FR3054241A1 - METHOD OF ISOLATING AND APPLYING RESIN OF "GALBA ® OIL" - Google Patents

Abstract

Process for the isolation of a total resin contained in an oil derived from a plant belonging to the genus Calophyllum, characterized in that it comprises the following stages: - said oil is mixed with cyclohexane and methanol up to obtaining two immiscible phases, a methanol phase and a cyclohexane phase; said two phases are separated from each other; - The total resin is taken in the methanol phase.

Description

Holder (s): PHYTOBOKAZ Limited liability company, UNIVERSITE DE MONTPELLIER Public establishment, NATIONAL CENTER FOR SCIENTIFIC RESEARCH Public establishment, HIGHER NATIONAL SCHOOL OF CHEMISTRY OF MONTPELLIER.

Extension request (s): Polynesia-Fr

Agent (s): CABINET BREV & SUD.

ISOLATION PROCESS AND APPLICATION OF THE RESIN OF “GALBA OIL”

FR 3 054 241 - A1 (6 /) Method for isolating a total resin contained in an oil from a plant belonging to the genus Calophyllum, characterized in that it comprises the following stages:

Said oil is mixed with cyclohexane and methanol until two immiscible phases are obtained, a methanol phase and a cyclohexane phase,

- Said two phases are separated from one another;

- Said total resin is taken in the methanol ic phase.

Title s Isolation process and application of the resin of "GALBA OIL" ®

The present invention falls within the field of chemical compositions comprising resins of natural origins.

In the present invention, the term “chemical composition” is understood to mean both the pharmaceutical compositions and the cosmetic compositions.

The purpose of pharmaceutical compositions, in particular medicaments, is to treat. Consequently, they contain at least one active principle which has a preventive or curative effect against a disease or a symptom developed by a patient. These pharmaceutical compositions are both therapeutic and diagnostic compositions.

The cosmetic compositions, in particular the dermatological, hair or use compositions on the integuments, are intended to remedy an aesthetic problem which does not endanger the health of the user.

The invention particularly relates to a method of isolating a resin contained in oil from a plant of the genus Calophyllum, the resin as such, as well as its use.

It is known to use, in chemical, especially cosmetic or dermatological compositions, oils derived from plants of the genus Calophyllum,

For example, Tamanu oil, from the seeds of Calophyllum inophyll-aja, is known for its healing, anti-ultraviolet, or anti-infectious skin properties.

Likewise, "HülLE DE GALBA ®" from Calophyllum calaha is also widely used in chemical compositions for its healing and soothing properties.

However, the biological and physiological activity of these oils is not entirely satisfactory. Indeed, part of the constituents of these oils has either no biological activity, or only very low activity.

These constituents with no specific activity or weakly active are restrictive because they have the drawback of reducing the biological effect of the active constituents of the oil.

Therefore, an effective way should be found to separate the biologically active components from those that are inactive in oil.

In general, the oils derived from Calophyllum have a saponifiable part comprising compounds of lipidic nature and an unsaponifiable part comprising non-lipidic compounds. The saponifiable part comprises in particular triglycerides while the unsaponifiable part constitutes the resin.

Scientific research has shown that for an oil, most of the biologically active compounds capable of physiologically interacting are present in the unsaponifiable part. In other words, the biological activities observed for plant webs are generally partly unsaponifiable.

For example, in an olive star or argan oil, tocopherol, a molecule with high biological activity, is present in a significant content in the unsaponifiable part and constitutes a guarantee of the quality of the oil.

In order to enhance these fabrics and increase their biological activity, it is necessary to find a simple, quick and inexpensive solution to dissociate the resin from its oil and concentrate the biological active ingredients within said resin.

It is also appropriate, in order to concentrate the biological active agents of the oil within its resin, to have a high resin extraction yield, with a quality of resin which contains a small portion of pollutants which are not biologically active.

The present invention aims to overcome the drawbacks of the state of the art, by proposing a method for isolating a total resin contained in a canvas from a plant belonging to the genus Calophyllum.

This isolation process includes the following steps:

- Said oil is mixed with cyclohexane and 1 i

methanol until two immiscible i phases are obtained, a methanol phase and a phase | cyclohexane, 1

Said two phases are separated from one another; o takes J said total resin in the metbanolic phase, According to a preferred mode I of the invention, said mixture produced comprises in j mass percentage relative to its total mass î j

- Between 7% and 20.5%, of oil, preferably 13.5%; j

- Between 46.5% and 60.5% of cyclohexane, preferably

53.5%; J

- Between 27.5% and 38.5% methanol, preferably 33%. ((

According to another characteristic of the invention, said plant consists of a Calophyllum calaba. The present invention also relates to the total resin obtained directly by the abovementioned isolation process, as well as to its use as a medicament, more specifically as a principle, active ingredient of a medicament, or even as an agent. anti-inflammatory and / or healing and / or anti-radical agent i and / or antioxidant and / or anti-collagenase and / or for stimulating the I |

melanogenesis and / or as an antimicrobial agent. J

I this iaveation also relates to the process of | fractionation of the total resin obtained by the implementation I of the process, characterized in that:

a) After removal of the total resin, a | organic solvent to said total resin to obtain a mixture; j

b) A solution | sodium hydroxide; until obtaining a first organic phase j and a first phase I i

aqueous;

I

c) The said first phases are separated from one another /

d) We see and then the first organic phase is evaporated until the neutral resin is obtained,

e) An organic solvent and a sulfuric acid solution are added to said first aqueous phase, until a second ethereal phase and a second aqueous phase are obtained i

£) The said second phases are separated from one another;

The second organic phase is dried and then evaporated until the acid resin is obtained. The present invention also relates to the acid resin obtained directly by the aforementioned fractionation process as well as to its use as a medicament, more specifically as an active principle. of a drug, or also as a healing agent and / or anti-radical and / or antioxidant and / or to stimulate the phenomenon of melanogenesis and / or antimicrobial agent.

The present invention also relates to the neutral resin obtained directly by the aforementioned fractionation process as well as to its use as a medicament, more specifically as an active principle of a medicament, or also as an anti-radical agent and / or antioxidant and / or anti-collagenase and / or healing agent and / or antimicrobial agent, in particular against strains

Staphylococcus aureus / Staphylococcus

Cor ^ ebacteriaai minutissimum; Prqpîonobacterium acnes; Propionobacterium g-ranulosust; Malassezia furfur and Candide albicans.

following t epidenaidis}

In addition, 1 / invention also relates to a chemical composition comprising the total resin obtained during the implementation of the isolation process and / or the neutral resin and / or the acid resin obtained (s) during the implementation of the fractionation process.

Other characteristics and advantages of the invention will emerge from the detailed description which follows of the modes:

non-limiting embodiments of the invention.

The present invention relates to a process for isolating a resin, contained in an oil obtained from a plant belonging to the genus Calophyllum. :

Preferably, according to the invention (the oil is an oil) derived from the GalopbyllÎaa calaba plant and is obtained by cold pressing, j

Plants of the genus Calophyllum are tropical trees belonging to the Clusiaceae family according to Crcuaquist classification 1. |

Trees belonging to the genus Calophyllum are cultivated in Guadeloupe, New Caledonia, Madagascar, Polynesia!

but also in Southeast Asia or South America, i

Several species belong to the genus Calophyllum, in particular the following species: C. piaetorwa; C, antillarum, C, rivalare; C. brasiliease "C. blaucoi, C,!

polyaa thuni. " C, iuophyllvun, C. eale & Hiîciun or even C. calaha. J

One can extract from all plants of the genus Calophyllum j an oil advantageously having active biological properties J. i l

Preferably, according to the invention, the Calophyllum oil j is extracted from the seeds of the plant, j

Indeed, the oils from the seeds of the plants of

Calophyllum, whatever the species, have a saponifiable part J with molecules or compounds of lipid nature, i as well as an unsaponifiable part with molecules or i compounds of non-lipid nature. 1

The unsaponifiable part, with molecules or compounds of a non-natural nature. lipid, consists at least in part of a residue I, called "total resin:". |

In the present invention, the term “!

total resin ", the compounds of a non-lipidic nature, that is to say all of the oil compounds which are not soluble in an apolar solvent and which are soluble in a polar solvent" these f compounds being present in the part unsaponifiable.

The object of the present invention is to concentrate the constituents with active biological properties of the oil within its total resin residue, in particular by isolating, that is to say by separating or dissociating, the total resin from its oil.

In order to optimize the isolation of the resin from its oil, to obtain a good extraction yield, to respect the environment, and to minimize the residual toxicity due to its extraction, the total resin is isolated from an oil from a plant of the genus Calophyllum by mixing said oil with cyclohexane and methanol.

It has been surprisingly demonstrated by the inventors that cyclohexane is an apolar solvent capable of separating the total resin from its oil without chemically modifying it and without interacting with the latter. Cyclohexane has the advantage of being slightly toxic because it does not form toxic reactive metabolites, unlike hexane which is a neurotoxic commonly used as an apolar extraction solvent.

Methanol is a polar solvent capable of dissolving the total resin of the oil from a plant of the genus Calophyllum.

According to the invention, the mixture of said oil with cyclohexane and methanol generates the formation of two immiscible phases of different densities: the "methanolic phase" and the "cyclohexane phase".

The “methanolic phase” designs the total resin of the oil which represents the main compound of this phase.

In most of the cases tested by the inventors, the “methanolic phase” is the lowest density phase and is present above the “cyclohexane phase”. However, an opposite position of the phases is possible depending on the density of the molecules present in each phase.

The total resin of the oil is soluble in said polar “methanolic phase”.

On the contrary, the "cyclohexane phase" co-carries, for the most part, all the oil residues which do not correspond to the total resin of the invention. In particular, the "cyclohexane phase" includes all the residues of the oil which have little or no biological activity of cosmetic or pharmaceutical interest, such as triglycerides.

In most cases, the "cyclohexane phase" is the highest density phase and is present below the methanolic phase.

According to the invention, once the separation into two phases is carried out according to a density differential, that is to say that one has dissociated the oil from its resin, the "methanolic phase" is taken. of the “methanolic phase” makes it possible to isolate the total resin, extracted from the oil, from its other constituents. Said other constituents being present in particular in the “cyclohexane phase”,

The removal of the "methanolic phase", that is to say its separation or dissociation from the "cyclohexane phase" can be carried out by any known method, in particular using a pipette or even by simple elimination or flow by gravity of the "cyclohexane phase",

According to a particular embodiment, in order to enrich and concentrate the "methanolic phase" in total resin, it is possible to carry out successive washes of said "methanolic phase" by adding cyclohexane.

Preferably, at least three successive washings will be carried out with a cyclohexane solution. These washes make it possible to remove any traces of non-resinous constituents, in particular the residual triglycerides, which have potentially remained in the “methanolic phase” obtained initially.

The addition of cyclohexane in the initial “methanolic phase” will allow a new separation into two phases, that is to say a new dissociation of the non-resinous constituents and of the total resin, distributed respectively between a “cyclohexane phase” secondary and a secondary “methanolic phase”. To isolate the total resin as much as possible and in order to obtain a good yield, the operation of adding cyclohexane can be repeated several times in the different secondary, tertiary “methaaolic, e” phases, etc. obtained after each separation.

Likewise, in order to recover the potential total resin residues which may be present in the “eyclohexane phase”, it is possible to carry out successive washes of said “eyclohexane phase” by adding “ethanol”

Preferably, at least three successive washings will be carried out with an “ethanol” solution. These washes make it possible to recover by sampling the total resin residues which may be present in said initial, secondary "cyclohexane phases", etc.

According to the invention, the mixture of oil with cyclohexane and methanol can be obtained according to three different embodiments.

In the first embodiment, © added cyclohexane to the oil in a first form. Stir, then add, in a second step, methanol. The mixture is again stirred and allowed to settle until the two phases are obtained.

In the second embodiment, methanol is first added to the oil. Stirred, then cyclohexane is added in a second step. The mixture is again stirred and allowed to settle until the two phases are obtained.

In the third embodiment, a preparation of a cyclohexane / methanol mixture is added to the oil, directly, simultaneously, in a single step. Then, it is again stirred and allowed to settle until the two phases are obtained. .

According to the invention, the first embodiment is a preferred embodiment of the mixture which makes it possible to obtain a clearer separation of the immiscible "methanolic" and "cyclohexane phases".

According to a particular characteristic of the isolation process of the invention, the mixture between the oil, methanol and cyclohexane will be produced so that the oil represents between 7¾ and 20.5%, the cyclohexane represents between 46 , 5% and 60.5% and methanol represents between 27.5% and 38.5% by mass relative to the total mass of said mixture.

According to a particular embodiment of the process for isolating the resin, the mixture comprises 13.5% of oil, 53.5% of cyclohexane and 33% of methanol by mass relative to its total mass.

According to a preferred embodiment of the invention, the process for isolating the resin is carried out for 1 gr of oil derived from Calophyllum calaba seed to which 3 ml of methanol and 5 ml of cyclohexane are added until d 'a "methanolic phase" immiscible with a "cyclohexane phase".

Said “methanolic phase”, generally found in the upper part after mixing, comprises the total resin of Calophyllum calaba, While said “cyclohexanic phase”, generally found in the lower part after mixing, comprises compounds other than the resin, in particular the triglycerides .

This preferred embodiment makes it possible to effectively isolate the resin from “GALBA® OIL” and to effectively remove the triglycerides which can pollute the total resin,

The present invention also relates to a process for fractionating said total resin.

Indeed, chemical analyzes have shown that the total resin is made up of two kinds of compounds: so-called “neutral” compounds and so-called “acid” compounds.

According to the present invention, neutral compounds define "neutral resin", while acidic compounds define "acid resin". Thus, the total resin of the invention can split into at least two residues, the "neutral resin" and the "acid resin".

The neutral resin fraction comprises all of the compounds of the total resin that are not soluble in a basic aqueous solution and that are soluble in an organic solvent.

The fraction of acidic resin comprises all of the compounds of the total resin soluble in a basic aqueous solution · the fractionation process of the invention is based on an acid-base treatment of the total resin.

According to the invention, said fractionation method comprises the following steps:

Step a): An organic solvent is added to said total resin to obtain a mixture;

Step b): A solution of sodium hydroxide (NaOH) is added to the preceding mixture until a first organic phase and a first aqueous phase are obtained;

Step c): The said phases are separated from one another;

Step d) î The first organic phase is dried and evaporated until neutral resin is obtained;

Stage e): An organic solvent and a sulfuric acid solution (H ₂ SO ₄ ) are added to the first aqueous phase, until a second organic phase and a second aqueous phase are obtained.

Step f): The said phases are separated from one another;

Step g): The second organic phase is dried and evaporated until the acid resin is obtained.

Preferably, the organic solvent of the above-mentioned steps consists of diethyl ether, ethyl acetate or dichloromethane.

Step a) consists of taking up the resin in an ether solution, preferably 99% diethyl ether. Preferably, for 1 gr of total resin, between 5 and 20 mL of a solution of ether.

Step b) consists in basifying the mixture of step a) with a strong base, preferably with a 10% NaOH solution. Preferably, the addition of NaOH is carried out drop by drop,

According to the invention, the first organic phase comprises the majority of the neutral products of the total resin.

In the same way, the first aqueous phase obtained during the treatment with the base, in particular NaOH, comprises the majority of the acid products of the total resin, in. especially in their sodium earboxylate form.

After obtaining a first organic phase and a first aqueous phase, to carry out step c), using any technique known to those skilled in the art, the said phases respectively contain the neutral resin and acidic resin.

Step d) consists in isolating the neutral resin present in the first organic phase by evaporating it after drying with a drying agent, for example magnesium sulfate (MgSQ <) or sodium sulfate (Na ₂ SO ₄ ).

Preferably, the drying of the organic phase is carried out with MgSO ₄ .

Preferably, the evaporation is carried out using a rotary evaporator.

The viscous residue obtained, after drying and evaporation, consists of the concentrated neutral resin resulting from the fractionation of the total resin.

Steps e), f) and g) consist in isolating the acidic resin present in the aqueous phase by adding an organic solvent, in particular diethyl ether, then by acidifying it.

During step e) »the addition of organic solvent and of a sulfuric acid solution on the first aqueous phase will make it possible to extract, from said first aqueous phase, the acidified compounds.

More particularly, during step e), the acidification on the first aqueous phase makes it possible to recover the acidic products in the COOH form in the second organic phase formed.

Preferably, the acidification is carried out with a 10% H ₂ SO ₄ solution.

Setting. work of this step e) makes it possible to obtain a second aqueous phase, devoid of acid resin and a second organic phase comprising said acid resin.

Step f) makes it possible to separate 1a. second organic phase comprising the acid resin of the second aqueous phase. It can be carried out by any technique known to those skilled in the art,

Step g) consists in concentrating the acid resin present in the second organic phase by evaporating it after drying with a drying agent, such as, for example, MgSO <or Na ₂ SO ₄ . Evaporation and drying can be carried out by any means known to those skilled in the art.

Thus, the process of fractionating the total resin into acidic and neutral resin makes it possible to separate, according to their acid-base properties, the biologically active compounds from the resin obtained from Calophyllum calaba oil. The fractionation process is a simple process, inexpensive and presenting a good one. The present invention also relates to the total resin obtained by the implementation of the resin isolation method according to the above-mentioned invention, as well as the acid resin and the neutral resin obtained by the implementation of the abovementioned method of connection,

According to a particular application, the total resin can be used as an active principle of a medicament.

Likewise, the neutral resin and / or the acidic resin can be used as active ingredient of a medicament.

Advantageously, the total resin and / or the neutral resin and / or the acid resin can be used as active ingredient of a medicament.

According to another characteristic of the invention, the total resin and / or the neutral resin and / or the acid resin can be used as an anti-radical, antioxidant, anti-inflammatory, anti-collagenase agent and / or as a healing agent. And / or to stimulate the phenomenon of melanogenesis and / or antimicrobial agent.

Indeed, the tests and experiments carried out on the total resin, the acid resin and the neutral resin are repeated in detail below. These tests made it possible to show the specific effect of these resins and their different biological activity compared to l 'oil. Consequently, any “chemical composition”, within the meaning of the invention, including the total resin, the neutral resin and / or the acid resin of the invention has the specific properties and effects linked; resin.

The term “chemical” composition is understood to mean a pharmaceutical or cosmetic composition, for example a dermatological or hair composition.

The present invention has the advantage of specifically isolating the most active Calophyllum oil compounds which are of pharmaceutical or cosmetological interest,

The isolation and the concentration of these active agents within the total resin, acid or neutral allow their use in chemical compositions in particular:

- for medical purposes, thanks to their healing or antimicrobial or anti-inflammatory action or to stimulate the phenomenon of melanogenesis, for example to promote the healing of skin wounds by preventing infections

- For cosmetic purposes, thanks to their anti-radical or antioxidant or anti-collagenase action, for example in cosmetic anti-aging compositions for the skin.

The advantage of the invention is illustrated by the results and experiments below in relation to the corresponding figures. However, these examples and results should not be considered as limiting the invention.

A / Efficiency of the isolation process of the invention

To show the efficiency of isolation of the total resin contained in 1 gr of oil from Calophyllum calaba called "GALBA ® OIL" ", a comparison was made from resin samples obtained by extraction at 1 ethanol, or in the presence of an ethanol / cyclohexane mixture.

la) isolation of the total resin with a methanol / cyclohexane mixture according to the process of the invention

3 ml of methanol are mixed "5 ml of cyclohexane with 1 g of galba oil" With this process, two immiscible and clearly separated phases are obtained "Then the total resin obtained present in the" methanol phase "is taken.

2b) Isolation of the total resin with ethanol

Mix, volume by volume »1 gr of GALBA® OIL with ethanol. Then the total resin is taken from the ethanol-soluble part. With this process, the separation of the soluble and non-soluble phase in ethanol is not clear.

c) isolation with an ethanol / cyclohexane mixture

3 mL of ethanol, 5 mL of cyclohexane are mixed with 1 g of galba oil. It is observed that the separation does not take place, the two solvents remain miscible.

The resins obtained were analyzed by liquid chromatography coupled to mass spectrometry using a column of the reverse phase type with a polar mobile phase. The results obtained are illustrated on the chromatogram of FIG. 1.

These results show that the isolation process of the invention makes it possible to obtain good separation of the total resin from the other compounds of the oil, in particular the triglycerides present at the end of the chromatogram. The triglycerides are visible by a "solid" and peaks between 2.31% and 2.86% at the end of elution on the chromatogram.

The “bulk” of the triglycerides being less important with the isolation process of the invention, this indicates that there are fewer triglycerides contaminating the total resin. Isolation of the resin by the process of the invention is therefore more efficient than isolation by an ethanol process or an ethanol / cyclohexane mixture.

B / Antioxidant and anti-radical activity of the total resin, the neutral resin and the acid resin

B1 / DPPH test

In the context of the DPPH test published by Yoshiaki et al, 2000, the anti-radical nature of a molecule or mixture of compounds is linked to its ability to give an H · radical to the synthetic DPPH · radical of violet coloring to transform it into DPPH ₂ of yellow-green coloring.

The measurement of the decrease in purple coloration over time makes it possible to determine the SC50 (Scavenging Concentration at 50%), that is to say the concentration of active agent allowing the trapping of 50% of the DPPH in solution for a defined time. .

The percentage of trapping for a given concentration is calculated according to the following formula:

% SG = [(White Abs T _{x -} Abs product T _x ) / (White Abs T _x ) 1 * 100

Or :

"Abs white" represents the absorbance measured for the control solution, that is to say the DPPH solution without the compound to be tested;

"Abs produced" represents the absorbance measured for the DPPH solution in the presence of the compound whose anti-free radical power is to be determined>

"Tx" represents the reaction time after which 1 · absorbance is measured.

This calculation makes it possible to draw a graph representing the percentage of trapping of DPPH as a function of the concentration of product to be tested. It is then possible, from the curve obtained, to determine the SC50 (g / 1 or pmole / l). The lower the SC50, the more active the product.

By testing a known reference denoted “A” in parallel, the results can be expressed in pmoles equivalent to A / g of product to be tested. This is the equivalent number of A (in pmoles) necessary to trap the same amount of DPPH as a gram of product to be tested. In this case, the higher the values, the better the antioxidant power.

This DDPH test was carried out for a given time Tx equal to 2 hours, taking as a reference the Trolox known for its strong antioxidant power.

The products tested are:

- "> GALBA OIL" from Calophyllum calaba, before and after esterification, with ethanol,

- the total resin of 1 "HüXDE DE GALBA" of Calophyllum calaba produced by the implementation of the isolation process of the invention,

- the neutral resin, resulting from the above-mentioned total resin, produced by the fractionation process of the invention>

the acid resin, resulting from the abovementioned total resin, produced by the fractionation process of the invention, before and after esterification with 1 ethanol.

Table 1 below summarizes the results of the DPPH test, showing the anti-free radical and antioxidant power of the products tested;

Table 1: Anti-radical power of Galba oil, its total resin, neutral and acidic resin vis-à-vis the compound reference test following the DPPH test SC50 (g / 1) SC50 (pmol / l) Antioxidant power in jimoles eq.Trolox / g of product Trolox 0.0065 26 4000 - Total resin 5.5 4.7 Neutral resin 1 26 Acid resin before sterification 7.5 3.4 Acid resin after esterification 0.8 32.5 "CANVAS OF GALBA ®" before esterification 9.2 2.8 "GALBA ® OIL" after esterification 2.2 11.8

1g of trolox - 4000 micrcsnoles of trolox

The table above shows that the different resins tested have good anti-radical or antioxidant activity just like Galba oil. In particular, the neutral resin has an improved anti-radical activity and about nine times greater "pie" GALBA ® OIL ”before esterification.

In addition, it is observed that the esterification with ethanol allows an improvement in the anti-free radical activity both for the acid resin and for the Galba oil,

In particular, the acid resin, after esterification, with ethanol, can be intended to be used as an anti-radical agent in chemical compositions.

The use of these resins in compositions for their anti-radical and antioxidant effect is therefore possible.

B2 / Test of the absorption capacity of oxygen radicals or "ORAC"

The “ORAC” test measures the capacity of an extract to inhibit the oxidation induced by peroxyl radicals.

The method is based on measuring the decrease in fluorescence of fluorescein in the presence of a chemical oxidant AAPH, a stable free peroxyl radical (Ou et al., 2001).

The product to be tested may be able to protect fluorescein by reducing its rate of degradation. It is therefore considered to have antioxidant power.

Measuring the intensity of fluorescence over time makes it possible to trace the kinetics of degradation of fluorescein in the presence of the product tested. Interpretation of the ORAC analysis data is obtained by:

• the calculation of the area under the kinetic curve in AUC and the reduced area (net AUC t AUC sample - white AUC) • the development of a net calibration curve AUC = £ ([sample]) • the calculation of the antioxidant activity of the sample, in equivalent compared to a referent, from the calibration curve

The AUC is calculated as follows;

AUC = (I tO / I tO) + (I tl / I tO) + (1 t2 / I t0) + ... + (I t _x / l tO)

I = fluorescence intensity, tO - texts at 0 min tl = tesps at 1 min t2 = time at 2 min tx = tees at x min from the experiment,

The higher the value of the net AW, the better the activity.

The calibration curve (net AUC = f ([sample])) makes it possible to determine the concentration of product giving an AUC of 50,

The lower the concentration, the better the activity.

This ORAC test was carried out during a given time Tx equal to 2 hours, by taking measurements every minute, and taking as a reference the Trolox.

The tested products are s

- the total resin of Calopphyllum calaba oil produced by the implementation of the isolation process of the invention,

- resin ; neutral. issue of the resin: total mentioned above, produced speak process fractionation of invention  1 - resin acid, issue of the resin total mentioned above. produced speak process of fresh

of the invention.

Table 2 below summarizes the results of the ORAC test showing the anti-radical and antioxidant power of the products tested.

Table 2: Anti-radical power of the total resin, neutral and acidic resin towards the reference compound Trolox following the ORAC test AUC50 (SZD AUC50 (pmoles / 1) Antioxidant power (pmoles eq.Trolox / g of product) Trolox 0.064 256 4000

Resin total 0.016 - 16000 Resin acid 0.016 - 16000 Resin neutral 0.011 - 23273

* lg of trolox - 4000 micromoles of trolox

The table above shows that the various resins tested do indeed have anti-radical or antioxidant activity. In particular, the neutral resin has an improved anti-radical activity and almost six times greater than the reference compound Trolox.

The use of these resins in compositions for their anti-radical and antioxidant effect is advantageous in particular in the field of cosmetics.

CZ Anti-inflammatory activity of total resin compared to Galba oil

The anti-inflammatory activity is evaluated using the nitrite dosage test according to the method described by Tsai © t al. (1999).

This test consists in determining the capacity to inhibit the production of nitric oxide NO by the activated macrophages after addition of lipopolysaccharides from Escherichia cols, or "LPS".

Inflammation, that is to say the production of NO, was stimulated on mouse macrophages (RAW 264.7) in culture using LPS.

The products to be tested dissolved in a solvent, preferably dimethyl sulfoxide (DMSO), are placed X hours before or X hours after stimulation of the inflammation, this in order to demonstrate a preventive or curative effect.

Negative controls, not stimulated by LPS, called "cell controls" allow to know the natural concentration of nitrite in the absence of inflammation.

Positive controls, stimulated by LPS, for which the product to be tested is replaced by the solvent make it possible to determine the nitrite concentration in the case of an inflammation induced by LPS.

The dosage of nitrites in the medium was carried out using a calibration curve obtained from standard solutions of nitrites.

The tests are carried out in triplicate.

After production, NO is rapidly metabolized in the medium to nitrite ions. The production of nitrites N0 ₂ 'ions in the culture medium is measured as an indicator of the production of NO according to the colorimetric test based on the reaction of Griess (Kim et al., 1995).

Nitrite concentration curves as a function of the amount of product make it possible to find the inhibitory concentration at 50% or IG _SO "This is the concentration at which 50% inhibition of the production of nitrites is observed.

The evaluation of the anti-inflammatory activity by the dosage of nitrites was carried out by carrying out the stimulation of the inflammation for 2 hours, and by taking as:

- cell control (white), a medium which has not been stimulated by LPS, which has a nitrite concentration of approximately 2.5 μM,

- as a positive control, a medium stimulated by LPS in the absence of product to be tested, the concentration of nitrites of which is approximately 15 μM

The products tested are:

- Calophyllum calaba oil or Galba oil, at concentrations varying from 100 to 300 pg / ml

- the total resin, of Calophyllum calaba oil, produced by the implementation of the isolation process of the invention, at concentrations varying from 50 to 125 pg / mL,

Figures 2A and 2B show the results obtained for

1 · "GALBA OIL" and FIGS. 3A and 3B show the results obtained for the total resin,

These figures show a dose effect since, the more the concentration in "GALBA OIL" or total resin increases, the more the nitrite concentration decreases. There is an effect that is both preventive and curative since the concentration of nitrites decreases whether the product is put in 2 hours before or 2 hours after.

Table 3 shows for the above results the

IC _5O obtained (Griess test). A first test was carried out with different concentrations of “galba oil” or total resin (tested in triplicate), a 2® “® test was carried out under the same conditions.

Product tested <-2h) (effect preventive) (+ 2h) (effect curative) Oil of Galba ÏC50 (ug / ml) 1st try 260 ± 11.6 295 * 6.2 2nd try 231 ± 6.8 280 ± 4.4 Resin total of Galba IC50 (ug / ral) 1st try 83.9 ± 7.7 82.5 ± 9.2 2nd try 78.8 ± 3.7 93.2 ± 4.6

The lower the iCso, the better the anti-inflammatory activity. These results confirm the previous observations, both "GALBA OIL" and its total resin exhibit anti-inflammatory activity with a preventive and curative effect on inflammation.

In the case of the 2nd test, the total Galba resin is slightly more effective for the preventive effect <-2h) than for the curative effect.

The IC ₅ o are lower for the total resin 25 compared to the oil of Galba, consequently the use of the total resin is approximately 3 times more effective · than the use of "GALBA OIL" for the 'anti-inflammatory activity'

D / Anti-collagenase activity of the total resin and of the neutral resin

The anti-collagenase activity was evaluated according to the method of Van Wart and Steihbrink (1981). The products to be tested are incubated for 10 minutes with Clostridium histolyticum collagenase. This forms the polar reaction medium since the collagenase is solubilized in a buffer. After incubation, ua substrate, FALGFA or Furylacryloyl-Leu-Gly-ProAla; Sigma F 5135 is added to the reaction medium.

The degradation of this substrate is followed by measuring the decrease in its absorbance at 340 nm.

If the product to be tested has anti-collagenase activity, the reaction on the substrate is inhibited and the absorbance does not decrease.

The measurement is made every minute for 20 minutes.

The percentage of inhibition is evaluated by comparison with a solution containing no active ingredient (white). It can be calculated according to the following formula:

% I - [((Abs white TO-Abs white T20) - (Abs product TO-Abs product T2Q)) / (Abs white TO-Abs white T20)] * 100.

The anti-collagenase activity was measured for the total resin and the neutral resin, the results of the percentage of inhibition are shown in FIG. 4.

Anti-collagenase activity is observed for the total resin and the neutral resin. In the total resin, it is the neutral resin which has the best anti-collagenase activity.

The acidic resin has not been shown because, at the maximum concentration tested (250 pg / ml), it does not show any activity. It is the same for Galba oil "

The total and neutral resins obtained from the isolation and fractionation process according to the invention and originating from a plant of the genus Calophyllum calaba have a collagenase inhibiting action.

E / Healing activity of the total resin, acid and neutral

The ability to stimulate the growth of HACAT keratinocytes is an interesting model for testing wound healing activity in vitro. It is a qualitative test which consists in observing under the microscope the proliferation and migration of keratinocytes in the presence or absence of samples to be tested.

Keratinocytes were cultured. A Cois at confluence, a cut is made in the cell culture and the products to be tested are diluted in the medium. Microscope photos of cell culture are taken at TO (top photos) and T24h (bottom photos). The control corresponds to a medium which, instead of the product to be tested, received the solvent used for the dilutions (DMSO).

The migration of the keratinocytes after treatment with the total resin, the neutral resin, the acid resin and the "GALBA OIL" is compared to that obtained for the control after 24 hours.

Figure 5 shows the healing activity of "GALBA OIL" at a concentration of 200 or 100 ug / ml before and after 24 hours.

FIG. 6 shows the healing activity of the total resin of "GALBA OIL" at a concentration of 50 pg / ml before and after 24 hours.

FIG. 7 shows the healing activity of the neutral resin of "GALBA ® OIL" at a concentration of 50 pg / ml before and after 24 hours.

FIG. 8 shows the healing activity of the acid resin of "GALBA ® OIL" at a concentration of 12.5 ug / ml before and after 24 hours.

F / Melanogenesis stimulation activity

Melanogenesis is the process of producing melanin, responsible for the color of the skin in many tissues. A disruption of melanin production can lead to skin disorders. Consequently, regulation of the phenomenon of melanogenesis by inhibitors or stimulators may be of interest in the cosmetic or pharmaceutical fields, in particular in order to prevent or cure disorders of the pigmentation of the skin.

Before studying the action of the different resins on melanogenesis, the effect of said resins on the cell viability of MDA-435 melanomas was verified. It was found that none of the total, acidic or neutral resins are cytotoxic at the following concentrations 100: 50; 25 and 12.5 pg / ml of product tested. For each of the above-mentioned concentrations, the percentage of cell viability of MDA-435 melanomas is greater than or equal to 100% according to the method of Balekar et al (2012).

The capacity of the total resin, acidic or neutral to stimulate melanogenesis was tested on a cell culture of MDA-435 melanomas, cultured in an atmosphere humidified with 5% CO ₂ at 37®C in a 24-well plate using a MffiM medium (Eagle medium modified by Dulbeceo) containing 4.5 g / 1 of glucose, 10% (v / v) of fetal bovine serum (FBS) and 1% (v / v) of antibiotic.

These MDA-435 melanomas were incubated in the presence of the test products diluted in DMSO at final concentrations ranging from 60 to 20 pg / ml for the different resins, and from 50 to 12.5 pg / ml for the negative control. vitamin C, an inhibitor of melanin production. The cell control consists of MDA-435 melanomas incubated in the presence of DMEM medium and DMSO in place of the product to be tested.

After 24 h of incubation, the supernatant is aspirated. The adherent cells were washed with phosphate buffer (PBS IX) and then peeled off with a trypsin (IX) solution. The detached cells are taken up in 500 μl of medium and then centrifuged. The supernatant is removed while the pellet is taken up in 120 μL of a solution of WaOH (1N) and incubated at 60 ° C. for 1 h. The absorbance is read at 405 nm. After reading the absorbance, the% of melanin is calculated relative to the negative control according to the following formula:

Relative percentage of melanin production = Absorbance sample X 100 / Absorbance blank

The tests were carried out in triplicate. A solution of melanin of known concentration was used in order to produce a standard range and to measure the melanin in the medium.

FIG. 9 illustrates the percentage of stimulation of melanogenesis as a function of the type of resin and the quantity used.

The results show that the total resin and the acid resin are compounds which stimulate the production of melanin in a dose-dependent manner. A stimulation of more than 20% is achieved with 60 pg / ml of acid resin. On the other hand, the neutral resin does not seem to provoke the stimulation of melanin production, the stimulating effect is therefore due to the acidic resin which represents more than 80% of the total resin.

The acid resin is an effective stimulator of the phenomenon of melanogenesis and can be used as such in a chemical composition, in particular of the drug type.

G / Antimicrobial activity of resins

The antimicrobial activity of “GALBA OIL” and of the resins obtained from the oil by implementing the method of the invention was measured on the following strains: Staphylococcus auréUS CIP 4.83 ι Staphylococcus epiderjaidis CIP 68.21 î Corynebacterium adnut î ssi muai

CIP

100652T "·

Propionobactex-ium acnes CIP 531171? ; Propioaobscterlom gr & iml & sum CI '103262T; Malassezia furfur IP 1634.86; Candida albicass DSM 1386, The term CIP or IP indicating that the strain comes from the collection of the Institut Pasteur (Paris) and DSM that the strain comes from the Deutsche Sammlung Von

Microoganismen in Germany.

The antimicrobial activity was evaluated for the different resins on the five aforementioned microbial strains using a method of diluting the product in an agar medium.

The table below summarizes the different agar media used and the incubation conditions.

Table 1: Media and incubation and test conditions for the different strains tested

S. aureus - S. epidermidis

P, acnes - P. granulosum

C. minutissimum

Columbia Agar + 5% of Agar Trypcase- soy blood of sheep Middle and conditions (Biamérieux- 1004415990) LOT (Biamérieux 1004457230) Lot incubation Incubation for 4 8H. at 36 ± 1 ° C Incubation at during 24H anaerobic 36 ± 1 ° C at 48H under Columbia Agar + 5% of Agar Mueller-Hinton blood of sheep C PREPARATION internal - Lot (Preparation internal Middle and 6355) Lot 6356) conditions test Incubation during 48H at 36 ± 1 ° C Incubation at during 96 anaerobic 36 H, ± 1 ° C under

From cultures obtained on agar medium (24H or 48H, at 30 ° C ± 1 ° C or 36 ± 1 ° C), a microbial suspension is prepared extemporaneously in Tryptone and adjusted to 10 'CFU / ml or “Formant Unit Colony per milliliter).

Series of Petri dishes containing decreasing concentrations of resin or "GALBA OIL ®" to be tested are prepared in DMSO.

After drying, the dishes are inoculated in the form of spots (1-3 μl) from the above-mentioned suspensions, corresponding to a deposit on the agar medium of the order of 10 ⁴ CFU. After drying, the dishes are incubated according to the conditions set out in the previous table.

Control boxes without, resin or "GALBA OIL ·" are inoculated and incubated under the same conditions, as controls for microbial growth on agar in the absence of resin or Galba oil. Controls are also prepared with 5% and 10% DMSO in order to validate the results obtained.

The MIC or “minimum inhibitory concentration” of a resin or “GALBA oil” is defined visually as the lowest concentration in the absence of growth versus growth control positive for DMSO. The “MIC” therefore corresponds to the smallest concentration, tested having an activity of inhibiting the growth of the strain.

The table below summarizes the MICs obtained after incubation, from 48 to 96 hours depending on the strains.

Table 2: Minimum inhibitory concentrations (MIC) for samples on the different bacterial strains (g / 1) Strain microbial tested Oil of Galba Resin total Resin acid Resin neutral DMSO 10% DMSO 5% Staphylococcus aureus 0.78 0.2 <0.05 0.39 + + Staphylococcus 0.78 0.2 <0.05 0.39 + +

epidermîdîs Corynebacterîun mi nu t i ssîmin 0.39 0.1 "0.05 0.2 + + Propionobacteriun acnes 0.78 0.2 0.2 0.78 + Propionobacteri um ffranulosum 0.78 0.2 0.1 0.78 - +

It has been found that the various resins and 1 'OIL i

DE GALBA ”have antimicrobial activity on the different strains tested, especially when their MIC is less than 25 g / l. This activity is very important when the MIC is less than 0.05 g / l. I "GALBA ® OIL" has great antimicrobial activity J on the Corynebacterium minufeissimum strain, the i

MIC being 0.39 g / 1. "Galba OIL" also inhibits the growth of other strains with an MIC of 0.78 g / 1. j

The total resin has a high antimicrobial activity on the Coxynebacteriuæ miuutïssiiznun strain with an MIC of 0.1 g / l. It also inhibits the other strains at an MIC of 0.2 g / 1. The total resin is four times more active than 1 "GALBA OIL" on the different strains. These results suggest that the activity of "GALBA OIL" is mainly due to the total resin which represents approximately 20%!

Galba oil.

Total resin is therefore an excellent microbial growth inhibitor, especially for StaphylococcuB epiderraïdis strains; Propionobacterium acnes; Propionobacterîum granuloswn; Malassezia furfur; Caadlda albicans and more specifically for Coxynehacterium where nu t i ssîaïuai.

The acidic resin has a high antimicrobial activity on the following bacterial strains Staphylococcus aureus, Staphylococcus epiderxnidis and CozynebacterÏŒ minatissïmaffi with an MIC lower than 0.05 g / 1. It also has antibacterial activity on the other two strains of

Propioaobacterî wn.

Neutral resin, like 1 "GALBA OIL" and total resin, has a strong aaiiaxexoMenne activity on the CterynehacCeriŒB miaufcis & istuia strain with an MIC of 0.2 g / 1 "It is active on Sfcaphylococcus strains at a concentration of 0.39 g / 1 and on Propi oaobact erî uai strains at a concentration of 0.78 g / 1.

To conclude, the total resin as well as the acidic and neutral resins resulting from the isolation and fractionation process of the invention have an antimicrobial activity with an MIC less than 0.8 g / L, which makes these resins effective in combating against microbial growth of the above-mentioned strains (bacteriostatic action) but also as a bactericidal agent, Total resin, acid resin and neutral resin have at least bacteriostatic activity, depending on their concentration, or even bactericide on the above-mentioned strains.

Claims (14)

1. A method of isolating a total resin contained in an oil from a plant belonging to the genus Calophyllum, characterized in that it comprises the following steps; - Said oil is mixed with cyclohexane and methanol until two immiscible phases are obtained, a methanol phase and a cyclohexane phase, - The two phases are separated from each other i - Said total resin is taken in the methanolic phase. 2. A method of isolating a total resin according to claim 1 characterized in that said mixture produced coüiprend a% by mass relative to its total mass; - Between 7% and 20.5%, of oil, preferably 13.5%; - Between 46.5% and 60.5% of cyclohexane, preferably 53.5%; - Between 27.5% and 38.5% methanol, preferably 33%. 3. A method of isolating a total resin according to claim 1 characterized in that said plant consists of a Calophyllum calaba. 4. Method for fractionating the total resin obtained by implementing the method according to claim 1, characterized in that: a) After removal of the total resin, an organic solvent is added to said total resin to obtain a mixture; to the previous mixture, a sodium hydroxide solution until an organic and a first phase are obtained b) hydroxide of the first aqueous phase is added j c) said first phases are separated from one another; d) The first organic phase is dried and then evaporated until the neutral resin is obtained, e) An organic solvent and a sulfuric acid solution are added to said first aqueous phase, until a second organic phase and a second aqueous phase are obtained t £} Said second phases are separated one on the other> g) it is dried and then the second organic phase is evaporated until the acid resin is obtained. 5. Total resin obtained by the implementation of the isolation process according to one of claims 1 to 3. 6. Neutral resin obtained by the implementation: implementation of the fractionation process according to claim 4. 7. Acid resin obtained by implementing the fractionation process according to claim 4. 8. Total resin obtained by implementing the isolation process according to one of claims 1 to 3 for its use as a medicament. 9. Total resin according to the preceding claim intended to be used as an anti-inflammatory agent and / or healing agent and / or anti-radical and / or antioxidant and / or anti-collagenase and / or as an antimicrobial agent and / or to stimulate melanogenesis. 10. Neutral resin according to claim G for its use ea as a medicament. 11 »Neutral resin according to the preceding claim intended to be used as anti-radical agent and / or antioxidant and / or anti-collagenase and / or healing agent 5 and / or antimicrobial agent. 12. Acid resin according to claim 7 for its use as a medicament. 13. Acid resin according to the preceding claim intended to be used as a healing agent and / or anti-free radical and / or to stimulate the phenomenon of melanogenesis and / or antimicrobial agent. 15 14. Chemical composition comprising the total resin obtained during the implementation of the isolation process according to one of claims 1 to 3, and / or, the neutral resin and / or the acid resin obtained during the implementation of the fractionation method according to claim 4. 1/5