KR20160028495A - Slimming composition - Google Patents

Abstract

The present invention relates to the use of preparations comprising sulfated oligosaccharides comprising spermine and / or spermidine as active slimming ingredients in cosmetic, pharmaceutical and / or health supplement food compositions. One aspect of the present invention is to provide a cosmetic, pharmaceutical or dietary supplement composition having a slimming effect.

Description

SLIMMING COMPOSITION [0001]

The present invention relates to active slimming ingredients and to industrial processes for making them.

The entire range of cosmetics and pharmaceuticals is associated with active slimming ingredients. The various active ingredients in these areas are designed to address the unpleasant tissue problem of "orange peel skin" caused by fat cells overloaded with fatty acids.

Suffermin and spumidine are polyamines present in fat tissue and especially fat cells, respectively, at concentrations of 0.16 and 0.30 nmol / mg, respectively (Pedersen et al., 1989, Mol. Cell. Endocrinol, 62, 161-166).

In vitro, these polyamines have been proven to stimulate three major enzymes in the "fat production" process, which is the process of storing fat in fat tissue in the form of triglycerides.

The three enzymes are as follows.

- sn-glycerol-3-phosphate acyltransferase or GPAT,

- 1,2-diacyl-sn-glycerol acyltransferase or DGAT,

- Mg ^{2 +} phosphatidate phosphohydrolase or MGPPH.

In 1996, Jamdar et al. (1966, Enzyme protein, 49, 222-230) found an association between the concentration of spermine and spumidine in fat tissues and increased body fat in obese rats. This increase is associated with a strong stimulation of the enzyme relating to the lipogenesis process as GPAT, or DGAT MGPPH, a significant amount of a polyamine associated them in the body (in vivo) via local store process is demonstrated.

In addition, lipoprotein lipase (LPL), the fourth enzyme responsible for the transport of fatty acids from the bloodstream to the fat cells, appears to stimulate such activity in the presence of spermine and spperidine (Giudicelli et al., 1976, FEBS Lett , 62, 1, 74-76).

Finally, spermine and spumidine have been shown to act as "insulin-like" factors in fat cell metabolism, which promotes glucose transport and stimulates pyruvate dehydrogenase to improve conversion to triglycerides (Rutter et (Olefsky et al., 1979, Horm. Metab. Res., 11, 209-213; Lockwood et al., 1992, Biochem, J., 285, 435-439) et al., J. Biol. Chem., 1974, 249, 24, 7717-7722; Richelsen et al., Biochem. J., 1989, 261, 2, 661-665).

Finally, it has been confirmed that the fibroblasts differentiate into adipocytes when the spumidine ratio is significantly increased through Bethell et al. (1981, Biochim. Biophys. Res. Commun., 102, 1, 272-278). Therefore, this polyamine appears to play an important role in the process of converting fibroblasts into adipocytes.

Thus, spermine and spermidine promote fat storage in adipocytes (through the stimulation of enzymes involved in the fat production process) and inhibit the release of these lipids (through the inhibition of fat melting). When these two polyamines are blocked, it is expected that the fat production process will be inhibited and fat melting will be promoted, resulting in an overall slimming action.

To date, there are no active ingredients known to act on spermine and / or spermidine.

≪ Purpose of the Invention &

The main object of the present invention is to solve a new technical problem of industrial process provision for active slimming ingredient manufacture.

The present invention is designed to address new technical problems in the action of spermine and / or spermidine, molecules involved in lipogenesis / thawing, particularly at the adipocyte level.

The present invention is specifically designed to solve the technical problems described above in an industrial, reproducible and reliable manner at the lowest cost in the cosmetic, health supplement and / or pharmaceutical fields.

Surprisingly, it is possible to produce a composition comprising one or more active ingredients capable of capturing spermine and / or spermidine, which composition is particularly capable of acting on fat production and / or fat melting at the fat cell level of humans .

Accordingly, the present invention relates to a substance that captures spermine and / or spermidine.

Since spermine and / or spermidine capture can reduce fat production, it can reduce fat storage and increase fat melting, thereby promoting fat breakdown and inducing a double action on slimming action.

According to the inventors, "capture of spermine and / or spumidine" means: any method, preferably a compound called "substance that captures spermine and / or spumidine " through complexation And the association of spermine and / or spermidine.

Advantageously, the substance that captures spermine and / or spermidine in an effective manner is a substance capable of inhibiting fat production by at least 50% and / or promoting fat melting by at least 50% as tested in suspension in normal human fat cells to be.

Inhibition of fat production is known to reduce fat (lipid) storage in adipocytes. Promotion of fat melting is known to increase the concentration of non-esterified fatty acids and release it from adipocytes.

Among the possible active substances selected, sulfated polysaccharides belonging to the sulfated galactan system such as carrageenan or agar showed unexpected effects.

Particularly, the sulfated polysaccharide exhibiting the best characteristics is a structure containing at most 20, preferably at most 10 saccharide units.

Such materials can advantageously be prepared by hydrolysis of one or more sulfated polysaccharides.

Of these preferred polysaccharides or oligosaccharides, kappa carrageenan is preferred for better results.

The following two methods are advantageously used for carrageenan hydrolysis.

● Pseudomonas Enzymatic hydrolysis by carrageenase extracted from carrageenovora (Knutsen et al., 2001, carbohydr. res., 331, 101-106).

Chemical hydrolysis: Basic hydrolysis is generally slow and can open the loop. A hot reaction in the presence of hydrochloric acid (Ekstroem et al., 1983, carbohydr. Res. 116, 89-94) or sulfuric acid (Rochas et al., 1981, Polym. Bulletin 5, 81-86) .

Oligosaccharides produced by hydrolysis of carrageenan are composed of carrabiose unit, neocarrabiose unit and neocarratetraose unit chain (see Chemical Formula 1).

(1) Carabios , Neokarabios  And Neokaratetraose

 Generally, enzymatic hydrolysis of carrageenan produces oligosaccharides containing neo-carabiose units, and acid hydrolysis is known to produce oligosaccharides based on carabiose units (Kono et al., US Patent 4,748,032).

Advantageously, the present invention relates to an improved hydrolysis process at a sufficient temperature and for a time sufficient to place the carrageenan in a solution of at least one carrageenan and to dissolve the carrageenan in a solvent medium (water is preferred). The temperature advantageously exceeds 30 < 0 > C.

The hydrolysis process advantageously involves hydrolysis in an acid medium, for example using inorganic acids such as hydrochloric acid. This hydrolysis is carried out at a sufficient temperature and for a sufficient period of time to allow the carrageenan to hydrolyze into oligosaccharides. The reaction pH is advantageously less than 3, with a pH of less than 2 being preferred. It is generally preferred to hydrolyze the carrageenan over an inorganic acid such as an acid, for example hydrochloric acid, for over 5 hours (over 10 hours preferred, over 15 hours preferred).

Advantageously, an inorganic salt such as a basic solution, for example sodium hydroxide, is added to stop the reaction. The final pH of the preparation is generally 3.5-5.

The inventors preferentially adopted an acidic hydrolysis process from kappa-carrageenan. This process involves an innovative change made by the inventor in such a way as to obtain the optimal amount of kappa carabiose unit (see Chemical Formula 2) containing a beta-1,4 bond between two saccharide units. However, the present invention is not limited to such a process. Thus, other hydrolytic changes from iota and / or lambda carrageenan are utilized.

(2) kappa Carabios  unit

The oligosaccharide solution prepared according to the characteristics and concentration of acid, temperature and reaction time shows various concentrations of carabiose and neokaratetraose.

In the present invention, the oligosaccharide solution capable of capturing spermine and spermine has a degree of polymerization or dp 2 (dp 1 = 1 carbaspic unit) to 20 (preferably 2 to 10, preferably 2 dp units Bios) is preferred.

After the (chemical or enzymatic) hydrolysis step, all insoluble fractions are removed from the mixture by centrifugation or filtration.

If necessary, the oligosaccharide solution can be purified, for example, by discoloration in activated carbon or ion exchange resin, or the oligosaccharide solution can be separated by a membrane or gel chromatography.

For example, the solution can be transformed into a powder, in particular through freeze drying, atomization, crystallization, and the like.

Advantageously, the solution of the sulfated oligosaccharide comprises at least 0.1% carabiose (advantageously at least 0.2% (p / p), preferably at least 0.5% (p / p) It does not generally contain more than 10% (p / p) of carabios. The sulfated oligosaccharide solution contains less than 1% of carabios. However, the solution of sulfated oligosaccharides can be lyophilized, so that a higher proportion of carabiose can be produced, for example, up to 30% or even up to 40% (p / p) of the freeze-dried composition.

Such solutions of sulfated oligosaccharides are considered to be active ingredients in the present invention and can be diluted in the final composition. Typically, a solution of the sulfated oligosaccharide is used at a concentration of 0.01 to 10% by weight (preferably 0.1 to 5% by weight) of the final composition. Thus, the final composition generally comprises a carabiose ratio of 1 x 10 ^-5 to 10 wt.% (Preferably 2 x 10 ^-5 to 0.1 wt.%).

The solution of sulfated oligosaccharide is advantageously below pH 7, preferably at pH 3.5 to 5.

Oligosaccharide solutions are known to be liquid compositions and may be in the form of gels or hydrogels, which are based on, but not limited to, water. The solution may be an aqueous solution, may consist of various types of ions or solutions, and may be composed of insoluble or partially soluble compounds. The solution may be a solution of a solvent other than water, in which the sulfated oligosaccharides may be dissolved. This solvent advantageously meets the criteria for dermatology.

The compounds according to the present invention are used in the preparation of compositions and, in particular, can be administered topically or orally, and are used in the form of cosmetic or dietary supplements, skin-pharmaceutical or pharmaceutical compositions. Thus, in the case of such compositions, excipients include, for example, preservatives, emollients, emulsifiers, surface active agents, moisturizers, thickeners, conditioners, matting agents, stabilizers, antioxidants, tackifiers, polishes, film formers, ≪ / RTI > and sunscreen. Such excipients include, but are not limited to, amino acids and derivatives thereof, polyglycerols, esters, polymers and cellulose derivatives, lanolin derivatives, phospholipids, lactoferrin, lactoperoxidase, sucrose based stabilizers, vitamin E and its derivatives, natural and synthetic waxes, The present invention relates to a process for producing an oil-in-water emulsion, comprising the steps of: extracting an oil from an oil, a triglyceride, an unsitled oil, a phytosterol, a plant ester, a silicone and a derivative thereof, a protein hydrolyzate, a jojoba oil and a derivative thereof, , Glycine, and paraben, and it is preferable to select from the group consisting of butylene glycol, stearic-2, stearic-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, Propylparaben, butylparaben, butylene glycol, natural tocopherol, glycerin, sodium dihydroxycityl phosphate Isopropyl alcohol, isopropyl alcohol, isopropyl hydroxycetyl ether, stearic acid glycol, triisononanoin, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, glycerol, bisabolol, dimethicone, sodium hydroxide , PEG 30-dipolyhydroxy stearate, capric / caprylic triglyceride, cetearyl octanoate, dibutyl adipate, grape seed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum, Propylene glycol isostearate, PEG 8 beeswax, glycerides of hydrogenated palm oil, lanolin oil, sesame oil, cetyl lactate, isopropyl palmitate, isopropyl palmitate, isopropyl palmitate, Lanolin alcohol, castor oil, titanium dioxide, lactose, saccharose, low density polyethylene and isotonic saline solution It is preferred to select from the stage.

Advantageously, the above-mentioned compositions are particularly suitable for use in bottles or tubes, especially shower gels, shampoos, lotions, especially oil in water or water in oil or multiple emulsions, micro-emulsions or nano-emulsions , Or a water or oil solution in silicone, a cream or water gel, or a flowable gel; Lotions, especially in glass bottles, plastics or pump dispensers or aerosols; Vial; Liquid soap; Skin bar; Ointment; Foam agent; For example, in the form selected from the group consisting of sticky anhydrous (liquid, paste or solid being preferred) identified in lipstick.

As used herein, the term " topical application "refers to the application or spraying of a composition according to the present invention to the skin surface and / or mucosal surface.

As used herein, the expression " dermatologically acceptable "refers to the use of a composition or component of a composition in contact with human skin without unacceptable toxicity, incompatibility, instability, allergic response, or the like .

Many cosmetically active ingredients are known by those skilled in the art to improve the health and / or appearance of the skin. The skilled person knows how to make a cosmetic or dermatological composition for the best effect. In addition, the compounds described in the present invention can exhibit a synergistic effect when they are used in combination with other substances. This combination is also covered by the present invention. The CFTA Cosmetic Ingredient Handbook Second Edition (1992) describes a variety of cosmetic and pharmaceutical applications that are particularly applicable topically and are frequently used in the cosmetic and pharmaceutical industry. Some examples of components of this lineage include, but are not limited to, abrasives, absorbents, esthetics such as perfumes, pigments, dyes, essential oils, astringents etc. (e.g., clove oil, menthol, camphor, Anti-acne agents, anti-odor agents, non-foaming agents, anti-microbial agents (e.g., iodopropyl butyl carbamate), antioxidants, antioxidants, (For example, hydroquinone, kojic acid, ascorbic acid, phosphoric acid phosphate, phosphoric acid, phosphoric acid, phosphoric acid, phosphoric acid, phosphoric acid, (E.g., magnesium, magnesium, ascorbyl glucosamine), conditioners (e.g., moisturizers), skin sedatives and / or access inhibitors (e.g., panthenol and its derivatives such as ethyl panthenol), aloe vera, And derivatives thereof, allantoin, bisabolol and dipotassium glycyrrhizinate), thickeners, vitamins and their derivatives or equivalents.

Advantages of the present invention include, in particular, at least another slimming active ingredient addition selected from the group consisting of an inhibitor of phosphodiesterase enzyme, an activator of adenylate cyclase, a cyclic AMP or lipophilic derivative and / or a mixture thereof .

Preferably, the present invention comprises the addition of at least caffeine and / or forskolin and / or theophylline and / or theobromine.

For example, various plant extracts commonly used by manufacturers in slimming preparations such as root extracts of Coleus Forskohlii, peach extracts of cecropia obtuse or uva lactuca, It can also be used as a slimming active ingredient.

In particular, it is advantageous to use the active ingredient of the present invention in combination with caffeine. Indeed, these components have been shown to exhibit an auxiliary mechanism of action. Preferably, caffeine derivatives and / or salts, in particular caffeine, in the form of a caffeine carboxylate as shown in French patent application FR2624010 (Pierre Fabre Cosmetique), are administered concomitantly with the active ingredient of the invention, 10% by weight (preferably 1-5% by weight).

Particularly advantageous is the combined use of the active ingredients of the present invention with plant extracts containing at least one active ingredient of adenylate cyclase, in particular forskolin or forskolin.

Preferably, forskolin, such as a Coleus Forskohlii extract or a Plectanthus barbatus extract, is administered in combination with the active ingredient of the present invention, 1% < / RTI > by weight (0.05 to 0.25% by weight is preferred).

It is particularly advantageous to use the active ingredient of the present invention in combination with one selected from the following.

(GAG) stimulators and / or elastin stimulators and / or collagen stimulators, in particular retinol and / or vitamin C and derivatives thereof, tetrapeptides available under the trade name Matrixyl TM from Sederma, France A tetrapeptide containing 50 to 500 ppm of palmitoyl-Gly-Gln-pro-Arg,

- lipoprotein lipase inhibitors, such as the extracts of Peru liana (Uncaria tomentosa) or St. John's wort extracts described in patent US2003086949 (Coletica)

- active ingredients mimicking beta endorphin action to improve skin barrier function, such as cacao bark extracts cited for example in patent application US 2006069032,

For example, to provide a feeling of well-being, such as Tephroline of the plant tephrosia purpurea (Soliance), an active ingredient that stimulates beta endorphin synthesis,

- active ingredients that stimulate cell proliferation and / or cell differentiation, such as the following molecules: NGF, a-MSH,? Endorphin or derivatives (for example, P3-endorphin as shown in patent application FR 2857874 ),

Active ingredients which protect fibroblast growth factor (FGF) against degradation, in particular extracts of FGF2, in particular Hibiscus Abelmoscus extract, such as the vegetable extract shown in the patent application by the applicant published as GB244036,

- active ingredients that stimulate the activity and / or proliferation of fibroblasts, such as fermented peptides of soybeans, such as the product sold by the Applicant under the trade name Phytokine ^TM , optionally in combination with Hibiscus Abelmoskus extract,

- active ingredients that stimulate hyaluronase synthase, especially hyaluronase synthase 2 as shown in patent FR2893252,

- an active ingredient which stimulates the activity and / or synthesis of Lysyl Oxidase, such as LOXL, especially dill extract, such as the ingredient indicated in patent number FR 2855968 for elastic fiber stimulation,

- inhibiting PLA2, in particular active ingredients of anti-inflammatory action such as the ingredients shown in patent FR2847267 and pueraria lobata root extract (Inhipase®)

Active ingredients which change skin color, such as active ingredients for lightening and / or whitening,

Active ingredient with drainage properties, such as hesperitine laurate (Flavagrum®) or quercitine caprylate (Flavenger®).

The invention relates to a process for the preparation of (i) sulfated oligosaccharides for capturing spermine and / or spermidine, wherein sulfated oligosaccharides are more particularly indicated in the full description and examples of the invention, and (ii) caffeine, theophylline, Or a derivative thereof and / or a salt thereof.

A derivative is a compound formed from a similar compound when one atom is replaced by another atom or group of atoms, or a compound that may be expected to be derived from another compound. This definition is common in organic chemistry.

The present invention relates to the use of (i) sulphated oligosaccharides to capture spermine and / or spermidine (sulphated oligosaccharides more particularly shown in the full description and examples of the invention being preferred) and (ii) Caffeine, theophylline, theobromine, and a combination of at least one of the group consisting of forskolin or derivatives and / or salts thereof.

Accordingly, the present invention relates to a method of slimming management comprising the use of at least one active ingredient, or a cosmetic composition comprising such an active ingredient, as mentioned above or below. Such cosmetic management includes topical application of the active ingredient to the skin site to be treated. Administration of such a cosmetic composition can be carried out through the administration of the health supplement composition (oral administration). The site of the skin to be treated in the subject is advantageous in the region where the fatty acid contains more fat cells than the remaining fat cells in the skin tissue.

These areas are usually orange peel skin areas.

The present invention also relates to a therapeutic method for reducing the amount of lipid stored in adipocytes, which comprises, as mentioned above or below, at least one active ingredient or one cosmetic composition comprising such active ingredient Administration or topical application. Therapy is advantageous to combine reduced fat production and increased fat loss. Such treatment may be limited to the area of the skin to be treated.

Other objects, features, and advantages of the invention will become apparent to those skilled in the art from a reading of the following description, given by way of example only, and not by way of limitation.

The embodiments are an important part of the present invention, and all of the features that appear to be new in view of all of the prior art circumstances recognized from the entire specification including the embodiments are an important part of the invention in terms of functionality and universality.

Thus, each embodiment is in the overall scope.

In addition, all percentages are expressed by weight unless otherwise specified in the examples, unless otherwise specified, temperatures are expressed in degrees Celsius, and pressure is atmospheric unless otherwise specified.

Example

Example  One : Production of oligosaccharides

38.5 mL of pure laboratory water was heated to 60 < 0 > C.

0.49 g of sodium chloride and 1.67 g of kappa-carrageenan were added.

The solution was stirred at 60 < 0 > C for 25 minutes. 8.33 g of a 1N hydrochloric acid solution was added to initiate the hydrolysis reaction and the mixture was maintained at a temperature of 60 < 0 > C during the whole process. At the predetermined hydrolysis point, 9 mL of 1 N sodium hydroxide solution was added to stop the reaction. Thus, the pH of the preparation was 4 to 4.5.

The solution may then be filtered through a 500 nm filter and preservative and gelling agent may be added to the preparation.

In most of the following examples, experiments were performed on the filtrate (without preservative and coagulant).

Example  2 : Development of the composition of oligosaccharide preparations in terms of hydrolysis time

Sulfated oligosaccharide solutions were prepared according to Example 1 with hydrolysis times of 2, 5 and 16 hours.

This solution was then analyzed by HPLC (Waters Alliance 2685, column TSK gel 2000 PWXL) connected to the refractometer detector.

A standard range of glucose was analyzed together to reveal the composition in the mixture of carabiose and neokaratetraose.

From the results shown in Table 1, it was confirmed that the longer the hydrolysis time, the higher the concentration of carabios.

Hydrolysis time (hours) % Carabios % Neokaratetraose 2 0.106 0.228 5 0.365 0.180 16 0.890 0.043

The% carabiose or neokaratetraose was expressed as% by weight based on the total weight of the filtrate, and the pH of the filtrate was adjusted to 4 to 4.5.

Example 3: spermine and spur vitro of oligosaccharides effective for pyrimidine captured (in vitro) demonstrated

principle:

When spermine and sppermin were placed in the presence of deoxyribonucleic acid (DNA), DNA precipitation was observed and the absorbance of the mixture read at 600 nm was significantly increased.

When a compound that "trapped" spermine or spumidine was added to the polyamine solution in advance, precipitation did not appear when the DNA solution was added.

Then, the protection ratio indicated by the active component tested in terms of polyamine-induced DNA precipitation could be calculated.

protocol:

0.4 g of fish DNA (Sigma) was added to 160 g of pH 6 phosphate buffer (0.06 M potassium phosphate and 0.001 M sodium phosphate).

10 uL of a 50 mM aqueous solution of spermine (Sigma) was dispensed into each well of a 96-well plate.

Then, we did the following.

- solution of sulfated oligosaccharides (active ingredient) or aqueous solution of 150 mM aqueous adenosine triphosphate (or ATP, Sigma)

- or 90 μL of pure laboratory water ("control" well or "100%" well)

The plate was stirred for 5 minutes at room temperature and then 100 μL of DNA solution was added to all wells.

After 5 seconds of manual stirring, the absorbance of the wells was read at 600 nm.

In the presence of spermine, the protection ratio of the active component in terms of DNA precipitation was calculated as follows.

% Protection = 100 - [(OD _{Active or ATP}  / OD _100%  ) x 100]

Refers to the absorbance of the DNA, spermine, or spermidine and the well after containing the active ingredient or ATP, via "OD _{Active or ATP} ".

"OD _{100 %} " means the absorbance of wells containing DNA or spermine or spermidine after precipitation.

The higher the protection ratio, the higher the "capture" of spermine by the active ingredient and the less DNA precipitation.

The same experiment can be performed by replacing 50 mM spermine solution with 300 mM aqueous solution of spermidine (Sigma).

Oligosaccharide solutions were prepared according to Example 1 with hydrolysis times of 2, 5 and 16 hours.

After diluting to 10% in pure laboratory water, several solutions were tested.

The results are shown in Table 2 below.

Hydrolysis time % Spermine  protect % Spudmin  Protection ratio 2 3.9 29.9 5 4.5 28.2 16 10.3 34.3

The longer the hydrolysis time, the greater the carabiosity of the oligosaccharide solution and the greater the capture of spermine and spermidine.

Thus, an active ingredient containing at least 0.2% carabiose, based on the weight of the sulfated oligosaccharide solution, is preferred and an active ingredient containing at least 0.5% carabiose is preferred.

Example  4 : variety Sulfation Galactan  After hydrolysis Carabios  Comparison of ratios

A solution of sulfated oligosaccharides was prepared using kappa, iota and lambda carrageenan and agar according to Example 1 with a hydrolysis time of 16 hours.

Then, the extract was analyzed by HPLC according to the technique shown in Example 2, except for the agar hydrolyzate which can not be analyzed under the same conditions (agarobiose unit).

After dilution to 3% in pure laboratory water, the capture assay of spumidine was performed according to the protocol shown in Example 3.

Hydrolyzed Galactan Capture% % Carabios % Neokaratetraose Kappa-Karajien 16.1% 0.878 0.155 Iota - Karajien 13.1% 0.292 0.344 Lambda-Karajien 13.5% 0.179 0.197 Agar 8.9% Unapplied Unapplied

Carazene hydrolysates (kappa, iota and lambda) showed the best effect in capturing spermine. This capture was related to the carabiose ratio in the product.

Example 5 : Effect of oligosaccharides on inhibition of adipocyte formation in normal human adipocytes

The suspension from the 42-year-old women who have abdominal surgery fat cells isolated from samples collected during the evaluation of the in vitro (ex vivo) Inhibition of lipogenesis.

(Hydrolysis time: 16 hours) or a standard test solution (1 mM theophylline or caffeine) in the absence of such solution (control) and an adipocyte suspension of 37 or 37% Lt; 0 > C for 1 hour.

Each test was performed three times.

The radioactive label [2- ¹⁴ C] acetate was then added to the various test samples at a concentration of 2.5 μCi / mL.

After incubation at 37 ° C for 4 hours, the lipids were extracted through methanol / chloroform / water and then dehydrated. The radioactivity in cpm per minute was quantified via liquid scintillography (LKB 1210 Rackbeta).

The results are shown in Table 4 below.

Statistical analysis was performed according to Dunnett's multiple comparison tests.

process Concentration in water cpm Standard Deviation % contrast P Control group - 472253 4474 100 - Theophylline 1 mM 27225 6195 6 P < 0.01 Caffeine 1 mM 20741 2932 4 P < 0.01 Sulfated oligosaccharides 5%
3% 109647
97139 7064
9691 23
21 P < 0.01
P < 0.01

Depending on the laboratory conditions, the solution of sulfated oligosaccharides tested at 3-5% significantly inhibited fat production in a dose-dependent manner.

Example 6 : Demonstration of effect of the product on lipofusion stimulation in normal human adipocytes

Fatty mild stimulation activity was assessed in a suspension of human adipocytes isolated from samples taken during the peritoneal plasty of a 34 year old female.

(Hydrolysis time: 16 hours) or a standard test solution (1 mM theophylline or caffeine) in the absence of such solution (control) in a proportion of 3 or 5% of the total weight of the culture prepared according to Example 1 And an adipocyte suspension were incubated at 37 [deg.] C for 2 hours. Each test was performed six times.

At the end of the incubation time, the non-esterified fatty acid (NEFA) present in the culture was determined with a commercial administration kit (OXOID 46 551).

The results are shown in Table 5 below.

process density NEFA  (μM) Standard Deviation % contrast P Control group - 96 6 100 Theophylline 1 mM 537 45 560 p < 0.01 Caffeine 1 mM 525 40 547 p < 0.01 Sulfated oligosaccharides 5%
3% 200
180 10
12 209
187 p < 0.01
p < 0.01

Depending on the experimental conditions, the solutions of sulfated oligosaccharides tested at 3% and 5% significantly promoted fat melting in a dose-dependent manner.

Example  7 : Invention of oligosaccharides in combination with caffeine Slimming  Proof of action

The slimming effect of the hydro-alcohol gel containing a mixture of 3% caffeine + 3% sulfated oligosaccharide solution (hydrolysis time 16 hours) prepared according to Example 1 was measured using a 3% caffeine-containing hydro-alcohol gel .

The product was applied to the thighs of healthy volunteers and was measured in centimeters over an 8 week period to assess slimming.

In fact, 25 applicants aged 21 to 49 were selected and tested according to specific criteria (body mass index, presence of cellulite). During the preclinical study, the weight of each volunteer was monitored and could not change at ± 2 kg. The product was applied randomly to the right femur / left femur as follows: one femur treated with a combination (caffeine + oligosaccharide solution), and one femur treated with caffeine. The gel was applied twice a day using a small round operation until the entire product was introduced for 8 consecutive weeks.

The formulations selected in this trial were hydro-alcohol gels based on 75% water and 25% denatured alcohol ratios.

During the study, the same technician measured the circumference of the femur in centimeters in the middle of the applicant's femur by taping without compression.

Using the laser range - identification system for the measurement site, the evaluation at the same position of each thigh was made reproducible and accurate.

Measurements were made before application (T0), then 14 days, 1 month, and 2 months later.

The results of this clinical trial are shown in Table 6 below.

time Caffeine Caffeine + Sulfation
Oligosaccharides Significance of results
("Caffeine + Sulfation  Oligosaccharides "vs." caffeine ") 14 days 0 cm -0.1 cm Not significant 28th -0.1 cm -0.4 cm p < 0.001 56 days -0.3 cm -0.6 cm p < 0.01

After 14 days of treatment, it was found that the applicant 's femoral circumference was reduced by applying a gel containing caffeine and sulfated oligosaccharides. It should be noted that the gel containing caffeine had no slimming action.

The excellent effects of the combination of caffeine and sulfated oligosaccharides were even more pronounced after 28 and 56 days of treatment.

In fact, on the 56th day, the average femoral circumference of the volunteers using caffeine + oligosaccharide gels decreased by 0.6 cm, whereas those who applied caffeine gel decreased by only 0.3 cm (significant difference, p <0.01).

Example 8: Preparation of "oil-in-water & Lt ; RTI ID = 0.0 &gt; emulsion & lt ; / RTI &gt; type of cosmetic or pharmaceutical formulation

Formulation 8a :

Formulation 8b :

Formulation 8c :

Example  9 : " Wastewater Quot; type of formulation of the invention &lt; RTI ID = 0.0 &gt;

Example  10 : Use of the product of the invention in the form of a "shampoo or shower gel" type

Example  11 : Mercury Gel  Use of the product of the invention in the formulation

Example 12: "triple emulsion (triple emulsion)" Use of the product according to the present invention in the formulation types

Primary emulsion W1 / O

Secondary emulsion W1 / O / W2

Example  13 : Preparation of Pharmaceutical Formulations Containing the Products of the Invention

Formulation 13a: Tablet preparation

* For example, the product of the present invention was prepared following the drying step after the extraction process (hydrolysis time 16 hours) shown in Example 1.

Formulation 13b: Ointment Manufacturing

* For example, the product of the present invention was prepared following the drying step after the extraction process (hydrolysis time 16 hours) shown in Example 1.

Formulation 13c: Injection preparation

* For example, the product of the present invention was prepared following the drying step after the extraction process (hydrolysis time 16 hours) shown in Example 1.

Claims (1)

The use of a cosmetic or health supplement composition comprising as active slimming component sulphated oligosaccharides capable of capturing spermine or spermidine, or spermine and spermidine.