SUBLINGUAL COMPOSITIONS COMPRISING NATURAL EXTRACTS AND USES THEREOF

Abstract

The present invention relates to a sublingual composition comprising a part of branch extract, preferably from at least one resinous tree comprising at least about 50% by weight of polyphenols and less than about 10% by weight of terpenes, relative to the total weight of the extract. The present invention further relates to the non-therapeutic uses of such sublingual composition for improving the respiratory wellness, relieving joint pain and/or muscular pain and cramps and/or improving endurance, and improving libido and/or fertility.

Claims

1-13. (canceled)

14. A sublingual composition comprising at least about 10% by weight of a part of branch extract, relative to the total weight of the composition, wherein said part of branch extract comprises at least about 50% by weight of polyphenols and less than about 10% by weight of terpenes, relative to the total weight of the extract, and wherein polyphenols comprise: from about 25% to about 50% by weight of hydroxymatairesinol, from about 5% to about 15% by weight of lariciresinol, and from about 5% to about 15% by weight of secoisolariciresinol, relative to the total weight of the extract.

15. The sublingual composition according to claim 14, wherein said part of branch extract comprises at least about 70% by weight of polyphenols, relative to the total weight of the extract.

16. The sublingual composition according to claim 14, wherein said part of branch extract comprises at least about 90% by weight of polyphenols, relative to the total weight of the extract.

17. The sublingual composition according to claim 14, wherein the part of branch extract is selected from the group consisting of Abies alba, Picea abies, Betula pendula, Pinus sylvestris, Abies sibirica, Pinus sibirica, Abies balsamea, Thuja occidentalis, Northern white-cedar, Pinus cembra, Pseudotsuga menziesii, Larix decidua, Picea glauca, Picea mariana, Picea pingens, Abies pectinata, Abies lasiocarpa, Pinus banksiana, Pinus nigra, Pinus pinaster, Pinus resinosa, Larix lariciana, Larix sibirica, Thuja plicata, Fagus Sylvatica, Populus alba, Populus nigra, Populus tremula, Populus tremulus, Tsuga hetewphylla, Pinus contorta, Juniperus virginiana, and mixtures thereof.

18. The sublingual composition according to claim 14, wherein the part of branch extract is selected from the group consisting of Abies alba, Picea abies and a mixture thereof.

19. The sublingual composition according to claim 14, wherein said composition comprises from about 10% to about 50% by weight, of said extract, relative to the total weight of the composition.

20. The sublingual composition according to claim 14, wherein said composition further comprises thickeners, sweeteners, disintegrating agents, dispersing agents, lubricants, and/or flow agents.

21. The sublingual composition according to claim 14, wherein said composition comprises: from about 10% to about 50%, by weight of said extract; from about 40% to about 90%, by weight of microcrystalline cellulose; from about 0.01% to about 2%, by weight of reticulated sodium carboxymethyl-cellulose; and from about 0.01% to about 2%, by weight of magnesium stearate; relative to the total weight of the composition.

22. The sublingual composition according to claim 14, wherein said composition further comprises at least one active ingredient for respiratory wellness and/or at least one dietary supplement.

23. The sublingual composition according to claim 14, wherein said composition further comprises at least one active ingredient for respiratory wellness selected from the group consisting of an extract from Abies, Abies siribica, Acerola, Achellea millefolium, Allium ursinum, Althaea officinalis, Andrographis paniculata, anise, apple, Artemisia cina, Arum korolkowii, Asperula humifusa, Astragalus, Avenca, Aalsam peru, Aiebersteinia multifidi, bishop's weed, black spruce, black seed, blueberry, borage, Brazilian peppertree, butterbur, Camellia sinensis, carapia, catnip, caraway, Cinnamomum verum, Cinnamomum zeylanicum, citrus, Citrus paradisi, Citrus sinenseschamomile, chaparral, chlorella, Coleus forskohlii, coltsfoot, copaiba, Curcuma longa, Cymbopognon citratus, Cychopelarrium intybus, dandelion, danshen, Drosera, echinacea, embauba, English ivy, eucalyptus, Cucapyptus radiate, Espinheira, Evening primrose, Filipendula ulmaria, fir, forsythia, Fucus vesiculosus, garlic, gelsemium, Genipa americana, Gotu kola, ginkgo, ginseng, great plantain, grindelia, Guaco, mikania glomerata spreng, guandu, hawthorn, Hedera helix, horseradish, horsetail, Indian frankincense, juazeiro, Juniperus seravschanica, Justicia paniculata, kalanchoe, khella, kiwi, larch, lavender, lavandula, lobelia, Malpighia glabra, Melaleuca quinquenervia, Malva neglecta, Origanum majorana, Marrubium vulgare, moringa, myrtus, nettle, Nigella sativa, Origanum vulgare, Panax ginseng, pau d'arco, pelargonium, melaleuca, mentha, Mentha piperita, Origanum majorana, Pelargonium sidoides, peppermint, Perilla, picrorhiza, pine, piper, Phyllanthus emblica, Plantago major, plantain, Plectranthus barbatus, Primula veris, quillaja, red clover, rosemary, rosmarinus, Salvia officinalis, samambaia, schisandra, Siraitia grosvenorii, Soia isoflavoni, Sophora japonica, sorrel, soy, Spirulina platensis, sweet orange, Syzygium aromaticum, Taraxacum officinale, Terminalia chebula, Terminalia belerica, Thymus serpyllum, Thymus vulgaris, tomato, Tussilgo frfara, vassourinha, Verbascum thapsus, Viburnum lantana, white horehound, and Yerba santa, alpha-linolenic acid, alpha-lipoic acid, arabinogalactan, arginine, balsam tolu, beta-glucan, bifidobacteria, brewer's yeast, bromelain, caffeine, choline, coenzyme Q-10, cordyceps, eicosapentaenoic acid (EPA), forskolin, fish oil, gamma-aminobutyric acid (GABA), glutathione, green mold, green-lipped mussel, inositol, lactobacillus, linoleic acid, lutein, lycopene, matico, N-acetylcysteine (NAC), pyridoxine, propolis, pycnogenol, quail egg, quercetin, resveratrol, saffron, selenium, serrapeptase, shark liver, umckaloabo, whey protein, yogurt, and zeaxanthin.

24. The sublingual composition according to claim 14, wherein said composition further comprises at least one dietary supplement selected from the group consisting of ascorbic acid, copper, calcium, magnesium, zinc, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B8, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, iron, iodine, manganese, and molybdenum.

25. A method for improving the respiratory wellness in a subject, comprising administering a sublingual composition as defined in claim 14 to said subject.

26. A method for relieving joint pain and/or muscular pain and cramps and/or improving endurance in a subject, comprising administering a sublingual composition as defined in claim 14 to said subject.

27. A method for improving libido and/or fertility in a subject, comprising administering a sublingual composition as defined in claim 14 to said subject.

28. A kit comprising a sublingual composition as defined in claim 14 and a package leaflet or user instructions including the information that said composition is to be used for the respiratory wellness, and/or for joint pain and/or muscular pain and cramps and/or endurance, and/or libido and/or fertility.

Description

FIGURES

[0118] FIG. 1: Evaluation of the anti-radical activity of the extract of the invention (natural extract), hydroxymatairesinol (HMR), enterodiol, enterolactone, and pycnogenol by DPPH method.

[0119] FIG. 2: Evaluation of the antioxidant activity of the extract of the invention (natural extract) and its main metabolites (enterodiol and enterolactone), expressed as equivalent quantity of trolox (square-patterned) and vitamin E (dotted line).

[0120] FIG. 3: Evaluation of the total antioxidant power of the extract of the invention (natural extract), pycnogenol, and hydroxymatairesinol (HMR), expressed as millimole of global antioxidant per gram of product, by Technology PAOT/POT.

[0121] FIG. 4: Evaluation of the total antioxidant efficacy of the extract of the invention (natural extract) and its main metabolites (enterodiol and enterolactone), expressed as millimole of global antioxidant per gram of product, by technology PAOT/POT.

[0122] FIG. 5: Diagram of manufacturing process for the tablets containing vegetable powder.

[0123] FIG. 6A: Evaluation of the anti-inflammatory activity of the extract of the invention by measuring the inhibitory effect of HMR (representative polyphenol compound of the extract of the invention) at 10, 30, and 100 M towards TNF- and IL-1 cytokines.

[0124] FIGS. 6B and 6C: Evaluation of the anti-inflammatory activity of the extract of the invention by measuring the inhibitory effect of HMR (representative polyphenol compound of the extract of the invention) at 10, 30, and 100 M towards TNF- (FIG. 6B) and IL-1 (FIG. 6C) cytokines after 5 g/mL LPS induction.

[0125] FIG. 7: Evaluation of the anti-inflammatory activity of the extract of the invention by measuring the inhibitory effect of the extract at 10 M and HMR at 10 M towards 5-LO and COX-2.

[0126] Further aspects and advantages of the invention will be disclosed in the following experimental section.

EXAMPLES

Example 1: Sublingual Compositions

[0127] 1. Extraction Process of the Part of Branch Extracts.

[0128] 54 kg of natural crude material made of Picea abies and Abies alba part of branch was placed in an extractor (e.g. filtering bottom extractor Lavergne type equipped with a nylon membrane) and was extracted using 15% ethanol in water (v/v) as a solvent with a ratio material/solvent of 1/10 at 40 C. for 24 hours. Separation liquid/solid was obtained by use of a spin dryer. Solid extract was placed in said extractor and was extracted using 15% ethanol in water (v/v) as a solvent with a ratio material/solvent of 1/10 at 40 C. for 1 hour. Separation liquid/solid was obtained by use of a spin dryer. The two liquid extracts were combined and concentration of liquid extract was performed under vacuum at 60 C. with an evaporator (e.g. falling flow Wiegand type). Drying of the extracts was performed by atomization using a stainless steel spray dryer (e.g. atomization tower APV PSD 52 type) to give 3.88 kg of dry extract.

[0129] 2. Process for the Preparation of Compositions of the Invention

[0130] The equipment used for the manufacturing of the tablets may comprise a Turbula mixer, a tableting machine Frogerais equipped with convex punches 10 mms in diameter, a balance of precision Ohaus. The tableting is carried out by direct compression of the mixture of various excipients and plant extract.

[0131] As shown in FIG. 5, the diagram of manufacturing of the composition B comprises a stage of weighing of the ingredients, a first stage of mixture followed by two successive mixtures, this one being followed with the additions of magnesium stearate and colloidal silica. Various controls carried out relate to the homogeneity of the mixture of powders, the weight of the tablets obtained, the tableting of the powder without the vegetable powder, and the angle of Haussner.

[0132] More specifically, the first mixing has a duration of 30 minutes. The 2.sup.nd and 3.sup.rd mixings have one duration respectively of 3 minutes. Industrially, it is possible that it is necessary to conceive a stage of sifting at the end of the various stages of mixtures.

[0133] The composition A is obtained according the process as illustrated in FIG. 5 using particular ingredients of the composition A.

[0134] 3. Sublingual Formulations

[0135] 3.1 Sublingual Composition A

TABLE-US-00001 Part of branch extract from Abies alba and Picea abies 50-150 mg Microcrystalline cellulose 230 mg Reticulated sodium carboxymethyl-cellulose 4 mg Magnesium stearate 3 mg Talc 3 mg Polyoxyethyleneglycol distearate 50 mg

[0136] 3.2 Sublingual Composition B

TABLE-US-00002 Part of branch extract from Abies alba and Picea abies 50 mg Microcrystalline cellulose 166.70 mg Reticulated sodium carboxymethyl-cellulose 3.33 mg Magnesium stearate 4.36 mg Fructose 26.70 mg Corn starch 16.70 mg Colloidal silica dioxide 2.70 mg

Example 2: Evaluation of Antioxidant and Antiradical Activities of the Part of Branch Extracts of the Invention

[0137] 2.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) Assay

[0138] (Burda and Oleszek: Antioxidant and anti-radical activities of flavonoids, J. Agric. Food Chem., 2001, 49, 2774-2779).

[0139] To study the anti-radical activity of the extract, DPPH test has been used. DPPH free radical has a violet color which turns to yellow when it is reduced in the presence of free-radical compounds. The color intensity, measured with the spectrophotometer, is inversely proportional to the anti-radical activity of the tested compounds. Different dilutions of honeybee samples dissolved in methanol were added to 3.9 ml of a 6.110.sup.5 M DPPH methanolic solution. The exact initial DPPH concentration (C DPPH) in the reaction medium was calculated by a calibration curve. The bleaching of DPPH was monitored at 517 nm (Spectrophotometer) for 30 min against a blank constituted by the sample containing all reagents except DPPH. The scavenging activity of the samples on the DPPH was expressed as IC50 (mg/ml) and was extrapolated from a dose-response curve. The assays were performed in quadruplicate (n=4) with the extract of the invention, hydroxymatairesinol (HMR), enterodiol, enterolactone, and Pycnogenol (French maritime pine bark extract, reference antioxidant compound). The anti-radical activity is expressed as a value of EC50 (the smaller EC50 value corresponds to the stronger antioxidant activity).

[0140] As displayed in FIG. 1, the antioxidant activity of the part of branch extract of the invention is better than HMR, enterodiol, enterolactone, and pycnogenol. Indeed, the part of branch extract of the invention surprisingly shows an EC50 (Effective Concentration to reduce 50% of DPPH radicals) of 0.13 mg/ml which is smaller than EC50 of HMR (EC50=0.20 mg/ml) and pycnogenol (EC50=0.21 mg/ml).

[0141] In addition, the extract of the invention shows an EC50 of 0.13 mg/ml which is smaller than EC50 of its metabolitesi.e. enterodiol (EC50=0.28 mg/ml) and enterolactone (EC50=0.30 mg/ml).

2.2. Trolox Equivalent Antioxidant Capacity (TEAC) Assay

[0142] (Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C.: Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radical Biology & Medicine, 1999, 26 (9), 1231-1237).

[0143] The total antioxidant activity of the extract of the invention derives from its ability to inhibit the radical ABTS+, obtained from ABTS (ammonium salt of 2, 2-azinobis (3-ethylbenzothiazoline-acid 6-sulfonic) acid) compared to a reference antioxidant compound (Pycnogenol, -tocopherol, Trolox) and its metabolites (enterodiol and enterolactone). ABTS radical cation (ABTS+) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12-16 h before being used. Next ABTS+solution was diluted with PBS (Phosphate Buffer Saline) (pH 7.4) to an absorbance of 0.5 at 734 nm and equilibrated at 30 C. 200 l of ABTS+ and (10, 20, 35, 50, 70, 80 l) of product solutions (0.5 mg/L) was mixed and adjusted to 300 l with buffer solution. After 15 min incubation, the optical density was measured. The assays were performed in quadruplicate (n=4). Efficacy is expressed as a value of EC50 (the smaller value of EC50 corresponds to the stronger antioxidant activity). The natural extract of the invention surprisingly shows the smallest EC50 (Effective Concentration to remove 50% of ABTS at equilibrium) with a value of 0.020 mg/mL in that assay as displayed in Table 1.

TABLE-US-00003 TABLE 1 EC50 values obtained in the TEAC assay trolox entero- entero- pycno- -tocopherol (vit. E Sample Extract diol lactone genol (vit. E) like) EC50 0.02 0.14 0.17 0.030 0.031 0.022 (mg/ml)

[0144] In addition, FIG. 2 emphasizes the striking discrepancy in antioxidant efficacy between the part of branch extract and its main metabolites. Indeed, antioxidant activity of 1 g of the natural extract is equivalent to 1.07 g of trolox (square-patterned) and 1.53 g of vitamin E (dotted line) whereas antioxidant activity of 1 g of enterolactone is equivalent to 0.13 g of trolox (square-patterned) and 0.19 g of vitamin E (dotted line), and antioxidant activity of 1 g of enterodiol is equivalent to 0.15 g of trolox (square-patterned) and 0.22 g of vitamin E (dotted line).

2.3. Oxygen Radical Absorbance Capacity (ORAC) Assay

[0145] (Ou B., Hampsch-Woodill M. and Prior R. L.: Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe, J. Agric. Food Chem., 2001, 49 (10), 4619-4626)

[0146] Measurement of antioxidant capacity by the ORAC method is based on the detection of fluorescence drop of fluorescein (FL) thanks to its reaction with peroxyl radical ROO*, in a food matrix containing antioxidant compounds. Fluorescence was read with an excitation wavelength of 485 nm and an emission filter of 528 nm. AAPH solution and FL were prepared in a phosphate buffer solution at 75 mM at pH 7.4. The control of this reaction is the trolox which is also prepared in the same buffer. Six different quantities (10, 20, 35, 50, 70, 80 l) of product with concentration of 0.5 mg/L were placed in the wells of the microplate with FL and adjusted at the same volume with buffer solution (80 l). The mixture was preincubated for 30 min at 37 C., before rapidly adding the AAPH solution (220 l) using a multichannel pipette. The microplate was immediately placed in the reader and the fluorescence recorded every 6 min for 240 min. A blank with FL and AAPH using sodium phosphate buffer instead of the antioxidant solution was used. The inhibition capacity was expressed as reference compound equivalents (M), and is quantified by integrating of the area under the curve (AUC) of the fluorescein. The assays were carried out in quadruplicate (n=4). Efficacy of the tested samples is expressed as reference ORAC units per gram of products (the higher value corresponds to the stronger antioxidant activity). Results are illustrated in Table 2.

TABLE-US-00004 TABLE 2 Antioxidant activity of the part of branch extract of the invention, pycnogenol, HMR, enterodiol, and enterolactone. Values are expressed as equivalent units (mol of reference compound per gram of tested product). Extract pycnogenol HMR enterodiol enterolactone mol Trolox/g 2400 500 1270 60 210 50 500 60 560 50 tested product mol Vit. C/g 1300 200 670 90 110 10 700 100 110 10 tested product mol Vit. E/g 1200 170 630 70 105 10 600 80 105 10 tested product mol - 310 20 165 5 27 2 165 5 30 2 carotene/g tested product

[0147] The results show that the antioxidant activity of 1 gram of the part of branch extract is respectively equivalent to the total antioxidant power of 2400 mol of trolox, 1300 mol of vitamin C, 1200 mol of vit. E (tocopherol), and 310 mol of -carotene. Surprisingly the antioxidant activity of the part of branch extract is largely greater than the antioxidant activity of 1 g of pycnogenol, 1 g of hydroxymatairesinol (HMR) and 1 g of the metabolites of the natural extract (enterodiol and enterolactone).

2.4. Total AntiOxidant Power (PAOT) Assay (Oxidative Stress)

[0148] Technology PAOT/POT (Total AntiOxidant Power/Total Oxidant Power) is based on the electrochemical nature of redox reactions: a balance between antioxidants and oxidants [www.ie-antioxydants.com]. The assays were carried out in quadruplicate (n=4) (The higher value corresponds to the best antioxidant efficacy).

[0149] As displayed in FIG. 3, the overall antioxidant efficacy of the natural extract (10.31 mmol/g equivalent of antioxidant) of the invention is surprisingly better than pycnogenol (8.82 mmol/g equivalent of antioxidant), and HMR (4.99 mmol/g equivalent of antioxidant).

[0150] Table 3 below summarizes values of antioxidant activity of the natural extract, pycnogenol, and HMR with regard to reference antioxidant compounds i.e. vitamin E (tocopherol), trolox, and -carotene. Surprisingly, the part of branch extract shows the best antioxidant activity among the tested products. Actually, 1 gram of part of branch extract of the invention is equivalent to the antioxidant efficacy of 2.2 g of vitamin E, 1.29 g of trolox, and 5.53 g of -carotene.

TABLE-US-00005 TABLE 3 Antioxidant activities with regard to reference compounds. Part of branch extract pycnogenol HMR Vitamin E* 2.22 1.90 1.07 Trolox* 1.29 1.10 0.62 -carotene* 5.53 4.74 2.68 *gram of reference compound per gram of tested product

[0151] As illustrated in FIG. 4, the discrepancy between efficacy of the part of branch extract of the invention and its main metabolites in that assay, expressed as millimole of global antioxidant per gram of product (measure of the global antioxidant feature of tested products; the higher value corresponds to the best antioxidant efficacy).

[0152] Indeed, the inventors have surprisingly shown that the total antioxidant efficacy of the part of branch extract (10.31 mmol/g equivalent of antioxidant) was at least ten times greater than the antioxidant efficacy of its metabolites (enterodiol (1.07 mmol/g equivalent of antioxidant) and enterolactone (1.06 mmol/g equivalent of antioxidant)).

2.5. Conclusions

[0153] The inventors have surprisingly shown that the natural part of branch extract according to the invention had a greater antioxidant activity compared to reference compounds as pycnogenol, -tocopherol (vit. E), and trolox (vit. E like), thereby demonstrating an improved efficacy for respiratory wellness, for relieving joint pain and/or muscular pain and cramps and/or for improving endurance, and for improving libido and/or fertility. The inventors have also surprisingly shown that the natural part of branch extract according to the invention had a greater antioxidant activity compared to its main metabolites (enterolactone and enterodiol), thereby demonstrating an improved efficacy when the composition is administered by a route avoiding metabolization such as sublingual route.

Example 3: Evaluation of Anti-Inflammatory Activity of HMR (Representative Polyphenol Compound of the Part of Branch Extracts of the Invention)

[0154] Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mediate the development of numerous inflammatory diseases. IL-1beta and TNF-alpha therefore constitute suitable biomarkers to characterize the anti-inflammatory property of compounds of the part of branch extracts of the invention.

[0155] Peripheral blood mononuclear cells (PBMCs) were prepared from the peripheral blood of healthy donors (Etablissement Franais du Sang) according to Kmmerle et al. (J Med Chem. 2012, 55, 7525-7545) with slight modifications. PBMCs were isolated using Histopaque gradient (Sigma), washed in Hanks' balanced salt solution (Sigma) and were then cultured in RPMI-1640 medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (PAA), 100 U/mL penicillin and 100 g/mL streptomycin (PAA). PBMCs were seeded into 96-well plates (8104 cells/well) and stimulated with 5 g/mL LPS (Lipopolysaccharides, Sigma), into a total volume of 120 L per well. HMR was re-suspended in DMSO and tested at 10, 30, and 100 M (DMSO 1%) in duplicate. Cells without LPS stimulation were considered as bio-inactive control (basal level of cytokine). Cells incubation was realized at 37 C. (5% CO.sub.2).

[0156] After 24 h, PBMCs supernatants were transferred into a 384-well plate. Cytokine detection was performed using HTRF technology (Cisbio bioassays) for TNF- (62TNFPEC), IL-1 (62IL1PEC) according to supplier recommendations. Reading was performed after 3 h of incubation using Envision multi-labelled reader (PerkinElmer) using supplier recommended parameters.

[0157] The results are represented in the table 4 below and in FIGS. 6A, 6B, and 6C.

TABLE-US-00006 TABLE 4 % inhibition TNF- and IL-1 with HMR at 10, 30, and 100 M HMR % inhibition TNF- % inhibition IL-1 concentration (Standard deviation) (Standard deviation) 10 M 7% (8%) 10% (3%) 30 M 15% (0%) 25% (0%) 100 M 26% (5%) 28% (10%)

[0158] The results show the inhibitory effect of HMR against IL-1beta and TNF-alpha, thereby demonstrating an anti-inflammatory effect for the natural part of branch extract and sublingual compositions comprising such extract. Also, FIGS. 6B and 6C particularly show a dose-dependent inhibitory effect for HMR against TNF-alpha and IL-1beta, respectively.

Example 4: Evaluation of Anti-Inflammatory Activity of HMR and a Part of Branch Extract of the Invention

[0159] Prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2; COX-2) and arachidonate 5-lipoxygenase (5-lipoxygenase; 5-LOX; 5-LO) are well known enzymes involved in inflammation.

Human COX-2 Inhibition Assay:

[0160] The test compound, reference compound or water (control) were pre-incubated for 20 min at room temperature with the enzyme (0.2 g) in a buffer containing 90 mM Tris-HCl (pH 8.0), 1.98 mM phenol and 1.02 M hematine. Thereafter, the reaction was initiated by adding 2 M arachidonic acid and the mixture was incubated for 5 min at room temperature. For basal control measurements, arachidonic acid was omitted from the reaction mixture. Following incubation, the reaction was stopped by the addition of 1 M HCl then 1 M Tris/HCl (pH 8.0) followed by cooling to 4 C. The fluorescence acceptor (d2 labeled PGE2) and the fluorescence donor (anti-PGE2 antibody labeled with europium Cryptate) were then added. After 120 min, the fluorescence transfer corresponding to the amount of residual PGE2 was measured at ex=337 nm, em=620 nm and em=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio).

[0161] The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is NS398, which was tested in each experiment at several concentrations to obtain an inhibition curve from which its IC50 value was calculated.

REFERENCE

[0162] Glaser, K., Sung, M. L., O'neill, K., Belfast, M., Hartman, D., Carlson, R., Kreft, A., Kubrak, D., Hsiao, C. L. and Weichman, B. (1995), Etodolac selectively inhibits human prostaglandin G/H synthase 2 (PGHS-2) versus human PGHS-1. Eur. J. Pharmacol., 281: 107-111.

Human 5-LO Inhibition Assay:

[0163] The test compound, reference compound or water (control) were preincubated for 5 min at room temperature with 10.sup.4 lysed cells/well in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM CaCl.sub.2, 2 mM EDTA, 1 M ATP and 50 nM fluorescent probe dihydrorhodamine 123. The fluorescence intensity was then measured at ex=485 nm and em=535 nm using a microplate reader (Envision, Perkin Elmer). This measurement at t=0 allowed the detection of any compound interference with the fluorimetric detection method at these wavelengths. Thereafter, the reaction was initiated by adding 25 M of the substrate arachidonic acid and the mixture was incubated for 20 min at room temperature. The reaction was stopped by adding a buffer containing 67% acetonitrile and 0.2% acetic acid and the fluorescence intensity emitted by the reaction product rhodamine 123 was measured at the same wavelengths (t=20). The enzyme activity was determined by subtracting the signal measured at t=0 from that measured at t=20.

[0164] The results are expressed as a percent inhibition of the control activity. The standard inhibitory reference compound is NDGA, which was tested in each experiment at several concentrations to obtain an inhibition curve from which its IC.sub.50 value was calculated.

REFERENCE

[0165] Pufahl, R. A., Kasten, T. P., Hills, R., Gierse, J. K., Reitz, B. A., Weinberg, R. A. and Masferrer, J. L. (2007). Development of a fluorescence-based enzyme assay of human 5-lipoxygenase. Anal. Biochem., 364: 204.

[0166] The results are presented in the table 5 below and in FIG. 7.

TABLE-US-00007 TABLE 5 % inhibition of 5-LO and COX-2 with HMR (10 M) and a part of branch extract (equiv. 10 M) Biomolecular target % Inhibition (reference compound) HMR Part of branch extract 5-LO (NDGA; 14.7 25.2 EC50 = 270 nM) COX-2 (NS398; 18.7 9.6 EC50 = 99 nM)

[0167] The results show the inhibitory effect of HMR and a part of branch extract against 5-LO and COX-2, thereby demonstrating an anti-inflammatory effect for the natural part of branch extract and sublingual compositions comprising such extract.

CONCLUSIONS

[0168] The inventors have surprisingly shown that sublingual compositions according to the invention, in which polyphenols are not metabolized, had a greater antioxidant activity, while maintaining an anti-inflammatory activity. Such sublingual compositions exhibiting such double activity are thus suitable for improving respiratory wellness, for relieving joint pain and/or muscular pain and cramps, and/or for improving endurance, and for improving libido and/or fertility.