JP6167267B2 - Skin and colonic mucosa improving agent - Google Patents

Description

  The present invention relates to an ameliorating agent for skin or large intestine mucosa that can be produced relatively easily without going through complicated steps and yet has excellent medicinal effects.

  Improvement agents such as skin and large intestine mucosa belonging to theme code 4C201 (therapeutic activity of chemicals or pharmaceuticals) are FI code A61P17 / 00 (therapeutic agent for skin diseases) and A61P1 / 04 (mucosal protective agent) of the Japan Patent Office. Classification and various inventions have been proposed (for example, Patent Documents 1 to 3).

JP 2006-526392 A JP 2011-105614 A JP 2010-63406 A

  However, the invention described in Patent Document 1 requires a process of inoculating and inoculating fungi on an animal lipid substrate, which is complicated and requires a long-term process. The invention of Patent Document 2 uses an antigen-antibody reaction by chickens, and the production process is not simple. In this respect, the functional material described in Patent Document 3 is mainly composed of an alcohol extract from bird fillet, and the manufacturing process is simple.

  However, the functional material described in Patent Document 3 has a problem that its medicinal performance is not sufficient. That is, Patent Document 3 states that “a pharmaceutical preparation containing a functional material improves atopic dermatitis, prevents or improves dementia, repairs burned skin, suppresses hyperglycemia, and lowers blood cholesterol. It is preferably used for applications such as lipid metabolism promotion, anti-fatigue, maintenance of blood albumin level, recovery of liver function, suppression of muscle loss during exercise, etc. " And performance against ulcerative colitis is not sufficient.

  This invention is made | formed in view of said problem, Comprising: It aims at providing the improving agent which exhibits an effect with respect to skin and a large intestine mucosa, although a manufacturing process is simple.

  In order to achieve the above object, the present inventor has studied various skin or large intestine mucosa improving agents that can be produced by a simple method, and led to the present invention.

That is, the present invention is an atopic dermatitis or colonic mucosa improving agent composed mainly of a non-heated ostrich fat melt. Here, the main component preferably means 90% (more preferably 95%) or more by weight. In fact, it may be almost 100%.

  In the present invention, the improving agent is not limited to pharmaceuticals but is a concept including cosmetics, health foods, and the like.

The melt of the present invention is preferably squeezed from ostrich fat in an unheated state, and the melt is preferably always maintained below 100 °. In this case, the melt of the present invention is typically squeezed out by naturally thawing the frozen ostrich subcutaneous fat.

Moreover, it is also suitable for the melt of the present invention to extract alcohol from non-heated ostrich fat .

The melt should preferably have a saturated fatty acid to unsaturated fatty acid content ratio of 1: 2.5 to 1: 3.5. Here, the saturated fatty acid should contain palmitic acid C ₁₆ H ₃₂ O ₂ or stearic acid C ₁₈ H ₃₆ O ₂ as the main component, and the unsaturated fatty acid can be oleic acid C ₁₈ H ₃₄ O ₂ or linoleic acid C. ₁₈ H ₃₂ O ₂ should be the main component.

  In this case, it is preferable that the total amount of oleic acid and linoleic acid is 80% or more, preferably 85% or more, based on the total amount of unsaturated fatty acids. In the case of an improver made from ostrich belly fat, the content ratio of oleic acid and linoleic acid is preferably about 1: 1.2 to 1: 1.4. However, in the case of an improver made from ostrich back fat, it has been confirmed that the content of linoleic acid is higher than that of oleic acid, and considering this point, a suitable content ratio of oleic acid and linoleic acid Is about 2: 1 to 1: 1.4.

  Further, it is preferable that palmitic acid and stearic acid are contained in a total amount of 94% or more, preferably 96% or more based on the total amount of saturated fatty acids. The content ratio of stearic acid and palmitic acid is preferably about 1: 2 to 1: 5, more preferably about 1: 2.5 to 1: 4.

  Although the usage mode of the present invention is not particularly limited, 0.02 mL to 0.15 mL is preferably applied as a skin improving agent for atopic dermatitis and the like in terms of an affected area of 2 cm × 3 cm. In the application test for atopic dermatitis in the example, the application amount of ostrich fat was 0.1 mL / mouse, and 0.1 mL was applied to each mouse in the area of about 2 cm × 3 cm from the ear to the back. did.

  On the other hand, as an agent for improving large intestine mucosa for ulcerative colitis and the like, preferably 2 mL to 120 mL is orally ingested per day in terms of 60 kg human. In the oral administration test for ulcerative colitis in the examples, experiments were conducted at 0.5 or 2 mL / Kg body weight / day, 0.05 or 0.5 mL / Kg body weight / day, and 0.05 mL / Kg body weight / day. However, a predetermined effect has been confirmed, but when this minimum intake is converted to 60 kg, ostrich fat becomes 3 mL / 60 kg body weight / day.

  According to the present invention described above, it is possible to realize an improving agent that exerts an effect on the skin and the large intestine mucosa while the manufacturing process is simple.

It is drawing which shows the score of skin inflammation and the affected part photograph in contrast. It is drawing which shows transition of a skin inflammation score about a comparative example. It is drawing which shows the quantitative result of an inflammatory cytokine regarding a comparative example. It is drawing which shows transition of the skin inflammation score about Example 1. 1 is a photograph showing a tissue-stained photograph in Example 1. 1 is a drawing showing the results of protein quantification with respect to Example 1. 1 is a drawing showing the results of protein quantification with respect to Example 1. It is drawing which shows transition of a skin inflammation score about Example 2. FIG. 6 is a drawing showing the results of protein quantification with respect to Example 2. It is drawing which shows transition of a skin inflammation score about Example 3. FIG. 6 is a drawing showing the results of protein quantification with respect to Example 3. 4 is a drawing showing another result of protein quantification regarding Example 3. It is drawing which shows transition (a) of the symptom score (DAI) regarding Example 4, the change (b) of the weight after one week, and the length (c) of the large intestine after one week. It is drawing which shows transition (a) of a symptom score (DAI) regarding Example 5, and length (b) of the large intestine after one week. It is the photograph (a) which shows inflammatory cytokine (staining with an anti- TNF- (alpha) antibody) regarding Example 5, and the comparison figure (b) of the number of TNF- (alpha) positive cells. It is the photograph (a) which shows the cell adhesion molecule which correlates with the severity of ulcerative colitis regarding Example 5, and the comparison figure (b) of the number of ICAM-1 positive cells. 10 is a drawing showing another result of protein quantification by Western blotting with respect to Example 5. FIG.

  Hereinafter, although an Example is described, the concrete description content does not limit this invention at all.

<Adjustment of ostrich fat>
The ostrich fat was separated from the mass of subcutaneous fat that was frozen and preserved after slaughtering and dismantling the ostrich. The heat-treated ostrich fat was melted by putting the fat mass in a small pot and heating it with a gas stove, and dispensing the supernatant 1 mL at a time.

  On the other hand, the non-heated ostrich butter is chopped into a 50 mL sterilized conical tube (polypropylene centrifuge tube) in a room at 24-27 ° C. After centrifugation at 4,000 rpm for 30 minutes at Kubota Shoji Co., Ltd., the supernatant was recovered, dispensed 1 mL at a time, and stored frozen. Chicken fat is peeled from chicken with skin in a room at 24-27 ° C, cut into 50 mL sterilized conical tubes, centrifuged at 4,000 rpm for 30 minutes in a centrifuge at 27 ° C, and the supernatant is removed. Collected, dispensed 1 mL each, and stored frozen.

<Contrast experiment with heated ostrich fat>
NC / Nga mice (female, 12 weeks old) with hair removal applied with oak salmon allergen-containing ointment (Biosta AD, Biosta Co., Ltd.) for 2 weeks to induce atopic dermatitis, and then heat treated ostrich for 1 week Fat was orally administered at 0.5 mL / kg body weight / day. We examined healthy mice and atopic dermatitis / non-treated groups as comparative subjects.

  As for the symptoms of atopic dermatitis, the degree of redness / bleeding, scab formation / drying, edema, scratches / tissue defects was quantified to obtain a skin inflammation score. After the rearing, the affected skin was collected and inflammatory cytokines were measured by Western blotting. Western blotting extracts proteins from skin tissue, performs sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfers to polyvinylidene fluoride (PVDF) membrane, and then detects using antibodies to inflammatory cytokines Went.

  Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard was also performed, digitized using image analysis software, relative values for GAPDH were determined for each inflammatory cytokine, and comparison between test groups was performed .

  As shown in Table 1, the skin inflammation score was evaluated in four stages from asymptomatic (0 points) to severe (up to 3 points), as shown in Table 1, and the dermatitis symptoms were totaled from 0 points to 12 points. It was expressed as a numerical value up to a point. FIG. 1 is a drawing showing a comparison between a skin inflammation score and an affected area photograph.

<Test results>
FIG. 2 shows the transition of the skin inflammation score, and FIG. 3 shows the quantitative results of inflammatory cytokines by Western blotting. As a result, it was confirmed that the skin inflammation score was markedly increased and the expression of inflammatory cytokines was increased in the atopic dermatitis / non-treatment group compared to the healthy mouse group, but it was not improved by the administration of heat-treated ostrich fat. The The error bar indicates the standard error SEM, and this point is the same in other drawings.

<Example 1 (application of non-heated ostrich fat)>
Biosta AD was applied to NC / Nga mice (female, 10-12 weeks old) for which hair was removed for 2 weeks to induce atopic dermatitis, and then ostrich fat was treated with 0.1 mL / mouse / day for 2 weeks. Applied to. We examined healthy mice and atopic dermatitis / non-treated groups as comparative subjects.

  A skin inflammation score was obtained every week, and the affected skin was collected after breeding, and tissue staining was performed to observe skin thickening and inflammation. Proteins related to fibrosis, endoplasmic reticulum stress, oxidative stress, inflammation, etc. were measured by Western blotting. Α-tubulin was also detected as an internal standard, a relative value to α-tubulin was determined, and comparison between test groups was performed.

<Test results>
FIG. 4 is a drawing showing the transition of the skin inflammation score. As shown in the figure, the healthy mouse group was 0 at the end of the breeding, but the atopic dermatitis / non-treated group increased to 10.2 ± 1.4 (mean ± SEM). It is confirmed that it has decreased significantly to ± 1.0.

  FIG. 5 shows a photograph of tissue staining. In the non-treated group, skin thickening appears markedly due to inflammation, but it is confirmed that it improves with the application of ostrich fat. Mast cells with granules containing histamine and protease, which are powerful inflammatory mediators, appeared prominently in the untreated group, but improved with the application of ostrich fat.

  6 to 7 show the results of protein quantification by Western blotting. The expression of proteins involved in fibrosis, endoplasmic reticulum stress, oxidative stress, inflammation, etc. was increased in the atopic dermatitis / non-treatment group compared with the healthy mouse group, but these were improved by administration of ostrich fat Is confirmed.

<Example 2 (oral intake of non-heated ostrich fat)>
Biosta AD was applied to NC / Nga mice (female, 10-12 weeks old) with hair removal for 2 weeks to induce atopic dermatitis, and then ostrich fat was orally administered at 0.5 mL / Kg body weight / day for 2 weeks. Administered. The comparison was conducted in a healthy mouse group, an atopic dermatitis / non-treatment group, and an atopic dermatitis / chicken fat group in which chicken fat was orally administered at 0.5 mL / Kg body weight / day.

  In addition, skin inflammation scores were obtained every week, and affected skin was collected after breeding, and proteins related to oxidative stress, endoplasmic reticulum stress, apoptosis, inflammation, etc. were measured by Western blotting. GAPDH was also detected as an internal standard, a relative value to GAPDH was determined, and comparison between test groups was performed.

<Test results>
FIG. 8 shows the transition of the skin inflammation score. As shown in the figure, the healthy mouse group was 0 at the end of breeding, but the atopic dermatitis / untreated group increased to 12.0 ± 0 (mean ± SEM), and 6.7 ± 1 by ostrich fat administration It was confirmed that the improvement was weak at 9.3 ± 0.7 by chicken fat administration.

  FIG. 9 shows the results of protein quantification by Western blotting. As shown in the figure, the expression of proteins involved in oxidative stress, endoplasmic reticulum stress, apoptosis, inflammation, etc. was increased in the atopic dermatitis / non-treated group compared to the healthy mouse group, but all improved with administration of ostrich fat did. However, administration of chicken fat did not improve the inflammation markers (Cox2, Il-1β).

<Alcohol extraction of ostrich fat>
100 g of a mass of frozen ostrich subcutaneous fat was chopped and ground in a mortar. It moved to the 3L Erlenmeyer flask, 2500 mL of methanol was added, the stirring bar was put, and it stirred with the magnetic stirrer (I06G, Mitamura Riken Kogyo Co., Ltd.) for 3 hours.

  Natural filtration was performed with a filter paper with folds (qualitative filter paper No. 2, Advantech Toyo Co., Ltd.), and the filtrate was concentrated under reduced pressure with a rotary evaporator (N-2 type, Tokyo Riken Kikai Co., Ltd.). Then, it puts into a desiccator and depressurizes with a vacuum pump (DAIVAC 1.2L, Tokyo Riken Kikai Co., Ltd.) to completely remove methanol to obtain 5.27 g of ostrich fat methanol extraction fraction (average, 110.266 g for 21 times). It was.

  The results of component analysis of whole fat and methanol extraction fraction of whole fat are shown in Table 1 (a). Although the total fat is almost fat, the ethanol extract fraction had a fat content of 70.3%. Therefore, for the ethanol extraction fraction, each component is shown with the fatty acid contained in 70.3% of fat being 100. Table 2 (b) shows the main components divided into saturated fatty acids and unsaturated fatty acids.

  As above-mentioned, the content ratio of a saturated fatty acid and an unsaturated fatty acid is about 1: 3.0-1: 3.1 among the whole fatty acid.

  And palmitic acid occupies 71% -77% in the whole saturated fatty acid, and the total amount of palmitic acid and stearic acid occupies 98% -97% of the whole saturated fatty acid. In addition, the content ratio of stearic acid and palmitic acid was about 1: 2.7 to 1: 3.79.

  Moreover, linoleic acid accounts for 53 to 50% and oleic acid accounts for 39% to 38% in the whole unsaturated fatty acid. Here, the content ratio of oleic acid and linoleic acid was about 1: 1.35 to 1: 1.31.

  Then, linoleic acid and oleic acid account for 92% to 89% of the whole unsaturated fatty acid, and when linolenic acid is added thereto, almost all of the unsaturated fatty acid (98% to 96%) is obtained.

<Example 3 (oral intake of alcohol-extracted ostrich fat)>
Biosta AD was applied to NC / Nga mice (female, 10-12 weeks old) for which hair was removed for 2 weeks to induce atopic dermatitis, and then for 2 weeks, the ostrich fat methanol extraction fraction was 0.5 mL / Kg / It was orally administered on the day. As comparison subjects, healthy mouse group, atopic dermatitis / non-treatment group, ostrich fat (total fat) orally administered at 0.5 mL / Kg / day, atopic dermatitis / ostrich fat group, chicken fat 0.5 mL / Kg / Atopic dermatitis / chicken fat group administered orally on day was studied.

  The skin inflammation score was obtained every week, and the affected skin was collected after the breeding, and inflammatory markers and the like were measured by Western blotting. GAPDH was also detected as an internal standard, a relative value to GAPDH was determined, and comparison between test groups was performed.

<Test results>
FIG. 10 shows the transition of the skin inflammation score. As shown in the figure, the healthy mouse group was 0.2 ± 0.20 (mean ± SEM) at the end of the breeding, but the atopic dermatitis / non-treatment group increased to 9.0 ± 1.68, and the ostrich It decreased to 3.8 ± 0.58 by the administration of the fat methanol extraction fraction and 3.8 ± 0.80 by the administration of ostrich fat (whole fat), but 5.4 ± by the administration of chicken fat (whole fat). It is confirmed that the improvement is weak at 0.93.

  FIG. 11 to FIG. 12 show the results of protein quantification by Western blotting. As shown in the figure, the expression of proteins involved in inflammation and oxidative stress increased in the atopic dermatitis / non-treated group compared to the healthy mouse group, and improved by administration of ostrich fat (whole fat). Further improvement by administration of the fraction. However, administration of chicken fat (whole fat) showed almost no improvement.

<Example 4 (test for improving colitis)>
B57BL / 6J mice (female, 10 weeks old) are ingested with water containing 3% dextran sulfate sodium (DSS) to induce colitis, and ostrich fat is 0.5 or 2 mL / kg body weight for 7 days. / Day was observed after oral administration. As a comparison target, a healthy mouse group that was swallowed with water not containing DSS, and a non-treated DSS control group that was swallowed with 3% DSS water were studied. After the breeding, the large intestine was taken out, the length was measured, and the degree of atrophy was compared.

  The severity of colitis was scored based on weight loss, fecal properties, and fecal occult blood, and a disease activity index (DAI) was determined. DAI was evaluated for three symptoms as shown in Table 3, and the total severity of colitis was expressed as a numerical value from 0 to 10 points.

<Test results>
FIG. 13A shows the transition of symptom score (DAI), FIG. 13B shows the change in body weight after one week, and FIG. 13C shows the length of the large intestine after one week. As shown in the figure, the symptom score markedly increased in the DSS control group compared to the healthy mouse group, and atrophy of the large intestine was observed.

However, administration of ostrich fat reduced the symptom score, and weight loss was suppressed in a dose-dependent manner. It is also confirmed that colon atrophy is improved by administration of ostrich fat. In addition, it is known that the colon is shortened by ulcerative colitis induced by DSS (*)
(*) Okayasu I, et al: A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98 (3): 694-702. (1990)

<Example 5 (test for improving colitis)>
B57BL / 6J mice (female, 10 weeks old) were ingested with 3% DSS water to induce colitis, and observed for 7 days by oral administration of ostrich fat at 0.05 or 0.5 mL / kg body weight / day. . Healthy mice group that was given DSS-free water as a comparison target, DSS control group that was given 3% DSS water and was not treated, DSS that was orally administered with chick fat 0.05 or 0.5 mL / kg body weight / day A study was conducted with a group of chicken fat 0.05% and a group of DDS chicken fat 0.5%. After obtaining the DAI every day, the large intestine was taken out and the length was measured, and then the inflammatory markers and the like were measured by immunohistochemical staining and Western blotting. In Western blotting, GAPDH was also detected as an internal standard, relative values to GAPDH were determined, and comparisons were made between test groups.

<Test results>
14A shows the history of the symptom score (DAI), and FIG. 14B shows the length of the large intestine after one week. FIG. 15 (a) is a photograph showing inflammatory cytokines (staining with anti-TNF-α antibody), and FIG. 15 (b) is a comparison diagram of the number of TNF-α positive cells. FIG. 16 (a) is a photograph showing an intercellular adhesion molecule (staining with anti-ICAM-1 antibody) that correlates with the severity of ulcerative colitis, and FIG. 16 (b) is a comparative view of the number of ICAM-1 positive cells. It is. FIG. 17 shows the results of protein quantification by Western blotting.

  From the above results, the symptom score and inflammatory markers are significantly increased in the DSS control group as compared with the healthy mouse group, and colon atrophy is seen. However, it is confirmed that administration of ostrich fat reduces the symptom score and improves colon atrophy and inflammation markers. However, chicken fat showed no improvement in the symptom score or atrophy of the large intestine, and the improvement of the inflammatory marker was also small.

  It is known that ICAM-1-positive cells increase depending on the degree (severity) of histological activity of ulcerative colitis (**). (**) Histological study of ulcerative colitis-Activation of lymphocytes in the lamina propria of the large intestine and ICAM-1-positive dendritic cells and macrophages-. Japanese Journal of Gastroenterology 89 (1): 17-27 (1992)

  From the results of Examples 2 to 4, it was revealed that the inflammatory marker of the affected area was reduced even at a site distant from the skin and large intestine by oral administration of ostrich fat. This suggests that oral administration of ostrich fat may be effective even in inflammation where the affected area cannot be identified.

  By the way, the fat used in the examples is ostrich belly fat (subcutaneous fat). In addition, instead of the abdominal fat, back fat may be used. However, the back fat is more suitable because it may be hardly removed depending on the breeding environment and the quality varies. is there.

Claims (7)

  An atopic dermatitis or colonic mucosa improving agent composed mainly of a melt of non-heated ostrich fat.   The improving agent according to claim 1, wherein the melt is squeezed out of ostrich fat in an unheated state.   The improving agent according to claim 1 or 2, wherein the melt is squeezed by naturally thawing the subcutaneous fat of an ostrich that has been stored frozen.   The improving agent according to claim 1, wherein the melt is extracted with alcohol from ostrich fat in an unheated state.   In the melt, a content ratio of a saturated fatty acid mainly composed of palmitic acid and stearic acid and an unsaturated fatty acid mainly composed of oleic acid and linoleic acid is 1: 2.5 to 1: 3.5. The improvement agent in any one of Claims 1-4.   The colonic mucosa-improving agent according to any one of claims 1 to 5, wherein 1 mL to 3 mL is orally ingested per day in terms of human 60 kg. The agent for improving large intestine mucosa according to claim 6 , which is applied to ulcerative colitis.