JP5912023B2 - Antibacterial peptide secretion inducer - Google Patents

Description

  The present invention relates to an antibacterial peptide secretion inducer.

Bacterial infections and fungal infections are major diseases that affect the longevity of mankind, and various drugs such as antibiotics and synthetic antibacterial agents have been developed. However, antibiotics and antibacterial agents always show resistant microorganisms. Once such antibiotic-resistant bacteria emerged, conventional antibacterial agents had no means of treatment, and the only countermeasures against resistant bacteria were to develop new antibacterial agents.
In recent years, infectious diseases caused by resident bacteria associated with decreased immunity have become a problem among the elderly. There are many so-called multi-drug-resistant strains, and once a multi-drug-resistant bacterial infection occurs in a nursing home for the elderly, there is no effective means of treatment. Depending on the situation, it may be necessary to close the facility or isolate the patient. For this reason, measures against multidrug-resistant bacteria have become a major social problem.
For this reason, treatment and prevention methods have been proposed in which polysaccharides or glycoproteins that induce secretion of interferons or interleukins are administered to enhance the immune system of the living body, but the effect is not so high.
By the way, various organisms such as animals, plants, insects, yeasts, and lactic acid bacteria are originally equipped with a self-biological defense mechanism for self-protection against the invasion of pathogenic microorganisms from the outside world. In order to prevent such invasion from outside, it has been revealed that organisms themselves produce antibacterial peptides consisting of about 10 to 50 or more amino acids. These antibacterial peptides have a wide range of antibacterial spectrum against bacteria, fungi, etc., and have no side effects or inhibitory effects on living organisms because they are produced by living organisms themselves. .

  Among these antibacterial peptides, human-derived antibacterial peptides include, for example, the defensin family (α-defensin, β-defensin, etc.), cathelicidin (LL-37), darmcidin, LEAP-1 (hepcidin) LEAP-2 and the like are known. In addition, Ominagan pentahydrochloride, a synthetic cationic antimicrobial peptide of an analog of indolicidin, which is an antimicrobial peptide consisting of 13 amino acids rich in tryptophan isolated from bovine neutrophils and exhibits a broad spectrum antimicrobial activity, It has been developed as an infection prevention drug (see Non-Patent Document 1, for example). Furthermore, nisin, an antimicrobial peptide produced by lactic acid bacteria, has already been used as a food additive.

If this antibacterial peptide can be artificially enhanced or secreted by humans, a greater effect can be expected by infection with multidrug-resistant bacteria. As such a technique, for example, a technique using an organic acid as an active ingredient of a human β-defensin secretion promoter (Patent Document 1), a technique for inducing expression of a defensin gene using a yeast-containing polysaccharide-containing composition (patent) Document 2), a technique for inducing expression of a defensin gene using an insoluble fraction derived from yeast (Patent Document 3), a technique for inducing expression of an antibacterial peptide using a mannan-containing component derived from yeast (Patent Document 4), isoleucine, leucine or Expression of antibacterial peptides using active ingredients such as yogurt, amazake extract, shochu mash, and miso concentrate A guidance technique (Patent Document 6) has been proposed.
Currently, the search for an inducer of an antibacterial peptide that can be easily used in daily life is being conducted.

International Publication No. 2005/027893 Japanese Patent Laid-Open No. 2003-197 JP 2003-262 A JP 2006-241023 A JP 2003-95938 A JP-A-2005-270117

Selsted, et al .; Biol. Chem. 267: 4292, 1992

  An object of the present invention is to provide an inducer of an antibacterial peptide such as α-defensin, which is a kind of antibacterial peptide that exhibits a self-biodefense mechanism provided in humans.

That is, the main configuration of the present invention is as follows.
(1) A defensin inducer comprising a germinated brown rice and / or sterol glycoside derived from brown rice bran as an active ingredient (except for food, food additives, and pet feed additives).

  Since the antibacterial peptide expression inducer or secretion enhancer according to the present invention can induce or promote the expression of the antibacterial peptide in the intestinal tract by oral administration, it has the effect that it can be used as a medicine, food or drink, and pet feed. is there.

The graph which shows the secretion promotion effect of the antimicrobial peptide by this invention

Germinated brown rice and / or sterol glycoside fraction extracted from brown rice used in the present invention (hereinafter sometimes referred to as “ASG”) is a neutral lipid obtained from germinated brown rice and / or brown rice with hexane. The residue obtained is further contained in a lipid fraction extracted with an organic solvent, and can be separated and concentrated by high performance liquid chromatography (HPLC).
The present invention is an α-defensin inducer containing the germinated brown rice and / or sterol glycoside derived from brown rice as an active ingredient. The ingestion method can be performed orally or by injection, and can be used as an additive for pharmaceuticals, foods and drinks, food additives, pet pharmaceuticals, and pet feeds. Since it is a component derived from germinated brown rice and / or brown rice, which is usually used for food, safety is high.
Since ASG is a very small amount extracted from germinated brown rice and / or rice bran components, the extracted ASG itself is used when ASG is applied to medicine, food and drink, pet medicine and feed. Use of germinated brown rice and / or brown rice per se or germinated brown rice and / or powder of brown rice prior to extraction of ASG is out of the scope of the invention.

The present invention can be used as an inducer of antibacterial peptides such as α-defensin, food additives, foods and pet foods, and veterinary drugs. The dosage form can be formulated into an arbitrary form together with an auxiliary agent by a known method and taken orally (administered). It can be ingested (administered) as a capsule or tablet, granule, fine granule, powder, or liquid.
The ingestion (administration) amount may vary depending on the ingestion (administration) method and the age, medical condition, general condition, etc. of the subject, but in adults, 0.1 to 600 mg as an active ingredient per day is usually appropriate per 1 kg of body weight.

The inducer of antibacterial peptides such as α-defensin of the present invention can be incorporated into general foods and health foods, and can also be used as a component of food additives. The food to be blended is not particularly limited, for example, bakery foods such as bread, confectionery bread, pies, Danish, donuts, cakes, noodles such as udon, soba, Chinese noodles, yakisoba, pasta, fries such as tempura, croquettes, curry, Sources such as stew and dressing, sprinkles, kneaded products such as kamaboko, beverages such as juice, snacks, rice confectionery, rice cake, confectionery such as gum.
Suitable for pets as feed for mammals such as dogs, cats, hamsters and squirrels. The form of the pet food of the present invention is not particularly limited, and examples thereof include dry type, wet type, semi-moist type, biscuit type, sausage type, jerky type, powder, granule, capsule and the like.

<Sprouted brown rice and / or sterol glycoside derived from brown rice>
1. Germinated brown rice from which the koji component is collected can be prepared by a known method. The present applicant has made a number of proposals for germinated brown rice, which can be obtained, for example, by the method for producing germinated brown rice disclosed in Japanese Patent No. 3423927, Japanese Patent No. 3611804, Japanese Patent No. 3738025, and the like.
The germinated brown rice and / or brown rice is refined by about 5 to 15% and the koji component is collected. The soot component is first degreased with hexane. This degreasing step is the same as the method for collecting rice bran oil by degreasing general rice bran. In the present invention, the sterol glycoside fraction is further extracted using this defatted koji as a raw material and using an organic solvent.

2. ASG used in the present invention is a sterol glycoside specified by the following chemical formula.

(i) X in the general formula (1) is selected from the following group, and Y is 5α-cholest-8 (14) -en3β-ol: palmitic acid (16: 0), stearic acid (18: 0), 2-hydroxy-octadecanoic acid (18: 0 (2h)), oleic acid (18: 1), linoleic acid (18: 2), or lignoceric acid (24: 0)
(ii) X in the general formula (1) is 2-hydroxy-octadecanoic acid (18: 0 (2h)), and Y is Campesterol, Stigmasterol, 5α-cholest-8 ( 14) -en-3β-ol or β-Sitosterol

The following examples and test examples further illustrate the present invention.
<Examples of ASG preparation>
After removing the neutral lipid in the lipid component from the germinated brown rice bran with hexane, a crude extract of ASG was prepared for each residue using hexane, chloroform and methanol. This ASG crude extract was extracted with a chloroform: methanol (2: 1) mixture, and ASG was prepared by silica gel carrier column chromatography.
The extraction of ASG used in the test was performed using 200 kg of koji (10% koji accuracy) obtained by scouring about 2,000 kg of germinated brown rice.
An amount of hexane soaked with rice bran (500 g) was added and stirred sufficiently, followed by filtration with gauze to obtain a defatted koji. Thereafter, the total fat fraction was extracted by adding chloroform: methanol 2: 1 (3 L) to 1.5 kg of defatted soot in which hexane was volatilized, and the extract was dried to dryness by an evaporator.

The dried product was dissolved in 300 ml of chloroform: hexane = 1: 1, and the whole solution was applied to a column 10 cm in diameter and 100 cm in length (packed with 80 cm of Merck silica gel 60) swollen with chloroform. After all the solution had soaked into the Iatro beads, chloroform: hexane = 1: 1 (7,840 ml), chloroform (20,160 ml), chloroform: methanol = 9: 1 (10,080 ml) were passed in this order. Only a dark green solution separated by passing chloroform: methanol = 9: 1 was collected.
The collected dark green solution was dried to dryness using an evaporator. Table 1 shows the yield at each point.

<ASG analysis>
The extracted ASG was analyzed under the following conditions. As a result of this analysis, the final dried product was found to contain 72.6% ASG.
Analysis conditions Detector: CoronaTM CADTM Charged Aerosol Detector
Column: LiChrospher Si 60 (5μm, 125 × 4mm id, Merck)
Column temperature: 40 degrees Flow rate: 1 mL / min.
Injection volume: 10μL
Sample solvent: chloroform: methanol (2: 1, vol / vol)
Calibration curve concentration: 10, 20, 40, 60 and 80 μg / mL
Mobile phase, gradient conditions (see Table 2)

<Defensin induction test>
I. Materials and Methods JP using α- defensin secretion assay system from Paneto cells using mouse small intestinal crypts disclosed in 2011-78318 Patent Publication No. ASG is an antimicrobial peptide from the Paneto cells α- defensins the secretion-inducing effect of, was confirmed by the antibacterial activity as an index.
1. Small intestinal crypt material collection:
A small intestine tissue containing crypt (small intestine crypt) was collected from an ICR mouse (Nippon Charles River) according to a conventional method, and suspended in 1 mL of PBS (−) to prepare a suspension containing crypt { Ayabe T et al., Nature Immunol. Volume 1, pp. 113-118, 2000}. When the number of crypts contained in this suspension was counted using a hemocytometer (Burker-Turk; Sunreed Glass), it was 5 to 8 × 10 ⁴ . Moreover, when the ratio which the cryptography occupied among the structures | tissues contained in this suspension was calculated | required by observing in a weak expansion visual field using a phase-contrast microscope (Olympus), the ratio was 70% or more.
2. Panetocyte secretion reaction and ex vivo bactericidal assay:
The recovered small intestine crypt and the above ASG-containing composition were co-cultured ex vivo to recover the crude secretion. The concentration of ASG was diluted 1: 10,000, 1: 1,000 or 1: 100 with physiological saline. At the same time, the ASG-unexposed PBS control supernatant of the small intestinal crypt was also collected. The peptide was extracted in 30% acetic acid and lyophilized following dialysis. The collected secretion and the unexposed control supernatant extract were dissolved in pure water so that 1 μL was derived from 100 small intestinal crypts. Next, the bactericidal activity of the secretion against Salmonella Typhimurium phoP- was analyzed.
Panetocyte secretion and unexposed control supernatant and extract were reacted with 1 × 10 ⁶ colony-forming units (CFU) S. Typhimurium phoP- at 37 ° C for 60 minutes and then cultured on TSA plate overnight . Viable CFU was counted and the bactericidal rate is shown in FIG.

II. result
Ex vivo bactericidal activity against S. Typhimurium phoP- by inducing secretion of antibacterial peptides such as α-defensin from ASG panel cells was analyzed. As shown in Fig. 1, secretions from 50 small intestinal crypts exposed to ASG (1: 10,000-1 : 100 ) were examined by changing the reaction concentration and number of small intestinal crypts in mouse small intestinal crypts. Compared with the control, a significant sterilization rate of 99% or more was recognized. This bactericidal activity was dependent on the concentration of ASG . Secretions from 100 small intestinal crypts showed stronger bactericidal activity with ASG (1: 100 ).
Secretions from 200 and 500 small intestinal crypts resulted in less than 1/100 bacterial count at all ASG concentrations examined.
From the above results, it was revealed that ASG induces ex vivo bactericidal activity release by antimicrobial peptides such as α-defensin from mouse small intestinal panel cells. In addition, it was confirmed that the bactericidal activity was ASG concentration-dependent.

The prescription example using ASG of this invention is shown below.
Formulation Example 1
[Capsule]
Composition ASG ... 100mg
Beeswax ... 10mg
Grape seed oil ... 110mg
The above ingredients were mixed and filled into a capsule base mixed with gelatin and glycerin to obtain soft capsules.

Formulation example 2
[tablet]
Composition ASG dried product ... 150mg
Cellulose ... 80mg
Starch ... 20mg
Sucrose fatty acid ester ... 2mg
The above components were mixed and tableted to obtain tablets.

Formulation Example 3
[Beverages]
(Composition) (Composition: Mass%)
Fructose butter sugar liquid sugar 5.00
Citric acid 10.4
L-ascorbic acid 0.20
Perfume 0.02
Dye 0.10
ASG dried product 1.00
Water 82.28

Claims (1)

Defensin inducer containing germinated brown rice and / or sterol glycoside derived from brown rice bran as an active ingredient (excluding food, food additives and pet feed additives).