Plant extracts with extremely high concentrations of saffronal, methods for obtaining them and their uses

Abstract

The object of the present invention is a plant extract containing safranal in a concentration of at least 0.2% by weight relative to the total weight of the dry material, as determined by HPLC. The object of the present invention is also a method for obtaining such an extract, a composition containing such an extract and its uses. [Selected Figure] Figure 1

Description

[Technical field]

[0001] The present invention relates to a novel extract obtained from saffron-containing plant material, in particular saffron, which has a higher safranal concentration than currently known plant extracts.

[0002] The present invention also aims at a unique process allowing the obtaining of such an extract, as well as at compositions containing such an extract and their uses. [Background technology]

[0003] Safranal can be extracted from several plants, including saffron (Crocus sativus), Centaurea sibthorpii, Centaurea consanguinea, Centaurea amanicola, Erodium cicutarium, Chinese tea, Calycopteris floribunda, Crocus heuffelianus, Sambucus nigra, Gardenia jasminoides, lemon (Citrus limon), cumin (Cuminum cyminum L.), and Achillea distans, but is most commonly extracted from Crocus sativus, also known as saffron.

[0004] Saffron is a traditional flavouring that is primarily grown in Iran and is used in many areas, including mood disorders, premenstrual syndrome, erectile dysfunction and even skin cosmetics, as presented in Javadi B et al., "A survey on saffron in major islamic traditional medicine books," Iranian journal of basic medical sciences 2013;16(1):1-11. Several clinical studies have been carried out to determine the efficacy of saffron in alleviating depressive and anxiety disorders, as detailed for example in Akhondzadeh S et al., "Comparison of Crocus sativus L. and imipramine in the treatment of mild to moderate depression: a pilot double-blind randomized trial" [ISRCTN45683816]. BMC Complement Altern Med 2004;4:12, or more recently in Hausenblas HA et al., "Saffron (Crocus sativus L.) and major depressive disorder: a meta-analysis of randomized clinical trials", Journal of integrative medicine 2013;11(6):377-83.

[0005] Saffron (Crocus sativus) contains several molecules, including safranal, a volatile molecule responsible for the flavoring aroma, whose efficacy in the above-mentioned fields of application is recognized. The extracts used in the prior art are mostly standardized to 2% safranal by ultraviolet spectroscopy according to ISO standard 3632-2:2010 (Spices-Saffron (Crocus sativus L.)-Part 2: Test methods ISO International standard; 2010: 1-42). This standard describes a very comprehensive analytical protocol that consists of dissolving the saffron extract in water, stirring, filtering, and then taking a spectrophotometric reading at 330 nm. However, this method is highly criticized because no specific solvents and / or reagents are used, which leads to the quantification of molecules other than safranal, and therefore does not at all reflect the actual safranal concentration of the extract.

[0006] In fact, analysis of saffranal in extracts of the prior art by HPLC (also called CLHP or high performance liquid chromatography) shows actual concentrations that are 30 to 1000 times lower than those obtained by analysis by ultraviolet spectrometry according to ISO standard 3632-2, and the correlation between the two methods varies greatly, as the results in Table 1 below show.

[0007] [Table 1]

[0008] In fact, known safranal-containing plant extracts, and in particular saffron extracts, obtained by the methods of the prior art have a very low safranal content. The measurement method is therefore important, since it leads to results that differ from those obtained by previous measurement methods by spectroscopic measurements that are systematically used to determine the safranal concentration in a product. This previous method, reference number ISO 3632-2:2010, overestimates the safranal content, as is clearly stated in the present application as well as in the publication Garcia-Rodriguez et al., 2017 "Comparative evaluation of an ISO 3632 method and an HPLC-DAD method for safranal quantity determination in saffron". In this publication, 390 saffron samples were studied. The overestimation can be 20-50 times. Thus, an extract with a safranal titer of X% according to the measurement method reference number ISO 3632-2:2010 actually contains much less safranal. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] ISO Standard 3632-2:2010, “Spices-Saffron(Crocus sativus L.)-Part2:Test methods”, ISO International standard;2010:1~42 [Non-Patent Document 2] Garcia-Rodriguez et al, “Comparative evaluation of an ISO 3632 method and an HPLC-DAD method for safranal quantity determination in saffron”, 2017 Summary of the Invention [Problem to be solved by the invention]

[0010] The aim of the present invention is to address these problems by proposing a saffron extract which has a higher content of safranal than existing extracts. [Means for solving the problem]

[0011] In particular, the present invention is directed to a plant extract obtained from saffron, containing safranal in a concentration of at least 0.2% by weight relative to the total weight of the dry material of the extract, as determined by HPLC methods.

[0012] The extract is particularly prepared by a process that includes heat treatment, in particular - optionally drying the raw materials; - grinding the dried raw material; - extraction with water or a hydroalcoholic or organic solvent, - impregnating a carrier with the extract obtained; - heat treating the extract; The method can be obtained by a specific method including carrying out the steps of: Effect of the Invention

[0013] This method is economical, easy to carry out and makes it possible to obtain a saffron extract containing a safranal concentration of at least 0.2% by weight relative to the total weight of the dry material of the extract, as determined by the HPLC method.

[0014] Advantageously, such extracts contain a higher actual concentration of saffron than those of currently known extracts and thus have greater efficacy, in particular for the treatment of depression, anxiety, mood disorders, erectile dysfunction and premenstrual disorders.The present invention is therefore also directed to saffron extracts for these uses, as well as cosmetic, food, nutritional or pharmaceutical compositions comprising said extracts.

[0015] Other features and advantages will become apparent from the following detailed description of the invention, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1] 1 is a chromatogram of the extract of the present invention in Example 1 obtained by UHPLC method. [Diagram 2] Chromatogram of the extract of the present invention in Example 2 obtained by UHPLC method. [Figure 3A] FIG. 2 is a chromatogram of the prior art extract according to Example 3A (obtained without heat treatment) obtained by UHPLC method. [Figure 3B] FIG. 2 is a chromatogram of the prior art extract according to Example 3B (obtained without heat treatment) obtained by UHPLC method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The object of the present invention is to provide a plant extract obtained from a safranal-containing plant (or plant material), characterized in that it contains safranal in a concentration of at least 0.2% by weight relative to the total weight of the dry material, as determined by the HPLC method.

[0018] "Plant extract obtained from a plant containing saffron" or "plant extract obtained from a botanical raw material containing saffron" means at least one molecule or a collection of molecules derived from the whole or part of a plant containing saffron. This may be a specific selection of natural molecules present in the plant or molecules obtained by any type of transformation from said natural molecules. The raw material used to obtain the extract may consist of the whole or part of a plant containing saffron. If the plant containing saffron is saffron, the plant extract according to the invention may be obtained in particular from the stigmas and / or petals and / or bulbs of saffron.

[0019] The extract according to the present invention is not a material per se, it is not a plant, plant part, dried plant, dried plant part or plant sample or dried plant sample, and it is not an essential oil.

[0020] "Plants containing saffron" or "materials containing saffron" (within the context of the present invention the terms plants and plant materials may be used indiscriminately) refers to any plant containing saffron, in particular Crocus sativus, Centaurea sibthorpii, Centaurea consanguinea, Centaurea amanicola, Erodium cicutarium, Chinese tea, Calycopteris floribunda, Crocus heuffelianus, Sambucus nigra, Gardenia jasminoides, Citrus limon, Cuminum cyminum L., Achillea distans, and the like. Preferably, the saffron-containing plant providing the extract according to the invention is saffron (Crocus sativus).

[0021] The extract according to the invention contains at least safranal, which is present in a concentration of at least 0.2% by weight of the dry material as determined by the HPLC (High Performance Liquid Chromatography) method, which is of great importance since the results obtained by other methods, in particular ultraviolet spectrometry (ISO standard 3632-2), do not correspond to the actual concentration of safranal, due to the non-specific nature of this method.

[0022] The method of molecular analysis by HPLC is known to those skilled in the art and allows precise identification and quantification of the individual molecules.

[0023] Preferably, the analytical method used to quantify the molecules contained in the extract of the present invention, in particular safranal, is a UHPLC (Ultra High Performance Liquid Chromatography) method, which further increases the resolution and separation of compounds and allows the detection of multiple compounds on the same chromatogram from a single sample.

[0024] According to one highly adaptable embodiment, the analytical method HPLC or UHPLC comprises a preliminary sample preparation step comprising the steps of: - introducing a weighed amount of saffron extract into a hydroalcoholic solution, - magnetic stirring for at least 1 hour; - performing an ultrasonic bath for at least 5 minutes; - Membrane filtration followed by injection.

[0025] Post-preparative analytical methods for samples traditionally include a preparation step followed by elution and then detection.

[0026] Elution is preferably performed using a binary gradient. The first solvent may be, for example, an organic solvent and an acid. Preferably, it is formic acid in acetonitrile. The second solvent may be, for example, an acidified water solvent. Preferably, it is formic acid in water.

[0027] In addition to saffronal, the extract according to the invention may contain other molecules, in particular derivatives of crocin and / or flavonoids and / or picrocrocin derived from kaempferol, especially in the case of saffron extract.

[0028] If crocin is present it preferably represents at least 1% by weight of the dry material of the extract as measured by HPLC methods.

[0029] If present, the flavonoid derived from kaempferol preferably represents at least 500 ppm by weight of the dry material of the extract as determined by HPLC methods.

[0030] If picrocrocin is present, it preferably represents at least 0.5% by weight of the dry material of the extract as determined by HPLC methods.

[0031] The extract according to the invention is preferably impregnated in a carrier. The term "carrier" or "filler" in the sense of the present invention means any food substance of vegetable, mineral or chemical origin that is used as a food ingredient or additive, allowing the impregnation and thus dilution of the extract according to the invention.

[0032] The carrier may in particular be chosen from the following constituents: maltodextrin, sugar, silica, gum arabic, preferably maltodextrin.

[0033] When the extract is impregnated with a carrier, the percentage of molecules present in the extract is given by weight of the dry material of the extract including the carrier.

[0034] The extract according to the invention can be obtained by any means making it possible to obtain at least 0.2% of safranal by weight of the dry material of said extract.Preferably, the extract according to the invention is obtained by a method including a heat treatment step, which can be carried out on the raw material at the beginning, during or at the end of the process for obtaining the extract.

[0035] Heat treatment in the sense of the present invention means heating at a temperature higher than ambient temperature. Preferably, the heat treatment step is a heat treatment carried out at a temperature between 30° C. and 95° C., preferably between 30° C. and 60° C., for at least 2 hours, preferably at least 24 hours.

[0036] The heat treatment can be carried out by any known means, in particular in an incubator, in an oven, by calcination, by pasteurization or by heat sterilization. Preferably, the heat treatment is carried out in an incubator.

[0037] According to one highly adaptable embodiment, the plant extract according to the invention comprises the implementation of the following steps, carried out from saffron raw material: - an optional drying step, - grinding, preferably to a size of 50 to 500 μm; - extraction with water or a hydroalcoholic or organic solvent, - impregnating a carrier with the extract obtained; - Heat treating step.

[0038] The steps of extraction with water or hydroalcoholic or organic solvents and impregnation of the carrier with the extract obtained constitute a known extraction method called Tech'Care Extraction®.

[0039] The heat treatment step can be performed on the saffron raw material at any point in the process, preferably at the end of the process, and complements the Tech'Care Extraction® process.

[0040] According to one highly adaptable embodiment, the heat treatment step in carrying out this method is a heat treatment step in an incubator at a temperature between 30°C and 95°C, even more preferably at a temperature between 30°C and 60°C, for at least 2 hours, even more preferably at least 24 hours.

[0041] The grinding may be carried out by any known adapted means, in particular by means of a knife mill, a spindle mill or a hammer mill, preferably by means of a spindle mill.

[0042] The extraction step may be carried out by any known adapted means.

[0043] In the case of water extraction, the ground material is introduced into water at a rate of 50 g / L.

[0044] In the case of hydroalcoholic extraction, the solvent may in particular be ethanol, preferably 60% v / v ethanol. The ground material is introduced into the hydroalcoholic solution in a proportion of 50 g / L.

[0045] In the case of extraction with an organic solvent, the solvent may in particular be methanol or ethyl acetate, preferably 30% v / v methanol. The ground material is introduced into the organic solvent at a rate of 100 g / L.

[0046] After extraction, the method can also include an acidification step, which consists of adding an acid to the aqueous or hydroalcoholic solvent, thereby lowering the pH of the extraction solution to between 3 and 5. This step can be carried out in particular under conditions of adding citric acid or hydrochloric acid to the aqueous alcoholic solvent to adjust the pH to 4.

[0047] The carrier impregnation step consists of adding to the extraction solution a filler, the carrier or filler being chosen in particular from the following components: maltodextrin, sugar, silica, gum arabic, preferably maltodextrin.

[0048] After this impregnation step, the method may also include an emulsification and / or encapsulation step of the extract obtained, which consists of high-speed stirring of the extract solution containing the fillers and possibly auxiliary substances, which may be carried out in particular with auxiliary substances such as gum arabic, cyclodextrin or lipids.

[0049] The extract according to the invention may be incorporated into a cosmetic, food, nutritional or pharmaceutical composition or may be used alone, in a proportion of 0.1 to 100% by weight of the dry material of the composition.Preferably, the extract according to the invention is present in the composition in a quantity that allows the administration to a human or animal of at least 0.07 mg of the extract according to the invention per kg of body weight per day, and even more preferably 1.4 to 4.2 mg of the extract according to the invention per kg of body weight per day.

[0050] The present invention is therefore directed to such compositions, preferably in the form of gelatin capsules, compressed tablets, soft capsules, sticks, sachets, ready-to-eat meals, oils, lotions, creams and emulsions.

[0051] The composition containing the extract according to the present invention may contain other components as known in the art, such as excipients or other active ingredients or molecules selected according to the intended form and use of the composition.

[0052] The extract according to the present invention and compositions containing it can be used in many fields of application, in particular for preventing or treating depression and anxiety, as well as for preventing or treating mood disorders (pathological mood disorders), erectile dysfunction, and premenstrual disorders.

[0053] The present invention also provides - preventing or treating depression in humans or animals, - prevention or treatment of anxiety in humans or animals, - preventing or treating mood disorders in humans; - Prevention or treatment of erectile dysfunction in humans (males) - prevention or treatment of premenstrual disorders in humans (females), The invention is also directed to an extract for use in

[0054] Advantageously, the extract of the present invention shows high efficacy in these fields of application due to its high safranal content, which is higher than that of any other extracts currently available.Moreover, it is important to use the extract rather than the raw material (whole or partial plant in dried or wet state), because the extract of the present invention can increase the bioavailability of these active molecules, since they are no longer entangled in the floral plant matrix and are more accessible to the organism.The presence of a carrier in the extract can also obtain a better homogeneity of the beneficial molecules in the final product. EXAMPLES

[0055] The invention will now be illustrated by examples of extracts and methods according to the invention (Examples 1 and 2), comparative examples of prior art extracts (Examples 3A and 3B) and compositions.

[0056] For all examples, the method for quantifying the molecules in the extract, specifically safranal, is a UHPLC method with the following characteristics: 1. Sample Preparation Extraction of the sample with a hydroalcoholic solution. Magnetic stirring for at least 1 hour followed by an ultrasonic bath for at least 5 minutes. Membrane filtration followed by injection. 2. HPLC Elution Binary gradient: Solvent A (Formic Acid in MeCN) Solvent B (formic acid in water) 3. Detection Crocin 440nm Picrocrocin derivative 250nm Flavonoid 350nm Safranal 310nm 4.Standard trans-crocin-4-gentobiose-gentobiose Trans-Crocin-3-Gentiobiose-Glucose Beta-Cyclocitral Kaempferol glucoside Safranal

[0057] Example 1: Extract according to the present invention A first embodiment of the extract is an extract obtained by carrying out a method consisting of carrying out the following steps: - using the stigmas of saffron (Crocus sativus), - grinding to a size of 250 μm using a spindle mill; - hydroalcoholic extraction with 60% v / v ethanol at a ratio of 50 g of saffron per liter of hydroalcoholic solution; - impregnating the maltodextrin introduced into a hydroalcoholic solution; - Heat treatment in an incubator at 40°C for 48 hours.

[0058] The extract obtained is weighed for several molecules using the UHPLC method described in the introduction to the Examples section.

[0059] The resulting chromatogram is presented in FIG.

[0060] The extract has the following characteristics: - Safranal concentration 0.238% - Crocin concentration 3.96% - picrocrocin derivative concentration 1.08%, and - Flavonoid concentration 0.25%

[0061] Example 2: Extract according to the present invention A second embodiment of the extract is an extract obtained by carrying out a method consisting of carrying out the following steps: - using the stigmas of saffron (Crocus sativus), - grinding to a size of 250 μm using a spindle mill; - a water extraction step acidified with hydrochloric acid at pH 4; - impregnating gum arabic introduced in an aqueous solution, - Heat treatment in an incubator at 40°C for 72 hours.

[0062] The extract obtained is weighed for several molecules using the UHPLC method described in the introduction to the Examples section.

[0063] The resulting chromatogram is presented in FIG.

[0064] The extract has the following characteristics: - Safranal concentration 0.73% - Crocin concentration 1.0% - picrocrocin derivative concentration 2.93%, and - Flavonoid concentration 0.57%

[0065] Example 3: Extract according to the present invention A third embodiment of the extract is an extract obtained by carrying out a method consisting of carrying out the following steps: - using the stigmas of saffron (Crocus sativus), - grinding to a size of 250 μm using a spindle mill; - the first step of heat treatment in an incubator at 105°C for 2 to 6 hours; - the second step of heat treatment in an incubator at 140°C for 2 to 6 hours; - hydroalcoholic extraction with 60% v / v ethanol at a ratio of 50 g of saffron per liter of hydroalcoholic solution; - impregnating the maltodextrin introduced into a hydroalcoholic solution; - Drying the liquid extract by freeze-drying.

[0066] The extract has the following characteristics: - Safranal concentration 0.39% - Crocin concentration 2.31% - picrocrocin derivative concentration 1.37%, and - Flavonoid concentration 0.24%

[0067] Example 3A: Extract obtained by spraying (not according to the invention) A first counter example of an extract is an extract obtained by carrying out a method consisting of carrying out the following steps: - using the stigmas of saffron (Crocus sativus), - grinding to a size of 250 μm using a spindle mill; - hydroalcoholic extraction with 60% v / v ethanol at a ratio of 50 g of saffron per liter of hydroalcoholic solution; - impregnating the maltodextrin introduced into a hydroalcoholic solution; - drying the liquid extract by spray drying.

[0068] The resulting chromatogram is presented in FIG. 3A.

[0069] The extract obtained has the following characteristics: - Safranal concentration 0.028% - Crocin concentration 4.98% - picrocrocin derivative concentration 1.26%, and - Flavonoid concentration 0.24%

[0070] Example 3B: Extract obtained by freeze-drying (not according to the invention) A first counter example of an extract is an extract obtained by carrying out a method consisting of carrying out the following steps: - using the stigmas of saffron (Crocus sativus), - grinding to a size of 250 μm using a spindle mill; - hydroalcoholic extraction with 60% v / v ethanol at a ratio of 50 g of saffron per liter of hydroalcoholic solution; - impregnating the maltodextrin introduced into the hydroalcoholic solution; - Drying the liquid extract by freeze-drying.

[0071] The resulting chromatogram is presented in Figure 3B.

[0072] The extract has the following characteristics: - Safranal concentration 0.038% - Crocin concentration 4.40% - picrocrocin derivative concentration 1.15%, and - Flavonoid concentration 0.22%

[0073] Example 4: Example of a nutritional composition for men Example 4 is a 150 mg gelatin capsule consisting of: - Extract according to the invention of Example 1: 15 mg - Maltodextrin: 135mg

[0074] The composition is obtained by mixing the ingredients under conventional conditions known to those skilled in the art and placing them into gelatin capsules under also conventional conditions.

[0075] The recommended dosage is two gelatin capsules per day.

[0076] Example 5: Examples of medicines for men Example 5 is a 1500 mg compressed tablet consisting of: - Extract according to the invention of Example 2: 20 mg - Sorbitol: 1430mg, - Magnesium stearate: 27mg, - Brilliant Blue Dye FCF, laque E133: 20mg - Acesulfame K (E950): 1.5mg - Sodium saccharinate (E954): 1.5 mg.

[0077] The composition is obtained by mixing the ingredients under conventional conditions known to those skilled in the art and is compressed according to also conventional conditions.

[0078] The recommended dosage is one compressed tablet per day.

[0079] Example 6: Examples of nutritional compositions for animals Example 6 is a 300 mg compressed tablet consisting of: - Saffron extract of Example 1: 6mg - Microcrystalline cellulose: 135mg - Magnesium stearate: 10mg

[0080] The composition is obtained by mixing the ingredients under conventional conditions known to those skilled in the art and is compressed according to also conventional conditions.

[0081] The recommended dosage is one compressed tablet per day.

[0082] Example 7: Cosmetic composition for topical application in the form of a day cream This is a composition in the form of a cream for application to the skin.

[0083] The composition comprises: - Extract according to the invention of Example 1: 10 mg - Preservative: 0.5% - Fragrance: 0.6% - Fat phase + water phase: 97.4%

[0084] The fat phase is composed of emulsifiers and triglycerides, and the aqueous phase is composed of water bound to pyrrolidone carboxylic acid.

[0085] The composition is obtained by adding the saffron extract of Example 1 to the aqueous phase, under simultaneous stirring with the preservatives and flavorings, for 10 minutes, then adding the aqueous phase itself to the fatty phase during mixing for 30 minutes.

Claims

1. Saffron (Crocus sativus), Centaurea sibthorpii, Centaurea consanguinea, Centaurea amanicola, Dutch geranium (Erodium cicutarium), Chinese tea, Tintan (Calycopteris floribunda), Crocus heuffelianus, Elderberry (Sambucus nigra), Gardenia jasminoides, Lemon (Citrus limon), Cuminum cyminum L.), Achillea distans., and a carrier, the composition comprising a plant extract obtained from a safranal-containing plant raw material selected from the group consisting of Achillea distans., wherein the concentration of safranal in the composition is at least 0.2% by weight relative to the total dry weight of the composition when measured by HPLC; - after a step of grinding the safranal-containing plant raw material, preferably previously dried, The following steps: - extracting a plant extract from the ground plant material obtained in the grinding step with water or a hydroalcoholic or organic solvent; - impregnating a carrier with the plant extract obtained in the extraction step; Including, a step of heat treating the ground plant material obtained in the grinding step in an incubator at a temperature between 30°C and 95°C for at least 2 hours can be carried out at any time after the grinding step.

2. 2. The method of claim 1, further comprising an acidification step after the extraction step.

3. 3. The method according to claim 1 or 2, characterized in that the drying is carried out at a temperature between 30° C. and 60° C. for at least 24 hours.

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