Nutriprotective diet

Abstract

The present document describes a nutriprotective diet comprising a nutriprotective amount of a sugar plant syrup, a sugar plant syrup extract, a sugar plant extract, or combinations thereof. The present document also describes a method of eliciting a nutriprotective effect in a subject, which comprises administering a nutriprotective amount of a nutriprotective diet according to the present invention. Also, the present document also describes a method of treating a subject with a disorder, by administering a nutriprotective amount of a nutriprotective diet according to the present invention. Also, the present document describes a process for the extraction of polyphenolic compounds from maple syrup using adsorbent materials, and the extracts obtained therefrom.

Claims

1. A composition having liver-protecting effects, consisting essentially of: (i) a nutriprotective amount of a maple syrup extract produced by first contacting a maple syrup with a styrene divinylbenzene copolymer resin and then eluting the maple syrup extract from the resin by an organic solvent, and (ii) a carrier selected from the group consisting of a coating on a dietary supplement and a drinking liquid, wherein the composition and the maple syrup are not the same.

2. The composition of claim 1, wherein the maple syrup extract has been heated to evaporate the organic solvent.

3. The composition of claim 1, wherein the dietary supplement is a tablet.

4. The composition of claim 1, wherein the carrier is a protein rich drink.

5. The composition of claim 1, wherein the carrier is an energy drink.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the diet schedule and biological sample analysis procedure for the tested subjects.

(2) FIG. 2 illustrates the body weight and total food intake of the tested subjects.

(3) FIG. 3 illustrates the subjects' blood biochemistry.

(4) FIG. 4 illustrates the methods of DNA microarray data analysis.

(5) FIG. 5 illustrates hierarchical clustering.

(6) FIG. 6 illustrates the down-regulated genes for amino acid metabolysing enzymes.

(7) FIG. 7 illustrates the down-regulation of the cellular amino acid metabolic process of ammonia-forming enzymes such as serine/threonine dehydratase and histidine ammonia-lyase.

(8) FIG. 8 illustrates the experimental design of a study described herein.

(9) FIG. 9 illustrates biochemical measurements analysis of a study described herein.

(10) FIG. 10 illustrates biochemical measurements analysis of a study described herein.

(11) FIG. 11 illustrates biochemical measurements analysis of a study described herein.

(12) FIG. 12 illustrates a summary of the biochemical measurements analysis of a study described herein.

(13) FIG. 13 illustrates the data of a DNA microarray analysis described herein.

(14) FIG. 14 illustrates the analysis strategy of a microarray analysis described herein.

(15) FIG. 15 illustrates the functional analysis of the microarray analysis described herein.

(16) FIG. 16 illustrates the lipid metabolism-related genes observed in the analysis described herein.

(17) FIG. 17 illustrates the inflammation and immunity related genes observed in the analysis described herein.

(18) FIG. 18 illustrates a summary of the functional analysis performed herein.

(19) FIG. 19 illustrates the effect of a MSE diet on the insulin signaling pathway.

(20) FIG. 20 illustrates the effect of MSE on the insulin receptor beta subunit.

(21) FIG. 21 illustrates the effect of MSE on the insulin receptor substrate 2.

(22) FIG. 22 illustrates the effect of MSE on the protein kinase B (Akt).

(23) FIG. 23 illustrates a summary of the effect of MSE on liver signaling and the associated phenotypes.

(24) FIG. 24 illustrates the expected mechanism of action of the effect of MSE intake on liver metabolism of KK-Ay mice.

(25) FIG. 25 illustrates the experimental design of a study described herein.

(26) FIG. 26 illustrates experimental results of a study described herein.

(27) FIG. 27 illustrates experimental results of a study described herein.

(28) FIG. 28 illustrates experimental results of a study described herein.

(29) FIG. 29 illustrates experimental results of a study described herein.

(30) FIG. 30 illustrates experimental results of a study described herein.

(31) FIG. 31 illustrates the experimental design of a future study.

(32) FIG. 32A illustrates the significantly enriched GO Term (FDR-corrected P-value<0.05) identified among the top 246 up-regulated genes.

(33) FIG. 32B illustrates the significantly enriched GO Term (FDR-corrected P-value<0.05) identified among the top 236 down-regulated genes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(34) A maple syrup product (medium grade), slightly brownish in color, is provided by the Federation of Maple Syrup Producers of Quebec (Quebec, Canada). It consists of 33% water, 61.0% sucrose, 0.5% glucose, 0.3% fructose, 1.8% saccharide (oligosaccharides and polysaccharides), 0.40% protein derived compounds, 0.25% minerals, 0.15% organic acid, 0.10% vitamins, 0.02% phenolic compounds, 0.002% amino acid, and 0.0001% phytohormones.

(35) Three-week-old male Wistar rats, weighting about 51 g in average, are purchased from Charles River Japan (Kanagawa, Japan). They are quarantined and conditioned by administration of the AlN93G diet (Research Diets, New Brunswick, NJ, USA) for 4 days under the following conditions: temperature, 24±1° C.; relative humidity, 48±4%; and artificial lighting, 12 hours/day (8:00-20:00).

(36) Rats had free access to the diet and drinking water during this acclimatizing period. For feeding tests, they are dichotomized (n=7 and 8) for maple syrup and sugar mix syrup group, respectively, and then fed for 11 days on either the AlN93G diet containing 20% maple syrup or on the 20% sugar mix syrup with a similar sugar composition (FIG. 1). The amount of maple syrup or the sugar mix syrup is arranged by taking into consideration the amounts of sucrose and β-corn starch added in each diet. Subsequently, rats in both diet groups are fasted for 16 hours, prior to being anesthetically sacrificed for dissection. Immediately after, blood sample is taken from carotid artery of each rat for the following biochemical investigations by Nagahama Life Science Laboratory (Shiga, Japan).

(37) No significant diet difference is observed either in the total food intake or in the time-course body weight gain (FIG. 2). It should be noted that rats preferred maple syrup as well as the simulated sugar mix syrup without any sensory rejection. The serum biochemical parameters of the rats fed the maple syrup-containing diet, when compared to those fed the control, did not show significant differences in the levels of serum glucose, total cholesterol and triglyceride. While these metabolic parameters are similar to each other, there are important differences in the serum levels of aspartate aminotransferase (AST) alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), as liver impairment markers (FIG. 3). In particular, there are significant decreases in AST and LDH (P<0.05) (Table 1).

(38) TABLE-US-00001 TABLE 1 Serum Biochemical Parameters.sup.† Investigated Sugar mix syrup Maple syrup AST, IU/L 219.4 ± 9.5   185.6 ± 10.1* ALT, IU/L 35.5 ± 2.3 31.9 ± 1.6 LDH, IU/L 3079.5 ± 173.8  2478.0 ± 179.1* Glucose, mg/dL 51.0 ± 7.1 51.7 ± 4.5 Total cholesterol, mg/dL 73.0 ± 3.4 80.2 ± 5.1 Triglyceride, mg/dL  66.1 ± 15.5 56.1 ± 9.8 Values are represented as the means ± SEM for n = 8 and n = 7 in the sugar mix syrup and the maple syrup, respectively. .sup.†AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase. *P < 0.05, for between-diet differences

(39) A total RNA sample is prepared from the liver and 6 randomized samples are subjected to microarray analysis using GeneChip Rat Genome 230 2.0 Array (Affymetrix, Santa Clara, CA, USA). The obtained microarray data (CEL files) are quantified with the distribution free weighted method (DFW) using the statistical language Rand Bioconductor.

(40) All the microarray data are submitted to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO Series ID GSE30532). To determine specific effects of maple syrup on gene expression, differentially expressed genes (DEGs) between the two groups are identified by applying the rank products (RP) method to the DFW quantified data.

(41) Using the false discovery rate (FDR) significance <0.05, 246 up-regulated and 236 down-regulated genes is selected. To identify over-represented Gene Ontology (GO) terms in the DEGs, a functional annotation tool in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and QuickGO are used. GO terms with Benjamini and Hochberg FDR-corrected P-value less than 0.05 are regarded as significantly enriched. Such GO terms for genes that are up- or down-regulated by maple syrup intake are summarized in FIGS. 5, AND 32A-32B.

(42) The up-regulated GO terms included antigen processing-presentation, carboxylic acid (including amino acid) metabolism, oxidoreduction, hormone metabolism, and response to external stimulus (FIG. 32A), as well as negative regulation of transcription, glutamine family amino acid metabolic process, response to organic substance, response to drug, whereas the down-regulated GO terms comprised amine catabolism, cellular amino acid metabolism, aspartate family acid metabolic process, antigen processing-presentation and response to chemical stimulus (FIG. 32B).

(43) It is noted that genes for cellular amino acid metabolic process is down-regulated, including those for ammonia-forming enzymes such as serine/threonine dehydratase and histidine ammonia-lyase (FIGS. 6-7). In general, the excessive formation of free ammonia in the body is harmful to the liver, resulting in increased values of AST, ALT and LDH.

(44) Some cases have been reported where administration of 20% casein diet activates serine/threonine dehydratase. Threonine is known as an amino acid that is difficult to metabolize and that can be better metabolized when it is ingested together with casein, leading to induction of threonine dehydratase activity for production of 2-oxobutyrate and ammonia. The high content of casein (20%) in AlN93G diet may probably enhance the activity of serine/threonine dehydratase to liberate ammonia (FIGS. 6-7).

(45) It is likely that the enhancement can be countered when the gene for this enzyme is down-regulated by administration of maple syrup. On the other hand, mRNA level of the gene for AST is down-regulated. This should also participate in down-regulation of serum AST. The present document is the first to describe a piece of body-protecting effects of ingested maple syrup.

(46) In embodiments there are disclosed phenolic extracts and compounds from Canadian maple syrup (MS) and from maple trees (e.g. red, silver, or sugar maple). The compounds and extracts may be used for their hepatoprotective properties.

(47) In other embodiments there are disclosed twenty-three phenolic compounds isolated from a butanol extract of Canadian maple syrup (MS) using chromatographic methods. The compounds are identified from their nuclear magnetic resonance and mass spectral data as seven lignans: lyoniresinol (1), secoisolariciresinol (2), dehydroconiferyl alcohol (3), 5′-methoxy-dehydroconiferyl alcohol (4), erythro-guaiacylglycerol-β-O-4′-coniferyl alcohol (5), erythro-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol (6), and [3-[4-[(6-deoxy-α-L-mannopyranosyl)oxy]-3-methoxyphenyl]methyl]-5-(3,4-dimethoxyphenyl)dihydro-3-hydroxy-4-(hydroxymethyl)-2(3H)-furanone (7); two coumarins: scopoletin (8) and fraxetin (9); a stilbene: (E)-3,3′-dimethoxy-4,4′-dihydroxystilbene (10), and thirteen phenolic derivatives: 2-hydroxy-3′,4′-dihydroxyacetophenone (11), 1-(2,3,4-trihydroxy-5-methylphenyl)-ethanone (12), 2,4,5-trihydroxyacetophenone (13), catechaldehyde (14), vanillin (15), syringaldehyde (16), gallic acid (17), trimethyl gallic acid methyl ester (18), syringic acid (19), syringenin (20), (E)-coniferol (21), C-veratroylglycol (22), and catechol (23).

(48) The antioxidant activities of the MS extract, pure compounds, vitamin C (IC.sub.50=58 μM), and the synthetic commercial antioxidant, butylatedhydroxytoluene (IC.sub.50=2651 μM), are evaluated in the diphenylpicrylhydrazyl (DPPH) radical scavenging assay. Among the isolates, the phenolic derivatives and coumarins showed superior antioxidant activity (IC.sub.50<100 μM) compared to the lignans and stilbene (IC.sub.50>100 μM).

General Experimental Procedures

(49) .sup.1H and .sup.13C Nuclear Magnetic Resonance (NMR) spectra are obtained either on a Bruker™ 400 MHz or a Varian™ 500 MHz instrument using deuterated methanol (CD3OD) as solvent. Electrospray ionization mass spectral (ESIMS) data are acquired on a Q-Star Elite (Applied Biosystems MDS) mass spectrometer equipped with a Turbo Ionspray source and are obtained by direct infusion of pure compounds. Analytical high performance liquid chromatography (HPLC) are performed on a Hitachi Elite LaChrom™ system consisting of a L2130 pump, L-2200 autosampler, and a L-2455 Diode Array Detector all operated by EZChrom™ Elite software. Semi-preparative scale HPLC are performed on a Beckman-Coulter HPLC system consisting of a Beckman System Gold™ 126 solvent module pump, 168 photodiode array (PDA)-UV/VIS detector, and 508 autosampler all operated by the 32 Karat 8.0 software. All solvents are either ACS or HPLC grade and are obtained from Wilkem Scientific (Pawcatuck, RI). Ascorbic acid (vitamin C), butylatedhydroxytoluene (BHT), and diphenylpicrylhydrazyl (DPPH) reagent are purchased from Sigma-Aldrich (St Louis, MO).

(50) Maple Syrup (MS) Butanol Extract

(51) Maple syrup (grade C, 20 L) is provided by the Federation of Maple Syrup Producers of Quebec (Canada). The syrup is kept frozen until extraction when it is subjected to liquid-liquid partitioning with ethyl acetate (10 L×3) followed by n-butanol (10 L×3) solvents, to yield ethyl acetate (4.7 g) and butanol (108 g) extracts, respectively, after solvent removal in vacuo.

(52) Analytical HPLC

(53) All analyses are conducted on a Luna C18 column (250×4.6 mm i.d., 5 μM; Phenomenex) with a flow rate at 0.75 mL/min and injection volume of 20 μL. A gradient solvent system consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (methanol, MeOH) is used as follows: 0-10 min, 10% to 15% B; 10-20 min, 15% B; 20-40 min, 15% to 30% B; 40-55 min, 30% to 35% B; 55-65 min, 35% B; 65-85 min, 35% to 60% B; 85-90 min, 60% to 100% B, 90-93 min, 100% B; 93-94 min, 100% to 10% B; 94-104 min, 10% B. FIGS. 1A and 1B show the HPLC-UV profiles of the butanol extract and all of the isolated phenolics (combined into one solution/injection), respectively.

(54) Isolation of Compounds from the MS Butanol Extract

(55) The butanol extract (108 g) is extracted with methanol (100 mL×3) to afford methanol soluble (57 g; dark-brown powder) and methanol insoluble (51 g; off-white powder) fractions. Analytical HPLC analyses of the methanol soluble extract revealed a number of peaks characteristic of phenolic compounds at 220, 280 and 360 nm (see above for details of methodology; see FIG. 1A for chromatogram). Therefore, this fraction is selected for further purification by repeated chromatography on a Sephadex™ LH-20 column (4.5×64 cm), eluting with a gradient system of MeOH: H.sub.2O (3:7 v/v to 7:3 v/v to 100:0 v/v), and then with acetone: H.sub.2O (7:3 v/v). Based on analytical HPLC profiles, twelve combined fractions, Fr. 1-12, are obtained. Fr. 4 (1.5 g) is subjected to column chromatography on a Sephadex™ LH-20 column (4.5×64 cm) using a gradient solvent system of MeOH: H.sub.2O (3:7 v/v to 7:3 v/v) to afford twelve sub-fractions, Fr. 4.1-4.12. These are individually subjected to a series of semi-prep HPLC separation using a Waters Sunfire Prep™ C.sub.18 column (250×10 mm i.d., 5 μm; flow 2 mL/min) and eluting with a MeOH:H.sub.2O gradient system to yield compounds 1 (4.6 mg), 3 (3.8 mg), 5 (4.0 mg), 6 (41.6 mg), 7 (6.6 mg), 11 (3.5 mg), 15 (0.3 mg), 16 (0.8 mg), 18 (0.2 mg), 20 (1.3 mg), 22 (1.5 mg) and 23 (3.0 mg). Similarly, Fr. 5 (0.47 g) is purified by semi-prep HPLC using a Waters XBridge Prep C.sub.18 column (250×19 mm i.d., 5 μm; flow 3.5 mL/min) and a gradient solvent system of MeOH:H.sub.2O to afford four subfractions Fr. 5.1-5.4. These subfractions are separately subjected to a combination of semi-prep HPLC and/or Sephadex™ LH-20 column chromatography with gradient solvents systems of MeOH:H.sub.2O to afford compounds 2 (1.9 mg), 4 (1.9 mg), 8 (2.0 mg), 9 (2.3 mg), 14 (2.5 mg), 17 (2.4 mg), 19 (1.8 mg) and 21 (1.3 mg). Similarly, Fr. 6 (0.2 g) afforded compounds 12 (1.4 mg) and 13 (1.3 mg) and Fr. 11 yielded compound 10 (4.8 mg).

(56) Isolation of Compounds from the MS Ethyl Acetate Extract

(57) Maple syrup (grade C, 20 L) is provided by the Federation of Maple Syrup Producers of Quebec (Canada). The syrup (20 L) is kept in the freezer (−20° C.), until extraction when it is subjected to liquid-liquid partitioning with ethyl acetate (10 L×3) followed by n-butanol (10 L×3) solvents, to yield ethyl acetate (4.7 g) and butanol (108 g) extracts, respectively, after solvent removal in vacuo. The ethyl acetate extract (4.7 g) is subjected to a series of chromatographic isolation procedures using XAD-16, silica gel, Sephadex™-LH 20, and C-18 column chromatography. Semi-purified fractions obtained from these columns are then further subjected to prep-HPLC to yield twenty pure compounds.

(58) Identification of Compounds

(59) All of the isolated compounds are identified by examination of their .sup.1H and/or .sup.13C NMR and mass spectral data, and by comparison of these to published literature reports, when available (Table 2). The NMR data for compounds 7, 12, and 13 are provided here.

(60) TABLE-US-00002 TABLE 3 Compounds identified in a butanol extract of Canadian maple syrup (MS) Identification Structure  1 Iyoniresinol  2 Secoisolariciresinol  3 dehydroconiferyl alcohol  4 5′-methoxydehydroconiferyl alcohol  5 guaiacylglycerol-β-O-4′-coniferyl alcohol (1,3-Propanediol, 1-(4-hydroxy-3- methoxyphenyl)-2-[4-[(1E)-3-hydroxy-1- propenyl]-2-methoxyphenoxy]-, (1R,2R)-)  6 guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- hydroxypropyl)-2-methoxyphenoxy]-propane- 1,3-diol  7 [3-[4-[(6-deoxy-α-L- mannopyranosyl)oxy]-3- methoxyphenyl]methyl]-5-(3,4- dimethoxyphenyl)dihydro-3-hydroxy-4- (hydroxymethyl)-2(3H)-furanone  8 Scopoletin  9 Fraxetin 10 (E)-3,3′-dimethoxy-4,4′-dihydroxystilbene 0 11 2-hydroxy-3′,4′-dihydroxyacetophenone 12 1-(2,3,4-trihydroxy-5-methylphenyl)- ethanone 13 2,4,5-trihydroxyacetophenone 14 Catechaldehyde 15 Vanillin 16 Syringaldehyde 17 gallic acid 18 trimethyl gallic acid methyl ester 19 syringic acid 20 Syringenin 0 21 (E)-coniferol 22 C-veratroylglycol 23 Catechol 54 Quebecol

(61) In another embodiment, there are disclosed thirty phenolics obtained from an ethyl acetate extract of maple syrup (MS-EtOAc).

(62) Chemicals and Reagents. All solvents are of ACS or HPLC grade and are obtained from Sigma-Aldrich through Wilkem Scientific (Pawcatuck, RI). Sephadex LH-20, ascorbic acid, butylated hydroxytoluene (BHT), and diphenylpicrylhydrazyl (DPPH) reagent are purchased from Sigma-Aldrich (St. Louis, MO).

(63) Extraction and Isolation of Maple Syrup Ethyl Acetate (MS-EtOAc) Compounds. Maple syrup (grade C, 20 L) is provided by the Federation of Maple Syrup Producers of Quebec (Canada). The maple syrup is shipped and kept frozen upon delivery. The maple syrup is subjected to liquid-liquid partitioning with ethyl acetate (10 L×3) to yield a dried ethyl acetate extract (MS-EtOAc; 4.7 g) after solvent removal in vacuo. The MS-EtOAc (4.5 g) is initially purified on a Sephadex LH-20 column (4×65 cm) with a gradient system of MeOH/H.sub.2O (3:7 to 1:0, v/v) to afford seven fractions, A1-A7. Fraction A1 (2.08 g) is then chromatographed on a C18 MPLC column (4×37 cm) eluting with a gradient system of MeOH/H.sub.2O (3:7 to 1:0, v/v) to afford sixteen subfractions, B1-B16. These sub-fractions are individually subjected to a series of semi-preparative HPLC separations using a Phenomenex Luna C18 column (250×10 mm i.d., 5 μm, flow=2 mL/min) with different isocratic elution systems of MeOH/H.sub.2O to afford compounds 25 (0.9 mg), 26 (2.5 mg), 27 (0.8 mg), 28 (0.5 mg), 29 (17.5 mg), 730 (0.7 mg), 31 (1.1 mg), 32 (3.9 mg), 33 (1.1 mg), 34 (2.1 mg), 35 (2.8 mg), 36 (3.2 mg), 38 (2.4 mg), 39 (5.2 mg), 40 (0.8 mg), and 53 (0.5 mg). Similarly, fraction A3 (0.71 g) is purified by semi-preparative HPLC using a Waters XBridge Prep C18 column (250×19 mm i.d., 5 μm; flow=3.5 mL/min) and a gradient solvent system of MeOH/H.sub.2O to afford four subfractions C1-C4. These subfractions are separately subjected to semi-preparative HPLC with isocratic solvents systems of MeOH/H.sub.2O to afford compounds 24 (2.2 mg), 37 (4.5 mg), 42 (4.5 mg), 43 (2.2 mg), 44 (4.2 mg), 50 (3.7 mg), and 51 (1.1 mg). Similarly, fraction A4 (0.097 g) is purified by semi-preparative HPLC to afford compounds 41 (1.4 mg), 45 (2.6 mg), 46 (8.0 mg), 47 (0.4 mg), and 49 (3.2 mg) and subfraction A5 (0.022 g) yielded compounds 48 (3.6 mg) and 52 (1.1 mg).

(64) TABLE-US-00003 TABLE 3 Total Compounds isolated from an Ethyl Acetate Extract of Canadian Maple Syrup (MS-EtOAc) compd Identification 1 Lyoniresinol 2 Secoisolariciresinol 6 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane- 1,3-diol (guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol) 8 Scopoletin 22 C-veratroylglycol 24 5-(3″,4″-dimethoxyphenyl)-3-hydroxy-3-(4′-hydroxy-3′-methoxybenzyl)-4- hydroxymethyl-dihydrofuran-2-one* 25 (erythro, erythro)-1-[4-(2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1- (hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol* 26 (erythro, threo)-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1- (hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol* 27 (threo, erythro)-1-[4-[(2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1- (hydroxymethyl)ethoxy]-3-methoxyphenyl]-1,2,3-propanetriol.sup.a 28 (threo, threo)-1-[4-((2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1- (hydroxymethyl)ethoxy]-3-methoxyphenyl]-1,2,3-propanetriol.sup.a 29 threo-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol 30 erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2,6- dimethoxyphenoxy]-1,3-propanediol.sup.a 31 2-[4-[2,3-dihydro-3-(hydroxymethyl)-5-(3-hydroxypropyl)-7-methoxy-2-benzofuranyl]- 2,6-dimethoxyphenoxy]-1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol 32 Acernikol 33 leptolepisol D.sup.a 34 buddlenol E.sup.a 35 (1S, 2R)-2-[2,6-dimethoxy-4-[(1S,3aR,4S,6aR)-tetrahydro-4-(4-hydroxy-3,5- dimethoxyphenyl)-1H,3H-furo[3,4-c]furan-1-yl]phenoxy]-1-(4-hydroxy-3- methoxyphenyl)-1,3-propanediol.sup.a 36 Syringaresinol 37 isolariciresinol.sup.a 38 icariside E4.sup.a 39 sakuraresinol.sup.a 40 1,2-diguaiacyl-1,3-propanediol.sup.a 41 2,3-dihydroxy-1-(3,4-dihydroxyphenyl)-1-propanone* 42 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone.sup.a 43 3-Hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)propan-1-one.sup.a 44 dihydroconiferyl alcohol 45 4-acetylcatechol.sup.a 46 3′,4′,5′-trihydroxyacetophenone.sup.a 47 3,4-dihydroxy-2-methylbenzaldehyde 48 protocatechuic acid 49 4-(dimethoxymethyl)-pyrocatechol.sup.a 50 Tyrosol 51 isofraxidin.sup.a 52 4-hydroxycatechol.sup.a 53 phaseic acid.sup.a .sup.aFirst report from maple syrup *New compounds

(65) Preparation of Preparation of a Food-Grade Approved Extract from Maple Syrup.

(66) According to another embodiment of the present invention, there is disclosed a food grade extract from maple tree, including maple tree parts as well as syrup (e.g. Maple Syrup-XAD extract). The generation of the extract requires the utilization of non-food grade solvents and methods, a ‘food-grade approved’phenolic-enriched extract of maple syrup for future nutraceutical applications is prepared. Towards this end, the maple syrup methanol extract (MS-MeOH) may be prepared using a FDA-food grade resin, such as polymeric resins that include but are not limited to styrene and divinylbenzene resins, and styrene-divinyl-benzene (SDVB) cross-linked copolymer resin. Examples of such resins include but are not limited to Amberlite™ XAD-4 (divinylbenzene copolymer), XAD-2 (polystyrene copolymer resin), XAD-7 (aliphatic ester), XAD 7HP (aliphatic ester), XAD16 (polystyrene-divinylbenzene), XAD16HP (polystyrene-divinylbenzene), XAD761 (Crosslinked phenol-formaldehyde polycondensate), XAD1180 (Polydivinylbenzene), XAD1600 (polystyrene-divinylbenzene), FPx-66 (macroreticular aromatic polymer), XFS-4257, XFS-4022 (unfunctionalized polystyrene beads), XUS-40323 and XUS-40322. Amberlite™ strong anion exchange (SAX) resin, Amberlite™ WAX Resin, a pentafluorophenyl derivatived silica gel, HLB (hydrophobic-lipophilic balanced) type SILIaPrepX phase, strong anion exchange (SAX) resin on silica or mixed-mode strong anion exchange (SAX)-C.sub.18, an aqueous C.sub.18 phase, a C.sub.18 phase, a C.sub.18 type SILIaPrepX™ phase, diatomaceous earth. According to an embodiment of the present invention, the polymeric may be Amberlite™ XAD-16 (Sigma) and adsorption chromatography is performed by adsorbing the maple syrup on the XAD-16 resin column, eluted with copious amounts of water to remove the natural sugars, then finally eluted with MeOH to yield the maple syrup methanol extract (MS-MeOH) after solvent removal in vacuo. Elution may also be effected with other solvents, which include ethanol. 1. 1 Kg of Amberlite™ XAD-16 (Sigma) soaked overnight and packed in a large glass column 2. Eluted the XAD-16 column with copious amounts of water. 3. Adsorb a certain volume (to be determined; ca. 500 mL; (make sure it is not over loaded), of maple syrup which was previously diluted in water so that the solution is not too sticky. 4. Leave maple syrup column on XAD-16 column for ca. 1 h. 5. Elute the column with copious amounts of water to remove sugar (check the eluent for color). 6. Elute with methanol to remove phenolics. 7. Dry the methanol fraction using a rotary evaporator in vacuo, the temperature of the water bath should be set from 37° C. and should not exceed 40° C. 8. The dried sample is maple syrup XAD extract also known as MSX. 9. Repeat the steps to prepare enough quantities.

(67) According to another embodiment, there is also disclosed a process for the extraction of polyphenolic compounds from maple syrup. The process comprises contacting an adsorbent material having a maple syrup polyphenolic fraction adsorbed thereon with an organic solvent, for a time sufficient and for a number of times sufficient, to elute and collect said maple syrup polyphenolic fraction.

(68) According to an embodiment, the time sufficient may be about 30 minutes. The number of time sufficient is from about 1 time to about 3 times.

(69) According to another embodiment, the maple syrup mixture is adsorbed on the adsorbent material for a time sufficient to adsorb the polyphenolic fraction on the adsorbent material, and the mixture comprises maple syrup diluted in water.

(70) The time sufficient to adsorb said polyphenolic fraction is from about 12 to about 20 hours, or from about 12 to about 19 hours, or from about 12 to about 18 hours, or from about 12 to about 17 hours, or from about 12 to about 16 hours, or from about 12 to about 15 hours, or from about 12 to about 14 hours, or from about 12 to about 13 hours. The time sufficient to adsorb said polyphenolic fraction may be 12, 13, 14, 15, 16, 17, 18, 19, 20 hours. Preferably, the time is 16 hours.

(71) Examples of absorbent material include but are not limited to Amberlite™ XAD-4 (divinylbenzene copolymer), XAD-2 (polystyrene copolymer resin), XAD-7 (aliphatic ester), XAD 7HP (aliphatic ester), XAD16 (polystyrene-divinylbenzene), XAD16HP (polystyrene-divinylbenzene), XAD761(Crosslinked phenol-formaldehyde polycondensate), XAD1180 (Polydivinylbenzene), XAD1600 (polystyrene-divinylbenzene), FPx-66 (macroreticular aromatic polymer), XFS-4257, XFS-4022 (unfunctionalized polystyrene beads), XUS-40323 and XUS-40322. Amberlite™ strong anion exchange (SAX) resin, Amberlite™ WAX Resin, a pentafluorophenyl derivatived silica gel, HLB (hydrophobic-lipophilic balanced) type SILIaPrepX phase, strong anion exchange (SAX) resin on silica or mixed-mode strong anion exchange (SAX)-C.sub.1, an aqueous C.sub.18 phase, a C.sub.18 phase, a C.sub.18 type SILIaPrepX™ phase, diatomaceous earth.

(72) According to another embodiment, the process may further comprise the step of diluting the maple syrup in water prior to adsorption on said adsorbent material.

(73) According to another embodiment, the process may further comprise the step of washing the adsorbent material with water prior to step a).

(74) According to another embodiment, the process may further comprise step b): heating the collected polyphenolic fraction to evaporate the organic solvent and obtain a dried polyphenolic fraction. Heating may be at a temperature of about 37° C. to about 40° C.

(75) According to another embodiment, the organic solvents suitable for the process of the present invention may be chosen from methanol, ethyl acetate, butanol, ethanol, methyl tert-butyl ether, and combinations thereof.

(76) According to yet another embodiment, the process of the present invention may further comprise prior to step a) an extraction with a mixture of solids for extraction of a sugar from said maple syrup. For example, such extraction with a mixture of solids comprises extraction with MgSO.sub.4, NaCl, and a solid absorbent. Preferably, the solid absorbent for this extraction may be chosen from an aminated silica resin, a C.sub.18 silica resin, or combinations thereof.

(77) According to yet another embodiment, the process of the present invention may further comprising prior to step a) a liquid-liquid extraction of the maple syrup. For example, the liquid-liquid extraction may comprise an ethyl acetate extraction, a butanol extraction, or combinations thereof, followed by adsorption on a silicon dioxide (SiO.sub.2)/magnesium oxide (MgO) solid phase having a ratio of about 85% SiO.sub.2 and about 15% MgO (Florisil®).

(78) According to yet another embodiment, there is disclosed an extract obtained from the process of the present invention.

(79) Preparation of Maple Syrup Butanol Extract without Sugar (MS-BuOH without Sugar)

(80) According to another embodiment of the present invention, there is disclosed an MS butanol extract without sugar. 1. A known volume of maple syrup (based on the size of your separatory funnel) is subjected to liquid-liquid partitioning with n-butanol (1:1 v/v; 3 times). The maple syrup is diluted with water before partitioning since it is too sticky. (Usually we add around 300 ml water to 1 L maple syrup). 2. Combine the butanol fraction and dry in vacuo as previously described. 3. The dried butanol fraction will be still very sticky and we usually freeze-dry or vacuum dry to make sure it has a powdery consistency 4. The dried butanol extract powder is reconstituted in methanol and the filtered to remove the white solid i.e. sugars. The liquid portion is part is dried in vacuo as previously described. 5. After removing the solvent from the liquid part, add certain methanol to remove sugar again. Repeat filtering and drying. 6. The final dried extract is the MS-BuOH extract without sugar. 7. Repeat steps to prepare enough quantities

(81) Preparation of Maple Syrup Butanol Extract with Sugar (MS-BuOH with Sugar)

(82) According to another embodiment of the present invention, there is disclosed an MS butanol extract without sugar.

(83) Follow steps 1-3 above. In this case, the sugars are not removed with methanol.

(84) Determination of total phenolic content by the Folin-Ciocalteau method

(85) The total phenolic contents of the maple syrup extracts are determined according to the Folin-Ciocalteu method and is measured as gallic acid equivalents (GAEs). Briefly, the extracts were diluted 1:100 with methanol/H.sub.2O (1:1, v/v), and 200 μL of each sample was incubated with 3 mL of methanol/H.sub.2O (1:1, v/v) and 200 μL of Folin-Ciocalteau reagent for 10 min at 25° C. After this, 600 μL of 20% Na.sub.2CO.sub.3 solution was added to each tube and vortexed. Tubes were further incubated for 20 min at 40° C. and after, incubation; samples were immediately cooled in an ice bath to room temperature. Samples and standard (gallic acid) were processed identically. The absorbance was determined at 755 nm, and final results were calculated from the standard curve obtained from a Spectramax plate reader.

(86) Methods of Solvent Removal

(87) According to some embodiments, solvent removal from the extracts of the present invention may be effected in vacuo. However, other known techniques may be employed, such as atomization, lyophilization, evaporation, crystallization, dehydration, precipitation, centrifugation, or any other suitable process to eliminate the aqueous phase from any of the extracts of the present invention.

(88) TABLE-US-00004 TABLE 4 Presence and relative levels of pure isolated phenolic compounds in the different maple syrup extracts* MS- MS- MS- Compound Name BuOH EtOAc MeOH 1 Lyoniresinol + + + 2 Secoisolariciresinol + + + 3 2,3-dihydro-3-(hydroxymethyl)-2-(4-hydroxy-3- + + methoxyphenyl)-7-methoxy-5-benzofuranpropanol (dihydrodehydrodiconiferyl alcohol) 4 5′-methoxydehydrocortferyl alcohol + 5 1,3-propanediol, 1-(4-hydroxy-3-methoxyphenyl)-2- + + [4-[(1E)-3-hydroxy-1-propenyl]-2-methoxyphenoxy]- (1R,2R) 6 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- + + + hydroxypropyl)-2-methoxyphenoxy]-propane-1,3- diol (guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol) 7 3-[(4-[(6-dexoy-α-L-mannopyranosyl)oxy]-3- + + methoxyphenyl)-5-(3,4-dimethoxyphenyl)dihydro-3- hydroxy-4-(hydroxymethyl)-2(3H)-furanone 8 Scopoletin + + + 9 Fraxetin + + 10 (E)-3,3′-dimethoxy-4,4′-dihydroxy stilbene + + 11 2-Hydroxy-3′,4′-dihydroxyacetophenone + + 12 1-(2,3,4-trihydroxy-5-methylphenyl)-ethanone + + 13 2,4,5-trihydroxyacetophenone + 14 Catechaldehyde + + 15 Vanillin + + + 16 Syringaldehyde + + 17 Gallic acid + + 18 trimethyl gallic acid methyl ester + 19 Syringic acid + + + 20 Syringenin + + 21 (E)-coniferyl alcohol (coniferol) + + 22 C-veratroylglycol + + + 23 1,2-benzenediol (catechol) + + 24 5-(3″,4″-dimethoxyphenyl)-3-hydroxy-3-(4′-hydroxy- + + 3′-methoxybenzyl)-4-hydroxymethyl-dihydrofuran-2- one 25 (erythro, erythro)-1-[4-[2-hydroxy-2-(4-hydroxy-3- + + methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5- dimethoxyphenyl]-1,2,3-propanetriol 26 (erythro, threo)-1-[4-[2-hydroxy-2-(4-hydroxy-3- + + methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5- dimethoxyphenyl]-1,2,3-propanetriol 27 (threo, erythro) 1-[4-[(1R,2R)-2-hydroxy-2-(4- + + hydroxy-3-methoxyphenyl)-1- (hydroxymethyl)ethoxy]-3-methoxyphenyl]-1,2,3- propanetriol 28 (threo, threo) 1-[4-[(1R,2R)-2-hydroxy-2-(4-hydroxy- + + 3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3- methoxyphenyl]-1,2,3-propanetriol 29 Threo-guaiacylglycerol-β-O-4′-dihydroconiferyl + + alcohol 30 erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- + hydroxypropyl)-2,6-dimethoxyphenoxy]-1,3- propanediol 31 2-[4-[(2S,3R)-2,3-dihydro-3-(hydroxymethyl)-5-(3- + + hydroxy propyl)-7-methoxy-2-benzofuranyl)-2,6- dimethoxyphenoxy]-1-(4-hydroxy-3- methoxyphenyl)-1,3-propanediol 32 Acernikol + + 33 Leptoliepisol D + + 34 Buddenol E + + 35 (1S, 2R)-2-[2,6-dimethoxy-4-[(1S,3aR,4S,6aR)- + + tetrahydro-4-(4-hydroxy-3,5-dimethoxyphenyl)- 1H,3H-furo[3,4-c]furan-1-yl]phenoxy]-1-(4-hydroxy- 3-methoxyphenyl)-1,3-propanediol 36 Syringaresinol + + 37 Isolariciresinol + + 38 Icariside E4 + + 39 Sakuraresinol + + 40 1,2-diguaiacyl-1,3-propanediol + + 41 2,3-dihydroxy-1-(3,4-dihydroxyphenyl)-1-propanone + + 42 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1- + + propanone 43 3-hydroxy-1-(4-hydroxy-3,5- + + dimethoxyphenyl)propan-1-one 44 Dihydroconiferyl alcohol + + 45 4-hydroxycatechol + + 46 3′,4′,5′-Trihydroxyacetophenone + + 47 3,4-dihydroxy-2-methylbenzaldehyde + 48 Protocatechuic acid + + 49 4-(dimethoxymethyl)-pyrocatechol + + 50 Tyrosol + + 51 Isofraxidin + + 52 4-acetylcatechol + + 53 phaseic acid + 54 Quebecol + 55 Ferulic acid + 56 p-coumaric acid + 57 Catechin + 58 Epicatechin +

(89) The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE 1

Assessment of Metabolic Syndrome-Preventing Effects of the Nutriprotective Diet

(90) The objective of the current example is to evaluate the metabolic syndrome-improving effects of the nutriprotective diet.

(91) Materials and Methods

(92) A maple syrup extract is used which has been prepared from the amber grade of maple syrup by treatment with n-butanol which may be useful to prepare an extremely low-sugar product. A total amount of 50 g butanol extract, 20 mg butanol extract basis, is necessary for 5 repetitions of a 60-day-feeding trial with 8 rats fed on 20 g diet/capita/day.

(93) Feeding

(94) Male rats (Wistar) are fed on an AlN93G-based high-fat diet with 0.1% butanol extract of maple syrup (n=8) or on the same diet without the extract (n=8) for 2 months. During the feeding, daily body weight gains and diet intakes are measured every day.

(95) Dissection

(96) Each rat after the feeding is subjected to dissection for sampling the systemic blood, small-intestinal epithelia, liver and adipose tissue.

(97) Blood Chemistry Analysis

(98) Systemic blood qualities are analyzed for 20-50 items.

(99) Genomics

(100) Transcriptomics is carried with for total RNA samples from each organ or tissue by DNA microarray analysis using affymetrix gene chip.

(101) Results

(102) The use of the butanol extract of maple syrup as a supplement to high-fat (or high-calorie) diets reduces the risk of metabolic syndromes by modulating the lipid and/or sugar metabolic pathways.

EXAMPLE 2

Global Analysis of Hepatic Gene Expression Profiles in Diabetes Model KK-Ay Mice

(103) Now referring to FIG. 8, the purpose of this study is to elucidate the effect of butanol extract of maple syrup (MSE, as described above) on the liver of T2DM model mice—KK-Ay mice (diabetic mice). Male KK-Ay mice, aged 4 weeks, as T2DM model mice for investigation of obesity, hyperglycemia and insulin resistance are selected and separated into two experimental groups as shown in FIG. 8: 16 males, control group (n=8) fed AlN-93G diet, and experimental group (N=8) fed MSE (AlN-93G diet+0.1% MSE). The animals are prefed the same normal diet for 7 days, then fed the experimental diets for 43 days. They are fasted for 16 hours and liver and serum samples are collected.

(104) Now referring to FIGS. 9 to 11. Measurement of serum glucose, insulin and glycoalbumin levels show that MSE intake tends to improve hyperglycemia (FIG. 9). Furthermore, as shown in FIG. 10, MSE intake significantly activates the lipid b-oxidation in the liver, resulting in increased concentration of total ketone bodies. Furthermore, as shown by the measurement of the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (FIG. 11), MSE intake significantly (p<0.01 and p<0.05 respectively) ameliorates hepatopathy.

(105) As summarized in FIG. 12, feeding of MSE to KK-Ay mice results in improved hyperglycemia, activation of beta-oxidation, and mitigation of hepatopathy.

(106) Next, hepatic transcriptome analysis is performed with Affymetrix Genechip (Mouse Genome 430 2.0). Microarrays are performed and results are normalized with the distribution free weighted method (DFW). The two experimental groups clustered separately from one another (FIG. 13), indicating that there are noticeable differences in gene expression between the two groups. Furthermore, there are 736 significantly up-regulated genes and 1078 significantly down-regulated genes in the MSE group (FDR<0.05).

(107) The significantly differentially expressed genes are then analyzed by gene annotation enrichment analysis, and insulin signaling is focused on by verifying the statistical significance of the genes of this signalling pathway, as well as by immunoblotting.

(108) With respect to gene annotation enrichment analysis, as shown in FIGS. 15 to 18, the results show significant changes in lipid metabolism, inflammation and immunity, material transport, amino acid metabolism, oxidoreduction, as well as epithelial cell development, chemical homeostasis, and cell redox homeostasis. With respect to lipid metabolism-related genes, MSE intake appears to down-regulate lipid accumulation in the liver (FIG. 16). With respect to inflammation and immunity related genes, MSE appears to suppress the onset of inflammation in the liver (FIG. 17). As summarized in FIG. 18, the suppression of lipid accumulation in the liver is consistent with an increase in the total ketone bodies in serum, while the suppression of inflammation is consistent with reduction is serum AST and ALT.

(109) Next, as shown in FIGS. 19 to 24 the insulin signalling pathway is analyzed (FIG. 19). Immunoblotting of insulin receptor and phospho-insulin receptor (IR) beta subunit in each group reveals an increase in phosphorylation of the insulin receptor beta subunit (FIG. 20). Immunoblotting of the insulin receptor substrate 2 (IRS2) reveals an increase in protein expression of IRS2 in MSE treated animals. Immunoblotting of the protein kinase B (Akt) protein and phosphoprotein revealed an increase phosphorylated Atk in the MSE treated group (FIG. 22). As shown in FIG. 23, MSE intake by KK-Ay mice lead to an overall improvement of the insulin signalling function of KK-Ay mice liver. This result is consistent with the observed downregulation of plasma glycosylated albumin for suppression of hyperglycemia. As shown in FIG. 24, the suggested mechanism of the effect of MSE on the liver of KK-Ay mice is through decrease lipid accumulation, improved insulin sensitivity, increased β-oxidationm and decreased inflammation.

EXAMPLE 3

Assessment of Metabolic Syndrome-Preventing Effects of a Maple Syrup Extract Supplemented High-Fat Diet

(110) Now referring to FIGS. 25 to 31, the aim of this study is to clarify the liver-protecting effect of maple syrup using mice fed with a high-fat diet with or with supplementation with a maple syrup extract (MSX). The MSX extract is prepared as described above with the use of a food-grade approved resin XAD-16 to prepare a food grade approved extract from maple syrup. The MSX extract is prepared from 50 L of maple syrup. The experimental groups are defined in Table 5 below:

(111) TABLE-US-00005 TABLE 5 Experimental groups and respective diets Group Diet LFD 10%-fat diet 45F 45%-fat diet 45F + 0.06MSX 45%-fat diet with 0.06% MSX 45F + 0.12MSX 45%-fat diet with 0.12% MSX LFD = low fat diet; 45F = high fat diet

(112) As shown in FIG. 25, 3-week old C57/BL/6J male mice (N=4 in each group) are fed a low fat diet for 7 days, and then each group is switched to their respective experimental diet for 4 weeks (28 days). Liver and blood samples are collected at the end of the 4 week period. The results in FIG. 26 show that body weight increase and energy intake are the same between all 4 experimental groups.

(113) However, as shown in FIG. 27, the level of plasma choline esterase is significantly decreased in animals treated with MSX. As shown in FIG. 28, the level of plasma AST, ALT and LDH all decrease in a dose dependent manner in the MSX treated groups, suggesting that MSX has a liver protecting effect, and that the liver-protecting factors are present in MSX.

(114) As shown in FIG. 29, the total plasma protein levels, albumin levels are approximately unchanged between each group, and the blood urea nitrogen levels appears slightly higher in the 0.12% MSX group. As shown in FIG. 30, total plasma potassium, calcium levels, total bilirubin and alkaline phosphatase levels do appears to trend toward the low fat diet levels in the 0.12% MSX group. And the total bilirubin level do appear to trend downward toward the low fat diet levels for each MSX treated group. In conclusion, there results show that the choline esterase levels are significantly decreased in the MST diet groups, and that AST, ALT and LDH levels are decreased in a dose dependent manner in the MSX diet groups. These results indicate that MSX has a liver protecting effects. To confirm these results, a new experiment is being carried out, in which experimental groups of each n=14 animals are being treated with low fat diet, a high fat diet, or a high fat diet supplemented with 0.6% MSX for a total of 8 weeks.

EXAMPLE 4

Preparation of Preparation of a Food-Grade Approved Extract from Maple Syrup

(115) Described is the methodology for the extraction of bioactive compounds from maple syrup without the sugar moiety, using two types of resin 1) FPX 66 and 2) XAD-16. These two resins both are divinylbenzene phases. The mechanism of adsorption of polyphenols on these phases is exclusively by means of hydrophobic interactions.

(116) Methodology:

(117) 1. Dilution of a 5 L (6 kg) portion of maple syrup with 2.1 L of deionized water. 2. Adsorption of the maple syrup mixture on 16.8 kg of wet Amberlite™ FPX66 or XAD-16 (5.0-6.7 kg dry mass) for 16 hours. 3. Wash of the column with deionized water (7×15 L). 4. Elution with denatured ethanol (3×15 L). Before each elution, let the ethanol in contact with the resin for 30 minutes. 5. Evaporation of ethanol on “rotary evaporator”. The temperature of the water bath is set from 37° C. and does not exceed 40° C. 6. Isolation of the MSX fraction (15.3 g). 7. Reconditioning of the resin with two portions of 10 L of deionized water.

(118) While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.