WO2019065962A1 - Caspase 3 activity inhibitor, apoptosis inhibitor, inhibitor of toxicity due to secondary bile acid, and intestinal tract disorder inhibitor - Google Patents

Abstract

This caspase 3 activity inhibitor uses horseradish leaf extract as an active ingredient, said extract being obtained by performing solvent extraction on horseradish leaves.

Description

Caspase 3 activity inhibitor, apoptosis inhibitor, secondary bile acid toxicity inhibitor and intestinal disorder inhibitor

The present invention relates to a caspase 3 activity inhibitor, an apoptosis inhibitor, a secondary bile acid toxicity inhibitor and an intestinal disorder inhibitor.

In recent years, along with the westernization of eating habits in Japan as well, as with other countries, there is an increase in the opportunity to consume lipid-rich food, and the amount of lipid intake is increasing.

Ingestion of lipids causes bile acids to be secreted into the duodenum. Bile acids play a role in promoting the formation of micelles in the digestive tract and facilitating absorption of ingested lipids. Bile acid is produced from cholesterol in liver cells of the liver, stored as bile in the gallbladder and secreted into the duodenum.

Human bile acids include primary bile acids (cholic acid (CA), chenodeoxycholic acid, etc.) synthesized in the liver, and secondary bile acids (deoxycholic acid ( DCA), lithocholic acid etc.). Secondary bile acids are known to cause dyslipidemia, hepatotoxicity, carcinogenesis, dysbiosis (absence of enteric bacterial species) and the like.

Horseradish (Horseradish, scientific name: Armoracia rusticana, Japanese name: horseradish) is a cold-resistant perennial of the cruciferous family, and is also called yamawasabi in Hokkaido. Yamawasabi roots are provided for food and used as a savory.

Patent Document 1 describes that horseradish has an anti-inflammatory effect.

JP, 2013-146271, A

However, there have been no reports on the site of yamawasabi which suppresses caspase 3 activity, which suppresses secondary bile acid toxicity, or which suppresses intestinal tract injury, or the extract or treated product of yamasabi.

The present inventors have found that pokeweed leaf extract obtained by a predetermined production method has an excellent inhibitory action on caspase 3 activity, an inhibitory action on apoptosis, an inhibitory action on secondary bile acid and an inhibitory action on intestinal tract injury. Completed the invention. The present invention provides a caspase 3 activity inhibitor, an apoptosis inhibitor, a secondary bile acid toxicity inhibitor and an intestinal disorder inhibitor obtained by solvent extraction of pokeweed leaves and having excellent effects. To aim.

In order to achieve the above object, the inhibitor of caspase 3 activity according to the first aspect of the present invention uses a horseradish leaf extract obtained by solvent extraction of horseradish leaves as an active ingredient.

The apoptosis inhibitor according to the second aspect of the present invention comprises, as an active ingredient, a horseradish leaf extract obtained by solvent extraction of horseradish leaves.

The agent for suppressing toxicity by secondary bile acid according to the third aspect of the present invention comprises, as an active ingredient, a horseradish leaf extract obtained by solvent extraction of horseradish leaves.

The intestinal tract injury inhibitor according to the fourth aspect of the present invention uses, as an active ingredient, a horseradish leaf extract obtained by solvent extraction of horseradish leaves.

According to the present invention, a caspase 3 activity inhibitor, an apoptosis inhibitor, a secondary bile acid toxicity inhibitor and an intestinal disorder suppressor obtained by solvent extraction of pokeweed leaves are provided. be able to.

FIG. 5 is a graph showing cell viability to DCA treatment in each sample. FIG. 3 is a graph showing caspase 3 activity in the yamasabi site and extraction conditions, where (a) shows hot water extraction (70 ° C., 2 hours), methanol extraction (MeOH extraction) and ethanol extraction (EtOH extraction), (b) Comparison with hot water extraction (121 ° C., 20 minutes). It is a graph which shows the caspase 3 activity in the extraction temperature of the horseradish leaf extract. (A) is a graph showing the concentration of fecal CA after administration of CA, (b) is a graph showing the concentration of fecal DCA after administration of CA. Figure of colon sections after administration of CA, (a) non-treatment group, (b) CA administration group, (c) colon section of Hematoxylin-Eosin (HE) staining of CA + pokeweed leaf extract group It is. It is a graph which shows the S100A8 gene expression level of large intestine epithelial cells after CA administration. It is a graph showing the measurement result of urinary lactulose / mannitol ratio (LA / MA ratio) in a mild diet stress test. It is a graph showing the measurement result of serum I-FABP (intestinal-derived fatty acid binding protein) in the light meal load test. It is a graph showing the measurement result of fecal calprotectin in a mild diet stress test. It is a graph showing the questionnaire result of the color of the stool in a light meal stress test. It is a graph showing the questionnaire result of the shape of the stool in a light meal load test. It is a graph showing the questionnaire result of the smell of stool in a light meal stress test. It is a graph showing the measurement result of fecal calprotectin in a single brand in crossover test. It is a graph showing the questionnaire result of the shape of the stool in a single brand ind crossover test. It is a graph showing the questionnaire result of the smell of stool in a single brand ind crossover test. It is a graph showing the questionnaire result of fatigueability in a single brand ind crossover test. It is a graph showing the questionnaire result of the degree of fatigue removal in a single brand ind crossover test. It is a graph showing the questionnaire result of skin roughness in a single brand ind crossover test.

First, the horseradish leaf extract according to the present embodiment will be described in detail.

The radish extract according to the present embodiment is obtained by solvent extraction of radish leaves.

Yamawasabi (Horseradish (Science name: Armoracia rusticana, Japanese name: horseradish)) is a hardy perennial of Brassicaceae and is composed of leaves (leaf and petiole), flower stems and roots. In the horseradish leaf extract according to the present embodiment, a portion of the horseradish leaf (including leaf, stalk or both leaf and stalk) is used, and the radish leaf can be used as it is or dried. For example, freeze-dried or dried yamasabi leaves (for example, with a water content of 0 to 10% by weight) can be used, and the freeze-dried or dried yamasabi leaves are further ground, for example, with a mixer, etc. You may use it as it is.

The solvent used for solvent extraction of the horseradish leaves can be optionally used if it is a solvent usually used for extraction. For example, water, ethanol, methanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, acetic acid, ethyl acetate, ether, hexane or the like, or a mixture thereof can be used. For example, water, ethanol or the like is used in consideration of the influence on the human body.

The amount of solvent used for solvent extraction of the horseradish leaf is not particularly limited, but is 1 to 30 times the amount of freeze dried or dried horseradish leaf, and may be, for example, 15 to 20 times.

The extraction temperature of the horseradish leaf is not particularly limited, and may be, for example, 10 ° C. to 130 ° C., for example, 10 ° C. to 70 ° C.

The time for solvent extraction of horseradish leaves is not particularly limited, and is, for example, 5 minutes to 3 hours, for example, an extraction temperature of 10 ° C to 70 ° C for 40 minutes to 2 hours, for example, 100 ° C to 130 ° C. The extraction temperature may be 5 minutes to 1 hour.

As a method of solvent extraction, for example, a method such as adding an extraction solvent to yamasabi leaves and leaving still or shaking them, or adding an extraction solvent to yamawasabi leaves and performing ultrasonic treatment etc. is exemplified.

After solvent extraction, solid content may be removed, and for example, after removing the residue with a centrifuge, decanter or the like, it may be separated by suction filtration, clarifier or the like to make it clear. Thereafter, for example, the solution may be concentrated 2 to 15 times with a rotary evaporator or the like, an excipient may be added, and sterilization may be performed (for example, 121 ° C., 15 to 30 minutes). Excipients include dextrin, starch, maltose, sorbitol and the like. Furthermore, after that, for example, after a drying process such as spray drying or lyophilization, a process of sieving may be performed.

An example of the preparation method of the horseradish leaf extract is shown below.
1. The freeze dried radish was ground with a mixer.
2. Add 15-20 volumes of water to the leaves and extract for 2 hours at 70 ° C or for 20 minutes at 121 ° C.
3. Filter
4. The filtrate is lyophilized.

Another example of the preparation method of the horseradish leaf extract is shown below.
1. The freeze dried radish was ground with a mixer.
2. Add 15 to 20 volumes of methanol or ethanol to the leaves and sonicate for 30 minutes.
3. Filter
4. The filtrate is concentrated to dryness on a rotary evaporator.

As described below, the pokeweed leaf extract according to the present embodiment can be used as a caspase 3 activity inhibitor, an apoptosis inhibitor, a secondary bile acid toxicity inhibitor and an intestinal disorder inhibitor.

Next, a caspase 3 activity inhibitor, an apoptosis inhibitor, a secondary bile acid toxicity inhibitor and an intestinal disorder inhibitor will be described according to the present embodiment.

The caspase 3 activity inhibitor, the apoptosis inhibitor, the secondary bile acid toxicity inhibitor and the intestinal tract injury inhibitor according to the present embodiment contain the horseradish leaf extract according to the present embodiment as an active ingredient.

The caspase 3 activity inhibitor according to the present embodiment suppresses, for example, the activity of caspase 3 in the intestinal tract.

The apoptosis inhibitor according to the present embodiment suppresses, for example, apoptosis in the intestinal tract, for example, by suppressing the activity of caspase 3 in the intestinal tract.

The secondary bile acid toxicity inhibitor according to the present embodiment suppresses the toxicity of secondary bile acid (DCA, lithocholic acid, etc.) produced from primary bile acid. Secondary bile acids are known to cause disorders such as dyslipidemia, hepatotoxicity, carcinogenesis, dysbiosis (absent enteric bacterial species) and the like, and the inhibitor for toxicity by secondary bile acids according to this embodiment is Can be used to prevent, ameliorate, treat, etc. these disorders. The secondary bile acid toxicity inhibitor according to the present embodiment suppresses the secondary bile acid toxicity, for example, by suppressing an inflammatory reaction caused by the secondary bile acid.

The agent for inhibiting intestinal tract disorders according to the present embodiment is large intestine injury (surface epithelial detachment, erosion), acute enteritis (drug enteritis, ischemic colitis, etc.) and chronic enteritis (ulcerative colitis, Crohn's disease, amebic dysentery, intestinal tuberculosis) , Radiation enteritis etc.). In addition, the intestinal tract disorder inhibitor according to the present embodiment may be used for the intestinal tract disorder not caused by the secondary bile acid, or to the intestinal disorder caused by the secondary bile acid It may be used. Moreover, the intestinal tract injury inhibitor according to the present embodiment can exhibit improvement effects such as fatigue and rough skin by suppressing intestinal tract injury. The intestinal tract injury inhibitor according to the present embodiment exerts an intestinal protective effect by, for example, suppressing an inflammatory reaction caused by secondary bile acid.

The caspase 3 activity inhibitor, apoptosis inhibitor, secondary bile acid toxicity inhibitor and intestinal disorder inhibitor according to the present embodiment may be used as a supplement or a medicine. In this case, it is processed into a form suitable for eating and drinking such as granules, granules, tablets, capsules, gels, creams, pastes, suspensions, aqueous solutions, emulsions, powders, etc. be able to. In addition, commonly used excipients, binders, lubricants, colorants, disintegrants, thickeners, preservatives, stabilizers, pH adjusters and the like can be added. Furthermore, in order to improve the taste quality, saccharides, sugar alcohols, salts, oils and fats, amino acids, organic acids, glycerin and the like can be added as long as the effects of the present invention are not impaired. In the case of pharmaceuticals, they can be prepared, for example, in the form of tablets, granules, powders, capsules, syrups, injections and the like by conventional methods. In addition, excipients, binders, lubricants, colorants, disintegrants, thickeners, preservatives, stabilizers, pH adjusters and the like that are commonly used in pharmaceuticals can be added. The administration method may be appropriately selected within the scope of achieving the effects of the present invention, such as oral administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration and the like.

The caspase 3 activity inhibitor, apoptosis inhibitor, secondary bile acid toxicity inhibitor and intestinal disorder inhibitor according to this embodiment are, for example, 50 mg to 2,000 mg / day, preferably in terms of solid content of yamawasabi leaf extract The dose may be 100 mg to 1,000 mg / day, more preferably 300 to 500 mg / day. The dose may be appropriately selected based on the purpose of administration, dosage form, age of patient, body weight and the like.

As described above, the caspase 3 activity inhibitor with excellent effects, an apoptosis inhibitor, a secondary bile acid toxicity inhibitor, and intestinal disorder inhibition containing an effective horseradish leaf extract obtained by a simple method as an active ingredient An agent is provided.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples.

Example 1
We investigated the inhibitory effect of pokeweed leaf extract on DCA-induced cell death.

It was examined how much the cell death caused by DCA, which is a secondary bile acid, was suppressed by the horseradish leaf extract, the black soybean extract and the banana amylase treated product.

The preparation method of each sample is shown below.
Yamawasabi leaf extract: A 15-fold amount of water was added to dried wasabi leaf and subjected to hot water extraction at 121 ° C. for 20 minutes. After extraction, centrifugation was performed at 5,000 × g for 10 minutes, and the supernatant was collected. The collected supernatant was suctioned and filtered, and the freeze-dried powder of the obtained filtrate was used as a horseradish leaf extract.
Black soybean extract: 20 times amount of water was added to black soybean seed coat, and hot water extraction was performed at 121 ° C. for 20 minutes. After extraction, centrifugation was performed at 5,000 × g for 10 minutes, and the supernatant was collected. The collected supernatant was suctioned and filtered, and the freeze-dried powder of the obtained filtrate was used as a black soybean extract.
Banana amylase treated material: 20 times amount of water was added to dry powder of banana, and hot water extraction was performed at 121 ° C. for 20 minutes. After extraction, α-amylase (product name: α-amylase, manufactured by Wako Pure Chemical Industries, Ltd.) was added to 3% of the dry powder and reacted at 45 ° C. for 3.5 hours. After inactivation at 100 ° C. for 15 minutes, the supernatant was recovered by centrifugation at 5,000 × g for 10 minutes. The collected supernatant was filtered by suction, and the freeze-dried powder of the obtained filtrate was used as a banana amylase-treated product.

RPMI 1640 medium (product name: RPMI 1640 medium “Nissui” added with 10% fetal bovine serum (FBS) with human intestinal tract-derived cells (DLD-1, provided by: Tohoku University Institute of Aging Medicine, Medical Cell Resource Center) 2) Suspended in powder (manufactured by Nissui Pharmaceutical Co., Ltd.), and seeded at 50,000 / well in a 96-well microplate, and cultured at 37 ° C. under 5% CO ₂ for 24 hours. Each sample dissolved in RPMI 1640 medium was added at a final concentration of 250 μg / mL and kept at 37 ° C. under 5% CO ₂ for 24 hours. Thereafter, the culture supernatant was removed and the cells were washed twice with PBS. The washed cells were added with DCA to a final concentration of 0.65 mM and maintained at 37 ° C. under 5% CO ₂ for 1 hour. The survival rate (vs. non-treatment%) of DCA-treated cells was measured by MTT method. The MTT method was carried out according to the usual method (Journal of immunological methods, 1983, 16, 55-63, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays). In addition, "non-treatment" in Fig. 1 represents RPMI 1640 medium instead of sample, and represents cells not treated with DCA, and "control" adds RPMI 1640 medium instead of sample, and DCA-treated cells Represent.

The results are shown in FIG. Compared to "no treatment", cell death was promoted and the survival rate was reduced in "control" to which DCA was added. It was confirmed that the survival rate was increased by adding the horseradish leaf extract compared to the black soybean extract and the banana amylase-treated product, and that DCA-induced cell death, which is a secondary bile acid, was suppressed.

(Example 2)
Caspase 3 activity after apoptosis-inducing treatment with DCA was measured for each extract prepared using horseradish leaf blade, petiole and root.

As samples, horseradish leaf extract, horseradish leaf stem extract and horseradish root extract by hot water extraction, MeOH extraction and EtOH extraction were prepared. Below, the preparation method of the horseradish radish leaf extract is demonstrated. The radish extract of radish and radish extract were also extracted in the same manner as described below.

・ Hot water extraction (Fig. 2 (a), (b))
15 to 20 times the amount of water was added to the ground freeze dried radish radish leaves and extracted at 70 ° C. for 2 hours (FIG. 2 (a)) or at 121 ° C. for 20 minutes (FIG. 2 (b)). The freeze-dried product of the filtrate obtained by filtration was used as a hot water extract of yamawasabi leaf.

-MeOH extraction, EtOH extraction (Figure 2 (a))
15 to 20 volumes of a room temperature solvent was added to the ground freeze dried radish leaf lyophilizate and sonicated for 30 minutes. The solvent in the filtrate obtained by filtration was removed by a rotary evaporator, and the obtained solid was used as a solvent extract of pokeweed leaf.

Methods of DCA induction and caspase 3 activity measurement are described. The following was used for the reagent. In addition, "non-treatment" in Fig. 2 represents RPMI 1640 medium in place of sample and represents cells not treated with DCA, and "control" represents DCA-treated cells in which RPMI 1640 medium is added instead of sample. Represent.
Cell lysis buffer (10 mM TRIS, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.01% TRITON X-100)
Reaction buffer (10 mM HEPES, pH 7.5, 50 mM NaCl, 2.5 mM DTT, 40 μM Ac-DEVD-AMC)

DLD-1 was suspended in RPMI 1640 medium supplemented with 10% FBS, seeded at 50,000 / well in a 96-well microplate, and cultured overnight at 37 ° C. in the presence of 5% CO ₂ . The culture supernatant was removed, and each sample dissolved in RPMI 1640 medium was added at a final concentration of 1,000 μg / mL and kept at 37 ° C. under 5% CO ₂ for 24 hours. As a control, RPMI 1640 medium was added instead of each sample. The supernatant was then removed and cells were washed twice with PBS. RPMI 1640 medium containing 200 μM DCA was added to the washed cells, and maintained at 37 ° C. under 5% CO ₂ for 30 minutes. After removing the supernatant and washing twice with PBS, a cell lysis buffer was added and treated on ice for 30 minutes to prepare a cell lysate.

Caspase 3 activity was measured by an enzyme reaction using Ac-DEVD-AMC (product name: Ac-Asp-Glu-Val-Asp-MCA, manufactured by Peptide Research Institute, Inc.) as a substrate and a cell lysate as a crude enzyme. After adding 75 μl of the reaction buffer containing the substrate and 25 μl of the cell lysate to a 96-well black microplate (product name: F96 NONTREATED BLACK MICROWELL, manufactured by Thermo Fisher Scientific) and reacting at 37 ° C for 2 hours, the microplate The excitation wavelength of 380 nm / the fluorescence wavelength of 460 nm was measured by a reader. A value obtained by correcting the measured fluorescence unit (FU) with the amount of protein in the cell lysate was defined as the caspase 3 activity value (FU / mg protein). The protein concentration of the cell lysate was quantified using a BCA protein assay kit (product name: Micro BCA Protein Assay, manufactured by Thermo Fisher Scientific).

In addition, the measuring method of caspase 3 activity was constructed | assembled with reference to the following literature.
EnzChek Caspase-3 Assay Kit # 2 Product Information
・ Federation of European Biochemical Societies letters, 1996, 395, 267-271, Ceramide induce apoptosis via CPP32 activation
The Biochemical journal, 1999, 344, 477-485, Heat-shock protein 70 antisense oligomers enhancement proteasome inhibitor-induced apoptosis

The results are shown in FIG. In Fig. 2 (a), caspase 3 activity is elevated and apoptosis is promoted in the "control" to which DCA is added, but compared to the radish extract and radish extract, the radish extract It was confirmed that the addition suppressed the elevation of caspase 3 activity by the addition of DCA and suppressed apoptosis. This tendency was confirmed in any of hot water extraction (70 ° C., 2 hours), methanol extraction and ethanol extraction. Also in the hot water extraction (121 ° C., 20 minutes) shown in FIG. 2 (b), caspase 3 activity is increased and apoptosis is promoted in the “control” to which DCA is added, but the ratio is higher than that of yamawasabi root extract. Then, it was confirmed that the addition of the radish extract has suppressed the increase of caspase 3 activity by the addition of DCA, and the apoptosis is suppressed.

From the above, the inhibitory effect on caspase 3 activity was different depending on the site of yamawasabi, and the highest inhibitory effect on caspase 3 activity was shown in pokeweed leaf extract, especially pokeweed leaf extract.

(Example 3)
The extraction conditions of yamasabi leaves were examined.

Caspase 3 activity after apoptosis-inducing treatment by DCA using each sample of pokeweed leaf extract obtained at extraction temperatures 20 ° C., 70 ° C., 80 ° C., 90 ° C., 105 ° C. using water as extraction solvent Was measured.

The radish extract was prepared by grinding the freeze dried radish leaves, adding 15 to 20 volumes of water, extracting at each temperature, filtering and lyophilizing the filtrate. The extraction time was 1 hour. The method of apoptosis induction and caspase 3 activity measurement is the same as in Example 2.

The results are shown in FIG. In the horseradish leaf extract obtained at extraction temperatures of 20 ° C. and 70 ° C., a relatively good inhibitory effect on caspase 3 activity was shown.

(Example 4)
The inhibitory effect of DCA on the toxicity of pokeweed leaf extract was examined.

In this example, a CA-administered mouse was used as a DCA production promotion model mouse. The primary bile acid, CA, is converted to secondary bile acid DCA by the action of intestinal bacteria that inhabit the large intestine. It is reported that DCA concentration in feces increases when rats are fed with a CA-supplemented feed (Gastroenterology, 2011, 141, 1773-1781, Bile acid is a host factor that regulates the composition of the cecal microbiota in rats ). Using a model mouse in which intestinal injury was induced by raising the DCA concentration in the large intestine, the toxicity inhibitory effect of pokeweed leaf extract on DCA was evaluated.

The animals used were ddY mice (male, 5 weeks old), purchased at 4 weeks of age and acclimated for 5 days. As a sample, horseradish leaf extract was used. The horseradish leaf extract was extracted by adding 15 volumes of water to the freeze dried leaves and extracting at 121 ° C. for 20 minutes. It was then filtered and the filtrate was lyophilized to prepare.

The grouping was as follows.
1) Untreated group: n = 10 (one animal every 4 weeks)
2) CA group (CA: 1,500 mg / kg administration): n = 15 (2 animals dissected every 4 weeks)
3) Horseradish leaf extract group (CA: 1,500 mg / kg, horseradish leaf extract: 500 mg / kg administration): n = 5

The administration schedule will be described. The mice were reared on a normal diet (product name: CE-2, manufactured by CLEA Japan, Inc.), and had free access to water and food. The weight of the mice was measured, and based on the measured values, grouping was performed so that there was no difference in weight among the groups. Intake of the horseradish leaf extract was performed by oral gavage, and the start of administration of the horseradish leaf extract was taken as week -1. The intake of the CA mixed feed was started 7 days after administration of the horseradish leaf extract (Week 0). One control group and two CA groups were dissected at weeks 4 and 8 after intake of the CA mixed feed, and the large intestine was removed. At 10 weeks, all individuals in each group were dissected and blood, large intestine and feces were collected.

In order to confirm that CA was converted to DCA in the intestine, fecal CA and DCA amounts at 0, 4, and 10 weeks were measured. Feces collected from the mice were lyophilized, and 50 times the weight of the feces was added to methanol, and sonicated for 15 minutes. Thereafter, it was centrifuged at 16,200 × g for 5 minutes. The supernatant was diluted 1,000-fold in the untreated group and 20,000-fold in the CA group and the horseradish leaf extract administered group. The diluted supernatant was analyzed using an electrospray ionization mass spectrometer (product name: Agilent 6470 Triple Quad LC / MS system, manufactured by Agilent). Mobile phase conditions of analysis are A: 10 mM ammonium acetate (manufactured by Kanto Chemical Co., Ltd.) aqueous solution, B: acetonitrile (manufactured by Kanto Chemical Co., Ltd.), elution conditions: 0-5.5 minutes: 27% B, 5.5-5 .6 minutes: 27-95% B, 5.6-7.5 minutes: 95% B, the flow rate was 0.6 mL / min. The detection conditions of CA and DCA were CA: m / z 407.3, Rt 1.19 minutes, DCA: m / z 391.3, Rt 4.64 minutes (both in negative mode). A Kinetex evo C18 100A (5 μm, 2.1 × 100 mm, manufactured by Phenomenex) column was used for analysis. The amount of fecal CA is shown in FIG. 4 (a) and the amount of fecal DCA is shown in FIG. 4 (b). This indicates that the amount of fecal CA and DCA increased by CA administration. Thus, it is shown that the ingested CA reaches the large intestine, and that the CA is converted to DCA in the large intestine, and that administration of the horseradish leaf extract does not inhibit the conversion of CA to DCA in the large intestine. It was done.

At 4, 8, and 10 weeks after CA administration, the large intestine was collected and sectioned specimens were prepared. At 4 and 8 weeks, sections of the untreated group and the CA group were prepared, and at 10 weeks, sections of all groups (2 samples each) were prepared. The preparation of a colon segment was performed at Compound Safety Research Institute, Inc. Section preparation was performed by HE staining. At 4 weeks, local inflammation and erosion were observed only in the CA group. At the 8th week, exfoliation of the surface epithelium and moderate to high degree erosion were confirmed in the CA group. In the 10-week CA group, exfoliation and erosion of the surface epithelium associated with the inflammatory reaction were observed (FIG. 5 (b)). In the horseradish leaf extract group, partial damage to the large intestine was mild (FIG. 5 (c)). Therefore, administration of the horseradish leaf extract suppresses the toxicity of DCA, which is secondary bile acid converted in the intestine, and suppresses exfoliation and erosion of the surface epithelium which is a disorder to the large intestine. Indicated.

To evaluate DCA damage to the large intestine, gene expression analysis of calprotectin in mouse large intestine epithelial cells was performed. Calprotectin is one of the markers of ulcerative colitis. In this example, the expression level of the S100A8 gene, which is one of the proteins constituting calprotectin, was measured. An RNA extract (product name: TRIzol reagent, manufactured by Life Technologies, Inc.) was added to a large intestine isolated from a mouse and disrupted using a pestle stick. The mixture was centrifuged at 9,560 × g for 5 minutes at 4 ° C., and the residue was recovered and subjected to mRNA detection of the S100A8 gene. The total RNA was extracted, and the concentration and purity were measured with an ultratrace spectrophotometer (product name: Nanodrop, manufactured by Thermo Scientific). cDNA was synthesized using a cDNA synthesis kit (product name: ReverTra Ace qPCR RT Master Mix with gDNA Remover, manufactured by Toyobo Life Science Co., Ltd.). The reaction solution after reverse transcription was diluted with Nuclease-free water so as to be 6 ng / μL, and used as a real-time PCR template.
In the PCR reaction, S100A8 forward primer (SEQ ID NO: 1) and S100A8 reverse primer (SEQ ID NO: 2) were used as primers. The Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene was used as an internal standard gene to correct the expression of the S100A8 gene, and GAPDH forward primer (SEQ ID NO: 3) and GAPDH reverse primer (SEQ ID NO: 4) were used as the primers. The Real-time PCR reaction was performed with a real-time PCR analysis system (Product name: CFX Connect, manufactured by BioRad Laboratories) using a real-time reaction kit (Product name: SsoAdvanced SYBR Green Supermix, manufactured by BioRad Laboratories). A total of 10 μL of the PCR reaction was incubated at 95 ° C. for 2 minutes (initial denaturation), followed by 40 cycles of denaturation at 95 ° C. for 10 seconds and annealing at 68 ° C. for 30 seconds.
Sequence number 1: 5'-TGAGTGTCTCCTAGTTTGTGCAG-3 '
Sequence number 2: 5'-TGTGAGATGCCACACCCACTTT-3 '
Sequence number 3: 5'-ACCCAGAAGACTGTGGATGG-3 '
Sequence number 4: 5'-TCAGCTCTGGGATGACCTTG-3 '
The primers (SEQ ID NO: 1 and SEQ ID NO: 2) used for expression level analysis of the S100A8 gene were constructed with reference to the following documents.
Proceedings of the National Academy of Sciences of the United States of America, 2013, 12768-12773, IL-22-producing neutrophils contribute to antimicrobial defense and restoration of colonial epithelial integrity colitis
Using the Cq value obtained by the Real-time PCR analysis system CFX Connect, the expression amount ratio of the S100A8 gene was calculated based on the following formula (ΔΔCt method).
Cq value of untreated group S100A8: A
Untreated group GAPDHCq value: B
Cq value of S100A8 of experiment group: C
Cq value of GAPDH in experimental group: D
ΔCq (untreated group) = A−B
ΔCq (experimental group) = C−D
Δ (ΔCq) = ΔCq (experimental group) -ΔCq (untreated group)
Expression ratio = 2 ^{-Δ (ΔCq)}

Although the expression of the S100A8 gene was significantly elevated in the CA group as compared to the non-treatment group, no significant expression elevation was observed in the pokeweed leaf extract group by CA administration (FIG. 6).

From the above, it has been shown that pokeweed leaf extract protects the large intestine from secondary bile acid toxicity by suppressing the inflammatory reaction (expression of calprotectin) caused by DCA, which is a secondary bile acid.

(Example 5)
A light meal stress test (open label test) was conducted with radish wasabi leaf extract.

The preparation method of the horseradish leaf extract is demonstrated. Twenty volumes of water were added to the dried leaves of horseradish radish, and the mixture was extracted with hot water at 70 ° C. for 120 minutes, and centrifuged at 5,000 × g for 10 minutes to recover a supernatant. The collected supernatant is filtered by suction, the same amount of excipient as the freeze-dried powder of the obtained filtrate is added, and sterilization is carried out at 80 ° C. for 30 minutes, and the obtained freeze-dried liquid is used as a radish extract did.

Mild meal load test (open label test) using capsules (400mg / day as solid content of yamasabi leaf hot water extract solid content), which was prepared by the above method, for four men Did. More specifically, an animal-based diet (ABD: Animal Based Diet) (see David LA et al Nature. 2014 505: 559-63.) Containing fat at least 1.66 times the dietary intake standard of the Japanese. The following evaluations 1 to 4 were performed before and after daily intake and before and after intake of ABD + radish wasabi leaf hot water extract.
(1) Evaluation 1: Urinary lactulose / mannitol ratio (LA / MA ratio) (If intestinal leakage occurs, LA with a large molecular weight leaks from the intestinal tract and is transferred to blood, and finally detected in urine ( LA / MA ratio increases))
Subject prohibited eating and drinking for at least 8 hours after dinner the day before urine collection. On the morning of urine collection, after urination, a solution (test sugar solution) in which 10 g of lactulose and 5 g of mannitol were dissolved with 200 mL of water or lukewarm water was orally taken without taking breakfast, and thereafter 300 mL of water or lukewarm water was additionally consumed. An additional 200 mL of water was taken 3 hours after ingestion of the test sugar solution. Total urine was collected 6 hours after ingestion of the test sugar solution. The determination of lactulose and mannitol in urine was performed according to the procedure using an intestinal permeability measurement kit (product name: EnzyChrom Intestinal Permeability Assay Kit, manufactured by BioAssay Systems).
(2) Evaluation 2: I-FABP in serum (Intestine-derived fatty acid binding protein) (If intestinal leakage occurs, I-FABP originally present in intestinal epithelial cells is detected in blood)
Measurement of I-FABP concentration in serum was performed according to the procedure using a human I-FABP measurement kit (product name: I-FABP, Human, ELISA Kit, manufactured by HycultBiotech).
(3) Evaluation 3: Fecal Calprotectin (Detecting the inflammatory marker calprotectin in feces is an indicator of inflammation specific to the intestine)
Measurement of fecal calprotectin concentration was carried out using a human calprotectin assay kit (product name: Calprotectin, Human, ELISA Kit, IDK, manufactured by Immunodiagnostik) according to the procedure.
(4) Evaluation 4: Status of flight questionnaire

The measurement results of urinary lactulose / mannitol ratio (LA / MA ratio), which is an indicator of intestinal leakage, are shown in FIG. The LA / MA ratio increased in 3 out of 4 subjects after ABD intake compared to before ABD intake, and the mean value of 4 subjects also showed an increasing tendency before and after intake (p <0.1) . On the other hand, it was confirmed that the intake of the horseradish leaf hot water extract decreases the average value of the LA / MA ratio before and after intake although there is no statistically significant difference. Furthermore, the LA / MA ratio is not significant and equal before intake of ABD, ABD + horseradish leaf hot water extract, but after intake of horseradish leaf hot water extract, the value tends to be lower compared to ABD alone (p <0.1) was recognized.

The measurement results of serum I-FABP (enteric-derived fatty acid binding protein), which is an indicator of intestinal leakage, are shown in FIG. The I-FABP value was increased in all subjects after ABD intake compared to before ABD intake, and the mean value was also statistically significantly (p <0.05) increased. On the other hand, it was confirmed that the increase in I-FABP value can be suppressed by ingesting the horseradish leaf hot water extract.

The measurement results of fecal calprotectin, which is an indicator of inflammation specific to intestine, are shown in FIG. Although fecal calprotectin levels were increased in all subjects after ABD intake compared with before ABD intake, ingestion of yamawasabi leaf hot water extract suppresses the increase in fecal calprotectin levels. Was confirmed.

Regarding the defecation situation, the questionnaire was conducted before and after intake of ABD for 5 days and before and after intake of ABD + radish wasabi leaf hot water extract for the items shown below.
-Color of stool The color of ordinary stool was set to “0”, and the value became larger when the color of stool deteriorated.
3: Blackish brown (rgb (25, 13, 12))
2: Brown (rgb (61, 23, 12))
1: Brown (rgb (112, 56, 34))
0: Ocher (ordinary stool color) (rgb (175, 111, 28))
1: Orange (rgb (226, 159, 13))
-Shape of stool The standard stool was set to "0" based on the Bristol scale, and it was set so that the value would increase if the shape of stool deteriorated.
3: Colo stool (hard, cocoon-like stool)
2: Hard stool (sausiform but hard stool)
1: Slightly hard stool (sausage-like stool with cracks on the surface)
0: Normal (Smooth surface with a soft sausage-like or snake-like towing coil)
1: Slightly soft stool (soft, half-solid stool with sharp wrinkles)
2: Mud (unstable boundary, irregularly shaped small-piece, muddy)
3: Watery stool (watery, solid free liquid stool)
・ The smell of feces 3: very bad smell 2: bad smell 1: little smell 0: no smell

The questionnaire results of stool color are shown in FIG. Compared with before ABD intake, 3 out of 4 people increased the value showing deterioration of stool color, and the average value increased from 0.8 to 1.5 after ABD intake. On the other hand, when the horseradish leaf hot water extract is taken in addition to ABD, the change of the average value is 0.7 to 0.9, and it maintains a value close to "0" indicating the color of normal stool. Was confirmed.

The questionnaire result of the shape of the stool is shown in FIG. The shape of feces deteriorated in all subjects after ABD intake compared to before ABD intake, and a tendency for deterioration (p <0.1) was observed even on average. On the other hand, when the horseradish leaf hot water extract was taken in addition to ABD, no statistically significant change in the values before and after the ingestion was observed. Furthermore, the average value after intake of ABD is 2.0, while the average value after intake of ABD + horseradish leaf hot water extract is 0.9, and after intake of horseradish leaf hot water extract, it becomes a normal "0". It was confirmed that they approached and had a good stool shape.

The questionnaire result of the smell of stool is shown in FIG. The odor of feces was statistically significantly (p <0.05) worse in all subjects after ABD intake compared to before ABD intake, but by taking the horseradish leaf hot water extract in addition to ABD, The values before and after intake were not statistically significant, and the average value after intake was 2.3 after intake of ABD, whereas the average value after intake of ABD + horseradish leaf hot water extract was 1.8 and It was confirmed that the smell of the feces was suppressed by approaching “0” of “no smell”.

(Example 6)
A single brand ind crossover test was conducted with radish wasabi leaf extract.

A capsule (400 mg / day as solid matter of yamasabi leaf hot water extract) containing yamasabi leaf hot water extract prepared by the method of Example 5 was used for a total of 13 persons of 8 men and 5 women. Conducted a single-brand India crossover test. More specifically, the following evaluations 1 and 2 were performed before and after 14 days of horseradish radish leaf hot water extract intake or 14 days of placebo intake.
(1) Evaluation 1: Fecal Calprotectin Fecal calprotectin concentration measurement was carried out according to a consignment analysis (Study item: Fecal calprotectin, JLAC10 code: 5C235-0000-015-023) of SAR Inc. .
(2) Evaluation 2: Status of flight questionnaire / physical condition questionnaire

The measurement results of fecal calprotectin are shown in FIG. Fecal calprotectin levels tended to increase after taking placebo compared to before taking placebo, but intake of hot water extract of pokeweed radish tended to suppress rising of fecal calprotectin levels Was confirmed.

The questionnaire result of the shape of the stool is shown in FIG. Although there was no statistically significant change before and after placebo intake, the statistically significant (p <0.05) value decreased before and after intake of the horseradish leaf hot water extract, approaching the normal “0” It was confirmed that it had a good stool shape.

The questionnaire result of the smell of stool is shown in FIG. Although there was no statistically significant change before and after placebo intake, the statistically significant (p <0.05) value decreased before and after intake of the horseradish leaf hot water extract; It was confirmed that the smell of stool was improved.

The results of the physical condition questionnaire are shown in Figure 16-18. Each questionnaire represents the subject's own state on a straight line with “0” as “very good” and “10” as “worst state” before and after ingestion of horseradish leaf hot water extract or placebo intake. It was evaluated by marking the numbers intuitively. In Fig. 16-18, assuming that the condition before intake of the horseradish leaf hot water extract or before intake of the placebo is 0, if the value proceeds in the negative direction, "state improvement" is represented, and if the value proceeds in the positive direction, " "Deterioration of the condition" In any of ease of fatigue (Fig. 16), fatigue condition (Fig. 17) and rough skin (Fig. 18), the value is in the negative direction after intake compared to before intake of the horseradish leaf hot water extract Going forward, it has been shown that intake of pokeweed leaf hot water extract improves fatigue, tiredness and rough skin.

The present invention is capable of various embodiments and modifications without departing from the broad spirit and scope of the present invention. In addition, the embodiment described above is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated not by the embodiments but by the claims. And, various modifications applied within the scope of the claims and the meaning of the invention are considered to be within the scope of the present invention.

The present application is based on Japanese Patent Application No. 2017-191247 filed on Sep. 29, 2017. The specification, claims, and whole drawings of Japanese Patent Application No. 2017-191247 are incorporated herein by reference.

Claims (4)

1. The caspase 3 activity inhibitor which uses the horseradish leaf extract obtained by solvent-extracting the horseradish leaf as an active ingredient.
2. An apoptosis inhibitor comprising, as an active ingredient, a horseradish leaf extract obtained by solvent extraction of horseradish leaves.
3. An inhibitor of secondary bile acid toxicity, which comprises, as an active ingredient, a horseradish leaf extract obtained by solvent extraction of horseradish leaves.
4. An intestinal disorder inhibitor comprising a radish extract obtained by solvent extraction of radish radish leaves as an active ingredient.

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