METHOD FOR TREATING AND PREVENTING ULCERATIVE COLITIS AND METHOD FOR DAI REDUCTION

Abstract

The invention discloses a use of a mulberry (Morus alba L.) extract, including a method for treating ulcerative colitis, a method for preventing ulcerative colitis, and a method for reduction in Disease Activity Index, DAI. The use is application of a mulberry extract to the preparation of a product for treating and/or preventing ulcerative colitis. The invention experimentally demonstrated that positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) could each significantly inhibit colonic atrophy in UC model mice, and the effects in SZ-A-2, SZ-A-3 and SZ-A-4 groups are comparable to that in positive drug group, with SZ-A-5 (pretreatment) achieving a superior effect to the positive drug. The mulberry extract of the invention, as a component derived from natural plants, has unique advantages of small toxic and side effects, mild and lasting actions etc.

Claims

1. A method for treating ulcerative colitis in a subject, comprising administering a mulberry extract or a main active ingredient thereof to the subject.

2. The method for treating ulcerative colitis in the subject according to claim 1, wherein administering the mulberry extract or the main active ingredient thereof results in at least one of: (1) inhibition of colonic atrophy in the subject; (2) inhibition of weight loss in the subject; (3) reduction in Disease Activity Index, DAI, of the subject; and (4) improvement in colonic structure, gland arrangement, and inflammatory cell infiltration in the subject.

3. The method for treating ulcerative colitis in the subject according to claim 1, wherein the mulberry extract comprises a mulberry twig extract, a mulberry root-bark extract and/or a mulberry leaf extract.

4. The method for treating ulcerative colitis in the subject according to claim 1, wherein the mulberry extract or the main active ingredient thereof is in a form of a medicament or a pharmaceutical formulation, and the medicament or the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.

5. The method for treating ulcerative colitis in the subject according to claim 1, wherein the mulberry extract or the main active ingredient thereof is prepared by a preparation method comprising: step 1) preparing a crude extract from a plant of Moraceae; and step 2) separating the crude extract with a cation resin and/or an anion resin, to provide the mulberry extract.

6. The method for treating ulcerative colitis in the subject according to claim 5, wherein the preparation method of the mulberry extract or the main active ingredient thereof further comprises: step 3) subjecting an effluent from the cation resin and/or the anion resin in step 2) to an alcohol precipitation and collecting a supernatant; and step 4) concentrating and drying the supernatant; alternatively, the preparation method further comprises: concentrating and drying the effluent from the cation resin and/or the anion resin in step 2).

7. The method for treating ulcerative colitis in the subject according to claim 1, wherein the main active ingredient of the mulberry extract comprises at least one of: 1-deoxynojirimycin, N-methly-1-deoxynojirimycin, fagomine, 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol, calystegin B2, calystegin C1, 2-O-(-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O--D-glucopyranosyl)-D-arabinitol.

8. The method for treating ulcerative colitis in the subject according to claim 1, wherein the subject comprises mammals, including rats, mice or humans.

9. The method for treating ulcerative colitis in the subject according to claim 1, wherein the mulberry extract comprises: alkaloids 15-98%, polysaccharides 0.2-35%, flavonoids 0.05-7%, amino acids 0-45%, based on a weight percentage content of a sum of each component in the mulberry extract.

10. The method for treating ulcerative colitis in the subject according to claim 1, wherein the mulberry extract comprises: alkaloids 30-98%, polysaccharides 0.2-35%, flavonoids 0.05-2%, amino acids 0-25%, based on a weight percentage content of a sum of each component in the mulberry extract.

11. A method for preventing ulcerative colitis in a subject, comprising administrating a mulberry extract or a main active ingredient thereof to the subject.

12. The method for preventing ulcerative colitis in the subject according to claim 11, wherein administrating the mulberry extract or the main active ingredient thereof results in at least one of: (1) inhibition of colonic atrophy in the subject; (2) inhibition of weight loss in the subject; (3) reduction in Disease Activity Index, DAI, of the subject; and (4) improvement in colonic structure, gland arrangement, and inflammatory cell infiltration in the subject.

13. The method for preventing ulcerative colitis in the subject according to claim 11, wherein the mulberry extract comprises a mulberry twig extract, a mulberry root-bark extract and/or a mulberry leaf extract.

14. The method for preventing ulcerative colitis in the subject according to claim 11, wherein the mulberry extract or the main active ingredient thereof is in a form of a medicament or a pharmaceutical formulation, and the medicament or the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.

15. The method for preventing ulcerative colitis in the subject according to claim 11, wherein the mulberry extract or the main active ingredient thereof is prepared by a preparation method comprising: step 1) preparing a crude extract from a plant of Moraceae; and step 2) separating the crude extract with a cation resin and/or an anion resin, to provide the mulberry extract.

16. The method for preventing ulcerative colitis in the subject according to claim 11, wherein the main active ingredient of the mulberry extract comprises at least one of: 1-deoxynojirimycin, N-methly-1-deoxynojirimycin, fagomine, 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol, calystegin B2, calystegin C1, 2-O-(-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O--D-glucopyranosyl)-D-arabinitol.

17. The method for preventing ulcerative colitis in the subject according to claim 11, wherein the subject comprises mammals, including rats, mice or humans.

18. The method for preventing ulcerative colitis in the subject according to claim 11, wherein the method for preventing ulcerative colitis in the subject further comprises administrating a mulberry extract or a main active ingredient thereof to the subject before onset of the ulcerative colitis.

19. A method for reduction in Disease Activity Index, DAI, for a patient with ulcerative colitis, comprising administrating a mulberry extract or a main active ingredient thereof to the patient.

20. The method for reduction in Disease Activity Index, DAI, for the patient with ulcerative colitis according to claim 19, wherein the mulberry extract or the main active ingredient thereof is prepared by a preparation method comprising: 1) preparing a crude extract from a plant of Moraceae; and 2) separating the crude extract with a cation resin and/or an anion resin, to provide the mulberry extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] In order to explain the specific embodiments in the invention or the technical solutions in the prior art more clearly, the accompanying figures required for the description of the specific embodiments or the prior art will now be briefly introduced below. It is evident that the accompanying figures in the following description are some of the embodiments in the invention. It is possible for one ordinary skilled in the art to get access to other figures according to these accompanying figures without doing creative work.

[0111] FIG. 1 shows the colon lengths of mice in each treatment group (using the mulberry extract in Preparative Example 1) with * representing p<0.05 compared with model group, ** p<0.01 compared with the model group, and *** P<0.001 compared with model group;

[0112] FIG. 2 shows the photographs of the colons of mice in each treatment group (using the mulberry extract in Preparative Example 1);

[0113] FIG. 3 is a trend diagram of weight change of mice in each treatment group (using the mulberry extract in Preparative Example 1);

[0114] FIG. 4 shows the results of scoring of Disease Activity Index, DAI, for mice in each treatment group (using the mulberry extract in Preparative Example 1) with *** representing p<0.001 compared with model group;

[0115] FIG. 5 shows the results of scoring of Disease Activity Index, DAI, for mice treated with the mulberry extract in each Preparative Example with *** representing p<0.001 compared with model group; and

[0116] FIG. 6 shows the results of the pathological photographs of mice in each treatment group (using the mulberry extract in Preparative Example 1).

DESCRIPTION OF THE EMBODIMENTS

[0117] The invention will now be further explained in detail by way of examples. The features and advantages of the invention will be more explicit by virtue of these exemplary illustrations. The invention, however, is not limited to the following examples. Unless otherwise specified, the methods as described are all conventional ones. Unless otherwise specified, the raw materials as described would be commercially available.

[0118] As used herein, the professional language exemplary means serving as an example, embodiment or illustration. Any of the examples described herein as exemplary is not necessarily to be construed as being superior or better than other examples.

[0119] In addition, the technical features involved in different embodiments of the invention described below can be combined with each other unless they conflict.

[0120] The contents of the components involved in the invention have been detected according to the published methods (with reference to the methods described in Patent Publications Nos.: CN111077247A and CN110393738A).

I. Preparative Examples of Mulberry Extract

Preparative Example 1

[0121] 1000 kg of fresh mulberry twigs (Yuesang No. 11, Morus serrata Roxb.) were taken for crushing, followed by addition of 4000 L of water for extraction under heat reflux for 2 h. The extraction liquids were combined and filtered to remove insoluble substances to provide a crude extract. The crude extract was thermally concentrated until the solid content reached 4% by mass, which was kept at 50 C. as a loading liquid onto a cation resin column.

[0122] 150 kg of D113-type macroporous weakly acidic cation exchange resin was packed in a column, followed by washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; washing with 1 mol/L sodium hydroxide solution until the pH of the effluent was 8.5; washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; and rinsing with 5 column volumes of deionized water to complete activation. The concentrated extraction liquids were loaded, followed by elution with 1000 L of 2.5 mol/L aqueous ammonia at an elution rate of 6 BV/h. The eluent was collected when the effluent from the cation column was detected to have pH>7. Collection was stopped when the collected liquid reached 900 L. The collected liquid was subject to direct purification by an anion column.

[0123] 62.5 kg of D218-type macroporous strongly alkaline acrylic based anion resin was packed in a column, followed by washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0; washing with 1.5 mol/L hydrochloric acid solution until the pH of the effluent was 3.5; washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0, to complete activation. The collected eluent from the cation resin was loaded onto an anion resin. Collection of the effluent was not stopped until it reached 870 L.

[0124] The collected liquid was centrifuged to remove impurities, and then concentrated through reverse osmosis membranes. As a result, the concentrated liquid had a specific gravity of 1.25 and was transferred to a tank for alcohol precipitation, to which 25 L of anhydrous ethanol was added at 500 rpm with a stirring paddle. Once the addition of ethanol was finished, stirring was stopped for alcohol precipitation for 24 h. Supernatant was removed and concentrated under reduced pressure to provide an extractum as a mulberry twig extract.

[0125] In the extractum as a mulberry twig extract, the content of alkaloids was 52%, polysaccharides 22%, flavonoids 0.8%, and amino acids 20%. In the alkaloids, the content of 1-DNJ was 60%, FAG 17%, and DAB 15%.

Preparative Example 2

[0126] 10 kg of fresh mulberry twigs (Sangteyou No. 2) were taken for crushing, followed by addition of 150 L of water in two portions for extraction for 3 h each time according to a decoction method. The extraction liquids were combined and filtered to remove insoluble substances. The extraction liquids were thermally concentrated until the solid content reached 8% by mass, and then transferred to a tank for alcohol precipitation, to which 2367.9 g of anhydrous ethanol (3 L) was added at 300 rpm with a stirring paddle. Once the addition of ethanol was finished, stirring was stopped for alcohol precipitation for 24 h. Supernatant was removed as a loading liquid onto a cation resin column. 5 kg of 002SC-type strongly acidic styrene based cation resin was packed in a column, followed by activation of the cation resin according to the method of Example 1. The extraction liquids that were subject to concentration and alcohol precipitation were loaded, followed by elution with 100 L of 5 mol/L potassium chloride at an elution rate of 5 BV/h. The effluent was detected with 20% silicotungstic acid. Collection began when white precipitate was generated. Collection was stopped when the collected liquid reached 25 L, and the collected liquid was subject to direct purification by an anion column.

[0127] 10 kg of 711-type strongly alkaline styrene based anion resin was packed in a column, and the anion resin was subject to activation according to the method in Preparative Example 1. The collected eluent from the cation resin was loaded onto an anion resin. Collection of the effluent was not stopped until it reached 15 L. The collected liquid was reloaded onto a cation resin and separated twice with a cation resin and an anion resin in sequence according to the above method.

[0128] The collected liquid obtained from column separation for three times was centrifuged to remove impurities, and then concentrated through reverse osmosis membranes. The concentrated liquid had a specific gravity of 1.25 and was transferred to a tank for alcohol precipitation, to which 125 g of anhydrous ethanol was added at 1000 rpm with a stirring paddle. Once the addition of ethanol was finished, stirring was stopped for alcohol precipitation for 24 h. Supernatant was removed and concentrated under reduced pressure to provide an extractum as a mulberry twig extract. Fresh mulberry root-barks and mulberry leaves (Sangteyou No. 2) were taken for additional extraction, the extraction method and parameters were same as above.

[0129] In the resulting extractum as a mulberry twig extract, the content of alkaloids was 98%, polysaccharides 0.2%, flavonoids 0.05%, and amino acids 0%. In the alkaloids, the content of 1-DNJ was 99%, FAG 0.5%, and DAB 0.4%.

[0130] In the resulting mulberry root-bark extract, the content of alkaloids was 95%, polysaccharides 2%, flavonoids 0.1%, and amino acids 1%. In the alkaloids, the content of 1-DNJ was 96%, FAG 1.5%, and DAB 1.4%.

[0131] In the resulting mulberry leaf extract, the content of alkaloids was 90%, polysaccharides 4%, flavonoids 0.1%, and amino acids 3%. In the alkaloids, the content of 1-DNJ was 91%, FAG 3.1%, and DAB 2.8%.

Preparative Example 3

[0132] 1000 kg of fresh mulberry twigs (Morus atropurpurea Roxb.) were taken for crushing, followed by addition of 11500 L of water for extraction under heat reflux for 2 h. The extraction liquids were combined and filtered to remove insoluble substances to provide a crude extract. The crude extract was first subject to centrifugation for impurity removal, and then to concentration through reverse osmosis membranes until the solid content reached 1% by mass as a loading liquid onto a cation resin column.

[0133] 300 kg of D001-type macroporous strongly acidic styrene based cation resin was packed in a column, and the cation resin was subjected to activation according to the method in Preparation Example 1. The concentrated crude extract was loaded, followed by elution with 5000 L of 0.04 mol/L ammonium nitrate at an elution rate of 5 BV/h. The effluent was detected with 20% silicotungstic acid. Collection began when white precipitate was generated. Collection was stopped when the collected liquid reached 1000 L.

[0134] The collected liquid obtained from cation column separation was concentrated through nanofiltration membranes, and concentrated under reduced pressure to provide an extractum of the extract.

[0135] In the resulting mulberry twig extract, the content of alkaloids was 15%, polysaccharides 20%, flavonoids 7%, and amino acids 45%. In the alkaloids, the content of 1-DNJ was 55%, FAG 23%, and DAB 10%.

Preparative Example 4

[0136] 333 kg of dry mulberry twigs (Yuesang No. 11) were taken for crushing, followed by addition of 4000 L of water for extraction in two portions under heat reflux, with each reflux for 1 h. The extraction liquids were combined, filtered, and concentrated to 1 kg of crude drug/L.

[0137] 150 kg of D113-type macroporous weakly acidic cation exchange resin was packed in a column, followed by washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; washing with 1 mol/L sodium hydroxide solution until the pH of the effluent was 8.5; washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; and rinsing with 5 column volumes of deionized water to complete activation. The concentrated extraction liquids were loaded, followed by elution with 1000 L of 2.5 mol/L aqueous ammonia at an elution rate of 6 BV/h. The eluent was collected when the effluent from the cation column was detected to have pH>7. Collection was stopped when the collected liquid reached 900 L. The collected liquid was subject to direct purification by an anion column.

[0138] 125 kg of D218-type macroporous strongly alkaline acrylic based anion resin was packed in a column, followed by washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0; washing with 1.5 mol/L hydrochloric acid solution until the pH of the effluent was 3.5; washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0, to complete activation. The collected eluent from the cation resin was loaded onto an anion resin. Collection of the effluent having a pH>8 was not stopped until it reached 870 L. The collected liquid obtained from anion column separation was subject to filtration through microfiltration membranes to remove impurities, and then concentrated through reverse osmosis membranes. The concentrated liquid had a specific gravity of 1.1 and was transferred to a tank for alcohol precipitation, to which 15 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. Once the addition of ethanol was finished, stirring was stopped for alcohol precipitation for 24 h. Supernatant was removed and concentrated under reduced pressure to provide an extractum as a mulberry twig extract. Contents in the sample are alkaloids 80%, polysaccharides 5%, flavonoids 0.1%, and amino acids 4%. In the alkaloids, the content of 1-DNJ was 75%, FAG 12%, and DAB 10%.

Preparative Example 5

[0139] 400 kg of dry mulberry twigs (Yuesang No. 11) were taken for crushing, followed by addition of 4000 L of water for extraction in two portions under heat reflux, with each reflux for 1 h. The extraction liquids were combined, filtered, and concentrated to 1 kg of crude drug/L.

[0140] 62.5 kg of D218-type macroporous strongly alkaline acrylic based anion resin was packed in a column, followed by washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0; washing with 1.5 mol/L hydrochloric acid solution until the pH of the effluent was 3.5; washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0, to complete activation. The collected, extracted concentrate was loaded onto an anion resin, and the effluent was collected.

[0141] The collected liquid obtained from anion column separation was subject to filtration through microfiltration membranes to remove impurities, and then concentrated through reverse osmosis membranes, and further concentrated under reduced pressure and dried to provide an extractum as a mulberry twig extract. Contents in the sample are alkaloids 3%, polysaccharides 70%, flavonoids 10%, and amino acids 10%. In the alkaloids, the content of 1-DNJ was 68%, FAG 17%, and DAB 8%.

Preparative Example 6

[0142] 1500 kg of fresh mulberry twigs (Yuesang No. 11, Morus serrata Roxb.) were taken for crushing, followed by addition of 6000 L of water for extraction under heat reflux for 2 h. The extraction liquids were combined and filtered to remove insoluble substances to provide a crude extract. The crude extract was thermally concentrated until the solid content reached 4% by mass, which was kept at 50 C. as a loading liquid onto a cation resin column.

[0143] 100 kg of D113-type macroporous weakly acidic cation exchange resin was packed in a column, followed by washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; washing with 1 mol/L sodium hydroxide solution until the pH of the effluent was 8.5; washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; and rinsing with 5 column volumes of deionized water to complete activation. The concentrated extraction liquids were loaded, followed by elution with 1000 L of 2.5 mol/L aqueous ammonia at an elution rate of 6 BV/h. The eluent was collected when the effluent from the cation column was detected to have pH>7. Collection was stopped when the collected liquid reached 900 L. The collected liquid was subject to direct purification by an anion column.

[0144] 62.5 kg of D218-type macroporous strongly alkaline acrylic based anion resin was packed in a column, followed by washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0; washing with 1.5 mol/L hydrochloric acid solution until the pH of the effluent was 3.5; washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0, to complete activation. The collected eluent from the cation resin was loaded onto an anion resin. Collection of the effluent was not stopped until it reached 870 L. The effluent was concentrated under reduced pressure to provide an extractum as a mulberry twig extract, in which the content of alkaloids was 30%, polysaccharides 35%, flavonoids 2%, and amino acids 25%. In the alkaloids, the content of 1-DNJ was 62%, FAG 20%, and DAB 13%.

Preparative Example 7

[0145] 1000 kg of fresh mulberry twigs (Yuesang No. 11, Morus serrata Roxb.) were taken for crushing, followed by addition of 4000 L of water for extraction under heat reflux for 2 h. The extraction liquids were combined and filtered to remove insoluble substances to provide a crude extract. The crude extract was thermally concentrated until the solid content reached 4% by mass, which was kept at 50 C. as a loading liquid onto a cation resin column.

[0146] 100 kg of D113-type macroporous weakly acidic cation exchange resin was packed in a column, followed by washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; washing with 1 mol/L sodium hydroxide solution until the pH of the effluent was 8.5; washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; and rinsing with 5 column volumes of deionized water to complete activation. The concentrated extraction liquids were loaded, followed by elution with 1000 L of 2.5 mol/L aqueous ammonia at an elution rate of 6 BV/h. The eluent was collected when the effluent from the cation column was detected to have pH>7. Collection was stopped when the collected liquid reached 900 L. The collected liquid was subject to direct purification by an anion column.

[0147] 62.5 kg of D218-type macroporous strongly alkaline acrylic based anion resin was packed in a column, followed by washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0; washing with 1.5 mol/L hydrochloric acid solution until the pH of the effluent was 3.5; washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0, to complete activation. The collected eluent from the cation resin was loaded onto an anion resin. Collection of the effluent was not stopped until it reached 870 L. The effluent was concentrated under reduced pressure to provide an extractum as a mulberry twig extract, in which the content of alkaloids was 40%, polysaccharides 25%, flavonoids 0.5%, and amino acids 25%. In the alkaloids, the content of 1-DNJ was 57%, FAG 24%, and DAB 16%.

Preparative Example 8

[0148] 333 kg of dry mulberry twigs (Yuesang No. 11) were taken for crushing, followed by addition of 4000 L of water for extraction in two portions under heat reflux, with each reflux for 1 h. The extraction liquids were combined, filtered, and concentrated to 1 kg of crude drug/L.

[0149] 150 kg of D113-type macroporous weakly acidic cation exchange resin was packed in a column, followed by washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; washing with 1 mol/L sodium hydroxide solution until the pH of the effluent was 8.5; washing with 2 mol/L hydrochloric acid solution until the pH of the effluent was 4.5; and rinsing with 5 column volumes of deionized water to complete activation. The concentrated extraction liquids were loaded, followed by elution with 1000 L of 2.5 mol/L aqueous ammonia at an elution rate of 6 BV/h. The eluent was collected when the effluent from the cation column was detected to have pH>7. Collection was stopped when the collected liquid reached 900 L. The collected liquid was subject to direct purification by an anion column.

[0150] 62.5 kg D218-type macroporous strongly alkaline acrylic based anion resin was packed in a column, followed by washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0; washing with 1.5 mol/L hydrochloric acid solution until the pH of the effluent was 3.5; washing with 1.5 mol/L sodium hydroxide solution until the pH of the effluent was 9.0, to complete activation. The collected eluent from the cation resin was loaded onto an anion resin. Collection of the effluent having a pH>8 was not stopped until it reached 870 L.

[0151] The collected liquid obtained from anion column separation was subject to filtration through microfiltration membranes to remove impurities, and then concentrated through reverse osmosis membranes. The concentrated liquid had a specific gravity of 1.1 and was transferred to a tank for alcohol precipitation, to which 15 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. Once the addition of ethanol was finished, stirring was stopped for alcohol precipitation for 24 h. Supernatant was removed and concentrated under reduced pressure to provide an extractum as a mulberry twig extract. Contents in the sample are alkaloids 63%, polysaccharides 23%, flavonoids 1%, and amino acids 5%. In the alkaloids, the content of 1-DNJ was 61.9%, FAG 16.6%, and DAB 11.1%.

II. Verification of Effects of Mulberry Extract

Experimental Example 9: Pharmacodynamic Assays of Mulberry Extract in Preventing and/or Treating Inflammatory Bowel Disease

1. Experimental Protocols

1.1 Experimental Animals

[0152] Mice aged 6-8 weeks were adaptively kept in an animal house for 3 days. After weighing, they were randomly divided into 8 groups (counted as Day 0), with 5-6 mice in each group.

[0153] Experimental grouping was as follows: group 1, normal control; group 2, model; group 3, positive control; and groups 4-8, SZ-A with different administration doses, respectively. See Table 1 for Details.

1.2 Experimental Drugs

[0154] 3% DSS was formulated as follows: 3 g of DSS (Dextran Sulfate Sodium Salt) was dissolved in 100 mL of sterile water;

[0155] The mulberry twig extract as prepared in Preparative Example 1 was formulated at different concentrations and dissolved in 1% CMC solution;

[0156] Drug as positive control: Sulfasalazine Enteric-Coated Tablets (NMPN H31020557).

[0157] The main ingredient in this product was sulfasalazine, having 5-[p-(2-pyridylsulfamoyl)phenylazo]salicylic acid as its chemical name.

1.3 Modelling and Dosing

[0158] On the day when grouping was done, the mice in group 8, SZ-A-5 (pretreatment) group, were administered with a mulberry twig extract by oral gavage (see the following table for dosages) once a day. All groups are normally available to food and water.

[0159] After 3 days from administration to the mice in group 8, each of the groups began to have free access to 3% DSS (Dextran Sulfate Sodium Salt) for modelling and gavage administration except for the normal control group, with the dosage for the positive control group of 5-[p-(2-pyridylsulfamoyl)phenylazo]salicylic acid, 500 mg/kg/d. After 5 days from the start of modelling, the access to 3% DSS was stopped, and all groups had normal drinking water instead, with the administration done normally. The mice were administered continuously and observed for 10 days, and euthanized by cervical dislocation. The colons of the mice were removed by dissection, and the lengths of the colons were measured and photographed. During the administration, the weights of the mice in each group were measured. During the modelling and administration, stool texture and blood in stool of the mice were monitored daily, and Disease Activity Index, DAI, for the mice was scored according to Table 2 below. The results were shown in FIG. 1 to FIG. 3 and FIG. 4.

TABLE-US-00001 TABLE 1 Experimental Groupings and Dosages Experimental Grouping Nos. Groups (abbr.) Dosages 1 Control Nomal Control 2 DSS 3% DSS 3 SASP Positive Drug Group 4 SZ-A-1 50 mg/kg/d on extract basis 5 SZ-A-2 100 mg/kg/d on extract basis 6 SZ-A-3 200 mg/kg/d on extract basis 7 SZ-A-4 400 mg/kg/d on extract basis 8 SZ-A-5 pre-administration for 3 days, (pretreatment) 200 mg/kg/d on extract basis

TABLE-US-00002 TABLE 2 Criteria for Scoring of Disease Activity Index, DAI, for Mice Scores Weight Loss (%) Stool Texture Blood in Stool 0 None Normal Negative for Fecal Occult Blood Test 1 1-5 / / 2 6-10 Mushy Positive for Fecal Occult Blood Test 3 11-15 / / 4 16-20 Liquid Blood in Stool Observed with Naked Eyes

[0160] Note: DAI=Score for Weight Loss+Score for Stool Texture+Score for Fecal Occult Blood; Normal Stool: Shaped Stool; Mushy Stool: Pasty, Semi-Shaped Stool without Adhering to Anus; Liquid Stool: Thin, Watery Stool that may Adhere to Anus.

[0161] Colonic tissues were removed for H&E staining, which specifically comprises the steps of:

[0162] 1. Tissue samples, 3 mm thick, were taken for dehydration with alcohol in a series of gradients, 70%, 80%, 95%, 100%, for 30 minutes each, followed by two bottles of xylene for 20 minutes each, impregnation with paraffin wax in two barrels for 12 minutes each, embedding, slicing for 4 microns, and baking.

[0163] 2. Hematoxylin eosin (HE) staining: (1) Dewaxing with three bottles of xylene for 8 minutes each; two bottles of 100% alcohol for 8 minutes each; 90% alcohol, 80% alcohol and 60% alcohol for 8 minutes each. (2) Staining with hematoxylin for 4 minutes, and washing with running water. (3) Differentiation with hydrochloric acid and alcohol for 2-3 seconds, and washing with running water. (4) 0.5% aqueous ammonia for 20 seconds, washing with running water, and observation on a microscope. (5) Staining with 0.5% eosin for 1 minute. (6) Differentiation with 80% alcohol and 90% alcohol differentiate for 3-5 seconds each; 95% alcohol for 5 minutes; three bottles of 100% alcohol for 5 minutes each; two bottles of xylene for 5 minutes each. (7) Sealing with neutral resin glues; observation and microscopy with an optical microscope. Fresh colonic tissues from each group were placed in 4% paraformaldehyde for fixation, and after 48 hours they were placed in alcohol at different concentrations for gradient dehydration, and then placed in xylene for clearing. The cleared tissues were placed in paraffin wax for embedding. The embedded blocks of paraffin wax were fixed on a slicer for slicing.

1.4 Comparison of Disease Activity Index (DAI) Scores Treating with Mulberry Twig Extracts in Various Preparative Examples.

[0164] Disease Activity Index (DAI) scores for mice were compared using the mulberry twig extracts in each preparative example with reference to the experimental steps in sections 1.1-1.3 above. The mulberry twig extracts in Preparative Examples 1, 3, 4, 6 and 8 were used as a drug in a dosage of 200 mg/kg/d based on the mulberry twig extracts. The results are shown in FIG. 5.

1.5 Experimental Results

[0165] As shown in FIG. 1 to FIG. 2, the colon length in model group decreased significantly compared with normal control group; positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) all significantly inhibited colonic atrophy in UC model mice compared with model group, and the effects in SZ-A-2, SZ-A-3 and SZ-A-4 groups were comparable to that in positive drug group, with SZ-A-5 (pretreatment) achieving a superior effect to the positive drug in inhibiting colonic atrophy.

[0166] As shown in FIG. 3, the weight of mice in model group decreased significantly compared with normal control group; positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) could each inhibit weight loss in UC model mice compared with model group, showing comparable effects.

[0167] As shown in FIG. 4, positive drug group, SZ-A-1, SZ-A-2, SZ-A-3, SZ-A-4 and SZ-A-5 (pretreatment) could each reduce Disease Activity Index, DAI, for UC model mice compared with model group.

[0168] As shown in FIG. 5, the specific mulberry extracts provided in Preparative Example 1 (the content of alkaloids was 52%, polysaccharides 22%, flavonoids 0.8%, and amino acids 20%) and in Preparative Example 8 (the content of alkaloids was 63%, polysaccharides 23%, flavonoids 1%, and amino acids 5%) could reduce Disease Activity Index for UC model mice, while the mulberry extracts provided in the other preparative examples showed certain treatment effects.

[0169] As shown in FIG. 6, tissue sections of UC model mice were observed under an optical microscope, with the colonic structure of control mice shown as intact and clear, and the glands arranged regularly; while in model group, the colonic tissue of mice that had 3% DSS all the time showed congestion and edema in colonic wall, serious inflammatory cell infiltration in mucosa and submucosa, epithelial cell damage and shedding, goblet cell loss, crypt swelling and disruption, and severe colonic ulcer. After treatment with the mulberry extracts, the colonic histopathology of mice was significantly improved, the colonic structure relatively intact, the glands arranged regularly, and the infiltration of inflammatory cells mild, with effects achieved superior to positive control group. It suggested that mulberry extracts could effectively prevent and treat UC, and had a good effect of preventing and treating inflammatory bowel disease.

[0170] The invention has been described above in connection with preferred embodiments, but these embodiments are simply exemplary and serve as no more than illustration. In light of this, various alternatives and modifications can be made to the invention, and each of these falls within the scope of protection of the invention.

INDUSTRIAL APPLICATION

[0171] The invention experimentally demonstrated that a particular mulberry extract could inhibit colonic atrophy in UC model mice, inhibit weight loss of UC model mice, and reduce Disease Activity Index, DAI, for UC model mice, and that colonic histopathology of mice was significantly improved, the colonic structure was relatively intact, the glands were arranged regularly, and the infiltration of inflammatory cells were mild, after treatment with the mulberry extract. The results above showed that a mulberry extract or a main active ingredient thereof could be used to prepare and treat ulcerative colitis.