ANTIVIRAL PREPARATION FROM CHESTNUT SHELLS OF CASTANEA SATIVA MILL

Abstract

The present invention refers to the use of an extract from chestnut shells (CSE), peeling waste of the chestnut, (fruit of the tree Castanea sativa Mill., European Chestnut), or fractions of said extract, with specific antiviral activity in the treatment and/or prevention of Herpesviridae, Retroviridae, and Coronaviridae infections.

Claims

1. A method for the treatment and/or prevention of Herpesviridae and/or Retroviridae and/or Coronaviridae infections, comprising administering an extract from shells of chestnut fruits (CSE) of Castanea sativa Mill. and/or fractions thereof to a human in need of such treatment and/or prevention.

2. The method according to claim 1 wherein the infection is an Herpes Simplex Virus 1 or 2 (HSV-1 or HSV-2), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) or the Human Immunodeficiency Virus (HIV) infection.

3. The method according to claim 1 wherein the chestnut shells are waste from chestnut fruits industrial peeling.

4. The method according to claim 1, wherein the extract is obtained using traditional methods for the extraction of polyphenolic compounds from Castanea sativa (e.g. boiling water, maceration, extraction with solvents) and/or not traditional methods (e.g. microwaves, ultrasounds, supercritical extraction).

5. The method according to claim 1 wherein the extract is obtained from industrial chestnut shells waste as burned shells (Brulage method), dried shells or by manual chestnut peeling.

6. A method for the treatment and/or prevention of Herpesviridae and/or Retroviridae and/or Coronaviridae infections, comprising administering a pharmaceutical composition or supplement or medical device comprising an extract from shells of chestnut fruits (CSE) of Castanea sativa Mill. and/or fractions and at least one pharmaceutically acceptable excipient and/or carrier thereof to a human in need of such treatment and/or prevention.

7. The method according to claim 6 wherein the infection is an HSV-1, HSV-2, HIV or Sars-CoV-2 infection.

8. The method according to claim 6, wherein the excipient is selected from the group consisting of: emollients (e.g. glycerin and/or other), gelling (e.g. polyacrylamide, isoparaffin, laureth 7 and/or other), preservatives (e.g. 2-phenoxyethanol, 3-(2-ethylhexyloxy)propane-1,2-diol and/or other), alcohols, surfactants, bactericides and solvents (e.g. aqueous solutions, buffered physiological solution and/or other) and combinations thereof.

9. The method according to claim 6, wherein the amount of the extract and/or of fractions thereof ranges from 0.000002% to 20% by weight of the total amount of the composition or supplement or device.

10. The method of claim 6, wherein the chestnut shells are waste from chestnut fruits industrial peeling.

11. The method of claim 6, wherein the extract is obtained using traditional methods for the extraction of polyphenolic compounds from Castanea sativa (e.g. boiling water, maceration, extraction with solvents) and/or not traditional methods (e.g. microwaves, ultrasounds, supercritical extraction).

12. The method of claim 6, wherein the extract is obtained from industrial chestnut shells waste as burned shells (Brulage method), dried shells or by manual chestnut peeling.

13. Non-therapeutic use of an extract from shells of chestnut fruits (CSE) of Castanea sativa Mill. and/or fractions thereof as an antiviral agent or as an active ingredient of an antiviral agent, wherein the virus belongs to a family selected from the group consisting of: Herpesviridae, Retroviridae and Coronaviridae.

14. The non-therapeutic use according to claim 13 wherein the virus is HSV-1, HSV-2, HIV or Sars-CoV-2.

15. Non-therapeutic use according to claim 13 wherein said the extract and/or fractions thereof are used in a cosmetic, cleaning or personal care product, preferably selected from: cleaning products and detergents, soaps, shampoos, detergents, personal hygiene products, laundry and household cleaning products, cloths and absorbent paper, mouthwashes, antiperspirants, skin deodorants, cosmetic products, creams, hair lotions and conditioners, toothpaste, products for intimate hygiene and hygiene of children, adhesive paste for dentures and absorbent diapers.

16. Non-therapeutic use according to claim 15 wherein the extract and/or fractions thereof ranges from 0.000002% to 20% by weight of the total amount of the product.

17. Non-therapeutic use of a composition comprising extract from shells of chestnut fruits (CSE) of Castanea sativa Mill. and/or fractions thereof as an antiviral agent or as an active ingredient of an antiviral agent, wherein the virus belongs to a family selected from the group consisting of: Herpesviridae, Retroviridae and Coronaviridae, wherein the virus is preferably HSV-1, HSV-2, HIV or Sars-CoV-2 and/or said composition is preferably selected from: cream, emulsion, dispersion, gel, ointment, lotion or serum.

18. Non-therapeutic use according to claim 17 wherein the extract and/or fractions thereof ranges from 0.000002% to 20% by weight of the total amount of the composition.

Description

[0081] The present invention will now be illustrated with non-limiting examples with reference to the following figures.

[0082] FIG. 1. Evaluation panel of the inhibition mediated by the extract of chestnut shells on HSV-1, HSV-2 and cell cycle interference models.

[0083] FIG. 2. Comparison of interference in HSV-1 mediated infection on Vero cells with extracts from Burned and Dried chestnut shells.

[0084] FIG. 3. Comparison of the inhibition in HSV-1 mediated infection on Vero cells with extracts from Burned and Dried chestnut shells, obtained through different extraction and treatment methods.

EXAMPLE 1

Materials and Methods

Used Strains

[0085] a) S. aureus ATCC 6538: Gram-positive, asporigenic, aerobic and facultative anaerobic bacteria, immobile and belonging to the Micrococcaceae family, included in the genus Staphylococcus; [0086] b) E. coli ATCC 11229: Gram-negative, asporigenic, aerobic and facultative anaerobic bacteria, motile for peritric cilia belonging to the Enterobacteriaceae family, included in the genus Escherichia. [0087] c) Candida albicans ATCC 90028: dimorphic saprophytic fungus, belonging to the Saccharomycetes family.

Production of the Chestnut Shell Extract (CSE)

[0088] The preparation of the CSE with a commonly used and published method is reported here as an example. The chestnut shells (external and internal), coming from the peeling process of Italian chestnuts by drying (Dried) or burning (Burned), were brought to a constant weight in the oven at 55° C., then blended with a kitchen blender before being subjected to the extraction of bioactive molecules. The extraction was carried out at 5% (w/v) in deionized water, for 60 minutes, at 100° C. To compensate for evaporation, every 20 minutes the volume of the mixture subjected to extraction was restored to the initial level (Squillaci et al. 2018).

[0089] The extract was then centrifuged at 4000 rpm, at 4° C. for 60 minutes (5810R, Eppendorf), the pellet removed, the supernatant frozen at -20° C., and subsequently lyophilized.

Antibacterial Assays

[0090] Antibacterial assays were performed using the plate microdilution method, according to the guidelines established by the National Committee on Clinical Laboratory Standards (NCCLS). To standardize the bacterial suspension for the antimicrobial assay, fresh colonies of each strain, previously obtained on Brein Heart Infusion agar, were inoculated in liquid Brein Heart Infusion and incubated at 37° C. over-night. The bacterial suspension was resuspended in fresh medium and further incubated at 37° C. until reaching the exponential growth phase (1×10.sup.8 CFU/mL). Serial dilutions were performed to determine the bacterial concentration required for the assay (1×10.sup.6 CFU/mL). The test was carried out in sterile 96-well plates in which the extract was diluted in Brain Heart Infusion for a final volume of 100 .Math.l in order to obtain the required concentrations (200 .Math.g/mL - 0.39 .Math.g/mL). Similarly, dilutions were performed for ampicillin, used as a positive control. Subsequently, 50 .Math.l of bacterial culture (5×10.sup.5 CFU/well) were inoculated into each well. The antimicrobial activity of the CSEs was evaluated after 20 hours of incubation at 37° C., by measuring the absorbance at 600 nm, through the TECAN Sunrise microplate reader. The experiments were performed in duplicate and the percentage of inhibition was calculated with respect to the strains not treated with the substance (Franci et al. 2018).

Antiviral Assays

[0091] Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Cell lines supporting the multiplication of RNA viruses were the following: CD4+ human T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATCC CCL 22 (NBL-1) Bos Taurus]; Baby Hamster Kidney (BHK-21) [ATCC CCL 10 (C-13) Mesocricetus auratus], Vero cells (CCL-81; American Type Culture Collection, Manassas, VA, USA)

[0092] Viruses were purchased from American Type Culture Collection (ATCC), with the exception of Human Immunodeficiency Virus type-1 (HIV-1 IIIB laboratory strain) obtained from the supernatant of the persistently infected H9/IIIB cells (NIH 1983); SARS-CoV-2 [Clinical isolate]; bovine viral diarrhoea virus (BVDV) [strain NADL (ATCC VR-534)]. The HSV-1 and HSV-2 viruses belong to the Herpesviridae family, the Alphaherpesvirinae subfamily, and the Simplexvirus genus. They express the gene for β-galactosidase, under the control of the cytomegalovirus IE-1 promoter and have been propagated as reported in the literature (Franci et al. 2017).

Viral Activity Assays

[0093] HSV-1 and HSV-2: To evaluate the effect of the CSE on the inhibition of HSV infectivity, a co-treatment experiment was performed, in which cells (4×10.sup.5/multiwell 12 well) were incubated, simultaneously, with both CSE at different concentrations (200, 100, 50, 20, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1 and 0.05 .Math.g/mL) and with the virus (10.sup.3 PFU/mL) for 1 hour at 37° C./5% CO.sub.2. After the incubation hour, time required to allow viral adsorption, the non-penetrated virus was inactivated with citrate buffer (pH 3.0). Subsequently, the cell monolayer was washed with 1X Phosphate Buffer Saline (PBS) and incubated for 48 hours in MEM supplemented with carboxymethylcellulose. After two days, the cells were fixed and stained with 0.5% crystal violet, and the plaques were counted. The experiments were performed in duplicate and the percentage of viral inhibition was calculated with respect to the HSV-1 and HSV-2 control not treated with the substance. We initially evaluated, by means of co-treatment experiments, the inhibitory capacity of this extract on HSV-1. The cells were incubated with different concentrations of the substance (200, 100, 50, 20, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1 and 0.05 .Math.g/mL) in the presence of the virus (10.sup.3 PFU/mL) for 1 hour at 37° C. / 5% CO.sub.2. After the incubation hour, time required to allow viral adsorption, the non-penetrated virus was inactivated with citrate buffer (pH 3.0). Later, further experiments were carried out to understand whether CSEs acted in an intracellular or extracellular phase of infection. To assess whether they inhibited viral replication and therefore acted inside the cell, a post-treatment assay was set up: the cells were first infected with HSV-1 virus (10.sup.3 PFU/mL) for 1 hour at 37° C. / 5% CO.sub.2. After one hour of incubation, the non-penetrated virus was inactivated with citrate buffer (pH 3.0) and the cell monolayer was washed with 1X Phosphate Buffer Saline (PBS). We continued by adding the extract to the cells at different concentrations (200, 20, 2 and 0.2 .Math.g/mL) and incubating for a further hour at 37° C. / 5% CO.sub.2 . To evaluate, instead, if the extracts acted in an extracellular phase, cell pre-treatment and virus pre-treatment experiments were carried out. Cell pre-treatment evaluates whether the substance acts on cells, for example by masking a binding receptor for the virus. In this experiment, the cells are first treated with substances at different concentrations (200, 20, 2 and 0.2 .Math.g/mL) for 1 hour at 4° C.: this temperature allows the substance to interact with the cells, but not to enter them. After the incubation time, the cells are infected with the virus (10.sup.3 PFU/mL) and incubated for 1 hour at 37° C. / 5% CO.sub.2. Virus pre-treatment, on the other hand, indicates whether CSEs act directly on the virus. In this experiment, the extract at different concentrations (200, 100, 50, 20, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1 and 0.05 .Math.g/mL) is incubated together with the virus (10.sup.4 PFU/mL) for 1 hour at 37° C. Subsequently the mixture is diluted and added on the cells for 1 hour at 37° C./5% CO.sub.2. For all experiments, at the end of the treatment, the cell monolayer was washed with 1X PBS and incubated for 48 hours in MEM supplemented with carboxymethylcellulose. After two days, the cells were fixed and stained with crystal violet and the plaques were counted. The percentage of viral inhibition was calculated with respect to the HSV control not treated with the substance.

[0094] HIV: CSE activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 .Math.L of RPMI containing 1 × 10.sup.4 MT-4 cells were added to each well of flat-bottom microtitre trays, containing 50 .Math.L of RPMI without or with serial dilutions of test compounds. Then, 20 .Math.L of a HIV-1 suspension containing 100 CCID50 were added. After a 4-day incubation at 37° C., cell viability was determined by the MTT method.

[0095] BVDV: CSE activity against BVDV was based on inhibition of virus-induced cytopathogenicity in BHK-21 and MDBK cells, respectively, acutely infected with a m.o.i. of 0.01. Briefly, BHK-21 and MDBK cells were seeded in 96-well plates at a density of 5 × 10.sup.4 and 3 × 10.sup.4 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37° C. in a humidified CO.sub.2 (5%) atmosphere. Cell monolayers were then infected with 50 .Math.L of a proper virus dilution in maintenance medium [MEM-Earl with L-glutamine, 1 mM sodium pyruvate and 0.025 g/L kanamycin, supplemented with 0.5% inactivated FBS] to give an m.o.i of 0.01, without or with serial dilutions of test compounds. After a 3-day incubation at 37° C., cell viability was determined by the MTT method (Pauwels et al. 1988).

[0096] SARS-CoV-2 assays: SARS-CoV-2 field isolated was obtained from the University of Study of Campania Luigi Vanvitelli, Italy and propagated in Vero E6 cells. Virus stocks were generated from clarified cell culture supernatants harvested 3 or 4 days post inoculation. Virus titer and inhibition were determined by plaque assay in Vero E6 cells grown in six-well tissue culture plates (https://mbio.asm.org/content/11/5/e01935-20.long#sec-8).

Cytotoxicity Assays

[0097] Cytotoxicity assays were run in parallel with antiviral assays.

[0098] Exponentially growing MT-4 cells were seeded at an initial density of 1 × 10.sup.5 cells/ml in 96-well plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G and 100 .Math.g/mL streptomycin. Cell cultures were then incubated at 37° C. in a humidified, 5% CO.sub.2 atmosphere, in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 96 hrs at 37° C. by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method (Pauwels et al. 1988).

[0099] MDBK cells were seeded in 96-well plates at an initial density of 6 × 10.sup.5 and 1 × 10.sup.6 cells/mL, respectively, in Minimum Essential Medium with Earle’s salts (MEM-E), L-glutamine, 1 mM sodium pyruvate and 25 mg/L kanamycin, supplemented with 10% horse serum (MDBK). Cell cultures were then incubated at 37° C. in a humidified, 5% CO.sub.2 atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 72 hrs at 37° C. by the MTT method. The use of flow cytometry made it possible to evaluate the effect of CSE on the cell cycle. Vero cells (1 × 10.sup.6) were plated in 6-well multiwells and incubated the next day with the substance at the same concentrations used in the antiviral assays. After one hour, the cells were trypsinized and centrifuged, and the cell pellets were resuspended in a solution containing RNase A, propidium iodide (50 .Math.g/mL), sodium citrate (0.1%) and Np40 (0.1%) in 1X PBS for 30 min in the dark. The analysis was performed at the FACScalibur flow cytometer (Becton Dickinson) as described previously (Franci et al. 2018).

Antifungal Assays

[0100] The antifungal activity was performed using the plate microdilution method, according to the EUCAST guidelines. The test was carried out in sterile 96-well plates in which the CSE was diluted in the growth medium (RPMI-1640 with the addition of 2% glucose and 3- (N-morpholino)-propanesulphonic acid at the concentration of 0.165 mol/L) for a final volume of 200 .Math.l in order to obtain the required concentrations (800 .Math.g/mL - 0.39 .Math.g/mL). Similarly, dilutions were performed for amphotericinB, used as a positive control. The fungal suspension used for the assay is prepared by resuspending colonies of Candida albicans (ATCC 90028), previously obtained from a 48 hour culture in Sabouraud Dextrose Agar medium, in sterile distilled water until reaching 0.5 McFarland. Serial dilutions were performed to determine the fungal concentration required for the assay (2.5×10.sup.5 CFU/mL). Subsequently, 100 .Math.l of fungal culture (1.25×10.sup.5 CFU/well) was inoculated into each well. The antifungal activity of CSEs was evaluated after 48 hours of incubation at 37° C., by measuring the absorbance at 600 nm, through the TECAN Sunrise microplate reader. The experiments were performed in duplicate and the percentage of inhibition was calculated with respect to the strains not treated with the substance (Cuenca-Estrella et al. 2003).

Antitumor Assays

[0101] NB4 tumor line [DSMZ, ACC 207], acute promyelocytic leukemia, is propagated in RPMI 1640 medium containing 4.5 g/L supplemental glucose (Euroclone) with 10% bovine fetal serum (FBS) (Euroclone), 100 U/mL penicillin-streptomycin (Euroclone) and 2 mM L-glutamine (Euroclone). Cell cycle analysis: cells were plated at a confluence of 2 × 10.sup.5 cells/mL and induced for 30h with different concentrations of the substance (400, 200, 100, 50, 25, 12.5 and 0.78 .Math.g/mL). They were then collected, centrifuged at 1200 rpm for 5 min and resuspended in a solution containing 1 × PBS, 0.1% sodium citrate, 0.1% NP40 and 50 mg/mL propidium iodide. After 30 min of incubation at room temperature, in the dark, the cell cycle is evaluated by FACS (FACSCalibur BD Biosciences, San Jose, CA, USA) and analyzed by ModFit v3 software (Verity Software House). The Pre-G1 phase was instead analyzed with CellQuestPro software (Becton Dickinson) (Lenoci et al. 2014; Franci et al. 2018).

RESULTS

[0102] Before performing the cell tests, the right concentrations of CSE to be used were determined by analyzing cell cytotoxicity. Although a concentration curve was used to reach 200 .Math.g/mL, we did not record changes in the cell cycle and in the increase in the Pre-G1 phase (death) of the cells treated for 24 h with the extract on Vero Cells (FIG. 1A). Other cell lines were tested for the toxic effect of CSE. Among them, MT-4 and MDBK were tested with a metabolic assay called MTT, values close to 100 .Math.g/mL were still not toxic (See Tables 1-3). A complete panel of assays to evaluate the microbiological inhibitory activity of CSE was performed in biological duplicate and in technical triplicate. The results demonstrated that there was no interference with the growth of Gram- and Gram + bacteria even at the highest concentrations tested (200 .Math.g/mL). Negative results were also acquired in response to C. albicans treatment (not showed). Unlike bacteria and fungi, treatment with CSE on viral models such as HSV-1, HSV-2, HIV-1, SARS-CoV-2 showed strong antiviral activity.

[0103] As can be seen from the experimental data obtained, the CSE shows a very strong inhibition of the infection of both HSV-1 and HSV-2 in the co-treatment experiments (FIGS. 1B and 1F) and an even more intense inhibition in the viral pre-treatment experiments (FIGS. 1C and 1G). The data indicate that in the cell pre-treatment the extract is no longer active, as well as in the post-treatment assay (FIGS. 1D and 1E). All the results obtained open a window on the mechanism of operation of CSE in the interference of viral infection: CSE works in an extracellular phase of HSV infection, and, more precisely, they act directly on the virus. We can hypothesize that the CSE interacts/binds a glycoprotein of the viral envelope, masking it from attack with its cell receptor and, therefore, preventing the attack on the target cell and its subsequent infection. The doses required to achieve 100% inhibition of viral replication are 2 .Math.g/mL on HSV-1 in the co-treatment and 10 times less in the viral pre-treatment. For HSV-2, viral inhibition by CSE is reduced by 10 times: the concentrations required to block viral replication by 100% are 20 .Math.g / mL in the co-treatment and 2 .Math.g / mL in the virus pre-treatment. Such low doses and such high efficiency offer the possibility of using this extract in the treatment of HSV-1 and 2 infections with large margins of success.

[0104] To check whether the antiviral activity of CSE had a broader spectrum, further experiments were carried out on other virus models. CSE efficacy in infection mediated by the human immunodeficiency virus (HIV), responsible for acquired immunodeficiency syndrome (AIDS), was evaluated. It is a retrovirus of the lentivirus genus, which is divided into two strains: HIV-1 and HIV-2. The first of the two is mainly located in Europe, America and Central Africa; HIV-2, on the other hand, is found mostly in West Africa and Asia and results in a clinically more moderate syndrome than the previous strain. Antiviral treatments were conducted on H9/IIIB cells. The virus used (HIV-1 IIIB laboratory strain) was obtained from the supernatant of persistently infected H9/IIIB cells. The CSE was able to inhibit at least at 50% the virus replication at about 13.5 .Math.g/mL. EFV and T20, two well known drugs able to inhibit HIV-1 replication were used as positive controls (Wang et al. 2011) (Table 1).

[0105] The CSE also inhibited at 50% the replication of SARS-CoV-2 at a value of 6.05 .Math.g/mL. For these experiments, the peptide EK1 was used as control, as published in the paper by Xia et al. (2020) (Table 2).

[0106] No effect on replication of BVDV was achieved by CSE even at higher concentrations. For the BVDV trials, the reference drug NM108 was used as control (Tonelli et al. 2011) (Table 3).

[0107] It is really interesting how the CSE is able to inhibit some specific virus families and it is not active on all the viruses tested. This leaves the potential application of CSE in a specific virus category.

TABLE-US-00002 Cytotoxicity and in vitro activity of chestnut’s shells extract against HIV-1 Extracts Cytotoxicity in MT-4 cells In vitro efficacy EC.sub.50.sup.b[.Math.g/mL] CC.sub.50.sup.a[.Math.g/mL] HIV-1.sup.b 40 mg/mL 97.8 13 20 mg/mL 96.0 14 Reference CC.sub.50.sup.a HIV-1.sup.b *EFV 40.0 0.002 T-20 (.Math.g/ml) >100 0.017 .sup.aCompound concentration (.Math.g/mL) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method. .sup.bCompound concentration (.Math.g/ml) required to achieve 50% protection of MT-4 cells from the HIV-1 induced cytopathogenicity, as determined by the MTT method. *.Math.M

TABLE-US-00003 Cytotoxicity and in vitro activity of chestnut’s extract against Coronaviridae Extracts Cytotoxicity in VERO cells In vitro efficacy EC.sub.50.sup.b [.Math.g/mL] CC.sub.50 .sup.a[.Math.g/mL] SARS-CoV-2.sup.b t 40 mg/mL >500 6.2 20 mg/mL >500 5.9 CC.sub.50.sup.a SARS-CoV-2.sup.b *EK1 >100 5.4 .sup.aCompound concentration (.Math.g/mL) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method. .sup.bCompound concentration (.Math.g/mL) required to achieve 50% protection of VERO cells from SARS-CoV-2. *.Math.M

TABLE-US-00004 Cytotoxicity and in vitro activity of chestnut’s extract against BVDV Extracts Cytotoxicity in MDBK cells In vitro efficacy EC.sub.50.sup.b [.Math.g/mL] CC.sub.50.sup.a [.Math.g/mL] BVDV.sup.b 40 mg/mL 106.5 >106.5 20 mg/mL 109.0 >109.0 Reference CC.sub.50.sup.a BVDV.sup.b *NM 108 >100 1.6 .sup.aCompound concentration (.Math.g/mL) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method. .sup.bCompound concentration (.Math.g/mL) required to achieve 50% protection of MDBK cells from BVDV. *.Math.M

[0108] To confirm that the antiviral activity was really ascribed to CSE, regardless of the origin of the shells (obtained by drying, and called Dried, or by burning, and indicated as Burned), the effectiveness of the extracts from the two different types of shells was compared. Both extracts led to the same results, with an inhibition of HSV-1 infection, at a concentration of 2 .Math.g/mL, close to 100% for the extract from Burned shells (FIG. 2A) and equal to 100% for the extract from Dried shells (FIG. 2B).

[0109] Furthermore, to confirm that the antiviral activity observed depended on the extract, regardless of the type of shells (Burned or Dried), and also of the type of extraction, CSEs produced with a method other than boiling were analyzed.

[0110] In FIG. 3, CSEs of different origin and prepared by different extraction and treatment methods were compared. In particular, sample 1 (FIG. 3A) refers to an extract obtained from Burned shells by Naviglio method (Naviglio extractor works at high pressure and moderate temperature); sample 2 (FIG. 3B) indicates an extract obtained from Dried shells by Naviglio extraction; sample 3 (FIG. 3C) refers to an extract prepared from Dried shells by boiling. As can be seen, all the three extracts show a trend in the inhibition of viral replication comparable to the reference extract. This indicates that the antiviral activity is independent not only of the type of extraction method used, but also of the origin of the extracts. Finally, even the sterilization process of the CSE, by filtration or passage in an autoclave, does not affect the antiviral power, when compared to the untreated extract.

EXAMPLE 2

[0111] The following composition was prepared and tested on volunteers with a clinical history of injury due to HSV-1 infections of at least 5 years with at least two events / year:

TABLE-US-00005 1) Chestnut shells freeze-dried 0.20% p/p 2) Glycerine 5.00% p/p 3) Polyacrylamide 1.40% p/p 4) Isoparaffin 0.70% p/p 5) Laureth 7 0.19% p/p 6) 2-phenoxyethanol 0.10% p/p 7) 3-(2-ethylhexyloxy)propane-1,2-diol 0.10% p/p 8) Purified water just enough to100 g Check the pH.

[0112] Indications to 20 volunteers about the use of the preparation were the following: apply as soon as the typical tingling that indicates the appearance of the lesion is felt on the lip, apply at least 3 times a day. All the volunteers, with the exception of one, confirmed the effectiveness of the preparation in preventing the onset of the lip lesion. This efficacy was in some cases higher than that of commonly used antiherpetic drugs.

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