Triterpenic saponin fraction for immunostimulant, antibacterial, antiviral, antifungal and antitumor formulations

Abstract

The present invention discloses a triterpenic saponin fraction denominated FR, isolated by preparative RP-HPLC from an extract of Quillaja saponaria Molina. FR fraction is characterized by its significant immunostimulant, antibacterial, antiviral and antifungal activity, as well as its cytotoxic effect against tumoral cells.

Description

[0001] TRITERPENIC SAPONIN FRACTION FOR IMMUNOSTIMULANT, ANTIBACTERIAL, ANTIVIRAL, ANTIFUNGAL AND ANTITUMOR FORMULATIONS.

[0002] BACKGROUND OF THE INVENTION.

[0003] Field of the invention.

[0004] The present invention relates to a new fraction of triterpenic saponin of Quillaja saponaria Molina suitable as component of vaccine adjuvant formulations as well as active ingredient in antibacterial, antiviral, antifungal and / or antitumoral formulations. Additionally, the present invention discloses the isolation of this new fraction from an extract of Q. saponaria Molina, by RP-HPLC chromatography.

[0005] DESCRIPTION OF RELATED ART.

[0006] Saponins are steroid or triterpenoid glycosides widely spread in the plant kingdom. These compounds have a wide range of medicinal and commercial uses (Hostettmann & Marston. 1995. “Saponins”. Cambridge University Press. Cambridge). The Chilean tree Quillaja saponaria Molina is an abundant source of triterpenic saponins. The saponins of this tree are a complex family of more than 50 different glycosides, most of them consisting of quillaic acid aglycone substituted with oligosaccharides at C-3 and C-28 positions (Kite et al. 2004. Rapid Communications in Mass Spectrometry; 18: 1-12). Kensil & Marciani (1991. US Patent N° 5,057,540) described the separation of 22 chromatographic fractions of saponins (QA-1 to QA-22) from an extract of quillaia bark by reverse phase HPLC. In a simpler procedure, Quillaja saponins were fractioned by reverse phase HPLC in three heterogenous fractions, QHA, QHB and QHC, being the most conspicuous components in these fractions saponins QS-7 (in QHA), QS-17 and QS-18 (in QHB) and QS-21 (in QHC) (Ronnberg et al. 1995. Vaccine, 13: 1375-1382). The chemical differences between the components of saponin fraction of Quillaja extracts are very subtle in some cases, restricted to changes in only one sugar in the structure (Kite K. 2004. Rapid Communications in Mass Spectrometry. 18: 2859-2870).

[0007] Although Quillaja saponins have been used as surfactants in cosmetic and food & beverage industry (Fleck etal. 2019. Molecules, 24: 171 ), nowadays there is an increasing interest in using Quillaja saponins as component in pharmaceutical formulations.

[0008] Previous studies have shown that Quillaja extracts containing unfractionated saponins have antibacterial, antiviral, antifungal and immunostimulant activity. Quillaja extracts have exhibited a low minimum inhibitory concentration (0.1 mg / mL) against different pathogenic microorganisms, such as Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli (Hassan et al. 2010. Applied Biochemistry and Biotechnology 162: 1008-1017). Regarding antiviral activity, Quillaja extracts have shown physiological activity against different viruses including vaccinia virus, herpes simplex virus type 1 , varicella zoster virus, human immunodeficiency viruses 1 and 2 (HIV-1 , HIV-2) and reovirus (Roner et al. 2007. Journal of General Virology 88: 275-285). As well, antifungal activity in vitro studies showed that saponin-rich Quillaja extracts inhibit the growth of common phytopathogenic fungi including Pythium ultimum, Fusarium oxysporum, and Verticillium dahlia (Chapagain et al. 2007. Industrial Crops and Products 26: 109-1 15). These antimicrobial and antiviral activities are related to the property of Quillaja saponins to increased cell membrane permeability (Berlowska et al. 2015. Enzyme and Microbial Technology 75-76: 44-48).

[0009] Regarding the immunostimulant activity of Quillaja extracts, it was demonstrated as early as 1932 by Galea & Tzortzakis (1932. “Essais d’immunisation anti-aphteuse du cobaye a I’aide d’un virus aphteux saponine”. Comptes Rendus des Seances de la Societe de Biologie et de ses Filiales. 109: 21 -23). These researchers injected healthy Guinea pigs with emulsions containing Quillaja extracts and aphtovirus rendered immune to foot and mouth disease, FMD.

[0010] As mentioned earlier, Quillaja extracts contain more than 50 different saponins, some of them having only minor chemical differences. The impact of those differences in the chemical structure on the immunostimulant properties was partially demonstrated by the study performed by Kensil et al. (1991. Journal of Immunology. 146: 431 -437). These researchers studied the immunostimulant activity of the four major saponin fractions in Quillaja extracts (QS-7, QS-17, QS-18 and QS-21 ). These purified saponins vary considerably in their toxicity, as assessed by lethality tests in mice. The most abundant saponin in Quillaja extracts, QS-18, is also the most toxic; its lethal effect in mice is observed even after injection of doses as low as 25 pg. Although QS-21 is very similar to QS-18 (it lacks one glucose residue present in the C-28 branch of QS- 18), showed very low toxicity in mice (lethal dose, 500 pg) and, in animal immunization tests, invoked highly neutralizing antibodies as well as cytotoxic T -limphocyte (CTL) responses (Cleland et aL, 1996. Journal of Pharmaceutical Sciences. 85: 22-28). The overall conclusion is that minor structural variations may generate non-predictable and significant changes in the biological activity of Quillaja saponins, particularly on their toxicity and immunostimulant activity (Sun, et al. 2009. Vaccine. 27: 1787-1796). Although no study related to the impact of chemical structure of Quillaja saponins on other biological properties has been published, it is expected that small changes in chemical structure have an impact on antibacterial, antiviral, antifungal and cytotoxic properties as well. In addition, it is expected that further research on minor saponin components in Quillaja extracts may reveal new compounds suitable for industrial applications.

[0011] The present invention discloses a triterpenic saponin fraction denominated FR, isolated by preparative RP-HPLC from an extract of Quillaja saponaria Molina, whose major components are saponins having a monoisotopic m / z of 2001.9 by negative electrospray ionization mass spectroscopy analysis (corresponding monoisotopic molecular weight of 2002.9 Da). FR fraction is characterized by its significant immunostimulant, antibacterial, antiviral and antifungal activity, as well as its cytotoxic effect against tumoral cells. SUMMARY OF THE INVENTION.

[0012] The object of the present invention is to provide a new fraction of Q. saponaria saponin, hereafter designated as “triterpenic saponin fraction FR”.

[0013] Another object of the invention is the application of “triterpenic saponin fraction FR” as immunostimulant ingredient for pharmaceutical formulations.

[0014] A further object of the invention is to expand the application range of purified Q. saponaria saponins to other pharmaceutical preparations by the use of “triterpenic saponin fraction FR” in antiviral, antibacterial, antifungal and antiproliferative formulations.

[0015] Still another object of the invention is to provide a method of purification of the “triterpenic saponin fraction FR”.

[0016] These objects are accomplished by the herein described invention which comprises a:

[0017] 1. A purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria, comprising: a. The preparation of saponin rich extract of quillaja saponins from Quillaja saponaria biomass. b. The chromatographic fractionation of the said saponin rich extract of Quillaja saponins by preparative RP-HPLC, on octadecylsilane column eluted with a solvent gradient of water, acetonitrile and / or methanol, with or without acidification , to render a “crude fraction FR”. c. The chromatographic fractionation of the “crude fraction FR” by preparative RP- HPLC, on octadecylsilane column eluted with a solvent gradient of water, acetonitrile and / or methanol, with or without acidification, to render a “triterpenic saponin fraction FR”.

[0018] 2. The triterpenic saponin fraction FR, obtained by the above purification method, whose major component has a monoisotopic m / z of 2001.9 by negative electrospray ionization mass spectroscopy analysis (corresponding to monoisotopic molecular weight of 2002.9 Da).

[0019] 3. The triterpenic saponin fraction FR characterized by its significant immunostimulant activity.

[0020] 4. The triterpenic saponin fraction FR characterized by its significant antibacterial activity.

[0021] 5. The triterpenic saponin fraction FR characterized by its significant antiviral activity.

[0022] 6. The triterpenic saponin fraction FR characterized by its significant antifungal activity.

[0023] 7. The triterpenic saponin fraction FR characterized by its significant cytotoxic effect against tumoral cells.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: FIGs. 1 A and 1 B show a chromatographic LIHPLC profiles of the “crude fraction FR” (FIG. 1A), and the “triterpenic saponin fraction FR” (FIG. 1 B) obtained by preparative chromatography of Q. saponaria extracts.

[0026] FIGs. 2A and 2B show a mass spectrum of: FIG: 2A) the isolated saponin “triterpenic saponin fraction FR”; FIG. 2B) the monoisotopic mass precursor ion [M-H]- at m / z 2001 .9.

[0027] FIGs. 3A-3D show the proposed chemical structures of the major components of the triterpenic saponin fraction FR having a monoisotopic m / z of 2001 .9 (Fig. 3A, corresponding to monoisotopic molecular weight of 2002.9 Da; residues 1A and 1 B, FIGs. 3B y 3C) and m / z of 1869.9 (corresponding to monoisotopic molecular weight of 1870.9 Da; residue 2, FIG. 3D) by negative electrospray ionization mass spectroscopy analysis.

[0028] FIG. 4 shows a bar graph comparison of the in vivo total IgG antibody production in final immune sera from groups of animals treated with “triterpenic saponin fraction FR” and QS-21 . Mean values relative to the negative control (PBS) with the error expressed as standard deviation. **P<0.01 ; ****P<0.0001.

[0029] FIGs. 5A-5C show minimum Inhibitory Concentration (MIC) assays bar graphs for Staphylococcus aureus strain ATCC 25923 (FIG. 5A), Klebsiella pneumoniae strain ATCC 1705 (FIG. 5B) and a clinical isolate of S. aureus resistant to methicillin (FIG. 5C) for “triterpenic saponin fraction FR”. Mean values of the percentage of bacterial growth are shown with error expressed as standard deviation for different concentrations in mg / L. A control with no addition of saponins is included. **P<0.01 ; ***P<0.001 ; ****P<0.0001 .

[0030] FIG. 6 shows a bar graph comparison of HeLa cell line subjected to human adenovirus 5 (VR-5) in the presence of “triterpenic saponin fraction FR”. Mean values of the percentage of cell viability are shown with error expressed as standard deviation for different concentrations in pg / L. Controls with HeLa cell cultures without virus infection (no infection control) and without saponin addition (infection control) are included (grey bars). ****P<0.0001 .

[0031] FIG. 7 show minimum Inhibitory Concentration assays bar graph for Candida albicans clinical isolate for “triterpenic saponin fraction FR”. Mean values of the percentage of yeast growth are shown with error expressed as standard deviation for different concentrations in mg / L. A control with no addition of saponins is included. *P<0.05; **P<0.01 .

[0032] FIGs. 8A and 8B show a curve graphs comparison of the IC50 assays for AGS (FIG. 8A) or HeLa (FIG. 8B) human cancer cell lines for “triterpenic saponin fraction FR” (black triangles). Mean values of the percentage of viability inhibition are shown with error expressed as standard deviation for different concentrations in Log [M], A control with no addition of saponins is included (black diamonds), ns P<0.05; **P<0.01 ; ****P<0.0001 . DETAILED DESCRIPTION OF THE INVENTION.

[0033] This invention aims to increase the availability of purified biologically active Quillaja saponins for their use in the preparation of immunostimulant, antibacterial, antiviral, antifungal and antitumoral formulations. To do so, the herein described invention comprises the following steps:

[0034] 1 . A purification method of the “triterpenic

[0035] 2. saponin fraction FR” of Quillaja saponaria biomass, comprising: a. A preparation of saponin rich extract of Quillaja saponins by: a.1 ) drying and milling Quillaja saponaria biomass, preferably, leafless Quillaja saponaria biomass, more preferably leafless Quillaja saponaria biomass containing less than 50% X-series saponin (on total saponin basis), even more preferably clonally grown leafless Quillaja saponaria biomass containing less than 50% X-series saponin (on total saponin basis), and submitting such dried and milled Quillaja saponaria biomass to a water extraction, preferably at a temperature within the range from 20 to 90 °C, more preferably within the range from 30 to 80 °C, and even more preferably within the range from 50 to 70°C, and preferably at a water: solid (dried and milled leafless biomass) ratio within the range from 2:1 to 15:1 v / wt, more preferably within the range from 4:1 to 8:1 v / wt, and even more preferably within the range from 4:1 to 6:1 v / wt to obtain a crude saponin extract; a.2) removing non-saponin impurities from the crude saponin extract resulting from step a.1 ) by adsorption on organic and / or inorganic adsorbents, by filtration, by selective precipitation with organic solvents, or by a combination thereof; b. a first chromatographic fractionation wherein the partially purified crude saponin extract resulting from step a) is fractionated by liquid chromatography (LC) using water, organic solvents or a combination thereof as eluents, either in isocratic or gradient elution mode. In addition, the mobile phase eluents might be adjusted with pH modifiers such as organic acids or organic bases, inorganic acids or inorganic bases or a combination thereof. The stationary phase employed in LC might be either hydrophobic or hydrophilic and column packed. LC separation is performed under atmospheric pressure, Medium Pressure Liquid Chromatography or High Performance Liquid Chromatography elution mode. First eluate is recovered as discrete fractions and selecting eluates containing a major component peak of “triterpenic saponin fraction FR” to then being pooled together and concentrated by vacuum and then freezing and freeze-drying to obtain a resulting residual aqueous solution of an intermediate “crude fraction FR”; and subsequently, c. A second chromatographic fractionation wherein such intermediate “crude fraction FR” is sub-fractionated by liquid chromatography (LC) using water and / or organic solvents as eluents, either in isocratic or gradient mode. In addition, the mobile phase eluents might be adjusted with pH modifiers such as organic acids or organic bases, inorganic acids or inorganic bases or a combination thereof. The stationary phase employed in LC might be either hydrophobic or hydrophilic and column packed. LC separation may be performed under atmospheric pressure, Medium Pressure Liquid Chromatography or High Performance Liquid Chromatography elution mode. Second eluate is recovered as discrete fractions and selecting eluates containing a major component peak of “triterpenic saponin fraction FR” to then being pooled together and concentrated by vacuum and then freezing and freeze-drying to obtain a “triterpenic saponin fraction FR” whose major component is a saponin having a monoisotopic molecular weight of 2002.9 Da as determined by combined liquid chromatography-mass spectroscopy analysis (negative ionization mode).

[0036] 3. A triterpenic saponin fraction FR, obtained by the above purification method, whose composition has a principal FR peak with at least 95% of total area as determined by Ultra- High Performance Liquid Chromatography (UHPLC) liquid chromatography coupled to 210 nm UV analysis.

[0037] 4. A triterpenic saponin fraction FR, obtained by the above purification method, whose principal FR peak contains more than 50% of a compound having a formula as shown in Figure 3A, with two isomers having formulas wherein R corresponds to as the residues shown in Figures 3B and 3C, each with a monoisotopic molecular weight of 2002.9 Da, more preferably within the range of 50 and 99%, and even more preferably within the range of 55 and 90%, and contains less than 40%, more preferably within the range of 1 and 40%, and even more preferably within the range from 10 to 39%, of a compound having a formula as shown in Figure 3A wherein R corresponds to the residue shown in Figure 3D having a monoisotopic molecular weight of 1870.9 Da, as determined by combined liquid chromatography-mass spectroscopy analysis.

[0038] 5. Use of the present triterpenic saponin fraction FR as immunostimulant agent. The triterpenic saponin fraction FR characterized by its significant immunostimulant activity. Having an immunostimulant activity with levels comparable to QS-21 , i.e, having a high immunostimulant activity.

[0039] 6. Use of the present triterpenic saponin fraction FR as antibacterial agent. The triterpenic saponin fraction FR characterized by its significant antibacterial activity. Preferably, having antibacterial effect against Staphylococcus aureus, Klebsiella pneumoniae and Staphylococcus aureus methicillin resistant.

[0040] 7. Use of the present triterpenic saponin fraction FR as antiviral agent. The triterpenic saponin fraction FR characterized by its significant antiviral activity. Preferably, having an antiviral effect against human adenovirus 5 (VR-5 non enveloped). 8. Use of the present triterpenic saponin fraction FR as antifungal agent. The triterpenic saponin fraction FR characterized by its significant antifungal activity. Preferably, having an antifungal effect against Candida albicans.

[0041] 9. Use of the present triterpenic saponin fraction FR as antitumoral agent. The triterpenic saponin fraction FR characterized by its significant cytotoxic effect against tumoral cells. Preferably, having a cytotoxic effect against gastric adenocarcinoma cells, more preferably, a cytotoxic effect against human gastric cancer (AGS) cells, and against cervical carcinoma cells, preferably, a cytotoxic effect against HeLa cells.

[0042] 10. A use of the triterpenic saponin fraction FR as adjuvant for vaccines. Preferably as adjuvant for antibacterial vaccines, antiviral vaccines, antifungal vaccines, or anticancer vaccines.

[0043] The term “Quillaja saponaria biomass” means any part or tissue coming from Quillaja saponaria Molina. For example, the Quillaja biomass can be selected from one of the following: bark, trunk, leaves, stems, roots, seeds, flowers, fruits or a combination of any of them coming from Quillaja saponaria Molina. In some embodiments, selected Quillaja biomass is used, “selected Quillaja biomass” means Quillaja saponaria biomass containing less than 50% X-series saponin on total saponin basis (equivalent to more than 50% R-series saponin on total saponin basis). In some embodiments the biomass is obtained from clonally grown whole plants. In some embodiments the biomass comprises bark, trunk, leaves, stems, roots, seeds, flowers and / or fruits of clonally grown whole plants. In some embodiments, the biomass is obtained from clonally grown whole plants which contains less than 50% X-series saponin (on total saponin basis) as described in US Patent 11 ,254,699 B2.

[0044] The term “X-series saponin” as used herein, refers to Quillaja saponins containing a p-D- galactopyranosyl-(1 -^2)-[p-D-xylopyranosyl-(1 — >3)]-p-D-glucuronopyranosyl trisaccharide attached to the C3position of the aglycone portion of a saponin.

[0045] The term “R-series saponin” as used herein, refers to Quillaja saponins containing a p-D- galactopyranosyl-(1 -^2)-[a-L-rhamnopyranosyl-(1 — >3)]-p-D-glucuronopyranosyl trisaccharide attached to the C3position of the aglycone portion of a saponin.

[0046] The term “leafless Quillaja saponaria biomass” means any part or tissue coming from Quillaja saponaria Molina with the exception of leaf tissue. In some embodiments, leafless Quillaja saponaria biomass can be harvested from an Ultra High Density (UHD) plantation as described in US Patent 11 ,254,699 B2.

[0047] The term “crude fraction FR” correspond to the partially purified fraction as shown in FIG. 1 A, whose major component is a saponin having a monoisotopic molecular weight of 2002.9 Da as indicated in FIG. 2A and 2B.

[0048] The term “triterpenic saponin fraction FR” correspond to the purified fraction as shown in FIG. 1 B, characterized by a principal FR peak, whose major component is a saponin having a monoisotopic molecular weight of 2002.9 Da as indicated in FIG. 2A and 2B. The term “immunostimulant” as used herein refers to the ability of the tested substance to trigger the adaptive immune response upon administration to a host, combined with an antigen.

[0049] The term “antibacterial effect” as used herein refers to the inhibition of the replication of bacterial cells and the decrease in the number of bacterial cells in vitro or in vivo.

[0050] The term “antifungal effect” as used herein refers to the inhibition of the replication of yeast or molds and the decrease of cells of yeast or molds in vitro or in vivo.

[0051] The term “antiviral effect” as used herein refers to the inhibition of the infection of cells by virus in vitro or in vivo.

[0052] The term “cytotoxic effect” as used herein refers to the inhibition of the replication of cancer cells and the decrease in the number of cancerous cells in vitro or in vivo.

[0053] The advantages of the present invention include, without limitation, the production of the triterpenic saponin fraction FR suitable as component of vaccine adjuvant formulations as well as active ingredient in antibacterial, antiviral, antifungal and / or antitumoral formulations. The present method may be further understood by reference to the following Examples, which are not intended to be limiting of the scope of the invention.

[0054] Example 1 : Analysis of triterpenic saponins by UHPLC liquid chromatography.

[0055] The chemical profile of the saponins present in Quillaja saponaria extracts, any intermediate fraction of their purification process, and “triterpenic saponin fraction FR” is shown in FIG 1 . B and was determined by ultrahigh performance liquid chromatography (UHPLC) in a Waters Acquity H-Class UPLC chromatography system coupled to a Waters PDA Acquity diode array absorbance detector. The analytical column used was an Acquity UPLC® BEH C-18 (particle size 1.7 pm, inner diameter 2.1 mm; length 100 mm) (Waters, MA, USA). The column was maintained at 30°C during each chromatographic run. The injected samples were eluted at constant flow rate (0.41 ml / min) in gradient elution mode employing the following mobile phases: Solvent A: Orthophosphoric acid 0.15% v / v in water, and; Solvent B: Ortho-phosphoric acid 0.15% v / v in acetonitrile. The solvent gradient employed is described as follows: Step 1 : 0.0-5.0 min from 34% to 45% of Solvent B; Step 2: 5.0-7.0 min 45% of Solvent B; Step 3: 7.0-10.0 min from 45% to 34% of Solvent B, and; Step 4: 10.0-15.0 min 34% of Solvent B. The eluted peaks were detected by monitoring of the absorbance at 210 nm.

[0056] Example 2: Preparation of saponin rich extract of quillaja saponins from plant material.

[0057] A liquid crude saponin extract was prepared from selected Quillaja saponaria Molina leafless biomass previously dried at 60 °C for at least 12 h and milled. The leafless biomass employed as raw material was harvested from an ultra-high density plantation (UHD) as described in US Patent 1 1 ,254,699 B2. Extraction was done in water at a water solid (milled leafless biomass) ratio of 6:1 v / wt, 60 °C for 4 h. The crude saponin extract was concentrated by evaporation at 60 °C up to 15 ° Brix. The concentrated crude saponin extract was frozen and freeze-dried for 48 h. The obtained solid fraction resulting from the extraction and concentration, was dissolved in methanol (solid fraction; methanol ratio 1 :10 wt / v), incubated for 30 min at 60 °C, centrifuged for 15 min at 4,000 rpm and sterile filtered with 0.45 pm nylon filter. The remaining solids obtained after dissolving, incubating, centrifugating and filtering were washed with methanol, sonicated for 15 min, centrifuged and filtered as before, and pooling all the resulting filtered crude saponin extracts. The resulting filtered crude saponin extract pooling was concentrated at 60 °C and 90 rpm in a rotary evaporator. Total saponin concentration in the concentrated methanol extract was determined by UHPLC chromatography according to the Example 1 .

[0058] Example 3: Isolation of “triterpenic saponin fraction FR” by preparative UHPLC liquid chromatography.

[0059] This concentrated methanol extract described in the Example 2 was processed through two steps of chromatographic separation:

[0060] First step of chromatographic separation: Aliquots of the liquid methanol extract were injected in a semipreparative reverse phase octadecylsilane HPLC column (inner diameter 21.2 mm; length 250 mm; particle size 7 pm) and eluted in gradient mode at constant flow rate (20 mL / min) employing the following mobile phases: Solvent A: Formic acid 0.15% v / v in water, and; Solvent B: Formic acid 0.15% v / v in methanol. The solvent gradient employed is described as follows: Step 1 : 0.0-19.0 min 72% of Solvent B; Step 2: 19.0-24.0 min 100% of Solvent B; Step 3: 24.0- 26.0 min from 72% of Solvent B.

[0061] The eluate was recovered as discrete fractions; the saponin profile of the collected fractions was determined off-line by UHPLC assay according to the Example 1. Fractions containing the principal FR peak of “triterpenic saponin fraction FR” were selected for further recovery. The selected fractions of the preparative runs were pooled together and concentrated by vacuum in a rotary-evaporator. The residual aqueous solution thus obtained was frozen and freeze-dried obtaining the intermediate “crude fraction FR” (see FIG. 1 A).

[0062] Second step of chromatographic separation: The “crude fraction FR” was dissolved in 30% acetonitrile in water to 100 g / L, sonicated for 20 min and centrifuged at 20,000 g for 15 min. One hundred (100) mL of this solution were injected in a semipreparative reverse phase octadecylsilane HPLC column (inner diameter 21.2 mm; length 250 mm; particle size 7 pm) and eluted in gradient mode at constant flow rate (17 mL / min) employing the following mobile phases: Solvent A: Formic acid 0.15% v / v in water, and; Solvent B: Formic acid 0.15% v / v in acetonitrile. The solvent gradient employed is described as follows: Step 1 : 0.0-17.0 min 34 to 52% of Solvent B; Step 2: 17.0-21 .0 min from 95% of Solvent B; Step 3: 21 .0-25.0 min from 34% of Solvent B.

[0063] The eluate was recovered as discrete fractions and their saponin profiles were determined offline by UHPLC assay according to the Example 1 . Fractions containing the principal FR peak of “triterpenic saponin fraction FR” as shown in FIG. 1 B were selected for further recovery of the product. The selected fractions of preparative runs were pooled together and concentrated by vacuum in a rotary-evaporator. The residual aqueous solution thus obtained was frozen and freeze-dried, rendering app 100 mg of “triterpenic saponin fraction FR” per Kg of selected Q. saponaria biomass.

[0064] Example 4: Characterization of “triterpenic saponin fraction FR” by liquid chromatography coupled to mass spectrometry.

[0065] The triterpenes present in the “triterpenic saponin fraction FR” were characterized by UHPLC- HRMS. The analytical column used was an Acquity UPLC® BEH C-18 (particle size 1 .7 pm, inner diameter 2.1 mm; length 100 mm) (Waters, MA, USA). The column was maintained at 30°C during each chromatographic run. The injected samples were eluted at constant flow rate (0.41 ml / min) in gradient elution mode employing the following mobile phases: Solvent A: Formic acid 0.15% v / v in water, and; Solvent B: Formic acid 0.15% v / v in acetonitrile. The solvent gradient employed is described as follows: Step 1 0.0-5.0 min from 34% to 45% of Solvent B; Step 2: 5.0-7.0 min 45% of Solvent B; Step 3 7.0-10.0 min from 45% to 34% of Solvent B, and; Step 4 10.0-14.0 min 34% of Solvent B.

[0066] The UHPLC chromatograph was coupled to a high-resolution electrospray ionization mass spectrometer (HRMS) Thermo Fisher Scientific Exactive Plus Orbitrap (Bremen, Germany) in the mass range 100 - 2100 Da (negative mode). The sheath, auxiliary and sweep nitrogen flow rate were set at 9, 2 and the capillary temperature at 320 °C. The S lens RF was set at 50 and 1 arbitrary units, respectively. Spray voltage was set at -5 kV and the heater temperature at 150 °C. Extracted compound chromatograms were processed employing Xcalibur™ Software. Observed results showed that the “triterpenic saponin fraction FR” has a principal FR peak compound with a characteristic monoisotopic molecular weight of 2002.9 Da as shown in FIG. 2A, with a precursor ion [M-H]- at m / z 2001.9 as shown in FIG. 2B, with the chemical structure described in FIG. 3A, with two isomers having formulas wherein R corresponds to the residues shown in Figures 3B and 3C, and a minor compound having a formula as shown in Figure 3A wherein R corresponds to the residue shown in Figure 3D, and with a monoisotopic molecular weight of 1870.9 Da as shown in FIG. 2A.

[0067] Example 5: Characterization of the immunostimulant effect of “triterpenic saponin fraction FR” in vivo.

[0068] Female BALB / c mice (8-12 weeks old, n = 7 per group) were immunized by intraperitoneal injection with four doses spaced 7 days apart (0, 7, 14 and 21 days), each containing 50 pg ovalbumin (OVA) and 25 pg of QS-21 or triterpenic saponin fraction FR in 100 pL of phosphate buffer saline solution (PBS). Three days after the last immunization the mice were sacrificed, and blood was obtained by intracardiac puncture and the serum was separated by centrifugation at 10,000 x g for 5 min at 4eC. Antibody production was determined by ELISA assay, attaching OVA (100 ng / well) to 96-well plates for 18 h at 4 °C. Non-specific binding was avoided by wash with PBS 0.1 % Tween-20 solution, and sequentially incubated with experimental sera dilutions (1 / 200, 1 / 400, 1 / 800 and 1 / 1600), and detection anti-mouse Ig antibody conjugated with horseradish peroxidase in the presence of 3,3',5,5'-tetramethylbenzidine as chromogenic substrate, determining antibody production by optical density at 450 nm in a plate reader. Total IgG production represented as “Relative amount total IgG” is shown in FIG. 4 as mean values relative to the negative PBS control with no saponin addition, where the triterpenic saponin fraction FR has comparable levels but with higher statistical significance than QS-21 , indicating its high immunostimulant activity.

[0069] Example 6: Characterization of the antibacterial effect of “triterpenic saponin fraction FR” in vitro.

[0070] To determine the antibacterial properties of triterpenic saponin fraction FR and QS-21 , Minimum Inhibitory Concentration (MIC) assays were performed on three bacterial strains with clinical significance: Staphylococcus aureus ATCC25923; Klebsiella pneumoniae ATCC 1705 and Methicillin-resistant S. aureus clinical isolate. All the antibacterial tests were carried out using fresh cultures in Mueller-Hinton broth, pelleted and suspended in 0.85 % NaCI to a turbidity of 0.5 McFarland (1.5 x 108CFU / mL). Then a dilution was made to have on each well 1 x106UFC / mL in a 96-well flat-bottom plate adding different concentrations of “triterpenic saponin fraction FR”. The bacteria were incubated at 37 °C, 150 rpm for up to 72 h. Bacterial growth inhibition was determined on an EPOCH spectrophotometer reading the absorbance at 600 nm. An equivalent comparison with saponin QS-21 was included as well as a control with no addition of saponins. Antimicrobial activity is the result of three independent experiments performed in triplicate. The antibacterial effect of the triterpenic saponin fraction FR is observed in FIG.5 where the values of MIC for Staphylococcus aureus ATCC25923 (FIG. 5A), Klebsiella pneumoniae (FIG. 5B) and Staphylococcus aureus methicillin resistant (FIG. 5C) are all below 0.25 mg / L.

[0071] Example 7: Characterization of the antiviral effect of “triterpenic saponin fraction FR” in vitro.

[0072] Antiviral activity evaluation assays were performed using HeLa cells (ATCC) grown in DMEM medium containing 10% FBS and 1 % penicillin-streptomycin in 25 cm2culture flasks at 37 °C in 5% CO2 and constant humidity. Cell culture was subjected to infection at a viral multiplicity of infection (MOI) of 0.01 with human adenovirus 5 (VR-5) previously tittered to use the appropriated viral load, under different non-cytotoxic concentrations of triterpenic saponin fraction FR prepared in 2% DMSO and added just before infection, incubating for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Plates were incubated for a period of time corresponding to four cycles of multiplication (72 h) and examined daily by inverted light microscopy for the appearance of a cytopathic effect (CPE). MTT assay was used to determine the cell viability post infection according to the manufacturer’s instructions. Briefly, 10 pL of MTT reagent was added to each well and incubated for 4 h at 37 °C. The medium was removed and 100 pL DMSO were added and the absorbance at 570 nm was recorded using an Epoch ELISA reader (ELx800, BioTek, VT, USA). An equivalent comparison with saponin QS-21 was included as well as controls of cell culture without virus infection (no infection control) and without saponin addition (infection control). Antiviral activity was the result of at least three independent experiments performed in triplicate. The antiviral effect of the triterpenic saponin fraction FR is observed in FIG.6, where protection of HeLa cells to infection with human adenovirus 5 (VR-5 non enveloped) is observed at doses equal or below 2 pg / L.

[0073] Example 8: Characterization of the antifungal effect of “triterpenic saponin fraction FR” in vitro.

[0074] To determine the antifungal properties of “triterpenic saponin fraction FR”, antifungal susceptibility testing was done by means of Minimum Inhibitory Concentration (MIC) assays, cultivating a Candida albicans clinical isolate yeast strain in RPMI broth. Suspensions with 1 x 104spores / mL were seeded on each well, in a 96-well flat-bottom plate, adding different concentrations of “triterpenic saponin fraction FR” and incubating at 37°C, 150 rpm for up to 72 h. Fungal growth inhibition was determined on an EPOCH spectrophotometer reading the absorbance at 600 nm. An equivalent comparison with saponin QS-21 was included as well as a control with no addition of saponins. Antifungal activity is the result of three independent experiments performed in triplicate. The antifungal effect of the triterpenic saponin fraction FR is observed in FIG.7 where the values of MIC for Candida albicans clinical isolate is equal or below 0.25 mg / L.

[0075] Example 9: Characterization of the cytotoxic effect of “triterpenic saponin fraction FR” in vitro.

[0076] MTT [3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide] assay was used to determine the inhibition of cancer cell proliferation with “triterpenic saponin fraction FR”. Assays were performed with 3,000 cells / well of human cancer cell lines AGS 1739 (gastric adenocarcinoma) or HeLa CRM-CCL-2 (adenocarcinoma) seeded into a 96-well plate in the respective medium and incubated during 24 h at 37 °C in a humidified 5% CO2 atmosphere. “Triterpenic saponin fraction FR” was prepared in 2% DMSO and serially diluted with media and added at different concentrations, incubating for 24 h at 37 °C in a humidified 5% CO2 atmosphere.

[0077] Separate control with no addition of saponins was included. MTT assay was performed removing “triterpenic saponin fraction FR” containing media and washing with 200 pL of PBS followed by addition of 20 pL of MTT reagent and incubating for 4 h at 37°C. The medium was removed and 100 pL of DMSO was added and the absorbance at 490 nm was determined using an Epoch ELx800ELISA reader. An equivalent comparison with saponin QS-21 was included as well as cells treated with DMSO 0.2% as vehicle control. The cell viability was expressed as percent of the viability of the cells not exposed “triterpenic saponin fraction FR” or QS-21 compared to the control condition. The experiment was performed in triplicate. The cytotoxic effect of the triterpenic saponin fraction FR is observed in FIG.8A and 8B where the values of the half maximal inhibitory concentration (IC50) for AGS and HeLa cancer cell lines are equal or below 3 pM.

Claims

CLAIMS1 . A purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass, comprising: a. A preparation of saponin rich extract of quillaja saponins by: a.1 ) drying and milling Quillaja saponaria biomass, and submitting such dried and milled Quillaja saponaria biomass to water extraction to obtaining a crude saponin extract; a.2) removing non-saponin impurities from the crude saponin extract resulting from step a.1 ) by adsorption on organic adsorbents, inorganic adsorbents or a combination thereof, by filtration, by selective precipitation with organic solvents, or by a combination thereof; b. A first chromatographic fractionation wherein the partially purified crude saponin extract resulting from step a) is fractionated by liquid chromatography (LC) using water, organic solvents or a combination thereof as eluents, either in isocratic or gradient elution mode, and further adjusting pH of the mobile phase eluents with pH modifiers, including organic acids, organic bases, inorganic acids, inorganic bases or a combination thereof, using in LC a stationary phase either hydrophobic or hydrophilic and column packed, being recovered a first eluate as discrete fractions and selecting eluates containing a major component peak of “triterpenic saponin fraction FR” to then being pooled together and concentrated by vacuum and then freezing and freeze- drying to obtain a resulting residual aqueous solution of an intermediate “crude fraction FR”; and subsequently, c. a second chromatographic fractionation wherein such intermediate “crude fraction FR” is sub-fractionated by liquid chromatography (LC) using water, organic solvents or a combination thereof as eluents, either in isocratic or gradient elution mode and further adjusting pH of the mobile phase eluents with pH modifiers such as organic acids or organic bases, inorganic acids or inorganic bases or a combination thereof, using a stationary phase in LC being either hydrophobic or hydrophilic and column packed, and recovering a second eluate as discrete fractions and selecting eluates containing a major component peak of “triterpenic saponin fraction FR” to then being pooled together and concentrated by vacuum and then freezing and freeze-drying to obtaining a “triterpenic saponin fraction FR” whose major component is a saponin having a monoisotopic molecular weight of 2002.9 Da as determined by combined liquid chromatography-mass spectroscopy analysis, negative ionization mode.

2. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein such Quillaja saponaria biomass of step a.1 ) is leafless Quillaja saponaria biomass.

3. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 2 wherein such leafless Quillaja saponaria biomass is leafless Quillaja saponaria biomass containing less than 50% X-series saponin, on total saponin basis.

4. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 3 wherein such leafless Quillaja saponaria biomass is clonally grown leafless Quillaja saponaria biomass containing less than 50% X-series saponin, on total saponin basis.

5. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein the water extraction of step a.1 ) is performed at a temperature within the range from 20 to 90°C.

6. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 5 wherein the water extraction of setp a.1) is performed at a temperature within the range from 30 to 80°C.

7. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 6 wherein the water extraction of step a.1) is performed at a temperature within the range from 50 y 70°C.

8. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein the water extraction of step a.1 ) is performed to a water: dried and milled leafless biomass solid ratio within the range from 2:1 to 15:1 v / wt.

9. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein the water extraction of step a.1 ) is performed to a water: dried and milled leafless biomass solid ratio within the range from 4:1 to 8:1 v / wt.

10. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein the water extraction of step a.1 ) is performed to a water: dried and milled leafless biomass solid ratio within the range from 4:1 to 6:1 v / wt.11 . The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein the LC separation of step b) is performed under atmospheric, MediumPressure Liquid Chromatography (MPLC) or High Performance Liquid Chromatography (HPLC) mode.

12. The purification method of the “triterpenic saponin fraction FR” of Quillaja saponaria biomass of claim 1 wherein the LC separation of step c) is performed under atmospheric, Medium Pressure Liquid Chromatography (MPLC) or High Performance Liquid Chromatography (HPLC) mode.

13. A triterpenic saponin fraction FR, obtained by a purification method as claimed in claim 1 .

14. The triterpenic saponin fraction FR of claim 13, having a principal FR peak with at least 95% of total area, as determined by UHPLC liquid chromatography coupled to 210 nm UV analysis.

15. The triterpenic saponin fraction FR of claim 13 having a principal FR peak containing more than 50% of a compound having a formula as shown in Figure 3A, wherein R corresponds to two isomers having formulas as showed in Figures 3B and 3C.

16. The triterpenic saponin fraction FR of claim 15 wherein each of the two isomers has a monoisotopic molecular weight of 2002.9 Da, as determined by combined liquid chromatography-mass spectroscopy analysis.

17. The triterpenic saponin fraction FR of claim 15 wherein the principal FR peak contains 50 - 99% of a compound having a formula as showed in Figure 3A as determined by combined liquid chromatography-mass spectroscopy analysis.

18. The triterpenic saponin fraction FR of claim 17 wherein the principal FR peak contains 55 - 90% a compound having a formula as showed in Figure 3A as determined by combined liquid chromatography-mass spectroscopy analysis.

19. The triterpenic saponin fraction FR of claim 13 having a principal FR peak containing less than 40% of a compound having a formula as Figure 3A, wherein R corresponds to a residue shown in Figure 3D, and the monoisotopic molecular weight of the compound is 1870.9 Da as determined by combined liquid chromatography-mass spectroscopy analysis.

20. The triterpenic saponin fraction FR of claim 19 wherein the principal FR peak contains a compound having a formula as Figure 3A within the range of 1 and 40% as determined by combined liquid chromatography-mass spectroscopy analysis.

21. The triterpenic saponin fraction FR of claim 20 wherein the principal FR peak contains a compound having a formula as Figure 3A within the range from 10 to 39% as determined by combined liquid chromatography-mass spectroscopy analysis.

22. Use of the triterpenic saponin fraction FR of claim 13 as immunostimulant agent.

23. Use of the triterpenic saponin fraction FR of claim 13 as antibacterial agent.

24. The use of claim 23 wherein the triterpenic saponin fraction FR is an antibacterial agent against Staphylococcus aureus, Klebsiella pneumoniae and Staphylococcus aureus methicillin resistant.

25. Use of the triterpenic saponin fraction FR of claim 13 as antiviral agent.

26. The use of claim 25 wherein the triterpenic saponin fraction FR is an antiviral agent against human adenovirus 5 (VR-5 non enveloped).

27. Use of the triterpenic saponin fraction FR of claim 13 as antifungal agent.

28. The use of claim 27 wherein the triterpenic saponin fraction FR is an antifungal agent against Candida albicans.

29. Use of the triterpenic saponin fraction FR of claim 13 as anticancer agent.

30. The use of claim 29 wherein the triterpenic saponin fraction FR is anticancer agaent against gastric adenocarcinoma cells and cervical carcinoma cells.31 . Use of the triterpenic saponin fraction FR of claim 13 as adjuvant for vaccines.

32. The use of claim 31 wherein such adjuvant is an adjuvant for antibacterial vaccines, antiviral vaccines, antifungal vaccine or anticancer vaccines.

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