral activity at the non-cytotoxic concentrations tested. The extracts of Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, were very active against an enteric coronavirus. A root extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. A Sambucus racemosa branch tip extract was also very active against respiratory syncytial virus while the inner bark extract of Oplopanax horridus partially inhibited this virus. An extract of Ipomopsis aggregata demonstrated very good activity against parainfluenza virus type 3. A Lomatium dissectum root extract completely inhibited the cytopathic effects of rotavirus. In addition to these, extracts prepared from the following plants exhibited antiviral activity against herpesvirus type 1: Cardamine angulata, Conocephalum conicum, Lysichiton americanum, Polypodium glycyrrhiza and Verbascum thapsus.

One hundred methanolic plant extracts were screened for antiviral activity against seven viruses. Twelve extracts were found to have antiviral activity at the non-cytotoxic concentrations tested. The extracts of Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, were very active against an enteric coronavirus. A root extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. A Sambucus racemosa branch tip extract was also very active against respiratory syncytial virus while the inner bark extract of Oplopanax horridus partially inhibited this virus. An extract of Ipomopsis aggregata demonstrated very good activity against parainfluenza virus type 3. A Lomatium dissectum root extract completely inhibited the cytopathic effects of rotavirus. In addition to these, extracts prepared from the following plants exhibited antiviral activity against herpesvirus type 1: Cardamine angulata, Conocephalum conicum, Lysichiton americanum, Polypodium glycyrrhiza and Verbascum thapsus.

After experimental contamination of bovine raw and heat-treated milks with bovine rotavirus and coronavirus strains, we observed a strong viral inhibition only with raw milks, from which virus recovery was 5× 10%. Between 30% and 80% of the virus was recovered from the heat-treated milks, depending on the level of inoculation. The antiviral substance is heat-labile (destroyed within 30 min at 100°C), precipitated by ammonium sulfate and filtrable (0.45 μm Millipore membrane). It also has neutralizing activity on tissue culture.

After experimental contamination of bovine raw and heat-treated milks with bovine rotavirus and coronavirus strains, we observed a strong viral inhibition only with raw milks, from which virus recovery was 5× 10%. Between 30% and 80% of the virus was recovered from the heat-treated milks, depending on the level of inoculation. The antiviral substance is heat-labile (destroyed within 30 min at 100°C), precipitated by ammonium sulfate and filtrable (0.45 μm Millipore membrane). It also has neutralizing activity on tissue culture.

BACKGROUND:Breast cancer is the most common cancer in women and affects 1.38 million women worldwide per year. Antiestrogens such as tamoxifen, a selective estrogen receptor (ER) modulator, are widely used in clinics to treat ER-positive breast tumors. However, remissions of breast cancer are often followed by resistance to tamoxifen and disease relapse. Despite the increasing understanding of the resistance mechanisms, effective regimens for treating tamoxifen-resistant breast cancer are limited. Antrodia cinnamomea is a traditional medicinal mushroom native only to Taiwan. In this study, we aimed to examine in vitro effect of antrodia cinnamomea in the tamoxifen-resistant cancer.

METHODS:Antrodia cinnamomea was studied for its biological activity against proliferation of tamoxifen-resistant breast cancer by XTT assay. Next, the underlying mechanism was studied by flow cytometry, qPCR and Western's blotting assay.

RESULTS:Our results revealed that the ethanol extract of antrodia cinnamomea (AC) can inhibit the growth of breast cancer cells, including MCF-7 cell and tamoxifen-resistant MCF-7 cell lines. Combination treatment with AC and 10 M tamoxifen have the better inhibitory effect on the proliferation of tamoxifen-resistant MCF-7 cells than only AC did. AC can induce apoptosis in these breast cancer cells. Moreover, it can suppress the mRNA expression of skp2 (S-phase kinase-associated protein 2) by increasing the expressions ofmiR-21-5p, miR-26-5p, and miR-30-5p in MCF-7 and tamoxifen-resistant MCF-7 cells.

CONCLUSIONS:These results suggest that the ethanol extract of antrodia cinnamomea could be a novel anticancer agent in the armamentarium of tamoxifen-resistant breast cancer management. Moreover, we hope to identify additional pure compounds that could serve as promising anti-breast cancer candidates for further clinical trials.

A chloroform extract of the edible mushroom Pleurotus eryngii showed an inhibitory effect on mammalian DNA topoisomerase I. The topoisomerase I inhibitory compound was purified and identified as ubiquinone-9. Ubiquinone-9 was shown to inhibit the activity of topoisomerase I with IC(50) of about 50 microM. Concentration of 110 microM ubiquinone-9 caused 50% growth inhibition of human leukaemia U937 cells, but not that of normal fibroblast NIH3T3 and 3Y1 cells. Ubiquinone-9-induced cell death was characterised with the cleavage of poly (ADP-ribose) polymerase and pro-caspase 3. Furthermore, ubiquinone-9 induced the fragmentation of DNA into an apoptotic DNA ladder, indicating that the inhibitor triggered apoptosis. The induction of apoptosis by ubiquinone-9 was also confirmed using flow cytometry analysis. Taken together, these results suggest that ubiquinone-9 may function by inhibiting oncogenic disease, at least in part, through the inhibition of topoisomerase I activity.

Hepatocellular carcinoma is a cancer of high mortality; therefore, the effective therapy on this cancer is an imperative issue. Recently, anticancer agent combined with natural products has been demonstrated to increase apoptosis of various cancer cells effectively. Accordingly, we investigated the apoptotic effect and possible mechanism of the ethanol extract from Taiwanofungus salmoneus (=Antrodia salmonea) mycelium (TsE) alone or in combination with cisplatin in SK-Hep-1 cells. In this study, the proliferation of SK-Hep-1 cells could be inhibited at various concentrations of TsE for 24 h whereas TsE combined with cisplatin would inhibit the cell proliferation more notably. Moreover, the DNA damage and the interruption of cell cycle of SK-Hep-1 cells would be effectively raised after incubation with TsE combined with cisplatin for 24 h. The apoptosis of cells was dramatically induced, and the expression of caspases 3, 8, and 9, apoptosis-related protein, were significantly upregulated. Therefore, we proposed that the TsE combined with cisplatin inhibited cell proliferation by elevating sub-G1 phase, inducing DNA damage, activating caspases 3, 8, and 9 activities, and triggering cells apoptosis. These results reveal that TsE could be a potential adjuvant chemotherapeutic agent.

Five new aromatic compounds, designed as lucidumins A-D (1-4) and lucidimine E (9), along with seven known aromatic compounds (5-8, 10-12) were isolated from Ganoderma lucidum. Their structures were determined by spectroscopic method. Bioactive evaluation showed that compounds 2-4 and 6-10 displayed remarkable neuroprotective activities against corticosterone-induced PC12 cell damage, with the cell viability ranging from 69.99% to 126.00%; and compounds 1-4, 9 and 10 exhibited significant anti-inflammatory activities against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, with ICvalues ranging from 4.68 to 15.49 μM. In particular, compound 10 showed remarkable neuroprotection with ECvalue of 2.49 ± 0.12 μM, and potent anti-inflammation with ICvalue of 4.68 ± 0.09 μM.

Murill (ABM), a medicinal mushroom, has beneficial effects on various human metabolic diseases. The objective of this research was to evaluate the antioxidant and antidiabetic properties of ABM extracts (ethanol extract and ethyl acetate extract). The antioxidant activities of ABM ethanol extract (EE) and ethyl acetate extract (EA) were analyzed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydroxyl radical scavenging assays and the reducing power using KFe(CN)in vitro. Moreover, the effects of EE and EA onα-glucosidase inhibitory activity and improving glucose uptake by HepG2 cells were investigated in vitro. The EA showed stronger antioxidant activity, as well as inhibition of α-glucosidase, compared to EE. The analysis of glucose uptake by HepG2 cells showed that EA had significant glucose-lowering activity and exhibited no difference compared to metformin. The results suggest that ABM extracts could improve the glucose uptake by HepG2 cells and thereby alleviate postprandial hyperglycemia. This investigation provides a strong rationale for further studies on the application of ABM to control type 2 diabetes.

Grifola frondosa (Maitake mushroom) is an edible and medicinal mushroom with versatile effects such as antitumor and immunomodulating actions. Here, we demonstrated that an ethanol extract of G. frondosa fruiting body (Maitake extract) augmented intracellular lipid droplet formation and the production of triacylglycerols (TG), a major component of sebum, along with the activation of diacylglycerol acyltransferase, a rate-limiting enzyme of TG synthesis in cultured hamster sebocytes. The topical treatment of Maitake extract on the skin of hamster auricles augmented sebum accumulation in sebaceous glands and ducts. However, in comparison with the Maitake extract, another ethanol extract prepared from Agaricus blazei Murill showed less activity in sebaceous lipogenesis in hamsters in vivo and in vitro. These results provide novel evidence that Maitake extract augments sebaceous lipogenesis in hamsters in vivo and in vitro. Thus, Maitake extract is likely to be a unique agent leading to the remission of dry skin.

Targeting autophagy may serve as a promising strategy for cancer therapy. Ganoderma lucidum polysaccharide (GLP) has been shown to exert promising anti-cancer effects. However, the underlying mechanisms remain elusive. Whether GLP regulates autophagy in cancer has never been reported. In this study, GLP induced the initiation of autophagy in colorectal cancer (CRC) HT-29 and HCT116 cells, as evidenced by enhanced level of LC3-II protein, GFP-LC3 puncta, and increased formation of double membrane vacuoles. However, GLP treatment caused marked increase of p62 expression. Addition of late stage autophagy inhibitor, chloroquine (CQ), further enhanced LC3-II and p62 level, as well as increased autophagosome accumulation, suggesting a blockage of autophagic flux by GLP in CRC cells. We then found GLP blocked autophagosome and lysosome fusion as determined by mRFP-GFP-LC3 colocalization analysis. Mechanistic study revealed that GLP-induced disruption of autophagosome-lysosome fusion is due to reduced lysosome acidification and lysosomal cathepsin activities. Cell viability and flow cytometry assays revealed that GLP-induced autophagosome accumulation is responsible for GLP-induced apoptosis in CRC cells. In line with this, inhibition of autophagy initiation by 3-methyladenine (3-MA), an early stage autophagy inhibitor, attenuated GLP-induced apoptosis. In contrast, suppression of autophagy at late stage by CQ enhanced the anti-cancer effect of GLP. Furthermore, we demonstrated that GLP-induced autophagosome accumulation and apoptosis is mediated via MAPK/ERK activation. Finally, GLP inhibited tumor growth and also inhibited autophagic flux in vivo. These results unveil new molecular mechanism underlying anti-cancer effects of GLP, suggesting that GLP is a potent autophagy inhibitor and might be useful in anticancer therapy.

Anti-coronaviral activity of a mixture of oleoresins and essential oils from botanicals, designated QR448(a), was examined in vitro and in vivo. Treatment of avian infectious bronchitis virus (IBV) with QR448(a) reduced the virus titer as measured in two laboratory host systems, Vero E6 cells and embryonating eggs. The effect of QR448(a) on IBV in chickens was also investigated. Administering QR448(a) to chickens at a 1:20 dilution by spray, 2h before challenge with IBV was determined to be the most effective treatment. Treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral RNA in the trachea. Treatment with QR448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of IBV over a 14-day period. Anti-IBV activity of QR448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal. In addition, QR448(a) had activity against both Massachusetts and Arkansas type IB viruses, indicating that it can be expected to be effective against IBV regardless of serotype. To our knowledge, this is the first report on the in vivo use of a virucidal mixture of compounds effective against the coronavirus IBV.

Anti-coronaviral activity of a mixture of oleoresins and essential oils from botanicals, designated QR448(a), was examined in vitro and in vivo. Treatment of avian infectious bronchitis virus (IBV) with QR448(a) reduced the virus titer as measured in two laboratory host systems, Vero E6 cells and embryonating eggs. The effect of QR448(a) on IBV in chickens was also investigated. Administering QR448(a) to chickens at a 1:20 dilution by spray, 2h before challenge with IBV was determined to be the most effective treatment. Treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral RNA in the trachea. Treatment with QR448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of IBV over a 14-day period. Anti-IBV activity of QR448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal. In addition, QR448(a) had activity against both Massachusetts and Arkansas type IB viruses, indicating that it can be expected to be effective against IBV regardless of serotype. To our knowledge, this is the first report on the in vivo use of a virucidal mixture of compounds effective against the coronavirus IBV.

BACKGROUND: Complications in diabetes mellitus are partially mediated by enhanced formation of reactive oxygen species. Among the factors involved in reactive oxygen species formation, advanced glycation end products play a key role. Owing to a reduced activity of the enzyme transketolase, which requires diphosphorylated thiamine (vitamin B(1)) as cofactor, an accumulation of those deleterious glucose metabolites especially in diabetic patients can be observed. Benfotiamine, a lipophilic thiamine diphosphate prodrug, prevented early renal and retinal changes in animal studies, and reduced neuropathic pain in clinical studies. Several mechanisms for these activities have been described. We investigated for the first time direct antioxidant abilities of benfotiamine. Additionally, a potential DNA protective effect of benfotiamine was analysed. METHODS: Oxidative stress was detected by flow cytometry, antioxidative capacity was measured with the ferric reducing ability of plasma (FRAP) assay, two endpoints for genomic damage were assessed: the comet assay and the micronucleus test, and the expression and activity of transketolase was quantified. RESULTS: Benfotiamine prevented oxidative stress induced by the mutagen 4-nitroquinoline-1-oxide (NQO), the uremic toxin indoxyl sulfate, and the peptide hormone angiotensin II in three different kidney cell lines. Cell-free experiments showed a direct antioxidant effect of benfotiamine, which might account for the protective effect. Oxidative DNA damage, induced by angiotensin II, was completely prevented by benfotiamine. Incubation with benfotiamine increased transketolase expression and activity in the cells. CONCLUSIONS: Benfotiamine shows a direct antioxidant action. This effect of benfotiamine may be involved in the improvement of diabetic late complications, including peripheral neuropathy. Copyright (c) 2008 John Wiley & Sons, Ltd.

We previously reported that [[N-[3beta-hydroxyllup-20(29)-en-28-oyl]-7-aminoheptyl]carbamoyl]methane (A43D, 4) was a potent HIV-1 entry inhibitor. However, 4 was inactive against HIV-2 virus, suggesting the structural requirements for targeting these two retroviruses are different. In this study, a series of new betulinic acid derivatives were synthesized, and some of them displayed selective anti-HIV-2 activity at nanomolar concentrations. In comparison to compounds with anti-HIV-1 activity, a shorter C-28 side chain is required for optimal anti-HIV-2 activity.

As part of our search for botanical sources of SARS-CoV 3CL(pro) inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CL(pro) inhibitory activity (62% at 100μg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1-8) and four biflavonoids (9-12) were isolated and evaluated for SARS-CoV 3CL(pro) inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone (9) (IC(50)=8.3μM) showed most potent 3CL(pro) inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure-activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CL(pro) activity with IC(50) values of 280.8, 20.2, and 23.8μM,respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3' of flavones, as biflavone had an effect on 3CL(pro) inhibitory activity.

As part of our search for botanical sources of SARS-CoV 3CL(pro) inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CL(pro) inhibitory activity (62% at 100μg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1-8) and four biflavonoids (9-12) were isolated and evaluated for SARS-CoV 3CL(pro) inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone (9) (IC(50)=8.3μM) showed most potent 3CL(pro) inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure-activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CL(pro) activity with IC(50) values of 280.8, 20.2, and 23.8μM,respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3' of flavones, as biflavone had an effect on 3CL(pro) inhibitory activity.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-beta-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL(pro) were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure-activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.