Method for obtaining l-arginine caffeate salt in aqueous solution and use

Abstract

The invention relates to a method for obtaining L-arginine caffeate salt, in aqueous solution, which comprises: (a) preparing a disperse, non-solubilised mixture of 10-15% by weight caffeic acid in water; (b) adding 10-15% by weight L-arginine to the disperse mixture obtained in step (a), always in an amount equimolar to the amount of caffeic acid; (c) heating to a temperature of preferably 40-50°C, agitating and applying ultrasound with a frequency of preferably 15-25 kHz until the caffeic acid is completely dissolved and L-arginine caffeate salt is formed. The invention also relates to a pharmaceutical composition comprising the L-arginine caffeate salt for use as a drug and for use as an antimicrobial compound and / or hygiene product.

Description

[0001] METHOD FOR OBTAINING L-ARGININE CAFFEATE SALT IN SOLUTION

[0002] AQUEOUS AND USE

[0003] DESCRIPTION

[0004] TECHNICAL SECTOR

[0005] The present invention falls within the general field of chemistry, more specifically organic chemistry, and has applications in the pharmaceutical and medical device sectors. More specifically, it falls within the field of compositions that act as antimicrobials.

[0006] BACKGROUND OF THE INVENTION

[0007] Biotechnology is a vast field of continuous technological advancement, applicable to various branches of science and industry, evolving to meet new legislative and consumer demands, generating new solutions to increasingly stringent needs. Its vast potential allows for the generation of new biologically active compounds with improved activity compared to current ones. In the pharmaceutical and cosmetic sectors, there is growing interest in the use of natural substances that are harmless or have reduced toxicity, that have therapeutic value and do not compromise secondary aspects related to human health.

[0008] The bioavailability of an active compound (a drug or a natural molecule with some effect on human metabolism) is defined as the degree and speed with which it enters the circulation and reaches its site of action. Therapeutic compounds administered orally must cross the intestinal wall and then travel through the portal circulation to the liver, where the first step of metabolism occurs. Reduced or nonexistent bioavailability is common in drugs that are poorly water-soluble. Therefore, the basic requirement for a compound to be bioavailable is that it be soluble in water and thus able to be absorbed as it passes through the digestive and intestinal tracts.

[0009] Caffeic acid is a phenolic acid found in a variety of foods, including fruits, vegetables, grains, coffee, tea, and wine. It is an antioxidant that protects cells from damage caused by free radicals. Its health-promoting properties include antioxidant protection (helping to protect cells from damage caused by free radicals, which can contribute to the development of chronic diseases), reducing inflammation, improving cognitive function (such as memory and attention), protecting the liver from damage caused by toxins, and reducing the risk of certain cancers, such as colon cancer, breast cancer, and prostate cancer.

[0010] Caffeic acid is generally safe for most people when consumed in moderate amounts. The richest dietary sources of caffeic acid include coffee (one cup of black coffee contains about 120 mg of caffeic acid), tea (one cup of black tea contains about 30 mg), fruits (including blueberries, blackberries, raspberries, strawberries, apples, grapes, and pears), vegetables (broccoli, cauliflower, peppers, tomatoes, carrots, and onions), and whole grains, such as oatmeal, brown rice, and whole-grain bread.

[0011] The solubility of caffeic acid in water is very low, at room temperature it is approximately 3 g / l and at 100°C it is 8 g / l. It is more soluble in polar organic solvents such as ethanol, where it reaches a solubility of 50 g / l.

[0012] Caffeic acid could be used in a variety of cosmetic products, such as makeup, facial serums, moisturizers, or sunscreens, if it were water-soluble. Associated skin benefits include reducing wrinkles and fine lines, improving skin elasticity, reducing inflammation, and protecting the skin from sun damage and pollution, as well as protecting the skin from damage caused by UV radiation.

[0013] L-arginine, one of the 20 amino acids that make up proteins, is essential for growing infants and children, but healthy adults can synthesize it in the body. It serves a variety of functions in the body, including:

[0014] • participates in protein synthesis;

[0015] • is converted into nitric oxide, a chemical that dilates blood vessels and improves blood flow;

[0016] • is necessary for the repair of muscle tissue after exercise;

[0017] • and intervenes in the regulation of the immune system.

[0018] L-arginine is found in a variety of foods, including red meat, fish, milk and dairy products, nuts and seeds, and legumes, among others. L-arginine has been linked to several health benefits, including improved circulation (as it helps dilate blood vessels, which can improve circulation and lower blood pressure), improved sexual function (it is necessary for sperm production and may help improve sexual function in men), improved wound healing (as it helps repair muscle tissue and speed up wound healing), and reduced cancer risk. The recommended daily intake of L-arginine is 5 grams for healthy adults. However, people with certain medical conditions, such as kidney disease, may need to limit their L-arginine intake.

[0019] In cosmetics, L-arginine is used for its moisturizing, anti-inflammatory, and regenerative properties. It helps keep skin hydrated by attracting and retaining water, reduces dryness and flaking, and has anti-inflammatory properties that help reduce skin inflammation (which can improve the appearance of acne, rosacea, or dermatitis). L-arginine also helps stimulate the production of collagen and elastin, proteins that help keep skin youthful and firm.

[0020] L-arginine is used in a variety of cosmetic products, including facial serums, moisturizers, anti-aging treatments, and hair care products.

[0021] Based on the above, there is a need in this technical field to develop new organic and / or ecological compositions that increase the bioavailability of their active components, avoiding the use of chemical emulsifiers that can be toxic to humans.

[0022] DESCRIPTION OF THE INVENTION

[0023] A first object of the invention is a method for obtaining a salt between caffeic acid and L-arginine, in aqueous solution, comprising the following steps: a) preparing a dispersed, non-solubilized mixture of between 10% and 15% by weight of caffeic acid in deionized or distilled water, preferably under stirring at a preferred speed of between 300 and 1000 rpm; b) adding between 10% and 15% by weight of L-arginine to the dispersed mixture obtained in step (a), always in an equimolar manner to the amount of caffeic acid, preferably maintaining stirring at an intense speed of between 300 and 1000 rpm;c) heating to a preferred temperature of between 40 and 50°C, stirring preferably at a stirring speed of between 300 and 800 rpm and applying ultrasound at a frequency preferably between 15 and 25 kHz, and more preferably between 20 and 25 kHz, until the total dissolution of the caffeic acid and the formation of the L-arginine caffeate salt is complete, which can be stabilized and homogenized by introducing the sonicator head of the ultrasonic equipment into the solution, avoiding the solution from heating above 50°C. Preferably, the sonication can be carried out for periods of 2 minutes each, and for a total time of 10 minutes; and d) optionally, allowing to cool until reaching a temperature equal to or lower than 25°C;

[0024] In a preferred embodiment of the invention, the method may comprise an additional step of lyophilization of the salt obtained according to the method described above.

[0025] Preferably, the method described above will be carried out without the addition of chemical compounds such as emulsifiers or surfactants.

[0026] The invention also relates to a composition characterized by comprising the caffeate salt of L-arginine as claimed for use as a medicine.

[0027] Additionally, the object of the invention is the use of the caffeate salt of L-arginine obtained by the claimed method as an antimicrobial compound and / or as a medical device.

[0028] One of the main advantages offered by the salt obtained using the method of the present invention compared to other products described in the prior art is the surprising improvement it presents in terms of the solubility of its active ingredients, caffeic acid and L-arginine, in water, significantly improving their bioavailability. In particular, it has been shown that the claimed salt allows the solubilization percentage of caffeic acid in water to be multiplied by 36 times, a completely unexpected value. Additionally, it offers the advantage of being a harmless product, based on natural compounds and whose formulation does not require the use of chemical compounds, such as surfactants or emulsifiers, unlike other common prior-art products.It is important to note that the components of the claimed salt are generally recognized as safe, thus favoring its use in pharmaceutical or cosmetic compounds. It is, therefore, an environmentally friendly product that does not harm ecosystems, as it does not contain toxic substances and is based on biodegradable compounds that are even food-grade.

[0029] It is, therefore, a safe alternative to the use of substances such as antibiotics which, as described above, have the disadvantage of promoting the development of resistance to antimicrobial compounds. This is a growing problem that the present invention provides a solution to.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1.- a) Image of the structure of the resonance forms of the guanidinium group; and b) average spatial structure of guanidinium.

[0032] Figure 2.- a) Molecular structure and b) conformational structure (average) of caffeic acid. Energy content of -7.32 kcal / mol.

[0033] Figure 3.- a) Molecular structure and b) conformational structure (average) of L-arginine. Energy content of 7.2 kcal / mol.

[0034] Figure 4.- a) Molecular structure and b) conformational structure (average) of the salt between caffeic acid and L-arginine, called L-arginine caffeate. Energy content of -211.21 kcal / mol.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] As previously described, the present invention offers a novel development for increasing the solubilization of caffeic acid in water that has not been described in the technical literature to date. The claimed new salt increases the solubility of caffeic acid in water by up to 36 times and is especially suitable for use in the preparation of pharmacological compositions and / or medical devices.

[0037] Although in the publication by Atsushi Hirano et al. “Molecular Dynamics Simulation of the Arginine-Assisted Solubilization of Caffeic Acid: Intervention in the Interaction" (J. Phys. Chem. B 2013, 117, 25, 7518-7527 ), the authors used a molecular dynamics-based computer simulation to examine the solubilization effects of caffeic acid and found that the solubility increased with the addition of L-arginine hydrochloride at all pH, in practice it is not convenient to use L-arginine hydrochloride due to the presence of hydrochloric acid (which is contraindicated for use in formulations that may come into contact with the skin) and, in addition, they do not indicate the amount of caffeic acid that can be solubilized in water.

[0038] The present invention consists of a practical and simple method, at low / moderate temperature, through the use of ultrasound, which favors and accelerates the formation of the salt between L-arginine and caffeic acid, lowering the temperature from 80° to 40°C, thanks to the use of the same, which results in an increase in the stability of the salt and the solubility of caffeic acid up to 108 g / liter in the final aqueous solution, that is, obtaining a solubility 36 times higher than the normal one of caffeic acid in water. In particular, the method developed allows to obtain a salt called L-arginine caffeate through a hot mixing process with the application of ultrasound.

[0039] In the context of the present invention, the formation of the salt occurs as a consequence of the loss of the proton by the carboxyl of caffeic acid, which is moved from this molecule to the resonant structure of the guanidinium group, which becomes positively charged.

[0040] Preferably, the salt obtained by the claimed method is characterized in that it comprises up to 0.6 moles of caffeic acid in 55 moles of water (an aqueous solution volume of approximately 1 liter). The amount of L-arginine present in the salt will preferably be equimolar to the amount of caffeic acid.

[0041] The efficacy of the salt obtained using the method of the present invention, combined with its high bioavailability, makes it a truly advantageous product compared to commercially available pharmacological compounds. Furthermore, since it is composed of effective natural components, it avoids the need to use chemicals that are toxic to the environment or have undesirable side effects on humans.

[0042] As described above, it is worth noting that, thanks to the chemical composition of the salt obtained using the claimed method, the solubility of caffeic acid has been surprisingly and unexpectedly increased. In fact, it has been shown that this increase is due to the formation of the salt known as L-arginine caffeate. The action of the ultrasound applied during its production is essential for the formation of the salt.

[0043] One of the main advantages of the salt obtained is that it helps solve the problem faced by the pharmaceutical sector regarding infections caused by bacteria, including resistant bacteria, such as methicillin-resistant Staphylococcus aureus. This bacterium (MRSA) can cause a variety of problems, from mild skin infections to serious, life-threatening infections such as bacteremia, pneumonia, and endocarditis. Skin infections can cause redness, swelling, pain, and drainage, and can cause fever, chills, difficulty breathing, and confusion. MRSA primarily causes nosocomial infections, i.e., infections contracted in a hospital. Its most serious manifestation is nosocomial pneumonia, a potentially fatal disease contracted through the insertion of a ventilator tube into the patient's body.The bacteria can cause deep tissue infections if it enters the body through a break in the skin.

[0044] In particular embodiments of the present invention, the composition comprising the salt obtained by the claimed method may be presented in aqueous solution with a pH between 7 and 8, derived from the presence of the guanidinium group of L-arginine, as shown in Figure 1. This guanidinium group has a great tendency to capture the proton of the carboxyl group of caffeic acid and become positively charged, which gives the slightly cationic character to the complex formed. In preferred embodiments, the pH of the aqueous solution will be between 7 and 7.6.

[0045] A major advantage of the salt obtained by the claimed method is that it does not require the addition of emulsifiers or surfactants to improve its solubility and bioavailability in aqueous solution, unlike other alternative products in the state of the art, which carry a significant risk of toxicity.

[0046] Therefore, it is a highly advantageous solution compared to the compounds currently on the market, since the simple preparation of the L-arginine caffeate salt produces a highly stable product, which is transported in a more elemental manner than other alternatives based on oils and emulsifiers, polyelectrolytes, as well as other more complicated encapsulation products, with the consequent economic savings. Although it is known that the stability of the solutions can be improved by adding an emulsifying agent, preferably, for reasons of environmental sustainability, its use should be avoided in the formulation of the composition comprising the salt obtained by the claimed method.

[0047] In preferred embodiments of the present invention, the salt obtained by the claimed method allows successive dilutions in high quantities of water, surprisingly maintaining its solubility, without precipitating in the water or without forming an isolated powder on the free surface of the liquid.

[0048] Because L-arginine forms a salt that increases the solubility of caffeic acid at least 36-fold, in particular embodiments of the invention, the salt will maintain its solubility in water even in quantities of 1 liter of salt diluted in water in an amount between 30 liters and 100 liters. Surprisingly, it has been found that the salt obtained by the claimed method does not end up precipitating in the mixture as the amount of water in the solution increases.

[0049] A computer model of the formation of the salt between caffeic acid and the amino acid L-arginine has been developed. This computer model has shown that the internal energy of the salt of the invention is -211.21 kcal / mol, which is much lower than the energy content of the individual compounds, demonstrating the formation of the salt and its stability.

[0050] In a particular embodiment, the developed salt can be used as a medicine and, more particularly, can be used for the treatment of nosocomial infections that may occur in patients after an operation.

[0051] The invention also relates to the use of the salt obtained by the claimed method as an antimicrobial compound and / or as a medical device.

[0052] EXAMPLES

[0053] 1. Procedure for obtaining and characterizing a salt as claimed

[0054] It starts with the production of the salt between the two compounds (caffeic acid and L-arginine), according to the following composition and procedure:

[0055] To 55 moles of deionized or distilled water, 0.6 moles of powdered caffeic acid are added. After 5 minutes of stirring, so that the powder is "wet" and evenly distributed in the water, 0.6 moles of the amino acid L-arginine are added under intense stirring (between 300 and 1000 rpm). The mixture is heated to a temperature of 40° to 50°C, and ultrasound is applied; these conditions are necessary and sufficient for the formation of the salt in water. The resulting salt is stabilized and homogenized by directly introducing the sonicator head of the ultrasonic equipment (at a frequency of 20 kHz) into the solution. Sonication is carried out for periods of 2 minutes each, and a total time of 10 minutes, not allowing the solution to heat above 50°C. The resulting solution, protected from light, changes color to a dark brown. The formed salt can then be packaged or freeze-dried.The pH of the resulting solution cannot be altered to prevent the formation of undesirable precipitates. The resulting aqueous solution can be lyophilized to form a powder that can be compacted into tablets, according to standard pharmacopoeial techniques.

[0056] 2. Analysis and computer determination of the salt formation between caffeic acid and L-arginine

[0057] Computational verification of salt formation is based on the study of its energy content, which was calculated using ChemBio3D Ultra 16.0 software (Perkin-Elmer Inc., Waltham, MA, USA) and the Molecular Mechanics (MM2) calculation method. Calculations were performed to obtain the energy of the 3D molecular structure, analyzing and displaying the ground-state energy. The lower the final energy, the greater the stability of the molecule. MM2 is a method used to determine the geometry, molecular energies, vibrational spectra, and enthalpies of formation of molecules in their ground state. It is commonly used to determine the behavior of large molecules of biological and pharmaceutical importance. It is often used to determine the geometries of large molecules, such as those of biological and pharmaceutical importance, which are beyond the reach of more intensive molecular orbital-based methods.For this reason, MM2 is not useful for modeling transition states of chemical processes with a large spectrum of experimental steps.

[0058] Several calculations were performed to obtain the energy of different salt conformations, analyzing and presenting the one with the lowest formation energy. The total energy content, expressed in kcal / mol, of each molecule is described by the sum of the following interactions:

[0059] Eyotal = Stretching(“stretching”)+ Bending(“bending”)'*' Torsion(' orsion") '*' Non-bonded interaction(“Non-bonded inte")

[0060] To verify the energy content of the individual compounds and the salt formed between them, their conformational structure and interrelationship were studied. The results were as follows:

[0061] For caffeic acid, an energy content of -7.32 kcal / mol was obtained. Its molecular and conformational structure is shown in Figure 2.

[0062] An energy content of 7.2 kcal / mol was obtained for L-arginine. The molecular and conformational structure of L-arginine is shown in Figure 3.

[0063] Several studies were carried out on the formation of the L-arginine caffeate salt, yielding an energy content of -211.21 kcal / mol in the most favorable case. The molecular and conformational structure of the caffeic acid and L-arginine salt are shown in Figure 4, indicating that the salt has formed and explaining the significant increase in the solubility of caffeic acid in water.

[0064] 3. Antimicrobial capacity of the described compound.

[0065] An MIC (minimum inhibitory concentration, or MIC) assay was conducted on several bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Cutibacterium acnes, Corynebacterium striatum, Corynebacterium minutissimum, and Streptococcus pyogenes) and fungi (Candida albicans and Trichophyton rubrum). The MIC value is expressed as grams of L-arginine caffeate per liter of water.

Claims

CLAIMS 1. Method for obtaining a salt between caffeic acid and L-arginine, in aqueous solution, comprising the following steps: a) preparing a dispersed, non-solubilized mixture of between 10% and 15% by weight of caffeic acid in deionized or distilled water; b) adding between 10% and 15% by weight of L-arginine to the dispersed mixture obtained in step (a), always in an equimolar manner to the amount of caffeic acid; c) heating to a preferred temperature of between 40 and 50°C, stirring and applying ultrasound at a frequency preferably between 15 and 25 kHz, until the total dissolution of the caffeic acid and the formation of the caffeate salt of L-arginine is complete; and where said method is carried out without the addition of emulsifiers or surfactants.

2. Method according to claim 1, wherein steps (a) and (c) are carried out under stirring at a speed of between 300 and 800 rpm and step (b) is carried out under stirring at an intense speed of between 300 and 1000 rpm.

3. Method according to claim 1 or 2, wherein said method comprises an additional step of lyophilization of the caffeate salt of L-arginine obtained in step (c).

4. Pharmaceutical composition characterized in that it comprises an L-arginine caffeate salt obtained by the method according to any one of claims 1 to 3 for use as a medicine.

5. Use of the caffeate salt of L-arginine obtained by the method according to any one of claims 1 to 3 as an antimicrobial compound.

6. Use of the caffeate salt of L-arginine arginine obtained by the method according to any one of claims 1 to 3 as a medical device.

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