Pharmaceutical Composition Comprising Indigo Pulverata Levis Extract or Fraction Thereof as Effective Ingredient for Preventing or Treating Inflammatory Bowel Disease

Abstract

The present invention relates to a pharmaceutical composition comprising an Indigo Pulverata Levis extract or a fraction thereof as an effective ingredient for preventing or treating inflammatory bowel disease. Exhibiting the effect of reducing the activity and expression of inflammatory activation markers and alleviating the disease activity index of inflammatory bowel disease, an Indigo Pulverata Levis extract or a fraction thereof according to the present invention may be useful as an agent for preventing and treating inflammatory bowel disease.

Claims

1-8. (canceled)

9. A method for preventing or treating inflammatory bowel disease, comprising a step of administering a pharmaceutical composition containing a pharmaceutically effective amount of an Indigo Pulverata Levis extract or a fraction thereof as an effective ingredient into subjects to a subject in need thereof

10-11. (canceled)

12. The method for preventing or treating inflammatory bowel disease according to claim 9, wherein the Indigo Pulverata Levis extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms or mixtures thereof.

13. The method for preventing or treating inflammatory bowel disease according to claim 12, wherein the lower alcohol is selected from the group consisting of ethanol, methanol and butanol.

14. The method for preventing or treating inflammatory bowel disease according to claim 13, wherein the lower alcohol is ethanol.

15. The method for preventing or treating inflammatory bowel disease according to claim 9, wherein the fraction is an organic solvent fraction.

16. The method for preventing or treating inflammatory bowel disease according to claim 15, wherein the organic solvent is at least one selected from the group consisting of chloroform, hexane, ethyl acetate and butanol.

17. The method for preventing or treating inflammatory bowel disease according to claim 9, wherein the fraction is a hexane fraction, a chloroform fraction, an ethyl acetate fraction or a butanol fraction, obtained by sequential fractionation with hexane, chloroform, ethyl acetate and butanol.

18. A method for preventing or alleviating inflammatory bowel disease, comprising a step of administering a health functional food containing an Indigo Pulverata Levis extract or a fraction thereof to a subject in need thereof.

19. The method for preventing or alleviating inflammatory bowel disease according to claim 18, wherein the Indigo Pulverata Levis extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms or mixtures thereof.

20. The method for preventing or alleviating inflammatory bowel disease according to claim 19, wherein the lower alcohol is selected from the group consisting of ethanol, methanol and butanol.

21. The method for preventing or alleviating inflammatory bowel disease according to claim 20, wherein the lower alcohol is ethanol.

22. The method for preventing or alleviating inflammatory bowel disease according to claim 18, wherein the fraction is an organic solvent fraction.

23. The method for preventing or alleviating inflammatory bowel disease according to claim 22, wherein the organic solvent is at least one selected from the group consisting of chloroform, hexane, ethyl acetate and butanol.

24. The method for preventing or alleviating inflammatory bowel disease according to claim 18, wherein the fraction is a hexane fraction, a chloroform fraction, an ethyl acetate fraction or a butanol fraction, obtained by sequential fractionation with hexane, chloroform, ethyl acetate and butanol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a graph of showing a change in weights as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease:

[0045] PXC is a group dosed with piroxicam; PXC+1 is a group dosed with piroxicam+70% ethanol extract of Indigo Pulverata Levis; PXC+2 is a group dosed with piroxicam+ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis; and PXC+3 is a group dosed with piroxicam+butanol fraction of 70% ethanol extract of Indigo Pulverata Levis: * indicates a remarkable difference from a normal group, while does a remarkable difference from a group with induced disease, which are hereinafter the same.

[0046] FIG. 2 is an image of showing a change in colon lengths as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease.

[0047] FIG. 3 is a graph of comparing colon lengths with each other as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease.

[0048] FIG. 4 is a graph of showing an inhibition in MPO expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease.

[0049] FIG. 5 is a graph of showing an inhibition in IL-6 expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease.

[0050] FIG. 6 is a graph of showing an inhibition in IL-1b expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease.

[0051] FIG. 7 is a graph of showing an inhibition in IL-17 expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with piroxicam-induced bowel disease.

[0052] FIG. 8 is a graph of showing a decrease in the disease activity index (DAI) of bowel disease as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with piroxicam-induced bowel disease.

[0053] FIG. 9 is a graph of showing an increase in survival rates as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with piroxicam-induced bowel disease.

[0054] Statistical analysis based on Log-rank (Mantel-Cox) test.

[0055] FIG. 10 is an image of identifying a decrease in pathological cell destruction in a bowel tissue with a microscope as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with piroxicam-induced bowel disease.

[0056] FIG. 11 is a graph of showing a score of pathological cell destruction in a bowel tissue as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with piroxicam-induced bowel disease.

[0057] FIG. 12 is a graph of showing a change in weights as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with dextran sulfate sodium (DSS)-induced bowel inflammation:

[0058] DSS is a group dosed with dextran sulfate sodium; DSS+1 is a group dosed with dextran sulfate sodium+70% ethanol extract of Indigo Pulverata Levis; DSS+2 is a group dosed with dextran sulfate sodium+ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis; and DSS+3 is a group dosed with dextran sulfate sodium+butanol fraction of 70% ethanol extract of Indigo Pulverata Levis: * indicates a remarkable difference from a normal group, while does a remarkable difference from a group with induced disease, which are hereinafter the same.

[0059] FIG. 13 is a graph of showing a decrease in the disease activity index (DAI) of bowel inflammation as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with dextran sulfate sodium (DSS)-induced bowel inflammation.

[0060] FIG. 14 is a graph of showing an inhibition in IL-6 expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with dextran sulfate sodium (DSS)-induced bowel inflammation.

[0061] FIG. 15 is a graph of showing an inhibition in IL-1b expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with dextran sulfate sodium (DSS)-induced bowel inflammation.

[0062] FIG. 16 is a graph of showing an inhibition in IL-17 expressions in a bowel tissue as a result of administering an Indigo Pulverata Levis extract or a fraction thereof into models with dextran sulfate sodium (DSS)-induced bowel inflammation.

[0063] FIG. 17 is an image of showing an increase in colon lengths as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with dextran sulfate sodium-induced bowel inflammation.

[0064] FIG. 18 is a graph of showing an increase in colon lengths as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with dextran sulfate sodium-induced bowel inflammation.

[0065] FIG. 19 is a graph of showing an inhibition in macroscopic scores of inflammations in a colon tissue as a result of administering an Indigo Pulverata Levis extract into models with dextran sulfate sodium-induced bowel inflammation.

[0066] FIG. 20 is an image of identifying a decrease in pathological cell destruction in a bowel tissue with a microscope as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into models with dextran sulfate sodium-induced bowel inflammation.

[0067] FIG. 21 is a graph of showing a score of pathological cell destruction in a bowel tissue as a result of administering an ethyl acetate fraction of 70% ethanol extract of Indigo Pulverata Levis into amodels with dextran sulfate sodium-induced bowel inflammation.

BEST MODE FOR INVENTION

[0068] Hereinafter, the configuration and effects of the present invention will be described in more detail through Examples. However, the following Examples are provided only for the purpose of illustrating the present invention, and thus the content of the present invention is not limited thereto.

Example 1

Preparation for an Indigo Pulverata Levis Extract and a Fraction Thereof

[0069] Indigo Pulverata Levis and 70% ethanol were stirred for 48 hours to carry out an extraction, and filtered to evaporate solvent and water therefrom, such that an Indigo Pulverata Levis extract was prepared in a form of soft extract. After that, water was added to said extract, after which hexane was added thereto and fractionated, such that an organic solvent layer and a water layer were separated from each other, firstly. Chloroform (CHCl.sub.3) was added again to said firstly-separated water layer and fractionated, such that an organic solvent layer and a water layer were separated from each other, secondly. Ethyl acetate (EtOAc) was added again to said secondly-separated water layer, such that an organic solvent layer and a water layer were separated from each other, thirdly. Butanol (BuOH) was added again to said thirdly-separated water layer, after which an organic solvent layer and a water layer were separated from each other, fourthly, such that a hexane fraction, a chloroform fraction, an ethyl acetate fraction and a butanol fraction were collected respectively. In the following experimental examples, a 70% ethanol extract of Indigo Pulverata Levis, an ethyl acetate fraction thereof and a butanol fraction thereof were used as samples as shown in a following table 1.

TABLE-US-00001 TABLE 1 Sample No. Sample Types 1 70% ethanol extract of Indigo Pulverata Levis 2 Ethyl acetate fraction of the 70% ethanol extract of Indigo Pulverata Levis 3 Butanol fraction of the 70% ethanol extract of Indigo Pulverata Levis

Experimental Example 1

Experiment on an Indigo Pulverata Levis Extract for Preventing and Treating Colitis Bowel Disease

[0070] An effect of an Indigo Pulverata Levis extract on preventing and treating inflammatory bowel disease was identified by using animal models for colitis bowel disease. The animal models for colitis bowel disease, which were used in the experiment, were prepared by treating IL-10 knockout mice with piroxicam, wherein such mice were known to be used to identify an effect of preventing and treating bowel disease (Holgersen et al, J Crohns Colitis, 2014;8;147-60; Hale et al. Inflamm Bowel Dis 2005;11:1060-9).

[0071] Particularly, while the IL-10-deficient laboratory mice were fed a diet mixed with piroxicam, an Indigo Pulverata Levis extract or a fraction thereof, which had been prepared according to Example 1 above, was orally administered into each of the mice by 25 mg once a day for 13 days as shown in a table 1. A body weight was measured every day for an experiment period of 13 days. After the 14th day of the experiment, the laboratory mice were sacrificed, after which a length of colons was measured and tissues were obtained to measure immunoreactivity factors with an ELISA kit.

[0072] As a result of comparing weights, in case of a normal group, a certain level of weights was maintained. However, in case of a group dosed with piroxicam, the weights were drastically decreased. However, in case of a group dosed with the Indigo Pulverata Levis extract and the fraction thereof, it was identified that a rate of decrease in weights was low compared to a group dosed with piroxicam only (FIG. 1).

[0073] Also, as a result of identifying a change in colon lengths, it was shown that the length of colons was decreased in a case of a group dosed with piroxicam-induced colitis bowel disease. However, in case of the group dosed with the Indigo Pulverata Levis extract and the fraction thereof, it was identified that the length of colons was increased compared to the group dosed with piroxicam only and was recovered up to a level of the normal group (FIGS. 2 and 3).

[0074] Furthermore, as a result of comparing the levels of immune factors associated with colitis disease, it was shown that the level of MPO, IL-6, IL-1b and IL-17 was increased in case of the group dosed with piroxicam-induced colitis bowel disease. However, in case of the group dosed with the Indigo Pulverata Levis extract and the fraction thereof, it was shown that said level of immune factors was decreased compared to the group dosed with piroxicam only (FIGS. 4 to 7).

[0075] Meanwhile, for the group dosed with an ethyl acetate fraction of the Indigo Pulverata Levis extract, its weights, stool forms and bloody stools were observed during the experiment period of 13 days to measure the disease activity index (DAI) of colitis bowel disease according to a method of a table 2.

TABLE-US-00002 TABLE 2 Decrease in Weights Score Stool Form Score Bloody Stool Score <1% 0 Hard and 0 None 0 firm 1-5% 1 Firm, but 1 Hidden 1 sticky 5-10% 2 Soft, but 2 Visible 2 form maintained 10-15% 3 Soft and 3 Rectal 3 form lost bleeding 15-20% 4 Liquid with 4 no form

[0076] Also, after the 14th day of the experiment, the laboratory mice were sacrificed to separate colons therefrom, after which the presence of histological damages to the colons was identified with a microscope and a score of damages to the colons was given according to a method of a table 3.

TABLE-US-00003 TABLE 3 Degree Pres- Destruc- and Pres- ence tion of existence Range of ence of Score normal of muscular of goblet (Total = 11) mucosa cells hypertrophy crypts cells 0 Normal Normal Normal Absent Absent 1 Slight Slight Slight Present Present damage level level 2 Moderate Moderate Moderate damage level level 3 Extensive Transmural Extensive damage infiltration damage

[0077] As a result of the experiment, it was identified that the disease activity index (DAI) of bowel disease, reportedly having reflected inflammations best in a colitis model, was significantly alleviated compared with the group dosed with piroxicam-induced colitis (FIG. 8).

[0078] As a result of investigating a survival rate of the animal models during the experiment period, it was identified for the group dosed with the ethyl acetate fraction that the survival rate was high compared to the group dosed with piroxicam to have induced colitis (FIG. 9). Also, it was identified for the group dosed with the ethyl acetate fraction that a degree of damages to colons was decreased to pathologically alleviate colitis (FIGS. 10 and 11).

[0079] From the experiments above, it was identified that the Indigo Pulverata Levis extract and the fraction thereof had an excellent effect on preventing and treating colitis bowel disease without any toxicity.

Experimental Example 2

Experiment on an Indigo Pulverata Levis Extract for Preventing and Treating Bowel Inflammation

[0080] An effect of an Indigo Pulverata Levis extract on preventing and treating inflammatory bowel disease was identified by using animal models with dextran sulfate sodium-induced bowel inflammation, which were known to be used to identify an effect of preventing and treating bowel disease (Mizoguchi et al., Prog Mol Biol Transl Sci, 2012:105;263-320; Yan et al., PLos One, 2009:4;e6073).

[0081] Particularly, an Indigo Pulverata Levis extract or a fraction thereof, which had been prepared according to Example 1 above, was orally administered into laboratory mice (C57BL6) once a day in an amount suitable for each group as shown in a table 1. In two days after administering a sample, drinking water or drinking water containing 4% dextran sulfate sodium was supplied to such animal models for seven days, which weights, stool forms and bloody stools were observed every day for the experiment period to measure the disease activity index (DAI) of bowel inflammation according to the same standards of the table 2 above as shown in the experimental example 1. And, after the 8th day of the experiment, the laboratory mice were sacrificed, after which colon tissues were obtained therefrom to measure immunoreactivity factors with an ELISA kit.

[0082] As a result of the experiment, it was identified that weights were slowly increased in a normal group, but drastically decreased in a group dosed with dextran sulfate sodium. However, it was also identified for a group dosed with the Indigo Pulverata Levis extract or the fraction thereof that a degree of decrease in weights was significantly decreased (FIG. 12).

[0083] Also, it was identified for the group dosed with dextran sulfate sodium that the disease activity index (DAI) of bowel inflammation was increased to exhibit the characteristics of an inflammatory bowel disease model, but it was also identified for the group dosed with the Indigo Pulverata Levis extract and the fraction thereof that the disease activity index was decreased compared to the group dosed with dextran sulfate sodium (FIG. 13).

[0084] Furthermore, as a result of comparing the levels of immune factors associated with inflammatory bowel disease, it was shown that the level of IL-6, IL-1b and IL-17 was increased in a case of the group dosed with dextran sulfate sodium-induced inflammatory bowel disease. However, in case of the group dosed with the Indigo Pulverata Levis extract and the fraction thereof, it was shown that said level of immune factors was decreased compared to the group dosed with dextran sulfate sodium only (FIGS. 14 to 16).

[0085] Meanwhile, in case of a group dosed with an ethyl acetate fraction of the Indigo Pulverata Levis extract, such fraction was administered at varying concentrations. After the 8th day of the experiment, the laboratory mice were sacrificed, after which a length of colons was measured and a macroscopic score of inflammations in rectum and appendix was measured according to the standards of a table 4 (by using a method for looking into and judging a surface of intestinal mucosa without the use of a microscope). Also, the presence of histological damages to colons was identified with a microscope, and a score of the damages was measured according to a method of a table 5.

TABLE-US-00004 TABLE 4 Mucosal Bleeding of Score Hypertrophy Ulcer Region Digestive Organ 0 Normal None Normal 1 Slight level Local bleeding Slight level 2 Moderate level Ulcer (1-3 regions) Moderate level 3 Ulcer (>3 regions)

TABLE-US-00005 TABLE 5 Infiltration of Inflammatory Cells Score Muscular/ (Total Degree of Mucosal Submucosal Mesenteric 12) Destruction Mucosa Layer Layer 0 Normal Normal Normal Normal 1 Hyper-proliferation, Slight Slight- Slight- irregular crypts and level moderate moderate goblet cells level level 2 Slight or moderate Moderate Moderate loss of crypts level level (10-50%) 3 Severe loss of crypts Moderate (50-90%) level 4 Complete loss of crypts and normal epithelial cells 5 Small or medium ulcers (<10 crypt width) 6 Large ulcers (10 crypt width)

[0086] As a result of the experiment, it was identified for the group dosed with dextran sulfate sodium that the length of colons was significantly decreased compared to the normal group, but it was also identified for the group dosed with the ethyl acetate fraction that the length of colons was significantly increased compared to the group dosed with dextran sulfate sodium (FIGS. 17 and 18). Also, it was identified for the group dosed with dextran sulfate sodium that a histological score of inflammations was increased to exhibit the characteristics of an inflammatory bowel disease model, but it was also identified for the group dosed with the ethyl acetate fraction that a histological score of inflammations was decreased compared to the group dosed with dextran sulfate sodium (FIG. 19). Also, it was identified for the group dosed with the ethyl acetate fraction that a degree of damages to colons was decreased to pathologically alleviate colitis (FIGS. 20 and 21).

[0087] From the experiments above, it was identified that the Indigo Pulverata Levis extract and the fraction thereof had an excellent effect on preventing and treating inflammatory bowel disease without any toxicity.

[0088] While specific portions of the present invention have been described in detail above, it is apparent to those skilled in the art that such detailed descriptions are set forth to illustrate exemplary embodiments only and are not construed to limit the scope of the present invention.

[0089] Thus, it should be understood that the substantial scope of the present invention is defined by the accompanying claims and equivalents thereto.