KR101180546B1 - Composition for treatment or prevention of colitis - Google Patents

Abstract

The present invention provides soyasaponin I, soyasaponin Ab, soyasaponogenol A, soyasaponogenol B, zipenoside LXXIV, zipenoside XLV, TN-2, carlopanax saponin H, carlopanax saponin B, carlopanax saponin A, Pharmaceutical composition for treating or preventing colitis, including improving or preventing colitis, comprising at least one saponin selected from lansemaside X, ikanocyst acid 3- O- β-D glucuronopyranoside, and ikanocyst acid as an active ingredient It provides a food composition and a pharmaceutical composition for treating or preventing colitis and a food composition for improving or preventing colitis, comprising as an active ingredient at least one natural product extract or fermented product selected from soybeans, bean sprouts, dolung, Haedongpi, and deodeok. The composition of the present invention is effective for the treatment (improvement) or prevention of colitis.

Description

Composition for treatment or prevention of colitis

The present invention relates to a composition for treating (improving) or preventing colitis, and more particularly to a composition for treating (improving) or preventing colitis comprising saponin, a saponin-containing natural product extract or a fermented product as an active ingredient.

Colitis is a disease that causes inflammation in the large intestine, and includes inflammatory bowel disease (IBD) and the like. Ulcerative colitis (UC) and Crohn's disease (CD) are chronic inflammations of the digestive tract that are called inflammatory bowel disease. Both ulcerative colitis and Crohn's disease can cause severe chronic diarrhea and bloody diarrhea with abdominal pain, which is difficult to cure, and improves and worsens. Ulcerative colitis can occur anywhere in the gastrointestinal tract. Ulcerative colitis and Crohn's disease differ in lesions and symptoms of inflammation, but because of similarities in many aspects, the distinction between the two diseases is often unclear.

Saponin is a term used to refer to the non-sugar portion of glycosides widely present in the plant system, which is composed of various ring compounds, and is included in natural products used as useful medicines and is known to be effective in various diseases.

Beans, on the other hand, belong to the legumes and are used for food. In oriental medicine, bean sprouts made by germinating soybeans are used as medicines, called soybean yellow rice (大豆 黃 卷). The main components of soybean are sugar unit (genistein) and dyedzein (sugar unit). ) Is contained in the form of glycosides combined. Soybeans contain several types of saponins in addition to the highly active isoflavones. Soybean saponins are divided into soyasaponins A, B, and E according to the type of aglycone, and are structurally a hydrophobic aglycone moiety of the sterol or triterpene family and an aglycone C-3 or There are glycosides in the form of chains of different sugar units at the C-22 position. Soybean is known to be effective in osteoporosis, cancer prevention, hyperlipidemia due to the high content of estrogen-like components and proteins, but is not known in relation to colitis.

Dol et al. Are perennial vines of the dicotyledonous plant, Pak pak, and the scientific name is Gynostemma pentaphyllum, and contains saponin, zipinoside. It is known that it is effective in hyperlipidemia and other drugs, but is not known in relation to colitis.

Haedongpi (海桐 皮) is a bark of the plant belonging to the family arborida is known to be effective in neuralgia, etc., but is not known in relation to colitis. Thawed skin also contains kalopanaxsaponin and pictosaponin as saponin.

Deodeok is a perennial vine plant belonging to the campanula, wild throughout Korea. Deodeok's enlarged root is widely used for medicinal purposes, and is known to have medicinal effects such as expectorants, and is not known in relation to colitis.

The present inventors have completed the present invention as a result of research on the efficacy of various saponins and saponin-containing natural products for the treatment or prevention of various saponins.

The problem to be solved by the present invention is to provide a pharmaceutical composition for treating or preventing colitis.

In addition, another problem to be solved by the present invention is to provide a food composition for improving or preventing colitis.

The present invention provides soyasaponin I, soyasaponin Ab [3-O- β- D-glucopyranosyl- (1 → 2) -β-D-galactopyranosyl- (1 → 2) -O- β- D-glucuronopyranosyl, 22-O- β- D-2,3,4,6-tetra acetyl-glucopyranosyl (1 → 2) -α-L-arabinopyranosyl soyasapogenol A], soyasapogenol A, soyasapogenol B, zipeno side LXXIV (gypenoside LXXIV) [2α, 3 β, 20 (S) -trihydroxydammar-24-ene-20-O- [β- D-glucosyl (1 → 2) - β- D-glucopyranoside], jipe furnace side XLV (gypenoside XLV) [2α, 3β , 20 (S) -trihydroxydammar-24-ene-3-O- [ β- D-glucopyranosyl20-O- [α-L-rhamnosyl ((1 → 6) -β - D -glucopyranoside), TN-2 [2α, 3 β, 20 (S) -trihydroxydammar-24-ene-20-O- [α-L-rhamnosyl (1 → 6) -β - D-glucopyranoside], carlopanax saponin (kalopanaxsaponin) H [3-O-beta-D-xylopyranosyl (1 → 3) -alpha-L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-23-hydroxyolean-12-en-28-O-alpha -L-rhamnopyranosyl (1 → 4) -beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl ester], carlophanaxasponin B [3-O-alp ha-L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-23-hydroxyolean-12-en-28-O-alpha-L-rhamnopyranosyl (1 → 4) -beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl ester], Carlophanaxa saponin A [3-O-alpha-L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-Hederagenin], lancemacide X [echinocystic acid 28- O- β-D-xylopyranosyl (1 → 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl ester], Icanocystic acid 3 O -β-D-glucuronopyranoside (echinocystic acid 3- O- β-D-glucuronopyranoside; It provides a pharmaceutical composition for treating or preventing colitis comprising one or more saponins selected from EAG) and echinocystic acid as an active ingredient.

The saponin may be isolated or synthesized from one or more natural product extracts or fermented products selected from soybeans, bean sprouts, dodol, thaw and deodeok.

The extract may be a solvent extract, and the solvent may be water, and / or an alcohol having 1 to 4 carbon atoms.

The fermented product may be extracted after fermentation of at least one selected from soybeans, bean sprouts, dol-o, Haedongpi, and Deodeok or fermented one or more extracts selected from soybean, bean sprouts, dol-o, Haedongpi, and Deodeok, the fermentation is lactic acid bacteria May be fermentation.

The colitis is an inflammation of the large intestine, and includes inflammatory bowel disease (IBD) and the like, and may be one or more selected from ulcerative colitis (UC) or Crohn's disease (CD). have.

In addition, the present invention provides a pharmaceutical composition for treating or preventing colitis comprising as an active ingredient at least one natural product extract or fermented product selected from soybeans, bean sprouts, dodol, thaw, and deodeok. The active ingredient may be preferably a fermentation product. The fermented product may be extracted after fermentation of at least one selected from soybeans, bean sprouts, dol-o, Haedongpi, and Deodeok or fermented one or more extracts selected from soybean, bean sprouts, dol-o, Haedongpi, and Deodeok, the fermentation is lactic acid bacteria May be fermentation.

The present invention also provides soyasaponin I, soya saponin Ab, soya saponogenol A, soya sapogenol B, gypenoside LXXIV, gypenoside XLV, TN-2 , Kalopanaxsaponin H, carlopanax saponin B, carlopanax saponin A, lancemacide X, ikanocyst acid 3- O- β-D glucuronopyranoside and icanocyst acid ( It provides a food composition for improving or preventing colitis comprising one or more saponins selected from echinocystic acid as an active ingredient.

In addition, the present invention provides a food composition for improving or preventing colitis comprising at least one natural product extract or fermented product selected from soybeans, bean sprouts, dolsop, thaw, and deodeok.

The food composition of the present invention may be a health functional food, the formulation is prepared according to a conventional method, and then dried with a carrier and encapsulated, or may be formulated in the form of other tablets, granules, powders, beverages, porridge, and the like. In addition to the above, it can be manufactured in any food form.

Saponins, extracts and / or fermentations in the compositions of the present invention have the effect of treating, improving and / or preventing colitis.

Accordingly, the present invention provides for the treatment, improvement and / or prophylaxis of colitis of said saponins, extracts and / or fermentations.

The present invention also provides a method for treating, ameliorating and / or preventing colitis comprising administering to a mammal (including human) a pharmaceutically effective amount of said saponin, extract and / or fermentation product. .

Unless otherwise stated, the content of the composition of the present invention is equally applicable to the uses and methods of the present invention.

The active ingredient in the composition of the present invention can be administered orally or parenterally to mammals including humans, and the active ingredient can be formulated in combination with a pharmaceutically acceptable carrier. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are commonly used may be used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, and It is prepared by adding gelatin and the like. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes and suppositories. Injectable solutions, suspensions and emulsions can be prepared by mixing water, non-aqueous solvents or suspending solvents with the active ingredient. As non-aqueous solvents and suspending solvents, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethyl oleate Injectable esters such as and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. The pharmaceutical composition of the present invention can be administered by subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

In the compositions, uses and methods of the present invention, a dose of 1 to 50 mg / kg per adult on a saponin basis is available, and a dose of 10 to 200 mg / kg per adult on a extract and / or fermentation basis. Can be used as

The active ingredient may be formulated by adding a pharmaceutically or food-acceptable carrier, excipient or diluent, etc., and the formulation may be found in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA et al. Reference may be made.

The active ingredient in the composition of the present invention may be included in 0.1 to 100% by weight based on the total weight.

The composition of the present invention is effective for the treatment (improvement) or prevention of colitis.

1 is a result of confirming the colitis inhibitory effect of soyasaponin I in colitis model animals.
Figure 2 is a graph confirming the inhibitory effect of Soyasaponin I on the expression of inflammatory markers in colitis model animals.
Figure 3 is a result confirming the colitis inhibitory effect of carlopanax saponin in colitis model animals.
Figure 4 is a graph confirming the inhibitory effect of carlopanax saponin on the expression of inflammatory markers in colitis model animals.
5 is a result confirming the colitis inhibitory effect of Sadeok-derived saponin in colitis model animals.
Figure 6 is a graph confirming the inhibitory effect of the deodeok-derived saponin on the expression of inflammatory markers in colitis model animals.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples and Preparation Examples are only for illustrating the present invention, and the content of the present invention is not limited by the following Examples or Preparation Examples.

Example 1 Confirmation of Colitis Inhibition Effect of Extracts and Fermentations

1-1. Soybean Extract and Saponin Preparation

Glycine max L. Merr, Kyungdong Market, Seoul, South Korea) was used as a sample by repeatedly grinding five times for 2 minutes with a Hallde cutter (VCB-61). 5 kg of soybean powder was extracted with methanol (15L) for 80 hours and filtered for 5 hours, and the remaining residue was again extracted with methanol (15L) at 80 degrees for 5 hours and filtered. The filtered methanol extract was concentrated in a rotary pressure reducer (Sunil Isla, Eyla KSB 201, Korea) to obtain 150 g of methanol. 150 g of methanol was suspended in distilled water (1.5 L), followed by shaking with an equal amount of n-hexane (1.5 L), followed by separation of the n-hexane and the aqueous layer. The n-hexane extraction layer was removed, and the aqueous layer was shaken and added with the same amount (v / v) of ethyl acetate (1.5L), and the ethyl acetate and the aqueous layer were separated. After removing the ethyl acetate extract layer and shaking the same amount (v / v) of n- butanol was added to the aqueous layer and left to stand, the butanol layer and the aqueous layer was separated to obtain an n- butanol fraction 40g. 40 g of n-butanol fraction was subjected to silica gel column chromatography (Merck, 10 cm x 50 cm, 70-230 mesh) using an eluting solvent of CHCl 3: methanol: water (65:35:10) to obtain 6 small fractions. Each separated subfraction was developed with 5% sulfuric acid using TLC (Thin layer chromatography silica gel 60 F254, Merck, Germany) with developing solvent CH3Cl: methanol: water (65:35:10), Rf 0.8, 0.65, 0.6, 0.5 , 0.36, 0.2 of the material was confirmed. In 5% sulfuric acid, fraction II (300 mg) and fraction III (250 mg) developed yellow color, and fraction V (300 mg) developed purple color. Recrystallization from methanol from small fractions yielded white acicular crystals of compound 1 and fraction 2 in fraction II and white powder of compound 3 and compound 4 in fraction V.

The separated compounds were identified by H1-NMR (Bruker, AVANCE digital 400) and 13C-NMR (Bruker, AVANCE digital 400). As a result, it was confirmed that compound 1 is daidzin, compound 2 is genistin, compound 3 is soyasaponin Ab, and compound 4 is soyasaponin I. As a result, it can be seen that saponins such as soyasaponin Ab and soyasaponin I can be produced by the above method.

1-2. Bean Sprout Extract and Saponin Preparation

Soybean sprouts instead of soybeans (soybeans of the same type as the soybeans used in 1-1. Are soaked in water for 24 hours, put into a shiru and germinated by germinating for 7 days while incubating 35 times a day while keeping 35 degrees Ex) was used, and the same method as in 1-1. As a result, soybean sprout extract (methanol extract, etc.) was prepared and soyasaponin Ab, soyasaponin I was isolated therefrom. Structural identification was carried out in the same manner as in 1-1.

1-3. Dodol Extract and Saponin Preparation

1 kg of dried Doltae (Gynostemma pentaphyllum Makino, Daegu Yangnyeong Market, Daegu, South Korea) was extracted with water in a 100 degree Celsius water bath for 3 hours and concentrated with a rotary decompressor to obtain 120 g of extract. This was dissolved in water and fractional extraction with butanol was performed to separate only butanol layer and concentrate it to give 85g of extract. This was divided into 10 fractions (Fr a 1-10) with silica gel as the stationary phase and CHCl 3: methanol: water = 65: 35: 10 (lower layer) as mobile phase, in which Fr a 4 was silica gel as the stationary phase and CHCl 3 : Methanol: Water = 70: 30: 5 (lower layer) was used as a mobile phase and TN-2 (112 mg) was isolate | separated, and silica gel was fixed as the fixed phase from Fr a 5, and CHCl3: methanol: water = 70:30: 4 (lower layer). Gypenoside LXXIV (95mg) and gypenoside XLV (108mg) were isolated as a mobile phase. Structural identification was carried out in the same manner as in 1-1.

1-4. Haedipi Extract and Saponin Preparation

3 kg of dried bark (Kalopanax Pictus NAKAI) was heated with methanol at 100 degrees Celsius for 3 hours, refluxed with a reflux extractor, and the extract was concentrated under reduced pressure with a rotary pressure reducer to obtain 110 g of extract. This was dissolved in water and fractionated with butanol to separate only butanol layer and concentrated under reduced pressure to obtain an extract of 18g. This was achieved by using silica gel as the stationary phase and using the mobile phase of the gradient elution system of dichloromethane and methanol (dichloromethane: methanol = 10: 1, 7: 1, 5: 1, 4: 1 3: 1, 2: 1, 1: 1, 300 ml each), and the fifth fraction (2.8 g) was separated by mobile phase (10% acetonitrile in water to MPLC (medium pressure liquid chromatography; Yamazen 540-SY-S2CSC, Japan)). 90% acetonitrile in water) 4 fractions were separated for 4 hours, and 80 fractions were obtained. Among them, 20 mg of kalopanaxsaponin H was obtained from fractions 15-18, and fractions 29-33 were concentrated under reduced pressure to obtain 236 mg. Kalopanaxsaponin B was obtained, and fractions 61-69 were concentrated under reduced pressure to obtain 69 mg of kalopanaxsaponin A. Structural identification was carried out in the same manner as in 1-1.

1-5. Deodeok Extract Manufacturer

5 kg of dried Deodeok (Codonopsis lanceolata Trautv) roots (Gyeongdong Market, Seoul, South Korea) was heated to 80 degrees Celsius for 3 hours with methanol, extracted under reflux with a reflux extractor, and the extract was concentrated under reduced pressure with a rotary decompressor to extract 439.24 g of extract. Got it.

1-6. Soybean Fermentation and Saponin Preparation

0.5 kg of soybean powder was soaked in water and sterilized, incubated in tryptic soy broth (1 L) for 24 hours, and Lactobacillus acidophilus (KCTC, Korea Institute of Bioscience and Biotechnology, Korea) and Leuconostoc mesenterodies (KCTC) , Korea Institute of Bioscience and Biotechnology, Korea) and Bifidobacterium longum (KCTC, Korea Institute of Biotechnology, Korea) were transplanted and cultured for 3 to 5 days. The culture solution was lyophilized with an lyophilizer (ELYLA, model name Japan) and powdered or extracted with 50% alcohol (ethanol) to prepare ethanol extract of soybean fermentation broth.

In addition, except that the soybean fermentation broth ethanol extract instead of the soybean extract, it was carried out in the same manner as the saponin production method of 1-1. As a result, soyasapogenol A and soyasapogenol B were isolated. Structural identification was carried out in the same manner as in 1-1.

1-7. Bean Sprout Fermentation and Saponin Preparation

Soybean sprout fermentation (freeze-dried powder, bean sprouts fermentation broth ethanol extract) and saponins soyasapogenol A and soyasapogenol B were prepared in the same manner as in 1-6. Structural identification was carried out in the same manner as in 1-1.

1-8. Manufacture of extra fermentation products

Except for using the dried doldol powder instead of soybean powder, it was carried out in the same manner as 1-6. To prepare the doldol fermentation (freeze-dried powder, doldol fermentation broth ethanol extract).

1-9. Manufacture of Thawing Ferment and Saponin

Except for using the dried oak bark powder instead of soybean powder, it was carried out in the same manner as in 1-6. Subsequent operations were carried out in the same manner as in the method for preparing saponin of 1-4. Except that 50% ethanol extract of the thawed skin fermentation broth was used instead of the thawed skin extract. As a result, kalopanaxsaponin A was isolated. Structural identification was carried out in the same manner as in 1-1.

1-10. Deodeok Fermentation and Saponin Preparation

The deodeok fermented product was prepared in the same manner as in 1-6. Except that the dried deodeok root (Gyeongdong Market, Seoul, South Korea) was used instead of the soybean powder. In addition, 50% alcohol extract of the deodeok culture fractions were extracted with butanol, only butanol layer was separated and concentrated under reduced pressure to obtain an extract. This was achieved by using silica gel as the stationary phase and using the mobile phase of the gradient elution system of dichloromethane and methanol (dichloromethane: methanol = 10: 1, 7: 1, 5: 1, 4: 1 3: 1, 2: 1, 1: 1, 300 ml each), and the 2nd, 5th, and 7th fractions were separated using MPLC for 4 hours at a rate of 4 ml / min of mobile phase (10% acetonitrile in water to 90% acetonitrile in water). Echinocystic acid (25 mg), echinocystic acid 3- O -β-D-glucuronopyranoside (EAG, 32 mg), echinocystic acid 28- O -β-D-xylopyranosyl (1 → 3)- β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl ester (lancemaside X, 43 mg) was obtained. Structural identification was carried out in the same manner as in 1-1.

1-11. Laboratory Animal Preparation

Four-week-old ICR male mice (20-22 g) were purchased from Orient Bio. All mice were bred under controlled environmental conditions of 50 ± 10% humidity and 25 ± 2 ° C, and lighting was repeated for 12 hours and then turned off for 12 hours. Feed was used as standard experimental feed (Samyang, Korea) and drinking water was freely consumed. In all experiments, one group had 6 animals.

1-12. Colitis Induction and Animal Testing

One group of the experimental animals of 1-11. Was taken as a normal group, and for each of the remaining groups, the specimens shown in Table 2 were dissolved (or dispersed) in 5% cremophores at the stated doses and orally administered. On the third day of the sample administration, the animal was lightly anesthetized with ether, and a 1 ml syringe with a round tip was used to administer 2.5 g / 50% ethanol (TNBS) in the large intestine through the anus. Inflammation was induced by holding vertically for 30 seconds. 0.1ml saline was administered to the normal group. On the fifth day of the sample administration, suffocated with carbon dioxide and killed, and the large intestine from the cecum to the anterior region of the anus was extracted. The extracted colon was observed by observing its length and appearance and scored according to the criteria of Table 1 (Hollenbach et al., 2005 criteria for colitis), and the results are shown in Table 2. Sulfasalazine 50 mg / kg administration group was used as a positive control. In addition, a portion of the colon contents were taken for intestinal microbial analysis and frozen and stored at minus 80 degrees Celsius. The colon tissue was removed from the colon contents, washed in physiological saline, and some samples were fixed in 4% formaldehyde fixative for use as pathological samples. The remaining samples were frozen at minus 80 degrees Celsius for molecular biological analysis. Stored.

[Table 1]

<Myeloperoxidase (MPO) analysis>

In 100 mg of colon tissue of the experimental animal, 200ul lysis buffer was added for homogenization. After centrifugation for 15 minutes at 13000rpm at 4 degrees Celsius, the supernatant was used for MPO analysis.

MPO in colon tissue was analyzed using a Mouse MPO assay ELISA kit (Hbt HK210, USA). 100ul homogenate of colon tissue was put in a 96 well plate and reacted at room temperature for 1 hour. After the reaction was completed, the plate was inverted and emptied, and then the washing process was repeated three times using 200 ul of washing buffer. Then, 100 ul of diluted tracer was added and reacted at room temperature for 1 hour. After the reaction was completed, the plate was turned upside down, and each well was washed with 200 ul of wash buffer. After washing three times using 200ul of washing buffer, 100ul of diluted streptavidin-peroxidase conjugate was added and reacted for 1 hour at room temperature. After the reaction was completed, the plate was turned upside down, and each well was washed with 200 ul of wash buffer. The washing process was repeated three times using 200ul of washing buffer, and then 100ul of TMB substrate solution was added, and the plate was reacted for 30 minutes at room temperature by blocking light with aluminum foil. The reaction was stopped by adding 100ul of stop solution and the absorbance was measured at 450nm using ELISA reader. The results are shown in Table 2.

[Table 2]

Samples were prepared in Examples 1-1. To 1-10. The sulfasalazine was purchased from Sigma, USA.

From the above results, it can be seen that the soybean, bean sprouts, dodol, Haedongpi, Deodeok extract, and / or fermented products have an excellent colitis improvement effect. In addition, saponins, soyasaponin I, Ab, soyasapogenol A, B, gypenoside, TN-2, kalopanaxsaponin H, B, A which are saponins isolated from the natural products , Lancemaside X, echinocystic acid and the like showed excellent effects.

1-13. Confirmation of colitis inhibition effect of Soyasaponin I

In order to confirm the inhibitory effect of soyasaponin I on colitis, animal experiments were conducted in the same manner as in 1-12. Except that the specimens of the ingredients and contents shown in Table 3 were used. The results are shown in the following table. In the table, NOR means normal group.

[Table 3]

Compared with the colitis model animals, the soyasaponin I group significantly inhibited myeloperoxidase (MPO) activity, which is an indicator of long length, appearance and inflammatory markers. It is more effective than sulfasalazine, which is currently used as a colitis treatment drug.

In addition, Soyasaponin I was tested in the following method to determine whether the inflammatory markers iNOS, COX-2 expression and transcription factor NF-kB activation is inhibited.

100 mg of colonic tissues of the test animals were homogenized by adding 250 ul of RIPA (Radio Immunoprecipitation assay) buffer containing a protease inhibitor cocktail. After centrifugation for 15 minutes at 4 degrees Celsius, 13000 rpm, the supernatant was stored at minus 80 degrees Celsius, while iNOS, COX2, p65 (NF-kappa B), pp65 (phosphor-NF-kappa B), beta-actin Was measured by Western blotting analysis. All antibodies were purchased from SantaCruz Biotechnology (USA).

The results are shown in Fig. 1 is a Western blotting assay confirming the inhibitory effect of soyasaponin I inflammatory markers (iNOS, protein expression of COX-2 and NF-kB activation (pp-65)) in TNBS-induced colitis model animals, NOR is normal Group, TNBS is a group administered only TNBS, S10 means soyasaponin I 10mg / kg administration group, S20 means soyasaponin I 20mg / kg administration group, SUL means Sulfasalazine administration group.

As shown in FIG. 1, iNOS, COX-2 expression and transcription factors were significantly higher in the group receiving soyasaponin I (S10, soyasaponin I 10 mg / kg; S20, soyasaponin I 20 mg / kg) compared to colitis model animals. Activation of phosphorus NF-kB was inhibited. It showed a better effect than the sulfasalazine currently used as a drug.

In addition, the experiment was performed by the following method to determine whether Soyasaponin I inhibits the expression of inflammatory markers IL-1beta, IL-6 and TNF-alpha.

100 mg of colonic tissue of the test animal was homogenized by adding 250 ul of RIPA buffer containing a protease inhibitor cocktail. After centrifugation for 15 minutes at 4 degrees Celsius, 13000 rpm, the supernatant was stored at minus 80 degrees Celsius, and IL-1beta, IL-6, TNF-alpha was prepared in 96-well ELISA plate kits (Pierce Biotechology, Inc.). , Rockford, IL, USA). The results are shown in Fig. Fig. 2 is a graph of IL-1beta expression level (top), IL-6 expression level (middle), and TNF-alpha expression level graph (bottom). In each figure, the y-axis represents the expression amount (pg / ml), the NOR of the x-axis is normal group, TNBS is TNBS-only group, S10 is soyasaponin I 10mg / kg administration group, S20 is soyasaponin I 20mg / kg administration group, SUL Means Sulfasalazine administration group.

As shown in FIG. 2, IL-1beta, IL-6, significantly increased in the group administered soyasaponin I (S10, soyasaponin I 10 mg / kg; S20, soyasaponin I 20 mg / kg) compared to colitis model animals. Expression of TNF-alpha was inhibited.

1-14. Inhibition of Colitis by Kalopanaxsaponin A

In order to confirm the inhibitory effect of Kalopanaxsaponin A on colitis, animal experiments were conducted in the same manner as in 1-12. Except that the samples of the ingredients and contents shown in Table 4 were used. The results are shown in the following table.

[Table 4]

As a result, it was found that myeloperoxidase (MPO) activity, which is a long length, appearance status and inflammatory markers, was significantly inhibited in the group administered kalopanaxsaponin A compared to the colitis model animal. It showed a better effect than the sulfasalazine currently used as a drug.

In addition, experiments to determine whether Kalopanaxsaponin A inhibits the expression of inflammatory markers iNOS, COX-2 and activation of the transcription factor NF-kB were tested in the same manner as in 1-13.

The results are shown in Fig. 3 is Western blotting assay confirming the inhibitory effect of inflammatory markers (iNOS, protein expression of COX-2 and NF-kB activation (p-p65)) of kalopanaxsaponin A in colitis model animals induced by TNBS. Group, T means TNBS only, KSA10 means Kalopanaxsaponin A 10mg / kg administration group, KSA20 means Kalopanaxsaponin A 20mg / kg administration group, S means Sulfasalazine administration group.

As shown in FIG. 3, iNOS, COX-2 expression and transcription factors were significantly higher in the group administered Kalopanaxsaponin A (KSA10, Kalopanaxsaponin A 10 mg / kg; KSA20, Kalopanaxsaponin A 20 mg / kg) compared to colitis model animals. Activation of phosphorus NF-kB was inhibited. It showed a better effect than sulfasalazine (S, 50mg / kg) currently used as a drug.

In addition, experiments to determine whether Kalopanaxsaponin A inhibits the expression of inflammatory markers IL-1beta, IL-6 and TNF-alpha were tested in the same manner as in the method of 1-13.

The results are shown in FIG. Figure 4 is a graph of IL-1 beta expression, IL-6 expression, TNF-alpha expression. In the figure, the y-axis represents the expression level, the x-axis represents the IL-1beta expression level, IL-6 expression level, and TNF-alpha expression level, and the histograms corresponding to the respective inflammatory markers are normal from the left to the right, and TNBS. Only group, Kalopanaxsaponin A 10mg / kg group, Kalopanaxsaponin A 20mg / kg group, Sulfasalazine group are shown. In the figure, # means that there is a significant difference (p <0.05) compared to the normal group, * means a significant difference (p <0.05) compared to the colitis model animal group.

As shown in Figure 4, compared to the colitis model animals, Kalopanaxsaponin A (Kalopanaxsaponin A 10 mg / kg; Kalopanaxsaponin A 20 mg / kg) group was significantly higher than IL-1beta, IL-6, TNF-alpha Expression was inhibited.

Therefore, it can be seen from the above results that the treatment (improved) or prevention of colitis by the active ingredient included in the composition of the present invention.

1-15. Inhibition of colitis by lancemaside X, echinocystic acid 3- O -β-D-glucuronopyranoside (EAG) and Echinocystic acid

Echinocystic acid 28- O -β-D-xylopyranosyl (1 → 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl In order to confirm the inhibitory effect of colitis of ester (lancemaside X), echinocystic acid 3- O- β-D-glucuronopyranoside (EAG) and Echinocystic acid (EA), except that the samples of the ingredients and contents shown in Table 5 were used. Animal experiment was conducted in the same manner as -12. The results are shown in the table below.

[Table 5]

As a result, myeloperoxidase (MPO) activity, which is an indicator of long length, appearance, and inflammatory activity, was significantly inhibited in the group to which saponin-derived derivatives were administered compared to colitis model animals. It showed a better effect than sulfasalazine, which is currently used as a drug.

In addition, experiments to determine whether lancemaside X, EAG, and EA inhibit the expression of COX-2, which is an inflammation marker, and the activation of the transcription factor, NF-kB, were tested in the same manner as in 1-13.

The results are shown in Fig. Figure 5 is an experiment to investigate the inhibitory effect of lancemaside X, EAG and EA inflammation markers (protein expression of COX-2 and NF-kB activation (pp-65)) in TNBS-induced colitis model animals by Western blotting assay As a result, NOR was normal, TNBS was TNBS-only, LX10 and LX20 were lancemaside X 10 mg / kg, 20 mg / kg, EAG10 and EAG20, EAG 10 mg / kg, 20 mg / kg, respectively. EA10 and EA20 are EA 10mg / kg administration group, 20mg / kg administration group, S means Sulfasalazine administration group, respectively.

As shown in FIG. 5, COX-2 expression and activation of the transcription factor NF-kB were significantly inhibited in the group to which Saderin-derived derivatives were administered compared to colitis model animals.

It showed a better effect than sulfasalazine (SUL50, 50mg / kg) currently used as a drug.

In addition, experiments to determine whether lancemaside X, EAG and EA inhibit the expression of inflammatory markers IL-1beta, IL-6 and TNF-alpha were tested in the same manner as in 1-13.

The results are shown in FIG. 6 is an IL-1 beta expression graph (top), a TNF-alpha expression graph (middle), and an IL-6 expression graph (bottom). In each figure, the y-axis represents the expression level (pg / ml), the NOR on the x-axis is normal, CON is TNBS-only group, LX10 and LX20 are lancemaside X 10mg / kg, 20mg / kg, EAG10 and EAG20 administration group is EAG 10mg / kg administration group, 20mg / kg administration group, EA10 and EA20 is EA 10mg / kg administration group, 20mg / kg administration group, respectively, S means Sulfasalazine administration group. In the figure, # means that there is a significant difference (p <0.05) compared to the normal group, * means a significant difference (p <0.05) compared to the colitis model animal group.

As shown in FIG. 6, expression of IL-1beta, IL-6, and TNF-alpha was significantly suppressed in the group to which Saderin derived from Detox than the colitis model animal was administered.

Therefore, it can be seen from the above results that the treatment (improved) or prevention of colitis by the active ingredient included in the composition of the present invention.

Claims (8)

A pharmaceutical composition for treating or preventing colitis, comprising one or more saponins selected from soya saponin I, soya saponin Ab, soya sapogenol A, and soya sapogenol B as an active ingredient. The composition of claim 1, wherein the saponin is isolated from at least one natural product extract or fermentation product selected from soybeans and bean sprouts. The composition of claim 1, wherein the colitis is at least one selected from ulcerative colitis or Crohn's disease. Pharmaceutical composition for treating or preventing colitis comprising as an active ingredient at least one natural product extract or fermented product selected from soybeans and bean sprouts containing one or more saponins selected from Soya Saponin I, Soya Saponin Ab, Soya Saponogenol A, and Soya Saponogenol B . The composition of claim 4, wherein the fermented product is obtained by fermenting one or more selected from soybeans and bean sprouts or fermenting one or more extracts selected from soybeans and bean sprouts. The composition of claim 5, wherein the fermentation is lactic acid bacteria fermentation. Food composition for improving or preventing colitis, comprising one or more saponins selected from soya saponin I, soya saponin Ab, soya sapogenol A and soya sapogenol B as an active ingredient. Food composition for improving or preventing colitis comprising soy saponin I, soya saponin Ab, soya saponenol A, and soya saponenol B including at least one natural product extract or fermented product selected from soybeans and bean sprouts as an active ingredient .

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