KR101216253B1 - Composition for treatment or prevention of transplant rejection or autoimmune diseases - Google Patents

Abstract

The present invention provides a composition for treating or preventing transplant rejection or autoimmune disease, including cotton swab extract as an active ingredient. The composition of the present invention has the advantage of being effective in treating or preventing transplant rejection or autoimmune disease.

Description

Composition for treatment or prevention of transplant rejection or autoimmune diseases}

The present invention relates to a composition for treating or preventing transplant rejection or autoimmune diseases, and more particularly, to a composition for treating or preventing transplant rejection or autoimmune diseases, including cotton swab extract as an active ingredient.

Killing the middle layer of the strip and / or the strip of the king was called (竹茹), generally used in combination without distinguishing what is collected from the rod and the king, there are reports that have been studied in the treatment of asthma.

Asthma is a disease associated with an overreaction of T helper (Th) 2 lymphocytes to antigen. That is, the Th 2 response is abnormally strong among the T helper (Th) lymphocyte and the T helper (Th) lymphocyte reaction. Th 1 and Th 2 reactions are mutually inhibiting reactions. When the Th 2 reaction is strong, such as asthma, the Th 1 reaction hardly occurs. The present inventors have studied the anti-asthma effect of the kill extract, the kill kill extract can increase the response of Th 1 to relatively suppress the response of Th 2 can treat the asthma by balancing the Th 1 and Th 2 response (Journal of Ethnopharmacology, Volume 128, No. 1, 2 March (2010), 2010.3.1).

Transplant rejection and autoimmune diseases, on the other hand, attack the transplanted tissues by non-self-recognition reactions or misrecognize one's own tissues as external tissues. It was difficult to use the extract to increase the response 1 and inhibit the Th 2 response (presumed to be a mixture of cotton and gingiva) for the purpose of treatment or prevention of transplant rejection or autoimmune disease.

In addition, the Republic of Korea Patent No. 442243, the powder (cotton cord) extract exhibits the NO inhibitory activity in the macrophages, exhibiting anti-inflammatory action is effective for inflammatory diseases, the inflammatory diseases specifically gastritis, colitis, arthritis, nephritis , Hepatitis, cancer or degenerative disease. Gastritis, colitis, arthritis, nephritis, hepatitis, cancer, or degenerative diseases are diseases caused mainly by the influx of external toxic substances, which are qualitatively different from transplant rejection or autoimmune diseases caused by nonmagnetic recognition.

The present inventors completed the present invention as a result of the study on the treatment or prevention of transplant rejection or autoimmune disease.

The problem to be solved by the present invention is to provide a composition for treating or preventing transplant rejection or autoimmune disease.

The present inventors have found that cotton swab extract is effective in inhibiting graft rejection reaction or in treating or preventing autoimmune diseases, thus completing the present invention. Accordingly, the present invention provides a therapeutic or prophylactic use of cotton wool extract rejection or autoimmune disease. In the present invention, 'treatment or prevention' means 'including improvement or prevention'.

In addition, the present invention provides a composition for treating or preventing transplant rejection or autoimmune disease, including cotton swab extract as an active ingredient.

The swab may be a cotton swab powder, the swab extract may be at least one solvent extract selected from water, alcohol having 1 to 4 carbon atoms, methylene chloride or ethyl acetate. Preferably, the extract may be ethyl acetate soluble extract of cotton pad. The ethyl acetate soluble extract may be an ethyl acetate soluble fraction extracted with ethyl acetate after removing the methylene chloride soluble fraction of the cotton extract of cotton pad.

The 'sponge' is meant to include the whole or part of the cotton pad, and preferably may be an intermediate layer (aka, killing the cotton pad) to remove the outer shell of the stem of the cotton pad.

The transplant rejection reaction may be a transplant rejection reaction of a tissue or an organ, and preferably the transplant rejection reaction of the tissue or an organ is a bone marrow transplant, a heart transplant, a cornea transplant, an intestinal transplant, a liver transplant, a lung transplant, a pancreas transplant, a kidney At least one rejection of transplantation and rejection of transplantation. The transplant rejection reaction is meant to include autologous or other tags.

The autoimmune diseases include progressive systemic sclerosis, Scleroderma, systemic lupus erythematosus, insulin dependent childhood diabetes with pancreatic cell antibodies, multiple sclerosis, autoimmune hemolytic anemia, At least one selected from Myasthenia gravis or Grave's disease.

Treatment or prevention of the transplant rejection reaction or autoimmune disease may be due to one or more selected from T helper 1 and T helper 2 reaction inhibition or T cell proliferation inhibition.

The composition of the present invention may contain 0.1 to 20% by weight based on the total weight of the active ingredient.

The composition of the present invention may be a pharmaceutical composition or a food composition. In the food composition, the treatment or prevention is meant to include improvement or prevention. The food composition may be included in a variety of foods, including beverages, may be in the form of beverages, gum, tea, health functional foods, etc. The health functional foods may be formulated in the form of tablets, capsules and the like.

The present invention also provides a method for treating or preventing transplant rejection or autoimmune disease, comprising administering a cotton extract to a mammal including a human, and preparing a preparation for treating or preventing transplant rejection or autoimmune disease of the cotton extract. Provides a use for

Unless stated otherwise, the contents of the composition of the present invention apply equally to the use, treatment or prevention method of the present invention.

The extract or composition may be administered orally or parenterally to mammals including humans, and the active ingredient may be formulated in combination with a pharmaceutically acceptable carrier. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are commonly used may be used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, and And / or by adding gelatin or the like. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions and syrups, and various excipients such as wetting agents, sweeteners, fragrances and / or preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. May be included. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes and suppositories. Injectable solutions, suspensions and emulsions can be prepared by mixing water, non-aqueous solvents or suspending solvents with the active ingredient. As non-aqueous solvents and suspending solvents, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethyl oleate Injectable esters such as and the like.

As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and / or gelatin may be used. Parenteral administration may be by subcutaneous injection, intravenous injection or intramuscular injection.

Cotton swab extract included in the composition of the present invention or used in the uses and methods may be used at a dose of 10 to 100 mg / kg per day on an adult basis.

The cotton swab extract may be formulated by adding a pharmaceutically or food-acceptable carrier, excipient or diluent, etc. For formulation, see Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA et al. Reference may be made.

The composition of the present invention is effective for treating or preventing transplant rejection or autoimmune disease.

1 is a graph (A) and a graph (B) showing a change in concentration of IFN-gamma according to the treatment of cotton swabs in splenocytes (A).
Figure 2 is a graph showing the cell growth rate according to the concentration of cotton swab extract administration.
Figure 3 is a graph showing the cell cycle arrest results according to the concentration of cotton swab extract administration.

Examples and preparation examples are provided to aid the understanding of the present invention. The following examples and preparations are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples and preparations.

<Example 1> Confirmation of treatment or prophylactic effect of transplant rejection or autoimmune disease of cotton rod extract-Confirmation by IFN-r and IL-4 measurement

1-1. Preparation of extract

1-1-1. Preparation of Water Extract

Kills originating from Phllostachys nigra var. Henosis (intermediate layers stripped of stems) were purchased through Omniherb (Daegu, Korea). After killing, 5 L of water was added to 360 g, heated twice at 100 ° C. for 2 hours, and extracted continuously with reflux cooling.

1-1-2. Preparation of Methylene Chloride Soluble Fraction

100 g of distilled water was added to 5 g of the cotton swab extract obtained in 1-1-1., Suspended, 100 ml of methylene chloride was added, mixed, and then repeated and fractionated three times to obtain 100 ml of a water-soluble fraction and a methylene chloride soluble fraction. After 300 ml of methylene chloride soluble fraction was filtered off and dried under reduced pressure to obtain 312 mg of cotton band dry powder of methylene chloride soluble fraction.

1-1-3. Preparation of Ethyl Acetate Soluble Fraction

100 ml of ethyl acetate was added to 100 ml of the water-soluble fraction obtained in 1-1-2., And the mixture was mixed three times. The mixture was repeated three times to obtain 100 ml of the water-soluble fraction and 300 ml of the ethyl acetate-soluble fraction. After filtration and drying under reduced pressure, 100 mg of cotton band dry powder of ethyl acetate soluble fraction was obtained.

1-1-4. Preparation of n-butanol soluble fraction

100 ml of n-butanol was added to 100 ml of the water-soluble fraction obtained in 1-1-3., And then mixed three times. The mixture was repeated three times to obtain 100 ml of the water-soluble fraction and 300 ml of the n-butanol-soluble fraction, followed by n-butanol. The soluble fractions were filtered and dried under reduced pressure to give 835 mg of a dry bed of n-butanol soluble fractions.

1-1-5. Preparation of Methanol Extracts

Kills originating from Phllostachys nigra var.henosis were purchased through Omniherb (Daegu, Korea). After killing, 5 L of methanol was added to 360 g, heated twice at 70 ° C. for 2 hours, extracted with reflux cooling, filtered, and lyophilized under reduced pressure to obtain a freeze-dried product.

1-1-6. Preparation of ethanol extract

A freeze-dried product was prepared in the same manner as in 1-1-5. Except that ethanol was used instead of methanol.

1-1-7. Preparation of Ethyl Acetate Soluble Extract

A freeze-dried product was prepared in the same manner as in 1-1-5. Except that ethyl acetate was used as a solvent.

1-2. Inhibition of transplant rejection or treatment or prevention of autoimmune diseases by measuring cytokines

Autoimmune diseases or transplant rejection reactions show that Th 1/2 cells are activated and secrete IFN-r and IL-4. The purpose of this study was to determine the effect of treating or preventing autoimmune diseases.

○ Reagents and Media

Penicillin-streptomycin solutions, RPMI 1640 medium, fetal bovine serum (FBS) were obtained from HyClone Laboratories, Inc. Phosphate buffered saline (PBS) was used by USB Corporation (Cleveland, OH, USA), 3- [4,5-Dimethylthiazol-2-yl] -2,5- Diphenyltetrazolium bromide (MTT), concanavalin A (Con A), and red blood cell (RBC) lysis buffer were used as Sigma-Aldrich (St. Louis, MO, USA).

○ experimental animals

Balb / C body weight 20-21 g (6 week old) mouse females were purchased from Central Animal Experiments Inc. and used for the experiments.

○ Splenocyte Preparation

All preparations were carried out in a sterile environment. Mice were euthanized after euthanasia by cervical dislocation before necropsy, spleen was removed and splenocytes were separated using two slide glasses prepared in advance. The splenocytes were centrifuge at 1500 rpm for 5 minutes, the supernatant was discarded, and after tapping, 0.75 ml of RBC lysis buffer was added. After 1 minute reaction, the splenocytes were washed three times using RPMI 1640 medium to obtain splenocytes.

○ Stimulate 2 ㅧ 10 ⁶ cells / ml of splenocytes isolated for IFN-gamma and IL-4 measurement with Con A 1 ug / ml in a 24 well plate and kill the cotton pads (1-1-1. Water extract) After treatment with a concentration of 1, 10, 100 ug / ml each 48 hours incubation. As a control, PBS was treated in the same amount as the extract. After incubation, the supernatants were collected and the concentrations of IFN-gamma and IL-4 were measured.

IFN-r and IL-4 were measured using a sandwich ELISA with a Mouse CytoSet Kit (Invitrogen, CA, USA), and the concentrations were calculated by drawing the respective standard curves. The results are shown in Fig. Figure 1 A is a graph showing the change in concentration of IFN-gamma according to the kill treatment of cotton swab, Figure 1 B is a graph showing the concentration change of IL-4 according to the extract treatment of cotton swab. In each graph, C is the control group, PT1 is the killing of cotton swabs (1-1-1. Water extract) drug treatment group treated with a concentration of 1 ug / ml, PT10 is the killing of cotton swabs (1-1-1 The water extract) treated with 10 ug / ml of the drug treatment group, PT100 is a drug treatment group treated with a cotton rod kill (1-1-1. Water extract) at a concentration of 100 ug / ml.

As shown in Figure 1, killing cotton pad (1-1-1. Water extract) inhibits IFN-r 3.58%, 25.34%, 58.42% at 1, 10, 100 ug / ml concentration, respectively, compared to the control, IL-4 was inhibited by 8.86%, 16.84% and 51.79%, respectively. Therefore, the extract inhibits IFN-r and IL-4 in a concentration-dependent manner, it can be determined to inhibit the Th 1 and Th 2 response. As a result, it is possible to treat or prevent transplant rejection or autoimmune diseases by inhibiting Th 1 and Th 2 responses.

<Example 2> Confirmation of treatment or prophylactic effect of transplant rejection or autoimmune disease of cotton rod extract-Confirmation by measuring the effect of inhibiting T lymphocyte proliferation

Since autoimmune diseases or rejection of transplantation go through the process of activating and proliferating T cells, cotton extracts can be used to treat or prevent transplant rejection or autoimmune diseases by measuring the inhibitory effect of T cell proliferation on cotton extracts. To confirm that it is effective.

○ Reagents and Media

Penicillin-streptomycin solutions, RPMI 1640 medium, fetal bovine serum (FBS) were obtained from HyClone Laboratories, Inc. Phosphate buffered saline (PBS) was used by USB Corporation (Cleveland, OH, USA), and Concanavalin A (Con A) and red blood cell (RBC) lysis buffer were obtained from (Logan, UT, USA). Aldrich (St. Louis, MO, USA) was used, and AlamaBlue solution was used by AbD Serotec (Oxford, UK).

○ experimental animals

Balb / C body weight 20-21 g (6 week old) female mice were purchased and used for the experiment.

○ T cell preparation and proliferation inhibitory effect experiment

After obtaining cells using two slide glasses from the mouse spleen, T cells were isolated using MACS (Qiagen, Germanntown, MD, USA). The isolated T cells were stimulated with Con A 3 ug / ml in a 96 well plate at a concentration of 1 × 10 ⁶ cells / ml at the same time, and at the same time, the rods were killed and extracted (1-1-1. 3. Ethyl acetate soluble fractions) are treated at concentrations of 25, 50 and 100 ug / ml, respectively. After 48 hours of incubation, 10 ul of AlamaBlue solution was added to each well, followed by 12 hours of incubation, and the absorbance was measured at 570 nm. PBS was treated in the same amount as the extract for the control.

The results are shown in Fig. Figure 2 is a graph showing the cell growth rate according to the concentration of cotton swab extract administration. 2, the x-axis shows the extract dosage concentration and the y-axis shows the cell growth rate. In the graph, C is the control group, PT50 is the killing of cotton swabs (1-1-1. Water extract) in the drug treatment group treated with a concentration of 50 ug / ml, PT100 is the killing of cotton swabs (1-1-1. Extract) treated with a concentration of 100 ug / ml drug treatment group, PE25 is 1-1-3. Drug treatment group treated with ethyl acetate soluble fraction at a concentration of 25 ug / ml, PE50 was 1-1-3. Drug treated group treated with ethyl acetate soluble fraction at a concentration of 50 ug / ml.

As shown in the figure, 1-1-1. T-cell proliferation was inhibited by 16.06% and 60.38% at 50 and 100 ug / ml water extracts, respectively. T cell proliferation was inhibited by 14.61% and 54.31% at 25 and 50 ug / ml concentrations of ethyl acetate soluble fraction, respectively. When comparing the concentrations 1-1-3 at the same concentration. Ethyl acetate soluble fraction was 1-1-1. It was about twice as effective as water extract. Therefore, by inhibiting the proliferation of T cells by the extract, it is possible to effectively reduce the immune response by T cells, and to inhibit or prevent transplant rejection or to treat or prevent autoimmune diseases, and the ethyl acetate soluble fraction is higher than the water extract. It can be seen that the double precision.

<Example 3> Confirmation of the treatment or prophylaxis of transplant rejection or autoimmune disease of cotton rod extract-Confirmation by measuring cell cycle arrest effect in T lymphocytes

Since autoimmune diseases or transplant rejection reactions go through the process of activating and proliferating T cells, the cotton extract extracts rejected the transplant by measuring the inhibition of T cell proliferation by cell cycle arrest in T cells in the following manner. The purpose of this study was to determine whether it is effective in suppressing response or treating or preventing autoimmune diseases.

○ Reagents and Media

Penicillin-streptomycin solutions, RPMI 1640 medium, fetal bovine serum (FBS) were obtained from HyClone Laboratories, Inc. Phosphate buffered saline (PBS) was used by USB Corporation (Cleveland, OH, USA), Concanavalin A (Con A), red blood cell (RBC) lysis buffer, and propidium iodide (Logan, UT, USA). Sigma-Aldrich (St. Louis, Mo., USA) was used.

○ experimental animals

Balb / C body weight 20-21 g (6 week old) female mice were purchased and used for the experiment.

○ T cell preparation and proliferation inhibitory effect experiment

After obtaining cells using two slide glasses from the mouse spleen, T cells were isolated using MACS (Qiagen, Germanntown, MD, USA). 1 × 10 ⁶ cells / ml of isolated T cells were stimulated with Con A 3 ug / ml in a 24 well plate and killed with cotton pad extract (1-1-1. Water extract or 1-1-3. Ethyl acetate soluble fraction ) Are treated at concentrations of 25, 50 and 100 ug / ml, respectively. After 48 hours of incubation, the cell cycle was analyzed by staining DNA with propidium iodide solution and measuring with a fluorescent activated cell sorter (FACS). PBS was treated in the same amount as the extract for the control.

The results are shown in Fig. Figure 3 is a graph showing the results of the cell cycle arrest by extract concentration, x-axis is the extract concentration, y-axis means G0 / G1 ratio (%). In the graph, C is the control group, PT50 is the killing of cotton swabs (1-1-1. Water extract) in the drug treatment group treated with a concentration of 50 ug / ml, PT100 is the killing of cotton swabs (1-1-1. Extract) treated with a concentration of 100 ug / ml drug treatment group, PE25 is 1-1-3. Drug treatment group treated with ethyl acetate soluble fraction at a concentration of 25 ug / ml, PE50 was 1-1-3. Drug treated group treated with ethyl acetate soluble fraction at a concentration of 50 ug / ml.

As shown in the figure, 1-1-1. Water extract and 1-1-3. Increasing the concentration of ethyl acetate soluble fraction was shown to increase the G0 / G1 phase of T cells, 1-1-1. Drug treatment group treated with water extract at a concentration of 100 ug / ml and 1-1-3. Drug treatment groups treated with ethyl acetate soluble fraction at a concentration of 25 ug / ml showed similar effects. This result is described in 1-1-1. Water extract and 1-1-3. The ethyl acetate soluble fraction indicates that the T cell stays in G0 / G1 phase by inhibiting the cycle of T cell division and, as a result, inhibits the proliferation of T cells. Therefore, by inhibiting the proliferation of T cells by the extract, it is possible to effectively reduce the immune response by T cells, and to treat or prevent transplant rejection or autoimmune disease, and the ethyl acetate soluble fraction is about four times higher than the water extract. It can be seen that the effect is excellent.

<Preparation Example 1> Pharmaceutical composition-cotton swab extract

20 mg of ethyl acetate soluble fraction of Example 1-1-3., 3 mg of crystalline cellulose, 14.8 mg of lactose, and 0.2 mg of magnesium stearate were filled into gelatin capsules to prepare a capsule.

Preparation Example 2 Food Composition-Cotton Ball Extract

Ethyl acetate soluble fraction (1 wt%), liquid fructose (0.5 wt%), oligosaccharide (2 wt%), sugar (2 wt%), and salt (0.5 wt%) in Example 1-1-3. After adding the remaining amount to adjust the homogeneous mixture was instantaneous sterilization to prepare a healthy drink.

Claims (11)

Pharmaceutical composition for the treatment or prevention of transplant rejection or autoimmune disease comprising an extract of killing cotton pad as an active ingredient. The pharmaceutical composition of claim 1, wherein the kill extract of the cotton pad is at least one solvent extract selected from water, alcohol having 1 to 4 carbon atoms, or ethyl acetate. The pharmaceutical composition of claim 2, wherein the extract is an ethyl acetate extract. The pharmaceutical composition according to claim 3, wherein the ethyl acetate extract is an ethyl acetate soluble fraction extracted with ethyl acetate after removal of the methylene chloride soluble fraction in the water extract by killing cotton pads. delete The pharmaceutical composition according to claim 1, wherein the killing of the cotton pad is an intermediate layer from which the outer shell of the stem of the cotton pad is removed. The pharmaceutical composition of claim 1, wherein the transplant rejection reaction is a transplant rejection reaction of a tissue or an organ. 8. The pharmaceutical composition of claim 7, wherein the tissue or organ transplant rejection reaction is at least one selected from bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation and skin transplant rejection. . The method of claim 1, wherein the autoimmune diseases include progressive systemic sclerosis (Scleroderma), systemic lupus erythematosus (lupus), insulin-dependent childhood diabetes due to pancreatic cell antibodies, multiple sclerosis, autoimmune hemolytic anemia Pharmaceutical composition comprising at least one selected from: autoimmune hemolytic anemia, myasthenia gravis, or Grave's disease. 10. The method according to any one of claims 1 to 4, and 6 to 9, wherein the treatment or prevention of transplant rejection or autoimmune disease is carried out among T helper 1 and T helper 2 reaction inhibitors or T cell proliferation inhibitors. Pharmaceutical composition according to at least one selected. Food composition for reducing or preventing transplant rejection or autoimmune disease comprising the extract of killing cotton pad as an active ingredient.

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