ES2464192A1 - Phenolic extracts of mangifera indica linn, procedure of obtaining and uses. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Phenolic extracts of mangifera indica linn, procedure of obtaining and uses. The invention contemplates phenolic extracts of mangifera indica linn, rich in mangiferin and quercetin, obtained by green solvents at high pressure. The obtaining process comprises in: a) a stage of preparation of the raw material comprising drying, freezing and milling operations, b) extraction at high pressures with green solvents such as supercritical carbon dioxide and water/alcohol mixtures in the absence of light and oxygen, c) a stage of supercritical concentration in column, d) an evaporation of the solvent under vacuum or with nitrogen. The use of extracts with antioxidant activity and content of mangiferin and quercetin, for cosmetic and/or food use is also contemplated. (Machine-translation by Google Translate, not legally binding)

Description

PHENOLIC EXTRACTS OF INDICA LLNN MANGIFERA, PROCEDURE OF OBTAINING AND USES.

SECTOR OF THE TECHNIQUE

Bioactive phenolic extracts for application in the food, nutraceutical and cosmetic industry.

STATE OF THE TECHNIQUE

Phenolic compounds constitute one of the groups of secondary metabolites of the most numerous plants, with more than 8000 known structures (Sobratte et al. (2005) Phenolics as potential antioxidant therapeutic agents: Mechanism and actions. Mutation Research 579, 200-213) . The interest in these compounds lies in the beneficial biological effects they possess, such as: anti-inflammatory, antiallergic, antimicrobial, antifungal, antiviral, anticancer, antidiabetic, among others

(Nijveltdt et al. (2001) Flavonoides: a review of probable mechanisms of action and potential applications. American Journal Clinical Nutrition 74, 418-25). Many of these functions are attributed to its antioxidant capacity and other mechanisms involved in the protection against oxidative stress such as its antiradical activity, the chelation of metal ions, the modulation of genetic expression and the prevention of oxidative DNA damage.

Due to these properties, it is currently considered that phenolic compounds can act as therapeutic agents in a wide number of pathologies related to free radical damage, including cancer, neurodegenerative diseases, diabetes, cardiovascular dysfunctions, immunological diseases , and aging (Soobratte et al. (2005) Phenolics as potential antioxidant therapeutic agents: Mechanism and actíons. Mutation Research 579, 200-213).

Phenolic compounds can be found in almost any part of plants, so agro-industrial by-products are a rich source of this type of compound. In the case of the agricultural by-products of the Mangifera indica Unn plant, its interest is attributed to its high concentration in phenolic compounds of high antioxidant power (Teng Un et al. (2009) Standardized Mangifera indica extract is an ideal antioxidant. Food Chemistry 113, 1154-1159), considered as the compounds responsible for the multiple pharmacological properties of this plant. The literature describes antispasmodic, antipyretic, anti-inflammatory, antimicrobial, antifungal, antidiabetic, immunomodulatory, analgesic, antioxidant and antidiarrheal properties

(Aderibigbe et al. (2001) Evaluation of antidiabetic action of Mangifera indica in mice. Phytotherapy Research 15, 456-458; Amazzal et al. (2007) Mangiferin protects against 1methyl-4-phenylpyridinium toxicity mediated by oxidative stress in N2a cel / s. Neuroscience Left 428, 159-164; Caravalho et al. (2007) Gastroprotective effect of mangiferin, a xanthonoid from Mangifera indica, against gastric injury induced by ethanol and indomethacin in rodents. Plant Medicine 73, 1372-1376; Garrido et al . (2004) In vivo and in vitro anti-inflammatory activity of Mangifera indica L. extract (VIMANG). Pharmacological Research 50, 143-149; Massibo et al. (2008) Major Mango Polyphenols and their Potential Significance to Human Health. Comprehensive review in food science and food safety 7, 309-318).

Among the most important polyphenols of the mango plant, in terms of antioxidant capacity and quantity, are mangiferin and quercetin; although this plant contains other polyphenols in a smaller proportion such as catechins, kaempferol, anthocyanins, gallic acid and benzoic acid.

Mangiferin, from the xanthones family, presents interesting nutraceutical and pharmacological properties, and is considered a promising agent in the treatment of degenerative diseases such as cancer. Quercetin is a flavonoid that has antioxidant and anticancer properties as well as effects on the regulation of genetic expression (Rocha et al. (2010) Bioactive Compounds in Mango (Mangifera indica L.). Bioactive foods in promoting health. Fruits and vegetables. Sevier Chapter 34, 507514; Massibo et al. (2008) Major Mango Polyphenols and their Potential Significance to Human Health. Comprehensive review in food science and food safety 7, 309-318).

The cosmetic applications of mango extracts and their major components (mangiferin and quercetin) have also been described in application patents.

EP0793476: Utilization of mangiferine or uses derivés pour une applicaction cosmetique, Rouillard, F; Josse, A, Robin, J-R. It describes different cosmetic properties of mangiferin as a photoprotective effect, anti-collagenase activity and anti-radical activity.

US 2006/0088560 A1: Cosmetic use of mangiferin, Charrier, L; Poirier, F; Maíllet, G; Lubrano, C. This patent refers to the use of mangiferin obtained from polar extracts of Mangifera indica to prevent and / or reduce the effects of thermal stress on the skin caused by temperature variations.

Commercial extracts of the leaves and bark of Mangifera indica Linn are mainly obtained by means of solid-liquid extraction techniques, as for example described in the patent P0793476: Utilization of mangiferine or uses derérés pour une applicaction cosmetique, Rouillard, F; Josse, A, Robin, J-R. However, the extraction with organic solvents presents several disadvantages such as long extraction processes, use of large amounts of organic solvents considered as harmful to health and the environment, as well as long concentration / evaporation processes that sometimes degrade the bioactive compounds and they do not allow the solvents to be completely removed from the product.

Methods for extracting mangiferin from Mangifera indica Linn using methods of extraction with water or hydro-alcoholic mixtures at high pressure and temperature have also been described (CN 101429222 A: Method for extracting mangiferin, 2009). This method has advantages over conventional extraction such as reduced extraction times and energy costs. But the extraction by means of pressurized liquids using organic solvents still has the disadvantage of obtaining products with solvent residues.

There continues to exist in the state of the art, therefore, the need for a procedure that allows obtaining Mangifera extracts indicates Linn enriched in phenolic compounds, such as mangiferin and quercetin, which overcomes the drawbacks of the prior art procedures.

The invention object of this patent consists of a method of extraction and purification of phenolic compounds from Mangifera indicates Linn using supercritical CO2 in the presence of polar modifiers such as water and ethanol. Supercritical CO2 extraction has been widely developed in recent years for a wide variety of applications due to the low pollution it gives rise to, which operates under conditions where oxidation and / or degradation reactions are avoided since its Solvation power can easily be modified with changes in pressure and temperature, or by adding polar solvents in the case of extracting polar compounds.

The process described in the present invention has additional advantages such as the use of green solvents (CO2, water and ethanol), the reduction of the amount of liquid solvents, the reduction of the concentration stages, and the obtaining of extracts with high concentration in Phenolic compounds without organic solvent residues.

The invention also includes a new purification process for Mangifera indica Linn extracts, based on chromatographic separation with supercritical fluids. This method of purification also offers the advantage of allowing a green and efficient concentration stage, which increases the concentration of the extract in polyphenols and therefore the antioxidant properties thereof.

DESCRIPTION OF THE INVENTION

The invention relates to extracts of the leaves and / or branches of Mangifera indica Linn, containing mangiferin, quercetin and other minor phenolic compounds, and having potent antioxidant properties. It also protects its process of obtaining, and its use as an ingredient in the cosmetic and / or food industry.

The problem to be solved is obtaining Mangifera indica Linn extracts, rich in phenolic compounds, through a green and efficient technology. In particular, you want to get 100% natural mango extracts, free of organic solvents and with high antioxidant properties.

Mangifera indica Linn extracts characterized by their content in polyphenols, particularly mangiferin and quercetin, and their potent antioxidant properties are described. The procedure for obtaining, which is also protected, is based on the use of green solvents, such as carbon dioxide, water, and ethanol, under sub conditions

or supercritical at high pressure and temperature. It is achieved with this new process to increase the efficiency of the process, reduce operating times and obtain extracts free of organic solvents. In addition, it is important to note that the use of carbon dioxide minimizes the use of liquid solvents, which reduces the concentration stages and the possible degradation of the phenolic compounds.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: LC-ES-MS analysis of phenolic compounds present in extracts of Mangifera indica Linn.

Figure 2: LC-ES-MS and LC-UV analysis for the patterns of phenolic compounds present in Mangifera indica Linn sub-or supercritical extracts.

Figure 3: HPLC analysis by Sinergy Hidro RP reverse phase column for the phenolic extract obtained in example 1, with identification of the chromatographic peaks corresponding to the mangiferin and 3-13-0-glucosyl-quercetin phenolic compounds according to their time retention (tr = 20min for mangiferin and tr = 34.4 min for 3-13-0glucosyl quercetin).

Figure 4: LC-ES-MS analysis in quadrupole-ES Scan mode for the extract obtained in example 1. The presence of the phenolic compounds of gallic acid, methyl gallate, 3-D-galactosyl-quercetin, 3- ~ - is checked. D-glucosyl-quercetin, 3-0-aL-arabinopyranosyl

quercetin and 1, 2,3,4,6-penta-O-galoyl- ~ -D-glucose (m / z = 169, 183, 421, 463, 463, 433 and 939 5 respectively). No presence of quercetin is observed (m / z = 301 and tr = 47.7 min).

Detailed description of the invention

The present invention consists of extracts of the leaves and / or branches of Mangifera indica Linn characterized by having a minimum phenolic content of mangiferin and quercetin of

10 1.93 and 0.88% respectively, as well as the presence of minority compounds. The synergy between the different compounds present in the extract gives it powerful antioxidant properties, with a minimum Antioxidant Activity Index of 4.0, higher than that of tocopherol (IAA = 3.65), considered a potent antioxidant.

The characteristics of the extracts obtained allow them to be used in the

15 cosmetic industry for skin and / or hair care, for example as agents to combat aging. And in the case of the food industry, as natural antioxidants instead of synthetic antioxidants such as BHT (butyrohydroxytoluene) or BHA (butyrohydroxyanisole).

The leaves and bark of Mangifera indica Linn are an agri-food by-product that

20 is usually burned after pruning. Therefore, this procedure for obtaining extracts would imply a recovery of said by-product when obtaining products with greater added value.

Characterization of sub-or supercritical extracts of Mangifera indica Linn

Mangifera indica Linn extracts are characterized taking into account their phenolic content and antioxidant capacity, to determine their possible use in the cosmetic and food industry.

i) Determination of the phenolic content of Mangifera indica Linn sub-or supercritical extracts:

The phenolic content of the extract is determined by means of HPLC (High Performance Liquid Chromatography) high performance liquid chromatography, on an Agilent HPLC 1100 series (Agilent Technologies, United States), with a C18 reverse phase column (Thermo Electron Corporation) and a UV / vis detector.

The separation method uses as a mobile phase a solution of 2% acetic acid in water (solvent A) and methanol (solvent B), at a flow rate of 0.9 mL / min, an injection volume of 50 IJmL, and using the following elution conditions: 0-2 min, 5% B isocratic; 2-7 min, linear gradient 5-25% B; 7-11 min, 25% isocratic B; 11-19 min, linear gradient 25-32% B; 19-27 min, 32% B isocratic; 27-28 min, linear gradient 32-40% B; 28-38 min, 40% B isocratic and finally a washing and reconditioning stage of the column is included (38-50 min, linear gradient 40-100% B; 50-60 min, 100% B isocratic; 60-70 min , linear gradient 100-5% B; and 5 min, 5% B isocratic).

Phenolic compounds are detected at a wavelength of 340 nm according to their retention time, and are quantified using calibration curves for the corresponding standards.

The calibration curves of the major compounds present in the extracts, mangiferin and 3- ~ -D-glucosyl-quercetin, are expressed as Abs = 54,252. e -100.12, and Abs = 87.077 · e -130.11, respectively. Where C is the concentration expressed in IJg / ml. Triplicate measurements are made for each sample.

Finally the extraction performance of the phenolic compounds is expressed in terms of g / 100g dry extract. Mangifera indica Linn extracts obtained according to the extraction procedure, have a minimum content of mangiferin and quercetin of 1.93% and 0.88% respectively.

However, the antioxidant activity of the extracts depends not only on the high content of these two compounds, but also on the synergy between the different phenolic compounds extracted during the process.

ii) Qualitative determination of the phenolic compounds present in the sub-or supercritical extracts of Mangifera indica Linn:

To confirm that the extracts contain mangiferin and 3-j3-D-glucosyl-quercetin, as well as the presence of other phenolic compounds, an analysis by liquid chromatography coupled to LC-MS mass spectrometry is performed using a quadrupole-ES mass detector high resolution (Quadrupolar-ionization time-of-flight mass spectrometry).

For this analysis there were commercial standards of gallic acid, methyl gallate, mangiferin, 3-D-galactosyl-quercetin, 3-j3-D-glucosyl-quercetin, 3-0-aL-arabinopyranosylquercetin, 1,2,3,4 , 6-penta-O-galoyl-j3-D-glucose, and quercetin. In order to optimize the 10 ionization conditions, the patterns were injected individually, one by one, directly into the mass spectrometer. A mobile phase consisting of a solution A of water wing, 1% formic acid and a solution B of acetonitrile wing, 1% formic acid, in 50:50 isocratic mode, was used at a flow rate of 1.0 mLlmin , and an injection volume of 10J.lL, in Negative ES (Electro Spray) ionization mode with a mIz range of

15 CU50-2000

The following table shows the masses of the corresponding ions for each pattern of phenolic compound (mIz) obtained from the corresponding LC-ES-MS analysis for each standard. (Figure 1: LC-ES-MS analysis of phenolic compounds present in extracts of

Mangifera indicates Linn).

20 LC-ES-MS Signals [M-Hr (mIz) In addition to LC-ES-MS analysis for standards, extracts were analyzed by liquid chromatography coupled to HPLC-MS mass spectrometry to separate the different phenolic compounds. An HPLC with a Phenomenex C18 Reverse Phase Synergi 4 IJm Hydro -RP 150x3mm column with C18 pre-column was used. As the mobile phase, a solution A of 0.1% water of formic acid and a solution B of 0.1% acetonitrile of formic acid was used, at a flow rate of 0.8 mUmin, and using an elution gradient of: 0-0.2 min, 0% B; 0.2-0.3 min, linear gradient 0-7% B; 0.3-14.7 min, linear gradient 7-8.5% B; 14.7-40 min, linear gradient 8.5-19% B; 40-45 min, linear gradient 1933% B; 45-48 min, linear gradient 33-50% B; 48-50 min, linear gradient 50-95% BY finally a washing and reconditioning stage of the column (50-57 min, linear gradient 95-0% B; 57-63 min, 0% B), at a length of fixed wave at t = O min 278 nm, t = 18 min at 340 nm, t = 43 min at 278 nm, t = 45 min 340 nm, an injection volume of 201JL. As a detector, a quadrupole was used in SIM ES Negative ionization mode and fragmentor windows of the ions selected from the mass spectra of the standards: 0-7 min: 100V (169,125), 7-18 min: 120V (183,124), 18-33 min: 150V (421.301) 33-41.1 min: 160V (463.301.300), 41.1-43 min: 150V (433.301.300), 43-45 min: 240V (939.769.617), 45-63 min: 150V ( 301,151,179).

Phenolic compound

mIz [M-Hr tr (min)

Gallic acid

169 4.06

methyl gallate

183 10.13

mangiferina

421 25.94

3-D-galactosyl-quercetin

463 38.81

3-j3-D-glucosyl-quercetin

463 39.66

3-0-a-L-arabinopyranosyl-quercetin

433 42.39

1,2,3,4,6-penta-O-galoyl-j3-D-glucose

939 43.50

quercetin

301 47.86

A type chromatogram of a mixture of the standards is shown in Figure 2: LC-ES-MS and LC-UV analysis for the patterns of possible phenolic compounds present in Mangifera indica Linn sub-or supercritical extracts.

iii) Determination of antioxidant activity with the a-a-diphenyl- {3-picrylhydracil (DPPH) radical of Mangifera indica Linn sub-or supercritical extracts:

To measure antioxidant activity with the radical aa-diphenyl- {3-picrilhydracil (DPPH) (Brand Williams et al. (1995) Use of a free radical method to evaluate antioxidant activity. Lebensmittel. Wissenschaft. Und Technologie 28, 25-30 ), the decrease in absorbance at 515 nm, every two minutes until steady state, of a solution of 3.9 mL of DPPH 6 10-5 M in methanol and 10 IJL of the antioxidant extract is measured. The extract is evaluated at different concentrations between 0 and 5000 ppm. To obtain the reference of an antioxidant, the antioxidant activity of the commercial pattern of Qtocopherol is also evaluated.

The concentration of DPPH * (COPPH) in the reaction medium is calculated from the calibration curve determined by linear regression with the following equation:

Abs = 12,709 CDPPH + 0,002

The percentage of remaining DPPH is calculated according to the following equation:

% DPPH remaining = CDPPH / CDPPHo x 100

The EC50 or concentration that inhibits 50% of the DPPH radical is calculated graphically

5 using a nonlinear regression of the concentration curve vs. % DPPH remaining at steady state. The antioxidant activity of the extracts is expressed as an antioxidant activity index (IAA) that is calculated considering the final concentration of DPPH and EC50 as expressed by the following equation:

AA = DPPH Final Concentration (~~) / EC50 (~~)

10 Plant extracts have low antioxidant activity when IAA <0.5, moderate when IAA is between 0.5 and 1.0, potent when IAA is between 1.0 and 2.0, and very strong when IAA> 2.0.

The extracts object of this patent have a minimum IAA value of 4.0, and higher than the IAA value obtained for a-tocopherol (AAI = 3.65). This indicates the potent antioxidant activity of Mangifera indica Linn sub-or supercritical extracts, and its potential use as an antioxidant in food and cosmetics. In any case, these extracts may also present other activities / properties of interest to the food, dietary, cosmetic and pharmaceutical industry that would also be included in this invention. Thus, for example, it is expected that extracts containing high mangiferin content

20 and quercetin present activities described for isolated molecules such as anticancer, antibacterial, antifungal activities, or some ability to prevent heart or neurological diseases.

These extracts could be used as ingredients in food, for the preparation of

25 dietary and cosmetic supplements, as well as a natural antioxidant for preservation of food products.

Obtaining procedure

Another aspect of the invention is the method of obtaining the extracts characterized in that it comprises the following steps:

i) Preparation of the leaves and branches of Mangifera indica Linn:

It leaves from leaves and bark of Mangifera indica Linn wet or dry. Drying can be done in the environment or in the oven. The raw material is stored under refrigeration or freezing conditions and subsequently ground to reduce its size.

ii) Extraction of leaves and branches of Mangifera indica Linn with sub-or supercritical solvents:

The bark and dried and ground leaves undergo a continuous extraction process with green solvents at high pressure.

As solvent system mixtures of supercritical carbon dioxide (C02-SC) with water and / or hydro-alcoholic and / or alcoholic solvents are used. Methanol or ethanol are used as alcohols, preferably using ethanol as a GRAS solvent (Generally Recognized as Safe). The percentage of CO2 is set between 0-100% w / w, that of water between 0-100%, and likewise that of ethanol, giving rise to different solvent mixtures. The solvent system is subjected to a flow between 5-40 g / min depending on the size of the extraction plant.

The use of CO2 in the extraction process increases the efficiency of the process, reduces the extraction times, minimizes the use of liquid solvents, reduces the concentration stages, and allows to preserve the properties of the bioactive compounds.

The extraction temperature is set between a range of 35-140 oC, considering a temperature above the critical point of CO2 and not very high to avoid degradation of thermolabile compounds. A range between 80-10 ° C is preferably used

The pressure is set between 10-40 MPa, considering a pressure higher than the critical point of CO2, preferably in a range between 100-200bar.

The extraction time is in the range of 1 to 3 hours, preferably between 1.5-2 hours, for a solvent flow in a range of 5-40g / min depending on the size of the extraction plant

The extractive process with CO2-SC is carried out protected from light and in the absence of oxygen, this prevents the onset of oxidation reactions and prevents possible degradation of phenolic compounds. Additional advantage offered by the use of CO2 in the solvent system.

The overall extraction yield obtained with the extractive process ranges from 1-50 g / 100g of dry matter. The use of CO2-SC as a single solvent allows very low extraction yields. The addition of polar modifiers such as water and / or ethanol allows to obtain higher extraction yields, reaching yields of 42% when a mixture of CO2 1: 0.5: 0.5 is used as a solvent system.

The extracts obtained at this stage can already be used in cosmetic and food applications because they have a minimum phenolic content between 1.93% and 0.88% of mangiferin and quercetin respectively, as well as potent antioxidant properties with a minimum 1M of 4.0 , greater than 1M of tocopherol.

iii) Fractionation or purification of Mangifera indica Linn extracts:

In order to increase the concentration of the extracts in antioxidant phenolic compounds, a subsequent fractionation step can be carried out. In the present invention a method of fractionation of phenolic compounds of Mangifera indica Linn extracts is described, using a preparative column fractionation process with supercritical fluids.

For the purification stage developed in the present invention different types of normal silica filler or C18 reverse phase silica of particle size between 5-10 IJm are used. Green solvents such as CO2, water or ethanol are used for the mobile phase. The separation method is established in an isocratic mode, or using a ramp that starts with pure CO2, gradually increasing the percentage of cosolvent (water, ethanol, or water / ethanol mixtures). It works at pressure conditions between 10-40 MPa, temperature conditions between 35-140 oC, and a mobile phase flow between 5-40 g / min

Throughout the process fractions of the extract are constantly collected. Finally the column is washed for later use. And the fractions are characterized in terms of phenolic content and antioxidant activity.

iv) Obtaining dried extracts of Mangifera indica Linn:

The sub-or supercritical extracts of Mangifera indica Linn obtained can be dried in various ways, taking into account that it is always carried out preferably at temperatures below 40 ° C. Vacuum evaporation, nitrogen drying, lyophilization or precipitation drying using supercritical fluids are contemplated as drying forms.

The extracts are stored refrigerated or frozen, preferably at -4 oC, and protected from light to avoid possible alterations in the phenolic content, as well as in the antioxidant / anti-radical properties of the extracts.

Examples

Example 1

Preparation of Mangifera indica Linn leaf extract obtained with a CO: water / ethanol mixture in a ratio of 1.0: 0.5: 0.5 as a solvent system.

The dried, frozen and ground leaves of Mangifera indica Linn of the Kent variety are subjected to a continuous extraction process in a Thar Tehcnologies Model SF100 high pressure equipment of 100 mL capacity. A mixture of CO2 / water / ethanol in a 1.0: 0.5: 0.5 ratio at a CO2 flow of 5 g / m in is used as the solvent system. The extraction conditions are set at a temperature of 80 oC and a pressure of 120 bar. The extraction is carried out for a period of 3 hours, in an inert atmosphere and protected from light.

After the extraction stage, the extract obtained is evaporated with nitrogen at room temperature to dryness. It is stored in freezing at -4 oC protected from light to prevent its degradation.

The extraction yield for this example 1 is 38.8g of extract / 100g dried leaves.

The content of mangiferin and quercetin in the extract of this example 1 was determined by injecting 50 ~ L of extract at 1000 ppm in an Agilent HPLC 1100 series equipment, with a C18 reverse phase Thermo Electron column. A mobile phase Solvent A: 2% acetic acid in water and Solvent B: methanol at a flow rate of 0.9 mLlmin was used as the separation method, and with a gradient of: 0-2 min, 5% B; 2-7 min, 5-25% B; 7-11 min, 25% B; 11-19 min, 25-32% B; 19-27 min, 32% B; 27-28 min, 32-40% B; 28-38 min, 40% B; 38-50 min, 40-100% B; 50-60 min, 100% B; 60-70 min, 100-5% B; 5 min, 5% B isocratic, at a wavelength of 340nm.

Each compound was identified according to its retention time (tr = 20min for mangiferin and tr = 34.4 min for 3- ~ -D-glucosyl-quercetin), and quantified according to the respective calibration curve, Abs = 54.252 . e-100.12 for mangiferin, and Abs = 87.077. e-130.11 for quercetin.

The extract obtained from example 1 had a content of 3.78 ± 0.01 9 of mangiferin / 100g of extract and 1.23 ± 0.02 9 of 3- ~ -D-glucosyl-quercetin / 1OOg of extract. (Figure 3: HPLC analysis by Sinergy Hidro RP reverse phase column for the extract obtained in example 1, with identification of the peaks of mangiferin and 3- ~ -D-glucosyl-quercetin).

Other phenolic compounds present in the extract were also identified by the LC-MS method. 100IJL of a dilution of the extract was injected at a concentration of 300 ppm in a high-resolution Q-TOF equipment, using a Phenomenex C18 reverse phase column Synergi 4 IJm Hydro -RP 150x3mm with C18 pre-column, a mobile phase of

A: 0.1% water of formic acid and B: 0.1% acetonitrile of formic acid, at 0.8 mUmin, using an elution gradient of: 0-0.2 min, 0% B; 0.2-0.3 min, 0-7% B; 0.3-14.7 min, 7-8.5% B; 14.7-40 min, 8.5-19% B; 40-45 min, 19-33% B; 45-48 min, 33-50% B; 48-50 min, 50-95% B; 50-57 min, 95-0% B; 57-63 min, 0% B; at a wavelength of 278 nm 0-18 min, 340 nm 18-43 min, 278 nm 43-45 min, 340 nm up to 50min. Ionization was performed in 100V negative scan mode. The identification of the phenolic compounds was carried out taking into account the retention time and the ions of each compound determined by their respective mass spectrum.

In the extract obtained in this example 1, the presence in addition to mangiferin y3- ~ -D-glucosyl-quercetin, gallic acid, methyl gallate, 3-D-galactosyl-quercetin, 3-0-a-Larabinopyranosyl-quercetin was identified and 1, 2,3,4,6-penta-O-galoyl- ~ -D-glucose, but not quercetin (Figure 4: LC-ES-MS analysis in Q-TOF Scan mode for the extract obtained in Example 1 )

The antioxidant capacity of the Mangifera indica Linn extract was also determined with the DPPH · assay, expressed in terms of the IAA Antioxidant Activity Index. This extract from example 1 showed a potent antioxidant activity with an IAA value = 4.84, higher than that of tocopherol IAA = 3.65.

Claims (19)

one.

Procedure for obtaining Mangifera indica Linn extracts, characterized by the use of green solvents, such as carbon dioxide, water, and ethanol, under sub-or supercritical conditions at high pressure and temperature.

2.

Method of obtaining extracts of Mangifera indica Linn, according to claim 1, characterized in that it comprises the following steps:

i) Preparation of the raw material, consisting of leaves and / or bark of Mangifera indica Linn, which includes drying, freezing and grinding operations. ii) Continuous sub-or supercritical extraction of the bark and / or dried and ground leaves of Mangifera indica Linn with mixtures of CO2 and aqueous, alcoholic or hydroalcoholic solvents. iii) Purification of Mangifera indica Linn extracts by means of a column fractionation with supercritical fluids, in cases where it is desired to increase the concentration in phenolic compounds of the obtained extracts. iv) Obtain the dry extracts of Mangifera indica Linn, this drying can be carried out preferably at temperatures below 40 oC, by any of the following procedures: vacuum evaporation, nitrogen drying, lyophilization or precipitation drying using supercritical fluids.

3.

Method of obtaining extracts of Mangifera indica Linn, according to claim 2, characterized in that the continuous extraction process is carried out with high-pressure green solvents, such as CO2, water and ethanol, in sub-or supercritical conditions;

Four.

Method of obtaining extracts of Mangifera indicates Linn, according to claim 2, characterized in that the extraction temperature exceeds that of the critical point of CO2, is in the range of 35-140 oC, preferably using a range between 80-100oC.

5.

Method of obtaining extracts of Mangifera indicates Linn, according to claim 2, characterized in that the extraction pressure is in the range of 100-400bar, preferably using a range between 100-200bar.

6.

Method of obtaining extracts of Mangifera indica Linn, according to claim 2, characterized in that the solvent flow is in the range of 5-40 g / min according to the size of the extraction plant.

7.

Method of obtaining extracts of Mangifera indica Linn, according to claim 2, characterized in that the extraction time is in the range of 1-3 hours, preferably between 1.5-2 hours.

8.

Method of obtaining extracts of Mangifera indica Linn, according to claim 2, characterized in that the extractive process is carried out in the absence of light and oxygen.

9.

Method of obtaining extracts of Mangifera indica Linn, according to claim 3, characterized in that the extraction solvent is a mixture of CO2 / water / ethanol, preferably using a percentage of co-solvent of 50% w / w, which mixes the advantages of the extraction with pressurized liquids and supercritical extraction.

10.

Procedure for obtaining purified extracts of Mangifera indica Linn obtained according to claim 1, characterized in that the purification process is carried out by means of a supercritical fractionation or concentration with a preparative column using normal silica filling types or C18 reverse phase silica of size of particle between 5 -10 IJm.

eleven.

Method of obtaining purified extracts of Mangifera indica Linn obtained according to claim 1, characterized in that the purification process is carried out using as mobile phase CO2, water or ethanol, in isocratic mode, or using a gradient of pure CO2, progressively increasing the percentage of co-solvent (water, ethanol, or water / ethanol mixtures).

12.

Method of obtaining purified extracts of Mangifera indica Linn obtained according to claim 1, characterized in that the operating conditions of the purification process are established in a pressure range between 10-40 MPa, a temperature range between 35-80 oC, and a mobile phase flow between 5-40 g / min

13.

Method of obtaining extracts of Mangifera indica Linn, according to claim 1, characterized in that the extraction of dry extracts of Mangifera indica Linn, is preferably carried out with nitrogen at room temperature to avoid degradation of the phenolic compounds present in the extract.

14.

Dry extracts of Mangifera indica Linn obtained according to claims 1 to

13.

fifteen.

Dry extracts of Mangifera indica Linn, according to claim 14, characterized by having a minimum phenolic mangiferin content of 1.93% and 3-13-0glucosyl quercetin of 0.88%, with the presence of other phenolic compounds such as gallic acid , methyl gallate, 3-0-galactosyl-quercetin, 3-0-aL-arabinopyranosylquercetin and 1,2,3,4,6-penta-O-galoyl-I3-0-glucose, and with potent antioxidant activity with minimal value of IAA of 4.0, superior allAA of tocopherol of 3.65.

16.

Use of Mangifera indica Linn extracts, according to claim 15, as a cosmetic agent for skin and / or hair care, intended to act as an anti-aging and antioxidant agent, for preventing alterations derived from cellular oxidation or oxidative stress, and for acting as a free radical inhibitor.

17.

Use of Mangifera indica Linn extracts, according to claim 15, in the food industry as a natural preservative replacing synthetic antioxidants such as BHA and BHT.

· / II $ l) 1'SK ...... ', ... ..... eS .... YES ..... ·, 00 {T ·) .W17C .........: H »2 <11» 1l \ 1 H.oIIP ~: S.I'j ". $ I •• 11II1QO J ! to '" '" RT = 4.093 min. RT = 10.319 min. OR) .. methyl gallate Gallic acid or or " " : Q .... ~ " " " " , , . ~ '.. ..... 1, "", .... ... ""; ID N , .. ... '" '" RT = 26.162 min. RT = 38.840 min. .. mangiferina 3-D-galactosyl-quercetin " or . ,,; " " :;: C! g ~ " " : ¡¡ " " , , ~ 7 "._ ~» ~ '~ M •• _n ~, _u ~~, ......., .. ,,, on '•• "" "._, _" ..... "" "'." ....... s. "'Ye '-! .; :: ¡ TR = 42,399 mino r '" '" TR = 39.676 mino ~ 3-0-a-L-arabinopyranosyl-quercetin .. .. 3-P-D-glucosyl-quercetin or ., or " ~ .... ~ " " p. f.1 ~ " " , , '~ 7C, _ ~ "~" _. "' ~~" '' '•• ~'. • ".'....." "on ~ ~ '" ii or .. TR = 43,436 min ... I 1,2,3,4,6-penta-O-galoyl-p-D-glucose "" or i ..: " , '" " " " ", '. "" ", -' '' '' ~ '' '' '.' ''. '' ', ...' '' '' '' "" ~ RT = 47.766 min. quercetin C! ~ ~ or  í.,; ... Figure 1 . ~, , · ,, ~ u '~ <•. '' ', ~ •. •• (", twenty.,.  (4) ~ (6) LC-ES-MS (2) "'000 ~ (1) '";;; (3) ~ (5) "000 d ~ .... (8) '" 1 ~ g '" (7) '"

-

i

! Eii ~ ace .... , ..; ... "" "  ~~ J A TO or ., 10; 0; m ' VWD1A, W2Wl¡ ;; m. TT (~ l ~ l ~ 11 ~ D) '"" " . (7) LC-UV . (2) (6) ~ f2C'i ",,,, ~ > J ~ ~ (5) (8) " (4) ~~ N .... . ' , ~ TO'" TO  ~ .A ~ ! or r ~ ,;,,;, .n " '" Where: 1) Gallic acid, 2) methyl gallate, 3) mangiferin, 4) 3-D-galactosyl-quercetin, 5) 3-13D-glucosyl-quercetin, 6) 3-0-aL-arabinopyranosyl-quercetin, 7) 1,2,3,4,6-penta-O-galoyl-l3D-glucose "and 8) Quercetin.  Figure 2  Figure 3  

"" "" "" ---- ~ "~~ -" - "" -

- ~ --- "" ~ --- " --------, mass 169 Balic Acid

'0 20

40

"'.)" Nog 10 15 -, ---, 20 20' 0 16 "Nog ~ ¡.) S, Neg," '"100 API · S, Neg. &.': In. 100 20

mass 183 methyl Balato mass 421 mangiferina mi mi

12 \ 11--0 3-D-galactosyl-quercetin, (5) 3-P-D-glucosyl-quercetin 1 "33, le-I 11 : l mass 433 ~ t ~ 3 ~~~ _-L-arabinoPiranOSil-qUcecetina or MS01 i39, EIC =: (1 1 mass 939 1,2,3,4,6-penta-O-galoll-p-D-glucose .. 1. I mass 463 MS01301, EIC = 300 7: 3017 (2012 \ 11-07-04.0) Quercetin 301 dough 10 16 20 30 Figure 4