阅读说明：本技术 免疫刺激剂和免疫刺激用食品饮料 (Immunostimulant and food and drink for immunostimulation ) 是由 阿部圭马 西冈浩 高野翔 高桥麻起子 松浦英幸 于 2020-03-26 设计创作，主要内容包括：一种免疫刺激剂,含有通式I(式I中,n表示4～12的整数)所表示的化合物或其药理学上允许的盐作为有效成分。(An immunostimulant comprises a compound represented by the general formula I (wherein n represents an integer of 4 to 12) or a pharmacologically acceptable salt thereof as an active ingredient.)

1. An immunostimulant comprising a compound represented by the general formula I or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 1]

In the formula I, n represents an integer of 4-12.

2. The immunostimulant according to claim 1, which comprises a compound represented by the formula II or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 2]

3. An immunostimulating food and drink containing a compound represented by the general formula I or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 3]

In the formula I, n represents an integer of 4-12.

4. The food or beverage for immunostimulation according to claim 3, comprising a compound represented by formula II or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 4]

Technical Field

The present invention relates to an immunostimulant and an immunostimulant food or drink.

Background

As immunostimulants for enhancing immunity, various substances have been used so far. For example, non-patent document 1 describes that fucoidan (fucoidan) has an immunostimulating effect.

On the other hand, various physiological activities of carboxylic acid esters have been reported. For example, patent document 1 reports an activity against leishmania, and patent document 2 reports an activity against diseases caused by filamentous fungi that are plant pathogens.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2016-160237

Patent document 2: japanese patent laid-open publication No. 2013-180995

Non-patent document

Non-patent document 1, yamaokamuhong et al, evaluation of the effectiveness and safety of fucoidan derived from laminaria japonica order in the immune function of healthy adults (ガゴメ function of the immune function へ of healthy adults derived from laminaria origin フコイダン. potently なら and に), japan supplement and alternative medical society, 2015, vol 2, pp 87-93, No. 12

Disclosure of Invention

Problems to be solved by the invention

Under the present circumstances, development of a novel immunostimulant is expected.

The purpose of the present invention is to provide a novel immunostimulant and an immunostimulating food and drink having an excellent immunostimulating effect.

Means for solving the problems

The present inventors have newly found that a predetermined compound has an excellent immunostimulating effect, and have completed the present invention.

In order to achieve the above object, an immunostimulant according to claim 1 of the present invention comprises a compound represented by the general formula I or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 1]

(in the formula I, n represents an integer of 4-12).

For example, a compound represented by the formula II or a pharmacologically acceptable salt thereof,

[ solution 2]

The food or drink for immunostimulation according to aspect 2 of the present invention comprises a compound represented by the general formula I or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 3]

(in the formula I, n represents an integer of 4-12).

For example, a compound represented by the formula II or a pharmacologically acceptable salt thereof,

[ solution 4]

Effects of the invention

According to the present invention, a novel immunostimulant and an immunostimulating food and drink having an excellent immunostimulating effect can be provided.

Drawings

FIG. 1 is a diagram illustrating a synthesis method of AU-1833C.

FIG. 2 shows synthesized AU-1833C¹³C NMR and¹graph of H NMR data.

FIG. 3 is a graph showing TNF-. alpha.production-inducing activity of AU-1833C, lentinan and fucoidan.

Fig. 4 is a graph showing the immunostimulatory activity of AU-1833C, (a) is a graph showing the number of PECs, (b) is a graph showing the amount of TNF- α production induction (pg/cell), and (C) is a graph showing the amount of TNF- α production induction (pg/mouse (mouse)).

Detailed Description

First, the immunostimulant of the present embodiment will be described in detail.

The immunostimulant of the present embodiment contains a compound represented by the general formula I or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 5]

(in the formula, n represents an integer of 4 to 12). In the formula, n is preferably 4 to 10, more preferably 4 to 8, and still more preferably 5 to 7.

In the present specification, "pharmacologically acceptable salts" means derivatives of the disclosed compounds, wherein the parent compound is modified by substitution of an acid or base moiety in order to form a salt. Examples of pharmacologically acceptable salts include, but are not limited to: minerals or organic acid salts of basic residues such as amines and bases, or organic salts of acidic residues such as carboxylic acids. In the present specification, pharmacologically acceptable salts include, for example, the existing non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. In the present specification, pharmacologically acceptable salts are sometimes synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts are prepared by reacting the free acid or free base of these compounds in water or in an organic solvent or in a mixed solution of 2, in a stoichiometric amount of the appropriate base or acid. Generally, nonaqueous media such as ether, ethyl acetate (EtOAc), ethanol, isopropanol, acetonitrile, and the like are suitable. A list of suitable salts is described in Remington's Pharmaceutical Sciences, 17 th edition, Mack publishing Co., Isston, Pa., p. 1418 1985 and Journal of pharmacy (Journal of Pharmaceutical Science)66,2 (1977).

The compounds of the general formula I can be prepared, for example, by known synthetic methods such as "Synthesis of novel macrocyclic Compounds (Synthesis of New Macrocycles) section IV.I", Two-step Synthesis of Dimeric phthalates (Two-step Synthesis of polymeric Phthalic Acid Esters), Journal of the Chemical Society of Perkin Transactions 1,1974,2578-2580 ", and the like.

The immunostimulant of the present embodiment may contain, for example, a compound represented by formula II or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 6]

The compound of formula II is a compound of the aforementioned formula I wherein n is 6. The chemical name of the compound of formula II is 7,8,9,10,11,12,19,20,21,22,23,24-dodecahydro-dibenzo [1,6,13,18] -tetraoxaoctadeca (slow) ring-2, 5,14, 17-tetraone (7,8,9,10,11,12,19,20,21,22,23,24-dodecahydro-dibenzo [1,6,13,18] -tetraoxathioctadine-2, 5,14, 17-tetrane). In this specification, the compound of formula II is sometimes referred to as "AU-1833C".

An example of a method for synthesizing the compound of formula II (AU-1833C) will be described (FIG. 1). The following compounds 1 to 5 are shown in FIG. 1. Compound 1 was dissolved in pyridine, compound 2 was added thereto, and stirred at a predetermined temperature for a predetermined time. Further, compound 1 was added and stirred at a predetermined temperature for a predetermined time. Then, the mixture was diluted with EtOAc, and then, liquid separation was performed in the order of HCl solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. Adding Na into EtOAc layer₂SO₄After drying, concentration gave compound 3. The compounds 3, 4-Dimethylaminopyridine (DMAP) and N, N' -Dicyclohexylcarbodiimide (DCC) were dissolved in anhydrous dichloromethane, to which the compound 4 was added, and stirred for a predetermined time. Then, the mixture was diluted with chloroform, and then, liquid separation was performed in the order of HCl solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. Adding Na into chloroform layer₂SO₄After drying, concentration and purification on a silica gel column with EtOAc and hexanes, compound 5 was obtained. Compound 5 and a predetermined catalyst were dissolved in anhydrous dichloromethane and stirred for a predetermined time. Next, after purification on a silica gel column with EtOAc and hexanes, compound 6 was obtained. Compound 6 was dissolved in methanol, to which was added a palladium-activated carbon ethylenediamine complex, and stirred under a hydrogen atmosphere for a predetermined time. Subsequent filtration through celite followed by purification on a silica gel column with EtOAc and hexanes afforded AU-1833C. The compound of formula II (AU-1833C) may be synthesized by a method other than this method, or may be isolated by processing, extraction, or the like of animals and plants.

The immunostimulatory effect of the immunostimulant of the present embodiment can be determined to have the immunostimulant effect, for example, when the degree of enhancement of cytokine production (e.g., enhancement of tumor necrosis factor (TNF- α) production) in mammalian cells administered with the immunostimulant is higher than the degree of enhancement in mammalian cells not administered, or when the degree of increase in the number of peritoneal infiltration cells (PEC) in mammals administered with the immunostimulant is higher than the degree of increase in mammals not administered. Examples of the mammals include human, mouse, monkey, horse, and cow.

The immunostimulant of the present embodiment can be prepared into dosage forms such as tablets, granules, powders, capsules, syrups, injections, and the like by a conventional method, and an appropriate administration system (DDS) can also be used. In addition, excipients, binders, lubricants, colorants, disintegrants, thickeners, preservatives, stabilizers, pH adjusters and the like which are used in general pharmaceuticals may be added. The dose of the immunostimulant of the present embodiment can be appropriately set according to the age, weight, indication, and the like of the subject. The immunostimulant of the present embodiment may be administered by any method such as administration at meal time, administration after meal, administration before meal, administration between meals, and administration before sleep.

Next, the immunostimulating food and drink of the present embodiment will be described.

The food or drink for immunostimulation of the present embodiment contains a compound represented by the general formula I or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 7]

(in the formula, n represents an integer of 4 to 12). In the formula, n is preferably 4 to 10, more preferably 4 to 8, and still more preferably 5 to 7.

The food or drink for immunostimulation of the present embodiment may contain, for example, a compound represented by formula II or a pharmacologically acceptable salt thereof as an active ingredient,

[ solution 8]

The compound of formula II is a compound of the aforementioned formula I wherein n is 6. The chemical name of the compound of formula II is 7,8,9,10,11,12,19,20,21,22,23,24-dodecahydro-dibenzo [1,6,13,18] -tetraoxaoctadeca (slow) ring-2, 5,14, 17-tetraone (7,8,9,10,11,12,19,20,21,22,23,24-dodecahydro-dibenzo [1,6,13,18] -tetraoxathioctadine-2, 5,14, 17-tetrane). In this specification, the compound of formula II is sometimes referred to as "AU-1833C".

In the food and drink for immunostimulation of the present embodiment, the detailed information on the "pharmacologically acceptable salt", the "immunostimulation" action and the like is the same as described above.

The food or beverage for immunostimulation of the present embodiment can be processed into a form suitable for eating and drinking by a conventional method, for example, a granule, a tablet, a capsule, a gel, a cream, a paste, a suspension, an aqueous solution, an emulsion, a powder, and the like. In addition, excipients, binders, lubricants, colorants, disintegrants, thickeners, preservatives, stabilizers, pH adjusters, and the like, which are generally used in food and beverage, may be added. Further, sugars, sugar alcohols, salts, oils and fats, amino acids, organic acids, glycerol, and the like may be added to improve the taste within a range not impairing the effect of the present invention. When the food or beverage for immunostimulation of the present embodiment is used in the form of being included in existing food or beverage, the food or beverage for immunostimulation of the present embodiment may be appropriately selected as long as the effect of the present invention can be achieved.

The immunostimulating food and drink of the present embodiment can be used as, for example, a specific health food, a nutritional functional food, a food exhibiting functionality, a supplement, a so-called drinking agent, a feed for livestock, and the like.

Examples

The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples.

(example 1)

As shown in FIG. 1, a compound of formula II (AU-1833C) was synthesized. Compounds 1-6 are shown in FIG. 1.

(Synthesis of Compound 5)

3g (20mmol) of Compound 1 (manufactured by Fuji photo film and Wako pure chemical industries, Ltd.) was dissolved in 40mL of pyridine (manufactured by Fuji photo film and Wako pure chemical industries, Ltd.), 1.9g (16mmol) of Compound 2 (manufactured by Fuji photo film and Wako pure chemical industries, Ltd.) was added thereto, and the mixture was stirred at 80 ℃ for 4 hours. Further, 3g (20mmol) of Compound 1 was added thereto, and the mixture was stirred at 80 ℃ for 20 hours. Then, the mixture was diluted with EtOAc, and then, a 1M HCl solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution were separated in this order. Adding Na into EtOAc layer₂SO₄After drying, concentration gave compound 3(5g, 12mmol, yield 63%). Compound 3(5g, 12mmol), 1.2g (1mmol) of 4-dimethylaminopyridine (DMAP, Fuji film and Wako pure chemical industries, Ltd.), and 10.3g (5mmol) of N, N' -dicyclohexylcarbodiimideImine (DCC, fuji film and wako pure chemical industries, ltd.) was dissolved in anhydrous dichloromethane (70mL), and 2.2g (30mmol) of compound 4 (fuji film and wako pure chemical industries, ltd.) was added thereto and stirred overnight. Then, the mixture was diluted with chloroform and separated into a 1M HCl solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution in this order. Adding Na into chloroform layer₂SO₄After drying, concentration and purification with a silica gel column (200g) of EtOAc: hexane ═ 40:60(v/v), compound 5 was obtained (3.5g, 6.7mmol, 56% yield from compound 3). Preparation of Compound 5¹The results of H-NMR analysis are shown below:¹H-NMR(CDCl_3,270MHz)δ7.68(4H，dd，J＝5.7,3.5Hz)，δ7.49(4H，dd，J＝5.4,3.2Hz)，δ5.80(2H，m)，δ5.10(4H，m)，δ4.30(8H，m)，δ2.46(4H，m)，δ1.73(4H，m)，δ1.49(4H，m)。

(Synthesis of Compound 6)

Compound 5(3.5g, 6.7mmol) and 150mg (177mmol) of Grubbs 2 nd generation catalyst (manufactured by Sigma-Aldrich) were dissolved in anhydrous dichloromethane (100mL) and stirred overnight. Next, after purification on a silica gel column (200g) with EtOAc: hexane ═ 35:65(v/v), compound 6(1.3g, 2.6mmol, 39% yield from compound 5) was obtained. Preparation of Compound 6¹The results of H-NMR analysis are shown below:¹H NMR(CDCl_3,270MHz)δ7.72(4H，m)，δ7.53(4H，m)，δ5.59(2H，m)，δ4.30(8H，m)，δ2.46(4H，m)，δ1.75(4H，m)，δ1.46(4H，m)。

(Synthesis of AU-1833C)

Compound 6(1.3g, 2.6mmol) was dissolved in methanol (60mL), and 60mg of palladium-activated charcoal ethylenediamine complex (Fuji film and Wako pure chemical industries, Ltd.) was added thereto, followed by stirring for 3 hours under a hydrogen atmosphere. Next, after filtration through celite, the product was purified with a silica gel column (100g) containing EtOAc: hexane ═ 40:60(v/v) to give AU-1833C (0.9g, 1.8mmol, yield from compound 6: 68%).

[ solution 9]

Will be with respect to the synthesized compounds¹³C NMR (100MHz) data and¹h NMR (400MHz) data are shown in FIG. 2 (solvent: MeOH-d)₄). From these data, it was shown that the synthesized compound was indeed the compound of formula II (AU-1833C).

(example 2)

AU-1833C was verified for its immunostimulatory effect.

(TNF-. alpha.production-inducing Activity in cells)

TNF-alpha production-inducing activity in a mouse-derived macrophage-like cell line (RAW264, provider: center for research on biological resources of Japan institute of physico-chemical research) was examined. As the test samples, AU-1833C (synthesized in example 1), lentinan (product name: lentinan, Fuji film and Wako pure chemical industries, Ltd.) and fucoidan (product name: fucoidan, Fucus vesiculosus (Fucus vesiculosus) source, Sigma-Aldrich Co.) were used, and the test sample solutions were adjusted so that the final concentrations of the test samples were 50, 100 and 200. mu.g/mL. First, all test samples were dissolved in dimethyl sulfoxide (DMSO) so as to have a final concentration of 1,000 times, and then diluted 100 times with deionized water to prepare a test sample solution having a final concentration of 10 times (containing 1% DMSO). RAW264 was suspended in DMEM medium (product name: Dulbecco's modified Eagle Medium "Nishui" 2 powder, manufactured by Nishui pharmaceutical Co., Ltd.) supplemented with 10% Fetal Bovine Serum (FBS), seeded in a 96-well microplate at a cell density of 10,000 cells (cells)/well (well)/100. mu.L, and incubated at 37 ℃ in a 5% CO atmosphere₂The cells were incubated for 16 hours. The sample solution was added in an amount of one tenth (20. mu.L) to each sample area, or 1% DMSO aqueous solution was added to the non-treated area, and then replaced with fresh DMEM medium (180. mu.L) at 37 ℃ with 5% CO₂And culturing for 24 hours. Then, the culture supernatant was recovered and used for evaluation test of TNF-. alpha.production-inducing activity. ELISA Kit (product name: Mouse TNF-alpha Quantikine ELISA Kit, R) was used for measuring the concentration of TNF-. alpha.produced&Manufactured by D systems) according to the experimental protocol. The TNF-alpha concentration in each test sample area was calculated from the measured TNF-alpha concentration relative to the TNF-alpha concentration in the untreated areaThe ratio, as TNF-. alpha.production-inducing activity.

The results of TNF-. alpha.production-inducing activity are shown in FIG. 3. 50. The TNF- α production inducing activity of AU-1833C was shown to be significantly higher than the corresponding untreated control at all concentrations of 100, 200. mu.g/mL. In addition, the TNF-. alpha.production-inducing activity of AU-1833C at each concentration was significantly higher than that of lentinan and fucoidan at the same concentration.

From the above results, it can be seen that AU-1833C has significantly higher TNF- α production-inducing activity than lentinan and fucoidan, which are conventional immunostimulants, and has excellent immunostimulant effect.

(number of PEC and TNF-. alpha.production Induction amount in mouse)

Next, the number of intraperitoneal infiltrating cells (PEC) and the amount of TNF-. alpha.production induction in the mouse were examined.

AU-1833C as a sample was dissolved in DMSO so as to be 50mg/mL, and then diluted 5-fold with physiological saline (product name: Otsuka Denshi Kabushiki Kaisha) to prepare a sample solution of 10mg/mL (containing 20% DMSO).

The animals used were Slc: ddY mice (female, 6 weeks old) and grouped as described below.

1) A non-treatment area: n is 6

2) AU-1833C region: n is 6

Mice purchased at 5 weeks of age were acclimatized for 5 days in a state of free water and feed (general food (product name: CE-2, manufactured by CLEA, Japan)). The body weights of the mice at 6 weeks after acclimation were measured, and based on the measured values, the mice were grouped so that no difference in body weight was produced between the groups. Mice were dosed with 200 μ L of test sample solution or 20% DMSO physiological saline as the no-treatment zone by intraperitoneal injection, respectively. Mice that had been administered for 22 hours were sacrificed to die under isoflurane anesthesia, 5mL of physiological saline was injected into the abdominal cavity, the abdomen was kneaded about 10 times, and then the physiological saline was recovered from the abdominal cavity, thereby recovering cells in the abdominal cavity. The PEC concentration (cells/mL) as a nucleated cell, such as leukocytes infiltrated into the abdominal cavity, was measured using a portion of the collected physiological saline and TUERK solution (product name: TUERK solution, Wako pure chemical industries, Ltd.). The number of PECs (cells/mouse) per mouse was calculated by multiplying the measured PEC concentration by the amount of physiological saline (5mL) injected into the abdominal cavity.

In order to measure the TNF-. alpha.production-inducing ability of PEC under Lipopolysaccharide (LPS) (product name: Lipopolysaccharide from Escherichia coli O111: B4, manufactured by Sigma-Aldrich Co., Ltd.), the remaining recovered physiological saline was centrifuged to recover PEC. The PEC was washed by suspending it in serum-free RPMI1640 medium (product name: RPMI1640 medium "Nishui" 2 powder, manufactured by Nishui pharmaceutical Co., Ltd.), centrifuging it again, and suspending it in RPM1640 medium supplemented with 10% Fetal Bovine Serum (FBS). After counting the number of cells after washing again using the TUERK solution, the cells were seeded in a 96-well microplate at a cell density of 800,000 cells (cells)/well (well)/180. mu.L with the concentration adjusted using serum-containing RPMI1640 medium. LPS was added to the medium at a final concentration of 100ng/mL, and the total medium volume was set to 200. mu.L, and the medium was incubated at 37 ℃ and 5% CO₂The cells were incubated for 3 hours. Then, the culture supernatant was recovered and used in an evaluation test of the amount of induction of TNF-. alpha.production. The determination of the TNF-. alpha.concentration was carried out using an ELISA kit according to the protocol. From the measured TNF-. alpha.concentrations (pg/1,000. mu.L), the amount of TNF-. alpha.per cell was calculated using the following equation 1, and the amount of TNF-. alpha.produced by PEC per mouse was calculated using the following equation 2.

Calculation formula 1

TNF-. alpha.concentration (pg/1,000. mu.L) × (200. mu.L/1,000)/800,000 cells (cells) TNF-. alpha.production (pg/cell)

Calculation formula 2

TNF- α concentration (pg/1,000 μ L) × (200 μ L/1,000)/800,000 cells (cells) × PEC number per mouse individual (cells/mouse)) ═ TNF- α production amount (pg/mouse)

The results are shown in FIG. 4. It was shown that the number of PECs was significantly greater in mice intraperitoneally administered with AU-1833C compared to untreated mice as a control (fig. 4 (a)). It was also shown that in mice dosed with AU-1833C, TNF- α production per cell in PEC was significantly greater (fig. 4(b)) and also TNF- α production from PEC per individual mouse was significantly greater (fig. 4(C)) compared to untreated mice as controls.

From the above results, it was observed that AU-1833C had an excellent immunostimulatory effect due to the increase in the number of PECs and the amount of TNF-. alpha.production-inducing substance in PECs and the amount of TNF-. alpha.production-inducing substance from PECs of individual mice.

It will be evident that the invention may be embodied and practiced in various other forms without departing from the broader spirit and scope of the invention. The above embodiments are examples for explaining the present invention, and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, rather than the embodiments. Furthermore, various modifications made within the meaning of the claims and equivalent inventive meanings are considered to be within the scope of the present invention.

The invention is based on Japanese patent application No. 2019-67663, which is proposed by 3.29.2019. The specification, claims, drawings of japanese patent application No. 2019-67663 are incorporated herein by reference in their entirety.