WO2014170740A2

WO2014170740A2 - New cosmetic combination

Info

Publication number: WO2014170740A2
Application number: PCT/IB2014/000555
Authority: WO
Inventors: Giuseppe Robin VARIATI
Original assignee: Variati S.R.L.
Priority date: 2013-04-16
Filing date: 2014-04-16
Publication date: 2014-10-23
Also published as: WO2014170740A3; ITMI20130619A1

Abstract

The invention relates to a novel association with bacteriostatic/bactericide/anti- inflammatory activity and its cosmetic usage in formulations having deodorizing action, in form of sprayable solution and in form of emulsion, formulations in form of cream, in form of emulsion, formulations in form of products for intimate hygiene, in form of products intended for the treatment of acne, in form of after sun, after shave and post-depilation products.

Description

"NEW COSMETIC COMBINATION"

OBJECT OF THE INVENTION

The present invention relates to an association comprising at least one compound with soothing, anti-inflammatory, antibacterial action derived from natural sources, and in particular to the usage of the association for the preparation of soothing creams and solar products in general, in particular after-sun products, creams with generic anti-inflammatory action, deodorants, anti-acne creams, products for intimate hygiene and the like.

STATE OF THE ART

Recently considerable attention has been addressed to a plant known as "Mangosteen" {Garcinia mangostana L.) whose fruit is commonly called Mangosteen and as such is indicated herein below. Said plant is found to be particularly interesting for the following reasons. It is known that its fruits contain substances characterized by various activities, among which the antibacterial and anti-inflammatory ones. It is also known that its fruits are not toxic but, conversely, they are eaten in large quantities as a dessert in Far East countries.

The victualling of the raw material (whole fruits, parts thereof or extracts) is not a big problem, also because powder extracts containing the active compounds to be used as such or following further processing (e.g. isolation of desired active compounds in a purified form) are currently available on the market.

Garcinia mangostana (Guttiferae) is a tree 6 to 20 meters tall and is cultivated in Indonesia, Thailand, India and China wet tropical areas. The fruit, Mangosteen, is eaten in large quantities as a dessert for its sweet and delicate taste. It is known that some substances are contained in the fruit which are responsible for a number of biological and pharmacological activities. Some of them are taken as an advantage by the folk medicine, but only on few of them have been conducted in-depth studies. Among the biological properties experimentally tested, an antibacterial and antifungal activity is known. Such activities are all related to compounds of molecular structure very similar to each other and belonging to a single biogenetic family, i.e. the one of xanthones:

Four well distinct parts can be distinguished in the fruit:

a calyx formed by four rounded leaves, thick and robust (hulls) a very robust peel (rind)

a juicy pulp which encloses the seeds and is called aril 5 to 7 big seeds

Xanthones are not evenly distributed among the various parts of the fruit but they are found particularly abundant in the leaves, which constitute the calyx.

From various parts of the Garcinia mangostana fruit about 16 xanthones have been extracted. In structure of their molecule the xanthone core, consisting of three to six- member rings, is well recognizable. The differences among the compounds of the same structural and biogenetic family are represented by the substituents on the two side rings, i.e. by the hydroxyl groups, prenyls, modified prenyls and methoxy groups.

The prenyl group is defined, according to the present invention, as a group of (CH₃)₂- C=CH-CH₂- structure.

Some extraction methods of the active compounds contained in the fruits of Garcinia mangostana are mentioned in literature (water, water-methanol, ethyl acetate, succession of increasing polarity solvents, etc.) but their usage has so far only been finalized to the research of novel compounds, and extraction processes of the active compounds on industrial scale are not known.

The active compounds present in Mangosteen are known to have antibacterial/ antimicrobial/ antifungal/ anti-inflammatory activity. They are also known to have anti-tumor properties, as inducers of apoptosis, with anti-proliferative effect demonstrated in cases of breast tumors, leukemias, liver tumors.

They are also known to influence the cyclooxygenase and, consequently, the formation of prostaglandins, with consequent anti-inflammatory action.

Their generic anti-tuberculous activity is also known.

OBJECTS OF THE INVENTION Objects of the present invention is to provide an association that may be formulated as a cosmetic and having optimal stability over time, in addition being able to be used as an antibacterial /antimicrobial/ anti-inflammatory agent.

Still object of the present invention is to provide an association that can be used as an ingredient in different typologies of cosmetic products.

Also object of the present invention is to provide an association that may be easily prepared and that is mainly based on natural origin products.

Still object of the present invention is to provide available an association that may be easily prepared on industrial scale and that is economically advantageous.

DESCRIPTION OF THE INVENTION

These and still other objects and related advantages that will be better clarified from the following description, are achieved by an association consisting of at least 80% pure mangosteen extract, preferably 90% pure and 1,3-propanediol of plant origin. Such an association is advantageously used in cosmetic formulations comprising additives and excipients of known type and which may vary according to the final usage of said cosmetic formulation. In practice, the association according to the present invention represents the "active compound" that characterizes the cosmetic formulation still according to the invention.

In particular, the 90% mangosteen extract is prepared by a process that is also object of the present invention, whereas the 1,3-propanediol of plant origin is of various origins. For example, a type of 1,3-propanediol of plant origin, that can advantageously be used according to the present invention, is that currently marketed under the trademark Zemea™ by Dupont Tate & Lyle Bio Products.

Zemea 1,3-propanediol is a colorless liquid and highly pure derivative from a corn fermentation process. It is a highly conservative process from the environmental protection stand point that allows providing a product of pure plant origin that shows a number of advantages from its usage stand point, primarily in cosmetics and pharmaceuticals sector.

1,3-propanediol of plant origin of different provenience is anyway available on the market, as valid in order to realize the association according to the present invention. The mangosteen extract according to the present invention is differentiating from the extracts according to the known art, that are characterized by an extract coming from the peel of the fruit made by a known process using water and alcohol. Such known process leads to obtaining a standard extract titrated at 40%, as described for example in WO 2006/055688.

On the other hand, the process according to the present invention allows obtaining more purified fractions, up to a 99% extract titrated in alpha and gamma mangosteen, by using solvents such as, for example, ethyl acetate, in order to get to higher concentrations of active compound, equal to about 80-90%.

Hence, the present invention relates to an association of mangosteen extract titrated at concentrations between 40% and 99% active compound and 1,3 -propanediol of plant origin (Zemea) at concentrations between 95% and 99.5%.

According to the invention, an association consisting of mangosteen extract between 0.5% and 1.5% and 1,3-propanediol between 99.5% and 98.5%, respectively, is preferred.

An association, according to the invention, consisting of 1% of 90% mangosteen extract (CAS N.90045-25-3) and 99% of 1,3 -Propanediol of natural origin (Zemea) (CAS N.504-63-2) is particularly preferred.

The percentages of mangosteen extract compared to 1,3-propanediol are expressed in weight compared to the total weight of the association.

Hence, the object of the invention is a novel association with bacteriostatic/bactericide/anti-inflammatory activity and its cosmetic usage in formulations having deodorizing action, in form of sprayable solution and in form of emulsion, formulations in form of cream, in form of emulsion, formulations in form of products for intimate hygiene, in form of products intended for the treatment of acne, in form of after sun, after shave and post-depilation products.

It has surprisingly been observed that the association according to the present invention shows a synergistic effect compared to its single components, when used as an antibacterial/antimicrobial agent. In fact, the activity of the mangosteen extract used alone, towards Gram-positive bacteria, is reported in the following Table 1 BIS: MIC (ppm)

Staphylococcus aureus 1

Krhizophyla 1

Staphylococcus epidermidis 1

B.spizizenii 5

Bacillus cereus 10

Enterococcus hirae 1-5

S.pyogenes 1

The association according to the present invention, tested in the same conditions, showed the results according to the following Table 2 BIS:

Antimicrobial properties of the association according to the present invention were evaluated too, towards some microbial strains isolated from underarms of a group of volunteer subjects.

The association has been evaluated at different concentrations and for two different contact times, respectively 6 hours and 18 hours. The obtained results show a good antimicrobial efficacy already after 6 hours of contact, with an increase of the logarithmic decrease after 18 hours (Figure 1), that suggests a long lasting effect of the deodorizing action.

The isolated strains are the following:

- Staphylococcus epidermidis

- Staphylococcus capitis

One of the advantageous aspects of the present invention is avoiding the usage of solvents and glycols or solubilizing agents derived by petroleum, and hence being able to use components of completely natural origin. Moreover, the utilization of the concentrated fraction obtained from the Mangosteen titrated at 90%, allows to obtain a clear product scarcely colored and perfectly soluble that, contrarily to the product titrated at 40% according to the known art, can hence be used in a wide typology of cosmetic formulations such as, for example, deodorants, creams, after-sun products, products for after shave use, products for intimate hygiene, products for skin care.

Still according to the present invention, a study has been carried out for the placebo - controlled evaluation of the soothing effect of the association according to the invention, for cosmetic use, on the skin radiated with solar radiation (UVA+UVB). The study described below has been carried out to evaluate the soothing effect of the association according to the invention, for cosmetic use, (SAMPLE A 0.5% mangosteen extract in association with 1,3-propanediol of plant origin according to the present invention and SAMPLE B 1% mangosteen extract in association with 1,3-propanediol of plant origin according to the present invention) on the skin radiated with solar radiation (UVA+B). Said mangosteen extract is made according to the invention and hence shows the advantageous characteristics already indicated above.

The action of the products under consideration is compared to the action obtained in an area treated with the PLACEBO product and to an untreated control area. The anatomical region of the study is the back. The test has been done on a panel of 20 volunteers, all of legal age, according to the contemplated ethical requirements.

The test has been done as follows.

- Determination of the MED (minimum erythemal dose): the subjects are radiated with a radiation pattern (consisting on six doses of solar radiation) by a solar simulator. The radiation pattern is selected based on the phototype of the subjects.

- Evaluation of MED: 20±4 hours after irradiation: MED is visually evaluated

- Induction of damage: subjects are radiated in 4 skin sites with a fixed dose of UV (1.5 MED) in order to induce a reaction 20±4 after irradiation.

- Application of the products to be tested (2 mg/cm²) depending on the randomization plan. - The instrumental evaluations (erythema index) have been acquired before (TO), after 20±4 hours since the induction of damage (Tuv), after 30 minutes (T30min), 1 hour (Tlh), 2 hours (T2h), 4 hours (T4h) and 24 hours (T24h) since the application of the products.

- The study is carried out under the supervision of the dermatologist according to ethical principles for the research in the medical field (Helsinki declaration and subsequent amendments).

Burns, mechanical pressure, scratching, heat, chemical substances can induce displaying of an erythema on the skin surface.

The MEXAMETER MX 18 instrument (Courage+Khazaka, electronic GmbH) specifically measures the hemoglobin content in the skin. The measurement is based on the adsorption principle. The special probe of MEXAMETER MX 18 emits light of three defined wavelengths. A receiver measures the light reflected by the skin.

The position of the transmitter and receiver ensures that only the diffused and scattered light is measured. Since the amount of emitted light is defined, the amount adsorbed by the skin can be calculated. For the measurement of the erythema two different wavelengths are used: one corresponding to spectral absorption of the hemoglobin absorption peak, the other has been selected to avoid colorimetric influences of other pigments (e.g. bilirubin).

The results reported in the following pages summarize the values recorded by the instrument. It's important to note that standard references do not exist since the value of erythema is individual and sometimes also depend upon the race and phototype.

The probe used in this type of instrumentation is anyway very sensitive and records very small color variations.

At the set experimental times, images of treated anatomical areas have been acquired by a digital camera. Photos have been acquired by using a professional reflex digital camera (CANON EOS 60D) on 10 subjects.

Results are reported in the respective measure units in the tables.

In particular, in Table 1 (Figure 2) the values of erythema obtained for each volunteer are reported , by using SAMPLE A. On the other hand, in Table 2 (Figure 3) the values of erythema obtained for each volunteer are reported, by using SAMPLE B.

On the other hand, in Table 3 (Figure 4) the values of erythema obtained for each volunteer are reported, by using PLACEBO.

On the other hand, in Table 4 (Figure 5) the values of erythema obtained for each volunteer are reported, considering the UNTREATED AREA.

Figure 6 shows the average values of the ERYTHEMA INDEX parameter obtained at all the monitored experimental times.

On the other hand, Figure 7 shows the average percentage variations vs. UV of the

ERYTHEMA INDEX parameter

obtained at all the monitored experimental times.

The data obtained can be summarized as follows.

Data Summary

Sample B shows an effect higher than Sample A.

Average values have been calculated based on the following formula:

where: P is the value of the analysis parameter.

The standard error (SEM) of data is calculated as:

whereas the average percentage variations have been calculated according to the following formula:

where:

Xo is the value of the parameter after UV (Tuv)

Xt is the value of the parameter after application of the products (30 min, 1, 2, 4 and 24 hours after the single application).

Data and percentage variations are subjected to bilateral Student's t test for paired data. Statistical significance: p value is < 0.05.

Therefore, based on the results obtained it can be stated that the products called as SAMPLE A (0.5% mangosteen extract in association with 1,3 -propanediol of plant origin according to the present invention) and SAMPLE B (1% mangosteen extract in association with 1,3 -propanediol of plant origin according to the present invention), show a soothing effect towards the damage experimentally induced by the skin exposure to UV radiation, in terms of decrease of the erythema index in all the monitored experimental times.

It has been further carried out an evaluation of the cytotoxicity of an association made up by 2% mangosteen extract in association with 1,3 -propanediol of plant origin according to the present invention, through the NRU test: cell survival test with human fibroblasts cultivated in monolayer for the evaluation of the skin irritation potential and biocompatibility.

The in vitro methods constitute an interesting alternative to classical in vivo methods for the evaluation of biological characteristics of ingredients and topical finished products, in particular cosmetics, according to what is imposed by current regulations requiring the cosmetic manufacturers to verify the safe use and efficacy of products avoiding the use of animals.

The cytotoxicity assay can be carried out in order to evaluate the skin irritation potential of a finished product or ingredient or mixture of ingredients by using cultures of keratinocytes taken from human skin. The in vitro test carried out on cells derived by skin or epithelial tissue was in general found to be a simplified experimental method, but able to give many information on relationships that may verify in vivo.

The cosmetic topical products are applied directly on the skin, often in contact with the mucous membranes, and hence should have a toxicological impact null or very low towards the cells of skin or epithelial derivation.

Cytotoxicity test performed on fibroblasts or keratinocytes in monolayer, have been widely used as predictive indicators of the skin irritation potential of products for cosmetic or biomedical use. The target of this test is to quantitatively define the effects of the tested products on cell viability through the coloring test with NRU. The NRU test is a chemo-sensitive method for cell viability, based on live cells ability to incorporate and bind the Neutral Red (RN), a vital dye. The RN is a weak cationic dye penetrating the cellular membrane by non-ionic diffusion and accumulating in lysosomes where it binds in the anionic sites of the matrix. Alterations of the cellular surface or sensibility of the lysosomal membrane lead to the fragility of the lysosome and other changes that gradually become irreversible. These changes, due to xenobiotic action, lead to a decrease of the "uptake" and binding of RN. With this method it is possible to distinguish among live, damaged or dead cells.

A limit to this methodology is consisting of the fact that substances completely insoluble in an aqueous medium cannot contact cells and hence their potential toxicity may be underestimated. In cases where this kind of conditions is suspected, hence it is convenient to deepen the study with tests on reconstituted epidermis or in vivo in the animal. The cellular model used for in vitro test is represented by normal human fibroblasts from human skin (HSF: Human Skin Fibroblasts) Source: ATCC CRL-2522 Lot: 59899913.

The cells have been seeded in 96- wells plates and allowed to grow for 24 h at 37°C and 5% C0₂.

Therefore, fresh culture medium containing the products to be tested has been added in order to achieve 6 final dilutions between 5 and 0.16 mg/ml. The sample has been dissolved in ethanol at a concentration of 100 mg/ml and then diluted in the culture medium.

For each dilution three replicates have been carried out. Untreated cells have been used as negative control, and the cells have been treated with a surfactant with known toxicity (Sodium Lauryl Sulfate - SLS) dissolved in the culture medium at concentrations between 0.5 mg/ml and 0.03 mg/ml as positive control.

Hence the cytotoxicity test (NRU) has been performed at the end of the incubation in order to evaluate the percentage of cell survival. The NRU test evaluates the toxic impact on cell viability of the concerned substances.

After the incubation, the medium is substituted with a 0.33% Neutral Red solution, the cells are incubated at 37°C for 3 h. Hence, they are subjected to different washings to eliminate the excess dye residue, whereas the dye incorporated in the cells is dissolved with a solution solubilizing NR and quantified spectrophotometrically at 550 nm.

The plate is stirred on a plate stirrer ensuring that all crystals are dissolved and have formed a homogeneous solution. The absorbance is read by a colorimeter (Tecan Sunrise remote model) equipped with a plate reader by subtracting the background reading.

The result is expressed as cell viability in percentage according to the formula:

% of cell viability = [OD(550 nm - 690 nm) product tested/ OD(550 nm - 690 nm) negative control] x 100.

The cytotoxicity data obtained with NRU tests are plotted vs. the tested product concentration, thereby creating a dose-response curve, which allows determining: - the theoretical regression curve

- the theoretical value of IC50 (concentration of growth inhibition by 50%) that is the concentration inducing a decrease of cell viability by 50% compared to the untreated cells.

The cytotoxicity results have been corrected by subtracting the absorbance readings due to the diluting mean.

The IC50 (Inhibiting Concentration 50) value indicates the concentration of product necessary to inhibit the cellular growth by 50%. IC50 is a parameter that allows evaluating the irritant potential of a compound.

For finished products, values < 1 mg/ml (or 1 μΐ/ml) can be considered as irritants, values > 3 mg/ml (or 3 μΐ/ml) show a good biocompatibility. For detergents and products for rinsing, the eventual dilution factor must be taken into consideration. For raw materials, the use concentration has to be taken into account.

The results are outlined as follows.

Based on the above reported results, the association consisting of 2% mangosteen extract in association with 1,3 -propanediol of plant origin according to the present invention, showed to have an IC50 of 2.70 mg/ml on human fibroblasts.

These data can be considered as predictive of lack of in vivo irritant effects.

A test has been further carried out for the evaluation of the phototoxicity of the association according to the invention, in particular an association where the mangosteen extract is at 10%, for topical use, via the usage of the human 3T3 fibroblasts line. The evaluation of the cytotoxicity has been carried out with the NRU test.

The sample under consideration is solubilized at 10% in ethanol.

In addition, a phototoxic substance (chlorpromazine) has been used as positive control in the experiment.

The phototoxicity through the 3T3/NRU test corresponds to a cell survival test on 3T3 fibroblasts in the presence of the product to be tested with and without UV exposure, compared to a known phototoxic substance (Chlorpromazine - CPZ).

The performed test is keeping with what prescribed by the official methodology published on Gazzetta Ufficiale delle Comunita Europee of 08/06/2000 (L.136/98), according to the 2000/33 EC directive of 25/4/2000 and OECD 432.

Different molecular mechanisms have been identified through which the absorption of light and UVA, in particular by a chromophore, can lead to phototoxicity. All of these mechanisms cause cellular injury and hence allow considering the cells viability as an indicative parameter of phototoxicity.

The 3T3 NRU methodology allows to verify if the chemical molecules contained in the product under consideration exposed to radiation (mainly UVA) show or not toxic effects on the cultured cells compared to the same substances not irradiated. For their chemical nature, different cosmetic ingredients, among which in particular the sunscreens, are agents capable of absorbing UVA and UVB light.

As a consequence of this absorption of light, molecules can change their configuration or transform into different chemical molecules. A molecule with altered stereochemical configuration may give rise to further biological reactions with toxicological relevance different from that represented by the original molecule. The phototoxicity (photoirritation) is defined as the toxic response that is displayed after primary exposure of the skin to certain chemical agents, with subsequent exposure to light, or similarly induced by irradiation of skin after systemic administration of a substance.

Since the toxicological purpose of the in vitro assay is the determination of the photocytoxicity induced by the combined action of a chemical agent and light, the assay allows to identify compounds that are phototoxic in vivo after systemic application and distribution on the skin and compounds with photoirritant effect after local application on the skin.

The in vitro phototoxicity 3T3 NRU assay has been compared to acute in vivo phototoxicity effects/ irritant effects observed on human individuals and is confirmed as an excellent predictive assay of such effects.

The assay is not suitable to predict other kind of adverse effects, such as photoallergy, photogenotoxicity and photocarcinogenesis, which may arise by the combined action of a chemical product with light. The test is not further indicated to allow for an evaluation of phototoxic potency.

The used cellular model is a secondary line of murine Balb/c 3T3 fibroblasts coming from the American Type Culture Collection (ATCC). The cells are plated at a density of about lxlO⁴ cells/well in 96- well plates, in DMEM culture medium (Dulbecco Modified Essential Medium) +10% FCS (Fetal Calf Serum) + 4 mM glutamine, penicillin and streptomycin.

The substance to be tested is dissolved in ethanol and then diluted in PBS (Phosphate Buffered Saline) at scalar concentrations between 1000 μΐ/ml and 0.46 μΐ/ml. Additional convenient negative controls (cells untreated with the substance) and, as positive controls, phototoxic substances of reference, such as chlorpromazine (CPZ), are inserted.

The experiment is carried out in two 96-well plates, in triplicate; one plate is irradiated with UVA via SUNTEST® TM instrument CPS PLUS model, the other is stored for the same period at room temperature in the dark. At the end of such a period the sample has been eliminated. Therefore, washings in PBS are performed. PBS is substituted with culture medium and the cells are incubated overnight at 37°C, 5% C02. The following day, after being morphologically examined on microscope, the cells are subjected to NRU (Neutral Red Uptake) assay.

The NR medium is prepared by adding 0.33% Neutral Red solution in PBS to standard culture medium, with a final concentration of 50 mg/ml.

The cells are washed with PBS and hence 100 ml of NR medium is added to each well and the cells are incubated for 3 h at 37°C, 7.5% C02. Hence the medium is removed, the cells are washed and the plates are dried. 150 ml of 1% glacial acetic acid solution in 50% ethanol (desorption solution of neutral red) is added to each well and allowed to react for 15 min under stirring to ensure that all dye has been extracted by the cells and makes an homogeneous solution.

The absorbance of this solution is measured at 540 nm with a spectrophotometer equipped with a plate reader. The background is read at 690 nm. The cell viability of samples treated with the substance is expressed as a percentage compared to the controls of cells treated with negative control (medium + solubilizer). A non-linear regression procedure is applied to the concentration-response data, by calculating the IC50 value for the substance under consideration, i.e. the concentration determining an inhibition of 50% of the cellular NRU (compared to the untreated cells). The values are obtained by the averages of three replicates per sample and per each concentration.

Once the concentration-response curves, both in presence and in absence of UVA irradiation, and the relative IC50 values have been obtained, we proceeded to the calculation of the photoirritation factor (PIF) according to the following scheme: PIF= IC50 (-UV)/ IC50 (+UV)

If PIF< 2, there is no phototoxic potential

If PIF< 2 and > 5, the sample likely is phototoxic

If PIF > 5 there is a phototoxic potential

If a substance is cytotoxic when exposed to UVA and is not cytotoxic when not exposed to UVA, the PIF cannot be calculated, however such a response is an index of potential phototoxicity. In this case, a ">PIF" is calculated if the cytotoxicity assay without irradiation (-UV) is performed up to the highest test concentration (Cmax), and such a value is used to calculate the ">PIF".

>PIF= C max (-UV)/IC50 (+UV)

If only one ">PIF" can be calculated, than each value >1 predicts a potential phototoxicity.

If nor IC50 (-UV) nor IC50 (+UV) can be calculated as the substance under consideration is not cytotoxic above the highest tested concentration, than such a substance is considered potentially not phototoxic. In this case "PIF=1 *"

PIF= 1 * = C max (-UV)/C max (+UV)

If only one "PIF=1 *" can be obtained, this doesn't predict any phototoxic potential. The absolute optical density (OD540 NRU), measured in neutral red extract of the negative controls (untreated cells) as an average, must be > 0.2 in order for the assay to be valid.

Regarding the CPZ standard, it must satisfy the following acceptance criteria:

» CPZ irradiated (+UVA): having an IC50 between 0.1 -2 mg/ml,

· CPZ non-irradiated (-UVA): having an IC50 between 7 and 90 mg/ml.

• Having Photo Inhibition Factor (PIF) > 6.

The results are outlined herein below.

Chlorpromazine (+ UVA)

Dose mg/ml 100.0 25.0 6.25 1.56 0.39 0.10 0.02 0.01

% cell viability (vs.

negative control) 8.34 9.33 10.65 9.95 67.65 91.49 94.08 100.49

ic₅₀ = = 0.75 μg/ml

Chlorpromazine (no UVA)

Dose mg/ml 100.0 25.0 6.25 1.56 0.39 0.10 0.02 0.01

% cell viability (vs.

negative control) 16.14 23.19 84.70 94.35 95.55 100.08 84.10 90.47

ICso = 16.83 g/ml

Results for the association consisting of 10% mangosteen extract in 1 ,3-propanediol of plant origin in the presence of UVA

Results for the association consisting of 10% mangosteen extract in 1,3-propanediol of plant origin in the absence of UVA

Based on the results reported above, the association consisting of 10% mangosteen extract in 1,3-propanediol of plant origin didn't show in vitro phototoxic effects on 3T3 fibroblasts.

Therefore, according to the data derived from the predictive in vitro model, it was found to be non-photo toxic/non-photoirritant.

The following Table 5 shows the solubility and color tests (Camera Lux) carried out on the association according to the invention compared to the association between the same mangosteen extract and different components in place of 1,3-propanediol (Zemea) used according to the invention.

Table 5: TEST % SOLVENT COLOUR RESULT

GARDNER

6-7 -Completely soluble,

Mangosteen 1 Finsolv PG Yellow clear without filtration.

90% 22 6-7 -After 10 days: clear sol. with

Batch: Yellow slight precipitate.

GME28- 6-7 -After 30 days: clear sol. with

100301 Yellow slight precipitate

6-7 -Completely soluble,

Mangosteen 1 ZEMEA Cedar clear without filtration.

90% Yellow -After 10 days: clear sol.

Batch: 6-7 -After 30 days: clear sol.

GME28- Cedar

100301 Yellow

6-7

Cedar

Yellow

Mangosteen 13 -14 -Turbid solution, remained turbid

40% 1 Finsolv Light Brown after filtration

Batch: PG22 12-13 -After 10 days: clear sol. with

GME26- Light Brown slight precipitate

101015 12-13 -After 30 days: opalescent sol. with

Light Brown a lot of precipitate

Mangosteen 13-14 -Almost completely soluble, but

40% 1 ZEMEA Brown turbid, clear after filtration.

Batch: 13-14 -After 10 days: clear sol.

GME26- Brown -After 30 days: clear sol.

101015 13-14

Brown From the chemical stand point, active compounds contained in the Mangosteen fruit are represented by well-characterizable, isolable, stable, purifiable compounds and eventually transformable in derivatives thereof. In fact, some xanthones can be derived by others through simple chemical reactions (e.g. acidic treatments, methylations, prenylations etc.).

The formula reported herein below is that of alpha-mangosteen. This is the most abundant xanthone in the fruit and the one appearing to have the highest antibacterial activity.

The present invention is better illustrated by means of the examples reported in the following, in no way being limitative.

Extraction Procedure

80.0 g of commercial Garcinia mangostana preparation has been placed in a Soxhlet thimble and extracted with 1 1 of ethyl acetate for 6 hours. At the end of 6 hours, 100 ml of extract has been taken and brought to dryness under reduced pressure. The residue has been ground with petroleum ether and again brought to dryness, in order to remove the residual acetate by co-distillation.

GMV1 Fraction: 3.89 g; scraped off the flask 3.72 g.

The volume of extraction solvent has been restored by the addition of 100 ml of fresh acetate and the extraction has been continued for additional 6 hours. At the end, 100 ml of solution has been taken from the recovery flask and brought to dryness under reduced pressure; the residue has been co-distilled with petroleum ether to eliminate acetate, obtaining the GMV2 Fraction: 5.14 g; scraped off the flask 5.00 g.

The volume of extraction acetate has been restored with the addition of 100 ml of fresh acetate and the extraction has been continued for further 6 hours. At the end, 100 ml of solution has been taken from the recovery flask and brought to dryness under reduced pressure; the residue has been co-distilled with petroleum ether to eliminate acetate, obtaining the GMV3 Fraction: 5.60 g; scraped off the flask 5.45 g. Hence, all the acetate used has been brought to dryness under reduced pressure; the black oily residue has been ground with hexane and again brought to dryness to give the GMV4 Fraction: 41.08 g; scraped off the flask 40.93 g.

The material present in the Soxhlet thimble, still soaked in solvent, has been brought on a Buchner funnel and washed with ethyl acetate until obtaining a colorless filtrate. Therefore, about further 600 ml of solution has been recovered that, as the latter has been brought to dryness under reduced pressure and ground with hexane, gave the GMV5 Fraction: 5.89 g; scraped off the flask 5.78 g.

The residue recovered on the filter has been allowed to dry and is the GMVR Fraction: 18.24 g.

Extraction Yield

Hence, in total, 3.89+5.14+5.60+41.08+5.89- 61.60 g of extract has been obtained, with a yield of 77%.

Moreover, considering the extraction residue, the recovered material amounts to 79.84 g, equal to more than 99% of theoretical value.

Evaluation of antimicrobial activity of the association according to the invention The antimicrobial activity of the preferred association according to the invention has been verified, which provides for 1% of 90% Mangosteen and 99% of Zemea, versus a mix of microbial strains isolated by underarm pads performed on volunteers.

Schedule of the study

Test method: At different concentrations of the product under consideration, in particular at 0.15-0.5-1.0-2.5%, a test suspension of the microorganism has been added. The mixture has been maintained at 20°C±1 °C for contact times of 6 and 18 hours (required test conditions). After the preset time was elapsed, aliquots have been taken whose microbicide activity is immediately neutralized or suppressed by the dilution-neutralization method, previously validated. In every sample the number of survived cells of microorganisms is determined and the decrease of vital cell load calculated.

Process

Isolation of test microorganisms: Underarm pads have been performed to four different volunteers.

The pad has been soaked in the eluting solution to ease the sampling and subsequently has been scratched by rotating the tip on the surface of the axillary basin of volunteers and, finally, the pad has been immersed in the test-tube containing the eluting solution of isotonic liquid taking care to break the pad into the test-tube. For each sampling, under a laminar-flow hood, aliquots of suspension obtained by dissolving the pad in the transport test-tube have been plated and inclusion seeded by adding TSA and melted SDA and cooled at 45°C±1°C. The plates containing bacteria have been incubated at 32.5°C±1°C for 48 hours and the plates for the determination of mycetes have been incubated at 25°C±1 °C for 5 days. At the end of the incubation period, from the various plates of interest, colonies have been selected having different morphology and, after a purification step, biochemical identification of the microbial species proceeded.

Preparation of test suspensions and test product: The suspension used in the test is consisting of 5 microorganisms previously identified (5 Gram-positive bacteria) and mixed in a single culture.

The different microorganisms have been dissolved in sterile saline in order to obtain a title equal to 107 UFC/ml, initially evaluated for turbidity, and subsequently verified by counting through inclusion of aliquots of 1.0 ml of serial dilutions (10^~5 and 10^"6) in Tryptone Soy Agar (TSA) and subsequent incubation at 32.5°C±1°C for at least 48 hours.

Dilutions of products have been prepared in sterile water immediately before the assay and, in particular, the sample representing the association according to the invention has been tested at the following concentrations: 0.15-0.5-1.0-2.5%; contact times for all selected concentrations are 6 and 18 hours.

As a control of the evolution of the bacterial load of reference, a test-tube containing tryptoned water and inoculum has been prepared.

Test: Before the beginning of the test, all the reagents (product test solutions, microbial suspension) have been placed in the double boiler and equilibrated to the test temperature (20°C±1°C). In a sterile test-tube 8.0 ml of one of the test solutions at different concentrations have been added, 1.0 ml of water and 1.0 ml of the bacterial test suspension; after having immediately started the chronometer, the test- tube content has been mixed and the test-tube has been placed in the double boiler at 20°C±1°C. Immediately before the end of the preset contact times, 6 and 18 hours, the test mixture has been mixed under stirring and 1 ml of the test mixture has been transferred into a test-tube containing 8.0 ml of neutralizing agent and 1.0 ml of sterile water. After mixing, the test-tube with the neutralized mixture has been placed in the double boiler at 20°C±1 °C for a neutralization period of 5 min ± 10 sec, at the end of which aliquots, in duplicate, of 1.0 ml of neutralized suspensions of the test substance have been transferred in petri plates separated and inclusion seeded by adding TSA melted and cooled at 45°C±1 °C.

The same process has been carried out for all the selected concentrations.

Count: The plates containing the bacteria have been incubated at 32.5°C±1°C for 24 hours. The developed colonies have been counted and the plates have been stored to incubate for further 24 hours before carrying out a new count.

Validation: Preliminarily to test, a validation of experimental conditions, efficacy and toxicity of the neutralizing agent has been performed. The mix of microbial suspension of the validation test has been prepared by diluting the test suspension with sterile saline to obtain a number of cells between 6.0x10 UFC/ml and 1.5x10 UFC/ml. The count of the suspension (N_v) has been carried out on 2 aliquots of 1.0 ml of the dilution 10^"1 inclusion seeded in TSA and incubated at 32.5°C±1°C.

Before the beginning of the test, all the reagents have been placed in the bath and equilibrated to the test temperature. Two aliquots of 8.0 ml of neutralizing agent have been added in two sterile test-tubes, which have been placed in the double boiler at 20°C±1°C. To this test-tubes

either 1.0 ml of water for controlling the toxicity of the neutralizing agent or 1.0 ml of the test solution, at a concentration higher (2.5% of association according to the invention) than the product for controlling the neutralization- dilution, have been added.

The content of the test-tubes has been mixed and these re-placed in the double boiler at 20°C±1°C for 5 minutes±10 seconds. To each of the two test-tubes, 1.0 ml of microbial suspensions containing 6.0xl0² UFC/ml to 1.5xl0³ UFC/ml has been added. The content of the test-tubes has been mixed and the test-tubes have been placed in the bath for 30 min±l 0 sec.

Immediately before the end of the incubation, the content of the test-tube has been mixed under stirring and samples of 1.0 ml of the test mixture have been taken therefrom, in duplicate; the samples have been transferred in petri plates separated and inclusion seeded by adding melted TSA, cooled at 45°C±1°C.

The petri plates have been incubated as described in Count of test mixtures. For each sample of 1.0 ml the highest number of colonies has been determined and Nx calculated, the number of UFC/ml in controlling the toxicity of the neutralizing agent and Ny in controlling the neutralization-dilution test.

Calculation and expression of the results:

The determination of the vital microbial load for the test process and for the validation test is carried out in the following manner:

a) for the count of the vital load, only the plates with a colony number lower than

300 UFC/plate are taken into account. The count of vital colonies should be carried out by using the count of the colonies coming from both plates. If at least one plate contains 15 or more colonies, the following formula is used:

Vital microbial load = c nxdxV

where

c is the sum of the colonies counted on both plates

n is the number of plates taken into account

d is the dilution factor corresponding to the dilution taken into account; the dilution factor is 10 for the test process of dilution-neutralization and for the fungal suspension

V is the sample volume; the volume is 1.0 ml for the test process of dilution- neutralization, for the validation and fungal suspension

Verification of the method

The method is validated if, for each tested microorganism, the following requisites are satisfied:

N between 1.5 x 10⁷ and 5.0 x 10⁷ UFC/ ml

Nv between 6.0 xlO² and 1.5 x 10³ UFC/ml

N_X > 0.05 N_V

N_y > 0.5 N_v

where

N is the number of UFC/ml of the test microbial suspension

N_v is the number of UFC/ml of the microbial suspension

N_x is the number of UFC/ml of the control over toxicity of the neutralizing agent or control of the filtration

N_y is the number of UFC/ml of the control over dilution-neutralization or control over the test filtration.

Results

I) Biochemical Identification of test microorganisms isolated from underarm pad:

- Staphylococcus epidermidis

- Staphylococcus capitis

2) Test and Validation Test

The results are reported in Tables 6 and 7.

Table 6 Sample Test Validation Validation of Validation of Microorganism Microbial Toxicity of Dilution- Suspension Neutralizing Neutralization (UCF/ml) Agent (UCF/ml)

(UCF/ml)

MANGOSTEEN Isolated

90 Microorganism N_v : 1.5xl0³ Nx : l.OxlO² Ny : 2.3x10²

Mix

Table 7

The validation test confirmed the efficacy of neutralization of the residual antimicrobial activity and the absence of toxicity of the used neutralizing agent.

Conclusions The obtained results suggest that the association according to the invention, at the tested concentrations, perform an antimicrobial activity with a logarithmic decrease compared to the initial inoculum equal to >2.0 log after a contact time of 6 hours already.

In particular, it is underlined that the logarithmic decrease doesn't increase as a function of the increase of the tested concentration, but it's noted an increase of the logarithmic decrease as the contact time, regarding concentrations >0.5%, increases. Still according to the present invention, it is herein below reported an example of cosmetic formulation of the association consisting of 2% mangosteen extract in 1,3- propanediol of plant origin.

Claims

1. An association consisting of mangosteen extract with purity between 40% and 99% and 1,3 -propanediol of plant origin, characterized in that said extract is present at concentrations between 5% and 0.5% and said 1,3 -propanediol is present at concentrations between 95% and 99.5%.

2. The association according to claim 1, characterized in that said extract is 90% pure.

3. The association according to claim 1 , characterized in that said extract is 80% pure.

4. The association according to claim 1, characterized in that said extract is present at a concentration between 0.5% and 1.5% and said 1,3 -propanediol is present at a concentration between 99.5% and 98.5%.

5. The association according to claim 4, characterized in that said extract is present at a concentration equal to 1% and said 1,3 -propanediol is present at a concentration equal to 99%.

6. The association according to claim 1, characterized in that said 1,3- propanediol is of the type currently marketed under the trademark Zemea™ by Dupont Tate & Lyle Bio Products.

7. The association of claim 1 for use as an antibacterial/antimicrobial.

8. The association of claim 1 for use as an anti-inflammatory.

9. The association of claim 1 for use as an active compound in cosmetic formulations.

10. The cosmetic formulations comprising the association of claims 1 to 6, in addition to usual additives and excipients.

11. The cosmetic formulations of claim 10 for use as deodorants, creams, emulsions, solar products, after sun products, products for after shave use, products for intimate hygiene.

12. The cosmetic formulations according to claim 10, characterized in that they are suitable for the treatment of acne.

13. The cosmetic formulations according to claim 10, characterized in that they

1 are suitable for after-sun applications.

14. The cosmetic formulations according to claim 10, characterized in that they are suitable for the sun protection.

2