JP7089726B2 - Dipeptidyl peptidase IV (DPPIV) derived from fish nodes and cathepsin S and L inhibitory compositions, as well as pharmaceutical compositions and health functional foods containing the above compositions. - Google Patents

Description

INDUSTRIAL APPLICABILITY The present invention is a dipeptidyl peptidase IV (DPPIV) derived from fish bonito, a catepsin S and L inhibitory composition, a pharmaceutical composition containing the composition, and a health functional food , which have been conventionally used as foodstuffs. It relates to a highly safe fish section, for example, the composition derived from bonito flakes, a pharmaceutical composition, and a food with health claims .

Recently, the number of patients suffering from adult diseases, especially diabetes, is increasing, and for diabetes, attempts have been made to relieve those symptoms by administering substances having dipeptidyl peptidase IV (DPPIV) inhibitory activity to the patients. ing. This is based on the principle of inhibiting the activity of DPPIV, which decomposes incretins that enhance insulin secretion.
Previously, chemically synthesized substances were the mainstream as substances having an inhibitory activity on DPPIV, but in recent years, from the viewpoint of safety, natural products or food-derived substances, specifically natural or food-derived substances, Tory or oligo-peptides are being recommended as such substances.
For example, Patent Document 1 by the present applicant relates to a DPPIV inhibitory active composition containing a peptide derived from fish bonito, for example, bonito flakes, which is a food, by inhibiting the DPPIV activity by administering the composition, and the blood glucose level. It is disclosed that it can be reduced.

Japanese Patent No. 5872725

The invention described in Patent Document 1 relates to a composition containing a peptide that expresses only DPPIV inhibitory activity as a physiological activity, but if a single peptide having a plurality of physiological activities is found. By administering the peptide as an active ingredient, it is considered possible to treat or prevent a plurality of diseases at the same time.
Further, since it is not necessary to use a plurality of active ingredients having different physiological activities in combination, there is no concern about antagonistic action between the active ingredients, and it is expected that each active ingredient is effectively exerted.
If such an active ingredient is found from food-derived ones, it is expected to be used for efficient health functional foods or pharmaceuticals which are highly safe and can exert multiple physiological activities with a single active ingredient. Will be done.
Therefore, it has been an object of the present invention to find such an active ingredient and a composition containing the active ingredient from foods such as peptides derived from fish ass.

As a result of diligent research, the present inventor has found a peptide derived from fish nodes having a relatively high DPPIV inhibitory activity as well as a cathepsin inhibitory activity at the same time, and a composition containing the peptide, and completed the present invention. I arrived.
This fact was very surprising because the existence of a single peptide having both DPPIV inhibitory activity and cathepsin inhibitory activity was not known so far.

That is, the present invention is a composition derived from cathepsin, and the amount of tryptophan-valine (Trp-Val) or a salt thereof as an active ingredient is 0.001 to 0.1% by mass based on the dried product of the composition. The present invention relates to a dipeptidyl peptidase IV (DPPIV) and a cathepsin S and L inhibitory composition, which are characterized by being contained in .
A preferred embodiment of the present invention relates to the DPPIV inhibitory composition according to claim 1, which is characterized by having a cathepsin inhibitory activity of at least 3000 μg / mL (IC50).
Another aspect of the present invention relates to a pharmaceutical composition or a food with health claims, which comprises the above-mentioned dipeptidyl peptidase IV (DPPIV) and a cathepsin S and L inhibitory composition .

Cathepsin is a type of proteolytic enzyme contained in intracellular lysosomes, and it is known that cysteine proteases called cathepsin S and cathepsin L are involved in the degradation of connective tissue [Esser et al., Artristis & Rheumatism, 37. (2), 236 (1994)].
Excessive increase of these cathepsins is the cause of diseases associated with destruction of connective tissues (called connective tissue diseases) such as osteoarthritis, rheumatoid arthritis, and osteoporosis, or bone diseases for which inhibition of bone resorption has been pointed out. Therefore, cathepsin inhibitors (cysteine protease inhibitors) are considered to be effective in the prevention or treatment of these diseases.
Cathepsin inhibitors are also used to treat cysteine cathepsin-dependent diseases and symptoms, including tumors (particularly tumor invasion and tumor metastasis), coronary artery disease, atherosclerosis (including atherosclerotic plaque destruction and destabilization). obtain.

On the other hand, DPPIV is an incretin-degrading enzyme that enhances insulin secretion. By inhibiting this DPPIV, the decomposition of incretin is suppressed and the concentration of incretin in the blood is increased, and as a result, insulin secretion is promoted and the blood glucose level is lowered.
Therefore, the DPPIV inhibitor is expected to be used as an antidiabetic agent.

The composition of the present invention has both DPPIV inhibitory activity and cathepsin inhibitory activity, and therefore, not only as an agent for suppressing an increase in blood glucose level, but also as a preventive and therapeutic agent for connective tissue disease, or as a tumor inhibitor. Is also expected.
In this case, it can be applied not only to pharmaceutical products but also to foods with health claims such as foods for specified health use.
Such a composition of the present invention can be obtained as follows.

Roughly speaking, the composition of the present invention can be obtained by enzymatically decomposing a fish section, then separating and purifying it, but from the economical point of view, the fish section is extracted with hot water rather than the fish section itself. It is preferable to use the fish knots that remain after refining.
Fish clause is a food that has been eaten for a long time and is very preferable in terms of safety. Examples of the fish bonito used in the present invention include bonito flakes, Soda bonito flakes, mackerel flakes, bonito flakes, horse mackerel flakes, and tuna flakes. It is sufficient that these fish sections are manufactured by a method known to those skilled in the art, and of course, they may be those on the market.

For fish-bushi lees, for example, water is added to an appropriately ground bonito flakes and heated to a temperature of about 50 ° C. or higher, preferably 80 ° C. to 100 ° C. for about 5 to 60 minutes. It can be obtained as a precipitate excluding the supernatant by extracting with water and then centrifuging. The supernatant obtained at the same time can be eaten as it is or used as a component of a seasoning.

The resulting fish lees are then hydrolyzed with the appropriate protease. From the viewpoint of safety, such proteases are selected for use in the food industry. In particular, a protease derived from a strain selected from the genus Aspergillus oryzae is preferable, and in particular, Sumiteam LP50D (manufactured by Shin Nihon Kagaku Kogyo Co., Ltd .; genus Aspergillus oryzae; optimum temperature 45 ° C to 60 ° C (50 ° C); optimum pH 5 to 8 (PH 7)) is preferable.

The above-mentioned protease was selected by the present inventors for the completion of the present invention, is highly safe for food industry use, and has higher DPPIV inhibitory activity when enzymatically decomposed cathepsin lees. And cathepsin inhibitory activity can be imparted to the enzymatically degraded composition.

The treatment conditions with the protease may be appropriately selected according to the characteristics of the protease. The amount of enzyme and the treatment time are not particularly limited, but the amount of enzyme is 0.1% to 2% relative to the raw material protein. The reaction time is preferably 2 to 20 hours. The reaction temperature is preferably 35 ° C to 60 ° C.
The protease treatment can be terminated by inactivating the enzyme by heating or the like. It is also desirable to neutralize the pH after the enzymatic reaction for subsequent commercial application.

The protease decomposition product obtained by the protease treatment is directly filtered or centrifuged by a known means to remove the undecomposed product to obtain a clarified solution (supernatant). From the viewpoint of obtaining highly active DPPIV inhibitory activity and cathepsin inhibitory activity in high yield, it is desirable to concentrate the obtained clear solution and further freeze-dry or spray-dry it. Further, by subjecting it to various purification methods, it is possible to obtain a fraction having higher DPPIV inhibitory activity and cathepsin inhibitory activity. Purification by a resin purification method is also preferable.

Examples of the resin used in the resin purification method include cation exchange resin, anion exchange resin, porous resin, special resin (chelate resin, synthetic adsorbent, protein separator) and the like, and the recovered fraction. It is preferable to use a synthetic adsorbent because the desalting treatment step of the above is not required. Examples of the synthetic adsorbent include, but are not limited to, aromatic (styrene-vinylbenzene) type, aromatic modified type, acrylic (methacrylic) type and the like.

Further, in the resin purification method, the protease decomposition product containing the DPPIV inhibitory component and the cathepsin inhibitory component may be adsorbed on the resin by a known method.
Next, for elution of the adsorbed protease decomposition product, an acid, an alkali or various organic solvents such as lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, acetone and the like are used. Ketones of, but are not limited to these. Alternatively, it may be used as a mixed solvent with an acid and an alkali. From the viewpoint of economy and safety, it is preferable to use an ethanol aqueous solution or water having a concentration of 50% or less for elution. The resin purification method can be carried out by a batch method or a column method. The recovered fraction may be concentrated by reduced pressure or ultrafiltration, and if necessary, the solvent may be completely removed and dried or freeze-dried.

Preferably, the clarified solution obtained after the protease treatment is purified and then subjected to elution with a stepwise gradient having an ethanol concentration of 10%, 25%, 50% and 99.5%. Thereby, a fraction containing tryptophan-valine (Trp-Val), which is an active ingredient of DPPIV inhibitory activity and cathepsin inhibitory activity, can be efficiently obtained.

In the present invention, the protease decomposition product is mixed with the organic solvent by an organic solvent precipitation method such as methanol or ethanol, the precipitate fraction and the supernatant fraction are separated, and the supernatant fraction is recovered. Therefore, a fraction having high DPPIV inhibitory activity and cathepsin inhibitory activity can be obtained. In this case, it is preferable to allow the organic solvent mixture to stand until the precipitate fraction and the supernatant fraction are separated.
The standing temperature is preferably low. In addition, the collected supernatant fraction may be concentrated by reduced pressure or ultrafiltration, and if necessary, the solvent may be completely removed and dried or freeze-dried.

The supernatant fraction thus obtained can be used as it is as the dipeptidyl peptidase IV (DPPIV) and the cathepsin S and L inhibitory composition according to the present invention, but preferably until the water content is about 5% by mass or less. Can be dried. Then, in the composition after drying, Trp-Val is contained in an amount of at least about 0.001% by mass, preferably at least about 0.01% by mass, or generally, based on the mass of the composition. It is contained in an amount of 0.001 to 0.1% by mass, preferably 0.01 to 0.1% by mass.
Such a composition according to the present invention has a DPPIV inhibitory activity value (IC50) of at least about 200 μg / mL (numerical value 200>). On the other hand, it also has a cathepsin inhibitory activity value (IC50) of at least about 3000 μg / mL (value 3000>).
These inhibitory activity values are considered to be sufficient values for the composition according to the present invention to be used as a food with health claims.

The dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory compositions according to the present invention can be ingested as a beverage as it is or as a mixture with various seasonings.
In addition, the health functional food containing the composition according to the present invention is produced by adding 100 mg to 400 mg, preferably 120 mg to 300 mg of the composition as a single ingestion.
Furthermore, the composition according to the present invention can be made into a stable solid or powder form that is easy to handle and is stable by drying, and the composition in the form is also good in solubility in water. It is also well absorbed from the gastrointestinal tract. Therefore, there are no particular restrictions on the timing and method of addition to the food composition, and it can be used in the food field as a powder, solution, suspension, etc. in the raw material stage, intermediate process, final process of food composition production. It can be added by conventional methods.

Examples of the form of foods with health claims, such as foods for specified health use, foods with nutritional claims, or foods with functional claims, include solid, semi-fluid, fluid, sheet-like, tablets such as tablets and capsules, and granule powder. can.
Examples of the semi-fluid form include a paste form, a jelly form, and a gel form.
In addition, examples of the fluid form include juice, soft drinks, tea drinks, and energy drinks.
By continuously ingesting these various forms of health functional foods as energy drinks and seasonings, the composition of the present invention suppresses the increase in blood glucose and / or prevents connective tissue diseases or tumors. It can be expected to be treated.

When the DPPIV inhibitor of the present invention is used as a pharmaceutical composition, for example, it may be administered to a patient with hyperglycemic symptoms before meals in order to inhibit the patient's DPPIV and exert an inhibitory effect on blood glucose elevation. Since the active ingredient of the pharmaceutical composition of the present invention is derived from a natural product, fish node, it can be continuously and safely used.
Oral administration agents such as tablets, capsules, granules and syrups are preferable as the form of the pharmaceutical composition. The liquid agent may be a dry solid that can be dissolved at the time of use.

Since the dipeptidyl peptidase IV (DPPIV) and the cathepsin S and L inhibitory compositions according to the present invention have moderately suppressed DPPIV inhibitory activity and cathepsin inhibitory activity as compared with other pharmaceuticals having a hypoglycemic effect. The risk of side effects is lower, and it can be said that it is more suitable as an active ingredient of foods with health claims such as foods for specified health use.
Such compositions according to the present invention are useful for the prevention or treatment of diabetes, connective tissue diseases or tumors in mammals.
Further, if the composition according to the present invention is derived from fish node, which is a food, the safety is further enhanced, and it can be said that the composition is more suitable as a food with health claims.
Further, in the present invention, since a dipeptidyl peptidase IV (DPPIV) and a cathepsin S and L inhibitory composition can be obtained by only one protease treatment step, there is an advantage that the production efficiency is high.

FIG. 1 is a graph showing changes in blood glucose level over time when 125 mg of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition according to the present invention are orally administered. FIG. 2 is a graph showing changes in blood glucose level over time when 250 mg of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition according to the present invention are orally administered. FIG. 3 is a graph showing changes in blood glucose level over time when 500 mg of dipeptidyl peptidase IV (DPPIV) and a cathepsin S and L inhibitory composition according to the present invention are orally administered.

Test example 1. Method for measuring DPPIV inhibitory activity The DPPIV inhibitory activity of each sample was as follows.
After lyophilizing the samples, 16 mg, 8 mg, 4 mg, 2 mg and 1 mg thereof were dissolved in 1 mL of 50 mM Tris-hydrochloric acid buffer (pH 7.5), respectively, and the DPPIV inhibitory activity of each sample solution obtained was determined by the DPPIV inhibitory activity. Measurement was performed using a measurement kit (manufactured by BioVision; K780-100 type).

After preparing DPPIV to a predetermined concentration within the reaction time range in which a linear calibration curve is observed, 50 μL of each Well is dispensed, each sample (freeze-dried product) is prepared to the above concentration, and then 25 μL is DPPIV. Was dispensed into each Well containing 50 μL (measured n = 4 times at each concentration of each sample) and reacted at 37 ° C. for 10 minutes.

Next, after adjusting the concentration of the pseudo sample substrate (incretin hormone) (H-Gly-Pro-7-amino-4-methylcoumarin) to a predetermined concentration, 25 μL was dispensed and the reaction time was 0 to 30 minutes. The DPPIV measurement inhibitory activity (%) was calculated from the control (without inhibitor) and the measured values of various samples from the rate of increase during the reaction time when the calibration curve was measured once, and 50% inhibition of the DPPIV. The concentrations of various samples in which activity was observed were calculated as IC50 values. In addition, each concentration of each sample was measured four times, and the average value of the obtained four IC50 values was calculated and used as the DPPIV inhibitory activity (%).
The DPPIV inhibition measurement was performed with a fluorescent microplate reader (manufactured by Thermo Fisher) having an excitation wavelength of 380 nm and a measurement wavelength of 460 nm under the measurement conditions.

Test example 2. Method for measuring cathepsin inhibitory activity The cathepsin inhibitory activity of each sample was, in principle, as follows.
A solution containing cathepsin L or cathepsin S and a sample (1 mg) were mixed and reacted at pH 5.0 for 30 minutes. Then, Z-Val-Val-Arg-MCA was added as a substrate, and the mixture was reacted at 37 ° C. for 30 minutes. After that, the fluorescence of the cleaved AMC was measured with a fluorescence spectrophotometer (the symbol MCA is an abbreviation for 4-methylkumalyl-7-amide, and the symbol AMC is an abbreviation for 7-amino-4-methylcoumarin. ).

Example. Production of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition according to the present invention Katsuobushi (bonito flakes) (protein 160 kg) was extracted with hot water under the conditions shown in Table 1 below (Table 1). ..

To the obtained bonito flakes (72% by mass of protein), 10 times the amount of water (against the substrate) and Sumiteam LP50D were added, adjusted to the optimum pH 7 of the Sumiteam LP50D, and then reacted at 50 ° C. for 16 hours. Then, it was deactivated at 98 ° C. for 15 minutes, centrifuged (15000 rpm, 5 minutes), and then a clarified solution was obtained (the clarified solution is called C1).

Next, the obtained C1 was separated into DPPIV inhibitory active fractions by stepwise gradients having ethanol concentrations of 10%, 25%, 50% and 99.5% (Table 2).
With the water elution fraction and the 10% ethanol elution fraction, the DPPIV inhibitory activity can be divided at a ratio of 9 times, and the IC50 = 102.93 μg / mL, which is 3 to 4 times higher than the IC50 of the enzymatic decomposition product (C1). A fraction was obtained (hereinafter, this obtained fraction is referred to as an A-2 fraction). The yield from the enzymatic degradation product (C1) was 20%, and the recovery rate of the inhibitory activity was about 100%.

The obtained A-2 fraction was purified by UPLC chromatography under the following conditions.
The analysis conditions of UPLC chromatography used are as follows.
Equipment: Waters UPLC
Column: Waters Accessy UPLC BEH C18 (2.1 mm ID x 150 mm L, 1.7 μm)
Mobile phase A: 5% acetonitrile in 0.1% trifluoroacetic acid
B: Isocraty of 25% acetonitril in 0.1% trifluoroacetic acid
The ratios of mobile phases A and B are shown in Tables 3 and 4 below.
Flow velocity: 0.2 mL / min Temperature: 40 ° C
Detector: Photodiode array Detection: UV 200nm-300nm
Sample concentration: Solid content 0.1 mg to 0.5 mg / 5 to 10 μL
Standard (synthetic dipeptide) concentration: 0.1 μg / 1 to 10 μL

Each fraction was repeatedly fractionated a plurality of times with different acetonitrile concentrations to increase the degree of purification (Table 5).

Then, the composition of the fraction obtained by UPLC rechromatography (third time) was subjected to N-terminal amino acid sequence analysis (protein sequencer: PPSQ-33A; Shimadzu Corporation). As a result, a peptide consisting of the amino acid sequence of tryptophan-valine (Trp-Val) was identified.

On the other hand, when the content of Trp-Val in the A-2 fraction was measured, it was 15.24 mg / 100 g based on the dry mass (water content of 5% by mass or less) of the fraction.

Furthermore, when the DPPIV inhibitory activity of the Trp-Val was measured as described in Test Example 1, the IC50 value was 36.99 μM (11.21 μg / mL).
The DPPIV inhibitory activity of the A-2 fraction was 102.93 μg / mL (IC50) as shown in Table 2 above.

Measurement example. Human oral test of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition according to the present invention The composition according to the present invention (dried product of A-2 fraction; water content 5) obtained in the above Examples. A human oral administration test was conducted on (mass% or less) to examine the dose suitable for a food with a health function having a decrease in blood glucose level.
First, the three subjects A, B, and C (all men in their 50s and 60s) were not affected by the accumulation of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory compositions in the body. , Plain hot water (peptide-free placebo) was ingested on the first day, and the day after the placebo effect disappeared, dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory compositions were ingested. Supper was eaten early and fasted until the test so as not to be affected by the diet.

The protocol for the effect of suppressing the increase in glucose load blood glucose is as follows.
On the first day, after fasting for 18 hours, 30 minutes after ingesting placebo (plain hot water 150 mL), the blood glucose level was measured every 15 minutes for 180 minutes from the time of glucose ingestion.
Day 2 After 18 hours of fasting, 30 minutes after ingestion of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition (125 mg / 150 mL, 250 mg / 150 mL, 500 mg / 150 mL), 180 every 15 minutes from the time of glucose ingestion. Blood glucose levels were measured up to minutes.
Intakes of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory compositions were assigned to subjects A, B and C as follows. This time, instead of a double-blind controlled trial, placebo and glycemic peptide were openly identified and ingested.
Subject A: 1st day placebo 150mL intake to 2nd day dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition 125mg / 150mL subject B: 1st day placebo 150mL intake to 2nd day dipeptidyl peptidase IV ( DPPIV) and cathepsin S and L inhibitory composition 250 mg / 150 mL subject C: Day 1 placebo 150 mL intake to day 2 dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition 500 mg / 150 mL intake

During the test, blood was collected from each subject and their blood glucose levels were plotted and graphed (FIGS. 1 to 3).
The vertical axis shows the blood glucose level, and the horizontal axis shows the time before and after ingestion, where placebo is indicated by a white circle and blood glucose peptide is indicated by a black circle.
As a result, it was shown that the dipeptidyl peptidase IV (DPPIV) and the cathepsin S and L inhibitory compositions according to the present invention have significant effects and dose dependence in small amounts (FIGS. 1 and 2). The safety was set up to a maximum safety repeat rate of 1 g / kg based on the results of animal experiments, but as is clear from Fig. 3, a high dose of 500 mg / human is slightly too effective in suppressing blood glucose levels. The result was shown.

Since the effect of suppressing the increase in glucose-loaded blood glucose was observed in humans at all intakes, the following can be considered.
Quality of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition 125 mg, one tablet solid 250 mg diameter 8 mm (125 mg dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition, excipient 125 mg) It turned out that it is possible to design a product for one tablet.
In the case of dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition 250 mg, one tablet solid 250 mg in diameter 8 mm (125 mg dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory composition, excipient 125 mg ), It was found that it is possible to design two tablets.
In the case of 500 mg of glycemic peptide, there was a concern from the results that the effect was slightly too effective.
Therefore, it was judged that the food having a health function is preferably in the range of 125 mg to 250 mg of dipeptidyl peptidase IV (DPPIV) and a cathepsin S and L inhibitory composition.
In the results of a similar human test with a conventional composition showing a blood glucose lowering effect as a control, for example, indigestible dextrin, no significant effect was obtained without using 5 g in a single dose. Considered, the above-mentioned effect of the composition of the present invention was about 40 times stronger.

Since the effect of suppressing the increase in glucose-loaded blood glucose was observed in humans at all intakes, the following can be considered.
In the case of 125 mg of the DPPIV inhibitory composition, it was found that the product design of one tablet is possible with the quality of one tablet solid 250 mg in diameter 8 mm (125 mg DPPIV inhibitory composition, excipient 125 mg).
Further, in the case of 250 mg of the DPPIV inhibitory composition, it was found that it is possible to design two tablets with the quality of one tablet solid 250 mg in diameter 8 mm (125 mg DPPIV inhibitory composition, 125 mg excipient).
In the case of 500 mg of glycemic peptide, there was a concern from the results that the effect was slightly too effective.
Therefore, it was determined that the DPPIV inhibitory composition in the range of 125 mg to 250 mg is preferable as the food with health claims.
In the results of a similar human test with a conventional composition showing a blood glucose lowering effect as a control, for example, indigestible dextrin, no significant effect was obtained without using 5 g in a single dose. Considered, the above-mentioned effect of the composition of the present invention was about 40 times stronger.

Claims (4)

A composition derived from cathepsin, which contains tryptophan-valine (Trp-Val) or a salt thereof as an active ingredient in an amount of 0.001 to 0.1% by mass based on the dried product of the composition. Characterized by dipeptidyl peptidase IV (DPPIV) and cathepsin S and L inhibitory compositions. The composition according to claim 1, wherein the composition has a cathepsin inhibitory activity of at least 3000 μg / mL (IC50). A pharmaceutical composition for inhibiting dipeptidyl peptidase IV (DPPIV) and cathepsins S and L, which comprises the composition according to claim 1 or 2 . A food with health claims for inhibiting dipeptidyl peptidase IV (DPPIV) and cathepsins S and L, which comprises the composition according to claim 1 or 2 .

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