ITMI20130619A1 - NEW COSMETIC ASSOCIATION - Google Patents

Description

DESCRIPTION

â € œNEW COSMETIC ASSOCIATIONâ €

OBJECT OF THE INVENTION

The present invention refers to an association that includes at least one antibacterial compound derived from natural sources, and in particular to the use of the association for the preparation of deodorants, anti-acne creams, intimate hygiene products and the like. .

STATE OF THE TECHNIQUE

Considerable attention has recently been paid to a plant known as â € œMangosteenâ € (Garcinia mangostana L.) whose fruit is commonly called Mangosteen and thus indicated below. The aforementioned plant was particularly interesting for the following reasons. It is known that its fruits contain substances characterized by various activities, among which the antibacterial one. It is also known that its fruits are not toxic but, on the contrary, are consumed in large quantities as a dessert in the countries of the Far East.

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

Garcinia mangostana (Guttiferae) is a tree 6 to 20 meters tall and is grown in tropical wetlands of Indonesia, Thailand, India and China. The fruit, Mangosteen, is consumed in large quantities as a dessert due to its sweet and delicate taste. It is known that some substances responsible for numerous biological and pharmacological activities are contained in the fruit. Folk medicine benefits from some of them, but in-depth studies have only been conducted on a few. Among the biological properties tested experimentally, an antibacterial and antifungal activity is known. These activities are all attributable to compounds of very similar molecular structure and belonging to a single biogenetic family, namely that of xanthones.

Four distinct parts can be distinguished in the fruit:

- a calyx formed by four rounded, thick and robust leaves (hulls)

- a very strong peel (rind)

- a juicy pulp that encloses the seeds and is called aril (aril) - from 5 to 7 large seeds (seeds)

The xanthones are not evenly distributed among the various parts of the fruit but are found particularly abundant in the leaves, which make up the calyx. About 16 xanthones have been extracted from the various parts of the Garcinia mangostana fruit. In the structure of their molecule the xanthonic nucleus constituted by the three rings with six terms is well recognizable. The differences between the compounds of the same structural and biogenetic family are represented by the substituents on the two side rings, ie by hydroxyl, prenyl, modified prenyl and methoxyl groups.

Some methods of extraction of the fruits of Garcinia mangostana are mentioned in the literature (water, water-methanol, ethyl acetate, succession of solvents with increasing polarity, etc.) but their use has so far been aimed solely at the search for new compounds and there are no known processes for extracting the active ingredients on an industrial scale.

The active ingredients present in Mangosteen are known to possess antibacterial / antimicrobial / antifungal activity. They are also known to have anticancer properties, as inducers of apoptosis, with an antiproliferative effect demonstrated in cases of breast tumors, leukemia, liver tumors.

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

Their generic anti-tuberculosis activity is also known.

AIMS OF THE INVENTION

The purpose of the present invention is to provide an association which can be formulated as a cosmetic and which has excellent stability over time, as well as being used as an antibacterial / antimicrobial.

The object of the present invention is also to provide an association which can be easily prepared and which is mainly based on products of natural origin.

Another object of the present invention is to make available an association which can be easily prepared on an industrial level and which is advantageous from an economic point of view.

DESCRIPTION OF THE INVENTION

These and other purposes and relative advantages which will be better clarified by the following description, are achieved by an association consisting of 90% pure Mangosteen extract and 1,3-propanediol of vegetable origin. This combination is advantageously used in cosmetic formulations which include additives and excipients of known type and which can vary according to the final use of said cosmetic formulation. In practice, the association according to the present invention represents the â € œactive ingredientâ € that characterizes the cosmetic formulation always according to the invention.

In particular, the 90% Mangosteen extract is prepared by a process which is also the subject of the present invention, while the 1,3-propanediol of vegetable origin is currently marketed under the Zemeaâ „¢ brand by Dupont Tate & Lyle Bio Products.

1,3-propanediol Zemea is a colorless and highly pure liquid derived from a corn fermentation process. It is a highly conservative process from the point of view of environmental protection that allows you to make available a product of pure plant origin that has numerous advantages from the point of view of its use, especially in the cosmetic and pharmaceutical fields.

The Mangosteen extract according to the present invention differs from the extracts according to the known technique, which are characterized by an extract coming from the peel of the fruit obtained by means of a known process, which uses water and alcohol. This known process leads to obtaining a standard extract titrated at 40%, as for example described in WO 2006/055688.

The process according to the present invention, on the other hand, allows to obtain more purified fractions, up to a 99% extract titrated in alpha and gamma mangosteen, using solvents such as, for example, ethyl acetate, to reach higher concentrations, equal to about 80-90%.

The present invention therefore refers to an association of mangosteen extract titrated in concentrations between 40% and 99% in active principle and 1,3-propanediol of vegetable origin (Zemea) in concentrations between 95% and 99.5 %.

Particularly preferred is 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 No 504-63-2).

It has been surprisingly observed that the association according to the present invention has a synergistic effect with respect to its individual components, when used as an antibacterial / antimicrobial. In fact, the activity of the Mangosteen extract used alone, against Gram positive bacteria, is shown in the following Table 1:

MIC (ppm)

Staphylococcus aureus 1

K.rhizophyla 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 under the same conditions, showed the results according to the following Table 2:

MIC MMC

Propionobacterium 0.005% 1.0%

acnes

Staphylococcus 0.01% 2.5%

epidermidis

The antimicrobial properties of the association according to the present invention were also evaluated against some microbial strains isolated from the armpits of a group of volunteers.

The association was evaluated at different concentrations and for two different contact times, 6 hours and 18 hours respectively. The results obtained show good antimicrobial efficacy already after 6 hours of contact, with an increase in logarithmic reduction after 18 hours (Figure 1), which suggests a long-lasting deodorant effect.

The isolates are the following:

- Staphylococcus epidermidis

- Staphylococcus epidermidis

- Staphylococcus epidermidis

- Staphylococcus epidermidis

- Staphylococcus capitis

One of the advantageous aspects of the present invention is that of avoiding the use of solvents and glycols or of solubilizing agents derived from petroleum, and therefore of being able to use components of completely natural origin.

Furthermore, the use of the concentrated fraction obtained from Mangosteen titrated at 90%, allows to obtain a limpid, slightly colored and perfectly soluble product which, contrary to that titrated at 40% according to the known technique, can therefore be used in a wide range of cosmetic formulations such as, for example, deodorants, creams, after-sun products, products to be used after shaving, intimate hygiene products, skin care products.

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

TEST% SOLVENT COLOR RESULT

GARDNER

6 â € “7 -Fully soluble, clear yellow without filtration. Mangostana 1 Finsolv PG

90% 22 6 â € “7 -After 10 days: Clear Batch: Yellow with slight precipitate. GME28-100301 6 - 7 - After 30 days : Sol. Clear Yellow with Slight precipitate 6 â € “7 -Completely soluble, clear Cedar Yellow without filtration.

Mangostana 1 ZEMEA

90% 6 â € “7 -After 10 days: Sol. Clear Batch: Yellow Cedar

GME28 - 6 - 7 - After 30 days : Sol.limpida 100301 Yellow Cedar

Mangostana 13-14 -Solution

40% Light brown turbid, remained cloudy Batch: 1 Finsolv PG22 after filtration GME26- 12 - 13

101015 Light brown -After 10 days: Opalescent sol with 12 - 13 precipitated

Light brown

-After 30 days : Sol. Opalescent with a lot of Mangostana precipitate 13 - 14 - Almost completely 40% Brown soluble, but turbid, Batch: 1 ZEMEA clear after filtration. GME26- 13 - 14

101015 Bruno - After 10 days: Clear sol

13 - 14 - After 30 days : Sol.limpida Bruno

From a chemical point of view, the active principles contained in the Mangosteen fruit are represented by compounds that can be well characterized, isolated, stable, purified and possibly transformable into their derivatives. In fact, some xanthones can be derived from others through simple chemical reactions (e.g. acid treatments, methylations, prenylations, etc.).

The formula below is that of alpha-mangosteen. This is the most abundant xanthone in the fruit and the one that seems to have the greatest antibacterial activity.

Me2C CH CH2O OH

MeO CH2CH CMe2

HO OR OH

The present invention is better illustrated by means of the examples, reported below, which in no way assume a limiting value.

Extraction procedure

80.0 g of commercial Garcinia mangostana preparation was placed in a Soxhlet thimble and extracted with 1 L of ethyl acetate for 6 hours. At the end of the 6 hours, 100 mL of extract was taken and dried under reduced pressure. The residue was ground with petroleum ether and dried again, in order to remove the residual acetate by codistillation. GMV1 fraction: 3.89 g; scratched from the balloon 3.72 g.

The volume of the extraction solvent was replenished with the addition of 100 mL of fresh acetate and the extraction was continued for a further 6 hours. At the end, 100 mL of solution were taken from the collection flask and dried under reduced pressure; the residue was codistilled with petroleum ether to eliminate the acetate, obtaining the GMV2 fraction: 5.14 g; scratched from the balloon 5.00 g.

The extraction acetate volume was replenished with the addition of 100 mL of fresh acetate and the extraction continued for a further 6 hours. At the end, 100 mL of solution were taken from the collection flask and dried under reduced pressure; the residue was codistilled with petroleum ether to eliminate the acetate, obtaining the GMV3 fraction: 5.60 g; scratched from the balloon 5.45 g.

All the acetate used was then brought to dryness at reduced pressure; the black oily residue was ground with hexane and dried again to give the GMV4 Fraction: 41.08 g; scratched from the balloon 40.93 g.

The material present in the soxhlet thimble, still soaked in solvent, was brought to a Buchner funnel and washed with ethyl acetate until a colorless filtrate was obtained. A further 600 ml of solution were thus collected which, brought to dryness at reduced pressure and ground with hexane, gave the GMV5 fraction: 5.89 g; scratched from the balloon 5.78 g.

The residue collected on the filter was left to dry and constitutes the GMVR Fraction: 18.24 g.

Extraction yield

In total, therefore, 3.89 + 5.14 + 5.60 + 41.08 + 5.89 = 61.60 g of extract were obtained, for a yield of 77%.

Also considering the extraction residue, the recovered material amounts to 79.84 g, equal to over 99% of the theoretical.

Evaluation of the antimicrobial activity of the association according to the invention

The antimicrobial activity of the preferred association according to the invention was verified, which provides 1% Mangosteen at 90% and 99% Zemea, against a mix of microbial strains isolated from axillary swabs performed on volunteers.

Study plan

Test method: A test suspension of the microorganism was added at different concentrations of the product under investigation, in particular at 0.15-0.5-1.0-2.5%. The mixture was kept at 20 ° C ± 1 ° C for the contact times of 6 and 18 hours (test conditions required). After the predetermined time, aliquots were taken whose microbicidal activity is immediately neutralized or suppressed by the previously validated dilution-neutralization method. In each sample the number of cells of surviving microorganisms is determined and the reduction of the viable cell count is calculated.

Method

Isolation of test organisms: Axillary swabs were performed on four different volunteers.

The swab was soaked in the eluent solution to facilitate the collection and subsequently it was rubbed by rotating the tip on the surface of the axillary basin of the volunteers and finally the swab was dipped into the tube containing the eluent solution of isotonic liquid, taking care to break the swab inside the tube. For each sampling, under a laminar flow hood, aliquots of suspension obtained by diluting the swab in the transport tube were plated and sown for inclusion by adding melted TSA and SDA and cooled to 45 ° C ± I ° C. The plates containing the bacteria were incubated at 32.5 ° C ± 1 ° C for 48 hours and the plates for the determination of the fungi were incubated at 25 ° C ± 1 ° C for 5 days.

At the end of the incubation period, colonies with different morphology were selected from the various plates of interest and, after a purification step, the biochemical identification of the microbial species was carried out.

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

The various microorganisms were diluted in sterile physiological solution in order to obtain a titer equal to 107 CFU / ml, initially assessed for turbidity, and subsequently verified by counting for inclusion of 1.0 ml aliquots of serial dilutions (10.5 and IO ") in Tryptone Soy Agar (TSA) and subsequent incubation at 32.5 ° C ± 1 <o> C for at least 48 hours.

The dilutions of the products were prepared in sterile water immediately before the test and in particular the sample representing the association according to the invention was tested at the following concentrations: 0.15-0.5-1.0-2.5%; the contact times for all the chosen concentrations are 6 and 18 hours.

As a control of the evolution of the reference bacterial load, a test tube containing tryptonated water and inoculation was prepared.

Test: Before starting the test, all reagents (product test solutions, microbial suspension) were placed in the water bath and equilibrated to the test temperature (20 ° C ± 1 ° C). 8.0 ml of one of the test solutions at different concentrations, 1.0 ml of water and 1.0 ml of the test bacterial suspension were added to a sterile test tube; after immediately starting the stopwatch, the contents of the tube were mixed and the tube was placed in the water bath at 20 ° C ± 1 <o> C. immediately before the end of the predetermined contact times, 6 and 18 hours, the test mixture was stirred and 1 ml of the test mixture was transferred to a tube containing 8.0 ml of neutralizer and 1.0 ml of sterile water . After mixing, the test tube with the neutralized mixture was placed in the water bath at 20 ° C ± 1 ° C for a neutralization period of 5 min ± 10 sec, at the end of which 1.0 ml aliquots, in duplicate, of the neutralized suspensions of the substance tests were transferred to separate petri dishes and seeded for inclusion by adding melted TSA and cooled to 45 ° C ± 1 ° C.

The same procedure was carried out for all the chosen concentrations.

Enumeration: Plates containing bacteria were incubated at 32.5 ° C ± 1 <o> C for 24 hours. The developed colonies were counted and the plates were placed back to incubate for a further 24 hours before re-counting.

Validation: Prior to the test, a validation of the experimental conditions, efficacy and toxicity of the neutralizer used was performed. The mix of the microbial suspension of the validation test was prepared by diluting the test suspension with sterile saline to obtain a number of cells between 6.0x10 <2> CFU / ml and l.5x10 <3> CFU / ml. The counting of the suspension (Nv) was carried out on 2 aliquots of 1.0 ml of dilution 10 <-1> sown for inclusion in TSA and incubated at 32.5 ° C + 1 ° C.

Before the start of the test, all the reagents were placed in the water bath and equilibrated to the test temperature. Two 8.0 ml aliquots of neutralizer were added to two sterile tubes, which were placed in the water bath at 20 ° C ± 1 <o> C. To these tubes were added

o 1.0 ml of water for the neutralizing toxicity check. o 1.0 ml of the test solution at the highest concentration (2.5% of association according to the invention) of the product for the neutralization-dilution check.

The contents of the tubes were mixed and these returned to the water bath at 20 ° C ± 1 ° C for 5 minutes ± 10 seconds. 1.0 ml of the microbial suspensions containing from 6.0x10 <2> CFU / ml to 1.5x10 <3> CFU / ml was added to each of the two tubes. The contents of the tubes were mixed and the tubes were placed in the water bath for 30 min ± 10 sec.

Immediately before the end of the incubation, the contents of the tube were mixed by shaking and from it, samples of 1.0 ml of the test mixture were taken in duplicate; the samples were transferred to separate petri dishes and seeded for inclusion by adding melted TSA, cooled to 45 ° C ± 1 <o> C.

The petri dishes were incubated as described in Counting the test mixes. The highest number of colonies for each 1.0 mI sample was determined and Nx, the number of CFU / ml in the neutralizer toxicity control and Ny in the neutralization-dilution test control were calculated.

Calculation and expression of results:

The determination of the viable microbial load for the test procedure and for the validation test is carried out as follows:

a) only plates with a number of colonies less than 300 CFU / plate are taken into account for the vital charge count. Viable colony counts should be performed using colony counts from both plates. If at least one plate contains 15 or more colonies, the following formula is used:

Vital microbial load = c

-----------------------

nxdxV

where is it

c is the sum of the colonies counted on both plates

n is the number of plates taken into consideration

d is the dilution factor corresponding to the dilution taken into consideration; for the dilution-neutralization test procedure and for the fungal suspension the dilution factor is ̈ 10 <-1>

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

Method verification

The method is validated if the following requirements are met for each microorganism tested:

N between 1.5 x 10 <7> and 5.0 x 10 <7> UFC / ml

Nv between 6.0 x10 <2> and 1.5 x 10 <3> UFC / ml

Nx> 0.05 Nv

Ny> 0.5 Nv

where is it

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

Nv is the number of CFU / ml of the microbial suspension

Nx is the number of CFU / ml of the neutralizer toxicity control or of the filtration control

Ny is the number of CFU / ml of the control on the dilution-neutralization or of the control on the filtration test.

Results

I) Biochemical identification of test organisms isolated from axillary swab:

- Staphylococcus epidermidis

- Staphylococcus epidermidis

- Staphylococcus epidermidis

- Staphylococcus epidermidis

- Staphylococcus capitis

2) Trial and validation test

The results are reported in Tables 4 and 5.

Table 4

Table 5

The validation test confirmed the effectiveness of the neutralization of the residual antimicrobial activity and the absence of toxicity of the neutralizer used. Conclusions

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

In particular, it is noted that the log reduction does not increase as a function of the increase in the concentration tested, but an increase in the log reduction is noted as the contact time increases with regard to concentrations â ¥ 0.5%.

Claims (10)

CLAIMS 1. Association consisting of Mangosteen extract of purity between 40% and 99% and 1,3-propanediol of vegetable origin. 2. Association according to claim 1, characterized in that said extract is present in concentrations between 5% and 0.5% and that said 1,3-propanediol is present in concentrations between 95% and 99.5% . 3. Association according to claim 1, characterized in that said extract is 90% pure. 4. Association according to claim 3, characterized in that said extract is present in a concentration equal to 1% and that said 1,3-propanediol is present in a concentration equal to 99%. 5. Association according to claim 1, characterized in that said 1,3-propanediol is currently marketed under the trademark Zemeaâ „¢ by Dupont Tate & Lyle Bio Products. 6. Association of claim 1 for use as an antibacterial / antimicrobial. 7. Association as per claim 1 for use as an active ingredient in cosmetic formulations. 8. Cosmetic formulations which include the association as per claims 1 to 5, as well as usual additives and excipients. 9. Cosmetic formulations as per claim 8 for use as deodorants, creams, after-sun products, products to be used after shaving, products dedicated to intimate hygiene. 10. Cosmetic formulations according to claim 9, characterized in that said creams are creams for the treatment of acne.