PHARMACEUTICAL COMPOSITION CONTAINING SAURURUS CHINENSIS FRACTION, AND PREPARATION METHOD THEREOF

Abstract

The present disclosure relates to a pharmaceutical composition for preventing, treating or ameliorating inflammatory bowel disease, containing, as an active ingredient, a specific solvent fraction obtained from Saururus chinensis; and a preparation method therefor.

Claims

1. A method for preparing a solvent fraction of Saururus chinensis effective for inflammatory bowel disease, the method comprising the steps of: (S1) extracting Saururus chinensis with a 50-90 volume% ethanol aqueous solution, (S2) adding purified water to the residue remaining after the extraction, and heating to 70-100° C., (S3) enzymatically treating the result of the step S2, (S4) removing the residue in the extract and filtering the extract, (S5) adding ethanol to the filtrate of the step S4 to form a precipitate, and (S6) obtaining the precipitate.

2. The method according to claim 1, wherein the enzyme is polygalacturonase, pectin lyase, or a mixture thereof.

3. The method according to claim 1, wherein the ethanol aqueous solution of the step S1 is a 60-80 volume% ethanol aqueous solution.

4. The method according to claim 1, wherein the step S5 is a step of concentrating the filtrate, adding more ethanol than the concentrate and stirring the mixture to form a precipitate.

5. The method according to claim 1, wherein the method further comprises a step of concentrating the precipitate after the step S6 and freeze-drying.

6. A method for preparing a solvent fraction of Saururus chinensis effective for inflammatory bowel disease, the method comprising the steps of: (S1) extracting Saururus chinensis with a 50-90 volume% ethanol aqueous solution, (S2) filtering and concentrating the extract, (S3) passing the concentrate through a resin-filled column to separate the components depending on whether they are adsorbed to a resin, and (S4) obtaining components eluted according to the water and ethanol concentration gradient among the components adsorbed on the resin.

7. The method according to claim 6, wherein in the step S4, the portion eluted from the 20-40 volume% ethanol aqueous solution section is recovered.

8. The method according to claim 6, wherein the ethanol aqueous solution of the step S1 is a 60-80 volume% ethanol aqueous solution.

9. The method according to claim 6, wherein the resin is a porous synthetic adsorbent of a copolymer of styrene and DVB (divinyl benzene) or a porous synthetic adsorbent of nonionic aliphatic acrylic polymer.

10-15. (canceled)

16. A method of treating inflammatory bowel disease, comprising administering a composition comprising solvent fraction of Saururus chinensis to an individual in need thereof.

17. The method according to claim 16, wherein the composition is administered orally or parenterally.

18. (canceled)

19. The method according to claim 16, wherein the solvent fraction of Saururus chinensis is extracted with water, or ethanol aqueous solution.

20. The method according to claim 16, wherein the solvent fraction of Saururus chinensis is enzymatically treated by polygalacturonase, pectin lyase, or a mixture thereof.

21. The method according to claim 16, wherein the solvent fraction of Saururus chinensis is eluted by polarity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following drawings attached to the present specification illustrate preferred embodiments of the present disclosure, and serve to further understand the technical concept of the present disclosure together with the above-described contents of the present disclosure, and the present disclosure should not be construed as being limited only to the matters set forth in such drawings.

[0056] FIG. 1 schematically shows the process of preparing a solvent fraction of Saururus chinensis.

[0057] FIG. 2a and FIG. 2b are the results of evaluating the mouse colon length on the 10th day of the intestinal inflammation model evaluation after administration of the Saururus chinensis fraction (n=8).

[0058] FIG. 3 is the results of observing the histological change of the colon after administration of the solvent fraction of Saururus chinensis.

[0059] FIG. 4 is the evaluation results of TNF-α content in colon tissue after administration of the solvent fraction of Saururus chinensis.

[0060] FIG. 5 is the evaluation results of MPO content in colon tissue after administration of the solvent fraction of Saururus chinensis.

[0061] FIG. 6 is the results of evaluating the slgA secretion ability and the related cytokine induction ability by Peyer’s patches cells of the intestinal inflammation model after administration of the Saururus chinensis fraction.

[0062] FIG. 7 is the results of evaluating the effect of the active ingredient fraction of Saururus chinensis on intestinal mucus-secreting cells (LS 174T, a human intestinal goblet cell line).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] Hereinafter, the present disclosure will be described in detail with reference to Examples and the like for better understanding. However, the examples of the present disclosure can be modified in various ways, and they should not be interpreted as limiting the scope of the invention. The examples of the present disclosure are just for better understanding of the invention to persons having ordinary skill in the art.

Preparation of Solvent Fractions of Saururus Chinensis

[0064] Various fractions were prepared as shown in FIG. 1.

Example 1: Preparation of Saururus Chinensis Hot Water extract (SCW)

[0065] The dried and pulverized Saururus chinensis was mixed with 20-fold volume of purified water, and extracted at 100° C. for 2 hours. After extraction, the residue was removed using an extraction cloth, and the extract was filtered with Whatman No.2 filter paper. The filtrate was concentrated using a vacuum concentrator, and then freeze-dried to prepare a Saururus chinensis hot water extract (SCW).

Example 2: Preparation of Various Saururus Chinensis Organic solvent extracts (SCE, SCESII-1, SCESII-2, SCESII-3, SCESII-4)

[0066] The dried and pulverized Saururus chinensis was mixed with 10-fold volume of 70% (v/v) ethanol aqueous solution, and extracted at 70° C. for 2 hours. After filtration with an extraction cloth, the filtered residue was extracted once more under the same conditions. The extract was filtered with Whatman No.2 filter paper and concentrated using a vacuum concentrator. This was called 70% ethanol extract concentrate SCE. The SCE extract was passed through a column filled with synthetic resin HP20. All components that were not adsorbed to the resin were recovered using purified water. This component was called SCESll-4. In the next step, the components adsorbed to the resin were eluted in the order of purified water ~ 30 volume% ethanol, 30-70 volume% ethanol, and 70-100 volume% ethanol, and was recovered according to the polarity of the elution solvent. These were called SCESll-1, SCESll-2, and SCESII-3 according to the order in which they were first eluted.

Example 3: Preparation of Saururus Chinensis Crude Polysaccharide SCEP1, Active Polysaccharide SCEP2 and Low Molecular Saccharide SCES Fractions

[0067] Saururus chinensis was extracted with 70% ethanol aqueous solution, and the remaining residue was again mixed with 20-fold volumes of purified water and stirred at 100° C. for 2 hours. Enzyme (1 : 1 weight ratio of Pectinex® Ultra SP-L and Pectinex® Ultra MASH) was added and reacted with stirring at 55° C. for 72 hours. The enzyme reaction solution was inactivated at 100° C. for 30 minutes. After removing the residue using an extraction cloth, the extract was filtered with Whatman No.2 filter paper. This filtrate was called crude polysaccharide SCEP1. After the filtrate was concentrated, ethanol of 4 times the volume of the concentrate was added, stirred for 30 minutes, and then left at room temperature for 24 hours to form a precipitate. The precipitate was centrifuged to recover the precipitate and the supernatant, respectively. The recovered precipitate was concentrated and then freeze-dried to prepare a SCEP2 active polysaccharide fraction of Saururus chinensis, and the supernatant was concentrated and then freeze-dried to prepare a low molecular-weight sugar fraction of Saururus chinensis SCES.

Experimental Example 1: Evaluation of Anti-Inflammatory Effect In Intestinal Inflammation Model

[0068] A study was conducted on the inhibition and therapeutic effect of the Saururus chinensis active ingredient fraction. Inflammatory bowel disease was induced with dextran sodium sulfate (DSS) in the BALB/c mouse model. One hundred 8-week-old male BALB/c mice were purchased for the experiment and randomly divided into 9 groups. To induce acute colitis in mice, DSS was dissolved at 5% in drinking water, and BALB/c mice were watered ad libitum for 7 days. Normal control group (NT: not treated) was provided with drinking water ad libitum. After 1 day of DSS watering, the normal control group and the negative control group (NC) ingested singly with 5% DSS were dissolved in distilled water, and the sample group was dissolved in distilled water at the doses listed in Table 1 below, and administered orally once a day for 9 days.

TABLE-US-00001 Group Dietary method Normal group Not treated(NT) group Group in which neither dextran sulfate sodium (DSS) nor sample was administered Negative control (NC) group Dextran sulfate sodium (DSS)-treated inflammatory bowel disease induction group Experiment group Saururus chinensis alcohol extract SCE SCE 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Saururus chinensis water extract SCW SCW 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Low molecular and non-polar SCESII-1 SCESII-1 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Low molecular and non-polar SCESII-2 SCESII-2 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Low molecular and non-polar SCESII-3 SCESII-3 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Low molecular and non-polar SCESII-4 SCESII-4 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Low molecular saccharide SCES SCES 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Crude polysaccharide SCEP1 SCEP-1 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated Experiment group Active polysaccharide SCEP2 SCEP-2 2.0 mg/mouse + dextran sulfate sodium (DSS) were treated

[0069] The disease activity index (DAI) was calculated based on the score shown in Table 2 below after visual confirmation of the weight loss, loose or soft stool, bloody stool, and the presence or absence of blood in the anus at intervals of 2 days. Equation:

[00001]$\begin{array}{l}\text{DAI}=\left(\text{weight}\text{loss}\text{score}\right)+\left(\text{stool}\text{consistency}\text{score}\right)+\\ \left(\text{blood}\text{in}\text{stool}\right)\left(\text{score}\right)+\left(\text{blood}\text{in}\text{anus}\text{score}\right)\end{array}$

Disease activity index (DAI) scoring system

TABLE-US-00002 Score Weight Stool consistency Blood in stool Blood in anus 0 - Normal Normal Normal 1 1-5% - trace trace 2 5-10% Loose stool Hemoccult + Hemoccult + 3 10-20% - Hemoccult ++ Hemoccult ++ 4 >20% Diarrhea Gross bleeding Gross bleeding

[0070] The evaluation results are shown in Table 3 below.

TABLE-US-00003 Average 2-day 4-day 6-day 8-day NT Not treated 0.2±0.4472 0.8 ± 1.0954 1 ±1 0.6 ± 0.8944 NC DSS treated 2 ± 0.7071 1.6 ±2.0736 10.2 ± 1.7889 14.8 ± 1.3038 SCE alcohol extract 1.4 ± 1.1402 1 ± 0.7071 9.4 ±2.3022 13.2 ± 1.7889 SCE water extract 1.8 ± 0.5477 2.4 ± 0.8944 9.2 ± 0.8944 12.8 ±2.1679 SCESll-1 low molecular and non-polar 0 ± 0 2.4 ± 0.8944 6.6 ± 0.5477 8.4 ± 1.1402 SCESll-2 low molecular and non-polar 0.4 ± 0.5477 2.6 ± 0.8944 6.6 ± 0.8944 9.2 ± 2.1679 SCES ll-3 low molecular and non-polar 1 ±1 2.2 ± 1.6432 8.2 ± 3.2711 12.4 ± 1.5166 SCESll-4 low molecular polar 0±0 2 ±0 6±0 9.2 ± 2.1679 SCES low molecular saccharide 1 ±1 1.6±0 6.6 ± 1.8166 9.6 ±2.0736 SCEP1 crude polysaccharide 1±1 1.2 ± 1.6432 6.2 ± 3.2711 10.4 ± 1.5166 SCEP2 active polysaccharide 2 ±0 1.2 ± 1.0954 5.8 ± 1.7889 7.6 ± 0.5477

[0071] As a result of measuring the DAl change of each administration group, the DAl score increased from day 2 of all groups except for the normal control group, but the low molecular active substance fraction SCESII-1 and the active polysaccharide fraction SCEP2 had a significantly lower DAI score than the negative control group (NC) on the 8th day, indicating an inhibitory effect on colitis. On the other hand, the other solvent fractions and Saururus chinensis water extract had almost no effect.

[0072] DAI is measured using factors such as body weight, stool condition, and bloodstain level, and thus, the DAI could be a more important evaluation factor.

[0073] Mice were sacrificed on the 10th day of the experiment, and the length of the large intestine and small intestine and the weight of the spleen were measured and evaluated. The results are collectively shown in FIGS. 2.

[0074] Mice were sacrificed and the length of the large intestine was measured. As a result, the NC ingested with 5% DSS alone has approximately 32% reduction in colon length as compared to the negative control group that did not ingest DSS, but the low molecular active fraction SCESII-1, SCESII-4, and the active polysaccharide fraction SCEP2 recovered to a length almost similar to that of the normal group NT. This shows that colitis has improved remarkably as compared to the negative control group. In particular, the active polysaccharide fraction SCEP2 and the low molecular active fraction SCESII-1 derived from Saururus chinensis had very similar colon lengths between mice, so it was considered that the reliability of the experiment was high. On the other hand, it was confirmed that the other extracts or fractions experimental groups, including Saururus chinensis water extract, alcohol extract, etc. showed the length of the large intestine similar to that of NC or showed a minute recovery, thereby having no effects.

[0075] In particular, in the 5% DSS single intake group (NC), all mice had diluted stool and bloody stool was observed, whereas the active polysaccharide fraction SCEP2 had less watery stool than NC, and only occult blood was observed in 3 mice. This is a result showing that the active polysaccharide derived from Saururus chinensis has an excellent therapeutic effect on colitis. Taken together, the above results show that the active polysaccharide fraction SCEP2 and the low molecular active fraction SCESll-1 had a DAl score of 5% DSS alone recovered to a normal level, unlike the negative control group (NC), and the length of the large intestine is relatively long, thereby having colitis inhibitory activity.

Experimental Example 2: Observation of Histological Changes in Large Intestine After Administration of the Solvent Fraction of Saururus Chinensis

[0076] After autopsy, the excised colon was fixed in a 10% formaldehyde solution for histological examination, and then paraffin-embedded through a general tissue treatment process. Each embedded tissue was sectioned 5.0 .Math.m with a microtome (Leica, Wetzlar, Germany), followed by Alcian Blue staining. Each slice was treated with canadian balsam and observed with an Olympus BX53 microscope (Olympus Corp., Tokyo, Japan). The results are shown in FIG. 3.

[0077] As shown in FIG. 3, in the group treated with dextran sodium sulfate, tissue damage due to inflammation was severe, inflammation continued to the mucous membrane, and the entire intestinal gland and epithelial cells were severely damaged.

[0078] However, in the group administered with the active polysaccharide fraction SCEP2, the low molecular active fraction SCESII-1, and the low molecular active fraction SCESII-4, markedly recovered tissue findings were found.

Experimental Example 3: Evaluation of MPO and TNF-α Contents in the Colon Tissue After Administration of Solvent Fraction of Saururus Chinensis

[0079] Analysis of TNF-α and myeloperoxidase (MPO) contents was performed in colitis-induced colon tissues. The colon tissue sample was placed in a lysis buffer (Intron, Seoul, Korea) and pulverized using a homogenizer (Scilogex, Rocky hill, CT, USA). This was centrifuged at 10,000 rpm, 4° C. for 20 minutes to recover the supernatant and the TNF-α and MPO content was determined using an ELISA kit according to the manufacturer’s instructions. The results are shown in FIG. 4 and FIG. 5.

[0080] As a result, in the case of the active polysaccharide fraction SCEP2 and the low molecular active fraction SCESII-1, the production of the major proinflammatory factor TNF-α was significantly reduced. In addition, in the case of inhibition of the production of MPO involved in macrophage activation, it was confirmed that polysaccharide fraction SCEP2 and low molecular active fraction SCESII-1, 3 were remarkably inhibited.

Experimental Example 4: Evaluation of slgA Secretion Ability and the Related Cytokine Induction Ability by Peyer’s Patches Cells in Intestinal Inflammation Model

[0081] 62 1-week-old C3H/HeN mice were divided into groups of 6 mice, and then, the solvent fraction of Saururus chinensis was orally administered a total of 10 times (once/2 days) for 20 days. Peyer’s patches cells of mice were recovered and cultured in a 96-well plate for 3 days, and slgA produced by Peyer’s patches cells activated by oral administration was analyzed by ELISA. The small intestine of the mice after oral administration was removed and feces were collected, and the slgA production ability was analyzed by ELISA. The results are shown in FIG. 6.

[0082] The evaluation results confirmed that slgA production increased in the active polysaccharide fraction SCEP2, crude polysaccharide SCEP1, low molecular active fraction SCESII-1, and low molecular active fraction SCESII-4.

Experimental Example 5: Evaluation of SCFA-producing Ability Induced in Feces Due to Oral Administration of the Active Solvent Fraction of Saururus Chinensis

[0083] The cecum of the mouse after oral administration was recovered, diluted with 80% methanol to have a final concentration of 1 g/mL, filtered with a 0.45 .Math.m syringe filter, and the ability to produce five short-chain fatty acids acids (acetic acid, propionic acid, butyric acid, valeric acid and heptanoic acid) was confirmed through gas chromatography (GC) equipped with a DBFFAP capillary column. The results are shown in Table 4 below.

[0084] It was confirmed that the active polysaccharide fraction SCEP2 and the low molecular active fraction SCESll-1 in feces increased the production of SCFA in a concentration-dependent manner. The positive control group, sulfasalazine, actually decreased the production of SCFA.

TABLE-US-00004 The production of short chain fatty acid in cecal extracts (short chain fatty acid production in cecum (.Math.M)) Sample Dose (mg/mouse) Acetic acid Propionic acid Butyric acid Valeric acid Caproic acid Heptanoic acid Total SCFA NT - 10758 (64.2) 759 (4.5) 4200 (25.0) 761 (4.5) - 290 (1.7) 16768 (100) NC - 5417 (63.4) 830 (9.7) 836 (9.8) 774 (9.1) 123 (1.4) 565 (6.6) 8545 (100) PC 2 3284 (56.5) 798 (13.7) 841 (14.5) 513 (8.8) - 372 (6.4) 5808 (100) SCESII-1 0.5 4201 (60.9) 886 (12.8) 1207 (17.5) 609 (8.8) - - 6903 (100) 1 4932 (63.5) 1001 (12.9) 1202 (15.5) 629 (8.1) - - 7764 (100) 2 5130 (62.4) 1098 (13.4) 1379 (16.8) 614 (7.5) - - 8221 (100) SCEP-2 0.5 4846 (63.2) 797 (10.4) 790 (10.3) 595 (7.8) 144 (1.9) 500 (6.5) 7672 (100) 1 5227 (64.6) 836 (10.3) 1419 (17.5) 607 (7.5) - - 8089 (100) 2 5307 (61.1) 903 (10.4) 1858 (21.4) 621 (7.1) - - 8689 (100)

Experimental Example 6: Effect of the Active Ingredient Fraction of Saururus Chinensis on Intestinal Mucus-Secreting Cells (LS 174T, a Human Intestinal Goblet Cell Line)

[0085] BALB/c mice were provided with water containing 5% DSS for 7 days, and the experimental sample was orally administered for 8 days. After sacrificing the experimental animals, tissue analysis was performed. Sections of the large intestine were fixed with 10% formalin solution, then filled with paraffin, and mounted with canada balsam. Each slide was observed using an optical microscope (40Xmagnification). images were analyzed using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). The results are shown in FIG. 7.

[0086] As a result, in the case of the active polysaccharide fraction SCEP2, the low molecular active fraction SCESll-1 , and the low molecular active fraction SCESII-4, the amount of Mucin (stained blue by Alcian Blue) secreted from the Goblet cells was recovered similar to that of the normal group, when compared with the normal group not treated with DSS.

INDUSTRIAL APPLICABILITY

[0087] The present disclosure relates to a composition for preventing, treating or ameliorating g inflammatory bowel disease containing a specific fraction prepared from Saururus chinensis as an active ingredient, and a method for preparing the same.