KR100896325B1 - Kalopanaxsaponins K And Metabolite And Inflammatory And Arthritis Agent Containing The Same - Google Patents

Abstract

The present invention relates to carlophanax saponin K and its metabolites and agents for treating inflammation and arthritis containing the same. Specifically, the present invention relates to carlopanax saponin K extracted from thawed blood and metabolites produced by culturing it with human intestinal microorganisms, and to treating inflammation and arthritis containing the same as an active ingredient.

Carlopanax saponin, thawing blood, anti-inflammatory, arthritis treatment, intestinal microflora

Description

Carlopanax saponin Ｋ and its metabolites and therapeutic agents for inflammation and arthritis containing them {Kalopanaxsaponins K And Metabolite And Inflammatory And Arthritis Agent Containing The Same}             

1 is a graph showing the metabolism by intestinal microorganisms of Carlopanax saponin K over time (◇, Carlopanax saponin K; ◆, Carlopanax saponin J; ○, Carlopanax saponin H; ●, Carlopanax saponin I; □ , Carlopanax saponin B; ■, Carlopanax saponin A; ▲, Heatheragen 3-O-α-L-arabinopyranoside (HA); X, Heatherlagage Heinagenin (H)).

Figure 2 shows the metabolic pathway by the rose organism of carlopanax saponin K (bold solid line shows the major metabolic pathway is the secondary metabolic pathway).

The present invention relates to carlophanax saponin K and its metabolites and agents for treating inflammation and arthritis containing the same. Specifically, the present invention relates to carlopanax saponin K extracted from thawed blood and metabolites produced by culturing it with human intestinal microorganisms, and to treating inflammation and arthritis containing the same as an active ingredient.

Thawing blood is a folk medicine that has been used to treat neuralgia, arthritis and diabetes (Sano K. et al ., Chem. Pharm. Bull., 39, 865-870, 1991). Haedongpi is the dried bark of the Araliaceae tree ( Kalopanax pictus Nakai). This herb contains several species of carlophanaxa saponin, liriodendrin, syringin, coniferylaldehyde (Sano K. et al ., Chem. Pharm. Bull., 37, 311-314, 1989). In particular, carlopanax saponin is a structure in which sugars are bound to C-3 and C-28 positions using hederagen as a mother nucleus, and delta-hederin (δ- hedrin), kalopanaxsaponin A (KPA), I (KPI), and J (KPJ). Saponin B (KPB), H (KPH), K (KPK), etc. have been reported (Lee M., Hahn DR, Arch. Pharm. Res., 14, 124-128, 1991). Conventionally known hederagenin, deltahederin, carlophanax saponin A, I, J, B, H are all sub-structures of carlopanax saponin K, the main component of thawing blood, which can be obtained by complete hydrolysis or partial hydrolysis. to be.

On the other hand, arthritis is a chronic disease that mainly produces persistent inflammation in the knee joint, most common between the 30's and 50's, and about three times more often in women. Arthritis patients are reported in developed countries as about 2.5 to 3% of the total population, and in Korea, about 1% (Park HJ et al. , Planta Med., 67, 118-121, 2001). There are many studies on pathophysiology and treatment methods of arthritis, and in particular, not only clinical studies of patients, but also experimental models of causing arthritis in experimental animals and applying various manipulations have been used. Kim DW et al. , Arch. Pharm. Res., 21, 688-691, 1998).

Drugs used to treat inflammatory diseases such as rheumatoid arthritis include nonsteroidal anti-inflammatory drugs such as aspirin and corticosteroids (Kim DW et al. , Biol. Pharm. Bull., 21, 360-). 365, 1998). However, nonsteroidal drugs such as aspirin have excellent medicinal effects, and their use is extremely limited due to side effects such as severe ulcerative effects expected during long-term use. Dexamethasone is known to have the best anti-inflammatory effect of corticosteroids, but it is currently prohibited for use as a side effect (Park HJ et al. , Arch. Pharm. Res., 21, 24-29, 1998) .

Therefore, there is an urgent need for the development of new drugs for treating inflammatory diseases such as rheumatoid arthritis.

The inventors of the present invention, while studying the metabolic pathways of carlopanax saponin K contained in the thawed blood anaerobic culture of carlopanax saponin K with the intestinal microorganisms of the human body, carlopanax saponin K is either carlopanax saponin H or carlopanax saponin After being metabolized to J it was finally found to be metabolized to Carlophanax saponin I, Carlopanax saponin A or Hederagenin. In addition, the last metabolites, carlophanax saponin A and carlopanax saponin I, were found to show significant activity in the inhibition of edema and anti-inflammatory effects of vascular permeability.

Therefore, in one aspect, the present invention is to extract the solvent by extracting the thawed blood, and to separate the extract into an aqueous layer and an oil layer to obtain each fraction, which is silica gel column chromatography, each fraction chromatography It provides a method for preparing Carlophanax saponin K, Carlopanax saponin J, Carlopanax saponin A or Carlopanax saponin I, characterized in that the purified by.

In another aspect, the present invention is mixed with carlopanax saponin K or carlopanax saponin J and human feces, and then cultured under anaerobic conditions, the solvent fractionation of the culture, concentrated fractions under reduced pressure, and concentrate the silica gel column Provided is a method for preparing Carlophanax saponin A or Carlopanax saponin I using a human intestinal microorganism characterized by chromatography.

In another aspect, the present invention provides an anti-inflammatory agent containing an effective amount of any one or two or more selected from Carlophanax saponin K, Carlopanax saponin J, Carlopanax saponin A, or Carlopanax saponin I.

In another aspect, the present invention provides a therapeutic agent for arthritis containing an effective amount of any one or two selected from carlophanax saponin K, carlopanax saponin J, carlopanax saponin A, or carlopanax saponin I.

The native plant of Kalopanax pictus Nakai refers to the cypress tree of the Agapiaceae . Yin-tree is a plant native to Korea, but is imported and used in considerable quantities. This drug is long or semitubular, 5-30 cm long, 1-3 cm wide, 1-3 mm thick. This medicine is odorless and tastes a bit bitter. In one room, the temper was written, and the dementia was called liver and god.

Thawing blood is a major component of carlophanax saponins of Bisdesmoside such as carlophanax saponins H, B and K. However, they are not absorbed by the intestine upon oral administration and come into contact with the intestinal bacteria, which are converted to monodermoside, carlophanax saponin A, I and non-saccharide hederagenin. The present inventors observed that when the human intestinal microorganism, Eubacterium A-44, is involved in metabolism, the main metabolite is carlophanax saponin A. Carlophanax saponin K, B, and H are naturally derived from carlopanax saponin A and I. It confirmed that it became a precursor.

Carlophanax saponin is represented by the following structural formula, Carlopanax saponin K and its metabolites are represented by the following formula.                     

In one aspect, the present invention is to extract the extract by solvent extraction of the thawed blood, and to separate the extract into an aqueous layer and an oil layer to obtain each fraction, which is purified by silica gel column chromatography, each fraction purified by chromatography It provides a method for producing Carlopanax saponin K, Carlopanax saponin J, Carlopanax saponin A and Carlopanax saponin I characterized by the above.

Thawed blood can be purchased and used commercially. Preferably, it is dried in the shade and then finely pulverized the dry sample.

The solvent used for the extraction may be alcohol, preferably methanol.

The fractionation step of the extract is preferably, the alcohol extract is concentrated, water is added thereto, ethyl acetate is added to separate the aqueous layer and the ethyl acetate layer, and the aqueous layer is n-butanol added to extract the active ingredient. Ethyl acetate layer or n-butanol layer can increase the extract yield of the active ingredient by adding each solvent in addition to the aqueous layer. The fractions are each concentrated in a vacuum concentrator.

Each fraction obtained above was subjected to silica gel column chromatography to obtain a fraction, which was purified by Sephadex or ODS column chromatography to purify Carlophanax saponin K, Carlopanax saponin J, Carlopanax saponin A and Carlopanax saponin I. Can be obtained.

In another aspect, the present invention, after mixing carlophanax saponin K or carlopanax saponin J and human feces and incubating them under anaerobic conditions, after the solvent fractionation of the culture, the fractions are concentrated under reduced pressure, the concentrate is silica gel column chromatography Provided is a method for preparing Carlophanax saponin A or Carlopanax saponin I using human intestinal microorganisms characterized by grading.

Human feces and carlophanax saponins are mixed in a weight ratio of 1 to 10: 1, preferably in a weight ratio of about 5: 1. The mixture is incubated at 30 to 40 ° C. for 10 to 48 hours under anaerobic conditions.

When the incubation is completed, the culture is subjected to solvent fractionation with ethyl acetate, and the ethyl acetate fraction obtained by concentration under reduced pressure is purified by silica gel column chromatography to separate and purify Carlophanax saponin A or Carlopanax saponin I. As the developing solvent used for the column chromatography, CHCl ₃ -MeOH-H ₂ O (7: 3: 1, lower layer) can be preferably used.

In a particular embodiment, the present invention is incubated with carlopanax saponin K or carlopanax saponin J and human intestinal microorganisms under anaerobic conditions, followed by solvent fractionation of the culture, concentration of the fractions under reduced pressure, and concentration of the silica gel column chromatography. It provides a method for producing Carlophanax saponin A or Carlopanax saponin I characterized in that.

The human intestinal microorganisms include Bacteroides HJ15, Bacteroides JY6, Bifidobacterium microorganisms ( Bifidobacterium K103, Bifidobacterium K110), Eubacterium microorganisms ( Eubacterium A44, Eubacterium L8) Genus microorganisms ( Fusobacterium K60), Lactobacillus microorganisms ( Lactobacillus L2), Streptococcus microorganisms ( Streptococcus S2, Streptococcus S10).

In another aspect, the present invention provides an anti-inflammatory therapeutic agent containing Carlophanax saponin K, Carlopanax saponin J, Carlopanax saponin A or Carlopanax saponin I with a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a therapeutic agent for arthritis containing an effective amount of carlophanax saponin K, carlopanax saponin J, carlopanax saponin A or carlopanax saponin I.

Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-blocking polymers, polyethylene glycols and wool, and the like.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal.

The term “therapeutically effective amount” refers to a dose level between the minimum effective amount and the maximum effective amount (ie, the extreme amount) for use in the treatment of the condition in humans.

The term "prophylactically effective amount" refers to a dose level of about 0.1 mg to about 5 mg per kg body weight per day for use in the prevention of such symptoms in humans.

In addition, carlopanax saponin K and J according to the present invention and the metabolites of the human intestinal microorganisms carlopanax A and I can be used in food, beverages, tea and other dietary supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1

Preparation of Samples of Thawing Blood and Preparation of Alcohol Extracts

Haedongpi ( Kalopanax pictus Nakai) was purchased from the market and the specimens used were stored in the Department of Plant Resources at Sangji University. The thawed blood used in the experiment was dried in the shade, and the dried sample was pulverized finely to be used as an extraction material.

The dried pulverized thawed skin (dry weight, 4.8 kg) was placed in a 5 liter round flask, and methanol as an extraction solvent was added to sufficiently cover the sample. A reflux cooler was mounted and the active ingredient was extracted by heating using a heating mantle for 5 hours. The methanol extract was filtered using filter paper (Watman 1, Wattman), then the filtrate was taken and extracted twice more with methanol in the same manner. The filtrate obtained through the above process was concentrated under reduced pressure using a rotary vacuum concentrator while warming in a 40 ° C water bath. As a result, 613 g of methanol extract was obtained.

Example 2

Fraction of extract

613 g of the methanol extract obtained in Example 1 were suspended in 1000 ml of distilled water. The solution was placed in a separatory funnel in a 2 L container, 800 ml of chloroform (Chloroform) was added thereto, and then shaken vigorously for about 5 minutes and left at room temperature for 3 hours. The chloroform layer located in the lower layer was taken, and the remaining aqueous layer was extracted twice with chloroform in the same manner. 800 ml of ethyl acetate was added to the aqueous phase and fractionated three times in the same manner as in the chloroform fraction. The upper ethyl acetate layer was collected and stored in another container. 800 ml of n-butanol was added to the aqueous phase and fractionated three times in the same manner as in the chloroform and ethyl acetate fractions. The upper n-butanol layer was collected and stored in another container. The fraction samples obtained through the fractionation process were separately concentrated in a vacuum concentrator to obtain 220 g of chloroform fraction, 30 g of ethyl acetate fraction and 230 g of butanol fraction.

Example 3

Isolation of Carlopanax saponin

① Separation of saponin from n-butanol fraction

A column with an inner diameter of 5 cm was fixed vertically to the stand and the bottom part of the column was closed with cotton. 500 L of pre-developed developing solvent chloroform-methanol-water (65:35:10, lower layer) and 600 g of silica gel (Merck, Art 7734, Germany) were suspended in an Erlenmeyer flask, and then quickly added to the column and the bottom end of the column Was opened and the developing solvent was removed. Using this, all remaining silica gel in the Erlenmeyer flask was washed and filled in the column. Elution was made until the developing solvent was on top of the silica gel and the column was capped.

In order to adsorb the sample onto silica gel, 15 g of butanol fraction prepared in Example 2 was mixed well with 100 ml of methanol to dissolve the fraction, and 10 g of silica gel was added thereto, and then the solvent was evaporated while warming to 50 ° C. in a vacuum concentrator. The dried powdery product obtained was added to mortar and finely ground with pestle. This was carefully loaded onto a preprepared column using a spoon. Aliquots of 50 ml each continued to obtain 100 fractions.

All obtained fractions were purified by Sephadex LH-20 or ODS column chromatography and eluted by thin layer chromatography (TLC) (TLC plate; Siliga gel 60F ₂₅₄ (1.05715), Merck). TLC was by the method described in the manufacturer's manual. UV spots, buried in 10% sulfuric acid and burned to identify the spots. The white needle formed when the fractions 20 to 35 were concentrated and dissolved in methanol, and was identified as carlophanax saponin B by spectroscopic analysis (yield: 1.9 g). 50 to 90 fractions (retention volume, 2.5 L-4.5 L) containing carlopanax saponin H and carlopanax saponin K were combined and concentrated, and then separated by column chromatography.

50 g of filler octadecylsilane (ODS) was added to the column having a diameter of 3 cm by mixing with a developing solvent (methanol-water (10: 2)). The solvent was slowly withdrawn and the stopper closed when the developing solvent came to the top of the column stop. Samples for separation were dissolved in 5 ml of the same developing solvent and then loaded onto the column. While developing the ODS column chromatography, a sufficient amount of developing solvent was injected when the sample solution came to the top of the stationary phase. The eluate was aliquoted in 10 ml portions to obtain 50 fractions. Eight fractions were TLC in the same manner as above and spots were identified by UV irradiation. The 12th to 26th fractions were concentrated and dissolved in methanol to yield a white amorphous powder that was produced. Spectrophotometric analysis confirmed as carlopanax saponin K (yield: 1.2 g). Fractions 50 to 68 were concentrated and left to dissolve in methanol. When the white amorphous powder produced at this time was filtered off, the compound carlophanax saponin H was obtained (amount: 1.3 g).

② Separation of Saponin from Ethyl Acetate Fraction

10 g of ethyl acetate fractions were subjected to silica gel column chromatography in the same manner as described above, except that the developing solvent was fractionated in 50 ml portions to 70 fractions. Among them, the fractions 22 to 57 containing saponin were concentrated and purified two times more using silica gel column chromatography and Sephadex LH-20 under the same solvent conditions. Carlophanax saponin A (yield: 1.2 g) and Carlophanax saponin I (amount: 650 mg) were obtained.

③ Preparation and Separation of Prosapogenin from Partial Hydrolysis of Saponin

20 g of thawed skin n-butanol fractions were dissolved in 2% -NaOH (prepared as a water: methanol = 8: 2 solution) and stirred at 80 ° C. for 50 minutes. The reaction solution was acidified with the same amount of HCl, washed with distilled water, and extracted with saturated n-butanol. The n-butanol fraction was concentrated under reduced pressure and silica gel column chromatography was carried out in the same manner as above using chloroform-methanol-water (7: 3: 1, lower layer) as a developing solvent. 50 ml aliquots were obtained in all 150 fractions and confirmed by TLC. 15 to 28 fractions were concentrated and column chromatography was carried out under the same conditions, and then recrystallized from MeOH to obtain hederin (HA, yield: 65 mg), and fractions 39 to 59 were concentrated to the same amount. Column chromatography and MeOH recrystallization under conditions yielded Carlophanax saponin A (amount: 1.8 g). In the same manner, fractions 75 to 90 were purified by separation to obtain carlophanax saponin H (yield: 54 mg), and fractions 102 to 130 were purified by separation to obtain carlophanax saponin J (yield: 1.2 g). .

④ Isolation of sapogenin from complete hydrolysis of saponin

10 g of n-butanol fraction was heated to 60 ° C. under reflux cooling with a 3% -HCl (MeOH: H ₂ O = 2: 8) solution for about 5 hours to undergo a complete hydrolysis reaction. The reaction was terminated, extracted three times with ethyl acetate, washed with distilled water and dehydrated with Na ₂ SO ₄ . This was separated by chloroform-methanol-water (7: 3: 1, lower layer) as a developing solvent by silica gel column chromatography, and recrystallized with MeOH to obtain 1.9 g of hederagenin (H).

Example 4

Structure Identification of Carlopanax saponin

Melting points of the three compounds separated were measured using a Thermoelectric Melting Point Apparatus (Electrothermal). The fluorescence was measured by the manufacturer's manual method using a Rudolph Autopol III automatic polarimeter. IR spectra were measured by KBr disk method using FT-IR spectrometer (Bomem MB-100 FT-IR spectrometer), and mass spectra were measured by Finnigan Mat TSQ-700. ¹ H- and ¹³ C-NMR spectra were measured on a spectrometer (Bruker AM-500 Spectrometer) with the addition of the internal standard tetramethylsilane (TMS). UV absorbance was measured by UV-160A UV-visible Recording Sepctrophotometer (Shmadzu). At this time, the wavelength and absorbance at the absorption maximum were read and the log ε value was calculated and recorded. Carlopanax saponin K, Carlopanax saponin J, Carlopanax saponin A and Carlopanax saponin I were identified by the above method.

The experimental results are as follows.

Carlophanax saponin K; Amorphous white powder

mp 217-219 ℃

IR V ^KBr _max cm ⁻¹ ; 3417 (broad, OH), 1729 (C = O), 1052 (O-CO)

¹ H-NMR (300 MHz, pyridine-d ₅ ): 5.38 (1H, brs, H-12), 0.95 (3H, s, H-24), 1.08 (3H, s, H-25), 1.08 (3H , s, H-26), 1.15 (3H, s, H-27), 0.85 (3H, s, H-29), 0.83 (3H, s, H-30), 5.05 (1H, d, J = 6.6 Hz, anomeric proton of 3-O-ara), 6.29 (brs, rhamnose anomeric proton of 3-O-ara-rham), 5.25 (1H, d, J = 7.7 Hz, brs, xylose anomeric proton of 3-O- ara-rham-xyl), 4.99 (1H, d, J = 7.9 Hz, brs, glucose anomeric proton of 3-O-ara-rham-xyl-glc), 6.23 (1H, d, J = 8.1 Hz, anomeric proton of 28-O-glc), 4.97 (1H, d, J = 7.7 Hz, glu anomeric proton of 28-O-glc-glc), 5.81 (1H, brs, rham anomeric proton of 28-O-glc-glc- rhamnose).

¹³ C-NMR (75.5 MHz, pyridine-d ₅ ): aglycone-38.9, 25.9, 81.0, 43.4, 47.5, 18.3, 32.6, 39.8, 48.3, 36.8, 23.7, 122.9, 144.0, 42.0, 28.2, 23.2, 46.1, 41.5, 46.1, 30.6, 33.9, 32.5, 64.0, 14.0, 16.6, 17.4, 25.9, 176.4, 33.0, 23.7, sugars at C-3-104.8, 75.3, 75.0, 69.2, 64.8, 101.3, 71.8, 83.1, 72.5, 69.5, 18.4, 107.1, 75.1, 76.3, 77.9, 66.0, 103.6, 75.2, 78.7, 72.9, 78.1, 62.6, sugar at C-28-95.6, 73.9, 78.3, 70.8, 77.1, 69.6, 104.5, 74.2, 76.5, 78.7, 78.0, 61.3, 102.7, 72.7, 73.7, 73.9, 70.3, 18.5.

Carlophanax saponin J; Amorphous white powder

mp 208-210 ° C

[α] _D -8.10

IR v ^KBr _max cm ^-1 ; 3400 (broad, OH), 1690 (C = O), 1050 (O-CO)

¹ H-NMR (300 MHz, pyridine-d ₅ ): 5.38 (1H, brs, H-12), 0.95 (3H, s, H-24), 1.08 (3H, s, H-25), 1.08 (3H , s, H-26), 1.15 (3H, s, H-27), 0.85 (3H, s, H-29), 0.83 (3H, s, H-30), 5.05 (1H, d, J = 6.6 Hz, anomeric proton of 3-O-ara), 6.29 (brs, rhamnose anomeric proton of 3-O-ara-rham), 5.25 (1H, d, J = 7.7 Hz, brs, xylose anomeric proton of 3-O- ara-rham-xyl), 4.99 (1H, d, J = 7.9 Hz, brs, glucose anomeric proton of 3-O-ara-rham-xyl-glc), 6.23 (1H, d, J = 8.1 Hz, anomeric proton of 28-O-glc), 4.97 (1H, d, J = 7.7 Hz, glu anomeric proton of 28-O-glc-glc), 5.81 (1H, brs, rham anomeric proton of 28-O-glc-glc- rhamnose).

¹³ C-NMR (75.5 MHz, pyridine-d ₅ ): aglycone-38.9, 25.9, 81.0, 43.4, 47.5, 18.3, 32.6, 39.8, 48.3, 36.8, 23.7, 122.9, 144.0, 42.0, 28.2, 23.2, 46.1, 41.5, 46.1, 30.6, 33.9, 32.5, 64.0, 14.0, 16.6, 17.4, 25.9, 176.4, 33.0, 23.7, sugars at C-3-104.8, 75.3, 75.0, 69.2, 64.8, 101.3, 71.8, 83.1, 72.5, 69.5, 18.4, 107.1, 75.1, 76.3, 77.9, 66.0, 103.6, 75.2, 78.7, 72.9, 78.1, 62.6, sugar at C-28-95.6, 73.9, 78.3, 70.8, 77.1, 69.6, 104.5, 74.2, 76.5, 78.7, 78.0, 61.3, 102.7, 72.7, 73.7, 73.9, 70.3, 18.5.

Carlophanax saponin A; Amorphous white powder

mp 265-268 ℃

[α] _D + 18 °

IR v ^KBr _max cm ^-1 ; 3415 (broad, OH), 1698 (C = O), 1058 (O-CO)

¹ H-NMR (300 MHz, pyridine-d ₅ ): 5.46 (1H, brs, H-12), 1.09 (3H, s, H-24), 0.90 (3H, s, H-25), 0.97 (3H , s, H-26), 1.21 (3H, s, H-27), 0.91 (3H, s, H-29), 0.98 (3H, s, H-30), 5.06 (1H, d, J = 6.6 Hz, anomeric proton of 3-O-ara), 6.26 (brs, rhamnose anomeric proton of 3-O-ara-rham), 5.26 (1H, d, J = 7.7 Hz, brs, xylose anomeric proton of 3-O- ara-rham-xyl), 4.98 (1H, d, J = 7.9 Hz, brs, glucose anomeric proton of 3-O-ara-rham-xyl-glc).

¹³ C-NMR (75.5 MHz, pyridine-d ₅ ): aglycone-38.9, 26.0, 81.1, 43.4, 47.7, 18.0, 32.8, 39.7, 48.1, 36.8, 23.6, 122.5, 144.1, 42.0, 28.2, 23.7, 46.5, 41.9, 46.5, 30.8, 34.1, 33.2, 64.0, 13.8, 16.0, 17.4, 26.0, 180.4, 33.2, 23.7, sugars at C-3-104.4, 75.3, 74.2, 69.4, 65.9, 101.4, 71.7, 83.1, 71.8, 69.5, 18.3, 107.5, 74.8, 76.2, 77.9, 64.7, 103.5, 75.2, 78.1, 72.9, 78.7, 62.6.

Carlophanax saponin I; Amorphous white powder

mp 218-220 ° C

[α] _D +11

IR v ^KBr _max cm ^-1 ; 3417 (broad, OH), 1695 (C = O), 1050 (O-CO)

¹ H-NMR (300 MHz, pyridine-d ₅ ): 5.46 (1H, brs, H-12), 1.09 (3H, s, H-24), 0.90 (3H, s, H-25), 0.97 (3H , s, H-26), 1.21 (3H, s, H-27), 0.91 (3H, s, H-29), 0.98 (3H, s, H-30), 5.06 (1H, d, J = 6.6 Hz, anomeric proton of 3-O-ara), 6.26 (brs, rhamnose anomeric proton of 3-O-ara-rham), 5.26 (1H, d, J = 7.7 Hz, brs, xylose anomeric proton of 3-O- ara-rham-xyl), 4.98 (1H, d, J = 7.9 Hz, brs, glucose anomeric proton of 3-O-ara-rham-xyl-glc).

¹³ C-NMR (75.5 MHz, pyridine-d ₅ ): aglycone-38.9, 26.0, 81.1, 43.4, 47.7, 18.0, 32.8, 39.7, 48.1, 36.8, 23.6, 122.5, 144.1, 42.0, 28.2, 23.7, 46.5, 41.9, 46.5, 30.8, 34.1, 33.2, 64.0, 13.8, 16.0, 17.4, 26.0, 180.4, 33.2, 23.7, sugars at C-3-104.4, 75.3, 74.2, 69.4, 65.9, 101.4, 71.7, 83.1, 71.8, 69.5, 18.3, 107.5, 74.8, 76.2, 77.9, 64.7, 103.5, 75.2, 78.1, 72.9, 78.7, 62.6.

Example 5

Metabolite Extraction, Isolation, and Structural Identification of Carlopanax saponin K or Carlopanax saponin J

In order to prepare metabolites by the intestinal microorganisms of carlopanax saponin K or J, a total volume of 500 ml was prepared by mixing 2 g of fresh stool of 20-year-old adult male with 0.4 g of carlopanax saponin K or J. Incubated for 24 hours in anaerobic conditions. Carlopanax saponin K or J was reacted with ethyl acetate and concentrated under reduced pressure. The ethyl acetate fraction was purified by silica gel column chromatography with CHCl ₃ -MeOH-H ₂ O (7: 3: 1, lower layer). .

The analysis of carlopanax saponin K, H, B, J, I, A, HA, hederagenin was performed on silica gel TLC plates (Merck, silica gel 60F ₂₅₄ ) with CHCl ₃ -MeOH-H ₂ O (65:35:10 , Lower layer) was carried out with a solvent. For analysis, 5% -H ₂ SO ₄ was sprayed, heated at 110 ° C. for 15 minutes, and quantitated by 0.5 to 10 μg with a TLC scanner. Isolated compounds were identified in their respective structures by synthesizing and comparing known isolated materials and physicochemical data. The experimental results are shown in Table 1.

Metabolism by Carlophanax saponin K and J by Human Intestinal Microorganisms ( Bacteroides sp.) Reactant KPK KPJ Metabolite KPH KPB KPJ KPI KPA HA H KPI KPA HA H Generation amount (μM) 32.3 7.70 <0.05 25.31 7.39 0 6.16 78.58 8.70 0 6.16

KPK: Carlopanax saponin K, KPJ: Carlopanax saponin J

KPH: Carlophanax saponin H, KPB: Carlophanax saponin B,

KPI: Carlophanax saponin I, KPA: Carlophanax saponin A

HA: Heatherlagenin 3-O-α-L-arabinofyranoside

H: headeragenin (H)).

Example 6

Metabolites Change over Time by Microorganisms of Carlopanax saponin K or J in Human Intestine

In order to observe the change over time of the metabolites of carlopanax saponin K or J of Example 5, this carlopanax saponin K or J 0.6 mM was mixed with 0.1 g of fresh stool of a 20 year old adult male to a total volume of 20 ml. After incubation for 24 hours at 37 ℃ anaerobic conditions. 1 ml of the reaction was taken over time, extracted twice with 5 ml of ethyl acetate, and the components of the matrix and its metabolites were analyzed by TLC. The experimental results are shown in Table 2.

Hourly Metabolism by Carlopanax saponin K and J by Microorganisms in the Human Intestine Reactant KPK Metabolite KPK KPH KPB KPJ KPI KPA HA H   Generation amount (μM) 0 hours 120 0 0 0 0 0 0 0 5 hours 5 64 One 3 53 0 0 0 10 hours 5 19 3 12 83 10 0.2 0 15 hours 5 0.3 0.3 2 75 21 0.5 30 20 hours 5 0.3 0.3 3 72 15 0.5 30 24 hours 5 0.3 0.3 6 77 14 0.5 30

Example 7

Screening of Enteric Microorganisms to Metabolize Carlopanax saponin K or J

Screening was performed for enteric microorganisms that metabolize carlopanax saponin K or J. Candidate's stool was diluted 10 ^{5-10 8} fold in GAM medium (Nissui Pharmaceutical Co. ltd.) And some were inoculated in GAM agar plates and incubated for 4 days under anaerobic conditions at 37 ° C. The resulting colonies were separately isolated and these colonies were incubated for one day in GAM medium containing 0.4% Carlopanax saponin K or J, respectively. The metabolite was solvent extracted with 5 ml of ethyl acetate and concentrated under reduced pressure and analyzed by TLC.

As a result of the experiment, as shown in Table 3, the intestinal microorganisms that metabolize carlophanax saponin K or J are Bacterides microorganism, Bifidobacterium microorganism, Eubacterium microorganism, Fusobacterium microorganism, Lactobacillus genus Microorganisms and microorganisms of Streptococcus genus were screened.

Human intestinal microorganisms that metabolize carlopanax saponin K or J microbe Metabolite (μM) KPK KPJ KPH KPB KPJ KPI KPA HA H KPI KPA HA H Bacteroides HJ15 0 2.13 1.43 0.78 0.68 0 5.03 0 1.99 0.93 7.0 Bacteroides JY6 32.30 7.70 <0.05 25.31 7.39 0 6.16 78.58 8.70 0 6.16 Bifidobacterium K103 2.79 1.31 0.96 1.37 1.62 0 3.04 6.86 2.42 0 4.46 Bifidobacterium K110 2.79 2.02 0.90 0.78 1.37 0 0 <0.05 0.81 0 0 Eubacterium A44 2.79 4.15 4.28 0 0.99 0 0.21 <0.05 5.78 0.93 4.46 Eubacterium L8 0 0 <0.05 2.29 1.24 0 3.33 <0.05 0 0 11.54 Fusobacterium K60 2.79 0.82 0.83 0 1.55 0.81 4.46 <0.05 0.99 0.75 4.74 Lactobacillus L2 0 0 <0.05 2.10 7.02 0.75 0 <0.05 2.42 1.12 0 Streptococcus S2 0 0 0.83 3.07 6.77 1.18 0 11.69 0 0.81 0 Streptococcus S10 0 0 <0.05 1.61 2.80 0 0 <0.05 0.99 0 0

Example 8

Anti-inflammatory Effects of Carlopanax saponin K Metabolites

Anti-inflammatory effects were confirmed by investigating changes in acetic acid-induced vascular permeability according to Whittle's method (Winter CA, Risley EA and Nuss GW, Exp. Biol. Med., 111, 544, 1962). Animals used in the experiment were collected from Korea Experimental Animal Development and reared for 2 weeks under constant conditions (temperature: 20 ± 2 ℃, humidity: 40-60%, contrast: 12 hours light / dark cycle). ICR male mice weighing 20-25 g and Sprague-Dawley male rats weighing 200-220 g were used. The experimental animals were supplied with a standard amount of food and water provided by Samyang Corporation.

The mice were intraperitoneally administered with Carlopanax saponin A or I. After 30 minutes, 0.1 ml / 10 g of 4% pontamine sky blue solution was injected into the tail vein of the rat. After 15 minutes, 0.5% histamine was administered intraperitoneally. After 20 minutes, the mice were killed and the pigments leaked into the abdominal cavity were collected and washed with 10 ml of distilled water. The amount of dye in the washing solution was measured at 570 nm using a spectrophotometer.

As a result, as shown in Table 4, the vascular permeability inhibition rate was significantly increased significantly in a concentration-dependent manner compared to the control group not administered with carlopanas saponin. This activity appeared in a small dose (20 mg / kg) of 1/5 of the aminopyrine (100 mg / kg) anti-inflammatory agent, and the vascular permeability inhibition rate was about 60% of the aminopyrine activity. These results indicate that the metabolites of carlopanax saponin according to the present invention, in particular, the major metabolites, carlopanas saponin A and I, have very good anti-inflammatory effects.

Inhibition of Vascular Permeability of Carlopanax saponin A and I group Dose (mg / kg) Permeability (Amount of Pigment, μg) Permeability inhibition rate (%) Normal - 106.7 ± 10.05 ^a - Control - 213.6 ± 14.53 ^b 0.0 KPA 5 189.9 ± 11.98 ^{b, c} 11.1 10 156.0 ± 9.95 ^{d, e} 27.0 20 150.9 ± 10.04 ^e 29.4 KPI 5 200.1 ± 13.91 ^b 6.3 10 186.9 ± 9.85 ^{b, c} 12.5 20 173.2 ± 9.85 ^{b, c} 18.9 Aminopyrine 100 109.9 ± 12.36 ^f 48.5

Values are mean ± SD (n = 10). Values of similar letters mean that they are not significantly different from the control. Inhibition rate values represent the average value of the inhibitory effect compared to the control

Example 8

Anti-inflammatory activity against edema of carlopanax saponin K metabolites

Anti-inflammatory action on the edema of carlopanax saponin K metabolites is described in Saito et al. (Saito B., Ohashi T., Togashi M., Kayanagi T., Nippon Hinyokika Gakkai Zasshi, 81, 993-996, 1990). To induce arthritis in the mice used in Example 7, 0.05 ml of Freund's complete adjuvant (FCA) reagent was injected into the right foot of the mouse. After 2 weeks, it was confirmed that edema was induced. Carlopanax saponin A or I was dissolved in physiological saline, and the arthritis was injected intraperitoneally at 5, 10, 20 mg / ml / day for 3, 5, 7, 10 days, and the resulting edema was suppressed according to the volumetric method. The activity was measured by obtaining Carlophanax saponin A (KPA) and Carlopanax saponin I (KPI) were administered intraperitoneally once a day over the duration of each experimental animal group.

As a result, as shown in Table 5, it was confirmed that the metabolite of carlopanas saponin K inhibited edema in a dose-dependent manner. Carlopanax saponin A significantly reduced edema in the two high dose groups 5 days after administration. When carlopanax saponin I was administered at 10 mg / kg or more, mild but edema inhibition was observed. Carlopanax saponin A and I were shown to be more effective as the dose was increased, and the effect of suppressing edema was increased even when the administration period was extended.

Edema Inhibitory Effect of Carlopanax saponin A and I group Dose (mg / kg) Edema rate (%) 0 3 5 7 10 Control 74.4 ± 5.58 ^{a, b} 78.6 ± 4.39 ^a 75.9 ± 3.27 ^{a, b} 71.3 ± 4.92 ^{a, b} 74.2 ± 5.49 ^{a, b} KPA 5 - 80.6 ± 11.3 ^b 66.5 ± 3.57 ^{c, d, e} 63.3 ± 2.82 ^{e, f} 61.8 ± 1.44 ^{e, f} 10 - 73.7 ± 3.44 ^{a, b} 67.9 ± 2.58 ^{c, a} 53.4 ± 3.73 ^{d, e} 50.6 ± 2.45 ^{d, e} 20 - 73.6 ± 4.78 ^{a, b} 63.0 ± 3.07 ^f 53.9 ± 3.86 ^{d, e} 49.5 ± 1.94 ^{d, e} KPI 5 - 75.3 ± 5.26 ^{a, b} 75.3 ± 2.41 ^{a, b} 73.9 ± 4.59 ^a 72.4 ± 2.80 ^{e, f} 10 - 76.2 ± 4.40 ^a 71.5 ± 2.00 ^{a, c, d} 62.1 ± 2.50 ^{e, f} 62.8 ± 2.17 ^{e, r} 20 - 4.7 ± 3.36 ^{a, b} 65.8 ± 3.42 ^{d, e, f} 62.1 ± 2.19 ^{e, f} 60.2 ± 2.50 ^f

The figures are mean SD (n = 10). Values of similar letters mean that they are not significantly different from the control.

Carlophanax saponin K isolated from thawed skin and metabolites produced by metabolizing it into human intestinal microorganisms has the effect of inhibiting anti-inflammatory effects and edema caused by vascular permeability changes. Carlophanax saponin K and its metabolites of the present invention can be usefully used for the treatment and prevention of anti-inflammatory and arthritis.

Claims (10)

delete delete Carlopanax saponin K or Carlopanax saponin J is mixed with human feces, and then cultured under anaerobic conditions, the culture is subjected to solvent fractionation, the fractions are concentrated under reduced pressure, and the concentrate is subjected to silica gel column chromatography. Method for producing Carlophanax saponin A or Carlopanax saponin I using human intestinal microorganisms 4. The culture according to claim 3, wherein the culture of carlopanax saponin K or carlopanax saponin J with human intestinal microorganisms is carried out under anaerobic conditions, the culture is subjected to solvent fractionation, the fractions are concentrated under reduced pressure, and the concentrate is subjected to silica gel column chromatography. Method for producing Carlophanax saponin A or Carlopanax saponin I characterized by the above-mentioned. According to claim 4, wherein the human intestinal microorganisms are Bacteroides sp., Bifidobacterium sp., Eubacterium sp., Fusobacterium sp. Fusobacterium sp., Lactobacillus sp., Streptococcus sp. An anti-inflammatory therapeutic agent containing an effective amount of carlophanax saponin K or carlopanax saponin J. A therapeutic agent for arthritis containing an effective amount of carlophanax saponin K or carlopanax saponin J. The therapeutic agent according to claim 7, wherein the arthritis is rheumatoid arthritis. The anti-inflammatory therapeutic agent according to claim 6, which further contains Carlophanax saponin A or Carlopanax saponin I. 8. The therapeutic agent for arthritis according to claim 7, which further contains Carlophanax saponin A or Carlopanax saponin I.

Images