IT202000015598A1 - EXTRACT FROM THE RESIN OF THE PROTIUM HEPTAPHYLLUM PLANT, FORMULATIONS INCLUDING THIS EXTRACT AND HYDROALCOHOLIC EXTRACTION PROCESS AT CONTROLLED PRESSURE AND TEMPERATURE - Google Patents

Description

?Extract from the resin of the Protium heptaphyllum plant, formulations comprising this extract and hydroalcoholic extraction process at controlled pressure and temperature?

DESCRIPTION

TECHNICAL FIELD

The present invention relates to the nutraceutical, pharmaceutical and cosmetic sectors. Specifically, the present invention relates to a hydroalcoholic extract from the resin of the Protium heptaphyllum plant enriched for the acid triterpenic component. The invention also relates to pharmaceutical formulations comprising the aforementioned extract.

The invention also relates to a hydroalcoholic extraction process at controlled pressure and temperature of the aforesaid extract.

The present invention finds advantageous applications in the inhibition of gene expression of the enzyme HMG-CoA reductase, in the alteration of gene expression in liver cells, in the alteration of gene expression of key factors involved in lipid metabolism and in the treatment of hypercholesterolemia.

Furthermore, the hydroalcoholic extraction process according to the present invention ? economically and qualitatively more? advantageous compared to known solutions thanks to the use of simple, inexpensive machinery and non-toxic solvents (water and ethanol) or in any case widely used in food and more? respectful of the environment.

STATE OF ART

The risk of atherosclerosis and coronary heart disease? increased in patients with elevated serum concentrations of low-density lipoprotein? of cholesterol (LDL), total cholesterol (TC) and triglycerides (TG).

The LDL when they are present in quantity? higher than the physiological ones tend to settle on the wall of the arteries, causing progressive thickening and hardening (atherosclerosis).

This process can lead over time to the formation of real plaques that can make it difficult for the correct blood flow and, in the most common cases, serious, lead to blockages.

For this reason ? felt the need to have available a technical solution able to exert an effect on the regulation of gene expression of the most? important enzymes involved in cholesterol metabolism.

Statins are organic molecules capable of inhibiting the action of hydroxymethylglutaryl-CoA reductase (or HMG-CoA reductase), a key enzyme in cholesterol metabolism as it converts the 3-hydroxy-3-methylglutaryl-CoA molecule into mevalonic. There are different types of statins both natural and synthetic; given their action they are used in the pharmaceutical field for the treatment of pathologies related to hypercholesterolemia as they are able to directly limit the biosynthesis of cholesterol; some molecules of this class are also able to interact directly with the LDL receptor, favoring its biosynthesis and thus preventing any damage caused by atherosclerosis. In this pharmaceutical class there are several molecules which are currently used as active ingredients; among the most important are simvastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin and fluvastatin.

In particular, monacolin K ? one of the molecules that can be obtained from the fermentation of red rice carried out with the Monascus purpureus mushroom, it has a structure similar to that of lovastatin and as such is used in supplements aimed at preventing damage caused by hypercholesterolemia.

The effectiveness of the molecule? been recognized to such an extent that in 2013 the EFSA attributed to monacolin K of fermented red rice in doses of 10 mg/day the claim 1924/2006 of ?maintenance of normal blood cholesterol levels?.

In addition to statins, there are other drugs on the market that can regulate hypercholesterolemia and the biosynthesis of fatty acids such as ezetimibe (selectively inhibits the absorption of exogenous cholesterol), resins that sequester bile acids (induce a reduction in blood levels of LDL ), acipimox (reduces blood levels of triglycerides and cholesterol), and fibrate drugs (regulates blood levels of cholesterol and triglycerides).

Like all drugs, even those overwritten for the treatment of hypercholesterolemia have various adverse effects when taken regularly; among the most ailments possible allergic reactions, gastrointestinal tract disorders, muscle weakness and pain, headache, taste alteration and skin disorders have been reported frequently. In the most cases serious ? It was possible to experience problems with the liver system.

The Italian phytosurveillance system (EpiCentro) revealed 52 reports of adverse reactions due to the intake of red yeast rice recorded between 2002 and 2015, which include myalgia and/or increased creatine phosphokinase (CPK), liver damage, increased of liver enzymes and hepatitis, damage to the gastrointestinal tract, skin reactions including urticaria or skin rash and other reactions including symptoms: INR increase due to interaction with warfarin, tachycardia, tingling in the extremities, dizziness, blurred vision.

These results led to the conclusion that the safety profile of red yeast rice is similar to that of statins (Mazzanti et al., 2017).

Furthermore, the fermentation process mediated by the Monascus purpureus fungus can generate as a secondary metabolite the mycotoxin known as citrinin, which has a nephrotoxic, hepatotoxic and probably carcinogenic action (source EFSA); for this reason on November 7, 2019 ? EU Regulation 2019/1901 has been issued, which requires that from 1 April 2020 the quantity? maximum amount of citrinin present in food supplements based on fermented rice with red yeast Monascus purpureus should? be 100 (?g/kg).

Given the?High quantity? of adverse reactions caused by drugs used to treat this pathology in recent years? more and more of study interest is the search for alternative bioactive molecules of natural origin that do not show particular complications; in particular, ? felt the need to have an extract deriving from a vegetable resin rich in secondary metabolites that give it different properties? pharmacologically interesting, with different potential effects on the body, high tolerability? and low toxicity? and also absent of adverse reactions.

Protium heptaphyllum resin? rich in secondary metabolites that give it different properties? pharmacologically interesting; it ? rich in molecules of natural origin with therapeutic effects whose exact nature is not known, but which could be used in order to generate supplements with a possibly preventive function that have high tolerability? and low toxicity? or adverse reactions.

The metabolites more known and studied of this resin are the molecules of ?-amyrin and ?amyrin: they are among the most common pentacyclic triterpenes? widespread in plant matrices and are particularly abundant in resin exudates and in the oils of various plants including in particular those of the Burseraceae family such as Protium heptaphyllum itself; in these resins the concentration of the two isomers pu? even reach 50%. In addition to this category of triterpenes and their oxidized derivatives in the resin of Protium heptaphyllum there are also molecules defined as acid triterpenes, in particular tetra and penta cyclic acid triterpenes (molecular weight 450-500 Da) which differ from the class of triterpenes described above (amirine and oxidized derivatives) for the presence of one or more? carboxylic substituents which give them different chemical-physical characteristics.

Historically the resin of Protium heptaphyllum? been studied for the presence of amyrins, these belong to the class of pentacyclic olean triterpenes and differ from each other for a different arrangement of two methyl groups positioned on the fifth ring.

The amyrins have found a lot of interest in the research field as they are found to be pharmacologically active molecules; have been done already? several studies on extracts of Protium heptaphyllum and on purified amyrins but to date the Inventors have never been investigated the activity? pharmacology of the triterpenic acid fraction cos? as the method for their enrichment starting from the resin of Protium heptaphyllum.

Amyrins and the related studies identified, in summary, focused on investigating the following:

? the resin extracts analyzed concerned only some selected parts of the Protium resin separated and purified to enrich the content in ?-amyrin and ?amirin;

? the activity? biological analysis of pentacyclic triterpene components (amirines) ? been tested in vivo in relation to activity? anti-nociceptive, activity anti-inflammatory, activity on the Central Nervous System, their hypoglycaemic action, on the metabolism and on the digestive tract;

? the few studies present in relation to the metabolism of cholesterol have been carried out on a purified fraction of ?-amyrin and ?-amyrin and have been carried out considering exclusively the variation of blood levels of LDL and HDL;

? no pharmacological studies? known to date about the triterpenic acid component of the resin of Protium heptaphyllum as well as? an economically and ecologically sustainable industrial method for the extraction and enrichment of this component with respect to the amyrin component.

Examples of known solutions are described in EP 2812005 B1, EP 3068412 B1, BR 102015012884-3 A2 and US 9233066 B2.

EP 2812005 B1 presents an in-depth study of the disorders that respond to the reduction of? of the enzyme HMG-CoA reductase in mammals; the invention consists of an extract of the Amelanchier alnifolia plant which can be used in the prophylaxis and/or therapy for the treatment of hypercholesterolemia, hyperlipidemia, HDL/LDL ratio.

The composition ? generated by the set of natural molecules with inhibitory action against the enzyme HMG-CoA reductase; protagonists are the polyhydroxylated pentacyclic triterpenes which include euscaphic acid, tormentic acid, myriatic acid, corosolic acid, oleanic acid, ursolic acid.

EP 3068412 B1 analyzes the problems relating to the metabolism of fatty acids and sugars; in particular the role of HDL is highlighted and how it can be considered a potential target for coronary heart disease; the invention consists of a medicinal composition obtained from the extract of the seeds of Emblica officinalis, with nutraceutical and pharmaceutical applications for the reduction of hypercholesterolemia, blood triglyceride level, blood glucose level, reduction of LDL and increase of HDL.

The treatment of dyslipidemia and hypercholesterolemia? carried out through triterpenoids (?-amyrin, ?-sitosterol, lupeol) and hydroxycinnamic acids (ferulic acid, p-coumaric acid).

BR 102015012884-3 A2 treats diseases associated with dysfunctions of the metabolism of fatty acids and sugars, such as type II diabetes and obesity; The invention consists of a plant protection product of natural origin capable of inhibiting digestive enzymes and exerting a hypoglycaemic, hypolipidemic and antiobesity action.

The natural source? the resin of the Protium paniculatum var. ?Nova?, which is subjected to extraction with mixtures of hexane and ethyl acetate, in different proportions, with polarity? growing. This type of processing allows to obtain an extract rich in pentacyclic triterpenes ?/? amyrins, which are then oxidized to obtain ?/? amironi; the latter are the claimed bioactive components and constitute up to 60% of the final product.

US 9233066 B2 addresses the difficulty? on the search for products with antimicrobial action and properties? biofilm removal; the invention consists of an essential oil obtained from at least one plant belonging to the genera Protium, Guatteria and Cyperus. This essential oil can be used to make a phytocosmetic and/or phytotherapic for personal hygiene, medicine and veterinary medicine. The decision to introduce the genus Protium in the invention? given by its properties? antimicrobial and its ability? to speed up wound healing.

A hydroalcoholic extract from the resin of the Protium heptaphyllum plant purified for the triterpenic acid component, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract, would satisfy the need to have a solution in able to have an effect on the regulation of gene expression of the pi? important enzymes related to the metabolism of cholesterol and lipids.

The present invention intends to respond to the above requirement.

In particular, the present invention intends to solve the technical problem of intervening on the modulation of the metabolism of cholesterol and lipids through the regulation of the transcription of the genes of enzymes and factors involved in these metabolic pathways and not on them as currently occurs in most of the drugs and supplements on the market that in some cases have potential adverse effects.

Furthermore, the present invention intends to solve the technical problem of identifying an economically, ethically and ecologically sustainable extraction and purification method of organic substances or mixtures having a pharmacological action on the metabolism of cholesterol and lipids. The substances that generally interact on these metabolic pathways are by their nature poorly soluble in polar solvents (water) and therefore strongly apolar solvents (for example hexane and ethyl acetate) are required for their extraction and purification, with a high degree of toxicity. , which therefore require subsequent very expensive solvent removal processes and the use of which presents a high environmental risk.

Furthermore, the present invention intends to solve the technical problem of producing drugs or supplements containing as active ingredient substances or molecules alternative to those obtained by synthesis, semi-synthesis or obtained by microbial fermentation processes, obtained from the intensive cultivation of vegetable organisms at the expense of forest soil or soil used for the cultivation of plants intended for basic nutrition.

Finally, the present invention intends to solve the technical problem of identifying a process for the extraction and purification of natural substances or mixtures for food and pharmaceutical use which is optimized, linear, inexpensive and easily feasible even in technologically disadvantaged geographical areas and in which it is difficult find manpower and highly advanced tools without reducing quality? and stability of the product and also obtaining an extract that can be easily used and integrated in the formulations of pharmaceutical products and supplements.

In summary therefore, up to the present moment, to the knowledge of the inventors, no solutions are known which allow to create - through a hydroalcoholic extract from the resin of the Protium heptaphyllum plant enriched for the acid triterpenic component, the corresponding pharmaceutical formulations comprising the aforementioned extract and the related hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract - an effect on the regulation of gene expression of the most? important factors related to the metabolism of cholesterol and lipids.

Therefore the Applicant, with the hydroalcoholic extract from the resin of the Protium heptaphyllum plant enriched for the triterpenic acid component, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract according to the present invention, intends to remedy this shortcoming.

PURPOSES AND SUMMARY OF THE INVENTION

? purpose of the present invention to overcome the drawbacks of the prior art related to the impossibility to obtain a direct effect in the regulation of gene expression of the pi? important enzymes involved in the cellular homeostasis of cholesterol.

These objectives are achieved with the hydroalcoholic extract from the resin of the Protium heptaphyllum plant, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract according to the present invention which, advantageously and thanks to the enrichment for the triterpenic acid component, allow to have an effect on the regulation of gene expression of the pi? important enzymes involved in cholesterol metabolism.

Specifically, the aforesaid and other objects and advantages of the invention, as will appear from the following description, are achieved with a hydroalcoholic extract from the resin of the Protium heptaphyllum plant according to claim 1.

Another independent aspect of the present invention relates to a pharmaceutical formulation comprising the extract and constitutes the object of claim 5. Another independent aspect of the present invention relates to a controlled pressure and temperature hydroalcoholic extraction process of the extract and constitutes the object of claim 15.

Preferred embodiments and variants of the present invention are the object of the dependent claims.

It is understood that all the attached claims form an integral part of the present description and that each of the technical characteristics claimed therein? possibly independent and usable autonomously with respect to the other aspects of the invention.

will result? It is immediately clear that innumerable modifications can be made to what has been described (for example relating to shape, dimensions, arrangements and parts with equivalent functions) without departing from the scope of protection of the invention as claimed in the attached claims.

Advantageously, the technical solution according to the present invention, which provides a hydroalcoholic extract from the resin of the Protium heptaphyllum plant enriched for the acid triterpenic component, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract, allows you to obtain a plant extract that is able to modulate the main enzymes and factors involved in the metabolism of cholesterol and lipids directly at the level of their gene transcription.

This particular hydroalcoholic extract from the resin of the Protium heptaphyllum plant? obtained through the use of solvents (water and ethanol) compatible with both food and the environment and the use of which during the process does not involve the adoption of further phases for their elimination below the admissible limits. The particular combination of these solvents and the control of certain temperature and pressure values during the process phases allows to selectively extract from the resin of the Protium heptaphyllum plant the class of molecules of interest for the modulating action on the metabolism of cholesterol and lipids .

The process of extraction and purification of the identified bioactive components ? economically advantageous as it uses inexpensive solvents, simple, widely used and low-cost machinery that can also be used by unskilled labour. Despite this, it guarantees speed, scalability, stability at the same time. of the components and the food safety of the extract during all the different stages of the process.

In addition, the collection of the raw material (resin) takes place from spontaneous and easily available trees within the entire area of the Amazon forest, thus not requiring the use of new areas for the cultivation or felling of the same producer trees for the exploitation of this resource.

The final extract obtained from this innovative process consists of an easily crushable solid material which, once pulverized, can be easily combined with the excipients needed for the final formulations.

The extract can be easily conveyed in its powder form (PULVERIZED EXTRACT OF PROTIUM) for the creation of products designed for oral intake (capsules/tablets/sachets). Any problems related to the intake of the aforementioned products can be solved through a liquid formulation (FLUID EXTRACT OF PROTIUM), for making syrups; in fact, the extract due to its lipophilic nature can? be easily solubilized in vegetable oils widely used for food purposes (e.g. rice oil). To overcome the problems relating to bioavailability? of the bioactive molecules, the extract lends itself to the creation of liposomal formulations (LIPOSOMAL EXTRACT OF PROTIUM) which exploit the creation of phospholipid micelles with an amphiphilic character containing the mixture or substance to be conveyed by increasing the quantity? assimilable in the intestine.

To overcome the problems relating to the passage through the gastric tract and the possible degradation or alteration of the biomolecules, the extract lends itself to the creation of formulations in microcapsules (PROTIUM MICROENCAPSULATED EXTRACT) obtained by gelation with alginate whose composition gives the necessary protection to the It is extracted during gastric transit and subsequent dissolution in the intestinal environment.

Further advantageous characteristics will appear more evident from the following description of preferred but not exclusive embodiments, provided purely by way of non-limiting example.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will come described hereinafter by means of some preferred embodiments, provided by way of non-limiting example, with reference to the attached drawings. These drawings illustrate different aspects and examples of the present invention and, where appropriate, similar structures, components, materials and/or elements in different figures are indicated by like reference numerals.

FIG. 1 ? a flow diagram of the extraction process according to the present invention;

FIG.2 ? a bar graph illustrating the expression of key enzymes in cellular cholesterol biosynthesis using the extract according to the present invention;

FIG. 3 ? a bar graph illustrating the expression of hepatic cholesterol homeostasis regulating factors using the extract according to the present invention;

FIG. 4 ? a bar graph illustrating the expression of blood cholesterol homeostasis regulating factors using the extract according to the present invention;

FIG. 5 ? a bar graph illustrating the expression of factors involved in lipid metabolism, in particular the ACC1 gene, using the extract according to the present invention;

FIG. 6 ? a bar graph illustrating the expression of factors involved in lipid metabolism, in particular the FASN gene, using the extract according to the present invention;

FIG. 7 ? a bar graph illustrating the expression of factors involved in lipid metabolism, in particular the PPAR? gene, using the extract according to the present invention;

FIG. 8 ? a bar graph illustrating the expression of factors involved in the suppression of the conversion of cholesterol to bile salts using the extract according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

While the invention? susceptible to various modifications and alternative constructions, some preferred embodiments are shown in the drawings and will be described in detail below.

It must be understood, however, that there is no no intention of limiting the invention to the specific embodiments illustrated, but, on the contrary, the invention is intended to cover all modifications, alternative constructions, and equivalents which fall within the scope of the invention as defined in the claims.

In the following description, therefore, the use of ?for example?, ?etc.?, ?or / or? indicates non-exclusive alternatives without limitation, unless otherwise indicated; the use of ?also? means ?including, but not limited to? unless otherwise indicated; the use of ?includes / includes? means ?includes / includes, but not limited to? unless otherwise indicated.

The hydroalcoholic extract from the resin of the Protium heptaphyllum plant, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract according to the present invention of the present invention are based on the innovative concept of carrying out an enrichment for the triterpene acid component.

The present invention finds advantageous applications in the inhibition of gene expression of the enzyme HMG-CoA reductase, in the alteration of gene expression in liver cells, in the alteration of gene expression of key enzymes involved in lipid metabolism and in the treatment of hypercholesterolemia.

In the present description, with the term ?extract? it means the mixture of organic molecules obtained at the end of the manufacturing process conceived starting from the resin of Protium heptaphyllum.

In the present description, with the term ?hydroalcoholic extract? means the mixture of organic molecules obtained at the end of the manufacturing process in which ? only a mixture of water and ethanol is used as a solvent.

In the present description, with the term ?hydroalcoholic extraction? it means the selective solubilization of organic molecules in a fluid consisting exclusively of a mixture of water and ethanol.

In the present description, with the term ?resin? refers to the organic material obtained from the incision of the trunk of Protium heptaphyllum.

In the present description, with the term ?acid triterpenic component / acidic triterpenes? means the set of organic molecules present in the extract characterized by 30 carbon atoms, by the presence in the structure of 4 or 5 cycles and in particular by one or more? carboxylic substituents which give the molecule a lower acid dissociation constant than organic molecules with the same basic triterpenic structure with 4 or 5 cycles, but possibly having non-carboxyl substituents. These acidic triterpenes have a molecular weight between 450 Da and 500 Da; in the present description the terms ?acid triterpene component? and ?acidic triterpenes? they will be used interchangeably, as synonyms.

In the present description, with the term ?pharmaceutical formulation? means the form in which any type of pharmaceutical product is presented, whether medicinal or non-medicinal. Pharmaceuticals are not per se? exclusively medicines or active ingredients, therefore? this classification also includes cosmetic, food and herbal products classified as pharmaceutical products or even having exclusively quality? or pharmaceutical grade.

In the present description, with the term ?inhibition of gene expression of the enzyme HMG-CoA reductase? you mean the reduction of the quantity? of intracellular messenger RNA (mRNA) subsequently involved in the translation processes for obtaining the HMG-CoA reductase protein (EC number -1.1.1.88).

In the present description, with the term ?alteration of gene expression in liver cells? do you mean the capacity? to change the quantity? of different sequences of messenger RNA (mRNA), present within cell lines of human hepatocytes, transcribed starting from DNA sequences of specific genes.

In the present description, with the term ?alteration of gene expression of key enzymes involved in lipid metabolism? do you mean the capacity? to change the quantity? of different messenger RNA (mRNA) sequences, transcribed from DNA sequences of certain genes involved in lipid metabolism.

In the present description, with the term ?hypercholesterolemia? means the condition in an organism in which LDL levels higher than normal physiological levels are present in the plasma.

In the present description, with the term ?controlled pressure and temperature? do you mean the possibility? to monitor and adjust the temperature and pressure parameters during the process phases, keeping them within certain ranges of values.

In the present description, with the term ?polarity? do you mean a property? chemical-physics for which a molecule (called polar) has one or more? partial positive charges on a part of the molecule and one or more? partial negative charges on the opposite side of it. Molecules that do not exhibit the phenomenon of polarity are called apolar or non-polar.

The main innovation that characterizes the extract which is the subject of this application? constituted by the combination between the method of obtaining the same from the starting resin, through a technology that allows the selective solubilization and the consequent enrichment of a certain class of molecules present in the starting resin (the fraction of acid triterpenes), and the significant action that this fraction has, even at low doses, in the regulation of cholesterol metabolism by regulating the gene expression of certain key genes in cholesterol metabolism.

The effect that is found on gene expression is also innovative compared to those commonly studied (blood measurements of LDL and HDL levels) because? it is based on the investigation of the molecular mechanisms upstream of cholesterol metabolism and its regulation, and not on effects resulting from changes in metabolism.

In addition to the above, does the extract comply with the hygiene and quality requirements? required for the production of pharmaceutical formulations.

The identified extraction process takes the form of green chemistry technologies, safeguarding the environment and avoiding the use of highly toxic or fossil-based solvents.

This extraction method? been developed to be particularly optimized and economical: ? composed of a few phases; involves the use of simple and easily available instruments on the market. The extraction? maximized in order to obtain the highest yield of extraction of the fraction of interest as a function of the costs necessary to obtain it.

The use of this extract derived from the resin of a spontaneous plant present in the area of the Amazon forest, with a view to an economy that respects the environment, solidarity and circular, favors the responsible use of natural resources by shifting the interest of local populations from various exploitations of the resources of the Amazon forest which include, for example, deforestation works and at the same time proposes an economic return for the local populations in line with their traditional lifestyle. Finally, the by-products obtained as a secondary result from the activity of extraction find application in the agricultural sector as natural biostimulants to replace, for example, pesticides and parasiticides.

A hydroalcoholic extract from the resin of the Protium heptaphyllum plant comprising acidic triterpenes in a quantity ? 40% by weight with respect to the total quantity of the extract obtained.

Preferably the extract comprises molecules of acidic triterpenes having a molecular weight in the range of 450 Da - 500 Da.

Preferably the extract also comprises other triterpenes, sequiterpenes and monoterpenes (with lower polarity than acid triterpenes) characteristic of the resin of Protium heptaphyllum in lower than the concentrations present in the resin as such.

Preferably the ratio by weight of acidic triterpenes to triterpenes ? And ? amyrina ? variable between 1.4 and 8, more? preferably ? equal to 4.

A pharmaceutical formulation comprising the extract as described above constitutes an independent aspect that can be used autonomously with respect to the other aspects of the invention.

Preferably, the pharmaceutical formulation further comprises one or more? Additional ingredients selectable from:

- a carrier agent of the extract chosen from:

? rice starch and tribasic calcium phosphate;

? rice oil;

- amphiphilic molecules such as for example phospholipids and derivatives;

- sodium alginate, calcium chloride and microcrystalline cellulose;

- additional botanical extracts such as:

? extract from the Commiphora mukul plant;

? extract from the Berberis vulgaris plant;

? extract from the Allium sativum plant;

- one or more flavoring and sweetening agents;

- one or more pharmaceutical, food or cosmetic grade bulking agents, excipients and diluents.

The extract as described above and the pharmaceutical formulation comprising this extract are intended for use in the inhibition of the gene expression of the HMG-CoA reductase enzyme.

The extract as described above and the pharmaceutical formulation comprising such extract are intended for use in the alteration of gene expression in liver cells.

The extract as described above and the pharmaceutical formulation comprising such extract are intended for use in altering the gene expression of key enzymes involved in lipid metabolism.

The extract as described above and the pharmaceutical formulation comprising this extract are intended for use in the treatment of hypercholesterolemia.

Referring to FIG. 1, which illustrates the preferred embodiment of the present invention, it can be observed that the hydroalcoholic extraction process at controlled pressure and temperature comprises the following steps:

- phase 100: prepare a quantity? predetermined resin of the plant Protium heptaphyllum;

- phase 101: crush the resin of the Protium heptaphyllum plant using a suitable device until obtaining a powder with a particle size in the range of 2-5 mm;

- phase 102: carry out the extraction of the terpenic component pi? polar contained in the resin by introducing a quantity? predetermined resin powder in a tank with mixer, adding a quantity? predetermined of a hydroalcoholic solution and stirring at low temperature; - step 103: separate the insoluble component from the mixture by means of a suitable low temperature filtration or centrifugation system;

- phase 104: concentrate the terpenic component more? polar extracted by evaporative removal of solvent under vacuum at low temperature; - phase 105: carry out the final drying and the removal by evaporation of the most? birds;

- step 106: obtaining a hydroalcoholic extract from the resin of the Protium heptaphyllum plant comprising acid triterpenes in a quantity ? 40% by weight with respect to the total quantity of the extract obtained.

Preferably the process involves

- phase 100: the quantity? predetermined resin of the plant Protium heptaphyllum ? variable between 80 kg and 160 kg, preferably ? equal to 110 kg;

- phase 101: the resin of the Protium heptaphyllum plant is crushed by means of a refrigerated hammer mill, with a temperature maintained between -20 ?C and 15 ?C;

- phase 102: the quantity? predetermined resin powder ? variable between 75 kg and 150 kg, preferably ? equal to 100 kg, the quantity? predetermined hydroalcoholic solution? variable between 350 liters and 1,000 litres, preferably ? equal to 450 liters and the extraction of the terpenic component more? polar content in the resin takes place at a temperature between -20?C and 15?C;

- phase 103: separate the insoluble component, i.e. the terpenic component more? apolar consisting mainly of ? And ? amyrin, from the mixture by filtration with Buchner filter, filter press or cellulosic passive filters having porosity? of 25 ?m at a temperature between -20?C and 15?C;

- phase 104: concentrate the terpenic component more? polar extracted by evaporation of the solvent under vacuum at a temperature between -20°C and 15°C and at a relative pressure value lower than -950 mBar with respect to the normal atmospheric pressure value;

- phase 105: carry out the final drying of the concentrated material together with the removal by evaporation in conditions of forced ventilation in a nitrogen atmosphere at a temperature between 40 and 130?C for a time between 1 and 12 hours of the components more? volatile, i.e. the residual content, still present in traces, of water and ethanol and most of the mono and sesquiterpene component still present in the extract.

Preferably the hydroalcoholic solution? choice between a mixture containing water and an alcohol miscible in water, pi? preferably ? a mixture consisting of water and ethyl alcohol.

Preferably 100 kg of powdered resin are introduced into a tank with mixer, into which a hydroalcoholic solution is added (450 l, weight/volume ratio between 1:4.5 and 1:5) having an ethanol concentration ranging from 50 % to 96% by volume.

Although the resin is made up of almost completely liposoluble elements (in fact only a small fraction of this is solubilized in water) these particular process conditions allow the extraction of a specific fraction of molecules from the resin, a fraction more ?polar?, avoiding to solubilize all the remaining material.

The hydroalcoholic extraction process at controlled pressure and temperature is further specified below.

The resin of Protium heptaphyllum (raw material), is crushed by means of a refrigerated hammer mill to obtain a powder with a particle size of 2-5 mm. During this phase the temperature is kept between -20?C and 15?C.

100 kg of powdered resin are introduced into a tank with mixer, into which a hydroalcoholic solution is added (450 l, weight/volume ratio between 1:4.5 and 1:5), which can have an ethanol concentration ranging from 50% to 96% by volume. The extraction process takes place under stirring, at a temperature between -20?C and 15?C.

The insoluble fraction is separated from the mixture by filtration (Buchner or filter press) with passive cellulose filters having porosity? of 25 ?m (or by centrifugation), at a temperature between -20?C and 15?C.

The filtered mixture is concentrated in a low temperature vacuum solvent evaporation system (pressure = -950 mBar, evaporation chamber temperature = 15°C).

The concentrated mixture is finally subjected to a process to remove the residual solvent and the volatile compounds still present (monoterpenes and sesquiterpenes) by placing it in a forced-ventilation cabinet at a controlled temperature and atmosphere (ambient pressure, temperature between 40° and 130?C, in a nitrogen atmosphere).

The final product obtained consists of an extract in solid-crystalline form. The extraction yield by weight? usually between 40% and 60% of the starting material. By obtaining the solid extract (name: ?Concentrated extract of Protium?), this can be finally worked with different modalities? with the aim of supplying an ingredient intended for the food, pharmaceutical and cosmetic industries that is technologically valid for the production of finished products.

The main forms in which the extract is supplied to the companies that will make the various finished products are as follows.

POWDERED PROTIUM EXTRACT

The extract and the excipients are crushed and homogenized by means of a refrigerated hammer mill (temperature between 10°C and 15°C) until obtaining a homogeneous powder with an average particle size of 200 ?m.

Composition example:

1- PROTIUM CONCENTRATED EXTRACT = 40-60%

2- Native rice starch = 20-40%

3- Tribasic Calcium Phosphate = 10-25%

PROTIUM FLUID EXTRACT

The extract is mixed with a low viscosity vegetable oil (for example rice oil) in a melter with mixer (temperature between 50 and 80°C, controlled atmosphere with nitrogen) until complete solubilisation and homogenization of the extract is obtained.

Composition example:

1- PROTIUM CONCENTRATED EXTRACT = 40-60%

2- Rice oil = 40-60%

Other possible (secondary) forms of commercialization of the ingredient are: PROTIUM LIPOSOMAL EXTRACT

Composition example:

1- PROTIUM CONCENTRATED EXTRACT = 40-60%

2- Liposomal blend containing Phospholipids = 40-60%

MICROENCAPSULATED PROTIUM EXTRACT

Composition example:

1- PROTIUM CONCENTRATED EXTRACT = 25-80%

2- Microcrystalline cellulose = 15-50%

3- Sodium alginate = 5-15%

4- Calcium Chloride = 1-5%

Below are some examples of use of the different forms of ingredient in final formulations (finished products) intended for consumption for the food supplement, pharmaceutical and cosmetic markets:

A. Capsule formulation example (amount per capsule):

1. MICROENCAPSULATED PROTIUM EXTRACT: 50-200 mg

2. Microencapsulated extract titrated in guggulsterones from Commiphora mukul: 50-200 mg

3. Pullulan based vegetable capsule

B. Example of tablet formulation (amount per tablet):

1. PROTIUM PULVERIZED EXTRACT: 50-200 mg

2. Berberine titrated extract from Berberis vulgaris: 50-200 mg

3. Microcrystalline cellulose: 0.1-50mg

4. magnesium stearate: 0.1-50mg

5. Hydroxypropyl methylcellulose: 0.1-50mg

C. Example of formulation of effervescent granules in sachets (quantity per sachet):

1. PROTIUM PULVERIZED EXTRACT: 50-200 mg

2. Tartaric acid: 100-200mg

3. Baking soda: 1-1.5g

4. Citric acid: 500-800mg

5. Sweeteners: 50-200mg

6. Natural flavors: 10-50mg

D. Example of formulation in syrups (quantity per 150ml bottle):

1. LIPOSOMAL PROTIUM EXTRACT: 1000 mg

2. Liposomal extract titrated in guggulsterones from Commiphora mukul: 1000 mg 3. Extract titrated in berberine from Berberis vulgaris: 500 mg

4. Glycerol: 20g

5. Natural flavors: 0.1-200mg

6. Steviol glycosides (sweeteners): 100-200 mg

7. Sodium Benzoate: 100mg

8. Water: just enough up to volume

E. Example of soft-gel formulation:

1. PROTIUM FLUID EXTRACT: 50-200 mg

2. Fluid extract titrated in guggulsterones from Commiphora mukul: 50-200 mg

3. Vegetable oil bulking agent: 10-200mg

4. Vegetable capsule based on hydroxypropyl methylcellulose

The hydroalcoholic extract from the resin of the Protium heptaphyllum plant enriched for the triterpenic acid component, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract according to the present invention are described below in greater detail detail with reference to the following experimental data, which are to be understood as illustrative but not limiting of the present invention.

To determine the effects of the extraction process, the resin matrix and the extract were quantified in order to characterize the bioactive molecules present.

Comparing the initial composition present in the raw material with the extract obtained? It is possible to observe how the extraction method used and the subsequent heat treatment allow the triterpene component to be concentrated with carboxylic substituents (with acid function), while the quantities are reduced from the ? And ? amyrins (during the low temperature extraction phase) and the volatile fraction (during the final heat treatment with an inerted atmosphere). In the extract ? however, present a minimal oxidized triterpenic component (amirones, ? 6%) exclusively due to the presence of these already? in the starting material and not to oxidative phenomena during the process.

The acid triterpenic component, the objective of the extraction process devised and with which the extract is enriched, is? constituted by a group of molecules all united by having at least one carboxyl function, characterized by having 30 carbon atoms and by the presence in the structure of 4 or 5 cycles and a molecular weight between 450 Da and 500 Da.

Through a qualitative analysis in HPLC-HRMS ? the acid triterpene fraction has been characterized, which appears to be mainly composed of acid triterpenes with a molecular weight particularly in the ranges 452 - 457 Da, 468 - 473 Da and

494 - 498 From.

The residual volatile component following the process? mainly made up of

monoterpenes and sesquiterpenes in which the molecules pi? representative are hulls,

limonene, terpineol, cymene and terpinene.

Comparison of the typical composition of the extract obtained with the starting resin

RESIN EXTRACT

Acid triterpene fraction 25% 60% ? amyrins 25% 12% ? amyrins 20% 9% ? amyron 2.5% 3% ? amirone 2% 3% brein 2.5% 1.5% maniladiol 3% 2.5% Volatile component 20% 9% Concentration ranges (maximum and minimum) obtainable for the various components

of the extract

minimum value Maximum value Acid triterpene fraction 40% 80% ? amyrins 5% 15% ? amyrins 5% 15% ? amyron 0.5% 4% ? amirone 0.5% 4% brein 0.5% 3% maniladiol 0.5% 3% volatile component 4% 10%

Characterization of the triterpenic acid fraction in the extract

* Molecular ion in ESI ionization polarity? negative

* *Characteristic fragmentation pattern

Maximum residual volatile component in the extract:

? 2.5% Hulls

? 1% Limonene

? 1% Terpineol

? 4.5% Cimene

? 1% Terpinene

ACTIVITY? PHARMACOLOGICAL

The extract obtained as reported above (hydroalcoholic extraction)? been tested on cells of the hepatic tumor line HepG2 characterized by the expression of a set of expressed genes typical of more? specialized liver tissues.

The concentrations of extract used were calculated according to the dilution that the pharmaceutical formulation undergoes in the volume of fluids present in the digestive tract and according to the absorption in the intestine.

Cells in control and treatment culture medium at different extract concentrations were incubated and used for subsequent gene expression analyzes and cytotoxicity evaluation.

The transcripts whose activity? ? been monitored following the administration of the resin extract are related to some of the genes that code for the main direct and indirect key mechanisms related to cholesterol metabolism and in particular:

- HMG-CoAR (HMG-CoA Reductase)

-PPAR? (Peroxisome Proliferator-Activated Receptors)

- FXR (Farnesoid X Receptor)

- PCSK9 (Proprotein Convertase Subtilisin/kexin Type 9)

- IDOL/MYLIP (Inducible Degrader of the LDL receptor)

- FASN (Fatty Acid Synthase)

- ACC1 (Acetyl-CoA carboxylase)

For qRT-PCR analysis (semi-quantitative PCR) yes ? taking into account the expression of the target genes in cells proliferating in media at different concentrations of the extract, in the range between 1 and 50 ?g/ml.

Transcripts in cells treated with the same volume of solvent without extract were analyzed as a negative control.

The measurement, in addition to these transcripts, of a reference gene constitutively expressed (housekeeping) such as beta-actin, whose activity? Not ? altered by substances such as those under study, ? been monitored in order to normalize the expression data of the other genes among all the treatments carried out excluding variables related to the repeatability? of the tests.

qRT-PCR analysis of target genes led to the identification of gene expression regulation events dose-dependently related to the treatment received by the cells.

The relative graphs are shown below, highlighting the dose-dependent effects (at concentrations of 1, 10, 25 and 50 mg/L) of the extract on the activity transcriptional analysis of the genes involved in cholesterol metabolism by comparing them to the normal levels present in the control (reference value on the ordinate equal to 1):

Expression of key enzymes in cellular cholesterol biosynthesis

The HMG-CoAR enzyme, present above all in hepatocytes (liver cells), appears to be the limiting, and therefore regulating, step of cholesterol synthesis. The downregulation of the gene that codes for this enzyme? related to a lower biosynthesis of intracellular cholesterol.

Referring to FIG. 2 it is observed that the messenger RNA of the HMG-CoA reductase gene ? strongly down-regulated following treatment with the extract, already? at values equal to 10 mg/L.

Expression of regulating factors of cholesterol homeostasis at the hepatic and blood level

The factors IDOL and PCSK9 both contribute to reduce the levels of LDLR, a membrane receptor involved in the uptake of LDL in the bloodstream, therefore the down-regulation of their expression results in a greater quantity of receptors capable of internalizing LDL.

Referring to FIG. 3 it is observed that the messenger RNA of the IDOL/MYLIP gene ? down-regulated in direct proportion to the increase in the amount of extract for treatment.

Referring to FIG. 4 it is observed that the messenger RNA of the PCSK9 gene ? significantly down-regulated following treatment with the extract, starting from extract concentration values of 10 mg/L.

Expression of factors involved in lipid metabolism

The metabolism of lipids is regulated by a class of enzymes called Acetyl-CoA carboxylase, among these ? the ACC1 isoform is present, which, when present and active, is responsible for the formation of malonyl-CoA, a key precursor in the biosynthesis of fatty acids. This is followed by the activity? of FASN, a multienzyme complex that uses malonyl-CoA and acetyl-CoA in the presence of NADPH for the synthesis of palmitic acid. Inversely to ACC1 and FASN, the class of peroxisome proliferator-activated receptors alpha (PPAR?) ? involved, particularly in the liver, in the processes related to the catabolism of fatty acids. The down-regulation of ACC1 and FASN and the up-regulation of PPAR? ? index of a reduction in the biosynthesis of intracellular fatty acids against an increase in their catabolism.

Referring to FIG. 5 it is observed that the messenger RNA of the ACC1 gene ? strongly down-regulated following treatment with the extract even at very low concentrations (1 mg/L).

Referring to FIG. 6 it is observed that the messenger RNA of the FASN ? significantly down-regulated following treatment with the extract, starting from extract concentration values of 10 mg/L.

Referring to FIG. 7 it is observed that the messenger RNA of the PPAR? ? significantly upregulated following treatment with the extract even at very low concentrations (1 mg/L).

Expression of factors involved in the inhibition of the conversion of cholesterol into bile salts

FXR? a receptor present in the intestine and liver which, once bound to bile acids, migrates as a heterodimer in the nucleus, inhibiting the activity in a cascade of the CYP7A1 enzyme responsible for one of the key steps in the conversion of cholesterol into bile acids. The down-regulation of the FXR gene consequently decreases the amount of receptor present, thus allowing the inhibition of CYP7A1 to be reduced.

Referring to FIG. 8 it is observed that the messenger RNA of the FXR gene ? downregulated following treatment with the extract even at very low concentrations (1 mg/L).

ASSESSMENT OF CYTOTOXICITY? OF THE EXTRACT

The evaluation of the potential cytotoxicity? on HepG2 cells as a function of the concentration of the extract administered allowed to exclude a potential cytotoxic effect; in fact for all the concentrations of extract tested (1, 10, 25, 50 ?g/ml) the cells have shown to maintain a vitality? equal to 100%.

As can be deduced from the above, the innovative technical solution described here has the following advantageous characteristics:

- the extract object of the invention does not exert any cytotoxic effect on the hepatocyte cell line, thus resulting to be safe at all tested concentrations, which include the concentration of extract intended for the preparation of pharmaceutical formulations;

- the bioactive molecules contained in the extract exert a significant modulatory action on the transcription of the pi? important genes related to the metabolism of cholesterol and fatty acids favoring, through an accurate administration and appropriate monitoring during treatment, the maintenance of normal physiological values of cholesterol and intracellular lipids.

From the description above ? evident, therefore, as with the hydroalcoholic extract from the resin of the Protium heptaphyllum plant enriched for the acid triterpenic component, the corresponding pharmaceutical formulations comprising the aforementioned extract and the relative hydroalcoholic extraction process at controlled pressure and temperature of the aforementioned extract according to the present invention allow to achieve the proposed goals.

? equally evident, to a technician of the branch, that ? It is possible to make modifications and further variations to the solution described with reference to the attached figures, without thereby departing from the teaching of the present invention and from the scope of protection as defined by the attached claims.

Claims (18)

1. Hydroalcoholic extract from the resin of the Protium heptaphyllum plant comprising acidic triterpenes in a quantity ? 40% by weight with respect to the total quantity of the extract obtained. 2. The extract according to claim 1, comprising molecules of acidic triterpenes having a molecular weight in the range of 450 Da - 500 Da. 3. The extract according to claim 1 or 2, further comprising other triterpenes, sequiterpenes and monoterpenes with polarity? lower than that of the acidic and characteristic triterpenes of the resin of Protium heptaphyllum in quantity? lower than the concentrations present in the resin as such. 4. An extract according to claim 3, wherein the ratio by weight of acidic triterpenes to ? And ? amyrins ? variable between 1.4 and 8, preferably ? equal to 4. 5. Pharmaceutical formulation comprising the extract according to any one of claims 1 to 4. 6. A pharmaceutical formulation according to claim 5, further comprising one or more? additional ingredients chosen from: - a carrier agent of the extract chosen from: ? rice starch and tribasic calcium phosphate ? rice oil ? amphiphilic molecules such as phospholipids and derivatives ? sodium alginate, calcium chloride and microcrystalline cellulose - additional botanical extracts chosen from: ? extracted from the Commiphora mukul plant ? extracted from the Berberis vulgaris plant ? extracted from the Allium sativum plant - one or more flavoring and sweetening agents - one or more pharmaceutical, food or cosmetic grade bulking agents, excipients and diluents. The extract according to any one of claims 1 to 4 for use in inhibition of gene expression of the enzyme HMG-CoA reductase. The pharmaceutical formulation according to claim 5 or 6 for use in inhibition of gene expression of the enzyme HMG-CoA reductase. 9. An extract according to any one of claims 1 to 4 for use in altering gene expression in liver cells. 10. Pharmaceutical formulation according to claim 5 or 6 for use in the alteration of gene expression in liver cells. The extract according to any one of claims 1 to 4 for use in altering the gene expression of key enzymes involved in lipid metabolism. The pharmaceutical formulation according to claim 5 or 6 for use in altering the gene expression of key enzymes involved in lipid metabolism. 13. An extract according to any one of claims 1 to 4 for use in the treatment of hypercholesterolemia. 14. Pharmaceutical formulation according to claim 5 or 6 for use in the treatment of hypercholesterolaemia. 15. Hydroalcoholic extraction process at controlled pressure and temperature comprising the following stages: - phase 100: prepare a quantity? predetermined resin of the plant Protium heptaphyllum; - phase 101: crush the resin of the Protium heptaphyllum plant using a suitable device until obtaining a powder with a particle size in the range of 2-5 mm; - phase 102: carry out the extraction of the terpenic component pi? polar contained in the resin by introducing a quantity? predetermined resin powder in a tank with mixer, adding a quantity? predetermined of a hydroalcoholic solution and stirring at low temperature; - step 103: separate the insoluble component from the mixture by means of a suitable low temperature filtration or centrifugation system; - phase 104: concentrate the terpenic component more? polar extracted by evaporative removal of solvent under vacuum and at low temperature; - phase 105: carry out the final drying and the removal by evaporation of the most? birds; - step 106: obtaining a hydroalcoholic extract from the resin of the Protium heptaphyllum plant comprising acid triterpenes in a quantity ? 40% by weight with respect to the total quantity of the extract obtained. The process according to claim 15, wherein - phase 100: the quantity? predetermined resin of the plant Protium heptaphyllum ? variable between 80 kg and 160 kg, preferably ? equal to 110 kg; - phase 101: the crushing of the resin of the Protium heptaphyllum plant takes place by means of a refrigerated hammer mill, with a temperature maintained between -20 ?C and 15 ?C; - phase 102: the quantity? predetermined resin powder ? variable between 75 kg and 150 kg, preferably ? equal to 100 kg, the quantity? predetermined hydroalcoholic solution? variable between 350 liters and 1,000 litres, preferably ? equal to 350 liters and the extraction of the terpenic component more? polar content in the resin takes place at a temperature between -20?C and 15?C; - phase 103: separate the insoluble component, i.e. the terpenic component more? apolar consisting mainly of ? And ? amirine, from the mixture by filtration with Buchner filter, filter press or cellulosic passive filters having porosity? of 25 ?m at a temperature between -20?C and 15?C; - phase 104: concentrate the terpenic component more? polar extracted by removal by evaporation of the solvent under vacuum and at a temperature between -20°C and 15°C and at a relative pressure value lower than -950 mBar with respect to the normal atmospheric pressure value; - phase 105: carry out the final drying of the concentrated material together with the removal by evaporation in conditions of forced ventilation in a nitrogen atmosphere at a temperature between 40?C and 130?C for a time between 1 and 12 hours of the pi components ? volatile, i.e. the residual content, still present in traces, of water and ethanol and most of the mono and sesquiterpene component still present in the extract. 17. Process according to claim 15 or 16, wherein the hydroalcoholic solution is choice between a mixture containing water and an alcohol miscible in water, preferably ? a mixture consisting of water and ethyl alcohol. 18. Process according to any of claims from 15 to 17, wherein 100 kg of powdered resin are introduced into a tank with mixer, into which a hydroalcoholic solution is added (450 l, weight/volume ratio between 1:4, 5 and 1:5) having an ethanol concentration ranging from 50% to 96% by volume.