JP2011182742A - Composition having both dipeptidyl peptidase iv inhibitory activity in blood and secretion promotion activity of glucagon-like peptide-1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material effective for preventing and treating diabetes and obesity by increasing endogenous GLP-1. <P>SOLUTION: Glucagon-like peptide-1 (GLP-1) being a digestive tract hormone is known to have an antidiabetic effect, however, only promotion of secretion of GLP-1 exhibits an insufficient effect, inhibition of decomposition as well as promotion of secretion is effective, concretely, a remarkable effect is obtained by inhibiting dipeptidyl peptidase-IV(DPP-IV) in blood. The preventive or therapeutic agent for diabetes or obesity comprises corn protein Zein hydrolyzate (ZeinH) having both secretion promotion action on GLP-1 and inhibitory action on DPP-IV or its amino acid sequence Leu-Pro-Phe as an active ingredient. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to corn protein Zein hydrolyzate which has both the action of promoting secretion of glucagon-like peptide-1 (hereinafter referred to as GLP-1), a gastrointestinal hormone having anti-diabetic action, and the activity of inhibiting blood dipeptidyl peptidase-IV. The present invention relates to a preventive or therapeutic agent for diabetes or obesity containing a degradation product (hereinafter referred to as ZeinH) as an active ingredient, or an amino acid sequence thereof as an active ingredient.

The spread of metabolic syndrome including impaired glucose tolerance is a serious social problem, and the development of effective preventive and therapeutic methods is desired.
Glucose intolerance is accompanied by a failure of insulin secreted from pancreatic beta cells (insulin resistance) or a secretion failure. Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone produced by gastrointestinal endocrine cells (L cells) that are present in large amounts in the ileum and large intestine. Secreted from L cells
GLP-1 (7-37), GLP-1 (7-36) Amides are active forms. Its activity has effects such as promotion of insulin secretion from pancreatic beta cells, promotion of proliferation of pancreatic beta cells and suppression of apoptosis, as well as an appetite suppression effect. Because of these effects, GLP-1 analogs (liraglutide, exenetide) that are not easily degraded in blood have been applied as therapeutic agents for type 2 diabetes and prevention of obesity.
Active GLP-1 is dipeptidyl peptidase-IV (DPP-IV) in blood
As a result, the N-terminal 2 residues are rapidly cleaved and become inactive, so that the half-life of active GLP-1 is as short as about 2 minutes (Non-patent Document 1). GLP-1 In the blood for the purpose of extending the half-life
Drugs that inhibit DPP-IV have also been introduced as anti-diabetic drugs. However, there have been no reports of food-based foods that inhibit DPP-IV in the blood.
On the other hand, carbohydrates, lipids, etc. are well known as substances that promote GLP-1 secretion. However, for the purpose of preventing obesity and improving glucose tolerance, excessive intake of these components causes the pathogenesis of metabolic syndrome. Therefore, it is not appropriate.
There are reported examples of drugs that promote the secretion of GLP-1 to suppress the increase in blood sugar (Non-patent Document 2) and free amino acids (glutamine) (Non-patent Document 3), but this is achieved by eating foods. There is no.
There have been reports on food peptides that promote GLP-1 secretion in the past (Non-Patent Document 4), but no effect of promoting insulin secretion or improving glucose tolerance via this has been reported. In the experiment of applicants
Although in vitro secretion-inducing activity can be confirmed, it has been found that no in vivo action occurs.
Many drugs that inhibit DPP-IV (DPP-IV inhibitors) have been developed for the purpose of extending the half-life of GLP-1. For example, sitagliplin is the first product that was launched in Japan in 2009
DPP-IV inhibitor. In foods, cheese-derived peptides have been shown to have DPP-IV inhibitory activity in vitro (Patent Document 1).
There are no reports showing enhanced effects by increasing the half-life of GLP-1 in vivo.
Zein is a molecular weight that accounts for about 50% of protein components in corn.
24,000 hydrophobic proteins belonging to the prolamin family which is insoluble in water and soluble in alcohol. It is known as a typical indigestible protein because it is less susceptible to degradation in the digestive tract. (Non-Patent Document 5)

Patent Publication 2007-39424: Dipeptidyl peptidase IV inhibitor Patent publication WO / 2009/107660, international application PCT / JP2009 / 053409: preventive or therapeutic agent for diabetes or obesity

Hansen L et al., Glucagon-like peptide-1- (7-36) amide is transformed to glucagon-like peptide-1- (9-36) amide bydipeptidyl peptidase IV in the capillaries supplying the L cells of the porcineintestine.Endocrinology. 1999 140 : 5356-5363. Chu ZL et al., A role for intestinal endocrinecell-expressed G protein-coupled receptor 119 in glycemic control by enhancingglucagon-like Peptide-1 and glucose-dependent insulinotropic Peptide release.Endocrinology. 2008, 149 (5): 2038-47. Greenfield JR et al., Oral glutamine increasescirculating glucagon-like peptide 1, glucagon, and insulin concentrations inlean, obese, and type 2 diabetic subjects. Am J Clin Nutr. 2009, 89 (1): 106-13. Dumoulin V et al., Peptide YY, glucagon-likepeptide-1, and neurotensin responses to luminal factors in the isolatedvascularly perfused rat ileum. Endocrinology. 1998, 139 (9): 3780-6. Shinji Kakinuma, Teruo Nakahisa, Kenichi Otsubo "Science of Corn" Asakura Shoten, 2009 Umezawa et al., Diprotins A and B, inhibitorsof dipeptidyl aminopeptidase IV, produced by bacteria.J. Antibiot (Tokyo) .1984, 37 (4): 422-5.

The problem to be solved is to develop foods that can be ingested for a long period of time as a preventive and therapeutic method for chronic diseases such as diabetes and obesity, and highly safe foods and useful substances based on foods.
As mentioned above, the gastrointestinal hormone GLP-1 has an effective physiological action for the prevention and treatment of diabetes and obesity, but it is not effective only by increasing secretion from the gastrointestinal endocrine cells, and it is not effective in blood. Suppressing the decomposition is essential for fully exerting the action. However, there has been no ingredient that has both of these and can be safely ingested as a food. An object of the present invention is to provide a safe and highly effective preventive or therapeutic agent for diabetes and obesity by using a material having both GLP-1 secretion promotion and degradation inhibition.

In order to solve the above problems, the inventors have eagerly searched in vitro and in vivo for a peptide that strongly promotes the secretion of GLP-1, and obtained the corn protein Zein.
Of papain hydrolyzate (ZeinH) was found to have this effect. Furthermore, it was found that administration of this peptide to the rat intestinal tract promotes GLP-1 secretion, thereby promoting insulin secretion, and as a result, has an effect of suppressing blood glucose elevation. At this time, it was found that administration of ZeinH simultaneously decreased the DPP-IV activity in the blood,
We found that inhibition of DPP-IV by ZeinH was involved, and that this inhibitory action was essential for the suppression of blood glucose elevation. As a peptide having this DPP-IV inhibitory activity,
A peptide recognized by DPP-IV contained in the Zein sequence was identified to complete the present invention. In addition, ZeinH also suppressed the increase in blood glucose even after oral administration.
Proline (Pro) contained in Zein as a peptide with DPP-IV inhibitory activity
Alternatively, a peptide having an amino acid chain length of 2 to 5 containing alanine (Ala) is effective. For example, a tripeptide called Leu-Pro-Phe can be used. In addition, GLP-1
It was also revealed that it has a secretory promoting effect.

In the present invention, in addition to the promotion of GLP-1 secretion, the blood DPP-IV inhibition is combined to effectively suppress an increase in blood glucose level. As an oral composition for use, and as an oral composition for anti-obesity, it can be used for the prevention and treatment of obesity and diabetes.
By inhibiting DPP-IV, in addition to GLP-1, GIP (Glucose
The actions of dependent insulinotropic polypeptide) and GLP-2 (Glucagon lilke-peptide-2) are also maintained. GIP
Can be used for the prevention and treatment of diabetes because it has an insulin secretion-promoting effect similar to GLP-1. GLP-2 has a proliferative action of the intestinal epithelium and a bone growth action, so DPP-IV
Inhibitory peptides can also be used for these.

Corn Zein hydrolyzate was administered into the rat intestine to prove the secretion promoting activity of glucagon-like peptide-1 (GLP-1).
In the experiment, water and a meat protein hydrolyzate (meat peptone) whose GLP-1 secretion activity was reported in the past (Non-patent Document 4) were used as a control composition.

<Experiment method>
<Administered composition>
Preparation of corn Zein hydrolyzate (ZeinH). Corn Zein (Tokyo Kasei) is suspended in pure water and adjusted to pH 7.0 to 7.2, and then papain (Papain F manufactured by Asahi Breweries) is added to the substrate at 0.5%, and 60 ° C. at 55 ° C. Shake for minutes. After shaking, heat treatment was performed at 100 ° C. for 20 minutes to deactivate papain. Thereafter, the precipitate is removed by filtration and centrifugation, and the final filtered product with a 0.45 micrometer membrane filter is lyophilized,
Zein hydrolyzate (ZeinH) was obtained. 500 mg of this was dissolved in 2 mL of water. Preparation of meat protein hydrolyzate (MHY). Meat peptone (Sigma)
500 mg was dissolved with 2 mL of water.
<Experimental animal and administration and sampling method>
The experimental animals used were SD strains, male rats, and 7 weeks old. In the rat jugular vein and ileum, blood collection and sample administration cannulas were placed under anesthesia, respectively, and fixed subcutaneously to the back of the neck. An intraperitoneal glucose tolerance test (IPGTT) was performed after an overnight fast. The test composition (500 mg dry weight dissolved in 2 mL of water) was administered directly into the intestine via the ileal cannula without anesthesia after the treatment was recovered. 30 minutes later, glucose (1 g / kg
(Body weight) was administered intraperitoneally (0 min).
<Collecting and analyzing blood>
Using a cannula placed in the jugular vein, jugular vein blood was collected over time.
Plasma was collected from the collected blood by centrifugation, and the blood glucose level was measured with a glucose measurement kit (Wako Pure Chemical Industries, Glucose C-II test kit). By enzyme linked-immuno solvent assay (ELISA), insulin concentration (Shibayagi), Total
GLP-1 concentration (Yanaihara Laboratory) and active GLP-1 concentration (Rinco) were measured.

<Result>
Thirty minutes before the intraperitoneal administration of glucose, jugular vein blood was collected (minus 30 minutes), and the following three sample solutions were respectively administered to the ileum site. The time when the glucose solution was intraperitoneally administered (1 g / kg) was set to 0 minute, and jugular vein blood was collected 15, 30, and 60 minutes after glucose administration.
ZeinH and meat peptone all administered to the ileum site, Total GLP-1
(Fig. 3). The ZeinH administration group showed a slightly higher value than the meat peptone administration group, but there was no statistically significant difference between the two groups except after 60 minutes of glucose load.
However, the increase in blood glucose level was observed only in the ZeinH administration group (FIG. 1). The insulin level at that time is ZeinH
The administration group showed a higher value than the other two groups (FIG. 2). Thus, it was shown that ZeinH strongly promotes insulin secretion and suppresses an increase in blood sugar.

Total
Since there was no difference in the variation of GLP-1 between ZeinH and meat peptone administration groups, active GLP-1 (Active GLP-1) was measured.
In the administration group, the value was significantly higher than that of the other groups at 0 minutes (30 minutes after administration of ZeinH) (FIG. 4). It is considered that insulin secretion was induced by this high active GLP-1 level.
Although the level of Total GLP-1 is similar, active GLP-1 is
High in the administration group indicates that ZeinH and meat peptone have similar GLP-1 secretion activities, but in the ZeinH administration group, GLP-1 secreted in the blood maintained the activity. ing. GLP-1
Is rapidly degraded by DPP-IV in the blood and becomes inactive. This result suggests that blood DPP-IV activity may be suppressed in the ZeinH administration group. So, ZeiH
A test was conducted to measure blood DPP-IV activity after administration.

Furthermore, the following experiment was conducted. Corn Zein hydrolyzate is administered into the intestine of rats under anesthesia and blood dipeptidyl peptidase-IV (DPP-IV)
Inhibitory activity was demonstrated.
In the experiment, water and a meat protein hydrolyzate (meat peptone) whose GLP-1 secretion activity was reported in the past (Non-patent Document 4) were used as a control composition.
The experimental animals used were SD strains, male rats, and 7 weeks old. After fasting overnight, the rats were recovered under anesthesia and a blood cannula was placed in the ileal vein. Make ileal ligation loop, water 2mL, ZeinH
Solution or MHY solution (500 mg dissolved in 2 mL water) was administered into the ligation loop. Thirty minutes later, glucose (1 g / kg body weight) was intraperitoneally administered (0 minute).
Ileal venous blood was collected over time from before the sample administration (minus 30 minutes) to 60 minutes after administration.
Plasma is separated from the collected blood, and Gly-Pro p-nitroanilide
Plasma DPP-IV activity was measured using p-toluenesulfonate salt.

As a result of the experiment, administration of ZeinH to the ileum site showed a significant decrease in DPP-IV activity in mesenteric venous blood at 0 minutes (immediately before intraperitoneal glucose administration) (FIG. 5). There was no change in the water and meat peptone groups.
It was suggested that the peptides contained in the drug were absorbed from the intestinal tract and inhibited DPP-IV in mesenteric venous blood. This decrease in DDP-IV activity is thought to contribute to the increase in active GLP-1. Because DPP-IV is constantly present in the blood,
Rather than suppressing the secretion and synthesis of ZeinH, it was suggested that a peptide fragment of ZeinH absorbed from the intestine inhibited DPP-IV in the blood. Ie
It was suggested that ZeinH promotes secretion of GLP-1 in the lumen and that the absorbed fragments inhibit DPP-IV to maintain and enhance GLP-1 action.

Effect of peptide sequence contained in Zein on blood DPP-IV activity

<Experiment method>
DPP-IV recognizes proline (Pro) or alanine (Ala), the second residue from the N-terminus, and hydrolyzes the substrate peptide. Diprotin
A (DPA) is a tripeptide having an Ile-Pro-Ile sequence and having a DPP-IV inhibitory action (Non-patent Document 6). The amino acid of corn Zein (Fig. 6, Non-Patent Document 7) contains Leu-Pro-Phe.
5 sequences (LPF, underlined in FIG. 6) were included, and it was considered that this tripeptide may be absorbed from the intestinal tract and contribute to DPP-IV inhibition in blood.
In order to examine the DPP-IV inhibitory activity of LPF, rat plasma DPP-IV activity was measured in the same manner as described above in the presence of various tripeptides.

1 MAAKIFSILM LLALSACVLD ATIFPQYSQA PIAALLPPYL PSMTASVCEN
PTLQPYRLQQ
61 AIATSNLPLS PLLFQQSPAL SLVQSLVQTI
RAQQLQQLVL PLINQVALAN LSPYSQQQQF
121 LPFNQLSTLN LAAYLQQQLL PFSQLATAYS
QQQQFLPFNQ LAALNPAAYL QQQILLPFGQ
181 LATTNRASFL TQQQLLPFYQ QFSANPATLL
QLQQLLPFVQ LALTNPAAFY QQHIIGGAIF

Fig. 6 Amino acid sequence of Zein (from NCBI Reference Sequence: NP_001105888.1)

NCBI Reference Sequence: NP_001105888.1

<Result>
In DPP-IV activity measurement using rat plasma, DPA, LPF, glutathione (gamma-Glu-Cys-Gly:
When GSH) was allowed to coexist with 300 microM, DPA showed an inhibitory action of over 80%, and LPF showed an inhibitory action of about 50% (FIG. 7). A similar tripeptide, GSH, showed no inhibitory effect. In addition, 50% inhibitory concentration (IC50) was found to be 18 for DPA.
In microM and LPF, it was shown to be 205 microM (FIG. 8). Thus, the tripeptide LPF contained in maize Zein was found for the first time to have DPP-IV inhibitory activity. It is unclear how much LPF has migrated into the blood and contributed to the DPP-IV inhibition seen in FIG. 5, but by inhibiting the activity of DPP-IV in the mesenteric vein, It was suggested that the degradation of active GLP-1 secreted in mesenteric venous blood may be suppressed by promotion of intracavitary ZeinH.

The GLP-1 secretion action of peptide LPF was examined.
GLP-1-producing cell line GLUTag derived from mouse large intestine, Dulbecco's containing 10% FBS
The cells were cultured in modified Eagle's medium at 37 ° C in the presence of 5% CO2. GLUTag cells were cultured in 48-well plates for 2-3 days until sub-confluent. Before adding the sample, add Hepes buffer (140
mM NaCl, 4.5 mM KCl, 20 mM Hepes, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM D-glucose,
The wells were washed with 0.1% BSA, pH 7.4), the sample was dissolved in the same buffer, 80 microliters of the sample solution was added, and the mixture was incubated at 37 ° C. for 60 minutes. As a sample, 3.33
mM LPF was used. This corresponds to the concentration of LPF contained in ZeinH 20 mg / mL. In addition, GLP-1 secretion is induced by Hepes buffer (Blk) as a negative control and depolarization stimulation as a positive control.
mM KCl solution was used. After recovering the supernatant, the cells were precipitated by centrifugation (800 × g, 5 minutes, 4 ° C.), and 70 microliters of the supernatant was recovered and stored frozen. The GLP-1 concentration in the supernatant was measured using a commercially available Enzyme.
Measured with an immuno assay kit (Yanaihara Laboratory).

<Result>
With peptide LPF (3.33 mM), significant GLP-1 comparable to 70 mM KCl
Increased secretion was observed (FIG. 9). In the case of 20 mg / ml ZeinH, GLP-1 secretion increased 3 to 4 times that in the control group (Blk).
Although GLP-1 secretion activity does not depend only on LPF, it was shown that LPF itself has GLP-1 secretion promoting action. That is, LPF promotes GLP-1 secretion and DPP-IV
It was revealed that the peptide has both inhibitory effects.
As oligopeptides that promote GLP-1 secretion from GLUTag cells, gamma-Glu-Cys-Gly (glutathione) and gamma-Glu-Cys (gamma EC) have been reported (Patent Document 2).
Compared to this, LPF has higher activity because it promoted GLP-1 secretion at a concentration of 20 mM.

<Experimental Method> An intraperitoneal glucose tolerance test (IPGTT) was performed after overnight fasting using SD male rats, 7 weeks old. Water (8 mL / kg body weight) and ZeinH solution (2 and 4 g / kg) were orally administered using a feeding tube, and a glucose solution was intraperitoneally administered 15 minutes later. Blood was collected from the rat tail vein at minus 15 minutes (immediately before sample administration), 0, 15, 30, 60, 90, and 120 minutes, and the glucose concentration in plasma was measured in the same manner as above. did.
<Results> ZeinH was orally administered to suppress the increase in blood glucose concentration-dependently (FIG. 10). At 15 and 30 minutes after glucose loading, the blood glucose level was significantly lower by administration of 2 g / kg compared to the water administration group (Control), and the 2 g / kg administration group was also significantly lower in the 4 g / kg administration group. The value is shown. Here, since glucose is administered intraperitoneally, the action of ZeinH is not suppressed glucose absorption, but is thought to be mediated by GLP-1 and insulin secretion. As a result, it was clarified that ZeinH has an inhibitory effect on blood glucose elevation even by oral administration.

Foods and therapeutic agents for preventing and treating diabetes and obesity

Change in glucose concentration in the jugular vein. The change of the glucose concentration in the jugular vein in the intraperitoneal glucose tolerance test after water, meat peptone, and ZeinH administration to a rat ileum is shown. Values are mean ± standard error (n = 6-7). + Indicates that there is a statistically significant difference with respect to a value of −30 minutes. There is a statistically significant difference between values with different alphabets at the same time. Both are Fisher'stest, P <0.05. Jugular blood insulin concentration. The change of the insulin level in the jugular vein blood in the intraperitoneal glucose tolerance test after water, meat peptone, and ZeinH administration to a rat ileum is shown. Values are mean ± standard error (n = 6-7). + Indicates that there is a statistically significant difference with respect to a value of −30 minutes. There is a statistically significant difference between values with different alphabets at the same time. Both are Fisher'stest, P <0.05. Total GLP-1 concentration in jugular vein blood. The change of the total GLP-1 density | concentration in the jugular vein blood in the intraperitoneal glucose tolerance test after water, meat peptone, and ZeinH administration to a rat ileum is shown. Values are mean ± standard error (n = 6-7). + Indicates that there is a statistically significant difference with respect to a value of −30 minutes. There is a statistically significant difference between values with different alphabets at the same time. Both are Fisher'stest, P <0.05. Active GLP-1 concentration in jugular vein blood. The change of the active GLP-1 density | concentration in the jugular vein blood in the intraperitoneal glucose tolerance test after water, meat peptone, and ZeinH administration to a rat ileum is shown. Values are mean ± standard error (n = 6-7). + Indicates that there is a statistically significant difference with respect to a value of −30 minutes. There is a statistically significant difference between values with different alphabets at the same time. Both are Fisher'stest, P <0.05. DPP-IV activity in ileal venous blood. The change of DPP-IV activity in the ileal venous blood after water, meat peptone, and ZeinH administration to a rat ileum is shown. Values are mean ± standard error (n = 6-7). Values are expressed as% relative to activity before sample administration (minus 30 minutes). * Indicates that there is a statistically significant difference for the minus minus 30 minute value (Dunnet'st-test, P <0.05). Statistically significant difference between values with different alphabets (Fisher's test, P <0.05). Amino acid sequence of maize Zein protein (from NCBI ReferenceSequence: NP_001105888.1) Blood DPP-IV activity in the presence of various tripeptides (DPA: Leu-Pro-Ala, LPF: Leu-Pro-Phe, GSH: gamma Glu-Cys-Gly). The value is the average of three measurements ± standard error. Blood DPP-IV activity in the presence of various tripeptides (DPA: Leu-Pro-Ala, LPF: Leu-Pro-Phe) at various concentrations. The value is the average of two measurements. GLP-1 secretion in a GLP-1 producing cell line GLP-1 secreting mouse colon-derived GLP-1 producing cell line GLUTag is exposed to a 70 mM KCl solution and a 3.33 mM LPF solution for 1 hour, and the GLP-1 concentration in the supernatant is shown. The value is the mean value of the measurement ± standard error (n = 3). Statistically significant difference between values with different alphabets (Tukey'stest, P <0.05). The blood glucose level at the time of oral administration of ZeinH in rats The change of the glucose concentration in the jugular vein in the intraperitoneal glucose tolerance test after oral administration of water and ZeinH solution to rats is shown. Values are mean ± standard error (n = 6-7). There is a statistically significant difference between values with different alphabets at the same time. Both are Fisher'stest, P <0.05.

Corn Zein Papain Hydrolyzate Corn Zein protein was suspended in water, papain was added at a substrate concentration of 0.5%, and reacted at 55 ° C. for 60 minutes. The reaction supernatant was lyophilized to obtain a Zein hydrolyzate (ZeinH).

Claims (4)

A composition having both dipeptidyl peptidase-IV (DPP-IV) inhibitory activity in blood and secretion promoting activity of glucagon-like peptide-1 (GLP-1), comprising hydrolyzate of corn Zein protein as an active ingredient A composition having both blood dipeptidyl peptidase-IV (DPP-IV) inhibitory activity and glucagon-like peptide-1 (GLP-1) secretion-promoting activity, comprising a peptide having the amino acid sequence Leu-Pro-Phe as an active ingredient A composition having both blood dipeptidyl peptidase-IV (DPP-IV) inhibitory activity and glucagon-like peptide-1 (GLP-1) secretion-promoting activity comprising a peptide containing the amino acid sequence Leu-Pro-Phe as an active ingredient Foods for preventing and treating foods and therapeutic agents for diabetes and obesity comprising the components according to claim 1, claim 2 or claim 3.