POWDERED FOOD COMPOSITION COMPRISING ERIOCITRIN

Abstract

A method for treating inflammation, cancer, neurodegenerative diseases, diabetes, cardiovascular diseases, vascular diseases, cutaneous diseases, in a subject in need thereof. The method includes administering to a subject in need thereof an effective amount of a powdered food composition, obtained from lemon containing between 1% and 30% (w/w) of eriocitrin, in a dose of eriocitrin between 5 and 35 mg per day.

Claims

1. A method for treating inflammation, cancer, neurodegenerative diseases, diabetes, cardiovascular diseases, vascular diseases, cutaneous diseases, in a subject in need thereof, the method comprising: administering to a subject in need thereof an effective amount of a powdered food composition, obtained from lemon comprising between 1% and 30% (w/w) of eriocitrin, in a dose of eriocitrin between 5 and 35 mg per day.

2. The method according to claim 1, wherein the eriocitrin is administered in a dose of eriocitrin between 7 and 30 mg per day.

3. The method according to claim 1, wherein the administered composition further comprises other flavonoids.

4. The method according to claim 1, wherein said flavonoids are apigenin 6,8-di-C-Glu (Vicenin-2), chrysoeryiol 6,8-di-C-Glu and/or limocitrin HMG-Glu.

5. The method according to claim 1, wherein the eriocitrin is extracted from a lemon extract.

6. The method according to claim 1, wherein the eriocitrin is extracted from the lemon fruit.

7. The method according to claim 1, wherein the eriocitrin is obtained from the fruit of Citrus limon (L.) Osbeck.

8. The method according to claim 1, wherein the method consists essentially of the administering to the subject in need thereof the effective amount of the composition.

9. The method according to claim 1, wherein the powdered food composition is administered daily, every other day, 3×/week, or once per week.

10. A method for obtaining a powdered food composition comprising between 1% and 30% (w/w) of eriocitrin comprising a solvent extraction step and a tangential flow filtration step.

11. The method according to claim 10, wherein the size of the membrane in tangential flow filtration is between 100 Da and 100,000 Da.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 represents a diagram of the comparative metabolism of hesperidin and eriocitrin.

[0030] FIG. 2 represents chromatograms obtained at 340 nm of the orange extract and the lemon extract, respectively. The number of each chromatographic peak refers to Table 1.

As indicated in FIG. 1, eriocitrin, being soluble, allows the intestinal microbiota to access it and break down the rutin residue. Once this happens, the resulting molecule, eriodictyol, is able to enter the cells of the intestine where it is converted, at least in part, to hesperetin by the action of the COMT enzyme (point 3, FIG. 1). The hesperetin is then conjugated and circulates through the body. Therefore, eriocitrin is an ideal way to obtain the same final active metabolite, hesperetin, but generating it in situ, in the intestine.
As can be seen in FIG. 2, the ‘orange extract’ consisted exclusively of hesperidin (compound 10, table 1) while the lemon extract presented a much richer profile of compounds. In addition to eriocitrin (compound 6, table 1) as the major compound, two other flavanones were identified in the lemon extract: eriodictyol-glucoside-rhamnoside-glucoside (compound 3, table 1) and naringin (compound 9, Table 1).

[0031] FIG. 3 represents a extracted ion chromatograms (EICs) of urine and plasma metabolites after consuming (A,C) lemon or (B,D) orange extract. Numbers designate the metabolites according to Table 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0033] The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise.

[0034] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0037] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0038] The term “treatment” or “treating” means any treatment of a disease or disorder in a subject, such as a mammal, including: preventing or protecting against the disease or disorder, that is, causing the clinical symptoms not to develop; inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder that is, causing the regression of clinical symptoms. In some embodiments, the term treatment is used for the treatment of inflammation, cancer, neurodegenerative diseases, diabetes, cardiovascular diseases, vascular diseases, cutaneous diseases.

[0039] It will be understood by those skilled in the art that in human medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, as used herein the term “prophylaxis” is intended as an element of “treatment” to encompass both “preventing” and “suppressing” as defined herein.

[0040] Hereinafter, the present invention is described in more detail with reference to illustrative examples, which does not constitute a limitation of the present invention.

EXAMPLES

Example 1: Obtaining a Composition of the Present Invention with a 10 Times Higher Eriocitrin Content

[0041] To obtain a composition of the present invention with eriocitrin, 10 kg of lemon ground was extracted with water at 50° C. for 4 hours. Subsequently, by centrifugation, the liquid was separated from the undissolved solid. The extract contained the compound of interest, eriocitrin, a flavonoid abundant in Citrus.

[0042] The micelle with a dry residue of 5% (w/w) was subjected to a purification process by cascade tangential filtration. For them, 3 chained stages were carried out:

[0043] 1. The micelle was filtered through a 100,000 Da membrane, passing 99% (w/w) of the eriocitrin to the permeate and obtaining 3% (w/w) of dry residue.

[0044] 2. The permeate from the first stage was filtered through a 10,000 Da membrane passing the compound of interest to the permeate at 99% (w/w) and obtaining 1% (w/w) of solids.

[0045] 3. The permeate from stage 2 was filtered through a 1,000 Da membrane passing the eriocitrin at 98% (w/w) to the permeate and obtaining 0.5% (w/w) of solids.

[0046] Overall, the three stages went from a liquid extract of 5% (w/w) of solids to one of 0.5% (w/w) of solids, maintaining the same amount of eriocitrin. Therefore, the eriocitrin was concentrated 10 times.

Example 2: Obtaining a Composition of the Present Invention with an Eriocitrin Content 18 Times Higher

[0047] To obtain a composition of the present invention with eriocitrin, 10 kg of lemon ground was extracted with water at 50° C. for 4 hours. Subsequently, by centrifugation, the liquid was separated from the undissolved solid. The extract contained the compound of interest, eriocitrin.

[0048] The micelle with a dry residue of 5% (w/w) was subjected to a purification process by cascade tangential filtration. For them, 3 chained stages were carried out:

[0049] 1. The micelle was filtered through a 100,000 Da membrane, passing 99% (w/w) of the eriocitrin to the permeate and obtaining 3% (w/w) of dry residue.

[0050] 2. The permeate from the first stage was adjusted to a basic pH by adding an alkaline substance (sodium hydroxide) and the solution was filtered through a 10,000 Da membrane passing the compound of interest to the permeate by 99% (w/w). The pH caused a greater retention of solids with which a 0.5% (w/w) of solids was finally obtained in the permeate.

[0051] 3. The permeate from step 2 was filtered through a 1000 Da membrane passing the eriocitrin at 98% (w/w) to the permeate and obtaining 0.25% (w/w) of solids.

[0052] Overall, the three stages went from a liquid extract of 5% (w/w) of solids to one of 0.25% (w/w) of solids, maintaining the same amount of eriocitrin. Therefore, the eriocitrin was concentrated a total of 18 times.

Example 3: Characterization of the Compounds Identified in the Lemon and Orange Extracts by HPLC-UV-MS/MS

[0053] For the identification and quantification of the compounds present in the extracts, the supernatants and precipitates were analyzed by HPLC-UV-MS/MS. For the lemon extract, a single extraction was sufficient since no precipitate appeared after centrifugation. In the case of the orange extract, the precipitate formed was dissolved in 5 mL of DMSO and after centrifugation, no precipitate formed again.

[0054] The compounds identified in both extracts are listed in Table 1, ordered by their retention time (TR), their identified mass information (negative ion, [MH]−), their fragmentation profile (MS/MS) and their Maximum absorbance in UV (Amax).

TABLE-US-00001 TABLE 1 Compounds identified in lemon and orange extracts through HPLC-UV-MS/MS. Num. Compounds TR [M-H].sup.− MS/MS λ.sub.max  1 Ferulic Acid O-Glu 16.94 355 193/160/134 294/328  2 Apigenin 6,8-di-C- 18.13 593 503/473/ 270/332 Glu(Vicenin-2) 383/353  3 Eriodictyol-Glu- 18.70 757 595/449/287 282/330 Ramn-Glu  4 Diosmetin 6,8-di- 19.00 623 605/533/ 270/346 C-Glu 503/383  5 Chrysoeryol 6,8-di- 19.65 623 533/503/383 270/346 C-Glu  6 Eriocitrin 22.90 595 459/329/287 284/334  7 Apigenin 8-C- 23.39 563 413/341/293 268/332 xylanopyranosyl-Glu  8 Diosmetin 8-C-Glu 24.88 461 371/341 268/334  9 Naringenin 7-O- 25.02 579 271 278/330 rutinoside (Naringin) 10 Hesperidin.sup.a 28.10 609 301 284/336 11 Diosmetin 7-O- 28.40 607 299/284 270/334 rutinoside(Diosmin) 12 Limocitrin 29.43 651 549/507/345 274/350 HMG-Glu .sup.aOnly compound present in orange extract. Glu, glucoside. Ramn, ramnoside. HMG, 3-hydroxy-3-methyl-glutaryl

[0055] The chromatograms of the supernatants analyzed by HPLC-UV-MS/MS of each of the extracts are shown in FIG. 2, where all the compounds identified in each extract can be seen numbered. The number of each chromatographic peak refers to Table 1.

[0056] As can be seen in FIG. 2, the ‘orange extract’ consisted exclusively of hesperidin (compound 10, Table 1) while the lemon extract presented a much richer profile of compounds. In addition to eriocitrin (compound 6, table 1) as the major compound, two other flavanones were identified in the lemon extract: eriodictyol-glucoside-rhamnoside-glucoside (compound 3, table 1) and naringin (compound 9, table 1).

[0057] Other phenolic compounds from different families were also identified. For example, the ferulic acid glycoside (compound 1, table 1) was identified which is a hydroxycinnamic acid, and different flavones were also identified, which were different C-glycoside derivatives of: apigenin (compound 2, table 1), diosmetin (compound 4, table 1), and chrysoeriol (compound 5, table 1). The C-glycoside derivatives showed a characteristic fragmentation pattern [(M-H) −90] and [(M-H) −120]. A mono-glucoside (compound 8, table 1) and its rutinoside derivative, diosmin (compound 11, table 1) were also identified from diosmetin. Furthermore, another identified apigenin derivative was apigenin 8-C-xylanpyranosyl-glucoside (compound 7, table 1). Like flavanones, flavones have an absorbance maximum around 340 nm, but with a very different UV spectrum, which makes the compounds of these two families easily differentiable.

[0058] Finally, a flavonol, limocytrin-3-hydroxy-3-methyl-glutaryl glucoside (compound 12, table 1) was identified, observing its absorption maximum around 360 nm characteristic of this family of phenolics.

[0059] Regarding quantification, it was done for those major compounds of both extracts (Table 2). The main flavanones detected in the lemon and orange extracts were eriocitrin (83.3±5.6 mg/g extract) and hesperidin (133.7±15.9 mg/g extract) respectively. Both compounds were quantified with their own standards.

TABLE-US-00002 TABLE 2 Compounds quantified in the orange and lemon extracts. Num Compounds mg/g extracto ± DE  2 Apigenin 6,8-di-C-Glu (Vicenin-2)  14.6 ± 0.4 (lemon)  5 Chrysoeryol 6,8-di-C-Glu  11.3 ± 0.2 (lemon)  6 Eriocitrin  83.3 ± 5.6 (lemon)  133.7 ± 15.9 (orange) 10 Hesperidin   6.3 ± 0.1 (lemon) 12 Limocitrin HMG-Glu  12.0 ± 1.5 (lemon) Values are expressed as the mean ± standard deviation (SD) of an n = 3

[0060] In the orange extract, hesperidin was only detected and quantified in the analyzed supernatant (after extraction with DMSO). No quantifiable amounts of the compound were observed in the orange extract precipitate.

[0061] Flavones were also detected in the lemon extract in significant quantities, such as apigenin 6,8-di-C-Glu (Vicenin-2) and chrysoeriol 6,8-di-C-Glu (Table 2). The flavonol, limocitrin HMG-Glu in an amount of 12 mg/g extract was also quantified. The flavones (compounds 2 and 5) were quantified using apigenin as a standard and the quantified flavonol, limocitrin HMG-Glu (compound 12), with rutin.

Example 4: Characterization of the Metabolites after Consuming Lemon or Orange Extract

[0062] Through a targeted metabolomic strategy, a total of 17 metabolites were tentatively identified (Table 3). No hesperidin or eriocitrin-derived metabolites were detected in any volunteer at baseline. All the identified metabolites were detected at least in one volunteer, independently of the extract consumed, except for the metabolites M9 (eriodictyol sulfoglucuronide) and M11 (hesperetin sulfoglucuronide), which only were detected after consuming lemon and orange extract, respectively (Table 3). The extracted ion chromatograms (EICs) of all the compounds detected are shown in FIG. 3. These representative EICs illustrate the metabolites identified in urine (F3 fraction) after consuming lemon (FIG. 3A) and orange (FIG. 3B) extracts, and plasma after consuming lemon (FIG. 3C) and orange (FIG. 3D) extracts. From the 17 metabolites, 16 were detected after consuming lemon extract (except M11), and 15 after consuming orange extract (except M9 and M10 in urine, and M9 and M12 in plasma) (Table 3).

TABLE-US-00003 TABLE 3 Plasma and urine metabolites identified in volunteers (n = 16) after consuming lemon (LE) or orange (OE) extracts. Occurrence.sup.2 Num Metabolites RT m/z.sup.− Urine LE Plasma LE Urine LE Plasma LE M1 Eriodictyol glucuronide-1 6.37 463.0882 16 16 5 3 M2 Eriodictyol glucuronide-2 7.31 463.0882 16 16 10 7 M3 Naringenin 7-O-glucuronide.sup.1 7.52 447.0933 16 16 16 14 M4 Eriodictyol glucuronide-3 7.68 463.0882 16 16 11 7 M5 Naringenin 4'-O-glucuronide.sup.1 7.70 447.0933 16 16 16 14 M6 Homoeriodictyol glucuronide 7.72 477.1038 16 16 3 3 M7 Hesperetin 7-O-glucuronide.sup.1 8.02 477.1038 16 16 16 16 M8 Hesperetin 3'-O-glucuronide.sup.1 8.20 477.1038 16 16 16 16 M9 Eriodictyol sulfoglucuronide 8.99 543.0450 5 15 0 0 M10 Homoeriodictyol sulfate 9.02 381.0286 16 16 0 3 M11 Hesperetin sulfoglucuronide 9.30 557.0607 0 0 7 8 M12 Eriodictyol.sup.1 9.50 287.0561 14 16 4 0 M13 Eriodictyol sulfate 9.75 367.0129 16 16 12 10 M14 Hesperetin 3'-O-sulfate.sup.1 9.89 381.0286 16 16 16 16 M15 Naringenin.sup.1 11.22 271.0612 9 6 5 2 M16 Homoeriodictyol.sup.1 11.42 301.0718 12 15 1 1 M17 Hesperetin.sup.1 11.73 301.0718 5 8 8 3 .sup.1The identification was achieved using authentic standards. .sup.2Number of volunteers in whom each metabolite was detected after consuming lemon (LE) or orange (OE) extracts.

[0063] Table 4 and Table 5 show the urinary excretion of quantified metabolites at the different fractions collected, i.e., 0-4 h (F1), 4-8 h (F2), 8-10 h (F3), and 10-24 h (F4), after extracts intake. The total excretion (24 h) of metabolites was significantly higher after consuming lemon extract than after consuming orange extract, considering the total amount of flavanones consumed.

TABLE-US-00004 TABLE 4 Metabolites quantified in urine fractions after consuming lemon extract. Lemon Extract Num Metabolites F1 F2 F3 F4 Total F M1 Eriodictyol glucuronide-1 3.2 ± 5.9  9.6 ± 15.5 4.5 ± 6.2 0.7 ± 0.7 18.0 ± 28.4 M2 Eriodictyol glucuronide-2  6.4 ± 11.0 18.0 ± 17.2 18.1 ± 19.7 4.2 ± 5.7  46.6 ± 53.6** M3 Naringenin 7-O-glucuronide 1.0 ± 0.7 2.6 ± 4.0 1.7 ± 1.9 0.3 ± 0.3   5.7 ± 6.9*** M4 Eriodictyol glucuronide-3  9.4 ± 16.9 33.8 ± 64.1 27.7 ± 40.1 4.3 ± 6.4  75.1 ± 128* M5 Naringenin 4'-O-glucuronide 0.8 ± 0.5 1.7 ± 2.7 1.3 ± 1.5 0.4 ± 0.3   4.2 ± 5.0*** M6 Homoeriodictyol glucuronide 13.9 ± 16.2 48.1 ± 90.0 37.2 ± 56.9 6.8 ± 8.0 106 ± 171 M7 Hesperetin 7-O-glucuronide 1.2 ± 1.4 4.0 ± 4.7 2.7 ± 2.5 0.6 ± 0.6   8.5 ± 9.2*** M8 Hesperetin 3'-O-glucuronide 2.1 ± 2.3 10.7 ± 11.3 9.2 ± 7.7 2.2 ± 2.4   24.2 ± 23.7*** M10 Homoeriodictyol sulfate 3.8 ± 3.2 4.0 ± 5.5 3.6 ± 4.1 0.5 ± 0.4  9.6 ± 11.3 M13 Eriodictyol sulfate 4.6 ± 5.0 14.6 ± 17.3 11.9 ± 12.6 1.5 ± 1.5   32.6 ± 36.5*** M14 Hesperetin 3'-O-sulfate 1.9 ± 2.6 4.3 ± 4.5 3.3 ± 3.1 0.7 ± 0.6  9.0 ± 9.1*  Total excretion (24 h): 339 ± 482*** Values expressed as μg/mg creatinine (mean ± SD). F1: Fraction collected from 0 to 4 h after consuming lemon or orange extract; F2: Fraction collected from 4 to 8 h; F3: Fraction collected from 8 to 10 h; F4: Fraction collected at 24 h; Total F (24 h), sum of the fractions F1, F2, F3 and F4. *p < 0.05; **p < 0.01; ***p < 0.001.

TABLE-US-00005 TABLE 5 Metabolites quantified in urine fractions after consuming orange extract. Orange Extract Num Metabolites F1 F2 F3 F4 Total F M1 Eriodictyol glucuronide-1 — 0.4 ± 0.3 0.1 ± 0.1 0.05 ± 0.05 0.6 ± 0.4 M2 Eriodictyol glucuronide-2 0.1 ± 0.1 0.5 ± 0.8 0.5 ± 0.6 1.4 ± 3.4 2.5 ± 4.9 M3 Naringenin 7-O-glucuronide 0.1 ± 0.1 0.2 ± 0.6 0.2 ± 0.2 0.2 ± 0.3 0.7 ± 1.5 M4 Eriodictyol glucuronide-3 0.1 ± 0.2 0.5 ± 0.9 0.7 ± 0.7 2.1 ± 4.5 3.4 ± 6.2 M5 Naringenin 4'-O-glucuronide 0.1 ± 0.1 0.3 ± 0.5 0.3 ± 0.4 0.3 ± 0.5 1.0 ± 1.5 M6 Homoeriodictyol glucuronide — — — — — M7 Hesperetin 7-O-glucuronide 0.2 ± 0.6 2.1 ± 5.7 2.2 ± 3.6 1.1 ± 3.3  5.6 ± 13.2 M8 Hesperetin 3'-O-glucuronide 0.5 ± 1.5  4.3 ± 11.5 3.3 ± 4.2 1.8 ± 1.6  9.9 ± 18.9 M10 Homoeriodictyol sulfate — — — — — M13 Eriodictyol sulfate 0.02 ± 0.01 0.1 ± 0.1 0.2 ± 0.4 0.3 ± 0.4 0.6 ± 0.9 M14 Hesperetin 3'-O-sulfate 0.1 ± 0.3 1.2 ± 3.3 1.9 ± 3.1 0.6 ± 0.6 3.8 ± 7.3 Total excretion (24 h): 28 ± 55  Values expressed as μg/mg creatinine (mean ± SD). F1: Fraction collected from 0 to 4 h after consuming lemon or orange extract; F2: Fraction collected from 4 to 8 h; F3: Fraction collected from 8 to 10 h; F4: Fraction collected at 24 h; Total F (24 h), sum of the fractions F1, F2, F3 and F4. *p < 0.05; **p < 0.01; ***p < 0.001.