Hydrolyzed claws of chicken legs, their peptides and uses thereof (Machine-translation by Google Tra

专利摘要:
Hydrolyzed claws chicken legs, their peptides and their uses. The present invention pertains to the field of protein hydrolysates. In particular, it refers to chicken leg claw hydrolysates and the peptides it comprises. Likewise, it relates to the use of said hydrolysates and/or peptides in the treatment and/or prevention of a condition in which the incretin system is altered. (Machine-translation by Google Translate, not legally binding)
公开号:ES2702617A1
申请号:ES201731065
申请日:2017-09-04
公开日:2019-03-04
发明作者:Vázquez Francisca Isabel Bravo；Armengol Montserrat Pinent；Martí Angela Casanova；Jornet María Margalef；Grau Anna Ardèvol；López Joan Josep Serrano；Ferrer Lluis Arola；Marquínez Begoña Muguerza
申请人:Universitat Rovira i Virgili URV；
IPC主号:

专利说明:

[0001]
[0002] Hydrolyzed claws of chicken legs, their peptides and uses thereof
[0003]
[0004] Field of the invention
[0005]
[0006] The present invention pertains to the field of protein hydrolysates, in particular it refers to chicken leg claw hydrolysates (GPP), to the peptides it comprises and to its use in therapy, in particular for the treatment and / or prevention of a condition in which the incretin system is altered.
[0007]
[0008] BACKGROUND OF THE INVENTION
[0009]
[0010] Diabetes mellitus type 2 (DMT2) is one of the fastest growing metabolic diseases worldwide. This endocrine disorder associated with obesity is characterized by alterations in insulin secretion and insulin resistance. The first objective in the treatment of T2DM is to maintain glucose levels within normal physiological ranges. In this sense, the incretin hormones: glucagon-like peptide type 1 (GLP-1) and gastric inhibitory polypeptide (GIP) represent a useful strategy for the prevention and / or treatment of DMT2. GLP-1 and GIP are important mediators of glucose homeostasis, accounting for approximately 50-70% of the total insulin secretion after glucose ingestion. The main strategy focuses on increasing levels of GLP-1 instead of GIP, because the beta cells of patients with DMT2 have a reduced response to the action of GIP. Accordingly, most incretin-based therapies focus on the use of GLP-1 analogues, as well as on the promotion of endogenous GLP-1 secretion or the use of inhibitors of the dipeptidyl peptidase-4 enzyme ( DPP-IV). This enzyme is expressed in different parts of the body, including in the cells of the intestinal wall and in endothelial cells, although it can also be found in a soluble form in plasma. The main action of the enzyme DPP-IV is to hydrolyze the hormone GLP-1 converting it into its inactive form, once it is secreted in the plasma, having an average life of 1-2 minutes. Because the DPP-IV enzyme is responsible for the inactivation of more than 80% of the GLP-1 secretion, the inhibition of this enzyme represents an alternative to increase the levels of active GLP-1 and thus improve homeostasis of glucose.
[0011]
[0012] Peptides with enzyme inhibitory activity are known in the state of the art DPPIV (iDPP-IV activity) obtained in hydrolysates of different protein sources. For example, such peptides have been described in milk hydrolysates (WO 2006/068480) and in egg hydrolysates (WO 2009/128713) and it has been described that said peptides with iDPP-IV activity have at least one proline in their sequence and normally said proline is in position 2 of the N-terminal end.
[0013]
[0014] Surprisingly, the inventors of the present invention have developed several GPP hydrolysates and characterized one of them with anti-hyperglycemic activity in vivo. Said hydrolyzate comprises peptides with iDPP-IV activity that do not have the typical pattern of the peptides with said activity, that is, lacking the proline in the 2-position of the N-terminal end.
[0015]
[0016] There are documents in the state of the art describing the sequence of some of the peptides of the present invention. For example, WO2008 / 063369 describes a LAADDFR sequence peptide and its use as a biomarker for the diagnosis of neurological diseases, such as Alzheimer's. However, it does not describe its use for the treatment and / or prevention of a condition in which the incretin system is altered.
[0017]
[0018] Thus, it is desired to provide new protein hydrolysates that can help in the prevention and / or treatment of the conditions in which the incretin system is altered.
[0019]
[0020] Object of the invention
[0021]
[0022] In a first aspect, the present invention relates to a protein hydrolyzate having DPP-IV enzyme inhibitory activity in vitro, comprising one or two sequence peptide (s) selected from the group consisting of SEQ ID No. 1 and SEQ ID NO. No. 2, and is a protein hydrolyzate of GPP.
[0023]
[0024] In a second aspect, the present invention relates to a medicament, a food supplement or food product comprising a hydrolyzate according to the first aspect of the invention. Also, it relates to a pharmaceutical composition comprising a hydrolyzate according to the first aspect of the invention and a pharmaceutically acceptable excipient.
[0025] In a third aspect, the present invention relates to a hydrolyzate according to the first aspect of the invention for use in therapy.
[0026]
[0027] In a fourth aspect, the present invention relates to a hydrolyzate according to the first aspect of the invention for use in the treatment and / or prevention of a condition in which the incretin system is altered.
[0028]
[0029] In a fifth aspect, the present invention relates to a method for preparing the hydrolyzate of the first aspect of the invention.
[0030]
[0031] In a sixth aspect, the present invention relates to a hydrolyzate of GPP obtainable by the method according to the fifth aspect of the invention.
[0032]
[0033] Other objects, features, advantages and aspects of the present application will be apparent to the person skilled in the art from the description and the appended claims.
[0034]
[0035] Brief description of the figures
[0036] Figure 1 is a reverse phase HPLC chromatogram on a semi-preparative scale of the active supernatant obtained after centrifugation and ultrafiltration, through a 3,000 Da membrane of pore size, of the "p38 hydrolyzate" obtained by enzymatic hydrolysis with Neutrase ®, 24h, at 25 ° C pH 7 from GPP powder treated at 50 ° C, 1.5 h pH 3, F.1 - F.11 correspond to the eleven (11) fractions collected.
[0037]
[0038] Figure 2A is a reverse phase HPLC chromatogram on a semipreparative scale of the most active fraction (F.3) that was obtained in the first HPLC separation of the "p38 hydrolyzate" of GPP powder, observed at 214 nm. Specifically, four (4) subfractions were collected from F.3 (F.3.1-F.3.4).
[0039]
[0040] Figure 2B is a reverse phase HPLC chromatogram on a semipreparative scale of the most active fraction (F.3) that was obtained in the first HPLC separation of the "p38 hydrolyzate" of GPP powder, observed at 280 nm. Specifically, four (4) subfractions were collected from F.3 (F.3.1-F.3.4).
[0041]
[0042] Figure 3A is a graph showing the evolution over time of the plasma glucose concentration of female Wistar rats fed standard diet (STD) after administration of 1 mL of water (■) or with cafeteria diet (CAF) after the administration of 1 mL of water (•) or 1 mL of "p38 hydrolyzate" (300 mg protein / kg animal weight) (▲) during the glucose tolerance test The data represent the mean ± ESM for a minimum of six-seven (6-7) animals Different letters indicate significant differences (Anova 2-way, post-Bonferroni test).
[0043]
[0044] Figure 3B is a graph showing the area under the curve of the glycemic response of female Wistar rats fed standard diet (STD) after administration of 1 mL of water (STD + water) or with cafeteria diet (CAF) after the administration of 1 mL of water (CAF + water) or 1 mL of "p38 hydrolyzate" (300 mg of protein / kg of animal weight) (Hp38) (CAF + Hp38) during the glucose tolerance test. The data represent the mean ± ESM for a minimum of six-seven (6-7) animals, different letters indicate significant differences (Anova 1 via, Bonferroni posthoc test).
[0045]
[0046] Figure 4A is a graph showing the variation over time of the plasma glucose concentration of 7-month-old male Wistar rats fed standard diet (STD) after administration of 1.5 mL of water (o), of 1 , 5 mL of "p38 hydrolyzate" (300 mg of protein / kg of animal weight) (▲) or 1 mL of vildagliptin (1 mg / kg of animal weight) (□) during the glucose tolerance test The data represent the mean ± ESM for six (6-7) animals, different letters indicate significant differences (Anova 2-way, post-Bonferroni test).
[0047]
[0048] Figure 4B is a graph showing the area under the curve of the glycemic response of male Wistar rats aged 7 months fed standard diet (STD) after administration of 1.5 mL of water (STD + water), of 1 , 5 mL of "p38 hydrolyzate" (300 mg of protein / kg of animal weight) (STD + Hp38) or 1 mL of vildagliptin (1 mg / kg of animal weight) (STD + Vildagliptin) during the tolerance test to glucose The data represent the mean ± ESM for six-seven (6-7) animals Different letters indicate significant differences (1-way Anova, Bonferroni post-hoc test).
[0049]
[0050] Figure 5 is a graph showing the area under the curve of the glycemic response of healthy female Wistar rats fed standard diet (STD) after administration of 1 mL of water (STD + water) or 1 mL of "hydrolyzed p38 "(300 mg protein / kg animal weight) (STD + Hp38) during the glucose tolerance test The data represent the mean ± ESM for a minimum of six-seven (6-7) animals.
[0051] Figure 6A is a graph showing the secretion of active GLP-1 from STC-1 cells treated with control (control) solution or "p38 hydrolyzate" (5 mg lyophilized hydrolyzate / mL) (Hp38) diluted with the same control solution . The data represent the mean ± ESM (n = 6). * indicates significant differences (T-Student).
[0052]
[0053] Figure 6B is a graph showing the secretion of active GLP-1 from segments of ileum tissue treated with control (control) or "p38 hydrolyzate" solution (15 mg lyophilized hydrolyzate / mL) (Hp38) diluted with the same solution control. The data represent the mean ± ESM (n = 6). * indicates significant differences (T-Student).
[0054]
[0055] Detailed description of the invention
[0056] As used in the present application, the singular forms "a / one", "an" and "the" include their plural plurals unless the context clearly indicates otherwise, unless otherwise defined, all the technical terms used herein have the meaning that one skilled in the art to which this invention pertains is commonly understood.
[0057]
[0058] In order to facilitate understanding and to clarify the meaning of certain terms in the context of the present invention, the following definitions and particular and preferred embodiments thereof are applicable, applicable to all embodiments of the various aspects of the present invention:
[0059]
[0060] In the present invention "condition in which the incretin system is altered", refers to any condition detrimental to a subject in which the incretin system is altered. The incretin system is formed by the GLP-1 and GIP hormones and the DPP-IV enzyme, and in the present invention the alteration in one, several or all of them is considered an altered incretin system. In particular, it is understood that the incretin system is altered when there is an increase in DPP-IV activity and / or a decrease in the levels of the active GLP-1 and / or GIP hormone (s) and / or / or a decrease in the response to GLP-1 and / or GIP.
[0061]
[0062] Non-limiting examples of said conditions are diabetes, more particularly diabetes mellitus type 2, conditions of lower glucose tolerance (IGT), altered fasting plasma glucose conditions, more particularly hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, obesity, overweight and osteoporosis. Thus, in a particular embodiment the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered conditions of fasting plasma glucose, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, obesity, overweight, osteoporosis and combinations thereof. Preferably, it is selected from diabetes mellitus type 2, IGT, hyperglycemia, obesity, overpressed and combinations thereof, and more preferably diabetes mellitus type 2, hyperglycemia, overweight, obesity and combinations thereof. The implication of the incretin system in these disorders is well described in the literature and these disorders are therefore the main target of the hydrolyzate or peptide according to the present invention.
[0063]
[0064] In the present invention "hyperglycemia" (or hyperglycemia) refers to blood glucose levels higher than those considered standard for a healthy individual (normoglycemia). In particular, it refers to a blood glucose concentration> 110 mg / dl in the fasted state. More particularly, it refers to a blood glucose concentration> 110 mg / dl in the fasting state and> 140 mg / dl after the oral glucose test. This test consists in the determination of plasma glucose 2 hours after the ingestion of 75 g of glucose in 300 mL of water after a night of fasting.
[0065]
[0066] In the present invention "obesity" and "overweight" refer to an abnormal or excessive accumulation of fat, higher than those considered standard for a healthy individual, which can be detrimental to health. The definition given by the WHO for "obesity" or "overweight", which is applicable to the present invention, indicates that one of the ways to define "obesity" or "overweight" is by determining the body mass index ( BMI, weight of a person divided by its height squared) considering "obesity" when the BMI is equal to or greater than 30 Kg / m2 and "overweight" when the BMI is between 25 and 29.9 Kg / m2.
[0067]
[0068] "Subject" refers to any member of the Mammalia class, including, without limitation, humans and non-human primates such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs, and the like. The term does not indicate a specific age or sex. Therefore, adult and newborn subjects, as well as fetuses, whether male or female, are intended for inclusion within the scope of this term. The subject is, preferably, a human being.
[0069]
[0070] Supplement (or supplement) food "refers to any food component that provides specific nutritive and / or functional components and does not provide the full energy value required (ie, generally less than 2000 or 2500 kcal / day) and includes food supplements in the form of powder or tablets, as well as dietetic products, such as beverages dietetics Also included are ingredients that can be added to the food product before consumption or a preparation that can be consumed as such.
[0071]
[0072] "Food product" refers to any product, solid or liquid, natural or processed, intended to be ingested by a subject In a preferred embodiment, the food product is a functional food product, which refers to any product, solid or liquid , natural or transformed, destined to be ingested by a subject that, apart from its basic nutritive role from the material and energetic point of view, is capable of providing a health benefit.
[0073]
[0074] The term "prevention" means the prophylactic administration of the hydrolyzate, peptide, medicament, food supplement, food product and / or pharmaceutical composition to healthy patients to prevent the outbreak of the condition in which the incretin system is altered. Moreover, the term "prevention" means the prophylactic administration of said hydrolysates, peptides, etc. to patients who are in a pre-stage condition, especially diabetes, to be treated. In the case of functional food products, in the present invention "prevention" refers to the reduction of the risk of suffering from a condition in which the incretin system is altered.
[0075]
[0076] The present invention relates in a first aspect to a protein hydrolyzate having DDP-IV enzyme inhibitory activity in vitro, comprising one or two sequence peptide (s) selected from the group consisting of SEQ ID No. 1 and SEQ ID N ° 2, and is a protein hydrolyzate of GPP. This hydrolyzate is hereinafter referred to as the hydrolyzate of the invention or HGPP (from chicken leg claw hydrolyzate).
[0077]
[0078] The hydrolyzate of the present invention also has anti-hyperglycemic activity in vivo (see Examples 6-8) and GLP-1 secretory activity in vitro (see Example 9). Thus, in a particular embodiment, the first aspect of the invention relates to a protein hydrolyzate having inhibitory activity of the DDP-IV enzyme in vitro, has GLP-1 secretory activity in vitro and / or antihyperglycemic activity in vivo, comprises one or two sequence peptide (s) selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and is a protein hydrolyzate of GPP.
[0079] In another particular embodiment according to any one of the above embodiments, the HGPP further comprises at least one peptide selected from the group consisting of peptides of sequence SEQ ID No. 3-7, preferably at least one peptide of sequence SEQ ID No. 3. In a particular embodiment comprises one, several or all of the peptides selected from SEQ ID No. 3-7, more particularly comprising 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7. In a preferred embodiment, the HGP comprises the peptides of sequence SEQ ID No. 1 - and / or SEQ ID No. 2, and SEQ ID No. 3 - SEQ ID No. 7.
[0080]
[0081] In another particular embodiment according to any one of the above embodiments, it comprises 1, 2 or 3 peptides selected from SEQ ID No. 8-10. In a preferred embodiment, the HGPP comprises the peptides of sequence SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 -SEC ID No. 10.
[0082]
[0083] The sequences described in the present invention are the following:
[0084]
[0085]
[0086] The first aspect of the invention also relates to the isolated peptides (hereinafter referred to as peptides of the present invention) of sequence SEQ ID NO: 1 or SEQ ID NO: 2, and to a combination of said peptides. Thus, it refers to an isolated peptide whose amino acid sequence is selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2. And also refers to a combination comprising a peptide of SEQ ID No. 1 and a peptide of SEQ ID No. 2. These peptides have iDDP-IV activity in vitro (see Example 5, Table VI ). In a preferred embodiment, the peptide consists of the sequence SEQ ID No. 1. In another preferred embodiment, the peptide consists of the sequence SEQ ID No. 2. As shown in Table VI , these peptides are those with the highest iDDP activity -IV in vitro have.
[0087] In a particular embodiment according to any one of the embodiments of the preceding paragraph, the isolated peptide of sequence SEQ ID No. 1 or 2, or the combination thereof is in combination (ie combined) with 1, 2, 3, 4 or 5 peptides selected from the sequences SEQ ID No. 3-7 (preferably at least one peptide of sequence SEQ ID No. 3), and optionally 1, 2 or 3 peptides selected from the sequences SEQ ID No. 8-10. Thus, in a particular embodiment the first aspect of the invention relates to a combination comprising a peptide of sequence SEQ ID No. 1 and / or a peptide of sequences SEQ ID No. 2, and 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, preferably at least one is the peptide of sequence SEQ ID No. 3, and optionally comprises 1, 2 or 3 peptides selected from SEQ ID No. 8-10. In a particular embodiment, the combination comprises a peptide of sequence SEQ ID No. 1 and / or a peptide of sequence SEQ ID No. 2, and the peptides of sequence SEQ ID NO: 3-SEQ ID No. 7. In another embodiment In particular, the combination comprises the peptides of sequence SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 -SEC ID No. 10.
[0088]
[0089] As indicated above, it has been described that peptides with iDPP-IV activity have at least one proline in their sequence and normally said proline is in the 2-position of the N-terminus. Surprisingly, the peptides of the present invention do not have said proline in the 2-position and in fact, the sequence SEQ ID No. 1 does not comprise any proline. Thus, it was not expected that said peptides would have iDPP-IV activity.
[0090]
[0091] The assays for measuring iDDP-IV activity in vitro are widely known to those skilled in the art. Examples of tests that can be carried out to determine it are those shown in the Examples of the present application. Thus, in a particular embodiment, the in vitro iDDP-IV activity is measured following the method of Tulipano et al (Whey protein as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase 4) inhibitors. "2011. Peptides, 32, 835-838) , as has been carried out in the examples of the present invention.
[0092]
[0093] In a preferred embodiment according to any one of the preceding embodiments of the first aspect of the invention, the peptide (s) consist (s) in the given sequence (s), i.e. SEQ ID No. 1-SEQ ID No. 10.
[0094]
[0095] The hydrolysates, isolated peptides and peptide combinations of the present invention can be incorporated into foods, food supplements, and in the manufacture of pharmaceutical products. Thus, in a second aspect , the invention relates to a medicament, a food supplement or a food product comprising a HGPP, an isolated peptide and / or a combination of peptides according to any one of the embodiments of the first aspect of the invention.
[0096]
[0097] In a preferred embodiment, the medicament, food supplement or food product comprises a HGPP comprising a peptide of sequence SEQ ID No. 1 or a peptide of sequence SEQ ID No. 2 or a combination of said peptides; and optionally comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, with at least one of them being the peptide of sequence SEQ ID No. 3. In a particular embodiment according to any one of the embodiments above, HGPP further comprises 1, 2 or 3 peptides of sequences SEQ ID No. 8-10. Preferably the medicament, food supplement or food product comprises an HGPP comprising the peptides SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 - SEQ ID No. 7, more preferably comprises the peptides SEQ ID NO. No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 - SEQ ID No. 10.
[0098]
[0099] In another preferred embodiment, the medicament, food supplement or food product comprises a peptide of sequence SEQ ID No. 1 or a peptide of sequence SEQ ID No. 2 or a combination of said peptides; and optionally comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, with at least one of them being the peptide of sequence SEQ ID No. 3. That is, the drug, food supplement or product food comprises a peptide of sequence SEQ ID No. 1 or SEQ ID No. 2, or a combination comprising the peptides of sequence SEQ ID No. 1 and of sequence SEQ ID No. 2, and optionally comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, with at least one of them being the peptide of sequence SEQ ID No. 3.
[0100]
[0101] In a particular embodiment according to any one of the preceding embodiments, the medicament, food supplement or food product further comprises 1, 2 or 3 peptides of sequences SEQ ID No. 8-10. Thus, preferably the medicament, food supplement or food product comprises the peptide SEQ ID No. 1 and / or the peptide SEQ ID No. 2, and the peptides SEQ ID No. 3-SEQ ID No. 7, more preferably comprises the peptide SEQ ID No. 1 and / or the peptide SEQ ID No. 2, and the peptides SEQ ID No. 3-SEQ ID No. 10.
[0102]
[0103] Also, the second aspect of the invention relates to a pharmaceutical composition comprising a HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, and a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition comprises the hydrolysates, isolated peptides and combinations described in the three preceding paragraphs for the drug, food supplement and food product.
[0104]
[0105] The pharmaceutically acceptable excipients are widely known to those skilled in the art, in a particular embodiment are such as those described in, "Remington's Pharmaceutical Sciences Handbook" Mack Pub. Co., NYUSA
[0106]
[0107] The medicament, food supplement and pharmaceutical composition can be formulated in any desired form of administration, for example, as tablets, capsules, syrups and the like for oral administration, as sterile solutions or suspensions in liquids acceptable for parental administration.
[0108]
[0109] In the food product of the invention, the HGPP or isolated peptide or combination of the invention according to the first aspect of the invention can be in itself a functional food product or an ingredient that can be combined with any common food ingredient.
[0110]
[0111] As indicated above, the peptides and hydrolysates of the invention have iDDP-IV activity, whereby the medicaments, food supplements, food products, or pharmaceutical compositions comprising them can serve for the treatment and / or prevention of a condition in which the increment system is altered. Thus, in a third aspect, the present invention relates to the use of an HGPP, isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for the preparation of a medicament, a food supplement or a food product, in particular for the preparation of a medicine.
[0112]
[0113] The third aspect of the invention also relates to a HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for use in therapy. Also, it refers to the medicament, food supplement, food product, or pharmaceutical composition according to any one of the embodiments of the second aspect of the invention, for use in therapy.
[0114]
[0115] In a particular embodiment according to any one of the embodiments according to the two preceding paragraphs, the HGPP comprises the peptide of SEQ ID No. 2. More particularly, the HGPP comprises the peptide of SEQ ID No. 2 and the peptide of SEQ ID NO. 1. Even more particularly, HGPP further comprises 1, 2, 3, 4 or 5 peptides selected from SEC ID No. 3-7, with at least one of them being the peptide of sequence SEQ ID No. 3, and optionally further comprising 1, 2 or 3 peptides of sequences SEQ ID No. 8-10. Preferably, the hydrolyzate comprises the peptides SEQ ID NO: 1-SEQ ID No. 7, more preferably comprises the peptides SEQ ID NO: 1-SEQ ID No. 10. In another particular embodiment according to any one of the embodiments according to the two paragraphs above, the isolated peptide is the peptide of sequence SEQ ID No. 2 and the combination of peptides comprises the peptide of SEQ ID No. 2 and the peptide of SEQ ID No. 1; and optionally 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, with at least one of them being the peptide of sequence SEQ ID No. 3. More particularly, the combination also comprises 1, 2 or 3 peptides selected from SEQ ID No. 8-10. Preferably, the combination comprises the peptides SEQ ID NO: 1-SEQ ID NO: 7, more preferably comprises the peptides SEQ ID NO: 1-SEQ ID NO: 10.
[0116]
[0117] In a fourth aspect , the present invention relates to the use of an HGPP, isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for the preparation of a medicament, a food supplement, or a product food for the treatment and / or prevention of a condition in which the incretin system is altered. More preferably, it relates to its use for the preparation of a medicament for the treatment and / or prevention of a condition in which the incretin system is altered.
[0118]
[0119] The fourth aspect of the invention also relates to an HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for use in the treatment and / or prevention of a condition in which the incretin system is altered.
[0120]
[0121] In addition, the fourth aspect of the invention relates to a method of treating a condition in which the incretin system is altered, comprising the administration of a therapeutically effective amount of an HGPP, an isolated peptide and / or a combination according to a any of the embodiments of the first aspect of the invention, or of a medicament, food supplement, food product or pharmaceutical composition according to any one of the embodiments described in the second aspect of the invention, to a subject in need thereof. In a preferred embodiment, the administration is carried out orally. In another preferred embodiment, the subject is a human.
[0122]
[0123] Also, the fourth aspect of the invention refers to a method of preventing a condition wherein the incretin system is altered, comprising administering a prophylactically effective amount of an HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, or a medicament, supplement food, food product or pharmaceutical composition according to any one of the embodiments described in the second aspect of the invention, to a subject in need thereof. In a preferred embodiment, the administration is carried out orally. In another preferred embodiment, the subject is a human.
[0124]
[0125] In a particular embodiment according to any one of the above embodiments according to the fourth aspect of the invention, the HGPP comprises the peptide of SEQ ID No. 1 and / or SEQ ID No. 2. More particularly, the hydrolyzate comprises the peptide of SEQ ID NO. No. 1 and the peptide of SEQ ID No. 2. In another particular embodiment according to any one of the preceding embodiments, the hydrolyzate further comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, being preferably at least one of them is the peptide of sequence SEQ ID No. 3, and optionally further comprises 1, 2 or 3, peptides selected from SEQ ID No. 8-10. Preferably it comprises the peptides SEQ ID NO: 1 and / or SEQ ID NO: 2, and SEQ ID NO: 3-SEQ ID NO: 7, and more preferably comprises the peptides SEQ ID NO: 1 and / or SEQ ID NO: 2 , and SEQ ID No. 3 -SEC ID No. 10.
[0126]
[0127] In a particular embodiment according to any one of the preceding paragraphs of the fourth aspect of the present invention, the condition in which the incretin system is altered is selected from the group consisting of diabetes, diabetes mellitus type 2, IGT, altered plasma glucose conditions fasting, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof. Preferably, it is selected from diabetes mellitus type 2, IGT, hyperglycemia, overweight, obesity and combinations thereof, and more preferably diabetes mellitus type 2, hyperglycemia, overweight, obesity and combinations thereof.
[0128]
[0129] The prevention and / or treatment with the HGPPs, peptides and / or a combination of the present invention is a great advantage over chemical treatments since the food protein hydrolysates are food ingredients of natural origin that lack toxicity or have a minimal toxicity.
[0130]
[0131] The posology will depend on several factors such as type and severity of the pathological condition to be treated, patient weight and sex, etc. and will be easily determined by the skilled. Preferably, the hydrolysates of the invention are administered at a dose of between 1 mg and 8 g of protein per day and per kg of the subject to which they are administered, preferably between 5 mg and 6 g of protein per day and per kg. As for the isolated peptides, the preferred dose of administration is between 1 mg and 4 g of peptide per kg per day, preferably between 10 mg and 3 g per kg per day. Thus, in a particular embodiment, the medicament, food supplement, food product or pharmaceutical composition, comprises the hydrolyzate and / or peptide or combination of the invention in an amount such that it allows administration according to the doses mentioned in this paragraph. This embodiment is equally applicable to the second aspect of the invention.
[0132]
[0133] In a fifth aspect , the present invention relates to the method for preparing the HGPP according to any one of the embodiments of the first aspect of the invention (method of the invention).
[0134]
[0135] The method of the present invention comprises the following steps:
[0136] a) crush GPP and lyophilize to obtain a powder material with particle size less than 2 mm;
[0137] b) adjusting an aqueous solution of the above powder to a pH of between 2-4;
[0138] c) heat at between 40 ° C and 120 ° C, for between 10 and 120 min;
[0139] d) cooling the above solution at a temperature between 20 ° C and 55 ° C;
[0140] e) adjusting the pH of the previous solution to a pH between 6.5 and 7.5;
[0141] f) adding a proteolytic enzyme selected from the group consisting of:
[0142] - proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as EC 3.4.21.62 and 3.4.24.28,
[0143] - a serine protease from Bacillus licheniformis identified as EC 3.4.21.62,
[0144] - a Zinc-dependent metalloprotease of Bacillus amyloliquefaciens identified as EC 3.4.24, and
[0145] - a mixture between them,
[0146] and perform an enzymatic hydrolysis for between 1 and 24 h; Y
[0147] g) determine the iDPP-IV activity in vitro and select a hydrolyzate with an iDPP-IV activity greater than 80%.
[0148]
[0149] In a preferred embodiment, the powder of step a) has a humidity of less than 5%.
[0150]
[0151] Surprisingly, the pH treatment of step b) (hereinafter referred to as "pre-treatment with pH") and the heat treatment of step c) (referred to herein as further on as "thermal pre-treatment"), result in a hydrolyzate with improved iDPP-IV activity (see Table I ). Also note that these pre-treatments are not excessively expensive and yet result in a large increase in the iDPP-IV activity.
[0152]
[0153] Thus, the combination of said steps b) and c) causes an improvement in the iDPP-IV activity of the hydrolyzate obtained by the method of the present invention. In a preferred embodiment according to any one of the above embodiments, step b) is carried out at pH 3. In another preferred embodiment, step c) is carried out at 50 ° C or at 100 ° C. More preferably, step b) is carried out at pH 3 and step c) is carried out at 50 ° C or at 100 ° C. In another preferred embodiment according to any one of the preceding embodiments, step c) is carried out for a time of between 70 and 100 minutes.
[0154]
[0155] In another preferred embodiment according to any one of the preceding embodiments, steps c) and / or f) are carried out under stirring.
[0156]
[0157] In another preferred embodiment according to any one of the above embodiments, the temperature of step d) is between 25 ° C and 50 ° C.
[0158]
[0159] In another preferred embodiment according to any one of the preceding embodiments, the pH of step e) is pH 7.
[0160]
[0161] In a preferred embodiment of the method of the invention the pH adjustments are made with HCl or NaOH, as appropriate.
[0162]
[0163] In another preferred embodiment according to any one of the preceding embodiments, the enzymes used in step f) are proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens, and identified as EC 3.4.21.62 and 3.4.24.28; or a zinc-dependent metalloprotease of Bacillus amyloliquefaciens identified as EC
[0164] 3.4.24. Commercial enzymes of these types are the following: Protamex® (Novozyme) which at the date of the present application are the proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens (EC 3.4.21.62 and 3.4.24.28) and Neutrase® (Novozyme) which is a Zinc-dependent metalloprotease from Bacillus amyloliquefaciens (EC 3.4.24). Thus, in a preferred embodiment the proteolytic enzyme is Protamex® or Neutrase®.
[0165]
[0166] In another preferred embodiment according to any one of the preceding embodiments, the hydrolysis of step f) is carried out for between 2 and 24 h, preferably for 2 or 24 hours.
[0167]
[0168] In a preferred embodiment according to any one of the above embodiments, the pH of step b) is 3, step c) is carried out at 100 ° C and the enzyme is Protamex®. Preferably, the hydrolysis reaction is carried out at pH 7.50 ° C for 2 hours. As shown in Table I , these conditions result in a hydrolyzate with an DPP-IV inhibition activity of 93.30%.
[0169]
[0170] In another preferred embodiment according to any one of the preceding embodiments, the pH of step b) is 3, step c) is carried out at 50 ° C, and the enzyme is Neutrase®. Preferably, the hydrolysis reaction is carried out at pH 7, 25 ° C and 24 hours. As shown in Table I , these conditions result in a hydrolyzate with an inhibition activity of DPP-IV of 83.22%.
[0171]
[0172] In another preferred embodiment according to any one of the above embodiments, the pH of step b) is 3, step c) is carried out at 100 ° C, and the enzyme is Neutrase®. Preferably, the hydrolysis reaction is carried out at pH 7.50 ° C and 24 hours. As shown in Table I , these conditions result in a hydrolyzate with 100% DPP-IV inhibition activity.
[0173]
[0174] In a particular embodiment according to any one of the previous embodiments, step g) is carried out by determining the iDPP-IV activity in vitro according to the method described in Tulipano et al (2011), and the selection of a hydrolyzate with activity> 80 % is made with the original hydrolyzate undiluted.
[0175]
[0176] Thus, in a preferred embodiment according to any one of the aspects of the present invention, it is considered that a hydrolyzate shows iDPP-IV activity in vitro when iDPP-IV activity is determined in vitro according to the method described in Tulipano et al (2011) , as is done in the examples of the invention, and the hydrolyzate shows an activity> 80%.
[0177]
[0178] In a particular embodiment, the method comprises an additional step h), in which the hydrolyzate obtained in step g) is dried, preferably by heating in an oven, oven, lyophilizer or atomizer.
[0179] In a particular embodiment of the method of the invention, the method consists of steps a) -g) as defined in any of the embodiments described in the preceding paragraphs. In another particular embodiment, the method consists of steps a) -h) as defined in any of the embodiments described in the preceding paragraphs.
[0180]
[0181] As indicated above, the raw material for preparing the hydrolyzate of the invention are GPPs, which are a by-product of the meat industry. Thus, the production method of the hydrolyzate of the present invention is a cheap and easy to industrialize method, which allows to use an industrial by-product normally discarded.
[0182]
[0183] In a sixth aspect, the present invention also relates to the GPP hydrolysates obtainable by any of the methods described in the preceding paragraphs of the fifth aspect. In addition, it relates to medicaments, food supplements, food products or pharmaceutical compositions comprising them, to their use in therapy, and to their use for the treatment of a condition in which the incretin system is altered, as defined in the second, third and fourth aspect of the invention.
[0184]
[0185] Said hydrolysates obtainable according to the methods of the fifth aspect of the invention have iDPP-IV activity in vitro, anti-hyperglycemic activity in vivo and are capable of inducing the secretion of GLP-1.
[0186]
[0187] Finally, it should be noted that it has been ruled out that the hydrolyzate of the present invention (according to any one of the embodiments of the first or sixth aspect of the invention) exhibits hypoglycemic activity by an in vivo study in which normoglycemic rats were used and to which they were administered a hydrolyzate of the invention, as shown in Example 8. This hypoglycemic activity is not a desirable activity since it is the lowering of the glucose of a healthy subject (person or animal), while the antihyperglycemic activity is the decrease of glucose in a subject with hyperglycemia. It is of great importance that the hydrolyzate of the invention show a therapeutic effect in subjects with hyperglycemia and not in healthy subjects.
[0188]
[0189] Examples
[0190] Following are some specific examples of embodiment of the invention that serve to illustrate the invention.
[0191] Example 1: Method of obtaining GPP powder.
[0192] 15 kg of GPP Gallus gallus domesticus were washed with water and crushed in an industrial Cato shredder using an 8 mm cutting plate. This crushed was frozen in thin layers (1-1.5 cm) and lyophilized for 5 days. The lyophilizate was milled with a Fagor model BV-850 mixer and the grind obtained was sieved with a 2 mm pore size sieve. A fine powder with particle size <2 mm and a moisture content <5% (GPP powder) was thus obtained. The powder of GPP that presented a percentage of humidity higher than that indicated was re-lyophilized or allowed to dry in an oven at 50 ° C for 15 h. Subsequently, it was stored at -20 ° C in closed bottles and surrounded by silica gel to avoid an increase in the humidity of the samples.
[0193]
[0194] Example 2. Method for obtaining hydrolysates from the GPP powder. Hydrolysates were obtained from the GPP powder following the following process, the conditions of which are summarized in Table I.
[0195]
[0196] Once the GPP powder was obtained, a pre-treatment was carried out with changes in pH and / or temperature as indicated in Table I. In round bottom plastic tubes, 0.1 g of GPP powder was weighed, a volume of distilled water was added, thus obtaining a solution with a pH of 7.5 (ie initial pH = 7.5). In those hydrolysates subjected to pre-treatment by pH, the pH was adjusted to 3 with HCl (0.1 M), obtaining a final volume of 4 mL. Subsequently, the tubes were introduced in a water bath at a temperature of 25, 50 or 100 ° C, and left stirring 1.5 h.
[0197]
[0198] After this time, the samples pre-treated at 50 or 100 ° C were allowed to cool and all the pre-treated samples were adjusted to pH 7 with 1 M NaOH or 0.1 N HCl. Subsequently, the samples were introduced in a stove for 5 min at 25 or 50 ° C in order that the samples reached the desired temperature for hydrolysis and then proceeded to perform enzymatic hydrolysis.
[0199]
[0200] To carry out said hydrolysis, 0.5 mL of a 0.8 L Neutrase® enzyme solution or Protamex® (dissolved in distilled water) were added to the GPP samples obtained at an enzyme / protein concentration of GPP of 0.4 AU. The final volume of reaction in all cases was 5 mL, so the volumes were adjusted with distilled water.
[0201]
[0202] The hydrolysis was carried out at 25 or 50 ° C in constant agitation at 250 rpm in orbital, for 2 to 24 h, as indicated in Table I. After the hydrolysis, the enzymes inactivated by heat in a water bath at 85 ° C for 10 min. The samples were chilled on ice for 10 min and centrifuged at 10000 xg, 20 min, 4 ° C. The supernatants obtained are those which in the present invention are called "hydrolysates", which were filtered with filters of 0.45 p, m and were frozen at -20 ° C until their subsequent analysis.
[0203]
[0204] Table I.- iDPP-IV activity (%) of the GPP powder hydrolysates obtained with Neutrase® and Protamex® (0.4 AU) under different hydrolysis and pre-treatment conditions.
[0205]
[0206]
[0207]
[0208]
[0209] Example 3: Selection of the hydrolysates obtained from the GPP powder according to their inhibitory activity of DPP-IV
[0210] The hydrolysates obtained according to Example 2 were selected according to their ability to inhibit the enzyme DPP-IV, which was determined as described in Example 4.
[0211]
[0212] Only hydrolysates were selected that showed a percentage of inhibition greater (>) to 80% (marked in bold in Table I ). These hydrolysates were determined the minimum amount necessary to inhibit 50% of the enzyme DPP-IV (IC50), expressed as pL of hydrolyzate or pg protein / mL of hydrolyzate ( Table II ). All the hydrolysates had an IC50 comprised between 4 and 4.82 pl of hydrolyzate or 270 and 303 pg of protein / mL of hydrolyzate. No significant differences were observed between the IC 50, expressed in pg / mL, of the three hydrolysates studied.
[0213]
[0214] Table II . IDPP-IV activity represented in IC50 and protein content of the GPP powder hydrolysates with higher inhibitions.
[0215]
[0216] IDPP-IV activity
[0217] Protein content Hydrolysates IC 50 IC 50
[0218] (mg / mL)
[0219] (HL) (Hg / mL)
[0220] p16 4.42 297.4 6.83
[0221] p38 4.82 302.9 6.24
[0222] p68 4.45 300.1 6.62
[0223] n = 6 obtained from the measurement of the activity of duplicates of each of the hydrolysates
[0224]
[0225] When there were no significant differences in their activity (expressed as IC50), the "p38" hydrolyzate was selected, since in its preparation, it was the one that required the lowest temperature and therefore the lowest cost from the industrial point of view. with Neutrase® for 24 h at 25 ° C pH 7 with pre-treatment prior to hydrolysis at 50 ° C for 1.5 h at pH 3 (IC50 = 4.82 pL hydrolyzate or 302.9 pg protein / mL hydrolyzate ).
[0226]
[0227] Example 4: Measurement of DPP-IV inhibitory activity of hydrolysates, fractions and peptides
[0228] The determination of the inhibitory activity of the DPP-IV enzyme in the hydrolysates was carried out using the method described by Tulipano et al (2011).
[0229]
[0230] The assay was performed in 96-well plates. For this, 15 pL of a porcine enzyme solution of DPP-IV (Sigma-Aldrich) in 100 mM Tris HCl pH 8.0 buffer (0.26 mU / well) and 10 pL of the hydrolyzate were introduced into the wells. to be analyzed, incubated for 10 min at 37 ° C. Subsequently, 50 pL of the chromogenic substrate Gly-Pro-pNA (final concentration of 0.2 mM) (Bachem) was added and the volume of the well was adjusted to 100 pL with 100 mM Tris HCl buffer, pH 8.0 and the mixture incubated for 30 min at 37 ° C. The iDPP-IV activity was measured at 405 nm every minute in the aforementioned incubation period. In addition, positive (substrate enzyme) and negative (substrate hydrolyzed) controls were also performed. The validity of the method was verified by testing the compound diprotin A (Ile-Pro-Ne) which is a known inhibitor of this enzyme. The analysis was done in triplicate.
[0231] From the absorbance data, kinetic curves were made, obtaining a slope. The inhibition of DPP-IV expressed as a percentage and was calculated following the following formula:
[0232]
[0233] % inhibition = ((A- (B-C)) / A) x 100
[0234]
[0235] A: slope obtained in the incubation of enzyme and substrate
[0236] B: slope obtained in the incubation of enzyme, substrate and hydrolyzate
[0237] C: slope obtained in the incubation of the substrate and hydrolyzate
[0238]
[0239] For the determination of the iDPP-IV activity of the fractions and the peptides, the same procedure described above was followed with some variations. The fluorimetric substrate H-Gly-Pro-AMC (Bachem) was used at a final concentration in the well of 0.01 mM and the porcine enzyme DPP-IV was supplied by Millipore. The DPPIV activity of the samples was recorded for 30 min at 37 ° C in a fluorimeter using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
[0240]
[0241] The protein concentration of the hydrolysates to determine the IC 50 value was determined by the Kjeldahl method (FIL-IDF, Milk, Determination of nitrogen content (Kjeldahl method), Standard 20B, 1993. Int.Dairy Fed., Brussels, Belgium. ) by multiplying the nitrogen percentage of the sample by 6.25. While the protein concentration of the fractions was determined by the bicinchoninic acid method (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions.
[0242]
[0243] Example 5: Isolation, identification and synthesis of peptides with iDPP-IV activity Analytical and semipreparative high-performance liquid chromatography (HPLC) equipment was used, as well as a tandem mass spectrometry (MS) equipment that allowed the sequencing of the peptides. The steps mentioned below were performed.
[0244]
[0245] 5.A. Obtaining the soluble fraction of the "p38 hydrolyzate" of GPP powder
[0246] Following the procedure described in Example 2, 250 mL of the protein hydrolyzate called "p38 hydrolyzate" was obtained, the supernatant of which was centrifuged again in filtering devices (Centripep, Amicon Inc) with a hydrophilic membrane of 3000 Da pore size. The permeate (fraction less than 3000 Da) obtained was collected, lyophilized and stored at -20 ° C until its subsequent fractionation.
[0247] 5.B Fractionation by reverse phase HPLC on a semipreparative scale
[0248] The semi-preparative HPLC used was from the Agilent 1260 series (Agilent Technologies) which consists of a quaternary pump, a gradient controller, an injector, a diode array detector, a fraction collector and data acquisition and processing software ( Agilent OpenLab CDS ChemStation Edition for LC & LC / MS systems A.01.04).
[0249]
[0250] The lyophilized permeate obtained in section 5A was dissolved in water at a concentration of 100 mg of lyophilized residue / mL and the peptides were separated by reverse phase chromatography using a C18 column (Europe peptide, 120 A °, 25 ° C). x 1.0 mm, 5 ^ m; Teknokroma). The solvents used were a mixture of water: trifluoroacetic acid (1000: 1) and acetonitrile: trifluoroacetic acid (1000: 0.8), solvents A and B respectively, and the elution was carried out at a flow of 4 mL / min using the following gradients in the order of appearance: 0 to 40% of solvent B in 50 min), 40-90% of B in 3 min, was maintained at 90% B for 2 min, 90-100% B in 1 min . The injected sample volume was 750 ^ L and the absorbance of the solvent was monitored at 214 nm.
[0251]
[0252] Eleven different fractions ( Figure 1 ) named F.1 - F.11 were harvested from the protein hydrolyzate called "p38 hydrolyzate", which were lyophilized and reconstituted in Milli-Q water at different volumes.The protein content of these fractions reconstituted was determined by the bicinchoninic acid method (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions, then diluted to a concentration of 0.20 mg / mL (with the exception of fraction 11 whose protein content was lower at this concentration) and the iDPP-IV activity was determined according to the protocol described in Example 4 (see Table 111. ) At the protein concentration tested, all the fractions showed some iDPP-IV activity although only 4 of them, F.1, F.3, F.8 and F.10, showed inhibitions greater than 80%.
[0253]
[0254] Additionally, for those fractions with a percentage of iDPP-IV activity higher than 40%, the IC50 was determined ( Table 111 ), thus discarding fraction F.6. The results obtained showed that only 2 of the fractions tested had an IC50 lower than 100 ^ g / mL (F.1 and F.3), being the fraction F.3 the one that showed the lowest value of IC50 (69 ^ g of protein / mL versus 82.96 ^ g protein / mL for F.3 and F.1, respectively), which indicated that it was the fraction with the highest iDPP-IV activity. Although fraction F.11 could not be determined due to the small amount of fraction collected and its low protein content, it was discarded as a fraction with good iDPP-IV activity, since according to the results obtained, at a concentration of 90 ^ g protein / mL, only showed a 32.80% activity, so to reach a 50% inhibition, it would need a higher concentration of protein, higher than 100 ^ g protein / mL. This value is higher than that shown by fractions F.1 and F.3.
[0255]
[0256] Fraction F.3 corresponded to the peptides eluted between approximately min 8.5 and 11.8 ( Figure 1 ).
[0257]
[0258] Table III . Protein content, percentage of iDPP-IV activity and IC50 of the fractions obtained by reverse phase HPLC from the permeate of size <3000 Da obtained from the protein hydrolyzate called "p38 hydrolyzate".
[0259]
[0260]
[0261]
[0262] n = 6 per fraction
[0263] ND: Not determined
[0264]
[0265] Fraction F.3, which was the fraction with the highest iDPP-IV activity, was subjected to a second fractionation by reverse phase HPLC on a semipreparative scale using the same equipment, column and solvents but eluting the sample with a linear gradient of 0- 12% of solvent B in A in 30 minutes and 12% of solvent B in A was maintained for 1 min. To carry out the separation of the peptides contained in this fraction, it was necessary to reconstitute it in Milli-Q water, obtaining a solution of 50 mg of lyophilized residue / mL. In this case, the injection volume was 250 mL and the absorbance of the solvent was monitored at 214 and 280 nm. Four subfractions were collected from the fraction F.3 (F.3.1, F.3.2, F.3.3 and F.3.4) ( Figure 2 ), which were lyophilized and kept at -20 ° C until further analysis.
[0266]
[0267] In order to determine which of the subfractions collected from fraction F.3 contained the peptides with iDPP-IV activity, these subfractions were determined their inhibitory activity following the method already described in Example 4 and the concentration of protein by the BCA method (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions (see Table IV ). To carry out these analyzes it was necessary to reconstitute the fractions in Milli-Q water and they were all diluted in the same solution until having a protein concentration of 0.1 mg / mL in each of them. Three of the four fractions showed inhibitions greater than 50% (F.3.2, F.3.3, F.3.4). These three fractions were also determined the concentration needed to inhibit 50% of the enzyme. Table IV shows the results of this analysis and, as can be seen, fraction F.3.3 showed the lowest IC50 values (26.85 ^ g protein / mL). This subfraction F.3.3 eluted approximately between minutes 11 and 13 ( Figure 2 ), using the separation method described above.
[0268]
[0269] Table IV. Protein content, iDPP-IV activity and IC50 of the subfractions obtained by semipreparative HPLC in reverse phase from fraction F.3.
[0270]
[0271]
[0272]
[0273]
[0274] 5.C. Identification of peptides with iDPP-IV activity by tandem mass spectrometry (MS / MS)
[0275]
[0276] For the identification of the peptides responsible for the iDPP-IV activity of subfraction F.3.3, obtained by a double chromatographic separation of the <3000 Da fraction of the "p38 hydrolyzate", a LTQ Orbitrap Velos mass spectrometer was used. (ThermoFisher Scientific) equipped with an ionization source connected on-line to a nano-HPLC Easy-II (Proxeon, Thermo Fisher Scientific) . For which, subfraction F3.3 was reconstituted in Milli-Q water and diluted with 0.1% trifluoroacetic acid until reaching a protein concentration of 0.1 ^ g / l. The subfraction peptides were first passed through a C18 EASY-Column precolumn (2 cm long, ID 100 ^ m, particle size 5 ^ m, Thermo Fisher Scientific) to remove the salts that the subfraction might have and separated into a reverse phase C18 nano column (15 cm long, ID 75 ^ m, particle 3 ^ m, Nikkyo technos Co. LTD) in which 10 ^ L of the diluted fraction was injected. Chromatographic separation was performed with a continuous gradient of phase B (0 min 2%, 2 min 2%, 42 min 20%, 72 min 32%, 82 min 50%, 92 min 95%, 102 min 95%, 103 min 2%) using water as mobile phases milli Q water containing 0.1% formic acid (phase A) and acetonitrile containing 0.1% formic acid (phase B) and at a flow of 300 nL / min.
[0277]
[0278] All mass spectra were acquired in positive ion mode. The scan (m / z 50-2000) was purchased with a targeted value of 1000000 at a resolution of 30000 to 400 m / z and the 10 most intense ions were selected for collision-induced dissociation fragmentation in the LTQ with a targeted value of 10000 and a collision energy of 35%.
[0279]
[0280] The identification of the proteins / peptides was carried out in the Proteome Discoverer program v.1.4.0.288 (Thermo Fisher). All the MS and MS / MS spectra were analyzed using the Mascot search engine (v.2.5). Mascot was programmed to search the Gallus_20170224.fasta database (2281 entries) belonging to the Swiss-Prot database and assuming there was no enzymatic digestion. An error of 0.8 Da was allowed for ion mass of IT-MS / MS fragments and 10.0 ppm for a mass of primary FT-MS ions. The oxidation of methionine was established as dynamic modifications and the false discovery rate (FDR) and the probabilities of the protein were calculated by Perclorator. Only peptides with a Mascot score higher than 20 and an AC n less than 0.05 were considered. For the relative quantification of the peptide, the area of the precursor ion of the peptide was used. The peptides that are collected in Table V were identified.
[0281] Table V. Peptides identified in subfraction F.3.3
[0282]
[0283]
[0284]
[0285] 5.D Synthesis of the peptides with iDPP-IV activity
[0286] The peptides of the invention were sent to chemically synthesize Caslo ApS (Lyngby, Denmark) and preparations with the purity values shown in Table VI were obtained .
[0287]
[0288] Once synthesized their iDPP-IV activity was determined in order to know which one or more of the peptides identified in subfraction F.3.3 were responsible for said activity in the fraction. For this, the peptides were reconstituted in Milli-Q water (SEQ ID N °: 1, 2, 3, 5, 6, 7 and 9) or in 1% DMSO (SEQ.ID.N °: 4, 8 and 10). ) depending on its solubility. The iDPP-IV activity of the peptides was determined according to the protocol described in Example 4. The percentages of inhibition shown by the different peptides identified in subfraction F.3.3 at a concentration of 500 [mu] M are shown in Table VI . At the tested concentration, few peptides could inhibit the enzyme, only three of them showed an inhibition higher than 10% (SEQ ID NOS: 1,2 and 3). Clearly, SEQ ID No. 1 showed the highest inhibition with 41.91% at a concentration of 500 ^ M, with its IC 50 of 818.04 ^ M. The IC 50 of the peptides SEQ ID No. 2 and 3, which also showed some iDPP-IV activity, could not be determined due to the low solubility of them at high concentrations. However, the results obtained indicate that its IC50 is higher than 2 mM since at this concentration, these peptides showed percentages of inhibition of 41 and 49 (SEQ ID N ° 2 and 3, respectively).
[0289] Table VI. Characterization of the identified peptides of subfraction F.3.3
[0290]
[0291]
[0292]
[0293]
[0294] Example 6: Determination of the antihyperglycemic effect of the "p38 hydrolyzate" of GPP powder in rats with diet-induced glucose intolerance.
[0295] To evaluate the antihyperglycemic effect of the selected hydrolyzate, "p38 hydrolyzate", an acute study was carried out, with animals that showed glucose intolerance induced by the diet by performing a tolerance test to the same after the administration of the "hydrolyzate" p38 ".
[0296]
[0297] The animal model used was Wistar rats (Harlan, Barcelona, Spain) obese females of 20 weeks. The animals used in the study were individually stablished, with free access to food and water, and with temperature conditions of 22 ° C and 12 hours of light and 12 hours of darkness. The development of obesity was due to a diet with cafeteria diet (CAF) composed of carrot, bacon and milk with sugar and also with standard feed for 10 weeks. After these 10 weeks, the animals were divided into two groups: the control group, which was treated with water, and the group treated with hydrolyzate. On the day of the experiment at nine in the morning, the treatments were administered to the animals with an intragastric probe (fasted from the previous night). The doses administered were 1 mL of water or 1 mL of hydrolyzate (300 mg of protein / kg of animal weight dissolved in water). After 40 minutes, blood was taken from the tail (extraction at time 0) and then a dose of glucose (2 g / kg of animal weight), also with intragastric probe. Subsequently, blood samples were taken from the tail at 15, 30, 60 and 120 minutes after the administration of the glucose dose. The blood samples were quickly centrifuged (2500 xg, 4 ° C, 15 min) to separate the plasma, which was stored at -80 ° C until further analysis.
[0298]
[0299] Prior to the development of diet-induced obesity and glucose intolerance in these animals. To these animals, fed a standard diet (STD) consisting of feed and water ad libitum, they were also subjected to a glucose tolerance test, as previously described, after having given them 1 mL of water or 1 mL of hydrolyzate. (300 mg of protein / kg of weight of the animal dissolved in water). Blood was also collected from the tail in the same conditions previously described.
[0300]
[0301] The procedure used in the study was approved by the Ethical Committee of the Rovira i Virgili University.
[0302]
[0303] The antihyperglycemic effect of the "p38 hydrolyzate" was evaluated by quantifying the plasma glucose levels of the animals treated with the hydrolyzate compared with the untreated ones. Glucose concentrations were determined using the enzymatic colorimetry kit (Glucose oxidase-Peroxidase method of QCA). The results obtained were grouped and represented as plasma glucose concentration in the different study times (mean ± the standard error of the mean (ESM) for a minimum of 6-7 homogeneous tests).
[0304]
[0305] The kinetics of the different groups of animals were compared in a 2-way analysis of variance (ANOVA) using the Bonferroni test and the difference was considered significant for values of p <0.05. In addition, the results were also represented by calculating the area under the curve described above. A 1-way variance analysis (ANOVA) was performed using the Bonferroni test at the mean of the areas in order to check if there were significant differences between groups. The statistical program SPSS (IBM SPSS Statistics software version 20.0) was used for all statistical analyzes.
[0306]
[0307] Figure 3A shows the plasma glucose levels observed in healthy rats fed standard diet (STD) and treated with water and in obese rats fed coffee diet (CAF) and treated with water or with the "p38 hydrolyzate", after of the Ingestion of a dose of 2 g / kg of glucose animal weight (glucose tolerance test). At 15 minutes of glucose intake an increase in plasma levels of this sugar was observed in all cases, the highest being that obtained in CAF rats treated with water. Treatment with the "p38 hydrolyzate" in the CAF rats reduced this glucose peak, being statistically lower than that observed in the coffee rats treated with water and similar to that shown by the STD rats. administration, the plasma glucose levels were decreasing in the STD rats until they recovered the initial values, whereas in the CAF treated with water, the original values (glucose intolerance) were not recovered, in the case of the CAF rats treated with the "hydrolyzed p38", glucose levels between 30 and 120 min showed no significant differences with glucose levels observed in STD rats.
[0308]
[0309] Figure 3 B shows the area under the curve calculated from the curve made with the plasma glucose levels obtained during the glucose tolerance test in female STD rats treated with water or CAF treated with water or the "p38 hydrolyzate." Thus, the glucose area under the observed curve was significantly lower in the CAF animals treated with the "p38 hydrolyzate" than in the CAF group treated with water, no significant differences being observed with the area obtained by the STD rats treated with water.
[0310]
[0311] Therefore, in the animal model of diet-induced glucose intolerance, the "p38 hydrolyzate" produced a decrease in plasma glucose peak and tended to normalize glucose values, demonstrating its antihyperglycemic effect.
[0312]
[0313] Example 7: Determination of the antihyperglycemic effect of the "p38 hydrolyzate" of GPP powder in rats with age-induced glucose intolerance.
[0314] The possible antihyperglycemic effect of the "p38 hydrolyzate" was also tested in another animal model with glucose intolerance, in this case, induced by age.
[0315]
[0316] Male Wistar rats (Harlan) of 7 months (30 weeks) of age g were used, which were fed standard diet (STD) based on standard feed and water ad libitum. The stabling conditions were similar to those described in Example 6. The animals were divided into three groups and treated with 1.5 mL of water, 1.5 mL of hydrolyzate (300 mg of protein / kg of dissolved animal weight). in water) or 1 mL of vildagliptin (1 mg / mL of water, commercial DPP-IV inhibitor, as a positive control) administered acutely using an intragastric tube. Subsequently, a glucose tolerance test was performed and blood was collected at different times. The methodology used is described in Example 6. The results obtained were grouped and represented as plasma glucose concentration in the different study times (mean ± ESM for a minimum of 6-7 homogeneous tests).
[0317]
[0318] The kinetics of the different groups of animals were compared in a 2-way analysis of variance (ANOVA) using the Bonferroni test and the difference was considered significant for values of p <0.05. In addition, the results were also represented by calculating the area under the curve described above. A 1-way variance analysis (ANOVA) was performed using the Bonferroni test at the mean of the areas in order to check if there were significant differences between groups. The statistical program SPSS (IBM SPSS Statistics software version 20.0) was used for all statistical analyzes.
[0319]
[0320] Figure 4A shows the curves and Figure 4 B area curves obtained when analyzing the plasma glucose levels of male rats treated with water high age, "hydrolyzed p38" or vildagliptin. As expected, the rats treated with water showed to be glucose intolerant by not lowering glucose levels after 120 min of post-administration. However, rats treated with vildagliptin, reversed this effect.
[0321]
[0322] The results show that in this animal model of age-induced glucose intolerance, treatment with the hydrolyzate tended to decrease the plasma glucose curve, normalizing plasma glucose levels at 120 min after ingestion of this sugar by which demonstrates its antihyperglycemic effect in this animal model.
[0323]
[0324] Example 8: Determination of the possible antihyperglycemic effect of the "p38 hydrolyzate" of GPP powder in healthy rats.
[0325] In order to rule out whether the hydrolyzate had an antihyperglycemic effect in healthy rats, female Wistar rats were used for 8 weeks on a standard diet (STD), and 1 mL of water or 1 mL of "p38 hydrolyzate" was administered by intragastric tube. (300 mg of protein / kg of weight of the animal dissolved in water). Subsequently, these animals were subjected to a glucose tolerance test and the plasma glucose content was measured. The methodology used in both cases is described in Example 6. The Results obtained were grouped and represented as plasma glucose concentration in the different study times (mean ± the standard error of the mean (ESM) for a minimum of 6-7 homogeneous tests). The obtained curve was represented and the area under the curve was quantified. A Student Test analysis was performed on the average of the areas in order to check if there were significant differences between groups through the statistical program SPSS (IBM SPSS Statistics software version 20.0).
[0326]
[0327] Figure 5 shows the area under the glucose curve obtained from the plasma glucose content during the sugar tolerance test in healthy female rats treated with water or the "p38 hydrolyzate." The area obtained by both treatments It was similar, and no significant differences were observed between them.This result indicates that the administration of the "p38 hydrolyzate" has no effect on the plasma glucose levels induced by the performance of a glucose tolerance test.
[0328]
[0329] Example 9: Measurement of the ability to induce GLP-1 secretion
[0330] In order to elucidate the mechanisms by which the "p38 hydrolyzate" has antihyperglycaemic effect in rats with glucose intolerance, in addition to its DPP-IV inhibitory effect demonstrated in vitro (Tables I and II), the capacity was evaluated of GLP-1 secretion by the "p38 hydrolyzate" by means of two studies, one in vitro and the other ex vivo.
[0331]
[0332] 9.1.- In vitro study
[0333] The cell line used was STC-1, supplied by Dr. B. Wice (University of Washington, St. Louis) with the permission of Dr. D. Hanahan (University of California, San Francisco). This cell line is enteroendocrine from transgenic mouse tumor (Rindi et al, "Developmentof Neuroendocrine Tumors in the Gastrointestinal Tract of Transgenic Mice." 1990. American Journal of Phatology, Vol. 136, No. 6). Cells were cultured with DMEM (Dulbecco's Modified Eagle Medium) containing GlutaMAX ™ and 4.5 g / L D-glucose, without sodium pyruvate (Gibco®), supplemented with 17.5% fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin (BioWhittaker). Cells were incubated at 37 ° C at 5% CO 2 in a humidified atmosphere.
[0334]
[0335] For secretion studies, the cells were cultured in 24-well plates at a density of 2.0 * 10 5 cells / well. After two days of growth (density of 80-90% confluence) the secretion study was carried out. Cells were washed twice with HEPES buffer (20 mM HEPES, 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl 2, pH 7.4). Subsequently, the "p38 hydrolyzate" was added to the wells at a final concentration of 5 mg of lyophilized hydrolyzate / mL, dissolved in HEPES buffer with 10 mM glucose and incubated for two (2) hours at 37 ° C at 5% of CO2 with a humidified atmosphere. HEPES with 10 mM glucose was used as a negative control. After incubation, the supernatants were collected, which were centrifuged (1000 xg) to remove cell debris and stored at -80 ° C until analyzed. The cells were also collected, which were reconstituted in lysis buffer to extract the total protein from them. The determination of the protein content was carried out using the BCA method (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions. Three replicates from different cell passages were analyzed, including three wells per condition in each replicate. All studies were done between passes 30 and 50.
[0336]
[0337] All the supernatants obtained were determined the concentration of active GLP-1 with the active ELISA GLP-1 kit (Millipore) following the manufacturer's instructions. The concentration levels of active GLP-1 were normalized with the concentration of total protein obtained from the lysis of the cells.
[0338]
[0339] Figure 6A shows the levels of active GLP-1 secreted by STC-1 cells after exposure for 2 hours to HEPES with 10 mM glucose (control) or to "p38 hydrolyzate" dissolved in HEPES with 10 mM glucose. The treatment with the "p38 hydrolyzate" produced a significant increase in the secretion of active GLP-1, being seven times higher than the content of GLP-1 secreted by the cells treated with the control solution.
[0340]
[0341] 9.2.- Ex vivo study
[0342] Segments of distal ileum from female Wistar rats were used. To obtain the segments, an incision was made in the ileocecal junction and from that union 8 cm was extracted from the distal part of the ileum. This segment was cut into 6 parts of 0.75 cm 2 in cold HBSS buffer (Hank's balanced salt solution, Termofisher). These ileum segments were individually placed in 24-well plates and incubated for one hour at 37 ° C and 5% CO2 with a KRBS solution pH 7.4 (which contained 5 mmol / L KCl, 138 mmol / L NaCl , 4.2 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2.6 mmol / L CaCl2, 1.2 mmol / L MgCl2 and 10 mmol / L HEPES) enriched with 10 mM glucose and 0.1 mM diprotin A, DPP-IV inhibitor, (control) or with the same solution (KRBS glucose diprotin A) but supplemented with "p38 hydrolyzate" (final concentration of 15 mg lyophilized hydrolyzate / mL).
[0343] After incubation, the supernatants were collected, centrifuged (1000 x g) to remove cell debris and stored at -80 ° C until analysis. To the supernatants obtained, the concentration of active GLP-1 was determined with the active ELISA GLP-1 kit (Millipore) following the manufacturer's instructions. The normalization of the results was made taking into account the milligrams of the ileum segment of each well.
[0344]
[0345] Figure 6B shows the levels of active GLP-1 secreted by the ileum segments after exposure for 1 hour to the "p38 hydrolyzate" and to the control solution, in a medium containing a DPP-IV inhibitor to ensure that the observed effects they are about the secretion of the hormone and not through modulation of said enzyme.The treatment with the "p38 hydrolyzate" stimulated the cells of the ileum to secrete active GLP-1, significantly increasing the levels of active GLP-1 in comparison with the levels induced by the control solution.

权利要求:
Claims (27)
[1]
1. - An isolated peptide of sequence SEQ ID No. 1 or SEQ ID No. 2, for use in the treatment and / or prevention of a condition in which the incretin system is altered.
[2]
2. The peptide for use according to the preceding claim, wherein the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered fasting plasma glucose conditions, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof.
[3]
3. - An isolated peptide of sequence SEQ ID No. 2, for use in therapy.
[4]
4. A combination comprising a peptide of sequence SEQ ID No. 1 and a peptide of sequence SEQ ID No. 2, for use in the treatment and / or prevention of a condition in which the incretin system is altered.
[5]
5. - A combination comprising a peptide of sequence SEQ ID No. 1 and / or a peptide of sequence SEQ ID No. 2, and one, two, three, four or five peptides whose sequence is selected from the group formed by SEQ ID NO. No. 3-7, for use in the treatment and / or prevention of a condition in which the incretin system is altered.
[6]
6. The combination for use according to claim 4 or 5, further comprising one, two or three peptides whose sequence is selected from the group consisting of SEQ ID No. 8-10.
[7]
7. The combination for use according to any one of claims 4-6, wherein the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered plasma glucose conditions fasting, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof.
[8]
8. - A combination comprising a peptide of sequence SEQ ID No. 2 and a peptide of sequence SEQ ID No. 1, for use in therapy.
[9]
9. A combination comprising a peptide of sequence SEQ ID No. 2 and one, two, three, four or five peptides whose sequence is selected from the group consisting of SEQ ID No. 3-7, and optionally a peptide of sequence SEQ ID No. 1, for use in therapy.
[10]
10. The combination for use according to claim 8 or 9, further comprising one, two or three peptides whose sequence is selected from the group consisting of SEQ ID No. 8-10.
[11]
11. - A protein hydrolyzate characterized in that it has DPP-IV enzyme inhibitory activity in vitro, comprises one or two sequence peptide (s) selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2, and is a protein hydrolyzate of chicken leg claws.
[12]
12. - The hydrolyzate according to the preceding claim, further comprising one, two, three, four or five peptides whose sequence is selected from the group formed by SEQ ID No. 3-7.
[13]
13. - The hydrolyzate according to claim 11 or 12, further comprising one, two or three peptides whose sequence is selected from the group formed by SEQ ID No. 8-10.
[14]
14. - The hydrolyzate according to any one of claims 11-13, comprising a peptide of sequence SEQ ID No. 2.
[15]
15. - The hydrolyzate according to the preceding claim, which further comprises a peptide of sequence SEQ ID No. 1.
[16]
16. - A hydrolyzate according to any one of claims 11-15, for use in therapy.
[17]
17. - A hydrolyzate according to any one of claims 11 to 15, for use in the treatment and / or prevention of a condition in which the incretin system is altered.
[18]
18. - The hydrolyzate for use according to the preceding claim, wherein the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered fasting plasma glucose conditions, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof.
[19]
19. A medicament, food supplement or food product characterized in that it comprises a hydrolyzate according to any of claims 11-15 and / or a peptide as defined in claim 1 or a combination as defined in any one of claims 4-6, 8-10.
[20]
20. - A pharmaceutical composition characterized in that it comprises a hydrolyzate according to any one of claims 11-15 and / or a peptide as defined in claim 1 or a combination as defined in any one of claims 4-6, 8-10, and a pharmaceutically acceptable excipient.
[21]
21. - A method for preparing a hydrolyzate according to any of claims 11-15, characterized in that it comprises the following steps:
a) shred chicken leg claws and lyophilize to obtain a powder material with particle size smaller than 2 mm;
b) adjusting an aqueous solution of the above powder to a pH of 2-4;
c) heating at a temperature between 40 ° C and 120 ° C, for between 10 and 120 min;
d) cooling the above solution at a temperature between 20 and 55 ° C;
e) adjusting the pH of the above solution to a pH of between 6.5-7.5;
f) adding a proteolytic enzyme selected from the group consisting of:
- proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as EC 3.4.21.62 and 3.4.24.28,
- a serine protease from Bacillus licheniformis identified as EC 3.4.21.62,
- a Zinc-dependent metalloprotease of Bacillus amyloliquefaciens identified as EC 3.4.24, and
- a mixture between them,
and perform an enzymatic hydrolysis for a time between 1 and 24 h; Y
g) determine the iDPP-IV activity and select a hydrolyzate with an iDPP-IV activity greater than 80%.
[22]
22. - The method according to claim 21, wherein step b) is carried out at pH 3.
[23]
23. - The method according to claim 21 or 22, wherein step c) is carried out at a temperature of 50-100 ° C.
[24]
24. - The method according to any one of claims 21 to 23, wherein step c) is carried out for between 70-100 minutes.
[25]
25. The method according to any one of claims 21 to 24, wherein the temperature of stage d) is between 25 ° C and 50 ° C
[26]
26. - The method according to any one of claims 21 to 25, wherein in step f) the enzyme used is:
- proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as EC 3.4.21.62 and 3.4.24.28, or
- a serine protease from Bacillus licheniformis identified as EC 3.4.21.62.
[27]
The method according to any one of claims 21 to 26, comprising an additional step, step h), in which the hydrolyzate of step g) is dried.

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同族专利:

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公开号 | 公开日

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ES2702617B2|2019-11-18|

引用文献:

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EP2252729A1|2008-02-07|2010-11-24|Siu, Michael K. W.|Biomarkers for head-and-neck cancers and precancers|

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US20140309401A1|2011-11-04|2014-10-16|Nippi, Incorporated|Dpp-4 inhibitor|

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WO2013125622A1|2012-02-22|2013-08-29|森永乳業株式会社|Dipeptidyl peptidase-iv inhibitor|

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ES2606954A1|2015-09-25|2017-03-28|Universitat Rovira I Virgili|Procedure for obtaining a hydrolyzation of chicken leg claws with antihypertensive activity, hydrolyzed obtained and peptides contained |

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ES201731065A|ES2702617B2|2017-09-04|2017-09-04|Hydrolysed chicken leg claws, their peptides and uses thereof|ES201731065A| ES2702617B2|2017-09-04|2017-09-04|Hydrolysed chicken leg claws, their peptides and uses thereof|