FATTY ACID COMPOSITION AND APPLICATION THEREOF

Abstract

The present application provides a phosphatidylcholine product represented by formula (I) and a fatty acid composition containing same. In formula (I), R.sub.1 and R.sub.2 are each independently selected from residues of saturated or unsaturated fatty acids having more than 12 carbon atoms; and R.sub.1 and R.sub.2 comprise an EPA residue and a DHA residue. The phosphatidylcholine product represented by formula (I) and fatty acid composition provided by the present application can improve damage of IBD model animal colon tissues to a certain extent, have certain anti IBD activity, and are superior to the first-line drug, sulfasalazine.

Claims

1. A phosphatidylcholine product represented by Formula (I): ##STR00008## wherein R.sub.1 and R.sub.2 are each independently selected from residues of saturated or unsaturated fatty acids having 12 or more carbon atoms; and EPA residue(s) and DHA residue(s) are comprised in R.sub.1 and R.sub.2, and wherein an area ratio between EPA and DHA is about 0.2 to about 1:1.

2. The phosphatidylcholine product according to claim 1, wherein the area ratio between EPA and DHA is about 0.33 to about 0.6:1.

3. The phosphatidylcholine product according to claim 1, wherein R.sub.1 and R.sub.2 are each independently selected from residues of saturated or unsaturated fatty acids having 12 to 24.

4. The phosphatidylcholine product according to claim 1, wherein an area percentage of EPA to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 10% to about 40%.

5. The phosphatidylcholine product according to claim 1, wherein an area percentage of DHA to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 40% to about 70%.

6. The phosphatidylcholine product according to claim 1, wherein the area percentage of EPA to the total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 10% to about 35%, and the area percentage of DHA to the total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 40% to about 70%.

7. The phosphatidylcholine product according to claim 1, wherein an area percentage of saturated fatty acids to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is not more than 20%, and an area percentage of monounsaturated fatty acids to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is not more than 10%.

8. The phosphatidylcholine product according to claim 1, wherein an area percentage of n-3 fatty acids to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 60% or more, and relative to the n-3 fatty acids, an area percentage of EPA is about 15% to about 40%.

9. The phosphatidylcholine product according to claim 1, wherein an area percentage of n-3 fatty acids to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 60% or more, and relative to the n-3 fatty acids, an area percentage of DHA is about 45% to about 75%.

10. The phosphatidylcholine product according to claim 1, wherein an area percentage of DPA to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is not more than 15%.

11. The phosphatidylcholine product according to claim 1, wherein an area percentage of n-3 fatty acids to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 60% or more, and relative to the n-3 fatty acids, an area percentage of DPA is about 2% to about 20%.

12. A fatty acid composition comprising a phosphatidylcholine product represented by Formula (I) according to claim 1: ##STR00009## wherein R.sub.1 and R.sub.2 are each independently selected from residues of saturated or unsaturated fatty acids having 12 or more carbon atoms; and EPA residue(s) and DHA residue(s) are comprised in R.sub.1 and R.sub.2, and wherein an area ratio between EPA and DHA is about 0.2 to about 1:1.

13. The composition of claim 12, wherein the area ratio between EPA and DHA is about 0.33 to about 0.6:1.

14. The composition according to claim 12, wherein an area percentage of EPA to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 10% to about 35%, and an area percentage of DHA to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 40% to about 70%.

15. (canceled)

16. A method for preparing a fatty acid composition of claim 12, comprising subjecting free fatty acids to a condensation reaction with glycerol phosphatidylcholine, and purifying resulted reaction product to obtain the fatty acid composition.

17. The phosphatidylcholine product according to claim 3, wherein R.sub.1 and R.sub.2 are each independently selected from residues of saturated or unsaturated fatty acids having 14 to 22 carbon atoms.

18. The phosphatidylcholine product according to claim 4, wherein the area percentage of EPA to the total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 15% to about 35%.

19. The phosphatidylcholine product according to claim 5, wherein the area percentage of DHA to a total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 40% to about 65%.

20. The phosphatidylcholine product according to claim 6, wherein the area percentage of EPA to the total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 15% to about 32%, and the area percentage of DHA to the total amount of fatty acids in the phosphatidylcholine product represented by Formula (I) is about 40% to about 65%.

21. A method for preventing or treating inflammatory bowel disease, comprising administering to patient a therapeutically effective amount of a phosphatidylcholine product represented by Formula (I) according to claim 1 or a fatty acid composition comprising the phosphatidylcholine product.

Description

EMBODIMENTS OF THE INVENTION

[0104] The technical solutions in the examples of the application will be described clearly and completely below. Obviously, the described examples are only a part of the examples of the application, not all of the examples. Based on the examples in the application, all other examples obtained by those skilled in the art without creative work shall fall within the protection scope of the application.

[0105] Unless otherwise indicated, the reagents, materials, and devices used in the following examples are all commercially available reagents, materials, and devices.

Examples 1 to 9

[0106] Using commercially available ethyl ester-type fish oils with different fatty acid contents as raw materials, the fatty acid composition was prepared according to the following method:

[0107] (1) The ethyl ester-type fish oils with different compositions of fatty acids were heated to 30° C., to which a solution of sodium hydroxide, ethanol and water (wherein, sodium hydroxide was weighed and dissolved in purified water, and 95% ethanol was added thereto to obtain the solution) was added dropwise. It was stirred quickly to react for 1.5 h; n-heptane with a weight ratio of 1:1 to ethyl ester-type fish oil was added into the reaction solution, and the pH value of the reaction solution was adjusted to 1.5 with 3N hydrochloric acid. It was stood for separation, and washed with water to pH 6-7, dried with anhydrous sodium sulfate and filtered, and the obtained filtrate was concentrated under reduced pressure to solvent-free to obtain free fatty acids; wherein the weight ratio among the ethyl ester-type fish oil, sodium hydroxide and ethanol was 1:0.15:0.75;

[0108] (2) After mixing and dissolving glycerol phosphatidylcholine (GEC), EDCI, 4-dimethylaminopyridine (DMAP), the free fatty acid obtained in step (1) and the solvent N,N-dimethylformamide (DMF), it was vacuumed (wherein, the molar ratio among GPC, EDCI, DMAP and free fatty acid was 1.0: 2.86: 0.3: 2.6), and stirred under N.sub.2; Ar constant pressure at room temperature to complete the reaction. HPLC purification was performed by using C18 column with 93% methanol/water as the mobile phase, the target components were collected, and the fatty acid composition, was obtained after vacuum concentration, wherein the phosphatidylcholine product represented by Formula (I) was included.

[0109] The contents of the phosphatidylcholine products in the obtained fatty acid compositions were determined by the method described above, and the contents of the phosphatidylcholine products (in wt %) were all 99% or more. The fatty acid constitutions of the phosphatidylcholine products in the fatty acid compositions obtained in Examples 1 to 9 were tested, and the results were shown in Table 1

TABLE-US-00002 TABLE 1 Fatty acid content in the phosphatidylcholine products Kinds of fatty acids (% area) 1 2 3 4 5 6 7 8 9 C14:0 — — 0.04 — — — — 0.32 — C16:0 0.1 0.08 0.04 0.06 0.05 0.11 0.15 1.93 0.19 C16:4 n-1 — — — — — 0.04 0.06 — — C16:1 n-7 0.04 0.08 0.07 0.05 0.07 0.05 0.08 0.83 0.05 phytanic acid — — 0.11 — — — — 0.11 0.04 C18:0 0.04 — — 0.05 0.03 — 0.05 0.27 — C18:1 n-9 0.23 0.41 0.05 0.07 0.03 0.19 0.27 3.21 0.18 C18:1 n-7 0.09 0.17 — — — 0.05 0.08 0.99 0.06 C18:2 n-9 0.03 0.06 — — — — — 0.08 — C18:2 n-6 0.32 0.67 0.21 0.07 0.12 0.06 0.08 0.31 0.08 C18:2 n-4 0.38 0.28 0.04 — — — — 0.14 0.24 C18:3 n-6 0.17 0.1 0.29 — — — — 0.06 — C18:3 n-4 0.12 0.07 0.13 — — — — 0.04 — C18:3 n-3 0.29 0.46 0.28 0.08 0.05 0.07 0.07 0.20 0.08 C18:4 n-3 2.07 0.9 3.09 0.69 0.14 0.54 0.66 0.43 0.83 C18:4 n-1 0.19 0.11 0.25 0.06 — 0.03 — — 0.07 C20:1 n-9 0.07 — — 0.06 — — 0.03 0.7 — C20:2 n-9 0.06 — — — — — — — — C20:3 n-6 0.2 0.53 0.09 — — — — — — C20:4 n-6 3.67 4.38 2.05 0.78 1.50 0.81 1.86 0.87 0.93 C20:4 n-3 1.44 2.63 1.09 0.97 0.57 0.99 1.34 1.17 0.98 C20:5 n-3 73.01 55.47 44.14 25.39 8.91 23.74 21.11 18.05 27.65 C21:5 n-3 1.07 2.33 1.99 2.34 0.57 2.40 2.83 2.03 2.26 C22:4 0.07 0.23 0.05 0.05 0.2 0.08 0.2 0.14 0.06 C22:5 n-6 0.51 0.66 1.16 1.81 1.45 2.03 3.C1 1.31 1.84 C22:5 n-3 1.42 4.24 6.36 10.71 8.55 11.36 6.66 10.33 10.3 C22:6 n-3 11.79 23.5 36.33 54.58 74.13 54.32 58.27 51.02 51.54 Maximum 0.24 0.28 0.28 0.58 0.9 0.68 0.53 0.44 0.66 undetermined fatty acid Total 1.2 1.56 1.47 1.45 2.26 2.22 2.23 4.68 1.4 undetermined fatty acids Other impurities 1.42 1.08 0.67 0.73 1.37 0.9 0.97 0.77 1.21 EPA + DHA 84.8 78.97 80.47 79.77 83.04 78.06 79.38 69.07 79.19 EPA:DHA 6.19 2.36 1.21 0.47 0.12 0.44 0.36 0.35 0.54 n − 3 + n − 6 95.96 95.87 97.08 97.42 95.99 96.33 95.89 85.78 96.48 Other 0.85 0.75 0.47 0.11 0.2 0.15 0.26 0.4 0.38 polyunsaturated fatty acids saturated fatty 0.14 0.08 0.19 0.11 0.08 0.11 0.2 2.64 0.23 acid monounsaturated 0.43 0.66 0.12 0.18 0.1 0.29 0.46 5.73 0.29 fatty acid Note: “—” means non-detected.

Example 10 Pharmacological Experiment of the Fatty Acid Compositions in Examples 1-9

[0110] I. Materials and Methods

[0111] 1. Animals: C57BL/6J mice, male (18-20 g); 10 mice per group; purchased from Beijing Huafukang Biotechnology Co., Ltd.; license number: SCXK (Beijing) 2014-0004

[0112] 2. Grouping:

[0113] (1) Normal control group (control group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0114] (2) IBC model group (model group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0115] (3) Positive drug sulfasalazine group (SASP group): Shanghai Sinepharm Factory Co., Ltd. (batch number: 036151102); dosage: 500 mg/kg, prepared with 0.5 wt % sodium carboxymethyl cellulose, stored at 4° C., administered intragastrically once a day.

[0116] (4) Test group: the fatty acid compositions of Examples 1 to 9, dosage: 500 mg/kg, prepared with 0.5 wt % sodium carboxymethyl cellulose, stored at 4° C., administered intragastrically once a day.

[0117] 3. Experimental Method

[0118] For the convenience of experimental operation, two experiments were conducted. The fatty acid compositions of Examples 1 to 5 were used for the first experiment; the fatty acid compositions of Examples 6 to 9 were used for the second experiment. Both experiments were operated according to the following methods. C57BL/6J mice were adaptively fed in an SPF animal room (laboratory animal license number: SYXK (Beijing) 2014-0023) for one week, and then randomly grouped. The mice in the model group and the administration groups were constructed as an IBC mouse model with DSS (MP, CA9011-18-1, US) by entrusting to the Institute of Materia Medica, Chinese Academy of Medical Sciences. The mice in the normal control group were not treated. The normal control group (control group) and model group (model group) were administered with 0.5 wt % sodium carboxymethyl cellulose intragastrically, once a day, with an administration volume of 10 mL/kg. The SASP group and the test group were administered intragastrically according to the above experimental scheme, once a day. The animals in the model group showed obvious listlessness, reduced activity, body weight loss, sparse stools, hematochezia and other typical IBD lesions after 7 days of modeling, and the experiment can be terminated. The animals in each group were sacrificed, and each evaluation indicator of colitis was tested (see the section of Experiment results), and the anti-IBD pharmacodynamic activity of each test sample was evaluated comprehensively.

[0119] II. Experimental Results

[0120] The results were shown in Table 2. The experimental results showed that, compared with the normal control group, the colonic mucosa of mice in the model group modeled by DSS was significantly shed. The inflammation implicated the mucosa and the submucosal Lamina propria, and crypts were destroyed; the infiltration of a large number of inflammatory cells, which are mainly lymphocytes and neutrophils, can be seen at the inflammatory lesion site; inflammatory ulcer formation, bleeding and blood cell exudation can be seen in local areas with significant inflammation; inflammatory fibrous tissue hyperplasia can be seen at the bottom of the ulcer foci; a large number of red blood cells and exudates can be seen in the intestinal cavity. The positive drug SASP can effectively improve the colon tissue damage of DSS-induced IBD mouse model animals to a certain extent, showing that the structure of the intestinal mucosa was basically maintained normal, and the epithelial cells were occasionally arranged disorderly and loss of polarity; scattered superficial ulcers were formed; mild edema and inflammatory cell infiltration occurred in the submucosa and muscle layer; no obvious serosal layer destruction and mucosa hemorrhage were observed. Compared with the model group, the test groups of Example 1, Example 2, Example 3 and Example 5 showed no significant decrease in histopathological score, and there was no statistically significant difference. The test group of Example 4 has relatively obvious anti-IBD activity, which is manifested as a decrease in histopathological score and an increase in DAI comprehensive index inhibition rate. Compared with the model group, the statistical difference is very significant (p<0.01). The histopathological score was assessed from intestinal epithelial mucosal injury and ulcer formation, edema, lymphocyte/monocyte/plasma cell infiltration, neutrophil infiltration, eosinophil infiltration and other indicators. The lower the histopathological score was, the closer the colon tissue was to the normal physiological state of the animal.

[0121] Table 2 showed the effect of the test groups of Examples 1 to 5 on the inhibition rate of the disease comprehensive index DAI and histopathological scores of IBD mice verified in Example 10 of the application.

TABLE-US-00003 TABLE 2 DAI comprehensive index inhibition rate and histopathological score DAI comprehensive histopathological Group index inhibition rate (%) score normal control group — 0.30 ± 0.67 IBD model group 0  8.80 ± 1.44** Positive drug group 23.08.sup.#  7.25 ± 2.14.sup.# Test group of Example 1 8.33 8.13 ± 1.27 Test group of Example 2 15.39 7.89 ± 1.45 Test group of Example 3 −2.69 7.70 ± 2.11 Test group of Example 4 48.08.sup.##   5.40 ± 1.43.sup.## Test group of Example 5 0 8.38 ± 0.99 Note: **p < 0.01 vs. Con; .sup.#p < 0.05, .sup.##p < 0.01 vs. Mod.

[0122] The experimental results showed that, compared with the normal control group (con), disease comprehensive index DAI of the model group (mod) animal increased significantly, and the statistical difference was very significant, indicating that the model was successfully built. Compared with the model group, the positive drug group (SASP group) and the test group of Example 4 can reduce the disease comprehensive index DAI score of the experimental animal significantly, and the difference was statistically very significant. There was no significant improvement effect in the test groups of Examples 1 to 3 and the test group of Example 5, and there was no difference in statistical test. The DA score was evaluated from the degree of body weight loss, stool character, hematochezia and other indicators of animal. The lower the DAI score was, the closer the animal was to the normal physiological state.

[0123] The DAI scoring criteria were as follows:

TABLE-US-00004 Percentage of body Score weight loss (%) Stool character Degree of hematochezia 0    0  Normal Normal 1    1~ Loose(+) Occult blood positive(+) 2    5~ Loose(++) Occult blood positive(++) 3   10~ Sparse stool(+) Bloody stool with naked eyes(+) 4 >15  Sparse stool(++) Bloody stool with naked eyes(++) ~Referring to the right open interval, for example, 1~ refers to [1%, 5%) Note: {circle around (1)} Normal stool: formed stool; {circle around (2)} Loose stool: mushy, semi-formed stool that does not adhere to the anus; {circle around (3)} Sparse stool: watery stool.

[0124] The histopathology scoring criteria table was as follows:

TABLE-US-00005 Scoring criteria 0 1 2 3 Epithelial injury and None Submucosa/ Muscle Serosal ulceration erosion layer/ulcers layer/heavy Edema None Mild Moderate Heavy Lymphocyte, monocyte, None Submucosa/ Muscle Serosal plasma cell infiltration mild layer/moderate layer/heavy Neutrophil infiltration None Submucosa/ Muscle Serosal mild layer/moderate layer/heavy Eosinophil infiltration None Submucosa/ Muscle Serosal mild layer/moderate layer/heavy

[0125] The experimental results of the test groups of Examples 6-9 showed that, compared with the normal group, the colonic mucosa of mice in the model group modeled by DSS was significantly shed. The inflammation implicated the mucosa and the submucosal lamina propria, and crypts were destroyed; the infiltration of a large number of inflammatory cells, which are mainly lymphocytes and neutrophils, can be seen at the inflammatory lesion site; inflammatory ulcer formation, bleeding and blood cell exudation can be seen in local areas with significant inflammation; inflammatory fibrous tissue hyperplasia can be seen at the bottom of the ulcer foci; a large number of red blood cells and exudates can be seen in the intestinal cavity (DAI comprehensive index inhibition rate was 0%, histopathological score was 8.45±0.62, statistically significant difference, p<0.01 vs. Con). The positive drug SASP can slightly improve the colon tissue damage of DSS-induced IBD mouse model animals, which was manifested by the improvement of intestinal mucosal structure destruction, the alleviation of epithelial cell disorder arrangement and loss of polarity; scattered superficial ulcer formation; moderate edema and inflammatory cell infiltration in the submucosa and muscle layer; alleviation of the serosal layer destruction and mucosal hemorrhage. Test groups of Example 6, Example 7, Example 8, and Example 9 all had obvious anti-IBD activity MAI comprehensive index inhibition rates were all higher than 25%, histopathological scores were all lower than 6, statistically significant difference, p<0.01 vs. Mod), and can improve the damage of the colon tissue in the model animal to a certain extent, which was manifested by the alleviation of edema, inflammation, exudation and infiltration and the like of the mucosal layer, submucosa, and muscle layer to a certain extent, and the activities thereof were greater than the positive drug.

Examples 11 to 13

[0126] For further investigating the effect of the total content of EPA and DHA in the fatty acid composition on the anti-IBD activity of the composition, the inventors used the methods of Examples 1 to 9 to prepare the fatty acid composition comprising the phosphatidylcholine products with the fatty acid constitutions in following Table 3 from the commercially available ethyl ester-type fish oils, and the contents of phosphatidylcholine products (wt %) in these compositions obtained in Examples 11-13 were all 99% or more.

TABLE-US-00006 TABLE 3 Fatty acid content in the phosphatidylcholine products Kinds of fatty acids (% area) 11 12 13 C14:0  0.253  0.066  0.017 C16:0 17.649  0.175  0.075 C16:4 n-1 —  0.052  0.025 C16:1 n-7  0.153  0.571  0.027 phytanic acid  0.029 — — C18:0  0.316  0.037  0.049 C18:1 n-9  7.438  2.601  0.055 C18:1 n-7  0.156  0.789 — C18:2 n-9 —  0.083 — C18:9 n-6  1.443  0.277  0.321 C18:2 n-4  0.290  0.120 — C18:3 n-6 —  0.052  0.038 C18:3 n-4 —  0.011 — C18:3 n-3  0.051  0.191  0.031 C18:4 n-3  0.109  0.594  0.352 C18:4 n-1 —  0.081  0.050 C20:1 n-9  0.198  0.611  0.024 C20:2 n-9 —  0.216 — C20:3 n-6  0.133  0.08   0.034 C20:4 n-6  1.179  0.829  1.113 C20:4 n-3  0.840  1.181  0.540 C20:5 n-3 15.584 20.278 25.658 C21:5 n-3  1.588  2.118  1.597 C22:4  0.302  0.137  0.095 C22:5 n-6  1.488  1.179  0.635 C22:5 n-3  7.078  9.646  2.623 C22:6 n-3 40.132 51.477 64.528 Total undetermined fatty acids  2.525  5.602  1.746 Other impurities  1.066  0.946  0.367 EPA + DHA 55.716 71.755 90.186 EPA:DHA  0.388  0.394  0.398 saturated fatty acid 18.247  0.278  0.141 monounsaturated fatty acid  7.945  4.572  0.106 n-3 65.38  85.49  95.33  n-6  4.243  2.417  2.141 Note: “—” means non-detected.

Example 14 Pharmacological Experiment of the at y Acid Compositions in Examples 11-13

[0127] I. Materials and Methods

[0128] 1. Animals: C57BL/6J mice, male (18-20 g); 10 mice per group; purchased from Beijing Huafukang Biotechnology Co., Ltd.; license number: SCXK (Beijing) 2014-0004

[0129] 2. Grouping:

[0130] (1) Normal control group (control group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0131] (2) IBD model group (model group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0132] (3) Positive drug sulfasalazine group (SASP group): sulfasalazine enteric-coated tablets; dosage: 500 mg/kg, prepared with 0.5 wt % sodium carboxymethyl cellulose; stored at 4° C. and administered intragastrically once a day.

[0133] (4) Test group: the fatty acid compositions of Examples 11 to 13, dosage: 500 mg/kg, prepared with 0.5 wt % sodium carboxymethyl cellulose; stored at 4° C. and administered intragastrically once a day.

[0134] 3. Experimental Method

[0135] C57BL/6J mice were adaptively fed in an SPF animal room (laboratory animal license number: SYXK (Beijing) 2014-0023) for one week, and then randomly grouped. The mice in the model group and the administration groups were constructed as an IBD mouse model with DSS (MP, CA9011-18-1, US, keeping away from light) by entrusting to the Institute of Materia Medica, Chinese Academy of Medical Sciences. The mice in the normal control group were not treated. The normal control group and model group were administered with 0.5 wt % sodium carboxymethyl cellulose intragastrically, once a day. The SASP group and the test group were administered intragastrically according to the above experimental scheme, once a day. The animals in the model group showed obvious listlessness, reduced activity, body weight loss, sparse stools, hematochezia and other typical IBD lesions after 6 days of modeling, and the experiment was terminated. The animals in each group were sacrificed, and each evaluation indicator of colitis (see the section of Experiment results) was tested, and the anti-IBD pharmacodynamic activity of each test sample was evaluated comprehensively.

[0136] II. Experimental Results

[0137] Table 4 showed the effect of the test groups of Examples 11 to 13 on the inhibition rate of the disease comprehensive index DAI of IBD mice verified in Example 14 of the application.

TABLE-US-00007 TABLE 4 DAI disease comprehensive index inhibition rate Group DAI comprehensive index inhibition rate (%) Normal control group — IBD model group 0 Positive drug group 38.7.sup.# Test group of Example 11 32.06.sup.## Test group of Example 12 57.3.sup.## Test group of Example 13 30.7.sup.## Note: .sup.#p < 0.05, .sup.##p < 0.01 vs. Mod.

[0138] The experimental results showed that, compared with the model group, all of the test groups of Examples 11, 12 and 13 can significantly increase the inhibition rate of the disease activity index of experimental animals. The test groups of Examples 11, 12 and 13 were compared, statistical analyses were performed on the test group of Example 12 with the test groups of Examples 11 and 13, respectively, and the results showed that the statistical difference was very significant. The disease activity index inhibition rate of the test group of Example 12 was significantly higher than that of the test groups of Examples 11 and 13, indicating that the efficacy of the test group of Example 12 was significantly superior to the test group of Example 11 and the test group of Example 13.

Example 15 Effect on TNBS-Induced Rat IBD Model

[0139] 1. Animals: SD rats, SPF grade, male (122.3 to 152.6 g), 15 rats per group.

[0140] 2. Grouping:

[0141] (1) Normal control group (control group): 0.5 wt % sodium carboxymethyl cellulose (CMC-Na) was administered intragastrically.

[0142] (2) IBD model group (model group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0143] (3) Positive drug sulfasalazine group (SASP group): sulfasalazine enteric-coated tablets, dosage: 600 mg/kg, prepared with 0.5% sodium carboxymethyl cellulose; administered intragastrically once a day.

[0144] (4) Test Group: The fatty acid composition of Example 4, which was divided into three groups of 1, 2 and 3 according to the dosage of 150, 300 and 600 mg/kg, and prepared with 0.5 wt % sodium carboxymethyl cellulose; administered intragastrically once a day.

[0145] 3. Experimental method: After being fasted but water allowed for about 24 hours, rats in each group were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution, and a plastic tube with a diameter of 2 mm and a length of about 12 cm was gently inserted into the rat body via the anus for about 8 cm deep. The ethanol solution containing TNBS was slowly injected with the dose of TNBS 75 mg/kg and the administration volume of 3 mL/kg body weight, and then about 0.1 mL of air was injected to discharge the medicine remained in the plastic tube into intestine. After the administration, the plastic tube was slowly pulled out, the anus was pinched with hand, and the tail of the rat was lifted and kept upside down for about 30 s, so that the modeling agent can fully penetrate into the intestinal cavity of the rat. The normal control group was injected with the same amount of normal saline, Based on the body weight thereof, 75 TNBS modeled rats were randomly divided into model group, positive drug group (sulfasalazine 600 mg/kg body weight), test groups 1 to 3, 15 rats/group, and a normal control group was set up, in which 15 rats were included. On the second day after modeling, the corresponding test samples were administered intragastrically. The normal control group and the model group were administered with 0.5 wt % CMC-Na once a day for 5 consecutive days. DAI activity score was performed on days 1 and 3 of administration and 24 hours after the last administration. Intestinal mucosal damage was observed 24 hours after the last administration for CMDI scoring. The colon tissue was taken for histopathological examination.

[0146] 4. Experimental results: 1 Dark red filth was observed near the anus of the rats in the model group after TNBS modeling, and some animals excreted obvious sparse stools and bloody stools. The body weight of d6 and the weight gain during the administration period were lower (p<0.05), DAI score values of each time point of d1, d3 and d6, and CMDI score value of d6 were higher (p<0.05), and the length of colon+rectum was shorter (p<0.05). Histopathological examination showed that intestinal wall inflammatory cell infiltration, intestinal crypt structure destruction and mucosal fibrosis were found in different degrees in 6 cases of 15 cases, mucosal ulcers and edema were observed in some animals. {circle around (2)} Positive drug group: the symptoms of sparse stools and bloody stools of each group were alleviated until disappeared during the administration period. The weight gain during the administration period was lower (p<0.05), and DAI and CMDI score values of d6 were lower (p<0.05). Histopathological examination showed histological changes of the colon in 5 cases of 15 cases, and the lesion degree was significantly reduced compared with the model group. {circle around (3)} Test groups 1 to 3: the symptoms of sparse stools and bloody stools of animals in each group alleviated day by day until disappeared during the administration period. DAI score value of rats in test group 3 at time point of d6 was lower (p<0.05), and CMDI score values of rats in test groups 1 to 3 all had a decreasing trend. Histopathological examination found that the degree of colon histological lesions in the rats of test groups 1 to 3 was alleviated compared to the model group.

Example 16 Effect on OXZ-Induced Mouse IBD Model

[0147] 1. Animals: Balb/c mice, male (20-22 g); 10 mice per group.

[0148] 2. Grouping:

[0149] (1) Normal control group (control group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0150] (2) IBD model group (model group): 0.5 wt % sodium carboxymethyl cellulose was administered intragastrically.

[0151] (3) Positive drug sulfasalazine group (SASP group): Dosage: 500 mg/kg, prepared with 0.5 wt % sodium carboxymethyl cellulose; stored at 4° C. and administered intragastrically once a day.

[0152] (4) Test group: The fatty acid compositions of Example 4, which was divided into two groups (test group 1 and test group 2) according to the dosages of 500 mg/kg and 1000 mg/kg, prepared with 0.5 wt % sodium carboxymethyl cellulose; stored at 4° C. and administered intragastrically once a day.

[0153] 3. Experimental Method

[0154] Balb/c mice were adaptively fed in an SPF animal room for one week and then grouped randomly. The mice in the model group and the administration groups were constructed as an IBD mouse model with OXZ by entrusting to the Institute of Materia Medica, Chinese Academy of Medical Sciences. Then, the administration was performed for 6 consecutive days; the normal control group was not treated. The normal control group and the model group were intragastrically administered with 0.5 wt % sodium carboxymethylcellulose, once a day. SASP group and test groups 1 to 2 were administrated intragastrically according to the above experimental scheme, once a day. Animals in each group were sacrificed on day 6 of modeling, and various indicators related to colitis were tested (see the section of Experimental results).

[0155] 4. Experimental Results

[0156] Table 5 showed the effect of the fatty acid composition in Example 4 of the application on the inhibition rate of the disease comprehensive index DAI and the histopathological score of IBD mice.

TABLE-US-00008 TABLE 5 DAI comprehensive index inhibition rate and histopathological score DAI composite index inhibition histopathological Group rate (%) score Normal control group — 0.60 ± 0.16 IBD model group —  9.38 ± 0.56** Positive drug group 50   4.38 ± 0.42.sup.## Test group 1 58.85.sup.#   6.00 ± 0.46.sup.## (500 mg/kg) Test group 2 67.71.sup.#   4.43 ± 0.62.sup.## (1000 mg/kg) Note: **p < 0.01 vs. Con; .sup.#p < 0.05, .sup.##p < 0.01 vs. Mod.

[0157] The experimental results showed that compared with the normal control group (con), the animal disease comprehensive index DAI of the model group (mod) increased significantly, and the statistical difference was very significant, indicating that the model was successfully built. Compared with the model group, test groups 1 to 2 can significantly reduce the score of the disease comprehensive index DAI of experimental animals, and the difference was statistically significant.

Example 17 Pharmacokinetic Experiment

[0158] Administration Route: Single Intragastric Administration

[0159] Test drug: medium-chain triglycerides (MCI) were added to the fatty acid composition, of Example 4 to prepare the test products of groups A to C, and the test product of group D was prepared by using the ethyl ester-type fish oil with the same fatty acid constitution as the commercially available Lovaza product.

[0160] Test animals: 24 male SD rats qualified for quarantine and adaptation period, with 300-350 g of body weight, were taken. PEMS 3.1 software was used to randomly assign them into 4 groups: group A (administration dose 100 mg/kg, administration concentration 10 mg/mL, administration volume 10 mL/kg), group B (administration dose 300 mg/kg, administration concentration 30 mg/mL, administration volume 10 mL/kg), group C (administration dose 900 mg/kg, administration concentration 90 mg/m L, administration volume 10 mL/kg), group D (administration dose 900 mg/kg, administration concentration 90 mg/ml, administration volume 10 mL/kg).

[0161] Administration method: After being fasted but water allowed overnight for at least 12 hours, they were fed high-fat diet the next morning, the food intake within 0.5 hours was recorded, and the test drugs of corresponding concentrations were administered intragastrically according to the above administration scheme after 0.5 hours. They were fasted within 4 hours after the administration, and all fed standard diet after 4 hours, and the food intake within 10 hours after the administration needed to be recorded (the food intake was measured every 2 hours). Test process: The time points of blood sampling were set as follows: Baseline (4 points): −24 h, −16 h, −8 h, 0 h before administration; after administration (11 points): venous blood of 1 h, 2 h, 4 h, 6 h, 7 h, 8 h, 10 h, 12 h, 24 h, 48 h, 72 h after administration; 15 points were sampled in total. 0.2 ml of blood was collected from the retroorbital venous plexus, anticoagulated with EDTA-2K, and centrifuged at 4° C. under 1700×g for 10 min, and the plasma was separated, and stored at 80° C. for testing.

[0162] Sample analysis: The plasma concentration of EPA total acid, DHA total acid, EPA free acid (EPA-FA), and DHA free acid (DHA-FA) in plasma were determined by LC/MS/MS method.

[0163] Conclusion: The results of area under the plasma concentration-time curve (AUC) showed that the fatty acid composition comprising the phosphatidylcholine product of the application had better absorption, compared with the control drug ethyl ester-type fish oil at the same dosage. The statistical analysis results of each pharmacokinetic parameter showed that the dosage of the fatty acid composition comprising the phosphatidylcholine product of the application was correlated with the drug system exposure.

[0164] Described above are only the preferred embodiments of the application. It should be pointed out that for those skilled in the art, without departing from the principles of this application, several improvements and modifications may be made, and these improvements and modifications also should be regarded as falling within the protection scope of this application.