High-performance Countercurrent Chromatography to Access Rhodiola rosea Influenza Virus Inhibiting Constituents.

In a cytopathic effect inhibition assay, a standardized Rhodiola rosea root and rhizome extract, also known as roseroot extract (SHR-5), exerted distinct anti-influenza A virus activity against HK/68 (H3N2) (IC50 of 2.8 µg/mL) without being cytotoxic. For fast and efficient isolation and identification of the extract's bioactive constituents, a high-performance countercurrent chromatographic separation method was developed. It resulted in a three-stage gradient elution program using a mobile phase solvent system composed of ethyl acetate/n-butanol/water (1 : 4 : 5 → 2 : 3 : 5 → 3 : 2 : 5) in the reversed-phase mode. The elaborated high-performance countercurrent chromatographic method allowed for fractionation of the complex roseroot extract in a single chromatographic step in a way that only one additional orthogonal isolation/purification step per fraction yielded 12 isolated constituents. They cover a broad polarity range and belong to different structural classes, namely, the phenylethanoid tyrosol and its glucoside salidroside, the cinnamyl alcohol glycosides rosavin, rosarin, and rosin as well as gallic acid, the cyanogenic glucoside lotaustralin, the monoterpene glucosides rosiridin and kenposide A, and the flavonoids tricin, tricin-5-O-ß-D-glucopyranoside, and rhodiosin. The most promising anti-influenza activities were determined for rhodiosin, tricin, and tricin-5-O-ß-D-glucopyranoside with IC50 values of 7.9, 13, and 15 µM, respectively. The herein established high-performance countercurrent chromatographic protocol enables fast and scalable access to major as well as minor roseroot constituents. This is of particular relevance for extract standardization, quality control, and further in-depth pharmacological investigations of the metabolites of this popular traditional herbal remedy.

Antiviral activity of hydroalcoholic extract from Eupatorium perfoliatum L. against the attachment of influenza A virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Eupatorium perfoliatum have been traditionally used by American natives as a treatment for fever and infections. Also modern phytotherapy in Europe documents the use of hydroalcoholic extracts of this herbal material for the treatment of infections of the upper respiratory tract. AIM OF THE STUDY: The aim of the present study was to characterize the anti-influenza A virus (IAV) potential of extracts derived from the aerial parts of E. perfoliatum and to identify their antiviral mode of action and potential active fraction's compounds of the extract. MATERIALS AND METHODS: The inhibitory effects of extracts obtained by different organic solvents with different polarities on the cytopathic effect induced by IAV replication was determined in a Madin-Darby Canine Kidney Epithelial (MDCK II) cell-based assay measuring cell viability by MTT stain (MTTIAV assay). Plaque reduction assays were used for investigation of antiviral activity. The mode of action was investigated by different incubation and treatment cycles as well as hemagglutination inhibition assays. Influence of the test extract on tumor necrosis factor (TNF-α) and epidermal growth factor (EGF)-induced cell signaling was analyzed in human lung epithelial (A549) cells. Analytical characterization of extract and fractions obtained from the extract was performed by UHPLC-MS. RESULTS: Hydroalcoholic extracts from the aerial parts of E. perfoliatum were shown to inhibit growth of a clinical isolate of IAV(H1N1)pdm09 I1 and the influenza virus A/Puerto Rico/8/34 (PR8; H1N1) with a half-maximal inhibitory concentration (IC50) of 7µg/mL and 14µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 52 and 26, respectively. Extracts prepared with dichloromethane and methanol were inactive. At concentrations >1-10µg/mL of the hydroalcoholic extract plaque formation of IAV(H1N1)pdm09 was abrogated. The extract was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. The extract blocked attachment of IAV and interfered with virus-induced hemagglutination. TNF-α-induced signal transduction in A549 cells was not affected, while the EGF-induced signaling to phosphorylated ERK was slightly upregulated by the extract. Bioassay-guided fractionation and subsequent LC-MS analysis indicated that the antiviral activity might be due to polyphenolic compounds with higher molecular weights, which strongly interact with stationary phases of different chromatographic systems. These still unknown compounds with probably high molecular weight could not be isolated in the present study. A variety of different flavonoid glycosides and caffeoyl quinic acids obtained from E. perfoliatum did definitely not contribute to the antiviral effect of the extract and its respective fractions. CONCLUSION: Hydroalcoholic extracts from the aerial parts of E. perfoliatum and its main active polyphenolic constituents protect cells from IAV infection by inhibiting viral attachment to the host cells. The extract appears to be a promising expansion of the currently available anti-influenza agents.

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines.

Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki=11 µM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn-3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity.

3-O-galloylated procyanidins from Rumex acetosa L. inhibit the attachment of influenza A virus.

Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of Rumex acetosa (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTTIAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC50) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4ß→8)-epicatechin-3'-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC50 approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; o-trihydroxylation in the B-ring increased the anti-IAV activity. In silico docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents.

The NF-kappaB inhibitor SC75741 protects mice against highly pathogenic avian influenza A virus.

The appearance of pandemic H1N1 and highly pathogenic avian H5N1 viruses in humans as well as the emergence of seasonal H1N1 variants resistant against neuraminidase inhibitors highlight the urgent need for new and amply available antiviral drugs. We and others have demonstrated that influenza virus misuses the cellular IKK/NF-kappaB signaling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here, we show that the novel NF-kappaB inhibitor SC75741 significantly protects mice against infection with highly pathogenic avian influenza A viruses of the H5N1 and H7N7 subtypes. Treatment was efficient when SC75741 was given intravenously in a concentration of 5mg/kg/day. In addition, application of SC75741 via the intraperitoneal route resulted in a high bioavailability and was also efficient against influenza when given 15 mg/kg/day or 7.5 mg/kg/twice a day. Protection was achieved when SC75741 was given for seven consecutive days either prior to infection or as late as four days after infection. SC75741 treatment showed no adverse effects in the concentrations required to protect mice against influenza virus infection. Although more pre-clinical studies are needed SC75741 might be a promising candidate for a novel antiviral drug against influenza viruses that targets the host cell rather than the virus itself.

A plant extract of Ribes nigrum folium possesses anti-influenza virus activity in vitro and in vivo by preventing virus entry to host cells.

Infections with influenza A viruses (IAV) are still amongst the major causes of highly contagious severe respiratory diseases not only bearing a devastating effect to human health, but also significantly impact the economy. Besides vaccination that represents the best option to protect from IAV infections, only two classes of anti-influenza drugs, inhibitors of the M2 ion channel and the neuraminidase, often causing resistant IAV variants have been approved. That is why the need for effective and amply available antivirals against IAV is of high priority. Here we introduce LADANIA067 from the leaves of the wild black currant (Ribes nigrum folium) as a potent compound against IAV infections in vitro and in vivo. LADANIA067 treatment resulted in a reduction of progeny virus titers in cell cultures infected with prototype avian and human influenza virus strains of different subtypes. At the effective dose of 100 µg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation. Further, viruses showed no tendency to develop resistance to LADANIA067 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of LADANIA067 appears to be mainly due to interference with virus internalisation. In the mouse infection model LADANIA067 treatment reduces progeny virus titers in the lung upon intranasal application. In conclusion, an extract from the leaves of the wild black currant might be a promising source for the development of new antiviral compounds to fight IAV infections.

Effect of a fat spread enriched with medium-chain triacylglycerols and a special fatty acid-micronutrient combination on cardiometabolic risk factors in overweight patients with diabetes.

BACKGROUND: Medium-chain triacylglycerols (MCT), omega-3 polyunsaturated fatty acids (n-3-PUFA) and micronutrients may be useful for weight and cardiometabolic risk management. However, studies analyzing the effect of a combination of both in individuals at increased cardiometabolic risk are lacking. Therefore, this randomized, controlled, double-blind study investigated the effect of a fat spread enriched with two different doses of MCT and a special long-chain fatty acid-micronutrient combination on cardiometabolic risk factors in overweight diabetic patients. METHODS: Fifty-four patients received either a fat spread with 6 g/d MCT (MCT30%) or 1.2 g/d (MCT6%). Forty-three completed the study. Analysis was performed according to the median of MCT intake (supplemented and food-derived MCT). Clinical, anthropometric, blood, 24 h-urine parameters and dietary intake were assessed at baseline and after 12 weeks. RESULTS: Total MCT intake > 7 g/d (MCT > 7 group) significantly reduced waist circumference (WC) by 1.81 ± 2.69 cm, whereas ≤ 7 g/d MCT (MCT ≤ 7 group) increased WC by 0.32 ± 3.03 cm (p = 0.027), which was supported by a change in waist-to-height ratio (WHtR) (p = 0.018). Fasting serum triglycerides (TG) increased in both groups over time due to dietary habits. In contrast, diabetic metabolic situation and urinary albumin excretion did not alter. Urinary pH differed significantly between groups after 12 weeks. CONCLUSION: An intake of >7 g/d MCT reduced WC in overweight diabetics, whereas the increase in the intake of fatty acids may have worsened fasting TG. Therefore, the suitability of a fat for nutrient enrichment remains to be challenged, and further studies in low-fat matrices are desirable.

A polyphenol rich plant extract, CYSTUS052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance.

Infections with influenza A viruses still pose a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses of the H5N1 subtype capable to infect and kill humans highlights the urgent need for new and efficient countermeasures against this viral disease. Here we demonstrate that a polyphenol rich extract (CYSTUS052) from the Mediterranean plant Cistus incanus exerts a potent anti-influenza virus activity in A549 or MDCK cell cultures infected with prototype avian and human influenza strains of different subtypes. CYSTUS052 treatment resulted in a reduction of progeny virus titers of up to two logs. At the effective dose of 50 microg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation, which is consistent with the fact that these plant extracts are already used in traditional medicine in southern Europe for centuries without any reported complications. Viruses did not develop resistance to CYSTUS052 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of CYSTUS052 appears to be mainly due to binding of the polymeric polyphenol components of the extract to the virus surface, thereby inhibiting binding of the hemagglutinin to cellular receptors. Thus, a local application of CYSTUS052 at the viral entry routes may be a promising approach that may help to protect from influenza virus infections.

CYSTUS052, a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice.

Influenza, a respiratory disease caused by influenza viruses, is still a worldwide threat with a high potential to cause a pandemic. Beside vaccination, only two classes of drugs are available for antiviral treatment against the pathogen. Here we show that CYSTUS052, a plant extract from a special variety of Cistus incanus that is rich in polymeric polyphenols, exhibits antiviral activity against a highly pathogenic avian influenza A virus (H7N7) in cell culture and in a mouse infection model. In vitro and in vivo treatment was performed with an aerosol formulation, because the bioavailability of high molecular weight polyphenols is poor. In MDCK cells, a 90% reduction of plaque numbers on cells pre-incubated with the plant extract was achieved. For in vivo experiments we used a novel monitoring system for influenza A virus-infected mice that allows measurement of body temperature and gross motor-activity of the animals. Mice treated with CYSTUS052 did not develop disease, showed neither differences in their body temperature nor differences in their gross motor-activity and exhibited no histological alterations of the bronchiolus epithelial cells.