KR100295366B1 - Pathogenic extract with anti-inflammatory activity - Google Patents

Abstract

The present invention relates to an extract of Gilgil (桔梗, Platycodi Radix) having an anti-inflammatory activity, the extract of Gilgil of the present invention is prepared by extracting the Gilyeong with a lower alcohol and extracted again with an organic solvent such as ethyl acetate or butanol, in particular Giltan butanol extract fractions and the like have excellent anti-inflammatory activity and can be used in various ways to treat inflammation. On the other hand, the present invention provides a pharmaceutical composition having an anti-inflammatory effect containing them as an active ingredient by separating and purifying the anti-inflammatory active ingredients Platicodin D and Platicodine D ₃ from the Gilyeong extract.

Description

Giltyeong extract with anti-inflammatory activity

The present invention relates to a Platycodi Radix extract having anti-inflammatory activity. More specifically, the present invention relates to a variety of gilyeong extract prepared by extracting gilyeong with a lower alcohol such as methanol and extracted again with an organic solvent such as ethyl acetate or butanol, in particular, butanol extract fraction of gilyeong is an anti-inflammatory activity This is excellent and can be usefully used for the treatment of inflammation. The present invention also relates to a method for preparing and separating and purifying the anti-inflammatory active ingredients of Platycodine D and Platicodine D ₃ from the gilyeong extract, and a pharmaceutical composition having an anti-inflammatory effect containing them as an active ingredient.

Bellflower (Platycodon grandiflorum A. DC.) Is a perennial herb belonging to the Campanulaceae. It has no hairs, the roots are enlarged, the stems are single or gathered, stand straight and the height is 50-100 cm. The leaves are regenerated or aquatic, almost without petioles, obovate or oval, with sharp ends and sharp hangers at both ends, and the back of the leaves is white like powder. Flowers are purple, bloom in July-August, and bell flowers grow at the end of stem or split. Corolla divided into 5 branches, calyx is bell-shaped and divided into 5 pieces. There are five surgeries, the ovary is subordinate. Fruits are capsules, obovate spherical, and the upper part is divided into 5 branches. A white flower (Platycodon grandiflorum for.albiflorum Hara) that grows or grows in Korea, Platycodon grandiflorum for.duplex Mak. for.leucanthum Hara).

Kilkyung has dried bellflower roots, and in the study of cultivation and preparation methods, Hosoda et al. Reported that soil's high humidity and low air permeability help to grow Kyonggyeong (Hosoda, K et al., Shoyakugaku Zasshi, 43). (4), 271 (1989). Domestically, the shelled bellflower is soaked in water and dried, and consumed in large quantities for food (herb).

As a study of Gil-Kyung, Chung et al. Reported on amino acid composition and lipid composition (Tae-Young Jung, Research Report, Pusan National University, 10, 41 (1984)). In addition, Hwang et al. Studied the composition of lipids, amino acids and dietary fiber of Gilkyung, Perilla seed, Evening Primrose seed, Aloe Vera, which are functional foods, and 14% of Phospholipid among the lipid content of Gilkyung. As a result of analysis of fatty acid composition in total lipid, Gilkyung reported that linolenic acid and palmitic acid accounted for 64.44% of all fatty acids, and the ratio of essential and non-essential amino acids was 0.9. It was reported to be the highest among the samples (Hwang, Seong-Won et al., Korean Journal of Nutrition and Nutrition, 23 (4), 647 (1994)). Kim et al. Reported that the fiber content of Gilkyung was 23.24% compared to dry matter, which was lower among vegetables and algae (Kim Eun Hee et al., Korean Journal of Nutrition, 26, 196 (1993)).

Gilkyung contains about 2% of triterpene saponins as its components, and among these saponins, glycosides of platycodlgenin are Platicodin A, C, D, D ₂ and two species. Monoacetate platidine D ₃ , deepio-platicodine D _3, have been reported. In addition, glycosides of polygalacic acrid include polygalacin D and two monoacetates, polygalacin D ₂ and monoacetate, and glycosides of platycogenic acid A are methyl platyconate A, Methyl-2-methoxy platyconate A and the like have been reported, and the presence of platicogenic acids B and C as other aglycones has been reported. Other 0,03% sterol strains include α-Spinasterol, α-Spinasterol Glucoside, stigmast-7-enol, betulin, α It is reported that it contains spinnasteryl-D-glycopyranoside, and in addition, vitamin A, inulin, and the like.

Kubota and Shibata have isolated major sapogenin, polygalacic acid, and placodigenin, respectively, and Kubota has minor sapogenin. And platicogenic acids A, B and C were separated, respectively. Also, Elyakov et al. Reported the separation of Platicoside C consisting of Platycodigenin as aglycone and glucose, xylose, rhamnose and arabinose (2: 1: 1: 1) as sugar. Saito et al. Reported the structure of a new acylated anthocyanin, Platyconin, from the flowers of Gilt. Tada et al. Identified the structure of isolated platicodine D in a study on ciliary saponins, and Ishii ^et al. Confirmed the structure of prosapogenins from ciliaceae by C ^l3 -NMR and H ^l -NMR. The structures of polygalacin-D and polygalacin-D ₂ , platicodine D and platicodine D _2, and other monoacetates have been elucidated. In addition, new triterpene glycosides have been isolated and identified, as well as the composition and structure of the arabinose moiety of platicodine. Inada et al. Have isolated new dihydroflavonol glycosides and 3-methyl-1-butanol glycosides from Gil-gil seeds, and Oka et al. Reported on the structural properties of Inulin oligomers. .

Inflammation is a bacterium-derived abscess that actively infiltrate tissues with purulent fungi that white blood cells devour, resulting in the death of white blood cells, tissues damaged by bacteria, and cell debris, which accumulate in the tissues. The pathological state of the place, and the anti-inflammatory action is to inhibit the growth of invading bacteria by antibacterial agents, or to activate the macrophage (macrophage) that phages foreign matter accumulated in the abscess, and to increase the ability to digest and excrete them Abscess is the process of treatment. In general, an inflammatory response is a defense process of a living body that attempts to repair and repair the damaged area when an invasion that causes some organic change in a cell or tissue of the living body is applied. Therefore, such a series of reactions are said to include local blood vessels, various tissue cells of body fluids, immune-involved cells, and the like.

To date, anti-inflammatory agents have been shown to inhibit the biosynthesis of prostaglandins activated by physicochemical and physiological stimuli from the cell membranes of damaged tissues or leukocytes inoculated by chemotactic factors in acute inflammation. (Moyazawa, K and Mikami, T., Japan J. Pharmacol., 38, 199 (1985)). However, these drugs have a therapeutic effect on the symptoms of acute inflammation, but in chronic inflammation such as rheumatoid arthritis, only the primary inflammation is reported, and immunological therapeutic effects are not expected. Recently, arachidonic acid has been studied for hydroxyeicosatetraenoic acid (HETE), leukotriene B ₄ , and leurotene C ₄ , which are substances produced by lipoxygenase. Has been shown to have some effect on chronic inflammation (Huskisson. ED et al., Inflammation, Mechanism and Treatment, MTP, Lancaster, England, p.55 (1980)).

The present inventors confirmed the anti-inflammatory action of Giltyeong, identified the chemical structure of the active ingredient having an anti-inflammatory action and found out that the active ingredients are Platicodin D and Platicodine D ₃ to complete the present invention.

It is an object of the present invention to provide an gilyeong extract having anti-inflammatory activity.

FIG. 1 shows a process for separating Platicodin D and Platicodine D ₃ from butanol fraction.

In order to achieve the above object, in the present invention, Gil-gil extract having anti-inflammatory activity obtained by extracting Gil-kyung with a lower alcohol such as methanol and Gil-gil extract having anti-inflammatory activity extracted with lower alcohol and then extracted again with ethyl acetate or butanol, etc. to provide.

In another aspect, the present invention provides a pharmaceutical composition having an anti-inflammatory effect as the active ingredient the Gilyeong extract.

In addition, the present invention provides a method for preparing and separating and purifying the anti-inflammatory active ingredients of platicodine D and platicodine D ₃ from the gilyeong extract and a pharmaceutical composition having an anti-inflammatory effect as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an gilyeong extract having anti-inflammatory activity.

Firstly, the present invention obtains an alcohol fraction of Gil-gil having anti-inflammatory activity by repeatedly extracting Gil-gil in lower alcohol. At this time, it is preferable to use methanol as the alcohol.

In another aspect, the present invention provides a gilyeong extract having anti-inflammatory activity obtained by extracting gilyeong with a lower alcohol, such as methanol in the above process and then extracted again using an organic solvent. At this time, the organic solvent includes ethyl acetate or butanol. In particular, the gilyeong extract obtained using butanol shows the best anti-inflammatory effect.

Specifically, the present invention is finely chopped or dried dried Gilyeong or put the lower alcohol so that the Gilyeong is submerged and extracted for 4-7 hours at a temperature of 80-90 ° C and then repeat this process 2-3 times, It is filtered again and concentrated to produce a lower alcohol extract. In addition, the present invention is to suspend the lower alcohol extract obtained in the lower alcohol, the fractions transferred to various solvents are collected, filtered and concentrated to prepare a variety of solvent extraction fractions.

At this time, the organic solvent is used in order from the nonpolar organic solvent to the polar organic solvent. Indeed, the present invention uses n-hexane, chloroform, ethyl acetate and butanol in this order to prepare a hexane extract fraction, a chloroform extract fraction, an ethyl acetate extract fraction, a butanol extract fraction and a water extract fraction.

At this time, the temperature for preparing the fraction is more preferably in the range of room temperature, that is, in the range of 4 to 30 ° C.

In addition, in the present invention, the method of preparing Platicodin D of Formula 1 and Platicodine D ₃ of Formula 1, which are active ingredients having anti-inflammatory action, are separated and purified by column chromatography several times. To provide.

Specifically, Platicodin D and Platicodine D ₃ were extracted by repeatedly dipping the pathoscope in lower alcohol, and then filtered and concentrated to obtain a lower alcohol extract. Then, ethyl acetate and butanol were added to obtain a solvent extraction fraction. A solvent extraction fraction is prepared by separating and purifying several times by column chromatography.

[Formula 1]

[Formula 2]

In addition, the present invention provides a pharmaceutical composition having an anti-inflammatory effect as an active ingredient obtained from the Gilyeong fraction.

In addition, the present invention provides a pharmaceutical composition having an anti-inflammatory effect as an active ingredient Platicodine D of Formula 1 and Platicodine D ₃ of Formula 2 which is an anti-inflammatory active ingredient prepared by separating and purifying from the Gilyeong extract to provide.

Specifically, the pharmaceutical composition may be used for inflammatory diseases and the like, and may be preferably used as an effective therapeutic agent for inflammation and rheumatism.

Toxicity experiments were carried out with Platicodin D and Platicodine D _{3, which} are the active ingredients of Gil-Gil extract. The test substance was dissolved in water and administered orally to mice (5 mice per group) and observed for 14 days to determine mortality. At this time, the 50% lethal dose (LD ₅₀ ) Platicodine D was 3,000 mg / kg or more, and Platicodine D ₃ was 3,000 mg / kg or more.

In order to use the gilyeong extract and its active ingredients Platicodin D and Platicodine D ₃ as a therapeutic agent, it can be prepared by a known method in the pharmaceutical field, itself or a pharmaceutically acceptable carrier It may be mixed with a carrier, a forming agent, a diluent, and the like to prepare a powder, granule, tablet, capsule, or injection formulation.

The human dose of the active ingredient according to the present invention is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the treated disease, etc. About 15 to 2000 mg per day, but about 200 to 800 mg is preferred. Therefore, the unit dosage form of the present invention is prepared to have an amount of 5 to 500 mg of the active substance of the present invention in consideration of the above-mentioned effective amount range. Preferably it is formulated to contain 5 to 300 mg. This formulated unit dosage form can be administered several times at regular time intervals using specialized dosing methods as needed. Preferably it may be administered once to 7 times a day.

In fact, the anti-inflammatory activity was investigated using the gilyeong extract of the present invention, the gilyeong extract was excellent in anti-inflammatory effect even in the crude extract state, in particular butanol extract fraction showed an anti-inflammatory effect similar to the existing anti-inflammatory agent ibuprofen. In addition, the anti-inflammatory effect of Platicodin D and Platicodine D ₃ , which are anti-inflammatory active ingredients of Gilgil extract, was also excellent.

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

Example 1

Preparation of Methanol Extracts from Gilkyung

2040 g of well-dried street-length was cut and immersed in 15 L of 95% methanol, followed by extraction for 5 hours in a water bath at 100 ° C. The extract was filtered and the residue was extracted again with methanol. This process was repeated three times. The filtrate after filtration obtained above was concentrated under reduced pressure at a temperature ranging from 80 to 95 ° C. to give 581 g of a methanol extract in powder form.

The anti-inflammatory action of the Gilyeong methanol extract prepared in the above process was investigated by performing the experiment according to the procedure of Experimental Example 1 below.

Example 2

Preparation of Hexane Extract Fraction from Methanol Extract

581 g of the methanol extract prepared in Example 1 was suspended in 0.5 L of methanol, placed in a 2 L fraction filter, 0.5 L of n-hexane was added thereto, shaken, and allowed to stand for 1 hour to collect a substance which is transferred to n-hexane. The n-hexane layer obtained by performing the above process three times was collected, filtered and concentrated to prepare 25 g of a hexane extraction fraction (yield 4.30%).

Anti-inflammatory action of the hexane extract fraction prepared in the above process was investigated by performing the experiment according to the procedure of Experimental Example 1 below.

Example 3

Preparation of Chloroform Extract Fraction from Methanol Extract

The hexane extraction fraction obtained in Example 2 was prepared, 1 L of chloroform was added to 0.5 L of the remaining water layer, and the mixture was stirred for 1 hour to collect a substance that is transferred to chloroform. The chloroform layer obtained by performing the above procedure three times was collected, filtered and concentrated to prepare 15 g (yield 2.58%) of chloroform extract fractions.

The anti-inflammatory action of the chloroform extract fraction prepared in the above process was investigated by performing the experiment according to the procedure of Experimental Example 1 below.

Example 4

Preparation of Ethyl Acetate Extract Fraction from Methanol Extract

The chloroform extract fractions obtained in Example 3 were prepared, 0.5 L of ethyl acetate was added to 0.5 L of the remaining water layer, and shaken. The ethyl acetate layer obtained above was collected three times, and concentrated to prepare 10 g of an ethyl acetate extract (yield 1.72%).

Anti-inflammatory action of the ethyl acetate extract fraction prepared in the above process was investigated by performing the experiment according to the procedure of Experimental Example 1 below.

Example 5

Preparation of Butanol Extract Fraction from Methanol Extract

The ethyl acetate extract fractions obtained in Example 4 were prepared, and 1 L of butanol was added to 0.5 L of the remaining water layer, followed by shaking for 1 hour to collect the material which was transferred to butanol. The butanol layer obtained above was collected, filtered and concentrated to give 189 g (yield 32.63%) of butanol extraction fraction.

Anti-inflammatory action of the butanol extract fraction prepared in the above process was investigated by performing the experiment according to the procedure of Experimental Example 1 below.

Example 6

Preparation of Water Extract Fraction from Methanol Extract

After preparing the butanol extract fraction obtained in Example 5, 0.5 L of the remaining water layer was collected, filtered, and concentrated to prepare 335 g (yield 57.66%) of the water extract fraction.

Anti-inflammatory action of the butanol extract fraction prepared in the above process was investigated by performing the experiment according to the procedure of Experimental Example 1 below.

Example 7

Separation and Purification of Platicodin D and Platicodine D ₃ from Butanol Extracts

(Step 1) Subfractionation of Butanol Extract Fraction

Column chromatography was performed on 189 g of butanol extract fraction obtained in Example 5. That is, the soluble part of the dry powder butanol was dissolved in methanol, coated on the same amount of silica gel (No.7734), and the powder was put in a column and eluted with CHC1 ₃ : MeOH: H ₂ O (15: 10: 2). Thereafter, the fractions showing the same R _f value on TLC were combined and separated into 15 fractions (see FIG. 1). Each fraction layer was concentrated, dried and powdered, and then tested by the method of Experimental Example 1, and the fraction having excellent edema inhibitory activity was fraction 8 and the amount thereof was 29.06 g.

(Step 2) sub-subfractionation of butanol extract fraction

29.06 g of fraction 8 (see FIG. 1) on a dry powder of the fraction obtained in step 1 was coated on an equal amount of silica gel (No. 7729), and the powder was placed in a column and mixed with an unsaturated ethyl acetate and methanol mixed solution. Gradient column chromatography gave each fraction. Fractions of the same R _f value on the TLC were combined and concentrated under reduced pressure, and separated into four fractions of 8-1, 8-2, 8-3 and 8-4. Each fraction layer was concentrated to dryness and powdered to give 20.72 g, 2. 76 g, 1.47 g and 4.13 g, respectively.

(Step 3) sub-sub-subfractionation of butanol extract fraction

Coated with an equal amount of silica gel (No. 7734) with fractions 8-4 (see Figure 1), the powders were placed in a column and CHC1 ₃ : MeOH. Column chromatography was performed using the lower layer of the H ₂ O: n-PrOH (5: 6: 4: 1.2) mixed solution to combine fractions having the same R _f value on TLC. According to the R _f value, two fractions of 8-4-1 (1.03 g) and 8-4-2 (3.09 g) were separated, and each fraction layer was concentrated to form a crystalline solid powder. (0.45 g) and Platicodine D ₃ (2.05 g) were obtained.

Experimental Example 1

Anti-edema test

In Winter, CA et al., Carrageenan-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs, Proc. Soc.Exp. Biol. Med., 111, 544 (1962) Therefore, carrageenan edema inhibition was investigated. Edema was induced by subcutaneous injection of 0.1 ml of 1% lambda -carrageenan into the posterior sole of 150-200 g male rats, and the specimens were orally administered 30 minutes before carrageenan injection. The volume of edema was measured by a plethysmometer four times at an interval of 1 hour after carrageenan injection, and then, based on the volume before injection, the increase rate was calculated by Equation 1 below to test the degree of edema inhibition. Ibuprofen (Ibuprofen) 200 mg / kg oral administration as a control drug was compared.

The degree of edema inhibition was evaluated by the following equation with the increase rate based on the volume of the foot before injection.

[Equation 1]

In the above formula, Vt is the volume of the foot after a certain time after injection, and Vn is the volume of the foot just before injection.

[Equation 2]

In the above formula, Vc is the edema growth rate of the control group, Vs is the edema growth rate of the dosage group.

The experimental results were calculated using the mean value and standard error, and the comparison between the groups was statistically significant when the P value was less than 5% using Student's t-test.

Table 1 shows the edema inhibitory effect of the methanol extract obtained in Example 1, Table 2 shows the edema inhibitory effect of each extract fraction obtained in Examples 2 to 6, Table 3 is the Example It shows the edema inhibitory effect of the primary fraction of the butanol extract fraction obtained in step 1 of 7, Table 4 shows the edema inhibitory effect of the secondary fraction of the butanol extract fraction obtained in step 2 of Example 7, Table 5 Shows the edema inhibitory effect of the third fraction of the butanol extract fraction obtained in step 3 of Example 7.

As can be seen in Table 2, it was confirmed that the butanol layer showing a 99.9% significance at 3 hours shows the strongest edema suppression effect.

As can be seen in Table 3, it was found that the primary fraction 8 of the butanol extract fraction was superior in edema inhibition rate compared to the control at 2 hours and 4 hours.

As can be seen in Table 4, it can be seen that the secondary fraction 4 of the butanol extract fraction has an excellent λ-carrageenan-induced edema suppression action.

As can be seen in Table 5, it was found that the plaquedin D and Platicodine D ₃ has an excellent λ-carrageenan induced edema inhibitory action.

The gilyeong extract of the present invention shows an excellent anti-inflammatory effect even in the state of crude extract, in particular butanol extract fraction has a strong anti-inflammatory activity can be usefully used as an anti-inflammatory agent in the future. In particular, by finding out that the anti-inflammatory active ingredients of Giltyeong extract are Platicodin D and Platicodine D ₃ , pharmaceutical compositions containing them may also be used as effective inflammatory treatments.

Claims (3)

(1) extracting the pathogen with a lower alcohol and then filtering and concentrating to obtain a lower alcohol extract; (2) extracting the lower alcohol extract with n-hexane to discard the soluble substance, and extracting the residue from chloroform and extracting the remaining fractions with ethyl acetate or butanol to obtain a butanol extract; (3) column chromatography of the butanol extract with a mixture of CHC1 ₃ : Me0H: H ₂ O at 15: 10: 2 to obtain a fraction having an Rf value of about 0.45; (4) A gilyeong extract having an anti-inflammatory effect, characterized in that obtained by the step of fractionating according to the Rf value by gradient column chromatography with a mixture of unsaturated ethyl acetate and methanol. (1) extracting the path length with lower alcohol, and concentrating to obtain a lower alcohol extract by filtration, (2) extracting the lower alcohol extract with n-hexane, discarding the soluble substance, and extracting the residue with chloroform to discard the remaining fractions. Extracting with ethyl acetate or butanol to obtain a butanol extract; (3) fractionating the butanol extract by column chromatography with a mixture of CHC1 ₃ : Me0H: H ₂ O at 15: 10: 2; (4) fractionating a fraction having an Rf value of about 0.45 among the fractions of step (3) by gradient column chromatography with an unsaturated ethyl acetate and methanol mixture solution according to the Rf value; And (5) performing column chromatography on the lower layer of the solution obtained by mixing the fraction obtained in step (4) with CHCl ₃ : MeOH: H ₂ O: n-PrOH 5: 6: 4: 1.2. Platicodine D of 1 and Platicodine D ₃ of the formula (2). [Formula 1] [Formula 2] A pharmaceutical composition having an anti-inflammatory effect containing Platicodine D of Formula 1 and / or Platicodine D ₃ of Formula 2 as an active ingredient.