KR101988882B1 - A composition comprising ginsenoside Rk1 or Rg5 for preventing or treating sepsis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating sepsis containing at least one selected from ginsenoside Rk1 or Rg5 as an active component. The ginsenoside Rk1 or Rg5 has an excellent effect of decreasing the expression of HMGB1 and decreasing mortality due to sepsis, thereby being able to be usefully used as the pharmaceutical composition for preventing or treating sepsis.

Description

A composition for preventing or treating septicemia comprising ginsenoside Rk1 or Rg5 {A composition comprising ginsenoside Rk1 or Rg5 for inhibiting or treating sepsis}

The present invention relates to a composition for preventing or treating septicemia comprising ginsenoside Rk1 or Rg5.

Sepsis is a condition in which microbes are infected and a serious inflammatory reaction occurs in the whole body. A hypertensive symptom that goes up to 38 degrees Celsius, hypothermia that goes down to 36 degrees Celsius, an increase in respiratory rate (more than 24 times a minute), a heart rate of more than 90 beats per minute (tachycardia), an increase or a decrease in leukocyte count A systemic inflammatory response syndrome is called a systemic inflammatory response syndrome. It is called sepsis when the systemic inflammatory response syndrome is caused by microbial infection. Although the infection pathways of microorganisms are not always known, appendicitis, otitis media, skin pyrexia, pressure ulcer, lung disease, cholecystitis, pyelitis, and osteomyelitis are known to cause sepsis.

Until now, the essential therapeutic agents for the treatment of sepsis have not been identified, and they are usually treated with antibiotics or antifungal agents. Therapeutic agents and duration may be determined by the type of microorganism or hemodialysis or transfusion depending on the condition of the patient. When antibiotics and antifungal agents are well tolerated, sepsis may be cured, but patients may die because they are difficult to treat when they are infected with microorganisms resistant to the drug or are weak in immunity, or when treatment is started too late.

The mortality rate of sepsis is as high as 50 to 70%, and it is known that it accounts for a high percentage of deaths worldwide. No treatment for severe sepsis has been approved to date, since the FDA-approved septicemia treatment, drotrecogin alfa (Xigris ^® ), proved ineffective and was withdrawn from the market in 2011 (Ranieri, VM et al., N Engl J Med., 366 (22), 2055-2064, 2012). Therefore, the development of a therapeutic agent for sepsis has a significant clinical significance, and researches for the development of a therapeutic agent for sepsis both locally and externally are actively conducted.

HMGB1 (high mobility group box 1) is one of the most important chromatin proteins as well as histone, and is expressed in vascular endothelium when there is vascular damage such as sepsis, and is monocyte, macrophage, It is secreted from the umbilical vein endothelial cell (umbilical vein endothelial cell).

As receptors for HMGB1, RAGE (receptor for advanced glycation end products), TLR2 (toll like receptor 2) and TLR4 (toll like receptor 4) are known. Adhesion molecules (VCAM-1, ICAM-1, E-selectin) on endothelial cells are induced by the action of HMGB1, which promotes inflammation through recruitment of leukocytes (Bae, JS et al. , 118 (14), 3952-3959, 2011).

It is known that HMGB1 is detected in the serum of sepsis patients and the serum level of HMGB1 is significantly increased in patients with bad prognosis and it is known that the increase of HMGB1 concentration in the plasma of severe sepsis patients is likely to cause death, HMGB1 is mainly used as a target for prevention or treatment of cancer (Lee, W. et al., Toxicol Appl. Pharmacol., 262 (1), 91-98, 2012; Yang, EJ et al., J Cell Physiol. , 228 (5), 975-982, 2013; Wang, H. et al., Science, 285 (5425), 248-251, 1999).

Ginsenoside is a compound of ginseng and glycoside. It is known to have a unique chemical structure and pharmacological effect, unlike saponin found in other plants.

Ginsenoside is a neutral glycoside conjugated with glucose, arabinose, xylose, rhamnose and the like on the triterpenoid-based dammarane skeleton. At present, more than 30 chemical structures have been identified. Depending on the nature of the chemical structure, protopanaxadiol (PPD) (19 species), protopanaxatriol (PPT) (10 species) Oleanane system (one species).

The pharmacological activity by the ginsenoside is indicated by the specific ginsenoside originating from the above-mentioned ginsenosides present in large quantities, and it is considered that the pharmacological activity by the ginsenoside is enhanced by the immunosuppressive effect, anti-inflammatory, antiallergic, anticancer, hypotensive, anti-cholesterol, (Tachikawa, E. et al., Biochem Pharmacol. 23 (5), 518-524, 2000), which is known to be a major potent antioxidant, Shibata, S., J Korean Med Sci., 16 (suppl.), 66 (11), 2213-2221, 2003; Tsai, SC et al., Chin J Physiol., 46 ), S28-37, 2001).

On the other hand, Korean Patent No. 10-1293278 and No. 10-1352635 disclose the use of a novel angiostatic blocker of a ginsenoside Rk1 or Rg3 analog as a prior art relating to a composition for treating sepsis of ginsenoside Rk1 or Rg5 , Korean Patent Laid-Open No. 10-2006-0095599 discloses ginseng extract polysaccharide having an ability to protect against infection and to treat septicemia and has been reported in a previous paper [Xing, W. et al., Biosci Rep., 37 (4) Discloses the therapeutic effect of ginsenoside Rg3 on sepsis-induced injury and mitochondrial dysfunction.

However, in the case of the present invention, it was confirmed that the ginsenoside Rk1 or Rg5 was treated at a low concentration of 0.031 to 0.061 mg / kg in an animal model in which sepsis was induced to exhibit a survival rate of 30 to 60% 10-1293278 and 10-1352635, the compounds of the present invention have similar basic skeletons to the ginsenosides Rk1 or Rg5, but use compounds having different structures to inhibit vascular permeability and vascular leakage of retinal vascular endothelial cells or diabetic retinopathy In the above-mentioned prior art, ginsenoside Rg3 was treated with 10-20 mg / kg concentration (corresponding to a concentration 100 times higher than the present invention) in an animal model in which sepsis was induced, , It was confirmed that the ginsenoside Rk1 or Rg5 has excellent selective therapeutic effect on sepsis as in the present invention.

Korean Registered Patent No. 10-1293278, New Vascular Leakage Blocker, Registered on July 30, 2013. Korean Registered Patent No. 10-1352635, New Vascular Leakage Blocker, registered on Jan. 10, 2014. Korean Patent Laid-Open No. 10-2006-0095599, Ginseng Extract Polysaccharide Having Infective Defense Ability and Ability to Treat Sepsis, published on Sep. 01, 2006.

Bae, J. S. et al., Activated protein C inhibits high mobility group box 1 signaling in endothelial cells, 118 (14), 3952-3959, 2011. Kim, YS et al., Differential expression of protein kinase C subtypes during ginsenoside Rh2-Induced apoptosis in SK-N-BE (2) and C6Bu-1 cells, Arch Pharm Res., 23 (5), 518-524, 2000 . Lee, W. et al., Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal models, Toxicol Appl Pharmacol., 262 (1), 91-98, 2012. Ranieri, V. M. et al., Drotrecogin alfa (activated) in adults with septic shock, N Engl J Med., 366 (22), 2055-2064, 2012. Shibata, S., Chemistry and cancer prevention activities of ginseng saponins and some related triterpenoid compounds, J Korean Med Sci., 16 (suppl), S28-37, 2001. Tachikawa, E. et al., In vitro inhibition of adrenal catecholamine secretion by steroidal metabolites of ginseng saponins, Biochem Pharmacol., 66 (11), 2213-2221, 2003. Tsai, S. C. et al., Stimulation of the secretion of luteinizing hormone by ginsenoside-Rb1 in male rats, Chin J Physiol., 46 (1), 1-7, 2003. Wang, H. et al., HMG-1 as a late mediator of endotoxin lethality in mice, Science, 285 (5425), 248-251, 1999. Xing, W. et al., Ginsenoside Rg3 attenuates sepsis-induced injury and mitochondrial dysfunction in liver via AMPK-mediated autophagy flux, Biosci Rep., 37 (4), 2017. Yang, E. J. et al., Barrier protective effects of rosmarinic acid on HMGB1-induced inflammatory responses in vitro and in vivo, J Cell Physiol., 228 (5), 975-982,2013.

It is an object of the present invention to provide a composition for preventing or treating septicemia comprising ginsenoside Rk1 or Rg5.

The present invention relates to a pharmaceutical composition for preventing or treating septicemia comprising at least one selected from ginsenosides Rk1 or Rg5 as an active ingredient. The ginsenoside inhibits the expression of HMGB1 (high mobility group box 1) .

The pharmaceutical composition according to the present invention can be formulated into a suitable form together with a commonly used pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " refers to a composition that is physiologically acceptable and, when administered to humans, does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, But are not limited to, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraoxybenzoate, propylparaxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, stabilizers, binders, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition comprising at least one selected from the ginsenosides Rk1 or Rg5 disclosed in the present invention as an active ingredient can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. The dosage will depend on the age, sex, body weight, the particular disease or condition being treated, the severity of the disease or condition, the time of administration, the route of administration, the absorption, distribution and excretion of the drug, It depends on judgment. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.001 mg / kg / day to 2000 mg / kg / day. A more preferred dosage is 0.01 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In another aspect, the invention provides a health functional food for preventing or ameliorating septicemia comprising at least one selected from ginsenosides Rk1 or Rg5 and a pharmaceutically acceptable food additive. The health functional food refers to a food prepared or processed by using a raw material or a component having useful functionality and includes, for example, a health supplement food, a functional food, a nutrient, and an auxiliary agent.

The ginsenosides may be added in an amount of preferably 0.001 wt% to 50 wt%, more preferably 0.001 wt% to 30 wt%, and most preferably 0.001 wt% to 10 wt% based on the total weight of the total food have. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the ginsenosides of the present invention can be added include various foods, beverages, gums, tea, Vitamin complexes and the like.

The present invention relates to a pharmaceutical composition for preventing or treating sepsis comprising at least one selected from ginsenosides Rk1 or Rg5 as an active ingredient. The ginsenoside Rk1 or Rg5 is excellent in the effect of decreasing the expression of HMGB1 and decreasing the mortality due to sepsis, and thus can be usefully used as a pharmaceutical composition for preventing or treating sepsis.

FIG. 1 shows the results of confirming cell survival rate by treatment with ginsenoside Rk1 or Rg5 in vascular endothelial cells.
Figure 2 shows the results of ELISA for HMGB1 expression levels following treatment with ginsenoside Rk1 or Rg5 in a sepsis-induced cell (Figure 2A) or animal (Figure 2B) model.
FIG. 3 shows the results of ELISA analysis of HMGB1 receptor (TLR2, TLR4 and RAGE) expression levels of HMGB1-treated vascular endothelial cells following treatment with ginsenoside Rk1 or Rg5.
FIG. 4 shows the expression levels of the adherent molecules VCAM-1 (FIG. 4A), ICAM-1 (FIG. 4B) and E-selectin (FIG. 4C) according to treatment with ginsenoside Rk1 or Rg5 in HMGB1-treated vascular endothelial cells Were confirmed by ELISA.
FIG. 5 shows the results of confirming the survival rate according to the concentration of ginsenoside Rk1 or Rg5 in an animal model of sepsis.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Preparation of ginsenosides Rk1 and Rg5 >

Example 1-1. Production of black ginseng extract

2 kg of black ginseng were accurately weighed and pulverized, and leached in 5 L of a 70% (v / v) aqueous methanol solution for one day, followed by ultrasonic shaking for 3 hours, followed by filtration. 8 liters of 70% (v / v) methanol aqueous solution was added to the black gum powder, and the process of leaching and ultrasonic shaking was repeated two more times. Then, the filtrate was combined with the first filtrate and concentrated under reduced pressure to obtain 474 g of a completely dried methanol extract.

The methanol extract was thoroughly dissolved in 2.5 L of water by shaking ultrasonically, and then 2 L of diethyl ether was added thereto, followed by stirring at room temperature for 2 days. After stirring, the solution was transferred to a separatory funnel and layered. After removing the diethyl ether layer, 5 liters of water-saturated butanol was added to the remaining water layer, and the mixture was stirred in a stirrer for 2 days. After stirring, the solution was transferred to a separatory funnel and layer separation was performed for more than one day. Only the butanol layer was collected and concentrated under reduced pressure to obtain 87.6 g of completely dried butanol extract.

Examples 1-2. Separation and purification of ginsenosides Rk1 and Rg5 from black ginseng extract

The crude saponin extract of 87.6 g of the butanol fraction of black ginseng obtained in Example 1-1 was dissolved in methanol and mixed with 500 g of silica gel. It is then concentrated under reduced pressure to obtain a fine powder. 250-400 mesh silica gel is kneaded in chloroform and poured into a 12 × 50 cm open column to a height of 25 cm. Then, a sea sand (silica sand) It was used to separate the powder in a state of being filled without being mixed with the solvent). The powder was raised by 2 cm. The solution was mixed in a ratio of chloroform: methanol: water = 10: 2: 0.2 (1500 ml: 300 ml: 30 ml), and the solution was mixed in a separatory funnel and used as a development solvent for silica gel column chromatography. Thus, five fractions were firstly isolated to obtain a fraction No. 3 containing ginsenosides Rk1 and Rg5. C18 reverse phase column chromatography was performed with the third fraction containing the ginsenosides Rk1 and Rg5. A C18 reversed phase filler kneaded with a 40% (v / v) aqueous methanol solution was filled in a 3.5 x 60 cm flash column to a height of 30 cm, and then a fraction containing the ginsenosides Rk1 and Rg5 was dissolved in a 25% (v / v) aqueous methanol solution (V: v), 3: 1 (v: v), 4: 1 (v: v), and 5: 1 (v: v). As a result, 225 mg of pure ginsenoside Rk1 and 255 mg of pure ginsenoside Rg5 were finally obtained, and its physicochemical properties were as follows.

One. ginsenoside Rk1

white powder;

mp: 178-181 ℃, [α] D 20: = 11.0 (c 0.2, MeOH), C 42 H 70 O 12, MS (m / z) 789 (M + Na) +;

^{1 H-NMR (400㎒, C} 5 D 5 N, ppm) δ 0.62 (d, J = 11.47 Hz 1H), 0.72 (s, 3H), 0.91 (s, 3H), 1.02 (s, 3H), 1.31 (d, J = 4.37, 11.7 Hz, 1H), 3.89 (s, 3H), 1.49 s, 3H), 4.83 (d , J = 7.53㎐, 1H), 4.86 (br s, 1H), 5.13 (br s, 1H), 5.23 (br.t, J = 6.68㎐, 1H), 5.33 (d , J = 7.71, 11.7 Hz, 1H);

¹³ C-NMR (100 MHz, C ₅ D ₅ N, ppm) δ 15.80, 16.45, 16.58, 16.98, 17.74, 18.45, 25.74, 26.75, 27.08, 28.11, 30.77, 32.60, 32.67, 33.89, 35.36, 39.30, , 37.03, 40.21, 48.23, 50.86, 51.21, 52.49, 56.43, 62.76, 62.87, 71.65, 71.72, 72.47, 77.08, 77.96, 78.06, 78.19, 78.34, 83.45, 88.95, 105.09, 106.01, 108.15, 125.33, 131.21, 155.55 .

2. ginsenoside Rg5

white powder;

mp: 188-192.5 ℃, [α] D 20: = + 4.7 (c 1.2, MeOH), C 42 H 70 O 13, MS (m / z) 789 (M + Na) +;

^{1 H-NMR (400㎒, C} 5 D 5 N, ppm) δ 0.81 (s, 3H), 0.95 (s, 3H), 1.01 (s, 3H), 1.09 (s, 3H), 1.27 (s, 3H J = 6.9, 7.0 Hz, 1H), 3.26 (dd, J = 4.0, 11.63 Hz, 1H), 1.58 (s, 3H), 1.62 (s, , 4.90 (d, J = 7.4 Hz, 1H), 5.21 (t, J = 6.9 Hz, IH), 3.90 (br s, , 5.33 (t, J = 7.6 Hz, IH), 5.49 (t, J = 7.0 Hz, IH);

¹³ C-NMR (100 MHz, C ₅ D ₅ N, ppm)? 13.07, 15.72,16.35,16.49,16.92,17.66,18.33,2560,26.64,27.35,28.00,28.73,32.10,32.54,35.24,39.60,36.91 , 39.17, 40.14, 50.33, 50.66, 50.80, 50.91, 56.29, 62.58, 62.73, 71.50, 71.53, 72.49, 77.00, 77.82, 77.98, 78.13, 78.21, 83.31, 88.18, 105.00, 105.91, 123.21, 123.54, 131.16, .

Example 2 Confirmation of Cell Survival Rate by Treatment of Ginsenoside Rk1 or Rg5 in Vascular Endothelial Cells [

HUVEC (human umbilical vein endothelial cells, Cambrex Bioscience, Charles City, IA) was used for experiments by subculturing 3-5 times at 37 ° C and 5% CO ₂ using EBM-2 basic medium .

Subsequently, the subcultured cells were cultured in a 96-well plate at a density of 5 × 10 ³ per well and cultured for 24 hours. Then, the cells were washed with fresh medium, and then ginsenoside Rk1 or Rg5 was added at concentrations of 0, 20, 40 and 80 μM And reacted for 48 hours. After 48 hours, 100 쨉 l of 1 mg / ml MTT solution was added to the cells, followed by reaction for 4 hours. Then, 150 쨉 l of DMSO was added to dissolve the forma salt produced in the cells. The amount of dissolved formazan was measured at 540 nm with a microplate reader (Tecan Austria GmbH, Austria) and is shown in Fig.

Referring to FIG. 1, it was confirmed that when vascular endothelial cells (HUVEC) were treated with ginsenoside Rk1 or Rg5, cell viability was equal to that of the control group (no treatment group) regardless of the concentration and cytotoxicity was not shown.

Example 3. Confirmation of Effect of Ginsenoside Rk1 or Rg5 on HMGB1 Expression in a Sepsis Model>

Example 3-1. Production of sepsis-induced cells

The HUVEC subcultured in Example 2 was treated with 100 ng / ml LPS (lipopolysaccharide, serotype: 0111: B4; L5293) for 16 hours and then cultivated with ginsenoside Rk1 or Rg5 20 and 40 [mu] M) for 6 hours to obtain a cell culture solution. The cell culture broth was diluted with a protein separation reagent (1 mM PMSF, 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 1 μg / ml leupeptin, (Containing 15% RIPA lysis buffer (Upstate Biotechnology, USA)) at 4 ° C for 1 hour and centrifuged (15000 rpm, 4 ° C, 20 minutes) to recover only the supernatant .

Example 3-2. Preparation of sepsis-induced animal models

Sepsis was induced in mice using a cecal ligation and puncture operation to produce an animal model in which sepsis was induced. The mice used in the experiment were approved by Kyungpook National University Animal Care Committee (IRB No. KNU 2016-54).

Male C57BL / 6 mice (6-7 weeks old, body weight 27 g, Orient Bio Co., Sungnam, Republic of Korea) were maintained at 20-25 ° C, 40-45% For 12 days. Then, the mixture of oxygen gas and 2% isoflurane (JW pharmaceutical, South Korea) was anesthetized with a rodent respiratory anesthesia machine (RC2; Vetequip, Pleasanton, CA) and anesthetized with a face mask So that they can breathe naturally during surgery.

Next, a 2-cm midline incision was performed to expose the adjacent cecum, and the 5-mm area from the end of the cecum was tightly bound with a 3.0-silk suture, and a 22-gauge needle I made one hole. Thereafter, the cecum is gently squeezed, a small amount of excretion is extruded from the hole, and then placed in the peritoneal cavity, and then the incision is sealed with a 4.0-cm suture. At this time, the sham (sham-operated mice) were exposed to the cecum, but they were placed in the abdominal cavity without performing the bundling and perforating steps. Each mouse was exposed to sepsis symptoms 24 hours after CLP surgery, with symptoms such as shivering, bristled hair or weakness.

The ginsenosides Rk1 or Rg5 (0.031 or 0.061 mg / kg) of the present invention were intravenously administered 12 hours after the CLP surgery. After 24 hours of CLP administration, mouse blood was collected and centrifuged at 2000 xg for 5 minutes to obtain serum samples ≪ / RTI >

Example 3-3. Identification of HMGB1 expression levels in the sepsis model

To confirm the effect of ginsenoside Rk1 or Rg5 on the expression of HMGB1 in the sepsis model, the expression level of HMGB1 was determined by ELISA using a supernatant of the cell culture solution of Example 3-1 and the serum sample of Example 3-2 enzyme-linked immunosorbent assays.

First, a 96-well plastic flat microtiter plates (Corning, NY, USA) were dissolved in 20 mM carbonate / bicarbonate buffer (pH 9.6) containing 0.02% sodium azide The HMGB1 protein (Abnova) was coated and reacted at 4 ° C for over 18 hours. Thereafter, the plate was washed three times with PBS-T (PBS-0.05% [w / w] Tween 20), and then PBS-T was added thereto.

On the other hand, the HMGB1 protein (Abnova, for protein quantification, diluted in PBS-T), the supernatant of the cell culture of Example 3-1 and the serum sample of Example 3-2 were incubated with anti-HMGB1 antibody (Abnova, 1: 1000 dilution in PBS-T) for 90 minutes at 37 < 0 > C.

The antibody-pretreated HMGB1 protein, cell culture supernatant and serum samples were transferred to a plate coated with HMGB1 and reacted at room temperature for 30 minutes. Each plate was then washed three times with PBS-T and then reacted with secondary antibodies (peroxidase-conjugated anti-rabbit IgG antibodies, 1: 2000 dilution in R & D Systems, PBS-T) for 90 minutes at room temperature.

Next, the plates were washed three times with PBS-T and treated with 200 μl of substrate solution (100 μg / ml o- phenylenediamine & 0.003% H ₂ O ₂ ) for 60 minutes in a dark room at room temperature. After 60 minutes, the reaction was stopped with 50 μl of 8N, H ₂ SO ₄ , and the absorbance at 490 nm was confirmed and shown in FIG.

2, treatment of ginsenoside Rk1 or Rg5 with sepsis-induced cells (FIG. 2A) and animal (FIG. 2B) models is excellent in the effect of inhibiting HMGB1 expression in a concentration-dependent manner, It can be used effectively as a composition.

Example 3-4. Identification of cell surface marker receptor expression levels in HMGB1-treated vascular endothelial cells

Expression levels of RAGE (receptor for advanced glycation end products), TLR2 (toll like receptor 2), and TLR4 (toll like receptor 4), which are HMGB1 receptors, were determined by ELISA.

When HUVEC subcultured in Example 2 was cultured in a 96-well plate to give a confluence of 90%, HMGB1 (1 μg / ml) was added and reacted for 16 hours. Then, the ginsenosides Rk1 or Rg5 of the present invention (0, 10, 20 and 40 μM) and reacted for 6 hours. The cells were then washed with PBS (phosphate buffered saline) and fixed with 50 μl of 1% (w / v) paraformaldehyde for 15 min at room temperature (25 ° C). After 15 minutes, the paraformaldehyde was removed and the cells were washed and then added with 100 μl each of TLR2 antibody A-9, TLR4 antibody H-80 and RAGE antibody A-9 (Santa Cruz, CA). After 1 hour, each cell was washed three times, then 100 μl of 1: 2000 peroxidase-conjugated anti-mouse IgG antibody (1: 2000 peroxidase-conjugated anti-mouse IgG antibody, Sigma, Saint Louis, MO) Lt; / RTI > After washing three times, the o-phenylenediamine substrate (Sigma, St. Louis, MO) was reacted with the o-PPD substrate, and the absorbance of the color reaction was measured at 490 nm.

Referring to FIG. 3, when HMGB1-treated vascular endothelial cells of the present invention were treated with ginsenoside Rk1 or Rg5, the expression of HMGB1 receptors TLR2, TLR4 and RAGE was inhibited.

Examples 3-5. Identification of cell adhesion molecule expression level in HMGB1-treated vascular endothelial cells

Expression levels of VCAM-1 (vascular cell adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1) and E-selectin, which are adhesion molecules induced by the action of HMGB1 and receptor, were confirmed by ELISA.

In order to confirm the protein expression level, the same procedure as in Example 3-4 was carried out except that VCAM-1 was used instead of TLR2, TLR4 and RAGE antibodies (TLR2 antibody A-9, TLR4 antibody H-80 and RAGE antibody A- , Mouse anti-human monoclonal antibody (1: 50 dilution, Temecula, CA, USA) for ICAM-1 and E-selection was used. The results of confirming the expression levels of the adherent molecules VCAM-1, ICAM-1 and E-selectin are shown in FIG.

4, when the HMGB1-treated vascular endothelial cells of the present invention are treated with the ginsenosides Rk1 or Rg5, VCAM-1, ICAM-1 and E-2, which are adhesion molecules induced by the action of HMGB1 and the receptor, and the expression of LPS was suppressed.

Thus, it has been found that ginsenoside Rk1 or Rg5 is superior in the effect of suppressing the expression of HMGB1 and HMGB1 receptors, which are increased in expression by sepsis, and is excellent in the effect of inhibiting the action of HMGB1 and receptors, It can be used.

Example 4. Confirmation of survival rate change by treatment of ginsenoside Rk1 or Rg5 in an animal model in which sepsis was induced>

In order to confirm the effect of ginsenoside Rk1 or Rg5 on the animal model in which sepsis was induced, first an animal model in which sepsis was induced by performing CLP surgery by the method of Example 3-2 was obtained. The ginsenosides Rk1 or Rg5 (0.031 or 0.061 mg / kg) of the present invention were administered to the tail vein twice at 12 hours and 50 hours after the CLP surgery, The survival rate was confirmed (Kaplan-Meier survival analysis) and is shown in FIG.

5, when the ginsenoside Rk1 or Rg5 of the present invention was administered at a concentration of 0.031 or 0.061 mg / kg after CLP surgery, the survival rate was 30 to 60% as compared to the no treatment group (only CLP surgery) , And it was found that the treatment effect of sepsis was excellent.

≪ Formulation Example 1: Preparation of powder &

2 g of the ginsenoside Rk1 or Rg5 of the present invention and 1 g of lactose were mixed and filled in an airtight container to prepare a powder.

≪ Preparation Example 2 > Preparation of Tablets >

100 mg of ginsenoside Rk1 or Rg5 of the present invention, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and tableted according to a conventional method for preparing tablets.

≪ Formulation Example 3: Preparation of capsules >

After mixing 100 mg of ginsenoside Rk1 or Rg5 of the present invention, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, the above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules .

≪ Formulation Example 4: Preparation of pill &

Maltodextrin, corn starch and microcrystalline cellulose (MCC) as additives for inhibiting hygroscopicity were mixed in a small amount, and the resulting mixture was subjected to a conventional method Thus, 100 mg of pills were made.

≪ Formulation Example 5: Preparation of injection solution &

1 g of the ginsenoside Rk1 or Rg5 of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

≪ Formulation Example 6: Preparation of health functional food &

20 g of the ginsenoside Rk1 or Rg5 of the present invention, a proper amount of vitamin A, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 g of biotin, 1.7 g of nicotinic acid amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate, 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare granules, which can be modified into various formulations depending on the application. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above components may be mixed according to a conventional health functional food manufacturing method.

< Formulation example  7. Preparation of Health Functional Drink>

1 g of the ginsenoside Rk1 or Rg5 of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, sterilized and refrigerated according to a conventional beverage preparation method.

Claims (5)

A pharmaceutical composition for preventing or treating sepsis comprising ginsenoside Rk1 as an active ingredient. The method according to claim 1,
Wherein the ginsenoside inhibits the expression of HMGB1 (high mobility group box 1). The method according to claim 1,
Wherein the composition is formulated into a pharmaceutical dosage form by addition of a pharmaceutically acceptable carrier, excipient or diluent. A health functional food for preventing or ameliorating sepsis including ginsenoside Rk1 as an active ingredient. 5. The method of claim 4,
Wherein the health functional food formulation is selected from the group consisting of tablets, capsules, pills, and liquid preparations.

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