JP2010126489A - Lipase inhibitor and anti-obesity agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lipase inhibitor and an anti-obesity agent which produce few side effects because they are originated from vegetable foods and can be administered over a long term. <P>SOLUTION: A lipase inhibitor, an anti-obesity agent, and a fat accumulation inhibitor each of which has an isothiocyanate compound of an origin of a plant of genus Brassica, Brassicaceae family, especially, Brassica rapa L. and/or a glucosinolate thereof as active ingredients, and a pharmaceutical, a food and drink, etc., formulated therefrom are provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is characterized in that it contains an isothiocyanate compound and / or a glycoside thereof (glucosinolate) derived from a Brassicaceae plant, in particular, a Brassica rapa L. The present invention relates to lipase inhibitors, obesity inhibitors and fat accumulation inhibitors. More specifically, it effectively inhibits pancreatic lipase, which is the key to obesity and dyslipidemia, which is not capable of digesting and absorbing lipids in vivo to inhibit obesity and fat accumulation, and is highly effective in inhibiting lipase. The present invention relates to an agent, an obesity inhibitor and a fat accumulation inhibitor, and a pharmaceutical and a food and drink obtained by blending them.

  In recent years, it has been pointed out that the number of patients with lifestyle-related diseases such as obesity is rapidly increasing due to an increase in fat intake, especially with the westernization of eating habits. Obesity is an energy metabolism disorder characterized by an imbalance between energy intake and energy consumption, and as a result, it is defined as a state in which neutral fat is excessively accumulated in fat cells. Fat ingested by meals is decomposed and absorbed in the intestinal tract and accumulated as energy in the living body. However, when the amount of absorbed energy exceeds the consumption due to excessive intake of fat or calorie consumption, excessive accumulation of fat occurs in tissues and organs in the body, and symptoms such as obesity and dyslipidemia occur. Arise. These symptoms are known to be involved in the development of lifestyle-related diseases such as arteriosclerosis, diabetes and dyslipidemia. Therefore, suppressing excessive accumulation of fat in the living body is considered to be effective not only in preventing or improving the onset of symptoms such as obesity and dyslipidemia but also in preventing or treating the lifestyle-related diseases. ing.

  Fat is decomposed into fatty acid and glycerol by lipase present in the intestinal tract, and then absorbed in the intestinal tract, but absorption of glycerol and fatty acid due to excessive fat intake leads to obesity. Therefore, by inhibiting the activity of lipase existing in the intestinal tract, it is possible to suppress the decomposition and absorption of fat in the intestinal tract. For these reasons, lipase inhibitors are thought to be useful as preventive or ameliorating agents for obesity, dyslipidemia, etc., or as preventive or therapeutic agents for the lifestyle-related diseases. In fact, pancreatic lipase inhibitor orilstat (Non-patent Document 1) is clinically used as an anti-obesity drug in many countries such as the United States. However, side effects such as prodigation, sudden constipation, and fat stool observed at an early stage of taking orilstat have been pointed out.

  In recent years, from a preventive viewpoint, functional foods that scientifically study human health and food functions, especially active ingredients thereof, have been actively studied, and isothiocyanate compounds and their glycosides derived from Brassicaceae plants. Attention has been drawn to various physiological activities such as its cancer prevention effect (Non-patent Document 2). On the other hand, there are also several patent applications for functional ingredients of foods characterized by lipase inhibitory action (Patent Documents 1, 2, 3, 4, 5), and anti-obesity agents containing wasabi ingredients as active ingredients ( There are patent applications related to the anti-obesity action (Patent Document 7) containing patent documents 6), maca or benzylglucosinolate as an active ingredient, but phenethyl isothiocyanate, which is an isothiocyanate compound derived from Yamato mana of the Brassicaceae Brassica family There is no specific disclosure of lipase inhibitory activity, obesity inhibitory action and fat accumulation inhibitory action for -butenyl isothiocyanates and their glycosides.

A. M.M. Heck et al., Pharmacotherapy, 20: 270-279, 2000 Y. Zhang et al., Proc Nutr Soc., 65, 68-75, 2006 JP 2001-120237 A JP 2001-299272 A JP 2002-53484 A JP 2004-161644 A Japanese Patent No. 3689099 JP 2006-328056 A JP 2007-230987 A

  An object of the present invention is to provide a lipase inhibitor that is derived from plant foods and has few side effects and can be ingested or administered continuously, as well as an obesity inhibitor and a lipid accumulation inhibitor, and a pharmaceutical and a food and drink comprising the same Is to provide.

  In order to achieve the above-mentioned object, the present inventors have intensively investigated a substance that is derived from plant foods and is highly safe and effective in inhibiting lipase. As a result, the isothiocyanate compound derived from Yamato mana and / or its glycoside (glucosino) Lipase inhibitory effect, obesity inhibitory effect and fat accumulation inhibitor, and the present invention was completed.

That is, the present invention includes the following inventions.
(1) a lipase inhibitor characterized by containing an isothiocyanate compound and / or glycoside thereof derived from a Brassicaceae plant (Brassicaceae);
(2) an obesity inhibitor characterized by containing an isothiocyanate compound derived from a Brassica family Brassica plant and / or a glycoside thereof;
(3) The agent according to (2), which is a fat accumulation inhibitor;
(4) The agent according to any one of (1) to (3), wherein the Brassicaceae Brassica genus plant is Yamato mana (Brassica rapa L.);
(5) The agent according to any one of (1) to (3), wherein the isothiocyanate compound is phenethyl isothiocyanate;
(6) The agent according to (2) or (3), wherein the isothiocyanate compound is 3-butenyl isothiocyanate;
(7) A lipase inhibitor or an obesity inhibitor characterized by containing Yamato mana itself or an extract thereof as an active ingredient;
(8) A pharmaceutical product comprising the agent according to any one of (1) to (7);
(9) A food or drink comprising the agent according to any one of (1) to (7);
And so on.

  According to the present invention, by containing an isothiocyanate compound derived from a Brassicaceae Brassica plant, in particular, Yamato Mana, and / or its glycoside as an active ingredient, there are few side effects, orally, sustained and preventive ingestion or An administrable lipase inhibitor, an obesity inhibitor and a fat accumulation inhibitor, and pharmaceuticals and foods and drinks comprising the same can be provided.

  Hereinafter, the present invention will be described in detail. The isothiocyanate compound or its glycoside (glucosinolate) in the present invention is derived from the Brassicaceae Brassica genus, and as the Brassicaceae Brassica genus plant, broccoli, Takana, cabbage, Chinese cabbage, Yamato mana , Komatsuna, Cauliflower, Turnip, Chingensai, Mizuna, Nozawana etc.

  The isothiocyanate compound of the present invention is derived from a plant belonging to the Brassicaceae family, but arylalkyl isothiocyanates and alkenyl alkyl isothiocyanates are preferred, and phenethyl isothiocyanate is more preferred as arylalkyl isothiocyanates, and alkenyl alkyl isothiocyanates. Is preferably 3-butenyl isothiocyanate.

  In Brassicaceae plants, isothiocyanate compounds exist as glycosides (glucosinolates). The isothiocyanate glycoside is converted to an isothiocyanate compound upon contact with an enzyme such as intracellular myrosinase. In addition, it is known that isothiocyanate glycosides are converted to isothiocyanate compounds by human intestinal bacteria.

  As described above, in Brassicaceae plants, since they are present as isothiocyanate glycosides, they may be used as isothiocyanate compounds, but the glycosides (glucosinolates) may be used as lipase inhibitors. . Further, when an isothiocyanate glycoside is used, an exogenous myrosinase may be added and used.

  Furthermore, Yamato mana itself or an extract thereof can be used as an active ingredient. When using Yamato mana itself as an active ingredient, the collected Yamato mana is washed with water and, if necessary, freeze-dried, hot-air dried or naturally dried, shredded or crushed into a chopped or powdered product, or paste-like In various forms, such as a filtrate obtained by homogenizing and filtering insoluble matter, or a filtrate obtained by concentrating the filtrate is used. When the extract is used as an active ingredient, Daiwa Mana shredded or powdered material extracted with water, an organic solvent, or a mixture thereof, or a concentrate obtained by concentrating the extract (extract) Or a concentrate obtained by pulverizing the concentrate by vacuum drying, spray drying or freeze drying. Examples of the extraction organic solvent include lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol and isobutanol, ketones such as acetone and methyl ethyl ketone, acetates such as methyl acetate and ethyl acetate, tetrahydrofuran, diethyl ether and the like. Ethers, and hydrocarbons such as hexane and toluene. These organic solvents and water may be used alone or in combination of two or more.

  Extraction of isothiocyanate compounds from cruciferous plants, particularly Yamato mana, for example, after pulverizing or shredding the plant body by grinding, etc., and then sufficiently isolating the isothiocyanate compound from the isothiocyanate glycoside by means of plant endogenous myrosinase. And then extracting with an appropriate solvent. Depending on the nature of the isothiocyanate compound, extraction solvents include esters such as ethyl acetate and methyl acetate, ethers such as diethyl ether and ethyl isopropyl ether, halogenated hydrocarbons such as chloroform and dichloromethane, methyl alcohol, and ethyl. Solvents such as alcohols such as alcohol and isopropyl alcohol, and ketones such as acetone and methyl ethyl ketone are used. Preferably, ethyl acetate is used.

  Although the isothiocyanate compound is commercially available as a reagent, it can also be obtained by various chemical syntheses. For example, it is obtained from an amine compound, a silylamine compound, etc. (Tetrahedron Letters, 26, 166-4 (1985), Synthesis, 539 (1970), etc.).

  On the other hand, the extraction of isothiocyanate glycosides (glucosinolates) from cruciferous plants, particularly Yamato mana, for example, inactivates endogenous myrosinase in the plant in advance by heating, followed by crushing by grate etc. After shredding, the mixture is filtered, and the filtrate is separated and purified by column chromatography or the like to obtain the desired isothiocyanate glycoside (glucosinolate). On the other hand, isothiocyanate glycosides (glucosinolates) can also be obtained by various chemical methods (Tetrahedron, 36, 779-783 (1980), etc.).

  The thus obtained isothiocyanate compound and / or glycoside thereof can be used alone or in combination of two or more. The isothiocyanate compound and / or glycoside thereof can be taken alone, but can be added to various foods or drinks or used as a pharmaceutical composition. Specifically, it is added to solid foods such as confectionery, breads and noodles, or beverages such as juices and health teas, or compositions such as pharmaceuticals, quasi-drugs such as powders, granules, capsules and tablets It can be used as a product.

  When the isothiocyanate compound of the present invention and / or its glycoside is used as a medicament for therapeutic or prophylactic purposes, its dosage form, administration method, purpose of use, and age, weight, and symptoms of patients to be administered Although it varies depending on the adult, it is preferably 0.001 to 2000 mg / kg body weight, more preferably 0.01 to 200 mg / kg body weight per day for an adult.

  On the other hand, the ingestion amount of the active ingredient when using the isothiocyanate compound and / or glycoside thereof and the Brassicaceae Brassica plant containing the same, particularly Yamato mana as food and drink, is preferably 0.0001 to 100 mg / kg body weight, more preferably 0.01-1 mg / kg body weight.

  Hereinafter, the present invention will be described based on examples. The present invention is not limited in any way by the following examples, and it goes without saying that the present invention can be implemented with ordinary changes in the technical field to which the present invention belongs.

Production Example 1 (Confirmation of 3-butenylisothiocyanate (BUITC) from Yamato Mana)
100 g of distilled water was added to 17 g of harvested Yamato mana, homogenized for 30 seconds, left at 37 ° C. for 2 hours, and reacted with in vivo myrosinase. Thereafter, the homogenized liquid was centrifuged (3100 rpm, 5 min) to obtain a mana water extract. Subsequently, the water extract was distributed with an equal amount of ethyl acetate to obtain an ethyl acetate fraction, which was used as a sample for HPLC analysis. HPLC analysis using the above sample (Cosmosil 5C18-MSII, CH ₃ OH: H ₂ O = 4 / 6- (5 min) -4 / 6- (5 min) -6 / 4- (7.5 min) -6 / 4-4- (7.5 min) -10 / 0- (5 min) -10 / 0- (10 min) -10/0, 1.0 mL / min, 254 nm). As a result, a sample of 3-butenyl isothiocyanate (from Tokyo Kasei) Purchased 3-butenylisothiocyanate).

Production Example 2 (Confirmation of phenethyl isothiocyanate (PEITC) from Yamato Mana)
The product was treated in the same manner as in Production Example 1 to obtain a phenethyl isothiocyanate fraction, and as a result of HPLC analysis, it was completely consistent with the standard product (phenethyl isothiocyanate purchased from Tokyo Kasei).

Production Example 3 (Isolation and purification of 3-butenyl glucosinolate (BUGLS))
119 g of harvested Yamato mana was treated with 100 mL of distilled water heated to 80 ° C. for 20 minutes to inactivate myrosinase. Then, the mana returned to room temperature was homogenized with distilled water for 30 seconds and centrifuged (3100 rpm, 5 min) to obtain a hot mana extract and residue. Further, the residue was once again subjected to hot water extraction, and the obtained hot water extract was also combined with the first hot water extract. Subsequently, 5 mL of 1M zinc acetate was added to the hot water extract, and the protein was precipitated by stirring, followed by centrifugation to remove proteins. The obtained supernatant was purified with an anion exchange resin (DEAE Sephadex A-25), and finally, preparative HPLC (Cosmosil Hilic, CH ₃ CN: 10 mmol / mL ammonium acetate buffer (pH 5.6)). = 1: 1, 1.0 mL / min, 254 nm) to isolate 3-butenyl isothiocyanate glycoside (3-butenyl glucosinolate). This product was obtained as a yellow amorphous powder and was soluble in water. From the molecular ion peak value [m / z 372.04285 (M−H) −Δ−0.04 mmu] obtained by high resolution ESI-MS, the molecular formula is C ₁₁ H ₁₉ NO ₉ S ₂ , and 3- It was consistent with the molecular formula of butenyl isothiocyanate glycoside.
The ¹ H-NMR and ¹³ C-NMR spectrum data of this product are shown below. And from the analysis of these spectra, this product was determined to be 3-butenyl isothiocyanate glycoside.
¹ H-NMR (D ₂ O): 5.94 (1H, m, H10), 5.17 (1H, dd, J = 1.25 & 17.24 Hz, H-11a), 5.09 (1H, dd, J = 1.25 & 10.50 Hz, H-11b), 5.04 (1H, d, J = 9.83 Hz, H1), 3.90 (1H, d, J = 12.59 Hz, H-6a), 3 .72 (1H, dd, J = 5.63 & 12.59, H-6b), 3.57-3.47 (4H, m, H2, H3, H4 and H5), 2.84 (2H, m, CH2 -9), 2.49 (2H, m, CH2-8).
¹³ C-NMR (D ₂ O): 166.6 (C7), 139.6 (C10), 118.8 (C11), 84.6 (C1), 82.9 (C5), 79.9 (C3) ), 74.8 (C2), 72.0 (C4), 63.4 (C6), 36.8 and 35.4 (C8 and C9).

Production Example 4 (Isolation and purification of phenethyl glucosinolate (PEGLS))
119 g of harvested Yamato mana was treated with 100 mL of distilled water heated to 80 ° C. for 20 minutes to inactivate myrosinase. Then, the mana returned to room temperature was homogenized with distilled water for 30 seconds and centrifuged (3100 rpm, 5 min) to obtain a hot mana extract and residue. Further, the residue was once again subjected to hot water extraction, and the obtained hot water extract was also combined with the first hot water extract. Subsequently, 5 mL of 1M zinc acetate was added to the hot water extract, and the protein was precipitated by stirring, followed by centrifugation to remove proteins. The obtained supernatant was prepared by using anion exchange resin (DEAE Sephadex A-25), followed by preparative HPLC (Cosmosil Hilic, CH ₃ CN: 10 mmol / mL ammonium acetate buffer (pH 5.6) = 1: 1,1. (0.0 mL / min, 254 nm), phenethyl glucosinolate (phenethyl isothiocyanate glycoside) was obtained as a yellow amorphous powder. This product has a molecular formula of C ₁₅ H ₂₁ NO ₉ S ₂ from the molecular ion peak value [m / z 422.05850 (M−H) ⁻ , Δ−0.0 mmu] obtained by high resolution ESI-MS. It was consistent with the molecular formula of phenethyl glucosinolate. ¹ H-NMR and ¹³ C-NMR spectrum data are shown below, and these data are in good agreement with literature values (Fitoterapia, 72, 760-764, 2001).
¹ H-NMR (D ₂ O): 7.38 (4H, m, H11 and H12), 7.31 (1H, t, J = 7.00 Hz, H13), 4.89 (1H, d, J = 9.71 Hz, H1), 3.84 (1H, d, J = 12.80 Hz, H-6a), 3.67 (1H, dd, J = 5.10 & 12.80 Hz, H-6b), 3.47 (4H, m, H2, H3 , H4 and H5), 3.05 (4H, m, CH 2 -8 and CH 2 -9).
¹³ C-NMR (D ₂ O): 166.2 (C7), 143.4 (C10), 131.6 (C11 and C12), 129.5 (C13), 84.5 (C1), 82.9 (C5), 79.9 (C3), 74.7 (C2), 71.9 (C4), 63.4 (C6), 36.8 and 35.4 (C8 and C9).

Example 1 (Lipase inhibitory activity test)
A phenethyl isothiocyanate (PEITC) manufactured by Tokyo Chemical Industry was used. The lipase inhibitory activity of PEITC is lipase kit S (Dainippon Sumitomo Pharma) [substrate solution: dimercaprol tributyrate (BALB), sodium dodecyl sulfate (SDS); esterase inhibitor: phenylmethylsulfonium fluoride (PMSF); Agent: 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB); buffer: trisaminomethane buffer; reaction stop solution: sodium dodecyl sulfate (SDS)]. As the enzyme, porcine pancreatic lipase (ICN Biomedicals) was used to prepare a 1.0 unit / mL pancreatic lipase solution.

  Pancreatic lipase solution (5 μL), esterase inhibitor (2 μL), and coloring solution (73 μL) were mixed on a 96-well plate and preincubated at 37 ° C. for 5 minutes. Then, distilled water or methanol (10 μL) and a substrate solution (10 μL) were mixed as a sample solution or a control, and incubated at 37 ° C. for 30 minutes. However, the substrate solution was added to the blank after adding the reaction stop solution. Thereafter, a reaction stop solution (200 μl) was added and mixed well, and the absorbance was measured at 405 nm. The reaction rate (%) of the sample with the lipase was calculated by Equation 1. The results are shown in FIG.

Example 2 (Obesity inhibitory activity and fat accumulation inhibitory activity test)
Four groups of 5-week-old mice (female, Slc: ddY) were prepared. Each group consisted of 8 mice, and each group was divided so that the average value of the weight of each group at the start of the test was the same after the preliminary breeding for 1 week. The standard diet group is given the standard diet shown in Table 1, the high fat occupation group is given the high fat diet of Table 1, and the test group (BUGLS intake group and PEGLS intake group) is 0 in the high fat diet of Table 1. Feeding was started with a feed supplemented with 0.03% BUGLS or PEGLS. From the start of breeding, body weight and food intake were measured twice a week. The breeding days were 56 days, blood was collected after fasting for 12 hours, and blood lipids were measured. The animals were sacrificed on the 57th day from the start of the breeding, and the weight of the parauterine adipose tissue was measured.

  The weight measurement results are shown in FIG. 2, and the food intake measurement results are shown in FIG. As shown in FIG. 2, the weight of the high fat diet group mice significantly increased compared to the standard diet group. On the other hand, the increase in body weight was suppressed in the mice of the test group given the high fat diet plus 0.03% BUGLS or PEGLS compared to the mice in the high fat diet group.

  Moreover, the para-uterine adipose tissue weight measurement result is shown in FIG. As a result, the PEGLS administration group showed a significant effect of suppressing fat accumulation compared to the high fat diet group, and BUGLS showed a tendency to inhibit fat accumulation.

  According to the present invention, a lipase inhibitor and an obesity inhibitor containing isothiocyanate derived from a Brassicaceae genus plant food or a glycoside thereof, having few side effects and capable of continuous or preventive intake or administration, and further comprising the same Can be provided.

Shows lipase inhibitory activity of phenethyl isothiocyanate (PEITC) The result of body weight measurement of mice in a high fat diet group, a standard diet group, a BUGLS administration group, and a PEGLS administration group is shown. The amount of food of the mouse | mouth of a high fat diet group, a standard diet group, a BUGLS administration group, and a PEGLS administration group is shown. The para-uterine fat tissue weight measurement result of the mouse | mouth of a high fat diet group, a standard diet group, a BUGLS administration group, and a PEGLS administration group is shown.

Claims (9)

  A lipase inhibitor characterized by containing an isothiocyanate compound and / or a glycoside thereof derived from Brassicaceae plants.   An obesity inhibitor comprising an isothiocyanate compound derived from a Brassicaceae Brassica plant and / or a glycoside thereof.   The agent according to claim 2, which is a fat accumulation inhibitor.   The agent according to any one of claims 1 to 3, wherein the Brassicaceae genus Brassica plant is Yamato mana (Brassica rapa L.).   The agent according to any one of claims 1 to 3, wherein the isothiocyanate compound is phenethyl isothiocyanate.   The agent according to claim 2 or 3, wherein the isothiocyanate compound is 3-butenyl isothiocyanate.   A lipase inhibitor or anti-obesity agent characterized by containing Yamato mana itself or an extract thereof as an active ingredient.   A pharmaceutical product comprising the agent according to any one of claims 1 to 7.   Food-drinks which mix | blend the agent of any one of Claims 1-7.