WO2023006662A1

WO2023006662A1 - Skin care comprising saponin-containing quillaja extract

Info

Publication number: WO2023006662A1
Application number: PCT/EP2022/070786
Authority: WO
Inventors: Amandine Scandolera; Romain Reynaud
Original assignee: Givaudan Sa
Priority date: 2021-07-29
Filing date: 2022-07-25
Publication date: 2023-02-02
Also published as: GB202110902D0

Abstract

A hand care composition comprising a virucidally-effective proportion of a saponin- containing quillaja extract. The composition is particularly effective against coronaviruses, including the SARS-CoV-2 virus.

Description

SKIN CARE COMPRISING SAPONIN-CONTAINING QUILLAJA EXTRACT

This disclosure relates to hand care compositions, and more particularly to such compositions containing certain active saponins.

Hand care compositions in the form of creams and lotions are widely used for the protection and treatment of hands, especially those exposed to harsh environments, such as frequent washing. They provide protective, moisturising and nourishing effects. The basic formulations are well known, and will vary according to the desired function - protective compositions generally have a more oily constitution to repel water, whereas moisturizing and skin repair creams are formulated to be absorbed more readily into the skin better to provide desirable ingredients.

In recent times, there has been considerable concern about the more frequent of zoonotic viruses, against which humans have no natural resistance. The most recent of these, SARS-CoV-2, has reached a pandemic level, which resulted in widespread disruption of normal activities. The so-called delta-variant is of particular concern, because of its extreme contagiousness. There is therefore an interest in preventing the spread of this virus.

One of the essential recommendations has been the frequent washing of hands, as the surfactants used in soap can neutralize the virus by disrupting the fatty layer surrounding the virus. However, there remains the possibility that the virus can be picked up on hands after washing.

It has now been found that certain natural materials used in hand care compositions are not only effective against viruses, including the SARS-CoV-2 virus, but also do not cause irritation. There is therefore provided a hand care composition comprising a virucidally- effective proportion of a saponin-containing quillaja extract.

There is additionally provided a method of neutralising viruses on the hands, comprising the application of a hand care composition comprising a viricudally-effective proportion of a saponin-containing quillaja extract.

There is further provided the use of a saponin-containing quillaja extract in the neutralization of viruses on the hands. Quillaja extract is obtained from the tree Quillaja saponaria Molina, native to Chile. The extract is rich in saponins and has found a number of uses in the food industry, particularly as natural surfactants, an important consideration in a business where natural products have become more important.

It is surprising to find that it provides long-term protection against viruses, particularly the SARS-CoV-2 virus, on the hands when incorporated into a hand care composition.

It has been found that exposure to a hand care composition as hereinabove described produces a 4 log reduction in virus content, particularly coronavirus content, after 2 minutes’ exposure.

Saponin-containing quillaja extracts are well-known items of commerce, and any such extract may be used. The proportion of saponins may vary, depending on the extraction treatment, and in a particular embodiment, the extract contains at least 65% by weight saponins on a dry basis.

The extract may be incorporated into a hand care composition by simply blending it with the other ingredients, all of which are standard ingredients and all of which may be added in art-recognised proportions. The proportion of quillaja extract used will be that able to provide a suitable degree of virucidal effect. This will depend on the degree of virucidal effect desired, but typical proportions in a hand cream are from 1-5% by weight, more particularly from 1-2%, even more particularly from 1.8-2% by weight of the composition.

An additional advantage of the use of saponins is that it is not a skin itrritant, and that therefore the presence of the composition on the hands for an extended time is no problem.

Laboratory testing has demonstrated a 4 log reduction in coronavirus, including the SARS-CoV-2 virus in 2 minutes, compared with oral care compositions lacking the extract.

The disclosure is now further described with reference to the following examples, which are purely exemplary, and which are not intended to be in any way restricting on the scope of the disclosure. Example 1

Basic hand cream formulation Component A % by weight Xyliance™* 4

Capric triglyceride 2 Stearic acid 3 Dimethicone 0.5

Component B

Water to 100%

Glycerol 8

Sodium hydroxide (30.5%) 0.19 p-anisic acid 0.5

Component C

Uniprosyn™ PS 18^** 3 Component D

BisaboLife™*** 0.2

Component E

Tocopherol 0.1

Component F

Water 2

Component G Fragrance 0.1

*A commercially-available emulsifier, ex Givaudan

**A commercially-available cosmetic ingredient, ex Givaudan

*** (-)-a-bisabolol ex Givaudan

This formulation is referred to as Formulation X. Two further formulations, Y and Z, were prepared. In Formulation Y, the 2% water of Component F is replaced with 0.1 % benzalkonium chloride (a well-known antimicrobial compound active against a wide variety of bacteria, yeasts and fungi) and 1.9% water, and in Formulation Z by 2% Sapnov™, a commercially-available blend of quillaja extract, sodium benzoate and phosphoric acid, and containing 65% by weight quillaja saponins.

The creams were prepared as follows:

(i) Components A and B were prepared separately and heated with gentle stirring to 80°C.

(ii) Component B was then added slowly to Component A under high speed stirring, which was continued for a few minutes.

(iii) The mixture was allowed to cool to room temperature under gentle stirring.

(iv) The temperature was raised to 35°C and Components C, D, E and F were added in order with gentle stirring.

The product was a cream having a viscosity of 30420 mPa.s (Brookfield DVIII Ultra, Spindle E, speed 12 at 20°C) and a pH of 5.54.

Example 2

Testing of Example 1 hand creams

TEST METHOD

1 ASSAY SYSTEM

Virus

Bovine Coronavirus (BCoV) RVB-0020

This commercially-available bovine coronavirus is accepted as an equivalent of the SARS- CoV-2 virus.

The virus was kept at <-196°C in liquid nitrogen; before use it was multiplied in the appropriate cellular line as shown in the table below:

2. CELLS, MEDIA AND REAGENTS

Cellular culture PT (calf kidney cells) CCLV-RIE 11

The cellular line was kept at <-196°C in liquid nitrogen; before viral inoculum, it appeared as confluent monolayer. The cell debris was removed by centrifugation (400 g_N for 15 minutes), and the supernatant containing the virus was used for the test (test virus suspension).

Culture Medium and reagent

EMEM Eagle’s Minimal Essential Medium DMEM Dulbecco’s Modified Eagle Medium FBS Foetal Bovine Serum

Pen-Strep (1%) Antibiotics PBS Phosphate Buffer Saline

Trypsine-Edta BSA Bovine Serum Albumin

Sheep Blood Trypan Blue Water for injection Growth Medium for cell multiplication: DMEM supplemented with 10% FBS. Maintenance Medium for virus propagation: EMEM supplemented with 2% FBS.

Interfering substances (dirty conditions for the standard method)

Bovine albumin solution (BSA) plus sheep erythrocytes with a final concentration 10 times higher than the final concentration of 0.3% (simulating dirty conditions for the medical area): bovine albumin 6 g water for injection 194 ml sterilized through a 0.45 mhi. filter

16 ml of defibrinated sheep blood have been centrifuged at 800 gn for 10 min; the supernatant was discarded and the erythrocytes were re-suspended in PBS. The procedure has been repeated until the supernatant was clarified.

6 ml of erythrocytes has been re-suspended in 194 ml of bovine albumin solution prepared as described above.

Interfering substances (dirty conditions for the modified method) Bovine albumin solution (BSA) plus sheep erythrocytes with a final concentration 50 times higher than the final concentration of 0.3% (simulating dirty conditions for the medical area): bovine albumin 3.8 g water for injection 21.2 ml sterilized through a 0.45 mhi. filter

10 ml of defibrinated sheep blood have been centrifuged at 800 g_N for 10 min; the supernatant was discarded and the erythrocytes were re-suspended in PBS. The procedure has been repeated until the supernatant was clarified.

3.8 ml of erythrocytes has been re-suspended in 21.2 ml of bovine albumin solution prepared as described above.

3. EXPERIMENTAL DESIGN

Test temperature The test was performed at 20°C± 1 °C.

Experimental conditions

The test was performed at the following conditions:

- final concentrations: 97% (maximum testable concentration in the modified method) - 50% - 5% in test

- contact time: 2 minutes

Interfering substance

This test was conducted using as interfering substance a solution of bovine albumin plus sheep erythrocytes with a final concentration of 0.3% (simulating dirty conditions for the medical area). 4 EXECUTION OF THE ASSAY

Preliminary evaluation of the test product cytotoxicity level Prior to testing, the product test solutions, the interfering substance, and the water were previously stabilised at the test temperature. Assuming a likely level of cytotoxicity of the test item, two cytotoxicity tests were conducted in order to permit the evaluation of a logarithmic reduction according to the requirements of acceptability criteria of the test: one with the unfiltered test item and one with the test item filtered with the S400 HR columns MicroSpin™.

For each test concentration, a cytotoxicity test was carried out by mixing 8 ml of the product test solution with 1 ml of water for injection and 1 ml of interfering substance two times so one test mixture was treated with filtration technique and the other one not.

For the modified method, a cytotoxicity test was carried out by mixing 9.7 ml of the product test solution with 0.1 ml of water for injection and 0.2 ml of interfering substance two times so one test mixture was treated with filtration technique and the other one not. Then serial decimal dilutions were performed with Maintenance Medium. 0.1 ml of each dilution of the test item was put on in 96 wells microplates in six replicates. In parallel, at least 6 wells in the microplate did not receive the viral inoculum, but only ice-cold maintenance Medium and they were used as control of cellular line. After 1 hour of incubation at 37°C ± 1°C, 0.1ml of Maintenance Medium was added to each well. The cellular cultures were observed with inverted microscope to detect any cytopathic effect (CPE) due to test item.

On the basis of the preliminary results, the filtration procedure with S400 HR columns MicroSpinTM was not applied for the complete test. For highly cytotoxic test product concentration, the Large-Volume-Plating (LVP) method has been applied.

Using the LVP, the lowest apparently non-cytotoxic dilution of the test mixture was added to ice-cold medium after the required contact time and this mixture was added to a defined number of wells containing the cell line in 0.1 ml of maintenance medium. The assay for checking the cytotoxicity of the test item has been repeated in parallel with the complete test according to the procedure above described for the preliminary check.

Prior the testing, the product test solutions, the interfering substance, and the water were previously stabilised at the test temperature. The test suspension was kept in the ice bath to avoid titre loss.

Assay of viral activity (virus titration)

A virus titration was performed for the virus suspension as such, then serial dilutions 1:10 were prepared, starting from virus stock solution of viral suspension, with culture Medium. 0.1 ml were put six-fold on a 96-wells microplate containing the cellular confluent monolayer (>90%) without culture Medium. The outline of the microplate did not receive the viral inoculum and were used as control of cellular line.

After 1 hour of incubation at 37°C ±1 °C, 0.1 ml of culture Medium were added to viral inoculum.

After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopathic effect (CPE) due to viral suspension. After this detection the infecting activity (TC I D₅o evaluation) was calculated by means of Spearman- Karber method.

Preparation of the test item

The test item was used neat and diluted with water for injection to concentrations requested in the assay. Test item dilutions were prepared at a concentration 1.25 times higher than the concentration required to perform the test.

Check of cellular sensitivity to virus

To verify if the test item modifies the cellular sensitivity to viral infection, these procedure was followed: 0.1 ml of each dilution, prepared for the cytotoxicity check, for each test concentration was put on 96 wells microplates containing the cellular confluent monolayer; the other microplate in parallel were treated with 0.1 ml of PBS.

After 1 hour of incubation at 37°C ± 1°C, the test item and PBS were removed and 0.1 ml of each dilution of the viral test suspension were added six-fold. In parallel at least 6 wells in the microplate did not receive the viral inoculum, but only ice-cold maintenance Medium and used as control of cellular line. After 1 hour of incubation at 37°C ±1°C 0.1 ml of culture Medium were added to the viral inoculum. After 5 days, the cellular culture was observed with inverted microscope to detect any cytopathic effect (CPE) due to viral suspension. After this detection the infecting activity (TCID50) was calculated by means of Spearman-Karber method both in the cellular culture treated with the test item and in that treated with PBS.

Check of suppression of disinfectant activity

For each test concentration, 1 part of interfering substance was added to 1 part of Maintenance Medium and to 8 parts of the product test solution at the required test concentrations.

For the modified method, 0.2 parts of interfering substance were added to 0.1 part of Maintenance Medium and to 9.7 parts of the product test solution (97%).

At time zero, 0.3 ml was withdrawn and pipetted into 2.4 ml of ice cold Maintenance Medium. Then 0.3 ml of virus test suspension was added to the previous mixture, and after mixing, the tube was incubated in the ice bath for 30 minutes ± 10 sec.

After this period, serial dilutions 1:10 with ice cold Maintenance Medium were performed. 0.1 ml of the each dilutions was transferred onto 96 wells microplates six-fold and incubated at 37°C ±1°C for 1 hour. Then, 0.1 ml of Maintenance Medium was added. In parallel at least 6 wells in the microplate did not receive the viral inoculum, but only ice-cold maintenance Medium and used as control of cellular line. After the appropriate incubation period, the cellular cultures were observed with inverted microscope to detect any cytopathic effect (CPE) due to viral suspension. After this detection, the infecting activity (TCID50) was calculated by means of Spearman-Karber method.

Check of viral inactivation (Test) and virus control vitality (for all test product concentrations)

For each concentration, 1 ml of interfering substance was mixed with 1 ml of virus test suspension, then vortexed. 8 ml of the product test solution was added to this solution and left in contact for the required contact times at the required test temperature. For the modified method, 0.2 ml of interfering substance was mixed with 0.1 ml of virus test suspension, then vortexed. 9.7 ml of the product test solution (97%) was added to this solution and left in contact for the required contact time at the required test temperature.

After the contact time, serial decimal dilutions with culture Medium were performed. 0.1 ml of this solution were put on a 96-wells microplate containing the cellular confluent monolayer. The outline of the microplate did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37°C ±1°C 0.1 ml of culture Medium were added to the viral inoculum.

After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopathic effect (CPE) due to viral suspension.

In a second time the same test described above, was performed with water for injection instead of the test item, after 0 and maximum contact time tested (virus control).

After this detection the infecting activity (TCID50) was calculated by means of Spearman- Karber method both in the cellular culture treated with the test item and in the virus control.

Determination of the residual virus titre by the Large Volume Plating (LVP) method (only for 97%)

0.2 ml of interfering substance was mixed with 0.1 ml of virus test suspension, then vortexed. 9.7 ml of the product test solution (97%) was added to this solution and left in contact for the required contact time at the required test temperature.

After the contact time, the appropriate volume of the mixture was diluted in ice-cold culture medium to the lowest apparently non-cytotoxic dilution determined in the preliminary check. 0.1 ml of this solution were put on a defined number of 96-wells microplates containing the cellular confluent monolayer.

The outline of the microplate did not receive the viral inoculum and were used as control of cellular line. After 1 hour of incubation at 37°C ±1°C 0.1 ml of culture Medium were added to the viral inoculum.

After the appropriate incubation period, the cellular culture was observed with inverted microscope to detect any cytopathic effect (CPE) due to viral suspension. The desired test result was a 4 log reduction (99.5%) reduction in virus after 2 minutes’ exposure. Only Composition Z achieved this, Composition X achieving 1.83 log and Composition Y 1.56 log.

Claims

Claims:

1. A hand care composition comprising a virucidally-effective proportion of a saponin- containing quillaja extract.

2. A hand care composition according to claim 1, in which the virus comprises coronavirus.

3. A hand care composition according to claim 2, in which the coronavirus comprises the SARS-CoV-2 virus.

4. A hand care composition according to claim 1, in which the virucidal effectiveness is such that there is a 4 log reduction in virus content after 2 minutes’ exposure.

5. A hand care composition, in which the saponin-containing quillaja extract is present in the composition in the weight proportion of from 1-5% by weight, more particularly from 1-2%, even more particularly from 1.8-2%.

6. A method of neutralising viruses on the hands, comprising the application of a hand care composition comprising a viricudally-effective proportion of a saponin- containing quillaja extract.

7. Use of a saponin-containing quillaja extract in preparation of an oral composition for the neutralization of viruses on the hands.

8. Use according to claim 8, in which the virus is a coronavirus, particularly the SARS-CoV-2 virus.