ES2861593B2 - PHARMACEUTICAL COMPOSITION INCLUDING A SOLUTION OF SILYMARIN IN A DEEP EUTECTIC SOLVENT OF NATURAL ORIGIN, METHODS OF OBTAINING AND USE IN MEDICINE - Google Patents

Description

DESCRIPTION

PHARMACEUTICAL COMPOSITION THAT INCLUDES A SOLUTION OF

SILYMARIN IN A DEEP EUTECTIC SOLVENT OF NATURAL ORIGIN,

METHODS OF OBTAINING AND USE IN MEDICINE

Technical sector of the invention

The invention falls within the field of Organic Chemistry and has its fundamental application in the pharmaceutical sector. It specifically deals with a deep eutectic solvent of natural origin (NADES, natural deep eutectic solvent) and the dissolution of milk thistle extract from the plant known as Silybum marianum in said NADES or other similar ones mainly made up of glycerol, thus forming a composition ingestible pharmaceutical as obtained and presented. The synthesis of said solution takes place in a continuous method, which includes preparing NADES by mixing glycerol heated to a temperature below 65°C with a certain amount of water and arginine, under stirring. Once the NADES is obtained, it is mixed progressively and continuously with the extract of milk thistle, shaking the complex until the complete dissolution of said extract in the NADES is achieved. This method allows the obtainable solution to be completely soluble in water and, consequently, its bioavailability and absorption within organisms is guaranteed.

Background of the invention

Organic Chemistry is an area of Science of considerable breadth and in continuous technological progress, which is applied to various sectors of the economy, such as the pharmaceutical, food, agricultural and cosmetic industries, among many others.

One of these essential areas is pharmaceuticals, which is constantly evolving to deal with new diseases and infections by viruses and bacteria, but in which old unresolved challenges also remain in force, such as diabetes, which has become a a chronic disease for the patient. In general, the basic rule for a bioactive compound to have a potential beneficial use for health is that it be soluble in water, in such a way that, after ingestion, it can end up being introduced into the bloodstream, which guarantees that ends up reaching the organ that is intended to be treated.

Indeed, bioavailability is essential to guarantee that the active compound to be supplied is used by the organism and is useful for the intended purpose. Unfortunately, the administration of many compounds often suffers from solubility problems and, as a consequence, their bioavailability is absent or very low. Therefore, one of the challenges of current Organic Chemistry is the development of new solubilization or vehicle techniques for these active compounds applicable to the pharmaceutical sector, for the purpose of improving bioavailability.

Among these techniques, the Spanish patent application P202030007, by the same inventors as the present technological innovation, can be highlighted, where the solubilization of different active compounds applicable to the nutraceutical field, specifically polyphenols, is analyzed. Said complex is formed by sonication, in a batch method by applying ultrasound for a minimum time of at least 10 minutes. Currently, these same authors have perfected what is now presented in this document: a method based on the development of new NADES of natural origin not previously described. The fundamental action of these complexes is to allow the complete solubilization of bioactive compounds, such as natural extracts, and to prevent the degradation of the molecular structure of the compounds that are part of silymarin.

Other techniques to improve the bioavailability of hydrophobic or partially insoluble compounds in water are the use of polyhydroxy alcohols as a carrier agent for said substances, a use analyzed in several publications: Shehata, Emad & Grigorakis, Spyros & Loupassaki, Sofia & Makris, Dimitris (2015 ): Extraction optimization using water/glycerol for the efficient recovery of polyphenolic antioxidants from two Artemisia species ; Separation and Purification Technology, 149(10); and Corneliu Tanase et al (2019): A Critical Review of Phenolic Compounds Extracted from the Bark of Woody Vascular Plants and Their Potential Biological Activity, Molecules, 24: 1182.

Application JP2016145207 A reveals that discoloration of a polyphenol usually occurs during the preparation of compositions that include it as an active agent, and this problem can be avoided with a compound with two or more hydroxyl groups. alcoholics, and also an organic reducing agent. However, in these polyphenol stabilization techniques an oily solvent, a fatty acid triglyceride, is used. From a compatibility, security, and storage stability standpoint, it has historically been difficult to use this solution at an industrial level.

Patent application EP2646041 A describes a method for extracting propolis (characteristic product due to the presence of polyphenols), characterized in that it comprises the formation of extractable components with hydroxypropyl-p-cyclodextrin as an inclusion agent during extraction. After defining the particle size of the propolis with a mechanical device, in the second stage said particles are dispersed in a mixture of water/glycerol, in whose aqueous phase the hydroxypropyl-p-cyclodextrin has previously been dissolved. The mixture of water and glycerol is carried out to reduce the viscosity of the medium, due to glycerol, but the addition of cyclodextrins later hinders the release and bioavailability of the polyphenols contained in the propolis.

On the other hand, the international patent application WO2008096343 A1 describes several methods of preparing a liquid (alcoholic) pharmaceutical composition based on turmeric or curcumin and an alcohol, preferably ethanol, as extractive compound, which can be dissolved in water to be ingested, its objective being medical treatment. The method describes the extraction by heating of the turmeric and curcumin components, and combining them to form the ethanolic composition. When using ethanol, the heating must be considerably high (reaching in practice its boiling temperature, 78°C) and, if glycerin is added as a solvent to replace part of the ethanol, the mixture heats up to close to 100°C. c. Therefore, these compositions can also contain glycerin, but without dispensing with ethanol as an essential component, since glycerin is barely used to replace a part of the ethanol. The required temperature is too high to preserve the chemical and organoleptic properties of the active component (in this case, curcumin), which affects its functionality and the beneficial results expected after ingestion. It has been verified that the dissolution carried out at high temperatures, similar to those indicated in this cited international patent application, produces a degradation in the chemical structure of the polyphenol, which implies the loss of its biochemical properties and, as a consequence, of its functionality. .

The international patent application WO2015094479 A refers to a composition for oral care, which includes rutin as an active agent and a solvent that can be propylene glycol, glycerin or a mixture of both (although in reality only propylene glycol is used with glycerin or sorbitol, never glycerin alone), therefore a mixture of compounds with which it is intended to increase the solubility of the active agent. The composition of this mouthwash can be prepared simply by pre-mixing both mentioned compounds and then combining the mixture with the rest of the ingredients of the final composition. On the other hand, the text does not define the conditions in which the process is carried out, such as the temperature and the quantities to be solubilized, which determine its success or failure, since it is not generally achieved under any condition.

On the other hand, in the last two decades there has been a growing interest in the development of applications of ionic liquids (ILs, solvents made exclusively of ions), especially in the field of catalysts, electrochemical processes, technology and analysis, biotechnology and functional fluids. Mainly, the synthesis of IL s can be divided into two distinct categories, namely those that are formed from eutectic mixtures of metal halides and organic salts, and those that contain discrete anions. Due to the increasing need for new organic solvents and the high cost of ILs, researchers have focused their interest on deep eutectic solvents (DES), introduced by Abbot et al. in 2003, Novel solvent properties of choline chloride/urea mixtures.

Basically, DES are mixtures of two or more compounds that ultimately have a lower melting point than the individual compounds. Basically, DES are made by mixing a salt and a hydrogen bond donor (HBD), bonding the hydrogen to the anion of the salt. DES can be formed between different types of salts (organic and inorganic) and different types of HBD. DES have many advantages over conventional ILs, including simplicity of synthesis, lower cost of production, low or negligible toxicity profiles, and sustainability with respect to environmental and economic benefits. Recently, the use of DES has been reported in many applications, such as the use of catalysts for biodiesel production from palm oil. low-quality, electrolytes in electrochemical processes such as metal deposition in electroplating and metal electrodeposition and as viable cosolvents for enzyme-catalyzed hydrolysis of epoxides.

As for the known HBDs (donors), glycerol is a conventional solvent that is defined in Organic Chemistry as a polyol and is widely used in many industrial applications, especially in the food and pharmaceutical industries. However, there is limited use of it in organic transformations due to its low solubility in organic compounds and its intrinsic reactivity, which leads to the formation of side products. To overcome these disadvantages, researchers have worked to improve the physicochemical properties of glycerol through different methods. One of them is the preparation of DES that contain glycerol in its composition, such as HBD.

A deep eutectic solvent is used in international application WO2015044139A1 where a process for obtaining a flavoring is described, by preparing a reaction mixture comprising said solvent and flavor precursors, heating the mixture to form flavoring compounds. The deep eutectic solvent is a liquid based on a combination of at least two solid compounds at 25°C and comprises water and/or glycerol in an insufficient amount, or in such an amount that all solid compounds at 25°C are simultaneously saturated. at that temperature. Flavors or flavor precursors may comprise the compounds solid at 25°C on which the deep eutectic solvent is based. A further aspect of the invention is a food product comprising the flavoring composition obtainable by the process of the invention. The process obtained through this patent is limited to obtaining a product with flavoring characteristics, not a DES that serves as a solvent despite the fact that it calls it that. At the same time, it is synthesized at 25°C, a temperature that is insufficient when DES is intended to act as a solvent and obtain a solution of other active agents with which it can be combined, such as polyphenols, which are insoluble in water.

Regarding natural bioactive compounds, it is well known that silymarin is made up of different molecules, such as silybin, silydianin, silycristin and their corresponding diastereoisomers, with a common characteristic: almost no solubility in water and very poor bioavailability, therefore, despite its potential, its use as an active agent is greatly reduced. Silymarin is a complex mixture of flavanolignans (70-80%), represented in the diagram below, among which are silybin (A and B; 50%), isosilybin (A and B; 5%), dehydrosilybin (A and B ), silidianin (10%), silicristin (20%), a flavanol derivative (taxifolin) and a large number of secondary components. Silybin is the major component and is made up of an equimolar mixture of two diastereoisomers: silybin A and silybin B.

All of them are enantiomerically pure, therefore presenting a defined stereochemistry in their stereocenters and this is of great importance because the two enantiomers of a compound may present different biological activity (different reaction rates, interactions with different receptors, secondary effects). , etc.). In order to rationalize the different active compound-receptor interactions observed for the two enantiomers of a compound biologically active, Easson and Stedman proposed in 1933 a " 3D point interaction model". The model is based on the fact that if three groups of the molecule of a given enantiomer interact with three complementary positions in a chiral receptor for the organism, the other enantiomer will not be able to interact in exactly the same way.As a consequence, the two enantiomers of a compound may exhibit different biological activity.

On the other hand, diabetes is a chronic disease suffered by many patients worldwide, which originates because the body does not synthesize the amount of insulin it needs. This hormone is produced in the pancreas and its main function is to maintain adequate blood glucose values. It allows glucose to enter the body and be transported into cells, where it is transformed into energy for muscles and tissues to function. It helps cells store glucose until its use is needed. In people with diabetes there is an excess of glucose in the blood (hyperglycemia), since it is not distributed properly. High glucose can be harmful to the entire body, but mainly to the heart, kidneys, and arteries, so people who have diabetes and don't know it or don't treat it have a higher risk of kidney problems, heart attacks, weight loss, vision loss and lower limb amputations.

Type I diabetes is caused by the destruction of insulin-producing cells and usually appears in childhood. Currently, it is not possible to prevent type I diabetes, despite many attempts that have been made. For its part, type II diabetes mellitus is the most frequent and preventable type and is produced by a deficit of insulin, which is added to a reduced action of this hormone in the tissues. This type, which is the most frequent, can be prevented. Since the most important cause is obesity, all actions that have to do with obesity prevention -avoid sedentary lifestyle, junk food, sugary drinks...- will have a positive result. A healthy lifestyle reduces the chances of type II diabetes.

The incidence of diabetes in Spain is 11.58 cases per 1,000 people per year; 1,057 new cases are generated every day. The current percentage of people with diabetes (prevalence) is 13.8%, about 6 million people in Spain alone.

Some scientific evidence has been found on trials that demonstrate a certain partial efficacy of silymarin in type II diabetes mellitus, but as so far it has not been possible to improve its solubility and bioavailability, none of these scientific articles have obtained conclusive results. and effective. Citing some of these works, we find:

- Best herbs for managing diabetes: A review of clinical studies. Ahmad Ghorbani. Pharmacological Research Center of Medicinal Plants, School of Medicine, Mashhad University of Medical Sciences, Iran. Brazilian Journal of Pharmaceutical Sciences vol. 49, no. 3, Jul/Sep 2013.

- Effect of silymarin in diabetes mellitus patients with liver diseases. Journal of Pharmacology and Pharmacotherapeutics, Oct.-Dec. (2011), Vol 2-Issue 4.

- Silymarin in Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Luminita Voroneanu, Ionut Nistor, Raluca Dumea, Mugurel Apetrii, and Adrian Covic. Hindawi Publishing Corporation. Journal of Diabetes Research. Volume 2016, Article ID 5147468, 10 pages. http://dx.doi.Org/10.1155/2016/5147468.

- Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells. Marta García-Ramíreza, Mireia Turcha, Olga Simó-Servata, Cristina Hernándeza, Rafael Simóa. Endocrinol Diabetes Nutr. 2018; 65(4): 200 205. https://doi.org/10.1016/j.endinu.2017.12.004.

- The Efficacy of Silybum marianum ( L.) ( Silymarin) in the Treatment of Type II Diabetes: A Randomized, Double-blind, Placebo-controlled, Clinical Trial. H. Fallah Huseini, B. Larijani, R. Heshmat, H. Fakhrzadeh, B. Radjabipour, T. Toliat and Mohsin Raza. Phytotherapy Research Phytother. Res. 20, 1036-1039 (2006) DOI: 10.1002/ptr.1988.

In view of these documents, and given the need to promote the appearance of new pharmaceutical compositions from natural formulations, whose bioavailability is high enough to guarantee that they are effective, the present invention proposes a new solubilizing vehicle, specifically a NADES to water base, glycerol and the amino acid arginine, of said active agents of natural origin, specifically silymarin, and the composition that is prepared from said vehicle, as well as the method for obtaining it and its use in medicine.

General description of the invention

By means of the method presented in this invention, without carrying out previous treatments of the substances or subsequent mixtures with other components, the solubility and aqueous bioavailability of silymarin is drastically improved, a compound obtained from the extract of the Silybum marianum plant, originally insoluble, and its direct application in the pharmaceutical field is viable. In order to achieve this objective, a technique has been developed that consists of initially producing a deep eutectic solvent of natural origin DES as a carrier agent, from glycerol (also called glycerin or propan 1,2,3-triol), and which in the best embodiment also contains the amino acid arginine and water. Once the carrier agent is formed, and after mixing it with the extract (silymarin) as active agent, vigorous stirring is carried out, until the solubilization of the active compound is ensured in a short period of time, giving rise to the formation of complexes in dissolution.

Specifically, the present invention refers to a naturally occurring deep eutectic solvent (NADES) whose formulation is composed of water, glycerin and an amino acid that is arginine in a molar ratio between 1:1:0.05 up to 2:1:0.05, which presents complexes that are macromolecules in solution formed by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin, water and the carboxyl group and the amino groups of the guanidinium group of arginine, and where glycerin and this amino acid are hydrogen bond acceptor and donor compounds at the same time.

Indeed, in addition to comprising water in the defined amount, a unique characteristic of this complex is that NADES usually identifies a substance that acts as a hydrogen bond donor (HBD) compound and the other as an acceptor compound (HBA, hydrogen bond acceptor). Glycerol for example is typically an HBD with choline chloride or amino acids other than arginine acting as HBA. Well, when the amino acid is arginine, due to the basic nature of the guanidinium group and the tendency to form hydrogen bonds from these amino groups, it can act as HBD and HBA simultaneously in the composition described, so it forces glycerin to act with this same duality. For the first time in the technical literature, a NADES with these characteristics is described, in which its forming compounds act as HBD and HBA simultaneously.

Indeed, the advantage of the presence of arginine is the corresponding presence of the guanidinium group in the molecule, which also fulfills blood glucose control functions, a fundamental objective of the present invention, as shown below. Thus, by mixing and dispersing the bioactive agent (such as silymarin) in NADES, the solution complexes of the compound that is prepared are formed not only by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin, the carboxyl and guanidinium groups of arginine and water, but also with the phenolic hydroxyl groups of the active agent.

The presence of water in NADES is not an accessory, since in addition to improving the viscosity of NADES and the compounds with which it is used to prepare compositions with bioactive agents, such as natural extracts, it further improves the solubility of said substances. active agents with which the NADES is combined, particularly if water appears in the amount defined in this invention.

The following images represent the molecular structure of arginine and its resonant forms: L-arginine is the most basic of the 20 naturally occurring amino acids because its guanidinium ion side chain is protonated under all conditions commonly found within amino acids. cells. This guanidinium ion can be represented by the following resonant forms:

As seen in the following average structure, this positive charge is distributed equally by the three nitrogens of the guanidine function.

The guanidine function is present in various compounds that have been used traditionally and even today for blood glucose control. Of note is galegin, which is extracted from the Galega officinalis plant, a toxic compound that was used in the past for the treatment of diabetes, until better non-toxic synthetic drugs such as metformin were developed. The chemical structure of galegin is represented below:

Metformin, or the commercial preparation metformin hydrochloride, is an antidiabetic drug of the biguanide type. It is commonly used in the treatment and prevention of type II diabetes mellitus. Since 2009, metformin is one of two oral antiglycemic agents that belong to the World Health Organization's model list of essential medicines, along with glibenclamide, and is the only known medicine capable of preventing cardiovascular diseases associated with diabetes. The structure of metformin is represented below:

There are other biguanides, such as phenformin and buformin, already withdrawn from the market:

These cited compounds have their historical origin in the Galega officinalis plant (rue goat or Galician) that was used in medieval times to reduce exaggerated urination in diabetics.

Below is the chemical structure of the interactions that form between arginine and glycerol to form NADES. Molecules with electrostatic charges are represented.

Examples of NADES formation: L-arginine-glycerol: In glycerol we find different possibilities to act as a hydrogen acceptor or donor:

- As an acceptor: The presence of the guanidinium ion in arginine leads to a delocalization of the electronic deficiency between the three nitrogens equally, as seen when studying the resonant forms. It turns out that arginine can act as a donor in the formation of hydrogen bonds with glycerin, with one, two or three glycerin units. Here are a few of the many possibilities that can be written:

o With a glycerin molecule:

o With two glycerin molecules:

o With three units of glycerin, with the primary alcohols or with primary and secondary alcohols.

- As a donor: Glycerin with the amino acid L-arginine can also act in the formation of NADES as a hydrogen donor:

In the same arginine molecule, the two previous possibilities and all the possible variations can occur:

Another object of the present invention is the preparation method of the deep eutectic solvent of natural origin described herein, which comprises:

a) preheat glycerin and water to a temperature between 50°C-65°C, in a molar ratio between 1:1 and 2:1; and

b) mix the mixture preheated in the previous stage with arginine in a molar ratio of water:glycerol:arginine that varies between 1:1:0.05 and 2:1:0.05, until complexes are formed that are macromolecules in solution through weak intermolecular interactions by hydrogen bonds between the hydroxyl groups of glycerin, water and the carboxyl and guanidinium groups of arginine, and where glycerin and arginine are hydrogen bond acceptor and donor compounds at the same time.

Preferably, the temperature to which the mixture of glycerin and water is preheated is 60°C, which is the temperature at which the NADES is prepared throughout the process.

Likewise, the present invention refers to the use of the NADES described in this specification as a carrier agent for bioactive compounds in the formation of pharmaceutical compositions, the active compound being preferably silymarin. The advantages of this use are described below in defining the bioactive compositions that are prepared from NADES.

Thus, another object of the present invention is constituted by a pharmaceutical composition characterized in that it comprises:

- a bioactive agent that is silymarin or any of its active constituents; dissolved in

- a vehicle agent that is a deep eutectic solvent of natural origin (NADES), which comprises at least 75% by weight of glycerin with respect to the total weight of the vehicle agent, that is, as the main constituent;

in a ratio of silymarin and carrier agent between 1:10 and 1:25 by weight, where the composition is a solution of macromolecular complexes formed at least through weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin and the phenolic hydroxyl groups of silymarin.

The technique presented in this document is based on the elaboration of a natural composition, without additives or emulsifiers, from the use of deep eutectic solvents of natural origin (NADES) based on glycerol, a natural and innocuous substance that can be ingested safely. quantum satis, usually marketed in its bidistilled form and obviously (due to its application) food grade.

Silymarin is a more or less standardized extract of milk thistle seeds, which contains a mixture of flavonolignans, thus including silibinin (silybin), isosilybinin, silicristin and silydianin, among other constituents (see definition in the Description of the invention section, which by reference is integrated here as well as definition of the invention), which act as active agents also independently of each other. Thus, this pharmaceutical composition, as described herein, can be prepared starting from the complete silymarin extract as a bioactive agent, as it is found and marketed, or using one or more of its active molecules or components as said active agent, as is the case. preferred of silybin.

As for the deep eutectic solvent of natural origin, it preferably contains glycerin in an amount between 75%-85% by weight of the total composition of NADES. Also preferably, the NADES comprises water, which improves its solvent properties. It should be noted that in this NADES glycerol acts clearly as HBD, since it is the type of molecular relationship that it establishes with the other components (as we have said, arginine is a very rare exception in which both glycerol and arginine act doubly as HBD and HBQ). Therefore, this NADES also includes an additional compound that is an HBA of natural origin, preferably an amino acid, which is more preferably selected from the group consisting of: arginine, betaine, choline chloride and citric acid.

The proportion of silymarin and NADES in the mixture is preferably between 1:15 by weight.

In the most preferred particular embodiment, the invention refers to a pharmaceutical composition as defined above but which particularly comprises:

- a bioactive agent that is silymarin or any of its active constituents, dissolved in:

- a carrier agent that is the deep eutectic solvent of natural origin described in this report, whose formulation is made up of water, glycerin and an amino acid that is arginine in a molar ratio between 1:1:0.05 to 2:1 :0.05, which presents macromolecular complexes in solution formed by weak intermolecular interactions by hydrogen bonds between the hydroxyl groups of glycerin, water and the carboxyl group and the amino groups of the guanidinium group of arginine, and where the glycerin and this amino acid are hydrogen bond acceptor and donor compounds at the same time;

in a proportion of silymarin and carrier agent between 1:10 and 1:25 by weight, where the composition is a solution of macromolecular complexes formed by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin, water , the carboxyl and guanidinium groups of arginine and the phenolic hydroxyl groups of silymarin.

The properties described above for NADES, both essential and preferred, are applicable to pharmaceutical compositions that are prepared from said solvents. Likewise, the proportion of silymarin and NADES in this specific mixture is also preferably between 1:15 by weight.

Solubility studies and characterization of the stability of silymarin in NADES when the composition is formed have been carried out with a UV-VIS spectrophotometer and/or an HPLC-Mass-mass chromatograph. It has also been verified through these analyzes that, unexpectedly, the solubility in water of the complexes that form part of the resulting solution is maintained even in successive aqueous dilutions. This result is key to formulating a concentrated product and, later, being able to consume it diluted in water. Thus, the composition obtained maintains its solubility in water in subsequent dilution steps, from 1 to 50 to even 1 to 100 by weight of compound:water. In a preferred case, the composition can be consumed diluted in water in a weight ratio of between 1:1 and 1:100.

The chemical structure of the macromolecules that are in solution in the pharmaceutical composition object of protection formed by NADES and silymarin is represented below, where, as has already been said in the preferred case of NADES, the amino acid of the composition is the arginine. Molecules with electrostatic charges are represented. There is a solvation of NADES with water and an interaction by hydrogen bonds with silymarin (the silybin A molecule has been chosen to represent this interaction, which, as has been said, could act as an independent bioactive agent, having been extracted from silymarin). The solvation of the complex has not been considered.

• Glycerol and water bound to guanidinium ion: In this first example, water acts as a hydrogen bond donor against glycerol and arginine, the same with respect to silymarin:

In this second case, the water donates hydrogen bonds to glycerin and arginine. Silybin gives them to the amino acid through flavone phenols:

Glycerol attached to the alpha zone of the amino acid:

Two glycerol units, one attached to the guanidinium ion and one to the amino acid:

In the previous images, a plurality of possibilities have been represented in the hydrogen bonding that takes place during the formation of NADES, with arginine and silymarin as active agents in the pharmaceutical composition. These structures are not exclusive of other analogous ones that may take place. They should simply be considered indicative of the electrostatic bridges between the different compounds and that take place for the formation of the total complex.

When the pharmaceutical composition described, after being obtained, is further diluted in water (for example, before being ingested), the hydroxyl and amino groups present in the complex are solvated with it, increasing the solubility of the entire complex, which It is an additional advantage if one thinks that the invention is based on a bioactive agent, which is a natural extract, silymarin, with a very low solubility in water, almost zero. Solvation is an interaction of a solute with a solvent that leads to the stabilization of the solute species in solution. We have verified that this fact occurs in the complex formed, which entails, in unexpectedly, a drastic increase in the solubility in water of the complex formed between NADES and silymarin.

Another object of the present invention consists of a method for obtaining the pharmaceutical composition described from the deep eutectic solvent of natural origin (NADES) as a carrier agent, which comprises:

- mix and dissolve a bioactive agent that is silymarin in the deep eutectic solvent of natural origin, by stirring for at least 3 minutes at a temperature between 50°C and 65°C, in a ratio of silymarin and NADES between 1: 10 and 1:25 by weight, until they form macromolecular complexes through weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin and the phenolic hydroxyl groups of silymarin.

When in the most preferred embodiment the NADES used is the one that is protected in the present invention, then the macromolecular complexes are formed by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin, water, carboxyl groups and guanidinium from arginine and the phenolic hydroxyl groups from silymarin.

During the composition synthesis process, the starting temperature of the mixture is preferably 60°C. In any case, it never exceeds 65°C, that is, it is the same temperature at which the deep eutectic solvent of natural origin is obtained separately. In this way, it is guaranteed that when the extract or active agent is dispersed in the DES, the molecular structure of the active compounds in the silymarin are not degraded by the negative effect of excessive temperature applied to the molecule. This prevents the degradation of the biochemical properties of the molecules. This result is of great importance so that the biochemical and organoleptic characteristics are maintained intact when it is used as a pharmaceutical.

In this method, the temperature of the mixture is preferably the same at which the NADES is prepared, and at which it is once obtained. As a consequence of the foregoing, in a particular embodiment of the invention, the method for obtaining the pharmaceutical composition, which includes silymarin as an agent bioactive, comprises combining the preparation of NADES following the indications given in this report with the subsequent preparation of the composition, also with the method described herein, in the same process. In this way, the last stage is carried out at the temperature at which the NADES has previously been obtained, avoiding the prior heating of the NADES before mixing with the active agent.

In a particular embodiment, the stirring time is between 3 and 7 minutes, and more preferably it is between 5 and 7 minutes. In a more particular embodiment, the agitation of the mixture to achieve the complexation of the active agent with the vehicle agent is carried out for 5 minutes.

All the limitations and variations described for the pharmaceutical composition are also applicable to the method of preparation thereof. Thus, said composition preferably has a ratio of silymarin and NADES of 1:15 by weight.

Another aspect of the present invention is the use of the described pharmaceutical compound in medicine, that is, the invention also contemplates a compound as described for pharmaceutical and medical use. Specifically, preferably, the described pharmaceutical compound is used for the prevention and treatment of diabetes and low molecular weight LDL cholesterol, having observed an effect of the compound in reducing both diabetes and cholesterol after its ingestion. low molecular weight LDL. This beneficial effect is due to its anti-inflammatory, antioxidant, and regenerative properties of the pancreatic Beta cells, located inside the islets of Langherans.

The dosage to be ingested daily for treatment is preferably 5 ml, for at least 3 months, which is the time it takes for a significant decrease in glycated hemoglobin to occur; however, the drop in blood glucose requires less time with this treatment, even in a matter of days. On the other hand, the dosage to be ingested daily for prevention is preferably 3 ml.

An analysis of the activity of the described pharmaceutical compound is presented below, specifically of its active agent, silymarin, on endocrine function. of the pancreas, including its mechanism of action, of special relevance in order to support the solidity of the research carried out and the importance of the results obtained.

The pancreatic beta cell is a highly specialized cell, located inside structures called islets of Langerhans. They play a unique role in the physiology of the organism, since it is the only one capable of synthesizing the hormone insulin. Insulin then travels through the bloodstream to reach peripheral tissues to promote the uptake, utilization, and storage of nutrients. Its direct effects are to allow the passage of glucose into all cells, and thus maintain the balance between glucose and insulin levels in the body.

Chronic exposure to elevated levels of glucose and free fatty acids (FFA) causes p-cell dysfunction and can induce apoptosis leading to type I diabetes and type II diabetes. Exposure to high glucose has dual effects, initially triggering "hypersensitization" and then apoptosis, through different mechanisms that are explained later.

Pancreatic islets produce a variety of cytokines and chemokines in response to physiologic and pathologic stimulation induced by ingested nutrients. The cellular source of these inflammatory mediators includes alpha, beta, endothelial immune cells... and the cytokines produced in the islets promote adaptation and repair of alpha and beta cells in the short term. Eventually, chronic metabolic stress can induce a deleterious autoinflammatory process in the islets that leads to failure of insulin secretion and eventual diabetes. Understanding the specific role of cytokines and chemokines allows for targeted clinical interventions aimed at remodeling islet inflammation and preventing its destruction.

In both type I and type II diabetes, an increase in the synthesis and release of proinflammatory cytokines is observed, which cause damage to the pancreatic islets and, also in type II, the development of insulin resistance. This process results from an ultimate imbalance between proinflammatory and protective cytokines. Proinflammatory, such as ip interleukin (IL-1P), necrosis factor tumor a (TNF-a) and interferon-y (IFN-y), as well as the PANDER factor, are involved in the activation of different metabolic pathways that can end in apoptosis of pancreatic p cells. Interleukin IL-ip activates mitogen-activated protein kinases (MAPKs), affects nuclear factor kappa-activated B-cell light chain enhancer (NF- ^kB ), and activates inducible nitric oxide synthase (¡NOS). TNF-a and IFN-y synergistically activate calcium channels, leading to mitochondrial dysfunction and caspase activation. Neutralization of proinflammatory cytokines, especially ip interleukin with IL-1 receptor antagonist (IL-1Ra) and/or IL-ip antibodies can cause extinction of the pancreatic islet inflammatory process and consequently normalize blood glucose concentration and decrease insulin resistance.

In type I diabetes, the invading immune cells produce cytokines such as IL-ip, tumor necrosis factor-α (TNF-α), and interferon (IFN-γ). IL-ip and/or TNF-α together with IFN-γ induce p-cell apoptosis through the activation of gene networks under the control of the transcription factors NF- ^kB and STAT-1. Activation of the NF- ^kB factor leads to nitric oxide (NO) and chemokine production and endoplasmic reticulum (ER) calcium depletion. Death of p cells occurs through activation of mitogen-activated protein kinases, through activation of ER stress, and by initiation of mitochondrial death signals. However, high glucose does not induce or activate IL-1p, NF- ^kB , or inducible nitric oxide synthase. P-cell death occurs through activation of mitogen-activated protein kinases, through activation of ER stress, and by the release of mitochondrial death signals.

Silymarin has marked antioxidant activity and is capable of capturing free radicals in order to prevent them from reacting with cellular components. This mechanism occurs through the phenolic hydroxyls and determines their ability to capture the free radicals that are being formed.

Of the three phenolic hydroxyls present in the silybin structure, those at position C-7 and C-20 are the only ones that capture free radicals; the C-5 phenolic hydroxyl is somewhat exceptional because of its strong hydrogen bonding with the adjacent C=O group, which is conjugated to the aromatic ring, which disables it for the function of capturing free radicals:

interaction with

free radicals

radicales libres 5-OH forma un interaction with

5-OH free radicals form a

strong bond of

hydrogen with C=0

The radical that is formed with the OH in position 7 is more stable, because the conjugation extends to the aromatic ring and to the carbonyl.

Regarding the radical with OH at position 20, delocalization only occurs in the aromatic ring.

In general, the body forms oxidizing species that can damage the lipids, proteins, and DNA of our cells. The antioxidant effect of flavanolignans has been widely demonstrated in numerous studies and is due to mechanisms of action on the three antioxidant levels of the body, both directly (they act on free radicals) and indirectly (they prevent their formation and propagation). ).

Under normal conditions, cells metabolize most of the oxygen (O2) with the formation of water without generating toxic intermediates, while a small percentage (about 5%) forms three highly toxic intermediates, two of which are literally free radicals. (superoxide anion and hydroxyl). Low levels of antioxidants or inhibition of antioxidant enzymes cause what is called oxidative stress, and their presence in the body can damage or kill cells.

Cells are surrounded by a membrane that separates them from the extracellular medium. The basic structure of all biological membranes is the lipid bilayer, which functions as a selective permeability barrier. These are rich in polyunsaturated fatty acids and therefore vulnerable to attack by free radicals that result in lipid peroxidation. It is generally induced by a hydroxyl radical, which subtracts a hydrogen from the side chain of a fatty acid. forming a carbon radical, which generates a chain of oxidative reactions as seen in the following scheme:

Sibyllin is an antioxidant due to the phenolic hydroxyl groups it possesses, which capture free radicals to generate the flavinic radical (previous diagram, where the delocalization of the radical along the molecule was represented), which is much less reactive, since in it the unpaired electrons are more delocalized. In this way, the lipid peroxidation process is prevented, purifying reactive oxygen species before they can damage cells.

It has been verified here how silymarin inhibits lipid peroxidation, but it has also been seen that it activates antioxidant enzymes that prevent DNA degradation and is effective against the oxidation of low-density lipoproteins LDL (called vulgarly bad cholesterol), among others. Among the activities carried out indirectly, the following stand out:

- Increases the activity of erythrocytes, with the consequent increase in the amount of oxygen that the tissues receive;

- It has the capacity to chelate free transition metal ions such as iron and copper, which catalyze peroxidation reactions of fatty acids and phospholipids that constitute cell membranes. The following image explains this behavior:

- Induces the synthesis of glutathione (main antioxidant agent in cells, specifically a peptide made up of three amino acids, glutamate, cysteine and glycine), increasing the availability of cysteine, essential for its synthesis;

- Prevents the decrease of glutathione in stressful situations, thus reducing liver damage. The decrease in damage markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and fatty acid peroxidation is detected;

- Maintains an optimal redox balance in the cell, of special importance in the mitochondria, where more reactive oxygen species (ROS) are generated. It prevents the formation of ROS as it contributes to the functioning of the electron transport chain. This activity is also attributed to silybin, the main component of silymarin;

- Prevents the formation of ROS by inhibiting enzymes that form them, such as Xanthine oxidase or NADPH oxidase; and

- Promotes the activation of vitagenes. This term arose in 1998 and refers to a family of genes responsible for maintaining cell homeostasis in stress situations. Several studies show that silymarin can act by activating different points in this vitamin network.

The induction of the explained mechanisms translates into a protective effect of DNA, proteins and lipids against oxidation and formation of free radicals, which causes structural alterations in the functionality of these molecules and ultimately induces the appearance of various types of pathologies. .

Simultaneously, an anti-inflammatory effect of silymarin has been described in liver tissue, in the pancreas or in inflammatory bowel disease. There is sufficient evidence that it regulates several inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin receptor antagonists (IL-ip, IL-6 and IL-1) and nitric oxide. In addition, it reduces the synthesis of prostaglandins and leukotrienes, two potent neutrophil chemoattractants, inhibits cyclooxygenase II, reduces cytotoxic activity, CD8 proliferation, and decreases neutrophil sequestration at the site of inflammation.

Oxidative stress and inflammation are considered the main pathways contributing to diabetic pathogenesis. Silymarin administration reduces the levels of inflammatory cytokines (TNF-a and IL-1P) and oxidative stress mediators such as myeloperoxidase activity, lipid peroxidation, carbonyl, and thiol content of pancreatic tissue in an almost dose-dependent manner.

Furthermore, silymarin possesses antifibrotic properties. Fibrosis has two causes: inflammation and oxidative stress, which are triggered by different factors. Numerous trials have studied the mechanisms by which silymarin is capable of inhibiting this fibrosis:

- Reduces inflammation by antagonizing the release of leukotrienes in cells; - Inhibits the release of cytokines such as TNF-α, adhesins such as E-selectin, nitric oxide (NO) and 5-lipoxygenase;

- They delay the activation of stellate cells by inhibiting the activation of NF-kB and a-SMA, delaying their transformation into myofibroblasts;

- They reduce the expression of components of the extracellular matrix such as progocollagen-a, collagen and fibronectin; and

- They modify the expression of different genes related to the organization of the cytoskeleton and to extracellular matrix metalloproteinases (TIMPs) (enzymes that degrade components of the extracellular matrix) by reducing TGF-p expression.

The analysis that has been presented here can be summarized in a very abbreviated way in that the antioxidant and anti-inflammatory action that silymarin possesses is capable of preventing the appearance of diseases such as diabetes, but also inducing its recovery through the progressive regeneration of the cells. cells p.

examples

Example 1: Procedure for obtaining the deep eutectic solvent of natural origin (NADES) according to the present invention pharmaceutical compounds according to the present invention

The synthesis process begins with the preparation of the NADES. Weigh 92 grams of glycerol, 18 grams of water, mix, and heat to 60°C. Subsequently, 9 grams of the amino acid arginine are weighed and added to the previous complex under strong stirring. This system is kept stirred for 5 minutes, at that same temperature.

The composition obtained and its properties have been illustrated in the Description of the invention.

Example 2: Procedure for obtaining the pharmaceutical compound according to the present invention, from the NADES prepared in Example 1 Once the NADES, which is the carrier agent, has been produced, the silymarin extract is mixed progressively under high stirring with said NADES, at the same temperature as it was obtained, in a 1:15 ratio (silymarin:NADES) in weight. This stirring is also maintained for 5 minutes, until complete dissolution of the silymarin.

The pharmaceutical composition obtained and its properties have been illustrated in the Description of the invention, where illustrative examples of the macromolecules that are formed between the components by hydrogen bonds are shown.

Example 3: Treatment of diabetes with the pharmaceutical compound prepared in Example 2

A small-scale trial was carried out with six patients, two with type I diabetes and four with type II diabetes, aged between 50 and 75 years, with a decrease in glycated hemoglobin of 0.5% every three months. , as well as a stabilization of the glucose level around 130 mg/dl (fasting) after 30 days of treatment, when initially they were at values above 250 mg/dl.

Claims (10)

1. A deep eutectic solvent of natural origin, characterized in that it is composed of water, glycerin and an amino acid that is arginine in a molar ratio between 1:1:0.05 to 2:1:0.05, which presents complexes macromolecular compounds in solution formed by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin, water, and the carboxyl and guanidinium groups of arginine, and where glycerin and arginine are hydrogen bond acceptor and donor compounds at once. 2. A method of preparing the deep eutectic solvent of natural origin defined according to claim 1, characterized in that it comprises: a) preheat water and glycerin to a temperature between 50°C and 65°C, in a molar ratio between 1:1 and 2:1; and b) mix the preheated mixture in the previous stage with the arginine in a molar ratio of water:glycerin:arginine between 1:1:0.05 and 2:1:0.05, until macromolecular complexes are formed in solution by intermolecular interactions weak by hydrogen bonding between the hydroxyl groups of glycerin, water, and the carboxyl and guanidinium groups of arginine. 3. The method of preparing the deep eutectic solvent of natural origin according to claim 2, wherein the temperature to which the mixture of glycerin and water is preheated is 60°C. 4. Use of the deep eutectic solvent of natural origin defined according to claim 1, as a carrier agent for bioactive compounds in the formation of pharmaceutical compositions. 5. A pharmaceutical composition characterized in that it comprises: - a bioactive agent that is silymarin or any of its constituent molecules; dissolved in - a carrier agent that is a deep eutectic solvent of natural origin that is composed of water, glycerin and an amino acid that is arginine in a molar ratio between 1:1:0.05 to 2:1:0.05, which presents dissolving macromolecular complexes formed by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerol, water, and the carboxyl and guanidinium groups of arginine, and where glycerol and arginine are hydrogen bond acceptor and donor compounds. hydrogen at a time; in a proportion of silymarin and carrier agent between 1:10 and 1:25 by weight, where the pharmaceutical composition is a solution of macromolecular complexes formed by weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin, the water, the carboxyl and guanidinium groups of arginine, and the phenolic hydroxyl groups of silymarin. 6. The pharmaceutical composition according to claim 5, wherein the proportion of silymarin and carrier agent is 1:15 by weight. 7. The pharmaceutical composition according to any one of claims 5 or 6, wherein the active agent is one or more constituents of silymarin selected from the group consisting of: silibinin, isosilybinin, silycristin and silydianin. 8. The pharmaceutical composition according to any one of claims 5 to 7, which is diluted in water in a weight ratio of between 1:1 and 1:100. 9. A method for obtaining the pharmaceutical composition defined according to any one of claims 5 to 8, characterized in that it comprises: - mix and disperse the silymarin in the deep eutectic solvent of natural origin, by stirring for at least 3 minutes at a temperature between 50°C and 65°C, in a ratio of silymarin and deep eutectic solvent between 1:10 and 1:25 by weight, until macromolecular complexes are formed in solution by means of at least weak intermolecular interactions by hydrogen bonding between the hydroxyl groups of glycerin and the phenolic hydroxyl groups of silymarin. 10. The method for obtaining the pharmaceutical composition according to claim 9, wherein the temperature is 60°C.