ES2567530B1 - High pressure xanthone concentration procedure on a semi-industrial scale - Google Patents

Abstract

High pressure xanthone concentration procedure on a semi-industrial scale from extracts of the Gentianaceae, Anacardiaceae, Rubiaceae, Moraceae, Clusiaceae, Polygalaceae and Thymelaeaceae families, especially of the Mangifera indica L species, on a semi-industrial scale combines the use of an apolar or hydrophobic stationary phase and a mobile phase composed of pure CO {sub, 2} or a mixture of CO {sub, 2} and a polar co-solvent such as methanol or ethanol in isocratic or gradient mode. # Consists of a stage of washing using a minimum percentage of co-solvent to initially elute polar compounds with low affinity for the mobile phase and / or soluble in CO {sub, 2}, and a xanthone concentration stage in which the percentage of co-solvent is increased by concentration that allows to elute this type of molecules which have a medium affinity for the stationary phase and are poorly soluble in CO {sub, 2}.

Description

SEMI-INDUSTRIAL SCALE PRESSURE CONCENTRATION PROCEDURE

FROM XANTONES TO HIGH

SECTOR OF THE TECHNIQUE

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Concentration of xanthones, especially mangiferin, from natural extracts of the plant families Gentianaceae, Anacardiaceae, Rubiaceae, Moraceae, Clusiaceae, Polygalaceae and Thymelaeaceae, especially extracts of by-products of Mangifera indica L., by means of a high concentration method pilot plant scale pressure that combines the use of an apolar or hydrophobic absorber and an eluent consisting mostly of carbon dioxide (CO,)

STATE OF THE TECHNIQUE

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Xanthones are a family of biologically active molecules belonging to the chemical group of phenols. Its polyphenolic structure has a nucleus consisting of three hexacyclic rings with six double carbon bonds that make them highly stable, and whose central ring includes a ketone group and an ether group. Its unique skeleton, along with the type and position of the chemical groups attached. define the specific properties of xanthones.

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Xanthones, unlike other phenolic compounds such as flavonoids or phenolic acids, are found only in a few plant families, mainly in the Genlianaceae, Anacardiaceae, Rubiaceae, Moraceae, Clusiaceae, Polygalaceae and Thymelaeaceae (Negy el al. (2013) families. ) Review Arlide Nalurally Occurring Xanlhones: Chemistry and Bi% g) ', Journal o / Applied Chemistry 2013, 1-9). They are very scarce compounds in nature. In total, about 1000 different xanthones with substituents in different positions have been described, which gives rise to a wide variety of biological and biological properties (Pinto el al. (2005) Xamhone Derivalives: New lnsights in Biological Activities, Current Medicinal Chemistry, 12 , 2517-2538).

Xanthones have been recognized as the most potent antioxidants that exist

in nature becoming classified as "super-antioxidants. Its great

antioxidant capacity and its many biological properties such as

antioxidant, anti-inflammatory, anti-histamine, anti-tumor properties,

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antidiabetics (Peres el al. (2000) Review Tetraoxygenated nalurally occurring

xanlhones, Phytochemistry 55, 683-710) are of great interest for applications

Cosmetic, pharmaceutical or nutraceutical.

Mangiferin is the most representative molecule of the xanthones family. It was

the first compound identified with a glucose residue attached to a nucleus

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polyoxygenated xantonoid. It was obtained from Mangifera indica Linn in 1908,

being the bark and leaves of this plant the main natural sources of this

Xanthone, although it has also been identified in many other plant species. The

Mangiferin represents the most important characteristics of all others

related substances known to date: the glucosyl residue is attached to carbon

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2 or 4 of the nucleus that is oxygenated in positions J, 3.6 and 5 or 7 respectively.

Its four aromatic hydroxyl groups determine its strong anti properties

radicals and antioxidants. It is also an efficient chelating agent, therefore

prevents the generation of hydroxyl radicals in Fenton type reactions.

Numerous phamacological properties have been demonstrated in in vivo and in vivo studies.

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vitro: analgesics, antidiabetics, antisclerotic, antimicrobial, antivirals, cardio,

hepato, and neuro-protective, anti-inflammatory, antiallergic. Therefore it is considered

a powerful natural drug beneficial for the treatment of diseases

degenerative such as cancer, Alzheimer's, diabetes, cardiovascular diseases,

as well as diseases related to iron surplus (Rocha el al. (20JO)

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Bioaclive Compounds in Mango (Mang¡fera indica L.). Bioaclive foods in promoling

health. Fruits and vegelables. Sevier Chapter 34,507-514; Massibo al. (2008)

Major Mango Polyphenols and Iheir Potential Significance lo Human Health.

Comprehensive review ¡nfood science andfood safety 7, 309-318: Teng Un el al.

(2009) Standardized Mangifera indica extraet is an ideal antioxidanl. Food

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Chemistry 113.1154-1159).

The complex phenolic structure of xanthones makes it difficult to synthesize them chemically,

limiting its industrial production to extraction and purification processes from

agroindustrial plants or wastes (Kulkarni el al. (20 / 4a) Extraction ofmangiferin

from Mangifera indica lea ves using three pitase partitioning coupled with

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ultrasound Industrial Crops and ProduclS 52. 292-297). Commercial extracts

of the by-products of Mangifera indica L. are obtained mainly through

solid-liquid extraction techniques, as for example described in the patent

P0793476: Utilization of mangiferine or uses dérivés pour une applicaction

cosmetic, Rouillard, F; Josse A, Rabin, J-R. However, in recent years it has been

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aroused great interest in the use of these molecules in pharmaceutical products,

cosmetics and nutraceuticals, due to their high biological properties, and have

made numerous efforts to develop more efficient extraction techniques and

environmentally friendly for the recovery of mangiferin from

agro-industrial plants or waste. These include: fluid extraction

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supercritical (Fernández-Ponee et al. (2012) Extraetion of antioxidant eompounds

/ rom different varieties ofangifera indicates lea ves using green technologies. Journal

of Superreritical F1uids 72, 168-175; Prado et al. (2013) Supereritical C02 and low

pressure solvent extraetion of mango (Mangifera indica) leaves: Global yieJd,

extraction kinetics, chemical composition and cost of manufacturing. Food and

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Bioproducts Processing 91, 656-664), pressurized liquid extraction (CN

101429222 A: Method for extracting mangiferin, 2009), water extraction

subcritical (Fernández-Ponce el al. (20J 2); Kim el al. (20JO) Exlraction of

mangiferin fro m Mahkota Dewa (Plwleria macrocarpa) using subcritical water.

Journal of Industrial and Engineering Chemistry 16, 425-430), extraction with

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ultrasound (US2011 / 0046077 Al: Process lor the extraction of manglferina and

isomangijerin; Zou et al. (20J3) Comparison of microwave-assisted and

conventional extraction of mangiforin from mango (Mangifera indica L) leaves.

Journal of Separation Science 36, 3457-3462), and microwave extraction

(Kulkarni el al. (2014a): Kulkarni el al. (20 14b) Mapping ofan ultrasonic bathfor

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ultrasound assisted extraction of mangiferin from Mangifera indica leaves.

Ultrasonics Sonochemistry 21,606-611).

The extraction techniques penniten yields of 30-40 mg mangiferin / g raw material, as well as mango leaf extracts with mangiferin contents between 6

12% (Fernández-Ponce el al. (2012); Ku / karni el al. (2014a); Zou el al. (20/3)). Despite the efficiency of the extraction methods developed, the use of standardized natural extracts with a high concentration of pertussis mangi is still required at the industrial level.

Liquid chromatography techniques (US2011 1004607 A J.-Process lar the extraction of mangiferin and isomangiferin; Luo el al. (2012) Quantification and Purification 01 Mangiferinfrom Chinese Mango are used for laboratory and industrial level purification) Mangifera indica L.) Cultivars and I [s Protective EjJect on Human Umbilical Vein Endothelial Cells under Hl0r induced Stress. International Journal of Molecular Sciences J3. JJ 260-11274). These techniques use large volumes of organic solvents, such as water, methanol and acetonitrile, and require long stages of concentration.

One of the greatest difficulties in concentrating xanthones is that they are very sensitive to temperature and oxidative processes driven by factors such as light and oxygen. Concentration processes that require long evaporation times of the solvent, such as separation processes by liquid chromatography, reduce the efficiency of the process since they lead to the degradation of this type of molecules. Thus, the need for an efficient and green process that allows natural extracts enriched in xanthones, such as mangiferin, on an industrial scale to continue to be applicable in pharmaceutical formulations continues to exist in the state of the art. nutraceutical or cosmetic.

An alternative to the separation processes by liquid chromatography, are those that use carbon dioxide (CO2) as the mobile phase. Carbon dioxide is a nonpolar solvent so it solubilizes non-polar or low molecular weight solutes. Due to its non-polar character, C ~ is commonly used for normal phase separation processes. This chromatographic separation mode uses an apolar mobile phase and polar stationary phases such as silica gel, or modified silica with amino-, cyano-, phenyl- or propanediol functional groups (Ramirez et

to the. (2006) Iso / alian of fune / ional ingredienls rom rosemary by preparativesupercritical fluid chromalography (Prep-SFC). Journal of Pharmaceutical and Biomedical Analysis 4 /, 1606-1613; Taylor (2009) Supercritical fluid chromatography for lhe 21st cenlury. Journal of Supercritical Fluids 47. 566-573; Li, al. (2007) Characterization and use o / hydrophilic interaction ¡iquid chromatography type stationary phases in supercritical fluid chromatography. Journa / of Chromatography B 846.29 / -297),

Different methods of nonnal phase separation using carbon dioxide have been developed. For example, the purification of camosic acid from Rosmarinus officinalis L. extract was carried out by a mixture of CO2 and ethanol OR deactivating / coating the stationary phase composed of silica gel (lbañez et al. (2000) Tuning olmobile and stationary phase polarity lor the separation 01 polar compounds by SFC. Journal ol Biochemical and Biophysical MetllOds 43, 25-43; Ramírez, et al. (2007) Use 01 specially designed columns for anlioxidants and antimicrobia / s enrichment by preparative supercritica / fluid chromatography Journal of ChromalOgraphy A 1/43, 234-242). For the fractionation of Thymus vu / garis L. extract, a silica column and C02 + 3% ethanol were used as the mobile phase (Garda-Risco el. (2011) Fractionation of thyme (Thymus vulgaris L.) by supercritical fluid extraction and chromatography, Journal al Supercri / ical F / uids

55, 949-954). Grape seed polyphenols were separated with a diol analytical column using CO2 + 0.25% citric acid in methanol as mobile phase

(Karnangerpour el al. (2002) Supercritica / fluid chromatography of polypheno / ic compounds in grape seed extract, Chromatography 55, 4 / 7-42 /). The analytical separation of polyhydroxylated flavones such as quercetin, fesitin and isoramnetine was optimized using phenyl columns and a mobile phase composed of COethanol-phosphoric acid (90: 9.98: 0.02, v / v / v) (Liu el al. (/ 999) Separate / ion of Polyhydroxyljlavonoids by Packed-Column Supercritical Fluid Chromatography. Journal of Chromatographic Science 37,155-J58),

However, these nonnal phase separation processes using CO2 as a mobile phase are applied to CO2 soluble molecules whose solubility can be improved

by adding reduced percentages of polar modifiers. In contrast, xanthones are molecules with poor solubility in CO; Due to their complex structure and high polarity, and the methods used for the separation of these molecules involve the use of reverse phase liquid chromatography techniques.

(Berardini el al. (2004) Characlerization of gallolannins and benzophenone derivotives [ram mango (Mangifera indica L. ev. 'Tommy Atkins') peels. Pulp and kerneL,' by high-performance liquid chromalOgraphy / eJectro. \ Pray ionization mass speclromerry Rapid Communication in Mass Spectrometry 18, 2208-2216; Berardini Garlic (2005) Screening ofmango (Mangifera indica L.) cultivarsfor lheir contents or [j! Avonol 0-and xantllOne C-glycosides, anthocyanins, and pectin. Journal o [ Agricultural and Food Chemistry 53, 1563-1570). However, the development of reverse phase separation processes using C02 as a mobile phase is complex (Guiocho el Tara [der (201 1) Fundamental chal / enges and opporluni1ies [or prcparativc supercrilical fluid chromalography, Journal or [ChromalographyA 1218, / 037-1114); lbañez al. (2000)) due to the low power of CO2 solvation for complex polar substances, such as xanthones.

On the other hand, most methods of separation of natural substances that use CO2 have been developed on a laboratory scale for analytical applications in which it is sought to isolate or purify a compound for identification, or for preparative applications in order to separate and collect fractions that require subsequent verification analyzes of the isolated compound such as Nuclear Magnetic Resonance Analysis (NMR) (Guiochon el Tara [der (2011); Karnangerpour el al. (2002); Uu el al. 1999; Wesl el Lesellier ( 2008) Orthogonal screening system or [columns [or supercritical fluid chromatography, Journal o [Chromatography A 1203, 105-1 / 3; Desmorlreux el al. (2009) lmproved separufion or [filrocoumarins o [essenlial oi / s by supercritical fluid chromalOgraphy, Journal of Chromatography A 1216, 7088-7095).

However, purification methods developed on an analytical scale are not feasible on an industrial scale. At a laboratory scale the feed load is reduced to small quantities, which means that for industrial applications

numerous cycles are required to recover a sufficient amount of product as well

as long stages of solvent separation (Guiochon el Tara / der (201 /).

In addition, the columns used at an analytical scale are not applicable to scales.

higher and it becomes necessary to design columns that can be used to

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industrial scale The development of separation methods using CO2 at scales

superiors is very scarce and the methods developed are based on separation in

Nonnal phase of compounds soluble in COz (Pettinello el al. (2000) Production of

EPA enriched mixtures by supercritical fluid chromatography: from rhe laboratory

scale lO the pilol planto Journal of Supercritical Fluids 19, 5 / -60; Lia the al. (2007)

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Efficient and sea / able method in isolation ofpolymethoxyjlavones from orange peel

extract by supereritical fluid chromatography. Journal of Chromatography B 846,

291-297). Therefore, in the state of the art methods are still required

industrial concentrators that use CO2 in the mobile phase and that are applicable

at polar substances and poorly soluble in this solvent.

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The invention object of this patent consists of a method of concentration at scale

semi-industrial of polar and insoluble substances in CO2, particularly

xanthones, through a high pressure concentration process that uses CO2 as

Major component of the mobile phase and a reverse phase column filled with

a apolar or hydrophobic absorbent. The xanthone concentration process,

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especially mangiferin, it is made from natural extracts of families

of plants Gentianaceae, Anacardiaceae, Rubiaceae, Moraceae, Clusiaceae,

Polyga / aeeae and Thymelaeaceae, especially extracts of by-products of

Mangifera indicates L. which have a high concentration in this type of

highly bioactive molecules.

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This technique has advantages such as: the majority use of a non-solvent

toxic and safe as is C02, reducing the consumption of liquid solvents and

the easy recovery of the analyte by simple decompression of CO2 or by

concentration in the solvent which minimizes the evaporation stages of the

so lvente. Therefore, the process reduces the degradation problems of

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xanthones thus obtaining an extract enriched in compounds with high

bioactivity In addition, due to the low viscosity and high diffusivity of CÜ: 2 under high pressure conditions it is possible to design a concentration process using an absorbent with a very fine particle size without causing loss of resolution and overpressure problems, commonly presented in liquid chromatography equipment on an industrial scale in which it is necessary to use particles with a larger size than the particles used in analytical applications to the detriment of the resolution and efficiency of the process (Guiocho and Tarafder (20/1)). The proposed method also offers the advantage of designing an in-line process for the extraction and concentration of mangiferin at the industrial level.

DESCRIPTION OF THE INVENTION

The invention relates to a method of high pressure concentration of xanthones, mainly of the "super-antioxidant" mangiferin highly valued in pharmaceutical, cosmetic and nutraceutical applications, from natural extracts of the plant families Gentianaceae, Anacardiaceae, Rubiaceae, Moraceae , Clusiaceae, Polygalaceae and Thymelaeaceae, especially of the species Mangifera indica L.

The process of obtaining that is protected is based on the high pressure concentration of xanthones on a semi-industrial scale that combines the use of apolar or hydrophobic absorbent and an eluent composed mostly of eCh. The xanthone concentration process uses a high-pressure equipment coupled to a pilot plant scale column packed with fine particles (5-10 µl) of a non-polar stationary phase, where overpressure problems, commonly presented in liquid chromatography equipment at industrial scale, they are minimized by the low viscosity and high diffusivity of the supercritical solvent and therefore it is possible to use faster flow rates than those used in large-scale high pressure liquid chromatography (HPLC). The process also minimizes the consumption of liquid solvents and reduces the concentration stages that cause losses in the bioactivity of the molecules. Similarly, it offers the possibility

to develop an online high-pressure mangiferin extraction-concentration process.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

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Figure 1: Diagram of the high pressure concentration equipment at pilot plant scale, which shows:

1) Refrigerant bath, 2) CO2 pump, 3) mixer, 4) co-solvent pump, S) heat exchanger, 6) packed column, 7) UV detector, 8) pressure regulating valve, 9) cyclone separators.

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Figure 2: HPLC chromatograms at 340 nm of the FI to F3 fractions obtained from the reverse phase high pressure concentration process of a mixture of polyphenols at a pilot plant scale using as operating conditions: 40 MPa pressure, 40 oC temperature, 20 g / min of mobile phase flow composed of COz +% modifier (methanol + 0.5% phonic acid) in gradient mode established as follows (% modifier, time): (5%, 270 min); (25%, 90 min); and (50%, 90 min).

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Figure 3: Chromatograms obtained by HPLC of: A) crude extract; B) first fraction F 1; C) second fraction F2; O) third fraction F3; and E) fourth fraction F4 collected through the process of concentration at high pressure in reverse phase and pilot plant scale of a supercritical extract of Mangifera leaf indica L. using as operating conditions: 40 MPa pressure, 40 oC temperature, 20 g / min of mobile phase flow composed of COl +% modifier (methanol + 0.5% phonic acid) in gradient mode established as follows (% modifier, time): (5%, 270 min) and (25% , 90 min). Phenolic compounds are numbered in Table 1.

Table 1: Retention times, mass spectra, relative peak area and concentration factor (relative area in the fraction / relative area in the extract) of mango polyphenols identified by HPLC-MS in the crude supercritical extract

of Mangifera leaf indicates L. and the fractions obtained by the high pressure concentration method in reverse phase and at pilot plant scale.

DETAILED DESCRIPTION OF THE INVENTION

The present invention consists of a method of concentration at high pressure in the reverse phase on a semi-industrial scale of xanthones, especially of the "super-antioxidant" mangiferin highly valued in the cosmetic, fannaceutical and nutraceutical industry, from plant extracts of Guttiferae families. Gentianaceae Anacardiaceae, Rubiaceae and Thymelaeacea, which combines the use of an apolar or hydrophobic absorbent and an eluent with a high CO2 content.

The separation method is characterized by a first stage of washing the column with pure COZ or with a mixture of CO2 and co-solvent in isocratic mode or in gradient mode, using an increase in the percentage of cosolvent between O · 15%, preferably between 3 · 5%, in order to reduce the consumption of organic solvents. In this first stage polar compounds are separated, with low or medium affinity for the apolar stationary phase and / or soluble in CO2. • This washing stage is carried out for the time necessary to reduce the contamination of the fraction of interest, rich in xanthones. , by compounds strongly retained in the column and soluble in CG.!.

After the washing stage, the xanthone concentration stage is passed. In this step the percentage of co-solvent in isocratic mode or gradient mode is increased, between 15 and 50%, preferably between 20 and 30%. Xanthones have a medium affinity for the hydrophobic stationary phase and a low solubility in CO2, therefore higher percentages of cosolvent are required for elution.

This method of concentration at high pressure means obtaining a fraction rich in xanthones, mainly in mangiferin whose concentration can be increased by a minimum factor of 5 with respect to the original extract, which can be increased by successive injections of the enriched fraction to raise the purity of it.

Other fractions rich in different families of phenolic compounds such as phenolic acids, benzophenones and flavonoids are also obtained during the washing step, which may also be of interest to the cosmetic, pharmaceutical and nutraceutical industry. The high-pressure concentration process of xanthones, where the mobile phase is mainly composed of CO2 • considered a green and safe solvent, substantially reduces the consumption of organic liquid solvents with the consequent reduction of the evaporation stages of the solvent and the decrease of bioactivity losses of fractional molecules.

Obtaining procedure

The process for obtaining a fraction rich in xanthones, especially in mangiferin, which the present invention proposes is characterized in that a method of concentration of high pressure xanthones for semi-industrial applications is described, from extracts of the Gentianaceae, Anacardiaceae families. Rubiaceae, Moraceae, Clusiaceae. Po ~ ygalaceae and Thymelaeaceae, especially of the Mangifera indica L species, using an apolar or hydrophobic absorbent and an eluent with a high CO2 content •

The high pressure concentration equipment consists of a preparative column at pilot plant scale (25 cm x 3 cm) tennostatized and filled with a apolar stationary phase

or hydrophobic such as CI8 reverse phase silica or hydrophobic silica. The filler material has an average particle diameter between 15 J.1m, preferably 3 to 5 J.1m, a pore size between 7 to 30 nm, preferably 8-10 nm, and a specific surface area that is not particularly limited and that can be found between 5 and 450 m2 / g, This column is previously packaged with the filling material using a pressure gradient between O and 40 MPa, and a subsequent slow depressurization of the system. The equipment also includes two high-pressure liquid pumps with maximum flow rates of 200 and 50 g / min that are used to introduce CO2 and co-solvent, respectively, a mixer located before the column inlet to ensure mixing homogeneous CO2 and co-solvent, a pressure regulating valve that controls the pressure of the

column, a Nis UV light detector, and two separate cyclones to collect the

fractions obtained. A schematic diagram of the pilot plant scale equipment is

shown in Figure l.

At the beginning of the procedure, the feed is dried with the filling material in

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a ratio between 1; 5 and 1: 10. Dry feed is charged at atmospheric pressure and

room temperature at the top of the column. The column closes and it

heat up by a tennostatized jacket until the temperature of

job. Before entering the mobile phase to the column, the CO2 and the co-solvent are

mixed in a small pre-mixed column to provide the composition

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of the required mobile phase. The mobile phase flows in downward flow through the

bed of filling material at a constant flow, dissolving and eluting the

compounds of interest The column pressure is controlled JXlr a valve

pressure regulator The system is coupled to a UVNis light detector (Linear

UVIS 250). The samples are collected in two cyclone separators arranged in

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parallel. After the concentration process, the column is washed for later

use with 100% co-solvent and a drying stage is used and

conditioning using 100% C02 at 70 ° C.

The mobile phase is composed of pure C02 or mixtures of CO2 and co-solvents

polar (methanol or ethanol). The concentration method is set to mode

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isocratic, or using a ramp that starts with pure CO2 increasing

progressively the percentage of cosolvent between 0-100%. The

addition or not of citric acid, phonic acid or acetic acid as additives

secondary to a concentration between 0.1-1, 0% v / v in order to improve the

process resolution The operating conditions of the concentration process

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in reverse phase they are set in a column pressure range between 10-40 MPa,

a column temperature range between 35-120 oC, and a mobile phase flow

between 5-40 g / m in, preferably at a pressure and temperature above the point

critical of the CO2 + co-solvent mixture, and more preferably at a pressure of

column between 30-40 MPa, a column temperature of 40-50 oC and a flow of

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the mobile phase of 10-20 g / min

The high pressure xanthone concentration process on a semi-industrial scale comprises a washing step that uses a minimum percentage of co-solvent to initially polar compounds with low affinity for the mobile and / or CO2 soluble phase • and a concentration stage of xanthones in which the percentage of co-solvent is increased to a concentration that allows this type of molecules to have an average affinity for the stationary phase and are poorly soluble in CO2 •

The washing step uses a mobile phase in Socratic mode or gradient mode composed of pure CO2 or a mixture of CO2 with co-solvent which must be a polar alcohol, more preferably methanol or ethanol, with the addition or not of a polar secondary additive (phonic acid, citric acid, acetic acid) in a concentration between 0.1 and 1% v / v, and more preferably between 0.25 and 0.5% v / v. A mobile phase is preferably used in gradient mode that increases the concentration of co-solvent in CO2 by a percentage between I and 15% and more preferably in a range between 3 and 5% of co-solvent, for a time between 90 and 350 min, preferably between 250-300 min, for a washing of the column and elution of phenolic compounds soluble in CO2 that can contaminate the fraction rich in xanthones.

During this washing stage, highly polar compounds, soluble in CO2 and with low affinity for the apolar stationary phase, cuttings such as phenolic acids and their derivatives are eluted initially, with collection times between 0-15 min. Subsequently eluted, with collection times between 15-90 min, CO2 soluble compounds with medium or high affinity for the apolar stationary phase such as benzophenones and flavonoids. These fractions may or may not be collected depending on the utility they have at the industrial level. The column washing step extends to the operating conditions so far used in order to clean the column of retained compounds and condition the column for elution of the xanthone-rich fraction.

Subsequently, the concentration stage uses a mobile phase with greater eluting force. The co-solvent concentration is increased in C02 between 15 and

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100%, preferably between 15 and 50% and more preferably between 20 and 30% of the cosolvent which is composed of a polar alcohol, preferably methanol or ethanol, with the addition or not of a secondary additive (phonic acid, citric acid, acetic acid) in a concentration between 0.1 and 1% v / v, and more preferably between 0.25 and 0.5% v / v. The concentration stage is carried out for a sample collection time between 0-180 min, preferably between 10-60 ml corresponding to a retention time between 15-30 min. In this last stage a fraction rich in xanthones is obtained, especially in rnangiferin, with a minimum concentration factor of 5 with respect to the crude extract, which can be increased with the successive injection of the enriched fraction to increase the purity of said fraction.

Examples

Example I

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High-pressure concentration in reverse phase of mixtures of pure phenolic compounds at pilot plant scale

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10 rng of a mixture of standard phenolic compounds with a wide range of polarities, composed of gallic acid, methyl gallate, mangiferin and quercetin in equal proportions, was mixed with the filling of the stationary phase in a 1: 1O ratio. The dried sample was loaded on top of the chromatographic column under atmospheric pressure and ambient temperature conditions. The 25crn x 3cm supercritical column was previously packaged with a 5) .lm Sinergy Hidro-RP C18 hydrophobic filler (Phenomenex, USA) using a pressure gradient between O and 40 MPa. After loading the feed the column is closed and the fractionation by CFS is carried out according to the procedure previously described. The experimental conditions used were: 40 MPa of column pressure, 40 oC of column temperature, 20 g / min of mobile phase flow and gradient elution with an increase in co-solvent concentration between 5-50% w / w. The initial washing stage was carried out using a mobile phase composed of CO2 + 5% co-solvent (0.5% phonic acid in

methanol). During this washing step two fractions were obtained. A first

fraction, consisting of 53.4 ± O.2% gallic acid and 46.6 ± O.3% methyl gallate

characterized by HPLC, was obtained after a collection time of 15 min

corresponding to a retention time of 6 min A second fraction, composed

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by 78.6 ± O.2% quercetin, 13.6 ± O.1% gallic acid and 7.9 ± O.1% methyl gallate

characterized by HPLC, was eluted with after a collection time between 15 to 60

min corresponding to a retention time of 17 min The washing time of the

column needed to remove retained compounds in the column was extended

up to 180 min After the washing stage, the percentage of co-solvent is increased

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(0.5% formic acid in methanol) at 25% for 90 min and 50% for 90 min and

obtains the fraction rich in mangiferin with a purity of 93.7 ± 0.4% characterized

by HPLC. A chromatogram of the fractions obtained is shown in Figure 2.

Example 2

High pressure concentration in reverse phase and pilot plant scale of xanthone

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manglferin from a crude supercritical extract of Mangifera indica leaf

L.

30 mg of a crude supercritical extract of Mangifera indica L. leaf, was

mixed and dried with the filling of the stationary phase in a 1: IO ratio. The

dry sample was loaded on top of the chromatographic column in

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atmospheric pressure and ambient temperature conditions. The supercritical column

25cm x 3cm was previously packaged with a Sinergy hydrophobic filler

Hydro-RP C 18 of 5 Jlm (Phenomenex, USA) using a pressure gradient between

O and 40 MPa. After loading the feed the column is closed and the process of

High pressure concentration is performed according to the procedure previously

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described The experimental conditions used were: 40 MPa of pressure of the

column, 40 oC column temperature, 20 g / min mobile phase flow and

Gradient mode with increasing co-solvent concentration

(0.5% phonic acid in methane!) Between 5-25% w / w.

For the initial washing stage a mixture of CO2 + 5% was used as the mobile phase

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cosolvent (0.5% formic acid in methanol). At this stage 3 were collected

fractions as follows: fraction 1 (F l) was collected from O at 20 min, fraction 2 (F2) from 20 to 55 min and fraction 3 (F3) from 55-270 min The semi-quantitative composition of Crude extract and the fractions obtained are described in Table 1, as well as the concentration factor f (relative peak area in the fraction / relative peak area in the extract), and the respective chromatograms obtained by HPLC in the Figure 2. These three fractions obtained in the washing stage may or may not be discarded depending on their subsequent application at the industrial level.

The first fraction obtained has an increase in the concentration of phenolic acids: gallic acid (1) and benzoic acid (2) (Figure 2B). The content of gallic acid and benzoic acid was increased by concentration factors of three and four, respectively (Table 1). The flavonoid quercetin (13) was also eluted in this first fraction and concentrated by a factor of four compared to the crude extract (Table 1).

The second fraction (F2) obtained has high contents of benzophenones 3-C-pD-glucosyl-iriflofenone (4) and 3-C- (2-0-galoyl) -PD-glucosyl-iriflophenone (7) (Figure 2C) , which showed an increase in the relative area percentages compared to those of the crude extract, 38.2 to 57.9% for the compound (4) and 7.3 to 13.0% for the compound (7) ( Table 2). The F2 fraction also showed an increase in the concentration of methyl gallate (3) which was concentrated by a factor of three compared to the crude supercritical extract (1, 84 to 5.5% relative area). As well as flavonoids 3-P-D-glucosyl-quercetin (9) and 3-D-galactosyl-quercetin

(8) whose concentration increased 2.2 and 1.5 times, respectively with respect to the crude extract (Table 1). At the end of the washing step a fraction F3 was obtained which was discarded since it has a composition similar to that of the crude extract.

After the washing step, the percentage of co-solvent is increased to 25%, which resulted in the elution of the fraction rich in mangiferin (F4). For its elution a collection time of 90 min was used. The semi-quantitative content of mangiferin was increased by a factor of 5 compared to the crude extract, and the percentage of relative area of xanthone increased by 5.1% in the 26% crude extract in the

fraction F4. This fraction also contains high concentrations of compound 3C- (2-0p-hydroxybenzoyl) - ~ -D-glucosyl-iriflophenone (5). The absence of phenolic acid and its derivatives as well as some flavonoids and galotanins such as 3-0-xylosyl-quercetin (10), 3-0-aL-arabinopyranosyl-quercetin (11), quercetin 5 (\ 3), and 1 , 2,3,4,6-penta-O-galloyl- ~ -D-glucose (12) in fraction F4 demonstrate the obtaining of a fraction concentrated in rnangiferin from a supercritical extract of Mangrove leaf indica L. through the process of concentration at high pressure in reverse phase at pilot plant scale. In addition, this process can be carried out in successive stages by consecutive injection of the rich fraction

10 in xanthone to obtain a fraction with high purity.

.

Claims (7)

l. High pressure xanthone concentration procedure on a semi-industrial scale characterized in that it is based on plant extracts from families Gentianaceae, Anacardiaceae, Rubiaceae, Moraceae, Clusiaceae, Polygalaceae and Thymelaeaceae, especially of the species Mangifera indica L, combine the use of a mobile phase composed of CO2 and a non-polar stationary or hydrophobic phase composed of reverse-phase silica Cl8 or hydrophobic silica .

2.

Method of concentration of xanthones at high pressure on a semi-industrial scale, according to claim 1, which comprises: a) a washing cap that uses a minimum percentage of codiso) vcntc to initially clude polar compounds with low affinity for the mobile and / or soluble fasc in CO2 • b) a subsequent stage of concentration of xanthones in which the percentage of co-solvent is increased to a concentration that pennite this type of molecules which have an average affinity for the stationary phase and are poorly soluble in CO2 •

3.

Method of concentration of high pressure xanthones on a semi-industrial scale, according to claim 2, characterized in that the initial washing stage uses a mobile phase in the Socratic mode or gradient mode composed of CO2 pUTO or a mixture of CO2 with co-solvent which must be a polar alcohol, more preferably methanol OR ethanol, with the addition or not of a polar secondary additive (phonic acid, citric acid, acetic acid) in a concentration between 0.1 and 1% v / v, and more preferably between 0.25 and 0.5% v / v.

Four.

Method of concentration of xanthones at high pressure on a semi-industrial scale, according to claim 3, characterized in that the washing step preferably uses a mobile phase in a good way that increases the concentration of co-solvent in CO2 by a percentage between 1 and 15% Y more preferably in a range between 3 and 5% co-solvent, for a time between 90 and 350 min, preferably between 250-300 min, for a column wash and elution of [enotics compounds soluble in CO2 that can contaminate the fraction Rich in xanthones.

5.

Method of concentration of xanthones at high pressure on a semi-industrial scale, according to claims 1 and 2, characterized in that after the washing step the concentration of co-solvent in the CO2 is increased between 15 and 100%, preferably between 15 and 50% Y more preferably between 20 and 30% co-solvent which is composed of a polar alcohol, preferably methanol or ethanol, with the addition or not of a secondary additive (phonic acid, citric acid, acetic acid) in a concentration between 0.1 and 1% v / v, and more preferably between 0.25 and 0.5% v / v.

6.

Method of concentration of xanthones at high pressure on a semi-industrial scale, according to claim 5, characterized in that the concentration step is carried out for a sample collection time between 0-180 min, preferably between 10-60 min corresponding to a retention time between 15-30 min

7.

Method of concentration of xanthones at high pressure on a semi-industrial scale, according to claim 5, characterized in that the operating conditions of the reverse phase concentration process are established in a column pressure range between 10-40 MPa, a temperature range of the column between 35-120 oC, and a mobile phase flow between 5-40 g / min,

preferably at a pressure and temperature above the critical point of the co-solvent + CO2 mixture, and more preferably at a column pressure between 30-40 MPa, a column temperature of 40-50 OC AND a 10-20 g / min mobile rase flow