EP4701645A1 - Compositions with antioxidant, antiviral and immunomodulating activity comprising hop (humulus lupulus l.) and elder(sambucus nigra l.) extracts titrated in flavonoids - Google Patents

Abstract

Combinations of Humulus lupulus L. and Sambucus nigra L. extracts in specific ratios exhibit synergistic effects in antioxidant, antiviral and anti-inflammatory activity models, and are therefore useful as active ingredients in supplements, functional foods, and foods and beverages, for the prevention of influenza and to promote the body's natural defences.

Description

COMPOSITIONS WITH ANTIOXIDANT, ANTIVIRAL AND IMMUNOMODULATING ACTIVITY COMPRISING HOP (HUMULUS LUPULUS L.) AND ELDER (SAMBUCUS NIGRA L.) EXTRACTS TITRATED IN FLAVONOIDS

The invention relates to the use of extracts obtained from the aerial parts, and in particular the inflorescences (strobili or cones), of Humulus lupulus L., titrated in polyphenols and in particular in tannins and/or flavonoids, combined with an extract obtained from the aerial parts (including the fruit), and in particular the inflorescences, of Sambucus nigra L., titrated in polyphenols and in particular in tannins and/or flavonoids, for use in supplements, functional foods, and foods and beverages, for the prevention of influenza and to promote the body’s natural defences.

STATE OF THE ART

Influenza is an acute respiratory disease caused by influenza viruses; it occurs in a seasonal epidemic pattern, mainly during the winter period, but can sometimes also become pandemic, as in the case of the “Spanish” influenza of 1918, which caused at least 20 million deaths. Influenza is a disease of great importance for public health because of its epidemiological, clinical and economic impact. This is mainly due to the fact that its etiological agents, namely influenza viruses, have a marked tendency to mutate, thereby avoiding the immune defences developed by individuals who have already contracted the infection or been immunised. In some categories of individuals (children, the elderly, and individuals with comorbidities and chronic disorders), they may also give rise to serious complications, and sometimes fatal outcomes. The social costs of said disease are also high, having regard to factors such as working days lost and the consequent loss of productivity for businesses, to name just a few examples.

Vaccination and preventive hygiene measures are still the only weapons available to counteract the spread of the disease. Moreover, once it has been contracted, the therapeutic possibilities are extremely limited, and only rest and possibly the use of symptomatic medicaments, such as antipyretics and anti-inflammatories, are generally recommended for healthy individuals. According to the WHO guidelines, antivirals should only be prescribed in special cases, after thorough evaluation of the risk-benefit ratio, and in any event within 48 hours of the onset of symptoms. The antivirals which can be used to treat influenza mainly belong to two categories: neuraminidase or NA inhibitors (zanamivir and oseltamivir) and viral M2 protein inhibitors (amantadine and rimantadine), molecules which are not lacking in toxicity. All this demonstrates the importance of researching new molecules with antiviral activity. As many of the metabolic pathways used by the virus to infect the cell are sensitive to redox reactions, scientific research has focused for some time on studying polyphenols, which are natural antioxidants present in many plants. However, a synergistic combination of hop and elder in specific proportions has never been studied for this purpose, nor are the immunomodulating properties of such a combination known.

Humulus lupulus L. (hop), fam. Cannabaceae, is a dioecious climbing plant native to the temperate zones of Eurasia. Hop inflorescences are universally known for the manufacture of beer, to which they give a characteristic bitter, aromatic flavour. The use of hops in beer dates back to the 11th century. Hops are also used in diet supplements as a stomachic remedy to promote the digestive function, and as a sedative for relaxation and sleep in case of stress; in fact, hop infusions and tinctures have traditionally been used as an aid to digestion and sleep [Chevallier A., The Encyclopaedia of Medicinal Plants, Dorling Kindersley, 1996]. The use of hops in menopausal disorders is also known due to their phytoestrogen content [Stulikova K et al., Molecules. 2018 Mar 15;23(3):660. doi: 10.3390/molecules23030660. PMID: 29543713; PMCID: PMC6017581]. The antiviral and antioxidant properties of hops were described in a recent article [Antonella Di Sotto et al., Oxidative Medicine and Cellular Longevity, vol. 2018, Article ID 5919237, 14 pages, 2018. https://doi.org/10.1155/2018/5919237], but not in combination with elder.

Sambucus nigra L. (elder), fam. Adoxaceae, is a tree typical of temperate Europe, which becomes very attractive in spring due to its abundant blossom. Elder is an excellent diaphoretic, i.e. regulator of the sweating process, and a diuretic indicated for drainage of bodily fluids; it has an anti-inflammatory action, due to which it is used to improve the functionality of the upper respiratory tract and the fluidity of bronchial secretions. Its traditional use in disorders of the respiratory system and influenza is mainly due to its diaphoretic action, because increased sweating helps to reduce fever, and to its anti-inflammatory action [Chevallier A., The Encyclopaedia of Medicinal Plants, Dorling Kindersley, 1996]. During the recent COVID 19 pandemic, considerable interest focused on elderberry supplements designed to prevent and treat the disease, but a recent review [Wieland LS et al., BMC Complement Med Ther. 2021 Apr 7;21(1): 112. doi: 10.1186/sl2906-021-03283-5] concluded that although it may reduce the duration of influenza, the evidence is uncertain. However, this remedy also relates to the use of elderberries alone.

Some beers on the market contain hops and elder, but in such cases elder is added for purposes similar to those of hops, i.e. as a flavouring, in particular to add a refreshing note to the drink, an application which is unrelated to the purpose of the present invention. DESCRIPTION OF THE INVENTION

It has now been discovered that extracts of aerial parts, in particular inflorescences, of Humulus lupulus L. (hop) and Sambucus nigra L. (elder), combined in weight ratios ranging from 1 :9 to 1:11, preferably 1:10, perform synergistic effects in tests of cytoprotective, antiviral and immuno stimulating activity.

The object of the invention is therefore compositions comprising extracts of aerial parts of Humulus lupulus L. and Sambucus nigra L. in weight ratios ranging from 1:9 to 1:11, preferably in the weight ratio of 1:10.

Another object of the invention relates to the use of said compositions for the treatment and prevention of viral infections, in particular viral infections caused by influenza viruses.

The extracts usable according to the invention are preferably obtained by extraction with mixtures of water and ethanol, and have a specific flavonoid content.

In particular, Humulus lupulus L. extract with a total flavonoid content, expressed as rutin, of not less than 0.4%, is obtained by a method comprising the following steps: the predried aerial parts are chopped, preferably to a length of 1-2 cm; they are then treated with an extractive solvent containing water, ethanol and/or mixtures thereof; the mixture is maintained under percolation for 3-5 hours; at the end of the first recycle, the extraction is repeated under the same conditions 3 to 5 times, using fresh solvent every time. All the water-alcohol extracts are combined, filtered and concentrated; the extractive solvents are removed by vacuum concentration, under heating, and subsequent spray-drying or another suitable drying technology, supported on maltodextrin, until a homogeneous brown powder is obtained.

Sambucus nigra L. extract with a total flavonoid content, expressed as isoquercitroside, of not less than 1%, is obtained by a method comprising the following steps: the pre-dried aerial parts are chopped, preferably to a length of 1-2 cm; they are then treated with an extractive solvent containing water, ethanol and/or mixtures thereof; the mixture is maintained under percolation for 3-5 hours; at the end of the first recycle, the extraction is repeated under the same conditions 3 to 5 times, using fresh solvent every time. All the water-alcohol extracts are combined, filtered and concentrated; the extractive solvents are removed by vacuum concentration, under heating, and subsequent spray-drying or another suitable drying technology, supported on maltodextrin, until a homogeneous brown powder is obtained.

Both the extracts were titrated in total flavonoids by a spectrophotometric method as specified in European Pharmacopoeia, 10th ed.

A chromatographic analysis (HPLC-DAD) was also performed to identify the main polyphenols contained in the extracts, demonstrating the presence of 29 compounds for hop and 26 for elder.

The analysis demonstrated that the main constituents of the hop extract were isoquercetin, thymol and benzoic acid, followed by catechin; the following compounds were also present in appreciable amounts, but less than those specified above: 3-hydroxytyrosol, gallic acid, loganic acid, t-cinnamic acid, hesperidin, naringenin and quercetin; and finally, caftaric acid, p-coumaric acid, rosmarinic acid, t-ferulic acid, kaempferol, resveratrol, syringaldehyde and rutin were found in smaller amounts.

In the elder extract, the chromatographic analysis demonstrated the presence of large amounts of isoquercetin, chlorogenic acid and rosmarinic acid; smaller but appreciable amounts of thymol, catechin, naringenin, benzoic acid and syringaldehyde were also identified; and finally, progressively smaller amounts of t-ferulic acid, gallic acid, resveratrol, epicatechin, 3- hydroxytyrosol, caffeic acid, p-coumaric acid, quercetin, caftaric acid and traces of 4- hydroxybenzoic acid were identified

The data obtained from the activity tests demonstrate cytoprotective antioxidant properties useful to counteract not only the oxidative damage induced by various stressors, but also the development and replication of viral infections such as influenza, due to the antiviral properties of the mixture (especially towards the primary infection), in order to support and promote the body’s natural defences.

For the intended uses, the compositions according to the invention are typically formulated as capsules, tablets, granulates, solutions or suspensions, using conventional techniques and excipients. The combination of extracts can also be used to prepare functional foods, beverages and nutraceutical supplements.

The unit doses of the two extracts will be determined by the skilled person on the basis of pharmacotoxicological and clinical tests. As an approximation, the unit doses of the hop extract can range from 10 to 100 mg and those of the elder extract from 100 to 1000 mg.

The cytoprotective activity tests were conducted on BEAS-2B bronchial epithelial cells and A549 lung cancer cells treated with concentrations of up to 50 pg/ml for the hop extract and up to 200 pg/ml for the elder extract. On the basis of the tests conducted with different weight ratios between the extracts (1:1, 1:2, 1:4 and 1:8, 1:10, 1:50 and 1:100), a clear, marked synergy emerged for a narrow range of ratios (from 1:9 to 1:11), in particular for the ratio of 1:10.

The methods and results of the tests conducted are described by way of example in the experimental section below. ANTIOXIDANT AND ANTIVIRAL ACTIVITY OF MIXTURES OF HOP AND ELDER EXTRACTS: CELL MODELS

A- Cytoprotective activity of the extracts and mixtures thereof towards the oxidative damage induced by t-butyl hydroperoxide (tBOOH) in BEAS-2B bronchial epithelial cells and lung cancer cells (A549). and reactive oxygen species (ROS) levels in the treated cells: the cells were exposed to a 24 h pre-treatment with the extracts and combinations thereof, followed by a 3 h co-treatment with the same substances and with the pro-oxidant agent t-BOOH 500 pM. At the end of the exposure, the cytoprotective activity was evaluated in terms of restoration of cell viability reduced by tBOOH, and of the intracellular levels of reactive oxygen species (ROS).

B- Antiviral activity of the extracts and mixtures thereof towards influenza virus PR8/H1N1 in A549 lung cancer cells: the treatment protocol involves exposure to samples 1 h before infection, during infection (1 h) and after infection (24 h) (before, during and postinfection). The supernatant of the infected and treated cells was used to determine, by RT-PCR analysis, the mRNA content of some viral proteins, i.e. haemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA) and matrix protein M2, which are involved in the entry of the virus into the host cell, viral replication, and transmission of the virus from the infected cell respectively. In some cases the cell supernatants were used to evaluate their infective capacity, inducing a “secondary” infection by exposing canine kidney cells (MDCK) (susceptible to infection by influenza viruses) to A549 cell supernatants infected with the virus and treated with the before, during and post-infection protocol. At the end of the secondary infection, the cells were fixed and labelled with antibodies to conduct the fluorescence reading of viral protein haemagglutinin (HA), involved in the entry of the virus into the host cell. The fluorescence intensity of said protein is proportional to the degree of infectivity of the viral particles.

The mixture consisting of hop (HOPE) and elder (SA_P4296) extracts in the proportion of 1 : 10 (10 pg/ml : 100 (pg/ml) demonstrated synergy in the cell models tested. The mixture exhibited a significant increase in cell viability towards tBOOH (antioxidant properties), but in particular exhibited a veritable synergy in relation to antiviral properties: as regards the mRNA content of the cell supernatants, the 1:10 mixture produced superior effects to those of the individual extracts, as will be seen from the histogram in Figure 1.

In particular, the reduction in mRNA levels of nucleoprotein (NP) and neuraminidase (NA), amounting to 53% and 52% respectively, is attributable to a more marked effect at the replication and transmission stage of the infection than at the viral attachment stage (HA). IMMUNOMODULATING PROPERTIES (BOOSTER EFFECT ON CELL DEFENCES)

The synergistic combination of hop and elder (1:10) on the activity of nuclear factor- erythroid 2-related factor 2 (Nrf2) was evaluated in detail. Nrf2 is a transcription factor that governs the gene expression of a wide variety of cytoprotective antioxidant enzymes; in the presence of oxidative stress, Nrf2 is mobilised by cytosol to the nucleus, wherein it binds to a DNA promoter to initiate transcription of antioxidant genes and the proteins thereof, which in turn leads to synthesis of enzymes and antioxidant factors, such as glutathione.

As in the preceding cytoprotection experiments, the A549 lung cancer cells underwent a 24 h pre-treatment, at the end of which they were exposed to co-treatment in the presence of tBOOH for 30 minutes, a sufficient time for activation of factor Nrf2. At the end of the exposure to pro-oxidant factor t-BOOH, the cells were analysed by fluorescence imaging. In particular, the A549 cells were labelled with fluorescent antibodies towards cytosolic Nrf2 and towards the nucleus.

After image acquisition, a semi-quantitative fluorescence analysis was conducted to quantify the fluorescence of factor Nrf2 in relation to the number of cells. The mixture of hop and elder extracts (1:10) significantly increased Nrf2 expression not only compared with the control, but also with the individual extracts. Said effect also proved evident in relation to damage caused by tBOOH; in fact, the mixture restored the normal levels of Nrf2 reduced by tBOOH. Conversely, the individual extracts were unable to cause significant variations in Nrf2 levels induced by tBOOH (Figures 2 A and 2B, under baseline conditions and in the presence of oxidative damage respectively). This demonstrates further synergy towards Nrf2. Increased expression of Nrf2, in both the absence and presence of the oxidative stimulus induced by tBOOH, may represent the mechanism underlying the protective effects of the synergistic combination.

Claims

1. Compositions comprising extracts of aerial parts of Humulus lupulus L. and Sambucus nigra L. in weight ratios of 1:9 to 1:11, preferably 1:10.

2. Compositions according to claim 1 wherein the extract of Humulus lupulus L. has a total flavonoid content expressed as rutin of not less than 0.4%.

3. Compositions according to claim 1 wherein the extract of Sambucus nigra L. has a total flavonoid content expressed as isoquercitoside of not less than 1%.

4. Compositions according to any one of claims 1 to 3 wherein the extracts are obtained by extraction with water and ethanol mixtures.

5. Compositions according to any one of claims 1 to 4 wherein the aerial parts of Humulus lupulus L. and Sambucus nigra L. are inflorescences.

6. Compositions according to any one of claims 1 to 5 in the form of capsules, tablets, granules, solutions or suspensions.

7. The compositions of claims 1 to 5 for use in the prevention and treatment of viral infections.

8. Compositions for use according to claim 7 wherein the viral infections are caused by influenza viruses.