• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    Inclusion complex to improve the bioavailability of non-water soluble biologically active compounds. Inclusion complex comprising at least one non-water soluble bioactive compound, steviol glycoside, chitosan oligomer and, optionally, flavonoid glycoside and/or metal nanoparticles. Also, the present invention also relates to a method for obtaining said inclusion complex by means of ultrasound. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2718225A1
    申请号:ES201731489
    申请日:2017-12-28
    公开日:2019-06-28
    发明作者:Gil Jesus Martin；Petruta Mihaela Matei；Lebena Eduardo Perez
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] TECHNICAL SECTOR
    [0005]
    [0006] The invention presented falls within the general scope of organic chemistry and finds its application in the pharmaceutical, food and cosmetic sector. More specifically, the present invention provides a new complex, in particular an inclusion complex, which increases the solubility and bioavailability of those bioactive compounds with which it is associated. Advantageously, the present invention does not require the incorporation of surfactants or other chemical compounds to improve the solubility or bioavailability of the bioactive compounds.
    [0007]
    [0008] BACKGROUND OF THE INVENTION
    [0009]
    [0010] The benefits that phytochemicals or bioactive compounds (CBAs) such as polyphenols, terpenes and essential oils, flavonoids, lignans and cumestanes present in plants, as well as phytoestrogens, prebiotics or vitamins, are widely known. In particular, polyphenols are substances that can generate a consumer health benefit. For example, the beneficial cardiovascular effect of red wine or virgin olive oil, whose polyphenol content arouses increasing interest, is well known. In the case of olive oil, its hydroxytyrosol content is the basis of the well-known Mediterranean diet, cardiovascularly healthier than those that use other vegetable oils in food such as butters, sunflower oil or palm. In addition, the technical literature shows that, in vivo or in vitro, polyphenols have the following beneficial characteristics: they reduce inflammation through inhibition mechanisms, slow the development of tumors, have proapoptotic and anti-angiogenesis properties, are modulators of the immune system, present Antibiotic properties against Gram-positive bacteria, increase the resistance of the capillaries acting on the constituents of blood vessels and protect the cardiovascular system due to its antioxidant properties.
    [0011] To ensure that the ingested compound has an advantage on the part of the body and is useful for the intended purpose it is essential that it has adequate bioavailability. Unfortunately, the administration of bioactive compounds (CBAs) orally often suffers from problems of solubility and / or permeability, as well as stability problems that limit the life of the bioactive compound are frequent.
    [0012]
    [0013] Similar problems can be mentioned in relation to the use of antioxidant compounds in the food industry, where the low bioavailability of these CBAs once ingested determines their use in food. To solve this problem it is desirable to develop new vehiculization techniques. Among the known techniques to improve the bioavailability of hydrophobic compounds is the use of encapsulation vectors. However, the encapsulation of polyphenols and other antioxidant compounds in food is very limited by the materials used, since some present toxicity problems and others have limited use in food. In particular, vehiculization agents must be substances recognized as safe or GRAS.
    [0014]
    [0015] The proclivity of synthetic polymers to produce toxicity problems makes them not recommended for use as encapsulation vehicles and the alternative of using natural polymers, which do not have this problem, requires, on the other hand, the development of particle production methods. complicated In most cases, the particle size obtained is difficult to control. Normally, the active substance is incorporated into the nanoparticles or remains adsorbed on its surface. Therefore, to be able to release them, non-reactive polymers or macromolecules must be used.
    [0016]
    [0017] A known encapsulating agent is cyclodextrin, which can be of type a (with a 6-glucosal ring), p (7-glucosal ring) and y (8 glucoses). The most soluble in water is a derivative of beta-cyclodextrin called hydroxypropyl. In cyclodextrins, the CBA is housed inside the toroid formed by glucoses, which has the disadvantage of the difficulty in making the CBA available and thus being active. There are numerous patent documents that describe this method, not sufficiently taken to the commercial scale due to the aforementioned inconvenience.
    [0018] Therefore, it is desirable to develop new encapsulation techniques of hydrophobic biologically active compounds (CBAs), in particular compounds whose bioavailability entails difficulties, such as polyphenols and in general, antioxidant compounds, in order to protect them and keep them stable during storage, also allowing a controlled release that increases their bioavailability. Other properties that would be desirable in this new technology are to reduce the amount of bioactive compound to be used, control its release time and / or disguise flavors.
    [0019]
    [0020] Stevia and more specifically steviol glycosides are substitutes for glucose or sucrose sweeteners, have up to 300 times the sweetness of sugar and have attracted the attention of consumers who demand low carb sweeteners. Because stevia has an insignificant effect on blood glucose, it is attractive to people with low glucose diets. However, the use of steviol glycosides as a carrier agent is barely described in the technical literature. It is specified in a limited number of articles signed by Tozuka et al and Nguyen et al, such as:
    [0021]
    [0022] • H. Uchiyama, Y. Tozuka, M. Nishikawa, H. Takeuchi: “Nanocomposite formation between alpha-glucosyl stevia and surfactant improves the dissolution profile of poorly watersoluble drug”, International Journal of Pharmaceutics 428 (2012) 183-186, in that the attempt to increase the solubility of the bioactive is limited to an order of magnitude of 10 times.
    [0023]
    [0024] • Y. Tozuka, M. Imono, H. Uchiyama, K. Tahara, S. Tazawa, Y. Araki, H. Takeuchi: “Dry powder formulation with a-glycosyltransferase-treated stevia for the effective absorption of hydrophobic bioactive compounds in crude drugs ”, Powder Technology 240 (2013) 2-6].
    [0025]
    [0026] • K. Kadota, D. Okamoto, H. Sato, S. Onoue, S. Otsu, Y. Tozuka, Hybridization of polyvinylpyrrolidone to a binary composite of curcumin / a-glucosyl stevia improves both oral absorption and photochemical stability of curcumine, in Food Chemistry 213 (2016) 668 674], where an increase in the solubility of the bioactive curcumin of x13000 is described, when the PVP polymer is incorporated into the stevia.
    [0027]
    [0028] • TTH Nguyen, J. Si, C. Kang, B. Chung, D. Chung, D. Kim: “Facile preparation of water soluble curcuminoids extracted from turmeric (Curcuma longa L.) powder by using steviol glucosides '', Food Chemistry 214 (2017) 366-373];
    [0029]
    [0030] However, none of these documents describe the formation (or using ultrasound or other alternative methodology) of an inclusion complex consisting of the bioactive compound to be vehiculized together with steviol glycosides. Nor is the incorporation of chitosan oligomers as an additional encapsulating agent described.
    [0031]
    [0032] DESCRIPTION OF THE INVENTION
    [0033]
    [0034] A first aspect of the present invention relates to a complex, in particular an inclusion complex, comprising at least one bioactive compound (CBA), steviol glycoside and chitosan oligomer. Preferably, the molecular weight of the chitosan oligomer is 1000 to 100000 g / mol, even more preferably, between 2000 to 6000 g / mol.
    [0035]
    [0036] The complex described herein may comprise one or more different steviol glycosides. The most important steviol glycosides are stevioside, rebaudioside A, B, C, D, E and F and dulcoside. While the stevioside molecule is a complex of three glucose molecules and an aglycone molecule called steviol, the rebaudioside A molecule contains a total of four glucose units, with the average glucose of the triplet connected to the central steviol structure.
    [0037]
    [0038]
    [0039] An important aspect of the present invention (found by DRX, DSC and SEM) is that the interactions between the CBA and the glycosides present in the inclusion complex are weaker than those shown with other complexes such as those formed by cyclodextrins, since the Complex size is larger. In particular, the interactions between glycosides and CBA in the complex of the invention, obtained by sonication by ultrasound as described hereinbelow, take place by hydrogen bonds. The hydrogen bond is a strong electrostatic force when many molecules are attached, as it provides great stability, but weaker than the covalent bond or ionic bond. Consequently, the compounds of the present invention have a greater ability to release CBAs than could be obtained with cyclodextrins.
    [0040]
    [0041] In the appearance of the bonds through hydrogen bonds, the action of the ultrasound that is applied is essential. Otherwise, these links do not appear or do so very slowly, making the process described in this patent unfeasible.
    [0042]
    [0043] The inclusion complex described in this document has advantages over other vehiculization systems because it allows increasing the solubility of CBAs, allowing their storage and administration, thus facilitating their application in the food, pharmaceutical and cosmetic sectors. In particular, with the inclusion complex of the invention, that is, using steviol glycoside and chitosan oligomers as encapsulation agents, it has been possible to increase the solubility of gallic acid by a magnitude x 65000. Another important advantage of this complex lies in the exclusive use of natural polymers and in the non-use of surfactants. Additionally, the complex of the invention has a high stability in solution, which allows maintaining the solubility for a period of time greater than 72 hours.
    [0044]
    [0045] Generally, the inclusion complex described herein comprises steviol glycosides, for example from stevia, with molecular weights between 800 to 1000 g / mol, together with chitosan oligomers with molecular weights between 1000 to 100,000 g / mol , more preferably between 2000 to 6000 g / mol.
    [0046]
    [0047] The biologically active compound, also called the bioactive compound or CBA herein, can be any organic compound with some activity beneficial to humans, in particular any organic compound with low solubility in Water. Among these compounds can be found polyphenols, terpenes, terpenic oils, phenylpropanoic acids, phytoestrogens, vitamins, carotenoids, xanthophylls, fatty acids, oils, lignans, cumestates, prebiotics or any combination of the above. Preferably, the CBA is a polyphenol or mixture of polyphenols.
    [0048]
    [0049] More specifically, the polyphenol can be a flavonoid such as catechin, epicatechin, isoramnetine, kaempferol, myricetin or quercetin; an anthocyanin such as cyanidine, delfinidine, malvidin, peonidine, petunidine; resveratrol; hydroxytyrosol; curcumin; silymarin or silybin. In addition, the phenylpropanoic acid can be ferulic or caffeic acid; Vitamins can be vitamin A or its derivatives such as retinoic acid, retinal or retinol; vitamin E or its derivatives such as tocopherol or tocotrienol; vitamin D or its derivatives such as vitamin D1, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin D4 (22-dihydroergocalciferol) or vitamin D5 (sitocalciferol); vitamin K (phytomenadione) or its derivatives such as vitamin Kl (phylloquinone), vitamin K2 (menaquinone) or menadinone. Additionally, the CBA can be a carotenoid such as alpha-carotene, beta-carotene, cryptoxanthin or lycopene; a xanthophyll such as astaxanthin, cantaxanthine, capsantin, cryptoxanthin, flavoxanthin, lutein, rodoxanthin, rubixanthin, violaxanthin or zeaxanthin; a fatty acid such as, for example, an omega-3 fatty acid such as a-linolenic acid (ALA)) eicosopentanoic acid (EPA) or docosahexanoic acid (DHA); an omega-6 fatty acid such as y-linoleic acid; an oil in whose composition one or more CBAs appear, for example Neem oil. Additionally, the inclusion complex described herein may comprise more than one CBA of those mentioned in this paragraph.
    [0050]
    [0051] In preferred embodiments, the complex described herein comprises steviol glycoside in combination with at least one flavonoid glycoside. Preferably, rutin, hesperidin or a combination of the above. More preferably, flavonoid glycoside is routine.
    [0052]
    [0053] The presence of the flavonoid fragment in flavonoid glycosides gives them the properties of polyphenols. Consequently, the incorporation of these compounds as an encapsulation agent in the complex of the present invention provides additional beneficial properties.
    [0054]
    [0055] In particular, rutin (molecular weight 610 g / mol) is a flavonoid glycoside found in some fruits, especially citrus fruits, and its name comes from Ruta graveolens, a plant that contains it. It is sometimes referred to as vitamin P, but it is not strictly a vitamin. Routine is the glycoside formed between flavonol quercetin and rutin disaccharide. It is formed by creating a bond between the disaccharide and the hydroxyl group of quercetin.
    [0056]
    [0057]
    [0058]
    [0059] Rutin is an antioxidant and a potent VEGF inhibitor (angiogenesis inhibitor), and also plays a role in inhibiting some cancers. The following characteristics can be highlighted from its beneficial effects on health:
    [0060] • Inhibits platelet aggregation and vascular permeability, making blood less thick and improving circulation.
    [0061] • It is an anti-inflammatory.
    [0062] • Inhibits the activity of aldose reductase, an enzyme that helps transform glucose into sorbitol.
    [0063] • Strengthens the capillaries and can reduce hemophilia symptoms. It helps prevent edema of the legs. Routine, such as ferulic acid, reduces the cytotoxicity of oxidized LDL cholesterol and reduces the risk of heart disease.
    [0064] • It has application in the treatment of hemorrhoids, varicose veins, and microangiopathies.
    [0065] • Produces analgesic and anti-inflammatory effects that involve the participation of opioid receptors and the modulation of brain areas of the descending pathway of pain, such as the ventrolateral gray periacueductal substance.
    [0066]
    [0067] Although the routine has a water solubility lower than that of steviol glycosides, its incorporation into the complex described here allows it to considerably increase its solubility in water and, consequently, its bioavailability.
    [0068]
    [0069] The complex described herein may comprise between 100% and 50% steviol glycoside and, additionally, between 0% and 50% antioxidant in the form of flavonoid glycoside, preferably routine and / or hesperidin, where the indicated amounts are expressed by weight with respect to the total weight of glycosides in the complex.
    [0070]
    [0071] In preferred embodiments, the complex described herein comprises: 70-80% steviol glycoside and preferably between 20-30% antioxidant in the form of flavonoid glycoside, in particular rutin and / or hesperidin, where you are amounts are expressed by weight with respect to the total weight of glycosides in the complex.
    [0072]
    [0073] In other preferred embodiments of the invention, the weight ratio between the different components of the complex is 1: 5: 1 to 1: 10: 1 between the CBA: GL: OQ, the optimum ratio being 1 bioactive compound (CBA) : 5 glycoside (GL): 1 of chitosan oligomers (OQ), where the amount of glycoside corresponds to all the glycosides present in the complex, that is, the sum of the steviol glycosides and, if applicable, the glycosides of flavonoids that form the first sphere of encapsulation of the complex.
    [0074]
    [0075] Additionally, the complex of the present invention may comprise metal nanoparticles, preferably with a size between 5 and 60 nm. These nanoparticles can be selected from the group consisting of Cu2O or ZnO nanoparticles; Ag nanoparticles as antibacterial, Au or Pt nanoparticles as antitumor; essential trace elements such as Cu, Zn, Ca, Mg, Mn, Fe and Co.
    [0076]
    [0077] In those embodiments in which the inclusion complex comprises metal nanoparticles, it is preferred that it also comprises a flavonoid glycoside, in particular routine, since these compounds can act as an antioxidant and, therefore, the stability of the complex against temperature and oxidation, which favors its storage and bioavailability.
    [0078]
    [0079] A second aspect of the present invention refers to a method for obtaining the complexes described above, where the method comprises the following steps:
    [0080] a) mixing by sonication, preferably 10 to 20 kHZ, the optimum value being 20 kHz, a bioactive compound (CBA) to be encapsulated with at least one steviol glycoside;
    [0081] b) isolate the complex formed in step a) in solid state, preferably by lyophilization; Y
    [0082] c) re-encapsulate the complex formed by CBA and glycoside with chitosan oligomers, preferably by techniques such as ionic gelation injection or precipitation.
    [0083]
    [0084] In those embodiments in which the inclusion complex comprises at least one flavonoid glycoside, step a) of the method of preparation described herein comprises mixing by sonication a bioactive compound (CBA) to be encapsulated with at least one glycoside of Steviol and at least one flavonoid glycoside.
    [0085]
    [0086] In particular embodiments of the present invention, step a) of formation of the CBA complex: sonication glycoside takes place at a temperature between 15 and 50 ° C, more preferably between 20 and 40 ° C. To control that the temperature of the solution does not exceed 50 ° C, ultrasonic application can be performed intermittently, for example, in application periods of 2 minutes each.
    [0087]
    [0088] The solid state CBA / glycoside complex can be isolated by lyophilization of the solution, preferably hydroalcoholic, the alcohol being methanol or ethanol, in a 1: 1 (v / v) ratio, where sonication takes place. This isolation can be done directly, without the need to adjust the pH.
    [0089]
    [0090] In preferred embodiments of the present invention, the method for obtaining the complex comprises a step b1), where the particle size of the complex isolated in step b) is reduced to between 20 nm and 200 nm, preferably by a grinding process or grinding. Additionally or alternatively, the method may comprise a step c1), where the particular size of the inclusion complex CBA: GL: OQ obtained in step c) is reduced to a particle size between 200 nm and 400 nm, preferably through a crushing or grinding process.
    [0091]
    [0092] Through a sonication process, that is, the direct application of ultrasound on the solution comprising the mixture of CBA and glycosides, preferably in hydroalcoholic medium, the subsequent lyophilization of the complex formed and, additionally, crushing or milling of the isolated complex can be obtained nanoparticles of 20 to 200 nanometers, with weak interactions by formation of hydrogen bonds between glycosides and, therefore, with greater capacity to release biologically active compounds. The size of the nanoparticles can be controlled by transmission electron microscopy (TEM) and / or scanning electron microscopy (SEM) and the strength of the bonds or interaction Among the functional groups it can be done by infrared spectroscopy with Fourier transform (FTIR-ATR). The crystallinity characterization of the complex obtained can be followed by differential scanning calorimetry (DSC) or X-ray diffraction (DRX). The solubility and characterization studies of CBAs can be followed with a UV-VIS spectrophotometer and / or a mass chromatograph (HPLC-Masses).
    [0093]
    [0094] The solubility of CBAs in the complex of the invention increases with the concentration of glycosides. Thus, in the present invention it is preferred that the CBA: glycoside ratio is between 1: 5 and 1:10 (weight: weight), it being even more preferable that this CBA: glycoside ratio be 1: 5 (weight: weight), since it is with which adequate solubility is achieved at a lower cost.
    [0095]
    [0096] By way of theoretical explanation, but without binding character, a complex formed by a bioactive compound (gallic acid) and a steviol glycoside (stevioside) in a 1: 5 ratio is represented, which is characterized by the presence of weak interactions by hydrogen bonds between the molecules that favor the solubilization and subsequent vehiculization of the CBA. Although not shown in Figure 1, the inclusion complex of the present invention comprises successive spheres of coordination or self-assembly with chitosan oligomers. These types of interactions contrast with the strong interactions that appear between cyclodextrins and CBA (ibuprofen) that hinder their vehiculization (S. Pereva, T. Sarafska, S. Bogdanova, T. Spassov, Efficiency of “Cyclodextrin-Ibuprofen” inclusion complex formation, Journal of Drug Delivery Science and Technology (2016)) (Figure 2).
    [0097]
    [0098] The process can be extended by increasing the size of the nanoparticles between 200 nm and 400 nm (CBA-ES / RU-OQ), for example, by methods already known and incorporating complex chitosan polyelectrolytes (QUI).
    [0099]
    [0100] In preferred embodiments of the present invention, the method for obtaining the inclusion complex described herein comprises mixing by sonication, preferably between 10 kHZ and 20 kHZ, the CBA: GL complex with chitosan oligomers in a hydroalcolic solution. Then, by lyophilization of the hydroalcoholic solutions (directly and without adjusting the pH), the CBA-GL-OQ complex that is subsequently subjected to grinding can be isolated, so that the resulting complex has a nanoparticle size of less than 400 nm .
    [0101] The method of obtaining the inclusion complex described herein may comprise a step of incorporating metal nanoparticles, preferably with a size between 5 and 60 nm. These nanoparticles can be Cu2O or ZnO nanoparticles; Ag nanoparticles as antibacterial, Au or Pt nanoparticles as antitumor; essential trace elements such as Cu, Zn, Ca, Mg, Mn, Fe and Co. In particular embodiments of the invention, the incorporation of the metal nanoparticles can be carried out together with the incorporation of chitosan oligomer, or at a later stage.
    [0102]
    [0103] Preferably, both in the CBA-GL complex isolated in step b), and in the CBA-GL-OQ complex obtained in step c) of the method described herein, the relationship between the weight of the metal nanoparticles (NPs) and the CBA-GL complex is 1 vs. 1: 5 and the ratio between the weight of the metal nanoparticles (NPs) and the CBA-GL-OQ complex is 1 vs. 1: 5: 1.
    [0104]
    [0105] As mentioned above, in those embodiments in which the complex comprises metal nanoparticles, it is preferred to incorporate a certain amount of flavonoid glycoside, preferably routine, since this compound acts as an antioxidant and, therefore, increases the stability of the nanoparticles. against temperature and oxidation, which favors its storage and bioavailability.
    [0106]
    [0107] In relation to obtaining nanoparticles by crushing or grinding, the FTIR spectra of the obtained complexes show the advantages of using the ball mill or grinding to control the particle size, improve the solubility and stability of the CBA and even the amortization of the product, the latter aspect that can also be observed by DRX, DSC and / or SEM. In addition, the method is easy, economical, environmentally friendly and can be applied on an industrial scale.
    [0108]
    [0109] Generally, the CBA-GL complex formed by the bioactive compound and the glycosides that is isolated in step b) of the method of the invention already exhibits a greater water solubility than the unencapsulated bioactive compound. However, the incorporation of another encapsulation sphere formed by chitosan oligomer significantly improves its water solubility, while improving the control of release of the CBA and, consequently, its bioavailability and intestinal absorption.
    [0110] In particular embodiments of the present invention, step c) of re-encapsulation with chitosan oligomer is carried out by ionic gelation or precipitation injection technology. In the case of precipitation re-encapsulation, step c) may comprise the use of chitosan oligomer polyelectrolytes and a cross-linking agent such as, for example, sodium tripolyphosphate, in particular 0.5% weight / volume to adjust pH as described, for example, in Seyed Fakhreddin Hosseini, Mojgan Zandi, Masoud Rezaei, Farhid Farahmandghavi, Two-step method for encapsulation of oregano essential oil in chitosan nanoparticles: Preparation, characterization and in vitro relée study, in Carbohydrate Polymers 95 (2013) 50-56. The nanoparticles of the inclusion complex (CBA-GL-OQ) obtained according to this procedure preferably have a size between 200 nm and 400 nm.
    [0111]
    [0112] BRIEF DESCRIPTION OF THE FIGURES
    [0113]
    [0114] Figure 1: Non-binding theoretical model of the formation of an inclusion complex or clathrate between stevioside and gallic acid, with 5: 1 stoichiometry (CBA: GL).
    [0115]
    [0116] Figure 2: Formation of toroid complexes of p-cyclodextrin with ibuprofen.
    [0117]
    [0118] Figure 3. FTIR of lyophilized complexes comprising stevioside and gallic acid (thin dotted line); stevioside and caffeic acid (thick dotted line); and stevioside and trans-cinnamic (continuous line).
    [0119]
    [0120] Figure 4. FTIR of the inclusion complex comprising lyophilized stevioside and cinnamic (thick dotted line), lyophilized and crushed (thin dotted line), as well as the stevioside FTIR (solid line).
    [0121]
    [0122] Figure 5: FTIR of stevia leaf extracts (solid line) versus the stevioside and gallic complex (thick dotted line); extracts of stevia leaves, goji fruit (thin dotted line); Stevia leaf extracts thistle seeds (line based on short strokes and dots).
    [0123]
    [0124] Figure 6: Scanning electron spectroscopy (SEM) image showing stevia microfibers that completely cover the gallic acid with which they form a Inclusion compound by means of hydrogen bond formation. Resolution 5 um.
    [0125]
    [0126] Figure 7: Scanning electron spectroscopy (SEM) image showing the stevia microfibers that completely cover the gallic acid with which they form an inclusion compound through the formation of hydrogen bonds. Resolution10 um
    [0127]
    [0128] Figure 8: Record of X-ray Fluorescence, where the peaks corresponding to Ag nanoparticles are clearly observed, within the stevia-gallic acid-NPAg complex.
    [0129]
    [0130] Figure 9: Stevia microfibers by scanning electron spectroscopy (SEM). Resolution 40 um.
    [0131]
    [0132] Figures 10a and 10b: Study of platinum nanoparticles, PtNPs, produced in a stevioside-gallic acid-chitosan medium.
    [0133]
    [0134] Figures 11a, 11b, 11c and 11d: Study of silver nanoparticles, AgNPs, produced in a medium of stevioside-extracts of milk thistle-chitosan seeds.
    [0135]
    [0136] Figures 12a, 12b, 12c, 12d, 12e and 12f: Study of gold nanoparticles, AuNPs, produced in a stevioside-gallic acid medium.
    [0137]
    [0138] EXAMPLES
    [0139]
    [0140] A. PROCEDURE OF OBTAINING
    [0141]
    [0142] Example 1.- Preparation of lyophilisates or stevioside extracts (ES)
    [0143]
    [0144] The preparation of the extract of leaves of Stevia rebadiauna Bertoni, goji berries and milk thistle seeds is carried out by dispersing 10 g of material that has been extracted and finely ground (size less than 400 microns) in 1 liter of methanol and subjecting the mixture to sonication for 60 min, for which the sonicator head is inserted into the solution. During this sonication stage the temperature is controlled of the solution is greater than 50 ° C. The sample is then filtered, centrifuged and subsequently lyophilized and stored at a temperature between 4 and 6 ° C.
    [0145]
    [0146] Example 2.- Preparation of the complex (CBA-ES / RU-OQ) comprising hydrophobic CBAs with a mixture of steviol glycoside and rutin (ES / RU) and chitosan oligomers (OQ)
    [0147]
    [0148] Section 1: Part of 25 mg of a mixture of gallic, caffeic and trans-cinnamic acid (CBAs), and dispersed in 15 mL of absolute ethanol On the other hand, 100 mg of stevioside and 50 mg of routine are dispersed in 15 mL of distilled and deionized water. Both solutions are mixed, resulting in a hydroalcoholic mixture comprising the bioactive compounds (CBAs) and the glycosides (ES / RU) in a 1: 5 ratio (CBA-ES / RU). This mixture is emulsified by directly introducing the sonicator head of the ultrasonic equipment (20 kHz frequency) into the solution. The sonication is carried out intermittently, in periods of 2 min each, and for a total time of approximately 20 min, checking that the solution is heated above 50 ° C. At the end of the sonication stage the ethanol has been removed, thus reducing the volume of the solution by 15 mL. The resulting solution, protected from light, is filtered and / or centrifuged immediately to remove precipitates or impurities and stored cold 4-6 ° C overnight. Subsequently, it is lyophilized to obtain the CBA-ES / RU complex. It is not recommended to alter the pH of the resulting solution so that undesirable precipitates do not form.
    [0149]
    [0150] Section 2 : To obtain CBA-ES / RU nanoparticles, 100 mg of the lyophilized material obtained as described in section 1 is taken and crushed for 5 min in an agate mortar or ball mill.
    [0151]
    [0152] Section 3: 100 mg of the CBA-ES / RU complex obtained as described in section 2 is dissolved in 50 mL of water. A solution of a 2000-100000 low molecular weight chitosan oligomer polyelectrolyte (100 mg), dispersed in a 50 mL solution of 0.4% tripolyphosphate (p / p) is added dropwise with this syringe v). The inclusion complex CBA-ES / RU-OQ is obtained using ionic gelation or precipitation technology. Subsequently, this complex is centrifuged and washed several times with distilled and deionized water, resulting in CBA-ES / RU-OQ nanoparticles with an average diameter between 200 to 400 nm.
    [0153] Example 3: Preparation of metal nanoparticles (NPs) and their incorporation into the complex (CBA-ES / RU-OQ-NPs)
    [0154]
    [0155] A solution containing 20 mg of a metal salt of AgNO3, H2AuCl4, H2PtCl4, CuCl2, ZnCl2, or MgCl2 is prepared and dispersed in 100 mg of the CBA-ES / RU complex obtained as described in Example 2, section 2 in 200 mL of water A solution of a low molecular weight chitosan oligomer polyelectrolyte 2000-6000 g / mol (20 mg) in 50 mL of water is added dropwise with this syringe. This mixture is emulsified by directly introducing the sonicator head of the ultrasonic equipment (20 kHz frequency) into the solution. The sonication is carried out intermittently, in periods of 2 min each, and for a total time of approximately 10 min, checking that the solution does not heat above 50 ° C. The resulting solution, protected from light, is filtered and / or centrifuged immediately to remove precipitates or impurities and stored cold 4-6 ° C overnight. The resulting solution appears completely stabilized and the bioreduced nanoparticles. Subsequently, it is lyophilized to obtain the CBA-ES / RU-OQ-NPs complex, in a 1: 5: 1: 1 ratio. It is not recommended to alter the pH of the resulting solution so that undesirable precipitates do not form. The quality of the lyophilized and ground material can be carried out by Fourier transform infrared spectrometry (FTIR) and the quality of the nanoparticles by SEM-TEM microscopy. The size of the NPs nanoparticles, observed with a TEM, is between 5 nm and 60 nm.
    [0156]
    [0157] B. CHARACTERIZATION OF THE COMPLEXES OF THE INVENTION
    [0158]
    [0159] The characterization of several complexes studied by different types of spectroscopy and microscopy is included below. In particular, the following inclusion complexes have been characterized:
    [0160] i. stevioside and gallic acid
    [0161] ii. Stevia leaf extracts and goji berry extracts
    [0162] iii. Stevia leaf extracts and thistle seeds
    [0163] iv. stevioside and cinnamic acid
    [0164] v. stevioside and caffeic acid
    [0165] saw. stevioside, gallic acid and chitosan oligomers with phosphates
    [0166] vii. Stevia leaf extracts and thistle seeds and phosphate chitosan oligomers
    [0167] viii Stevia leaf extracts and goji seeds and phosphate chitosan oligomers.
    [0168]
    [0169] 1. - Characterization by infrared spectroscopy with Fourier transform and Attenuated Total Reflectance (ATR) of the complexes
    [0170]
    [0171] In Figure 3, the FTIR spectra of the complexes formed between stevioside and gallic acid are compared; stevioside and trans-cinnamic acid; and stevioside and caffeic acid.
    [0172]
    [0173] The greatest differences occur for gallic acid, since it is an organic acid, also known as 3,4,5-trihydroxybenzoic acid, or polyphenol. On the other hand, the spectra of the inclusion complexes of caffeic and cinnamic acids are very similar, since they are two polyphenols and caffeic acid is an organic compound classified as hydroxycinnamic.
    [0174]
    [0175] Additionally, the FTIR spectra of stevioside and the complex formed by stevioside and cinnamic acid both lyophilized and lyophilized and crushed are shown in Figure 4. In this figure it can be seen that there are some advantages of the material subjected to grinding, since it is somewhat more amorphous than the only lyophilized material. On the other hand, major changes are observed in the absorption bands of inclusion complexes against stevioside alone.
    [0176]
    [0177] Figure 5 compares the FTIR spectra corresponding to stevia leaf extracts against stevioside and gallic complexes, and against stevia leaf extracts with goji fruit and / or thistle seeds. As can be seen in this figure, stevia extracts show displacements of their bands except in the area of 1000 to 1080 cm "1, compared to stevioside and gallic complexes; stevia and goji; stevia and thistle showing a similar spectrum , so it follows that both goji fruit and thistle seeds contain polyphenol gallic acid.The dry extract of stevia leaves contains flavonoids, alkaloids, chlorophylls, xanthophylls, hydroxycinnamic acids (caffeic, chlorogenic, etc.) , oligosaccharides, free sugars, amino acids, lipids and trace elements (NF Komissarenko, AI Derkach, IP Kovalyov, NP Bublik, Rast. Research 1 (1994) 53.)
    [0178]
    [0179] 2. - Characterization by scanning electron microscopy (SEM) of the new composites
    [0180] In figures 6 to 9, the formation of inclusion complexes is shown by scanning electron spectroscopy (SEM). The stevia microfibers that completely cover the gallic acid by hydrogen bridge formation are visualized in Figure 6 and 7. In Figure 8, of X-ray Fluorescence, Ag nanoparticles are clearly seen within the stevia-gallic acid-chitosan complex. In Figure 9, the microfibers of the inclusion complexes are perfectly reflected.
    [0181]
    [0182] 3.- Characterization by Transmission Electron Microscopy (TEM) of the size of the nanoparticles
    [0183]
    [0184] 3.1. Study of platinum nanoparticles, PtNPs, produced in a stevioside-gallic acid-chitosan medium (Figures 10a and 10b).
    [0185]
    [0186] The size of the platinum nanoparticles is between 10 nm and 16 nm, they are quasi spherical and sometimes they are added two by two.
    [0187]
    [0188] 3.2. Study of silver nanoparticles, nAgNPs produced in a stevioside medium extracts of milk thistle-chitosan seeds (Figures 11a, 11b, 11c, 11d)
    [0189]
    [0190] The size of the silver nanoparticles is between 10 nm and 30 nm. Some of the particles are isolated or embedded in the matrix in the form of clusters. The nanoparticles are crystalline and the planes of maclas are observed in some cases. Most of the particles are spherical.
    [0191]
    [0192] 3.3. - Study of gold nanoparticles, AuNPs, produced in a medium of gallic stevioside (Figures 12a, 12b, 12c, 12d, 12e, 12f)
    [0193]
    [0194] The size of the gold nanoparticles is between 4 nm and 25 nm. Most of the particles have a size between 7-9 nm and 20 nm. They exhibit many geometries: triangular base prism (layered) and pentagonal and hexagonal icosahedra.
    权利要求:
    Claims (12)
    [1]
    1. - Inclusion complex comprising at least one bioactive compound, steviol glycoside and chitosan oligomer.
    [2]
    2. - Inclusion complex according to claim 1, wherein the bioactive compound is selected from the group consisting of polyphenols, terpenes, terpenic oils, phenylpropanoic acids, phytoestrogens, vitamins, carotenoids, xanthophylls, fatty acids, oils, lignans, cumestates, prebiotics and any combination of the above.
    [3]
    3. - Inclusion complex according to claim 2, wherein the bioactive compound is a polyphenol or mixture of polyphenols.
    [4]
    4. - Inclusion complex according to any one of claims 1 to 3, wherein the complex comprises at least one steviol glycoside in combination with at least one flavonoid glycoside.
    [5]
    5. - Inclusion complex according to claim 4, wherein the flavonoid glycoside is selected from the group consisting of routine, hesperidin and a combination of the above.
    [6]
    6. - Inclusion complex according to any one of claims 4 and 5, wherein the complex comprises between 70% and 80% steviol glycoside and between 20% and 30% flavonoid glycoside, the amounts of which are expressed by weight with respect to to the total weight of glycosides in the complex.
    [7]
    7. - Inclusion complex according to any one of claims 1 to 6, wherein the ratio between the bioactive compound (CBA), the total glycosides (GL) and the chitosan oligomer (OQ) in the complex is 1: 5 : 1 to 1: 10: 1 (CBA: GL: OQ).
    [8]
    8. - Inclusion complex according to any one of claims 1 to 7, further comprising metal nanoparticles.
    [9]
    9. - Method for obtaining the inclusion complex described in any one of claims 1 to 8, wherein the method comprises:
    a) sonicating a bioactive compound to be encapsulated with at least one steviol glycoside;
    b) isolate the complex formed in step a) in solid state; Y
    c) re-encapsulate the compound formed by CBA and glycoside with chitosan oligomers.
    [10]
    10. - Method for obtaining the inclusion complex according to claim 9, comprising a step b1) wherein the particle size of the complex isolated in step b) is reduced to between 20 nm and 200 nm.
    [11]
    11. - Method for obtaining the inclusion complex according to any one of claims 9 and 10, wherein the sonication takes place between 10 kHZ and 20 kHZ.
    [12]
    12. - Method for obtaining the inclusion complex according to any one of claims 9 to 11, comprising a step c1) wherein the particle size of the complex isolated in step c) is reduced to between 200 nm and 400 nm.
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    同族专利:
    公开号 | 公开日
    ES2718225B2|2019-11-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009126950A2|2008-04-11|2009-10-15|Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College|Diterpene glycosides as natural solubilizers|
    WO2016066876A1|2014-10-30|2016-05-06|Universidad De Valladolid|Composite with anti-microbial activity, comprising two self-assembled components of natural origin, and optionally a component of nanometric size|
    MX2014015444A|2014-12-16|2016-06-15|Ct De Investigación Y Asistencia En Tecnología Y Diseño Del Estado De Jalisco A C|Drying process by micro aspersion and preparation of the hesperidin/cyclodextrin inclusion complex.|ES2834732A1|2019-12-17|2021-06-18|Univ Valladolid|AQUEOUS SOLUTION THAT INCLUDES AN INCLUSION COMPLEX, OBTAINING METHOD AND ITS USE FOR CROP APPLICATION AND THE IMPROVEMENT OF ITS PERFORMANCE |
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