IT202100014186A1 - COMPOSITION FOR THE TREATMENT OF OBESITY AND METABOLIC SYNDROME BASED ON GARCINIA CAMBOGIA EXTRACTS - Google Patents

Description

?COMPOSITION FOR THE TREATMENT OF OBESIT? WHAT ABOUT THE METABOLIC SYNDROME BASED ON GARCINIA CAMBOGIA EXTRACTS?

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to the use of an extract of Garcinia Cambogia for the treatment of obesity? and metabolic syndrome.

STATE OF THE ART

In recent years, the requests for preparations and formulations to reduce excess weight and to combat disorders due to poor nutrition have increased. In fact? more and more there is a high percentage of overweight people with consequent health problems due precisely to an incorrect, high-protein and high-calorie diet. In this scenario, nutraceuticals can be particularly useful molecules as, thanks to their effectiveness and reduced toxicity, they (compared to conventional drugs) are more? indicated for long-term therapy. For this reason, the creation of formulations capable of reducing obesity? can represent a significant improvement in the well-being of the population.

SUMMARY OF THE INVENTION

Purpose of the present invention? therefore that of providing a new and more? targeted approach to the treatment of obesity? and metabolic syndrome.

In accordance with the present invention, the Applicant has surprisingly found that ? It is possible to pursue the aforementioned purpose using an extract of Garcinia Cambogia, which acts by stimulating the browning of white adipose tissue.

Therefore, the present invention relates in its first aspect to an extract of Garcinia Cambogia comprising at least 50% by weight of calcium hydroxycitrate, for use in the treatment of obesity. and metabolic syndrome, in which said extract stimulates the browning of white adipose tissue.

In fact, the Applicant has surprisingly found that the Garcinia Cambogia extract according to the present invention stimulates the so-called browning, ie the browning of the white adipose tissue into brown adipose tissue (the so-called Brown Adipose Tissue, BAT), thereby allowing to implement an effective treatment of obesity? and the metabolic syndrome which differs from the treatments known in the prior art, which are based on dietary approaches, which are not always effective, and pharmacological approaches which are burdened by numerous side effects. In particular, the Applicant has found that the treatment with the extract according to the present invention advantageously and unexpectedly results in showing a reduced and limited hepatotoxicity, thereby presenting minor critical profiles? in this respect for the subjects subjected to the treatment itself.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the optical microscopies obtained from the vitality test. with trypan blue for the control sample and for the 3T3-L1 cells after 24 hours of administration of Garcinia Cambogia extract according to Example 1 at 30, 60, and 120 mg/ml;

in Figure 2 ? reported the expression of genes UCP-1, DIO-2, CIDEA and PPAR-? in the fat cells treated with the Garcinia Cambogia extract according to Example 1;

in Figure 3 are reported the results of vitality? of cells with TCC test after treatment with Garcinia Cambogia extract according to Example 1 ;

Figure 4 shows the images (20x magnification) of the histological tests of Example 4 on the adipose cells of the Control Group and of the Group treated with the Garcinia Cambogia extract according to Example 1, highlighting a switch of the phenotype of the cells of the white (control animals) to brown adipose tissue in the case of treated animals;

Figure 5 shows the results of the Malondialdehyde (MDA) test of Example 4 carried out on the liver cells of the Control Group and of the Group treated with the Garcinia Cambogia extract according to Example 1; And

Figure 6 shows the histological images of Example 4 obtained from liver samples fixed in formalin (serial sections 5?m thick) from the Control Group and from the Group treated with the Garcinia Cambogia extract according to Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in its first aspect, to an extract of Garcinia Cambogia comprising at least 50% by weight of calcium hydroxycitrate, for use in the treatment of obesity. and metabolic syndrome, in which said extract stimulates the browning of white adipose tissue.

In fact, the Applicant has surprisingly found that the Garcinia Cambogia extract according to the present invention stimulates the so-called browning, i.e. the browning of the white adipose tissue into brown adipose tissue (the so-called Brown Adipose Tissue, BAT), thereby allowing to implement an effective treatment of obesity? and the metabolic syndrome which differs from the treatments known in the prior art, which are based on dietary approaches, which are not always effective, and pharmacological approaches which are burdened by numerous side effects. In particular, the Applicant has found that the treatment with the extract according to the present invention advantageously and unexpectedly results in showing a reduced and limited hepatotoxicity, thereby presenting minor critical profiles? in this respect for the subjects subjected to the treatment itself.

In the context of the present description and in the subsequent claims, all the numerical quantities indicating quantities, parameters, percentages, and so on? via are to be intended in all circumstances preceded by the term ?about? unless otherwise indicated. Furthermore, all ranges of numerical quantities include all possible combinations of maximum and minimum numerical values and all possible intermediate ranges, in addition to those specifically indicated below.

In the context of the present description and in the subsequent claims, with the expression:

- ?Garcinia Cambogia? means the subtropical plant of the Clusiaceae family, also called Garcinia gummi-gutta.

The present invention can present in one or more of its aspects one or more? of the preferred characteristics shown below, which can be combined with each other according to the application requirements.

Preferably, the Garcinia Cambogia extract? an extract of the flowers of said plant.

The Garcinia Cambogia extract according to the present invention comprises at least 50% by weight, preferably at least 55% by weight, even more? preferably at least 60% by weight of calcium hydroxycitrate.

Said excerpt ? advantageously obtainable through the phases of:

to. subject to extraction in a hydroalcoholic solution a quantity? of Garcinia Cambogia fruit;

b. add to the hydroalcoholic mixture obtained in phase a. at least a base up to a pH of at least 7, thus obtaining? an aqueous phase and a precipitate;

c. drying the precipitate obtained from step c.

Preferably, in phase a. said hydroalcoholic solution? an aqueous solution comprising 10 to 30% by weight of at least one alcohol, plus? preferably an alcohol selected from the group consisting of: methanol, ethanol.

Preferably, said step a. is carried out at a temperature ranging from 50 to 80 ?C, more? preferably in the range from 55 to 75°C.

Preferably, said extraction of step a. is carried out at pressure greater than atmospheric pressure.

Preferably, in said step b. say at least one basis? a hydroxide of an alkali or alkaline earth metal, more? preferably ? calcium hydroxide.

Preferably, said step c. comprises separating said precipitate from said aqueous phase.

Preferably, said separation is carried out by filtration.

The Garcinia Cambogia extract according to the present invention surprisingly stimulates the browning of the white adipose tissue, leading to its conversion into brown adipose tissue, also called BAT.

Surprisingly, the Applicant has found that the extract according to the present invention induces the expression of at least one gene selected in the group consisting of UCP-1 , DIO-2 and CIDEA, said genes being in particular specific to brown adipose tissue. Equally surprisingly, the Applicant has also found that the extract according to the present invention does not instead induce the expression of PPAR-? (PPAR-gamma), which instead appears to be the main target of many dietary components of the prior art.

Therefore, the Applicant has observed that the properties of the extract according to the present invention allow to implement an innovative and better treatment of obesity? and metabolic syndrome, compared to the treatments of the prior art.

Preferably the treatment with the extract according to the present invention is aimed at an obese subject, even more? preferably to an obese human being.

The extract according to the present invention can be administered by systemic administration including oral administration and parenteral administration.

The extract according to the present invention can be administered once or according to a dosing regimen in which a number of doses are administered at varying time intervals over a given period of time. Suitable dosage regimens for the extract of the invention depend on the properties? pharmacokinetics of the extract and in particular of calcium hydroxycitrate, such as absorption, distribution and half-life, which can be determined by the person skilled in the art. Furthermore, suitable dosage regimens, including the duration for which these regimens are administered, for the extract according to the invention depend on the condition being treated, the severity of the condition being treated, from the age? and the physical condition of the patient being treated, the medical history of the patient to be treated, the nature of the concurrent therapy, the desired therapeutic effect, and similar factors which are within the knowledge and experience of one skilled in the art.

Preferably, the treatment with the extract according to the present invention provides for the administration of said extract at a dosage ranging from 5 mg to 100 mg per day, per kilogram of body weight of the subject subjected to said treatment.

The extract of the invention will be? normally, but not necessarily, formulated into a pharmaceutical composition prior to administration to a patient. The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art.

The pharmaceutical compositions of the invention can be prepared and packaged in bulk form in which a quantity efficacy of the extract of the invention given to the patient as with powders, syrups and solutions for injection. Alternatively, the pharmaceutical compositions of the invention can be prepared and packaged in the form of unit dosages. A dose of the pharmaceutical composition contains at least one quantity? therapeutically effective of the extract according to the present invention.

Preferably, the treatment with the extract according to the present invention is conducted with a composition comprising said Garcinia Cambogia extract and at least one pharmaceutically acceptable carrier.

Preferably, said composition? administered orally to a person who needs it.

Preferably said pharmaceutically acceptable vehicle ? water or an aqueous vehicle (ie: saline, dextrose) and/or oil emulsions.

The extract of the invention and any pharmaceutically acceptable excipient(s) present will typically be formulated in a dosage form adapted for administration to the patient by the desired route of administration.

Conventional dosage forms include those adapted for (1) oral administration such as tablets, capsules, oval tablets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile reconstruction solutions, suspensions and powders.

Pharmaceutically suitable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, granulating agents, glazing agents, wetting agents, suspending agents, emulsifiers, sweeteners, flavor masking agents, coloring agents, anti- binders, humectants, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants and buffering agents. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch, cellulose, calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form can also include a binder. Suitable binders include starch, gelatin, sodium alginate, alginic acid, guar gum, povidone and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form can also include a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycollate, alginic acid and sodium carboxymethyl cellulose. The oral solid dosage form can also include a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate and talc. Suitable carriers for oral dosage form include, but are not limited to, magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, methyl cellulose, sodium carboxymethyl cellulose, and the like. The techniques used to prepare oral formulations are conventional mixing, granulating and compressing or filling capsules.

Further characteristics and advantages of the invention will become more evident from the following Examples, to be intended for illustrative and non-limiting purposes of the same.

EXPERIMENTAL PART

Example 1 - preparation of an extract of Garcinia Cambogia

The preparation of the Garcinia Cambogia extract was carried out as follows: a sample of dried fruits of Garcinia Cambogia was ground into small pieces of a few millimeters and subsequently extracted with the high pressure method in a mixture of hot demineralised water (60 -70°C) containing 20% by weight of methanol. What is the mixture? obtained was then subjected to subsequent precipitation, by adding a solution of calcium hydroxide up to pH 7.0, the precipitate so? obtained comprising the calcium salt of hydroxycitric acid, it was then dried and weighed. To quantify the extraction yield, an HPLC analysis was carried out using a 1:9 solution of 30% phosphoric acid and water as solvent.

An extract was obtained (6.5% by weight with respect to the weight of dry Garcinia Cambogia) with a calcium hydroxycitrate content of 61% by weight.

Example 2 - treatment of fat cells

3T3-L1 cells were differentiated into white adipocytes using a differentiation protocol involving the use of media spiked with isobutylxanthin, insulin, and dexamethasone. Once the differentiation into white adipocytes was completed, the Garcinia Cambogia extract according to Example 1 was administered to the cells, at a dose of 30, 60, and 120 mg/ml for 24 hours.

What are the cells? treated together with a control sample were tested with trypan blue, which revealed? the non-toxicity? of the extract at the doses tested (Figure 1).

At the same doses the cells also showed a phenotypic switch confirmed by Real-time PCR analysis of the expression of specific brown adipose tissue genes such as UCP-1, DIO-2 and CIDEA (Figure 2). Particularly surprisingly, it was also found that the Garcinia Cambogia extract according to Example 1 did not cause an increase in the expression of PPAR-gamma, which instead appears to be the main target of many dietary components.

Example 3 - tests of toxicity? in vitro

The Quasi Vivo 500? system was used for these tests, which allows for the same culture medium to circulate between different types of cells to allow for the identification of any toxic effects of the substances. Specifically, neuronal (SH-SY5), hepatic (HepG2) and adipocyte (3T3L1) cells were used, all kept in an incubator at a constant temperature of 37°C and at a concentration of CO2 equal to 5%. The cells were observed under the microscope and the media specially replaced, when deemed necessary, in such a way as to favor the adhesion of the cells and to possibly eliminate the non-adherent cells. 4-well plates were used, into which slides supplied with the system were placed. The slides were then placed in the silicone chambers of the system. The system consisted of a network of pipes which allows the soil to flow into the chambers. The cells were therefore treated with the association of different doses of Garcinia Cambogia extract according to Example 1 (30 mg/ml, 60 mg/ml and 120 mg/ml). The cells were treated for 48 hours and subsequently the toxicity was evaluated. of compounds.

At the end of the incubation, was the viability evaluated? cell with the 3% Triphenyltetrazolium Chloride (TCC) test for 20 minutes. The red reaction product formed was extracted with isopropanol; approximately 250 µl of supernatant was transferred to 96-well plates and the absorbance at 492 nanometers was measured with a spectrophotometer.

The graphs in Figure 3 report the results obtained and show that the treatments based on Garcinia Cambogia extract did not cause cell death on the cell lines tested.

Example 4 - In vivo study

For the animal study, 14 male rats belonging to the Zucker Crl:ZUC-Leprfa strain of 12 weeks of age were used. weighing between 250-300g (Calco, Milan, Italy). The animals were housed in the animal facility of the Department of Clinical and Experimental Medicine of the University of Messina and kept in standard environmental conditions (12 hours day/night cycle, at a temperature of 25?C) and fed with water and food ad libitum.

The animals were divided into 2 groups of 7 animals each. One group was used as a control group and represented the group of untreated obese animals; the other group was treated orally, by gavage (volume of 300??) with the Garcinia Cambogia extract according to Example 1, at a dose of 1000 mg/kg per day. The duration of treatment was 28 days.

At the end of the 28 days, the animals were sacrificed and the visceral adipose tissue and the liver were removed for histological and biochemical investigations.

Also in this case in the visceral fat subjected to histological investigation and stained with hematoxylin and eosin, a switch of the phenotype of the adipose tissue cells from white (control animals) to brown (treated animals) was highlighted as demonstrated in Figure 4 (20x magnification).

In Figure 4, the photo labeled ?Control? the adipocytes that characterized the visceral adipose tissue of the rats of the control group are shown: the cells showed a polygonal shape and appeared uniform in shape and size. In the photo labeled ?Garcinia? on the other hand, adipocytes of visceral adipose tissue of rats subjected to the treatment with the extract according to Example 1 are shown; the adipocytes, more? small in size when compared to those of the control group, they exhibited an irregular shape and did not appear uniform with each other in terms of size. This morphological aspect? comparable to the morphological aspects found in the adipose tissue in the browning phase.

The liver was used to study the mechanisms of oxidative stress and, in particular, lipid peroxidation, using the Malondialdehyde (MDA) test. The malondialdehyde? a marker of lipid peroxidation and its levels indicate the activation of peroxidation processes both in vitro and in vivo. Therefore, the liver taken from each experimental group was mechanically homogenized using a MICCRA D-1 homogenizer in a 1.15% KCI solution and centrifuged at 4000rpm for 15min; an aliquot of about 0.1 ml of supernatant was taken and added to a reaction mixture containing 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid, 1.5 ml of 0.8% thiobarbituric acid and 700 ml of distilled water. The samples were boiled for 1 hour at 95°C and centrifuged at 3000xg for 10 min. The absorbance of the supernatant was measured by a spectrophotometer at a wavelength of 650 nm. Liver tissue taken from untreated obese Zucker rats showed elevated levels of MDA compared to the levels generally observed in animals of normal weight. Treatment with Garcinia Cambogia extract at a dose of 1000mg/kg determines? a significant reduction in MDA levels compared to the obese group of animals (Figure 5).

To carry out a careful histological investigation and evaluate whether the extract used could cause hepatotoxicity, the liver was fixed in 10% formalin for 24 hours and subsequently embedded in paraffin. The samples were then cut obtaining serial sections of 5 pm thickness, stained with Hematoxylin-Eosin and finally observed under an optical microscope. Histological images demonstrated (Figure 6, 200x magnification) that the control group (image labeled ?CONTROL") had diffuse microvesicular steatosis characterized by enlarged hepatocytes with flocculating cytoplasm due to the presence of small fat droplets, and macrovesicular steatosis characterized by clear, rounded and well-defined fat droplets within the cytoplasm, as well as nuclei displaced in peripheral areas.The images obtained from the liver sections of the animals treated with the Garcinia Cambogia extract according to example 1 (image labeled ? GARCINIA 1000 mg/kg?), instead showed a significant reduction of macrovesicular steatosis in the central and periportal area, with a lower percentage of microvesicular steatosis (<33%).

Claims (10)

1. An extract of Garcinia Cambogia containing at least 50% by weight of calcium hydroxycitrate, for use in the treatment of obesity and metabolic syndrome, in which said extract stimulates the browning of white adipose tissue. 2. The Garcinia Cambogia extract for use according to claim 1, wherein said extract? obtainable through the phases of: to. subject to extraction in a hydroalcoholic solution a quantity? of Garcinia Cambogia fruit; b. add to the hydroalcoholic mixture obtained in phase a. at least a base up to a pH of at least 7, thus obtaining? an aqueous phase and a precipitate; c. drying the precipitate obtained from step c. The Garcinia Cambogia extract for use according to claim 2, wherein said extraction of step a. is carried out at pressure greater than atmospheric pressure. The Garcinia Cambogia extract for use according to any claim 1 to 3, wherein said extract induces the expression of at least one gene selected from the group consisting of UCP-1, DIO-2 and CIDEA. 5. The Garcinia Cambogia extract for use according to any claim 1 to 4, wherein said extract does not induce PPAR-? (PPAR-gamma). 6. The Garcinia Cambogia extract for use according to any claim 1 to 5, wherein said treatment of obesity? What about metabolic syndrome? aimed at an obese person. 7. The Garcinia Cambogia extract for use according to claim 6, wherein said obese subject is a human being. 8. Garcinia Cambogia extract for use according to any claim from 1 to 7, wherein said treatment provides for the administration of said extract at a dosage ranging from 5 mg to 100 mg per day, per kilogram of body weight of the subject subjected to said treatment. 9. The Garcinia Cambogia extract for use according to any claim from 1 to 8, wherein said treatment is conducted with a composition comprising said Garcinia Cambogia extract and at least one pharmaceutically acceptable carrier. 10. The Garcinia Cambogia extract for use according to claim 9, wherein said composition is administered orally to a person who needs it.