JP2010229118A - Lipase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lipase inhibitor applicable to foods. <P>SOLUTION: A lipase inhibitor composed of milk-derived proteins such as lactoferrin, apolactoferrin and a casein enzymatic decomposition product is provided. The lipase inhibitor can be applied to a cosmetic, an animal feed, drink and food products, a medicine, etc. A substance composed of milk-derived protein and a composition including the substance useful for inhibition or prevention of obesity are also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a lipase inhibitor.

  Lipids taken orally are hydrolyzed by digestive enzyme lipase secreted from the pancreas and absorbed into the body. Lipids are high in energy and tend to increase in recent years, which contributes to obesity. Obesity is easily associated with hypertension, impaired glucose tolerance, hyperlipidemia, etc., and is considered to be a cause of ischemic heart disease, stroke, diabetes, arteriosclerosis, fatty liver, cholelithiasis, kidney disorders, etc. .

  Therefore, in order to suppress lipid absorption, various lipase inhibitors having a lipase activity inhibiting function have been developed. For example, orilstat (Xenical) has recently been clinically used in many countries, such as the United States, as a pancreatic lipase inhibitor for anti-obesity.

  In addition, polymers of polyphenols obtained by oxidative polymerization of naringenin, hesperetin dihydrochalcone, phloretin, resveratrol, rosmarinic acid or their glycosides (Patent Document 1); yerba mate, amla fruit, cassis fruit, mugwort Solvent extract of leaves, Melissa leaves, and propolis (Patent Document 2); Protocatechuic acid and its derivatives (Patent Document 3); Kale from which water-soluble components are removed or a lower alcohol extract of pretreated kale that is a dried product thereof (Patent Literature 4): Various lipase inhibitors such as lotus root solvent extract (Non-Patent Literature 1) have been found.

JP 2008-253256 A JP 2008-50301 A JP 2008-74735 A JP 2006-62985 A

Saga Prefectural Industrial Technology Center Research Report, 2006, pages 36-38

  An object of this invention is to provide the lipase inhibitor which can be applied to a foodstuff.

  The present invention provides a lipase inhibitor comprising a milk-derived protein.

  In one embodiment of the lipase inhibitor of the present invention, the milk-derived protein is selected from the group consisting of lactoferrin, apolactoferrin, and casein enzyme degradation product.

  The present invention also provides an anti-obesity agent comprising a milk-derived protein.

  In one embodiment of the anti-obesity agent of the present invention, the milk-derived protein is selected from the group consisting of lactoferrin, apolactoferrin, and casein enzyme degradation product.

  According to the present invention, a lipase inhibitor comprising a highly safe milk-derived protein is provided. Also provided are milk-derived proteins useful as anti-obesity agents.

  As lipase inhibitors, lactoferrin and apolactoferrin, which are milk-derived proteins, are useful.

  Lactoferrin consists of about 690 chain amino acids, and its three-dimensional structure has two iron binding pockets, and iron is bound to each pocket. In particular, 100% iron bound to this pocket is called “hololactoferrin”. Normal bovine lactoferrin contains iron in 15 to 20% of the pockets, and the powder and solution are pink, and the higher the degree of iron binding, the more reddish. It approaches white as the iron content decreases.

  In the present specification, lactoferrin having an iron content of 4% or less in the molecule is particularly referred to as “aporactoferrin”. With the iron content bound to the pocket, apolactoferrin can be distinguished from lactoferrin. Here, the iron content refers to the ratio of the number of moles of iron to the number of moles of apolactoferrin. The iron content can be determined by measuring the absorbance of apolactoferrin by spectroscopic analysis, or by directly measuring the amount of iron in apolactoferrin by atomic absorption analysis or ICP spectroscopic analysis. In the present invention, the iron content refers to a value obtained by dissolving apolactoferrin powder in pure water to obtain a 1 w / v% solution, which is measured by absorbance at 470 nm. Usually, apolactoferrin is white and it can be recognized that it is apolactoferrin in its appearance.

  Apolactoferrin may further have the following properties. When an aqueous solution containing apolactoferrin at a concentration of 1 w / v% is prepared, the total cation concentration in the aqueous solution is 5 mmol / L or less. The total cation concentration is determined by dissolving apolactoferrin powder in 0.1N hydrochloric acid to prepare a 0.1 w / v% solution, and measuring the amount of each cation by atomic absorption photometry. And add them together. The total cation concentration may correspond to a salt (ion) contained as an impurity in the apolactoferrin powder. This is because not the ions bound to lactoferrin but only the salt mixed in the powder can be dissolved by the above 0.1N hydrochloric acid. The total cation concentration is preferably 3.0 mmol / L or less, and more preferably 1.0 mmol / L or less.

  Apolactoferrin can be usually produced by adjusting the pH of an aqueous solution containing lactoferrin to the acidic side to dissociate divalent iron ions of the lactoferrin molecule.

  Lactoferrin is separated and purified from mammalian secretions such as milk (eg, milk) or processed milk products such as skim milk and whey (eg, whey). Or a separation method using electrophoresis, a separation method using affinity chromatography, etc.). Furthermore, it may be produced by various cells (including microorganisms, plant cells, animal cells, insect cells, etc.) obtained by genetic recombination, plants, animals and the like. Lactoferrin is preferably derived from natural products. Preferably, it is derived from whey. Whey obtained as a by-product generated when producing a dairy product (for example, cheese, casein, etc.) from cow milk or skim milk can be suitably used as a source of lactoferrin. Lactoferrin can also be marketed as pharmaceuticals, reagents and the like.

Apolactoferrin can be preferably produced, for example, by adding an acid to the solution when ultrafiltration of the lactoferrin-containing solution to dissociate iron ions bound to lactoferrin. Examples of the acid that can be used here include citric acid, hydrochloric acid, phosphoric acid, malic acid, and acetic acid (0.4 M or more), and citric acid is preferable. Alternatively, for example, apolactoferrin is partitioned by these membranes in which bipolar membranes and cation exchange membranes, which are composite ion exchange membranes having a structure in which a cation exchange membrane and an anion exchange membrane are laminated, are alternately arranged. It can be suitably manufactured also by using an electrodialyzer having an acid chamber and a base chamber. In this case, hydrochloric acid produced during the manufacturing process in the electrodialyzer is used as the acid. As an ultrafiltration device, for example, Microza UF laboratory test machine (for example, LX-22001; Asahi Kasei Chemicals Corporation), LOV (hollow fiber module: membrane inner diameter 0.8 mm, effective membrane area) manufactured by the same company. 41m ² , membrane material: polyacrylonitrile, nominal molecular weight cut off: 50,000) may be used.

  In the production of apolactoferrin, the pH on the acidic side to be adjusted is preferably 0.5 to 3.0, more preferably 1.5 to 2.5. When the pH is close to neutral (for example, 5.5), the antibacterial properties of the obtained apolactoferrin may be weakened. As a pH adjuster of an aqueous solution containing lactoferrin, not only the acid but also phthalic acid, glycine and the like can be used. These pH adjusting agents are added to an aqueous solution containing lactoferrin in an amount suitable for adjusting the pH to the above value.

  The temperature at which the pH of the aqueous solution containing lactoferrin is adjusted to the acidic side is preferably not high considering protein denaturation. Usually, it is 5 to 60 ° C, more preferably 15 to 35 ° C, and even more preferably room temperature.

  In the production of apolactoferrin, apolactoferrin can usually be obtained in the form of an aqueous solution, but it may be used in the form of an aqueous solution as it is, or may be used in a powdered form after removing the solvent.

  In the present invention, apolactoferrin may be modified from the commercially available “lactoferrin” or “apolactoferrin” so as to have the above iron binding degree and, if necessary, the total cation concentration.

  Examples of apo-lactoferrin satisfying the above iron binding degree include products available from Tatsua Japan Co., Ltd.

  As the lipase inhibitor, a degradation product obtained by enzymatic treatment of casein which is a milk-derived protein (hereinafter referred to as “casein enzymatic degradation product”) is also useful.

  The casein enzyme degradation product may be a mixture containing peptides or peptide salts of various molecular weights. Examples include casein enzyme degradation products having the following molecular weight distribution: 20000 Da or more 20-30%; 10,000-20000 Da 30-40%; 5000-10000 Da 10-20%; 2000-5000 Da 10-20%; 500-2000 Da 5 to 10%; 500 Da or less. Measurement of molecular weight distribution can be made by the procedure described below: Test solution (degraded product) is treated with prefilter DISMIC-03CP (Advantech), injected into HPLC (Gilson), and separated by gel filtration . The column uses TSK-GEL G3000SWXL (Tosoh Corporation), and the eluent uses phosphate buffered saline (PBS). The flow rate is 1 mL / min, and the degradation product is detected using a UV detector using 215 nm.

  As an example, a substance obtained by hydrolyzing an aqueous solution of sodium caseinate with a protease at 37 ° C. for 1 hour can be mentioned. Since casein is water-insoluble, water-soluble sodium caseinate can be used as a starting material for enzyme degradation products in order to facilitate enzyme treatment. Casein sodium may be commercially available. As the protease, a combination (more preferably a combination of equal amounts) of three kinds of enzymes, that is, endoprotease, exopeptidase, and endopeptidase (for example, derived from microorganisms, manufactured by Sigma) is preferably used. Protease types or treatments that give a degradation product that satisfies the molecular weight distribution are preferred.

  Milk-derived proteins such as lactoferrin, apolactoferrin, and casein enzyme degradation products can be used to inhibit lipases produced by microorganisms resident in the skin based on their lipase inhibitory function. For example, acne, dermatitis, dandruff, and the like, can increase as sebum accumulates due to hypertrophy of sebaceous glands, hyperkeratosis of hair follicle pores, etc. Alternatively, the lipase produced by staphylococcus epidermidis may be produced by the degradation of triglycerides in the cortical components constituting sebum and converting them into free fatty acids, and the free fatty acids act on the epithelium. Therefore, such inhibition of lipase produced by microorganisms resident in the skin is useful for suppressing or preventing acne, dermatitis, dandruff and the like.

  The lipase inhibitor of the present invention is an active ingredient for external cosmetics, pharmaceuticals, quasi-drugs, toiletries and the like that suppress or prevent acne, dermatitis, dandruff and the like (for convenience, collectively referred to as “external preparations”). Can be used as

  The form of the external preparation is not particularly limited, and is a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, an ointment, a gel, etc. And can take any form that is applicable to the skin. The dosage form of the external preparation can be, for example, a lotion, emulsion, gel, cream, ointment, powder, granule, tablet and the like.

  In addition to the said lipase inhibitor by milk-derived proteins, such as lactoferrin, apolactoferrin, and a casein enzyme degradation product, a person skilled in the art usually uses a topical external preparation as long as the lipase inhibitory effect is not impaired. Other components such as oily components, surfactants, alcohols, water, humectants, whitening agents, antioxidants, ultraviolet absorbers, various skin nutrients and the like can be appropriately blended as necessary.

  Milk-derived proteins such as lactoferrin, apolactoferrin, and casein enzyme degradation products can be edible. For example, it can be used as an additive to foods such as oral health supplements or foods and drinks. In particular, it can be used for foods and drinks for the prevention or prevention of obesity, and pharmaceuticals for internal use.

  Foods such as oral health supplements include glucose, fructose, sucrose, maltose, maltitol, sorbitol, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, casein, gelatin, pectin, agar, amino acids , Excipients, extenders, binders, thickeners, emulsifiers, colorants, fragrances, food additives, seasonings, preservatives, and the like may be appropriately blended. Such foods can be formed into powders, granules, capsules, tablets, syrups, suspensions and the like depending on the application. Production of foods (for example, oral health supplements) can be carried out by methods commonly used by those skilled in the art, and the blending amount, blending method, and blending time can be appropriately selected.

  It can be ingested as it is, or dissolved or suspended in a solvent such as water and taken orally before or after a meal or between meals. It can also be added to foods and drinks for the purpose of suppressing or preventing obesity and eating and drinking.

  Additives to foods and drinks include, for example, home-use diabetic foods, liquid foods, foods for the sick (such as combination foods for adjusting diabetic foods), foods for specified health use, diet foods, or foods based on carbohydrates. For example, but not limited to. Specific food forms include, for example, cooked rice products, wheat products, vegetable products, milk drinks, soft drinks, and the like, but are not limited thereto. Addition or processing to foods can be performed by methods commonly used by those skilled in the art, and the blending amount, blending method, and blending time can be appropriately selected. Addition to non-human animals such as livestock or pet feed is also possible.

  When used as a pharmaceutical, pharmaceutically acceptable ordinary carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrating agents, solubilizers, solubilizers, isotonic agents, etc. Various blending ingredients can be added. It can also be administered orally in dosage forms such as powders, granules, capsules, syrups and suspensions. The production of pharmaceuticals can be carried out by methods commonly used by those skilled in the art, and the blending amount, blending method, and blending time can be appropriately selected. The dose can be appropriately selected depending on the degree of obesity, the weight of the patient, the dosage form, and the like. It can be administered orally as it is, taken in a solvent such as water or suspended. Administration can be performed once or divided into several times a day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited by these Examples.

  Lactoferrin was obtained from Wako Pure Chemical Industries, Ltd. and apolactoferrin was obtained from Tatsua Japan Co., Ltd. The iron content of this apolactoferrin is 3%. The determination of the iron content was made by dissolving the apolactoferrin powder in hydrochloric acid (Wako Pure Chemical Industries) and pure water so that the pH was 1.5 w / v%, and the resulting solution was FeC Test Wako (Wako Pure Chemical Industries). The iron binding degree (%) = (iron molar concentration in 8 w / v% solution / 8 mol / mol of apolactoferrin in solution) × 100.

  Water was added to sodium caseinate (Tatsua Japan Co., Ltd.) at a volume of 1:10 and dissolved sufficiently, and sterilized at 80 ° C. for 1 minute. To hydrolyze these proteases at 37 ° C. for 1 hour using equal combinations of endoproteases / exopeptidases / endopeptidases (all products derived from microorganisms and obtained from Sigma) and then inactivate these proteases Were treated at 90 ° C. for 2 minutes and then lyophilized. Hereinafter, this lyophilized product is referred to as “casein sodium enzymatic degradation product”. This casein sodium enzyme degradation product was a composition comprising peptides of various molecular weights shown in Table 1 below. Measurement of molecular weight for molecular weight distribution was performed according to the procedure described below: Test solution (degraded product) was treated with prefilter DISMIC-03CP (Advantech Co., Ltd.), injected into HPLC (Gilson), gel Separated by filtration. The column used was TSK-GEL G3000SWXL (Tosoh Corporation), and the eluent was phosphate buffered saline (PBS). The flow rate was 1 mL / min, and the degradation product was detected using a UV detector using 215 nm.

(Example 1: Pig-derived lipase inhibition test)
Using a measurement kit (Lipase Kit S; DS Pharma Biomedical Co., Ltd.), a lipase inhibition test was performed according to the attached measurement method. The principle of this method is that mercaprol tributyrate (BALB) is used as a substrate, and mercaprol tributyrate (BALB) is hydrolyzed by lipase activity to produce dimercaprol (BAL). Dimercaprol (BAL) reacts quantitatively with 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) to produce 5-thio-2-nitrobenzoic acid (TNB). The absorbance at the absorption wavelength (412 nm) of 5-thio-2nitrobenzoic acid (TNB) is measured and quantified to calculate the activity of lipase.

  The composition of the test solution is shown in Table 2 below. Orilstat (Xenical) was used as a condition containing a lipase inhibitor (“positive control”). As the enzyme solution (lipase), porcine-derived lipase (Wako Pure Chemical Industries, Ltd.) was used.

Abbreviations in the table represent:
BALB = mercaprol tributyrate, SDS = sodium dodecyl sulfate, PMSF = phenylmethylsulfonyl fluoride, DTNB = 5,5-dithiobis (2-nitrobenzoic acid).

  The test was conducted as described below. First, a sample (lactoferrin, apolactoferrin, or sodium caseinate enzymatic degradation product) or a positive control and buffer 2 were placed in each well of a 96-well plate (capacity meter 40 μL). On the other hand, as a negative control, “conditions containing no lipase inhibitor” were set, and a 0.1 M Tris-HCl (pH 7.0) solution containing the same volume (40 μL) of 0.1 M NaCl was placed in the well. Next, 1 μL of esterase inhibitor and 10 μL of buffer solution 1 (added to wash away the esterase inhibitor on the wall surface) were added and stirred sufficiently. Subsequently, 4 μL of an enzyme solution (lipase) was added, stirred, and preheated at 30 ° C. for 5 minutes, and 50 μL of a coloring solution was added. Next, 5 μL of the substrate was put in the dark and incubated at 30 ° C. for 30 minutes, and then 100 μL of the reaction stop solution was added. Within 1 hour after addition of the reaction stop solution, the absorbance was measured at a wavelength of 405 nm to determine lipase activity.

  The lipase inhibition rate was expressed as a percentage (%) relative to the lipase activity in the negative control. The inhibition rate of each test substance is shown in Table 3 below.

(Example 2: Human-derived lipase inhibition test)
In the same manner as in Example 1, a human-derived lipase inhibition test was performed using a measurement kit (Lipase Kit S; DS Pharma Biomedical Co., Ltd.). The test solution used was human-derived lipase (Wako Pure Chemical Industries, Ltd.) 10 units / mL (0.1 M Tris-HCl (pH 7.4) solution containing 0.1 M NaCl) as the enzyme solution (lipase), and the sample concentration was 0 to 64 mg / It is the same as that of the said Table 2 except having set it as mL. The test procedure is the same as in Example 1.

  The lipase inhibition rate was expressed as a concentration (IC50) that inhibits the activity of lipase by 50%. The inhibition rate of each test substance is shown in Table 4 below.

(Example 3: Binding test with porcine-derived lipase)
Lactoferrin and apolactoferrin were tested for binding properties with porcine-derived lipase (Wako Pure Chemical Industries, Ltd.) using an intermolecular interaction quantitative quartz crystal microbalance measurement (QCM) apparatus Affinix Q (Initium Co., Ltd.).

  After binding 1 μL of 100 μg / mL porcine lipase to the sensor chip, a sample (1 mg / mL lactoferrin or apolactoferrin) was added to the sample cup 8 μL × 3 times, and the Kd value and Bmax value were calculated.

The binding results of porcine lipase and lactoferrin are Kd = 2.89 × 10 ⁻⁶ , Bmax = 1500, r ² = 0.959, and the binding results of porcine lipase and apolactoferrin are Kd = 9.02 × 10 ⁻⁷ , Bmax = 1750, r ² = 0.951.

(Example 4: Binding test with human-derived lipase)
Example 3 except that lactoferrin, apolactoferrin, and casein sodium enzymatic degradation product were used for binding to human-derived lipase (Wako Pure Chemical Industries, Ltd.), and 1 mg / mL sodium casein enzymatic degradation product was also used as a sample. In the same manner as above, the intermolecular interaction quantitative quartz crystal microbalance measurement (QCM) apparatus Affinix Q (Initium Co., Ltd.) was used for the test.

The binding results of human-derived lipase and lactoferrin are Kd = 2.11 × 10 ⁻⁶ , Bmax = 1340, r ² = 0.983, and the binding results of human-derived lipase and apolactoferrin are Kd = 1.15 × 10 ⁻⁶ , Bmax = 1630, a ^{r 2} = 0.937, binding results for human-derived lipase and sodium caseinate enzymatic decomposition product, Kd = 3.03 × 10 ^-5, was ^{Bmax = 1020, r 2 = 0.999} .

  It was found that in lactoferrin, apolactoferrin, and sodium caseinate enzymatic degradation product, binding occurs between porcine-derived lipase or human-derived lipase.

  In addition to the results of the lipase inhibition test in Examples 1 and 2, it is understood from the results of the lipase binding test described in Examples 3 and 4 that these substances exhibit an inhibitory effect on lipase.

(Example 5: Weight loss effect clinical trial)
<Test method>
A double-blind study was conducted on 12 healthy subjects aged BMI 25 or older who participated voluntarily (both male and female; 6 in the test product intake group and 6 in the control product intake group). The assignment was requested from a third party belonging to a public research institution.

  Subjects ingested 400 mg of the test product (apolactoferrin) or control product (lactose) daily for 6 weeks within 30 minutes after each meal. Both the test product and the control product were coated with a tableting coating so that the appearance was indistinguishable.

  Subjects received a general physician diagnosis at the start of the study and at the end of the study (day 42). In addition, blood was collected on the same day, and the following test items were measured. After all the tests were completed, the key was opened and the test product intake group and the control group were compared. In conducting this clinical trial, approval was obtained from the Upwell Clinical Trial Ethics Committee, and informed consent was obtained from the subjects.

(Evaluation item)
General condition: weight, blood pressure, pulse;
Blood glucose marker: HbA1C, fasting blood glucose level;
Vascular inflammation markers: soluble VCAM-1 (vascular cell adhesion molecule-1), MCP-1 (monocyte chemotactic protein-1);
Blood test items: red blood cells, white blood cells, platelets, TG, HDL-CHO, LDL-CHO, uric acid level, AST (GOT), ALT (GPT), BUN, creatinine.

<Test results>
In the blood glucose marker and blood test items, there was no difference between the test product intake group and the control product intake group. As for vascular inflammation markers, MCP-1 was clearly decreased in the test product intake group, but was not statistically significant. In the general condition, blood pressure and pulse were not different between the two groups, but the body weight decreased significantly (P <0.01) in the test product intake group compared to the control product intake group (Table 5).

  According to the present invention, a milk-derived protein having lipase inhibitory activity is provided. The lipase inhibitory activity is useful in various fields including the cosmetics and food fields. Milk-derived proteins having anti-obesity action are also provided. Milk-derived proteins are highly safe for the human body, and are useful for the suppression or prevention of obesity by being contained in foods and drinks, animal feeds, and pharmaceuticals.

Claims (4)

  Lipase inhibitor consisting of milk-derived protein.   The lipase inhibitor according to claim 1, wherein the milk-derived protein is selected from the group consisting of lactoferrin, apolactoferrin, and a casein enzyme degradation product.   An anti-obesity agent consisting of milk-derived protein.   The anti-obesity agent according to claim 3, wherein the milk-derived protein is selected from the group consisting of lactoferrin, apolactoferrin, and a casein enzyme degradation product.