KR20030082763A - Articulation protective ingredients from medicinal plant and their composition - Google Patents

Abstract

PURPOSE: Provided are articulation protective ingredient from medicinal plant and their composition. Particularly, provided are an extraction method of Clematis mandshurica and a composition containing the extract of Clematis mandshurica which is useful as an articulation protection agent. CONSTITUTION: A composition containing an extract of Clematis mandshurica for the protection of articulation further contains 2.0 wt.% of oleanolic acid and 0.1-0.5 wt.% of isoferulic acid, as active ingredient.

Description

Articulation protective ingredients from medicinal plant and their composition

The present invention relates to a herbal composition having a protective effect on the joint tissues, and more particularly, a method for preparing a herbal extract from the gastric glands and the gastric herbaceous extract, containing the content of oleanolinic acid and isoferulic acid A composition optimized in a predetermined amount range, and relates to a herbal composition useful as an agent for protecting joint tissues, having an excellent effect on inhibiting articular cartilage tissue degradation and excellent protective activity for joint tissues.

Gastric glands are widely known as herbal medicines, and have been widely used for various inflammations such as boils, wounds, bronchitis, mastitis, tonsillitis, and gingivitis, as well as eliminating moist secrets. It does not, but pain in the lower back and shoulders, the whole body is weak and has been used as a prophylactic or herbal powder for the treatment of symptoms such as fat pain. These symptoms are similar to those of general arthritis, including chronic rheumatoid arthritis in modern pathological concepts.

The yeongyeongseon is the root of the ecstasy and coriander plant growing in the bush in various parts of our country. to be. In traditional medicine, it has been used for diseases involving limb joint pain, joint movement disorder, and hand and hand paralysis. In particular, it is a spiritual medicine that treats cases in which the urine, alcohol, and cold are hard to reach the ground. It has been widely used. Ingredients contained in soybeans and related plant species known to date include flamontone glycosides such as creatine, cremontanoside B, cremontanoside C, Saponins, such as cremontanoside S, are known, and sugars, glucose and steols, are also known. [Korea Useful Plant Resources Research Institute, Korea Chemical Research Institute, pp, 780 ~ 781] (1988); Dohae Hyangdae Dictionary, Younglimsa, pp. 489-490 (1990).

Most traditional Chinese medicines and related literatures, such as Dongbobogam, Herbal Medicine Collective, and Lactobacillus, refer only to the method of differentiating the form as a single herbal medicine for herbal medicine, and to the method of manufacturing herbal medicine and liquid solution. No knowledge regarding active active ingredients expressing medicinal effects was obtained.

Therefore, the present inventors obtained the extract from the gastric gland, as a result of trying to maximize the extraction efficiency of the active ingredient and more scientifically using the gastric gland known to improve anti-inflammatory effect as well as anti-inflammatory effect due to wind, cold, and wet, It was found that the joint protective action is exhibited by the active ingredient constituting the same, and the present invention was completed by developing an excellent herbal composition containing the extract.

Accordingly, the present invention aims to provide a herbal composition having a cartilage degradation inhibitory effect and a joint protection effect by standardizing and standardizing the preparation method of the herbal extracts from the gastric glands and the active fractions obtained from the herbal extracts with oleanolinic acid and isoferulic acid content. have.

Figure 1 shows the HPLC chromatogram of the gastric extract extracted by Example 1.

The present invention is characterized by the gastric medicinal herb extract with cartilage inhibition and joint protection.

Such herbal extracts are extracted with water or an aqueous alcohol solution to prepare a herbal medicine, extracting the gastric glands with water or an aqueous alcohol solution, separating the filtrate obtained in the extraction step with the same amount of lower alcohol and a non-polar solvent to concentrate under reduced pressure. The extract obtained in the step and the concentration step is azeotropically concentrated with water and lyophilized to prepare through a step of preparing a powder extract.

In addition, the present invention includes a joint protector containing the herbal composition.

Referring to the present invention in more detail as follows.

The herbal composition according to the present invention is prepared by limiting the content standards of oleanolinic acid and isoferulic acid, which have not been proposed in the past, to a certain level, and thus are very useful as an arthritis protective agent because they have an excellent effect on inhibiting articular cartilage tissue degradation and protecting joint tissue. Do.

Since the content of the effective bioactive substances contained in the herbal medicines can vary greatly depending on the place of origin, harvesting time, storage period and storage condition, the weight ratio of the ingredients used in defining the composition of each herbal composition often does not have a great meaning. .

Therefore, the present invention is characterized by selecting oleanolinic acid and isoferulic acid as an indicator for obtaining a herbal composition maximizing drug efficacy. When oleanolinic acid was hydrolyzed, the sugars of saponin were quantified in free form, that is, sazenin form. As a result, oleanolinic acid was found to exist in glycosides combined with various forms of sugar. The known physiological activity of oleanolinic acid is known to be excellent in antioxidant activity, inhibiting lipid peroxidation, anti-inflammatory and analgesic activity, and also excellent in chronic arthritis disease model induced by mycobacterial butyricum. [J. of Pharm. Pharmacol., 44, No. 5, pp 456-458 (1992); Chung-Kuo-Li-Hsueh-Pao, 10, No. 4, pp 381-384 (1984); Chem. Pharm, Bull., 28, No. 4. pp 1183-1188 (1980); Biochem. Int., 24, No. 5, pp 981 to 990 (1991)]. Isoferulic acid is known to have anti-inflammatory effects (Mediators Inflamm., 8 (3), 173-175 (1999)). In addition, the content ranges of oleanolinic acid and isoferulic acid contained in the herbal medicines of the present invention vary greatly depending on the place of origin, harvesting time, storage period and storage condition, as well as a great difference in the joint protection physiological activity according to the content of this component. Therefore, in the present invention, oleanolinic acid and isoferulic acid are selected as indicators. Therefore, the herbal composition of the present invention can be prepared by extracting the herbal extract obtained by extracting from the gastric glands, or by extracting the active fraction by extracting the crude drug, the compounding ratio at this time is oleanoline regardless of the weight ratio of the crude drug It is determined by the content in the herbal composition of acid and isoferulic acid.

As a result, the herbal composition of the present invention was able to obtain a desired drug efficacy when contained in an amount of 2.0 wt% or more, preferably 2.0 to 10.0 wt%, based on the total extract of oleanolinic acid as an indicator. The herbal composition containing less than 2.0% by weight of oleanolinic acid has relatively low results in inhibiting articular cartilage tissue degradation and protecting joint tissue. In addition, in the present invention, there is no particular limitation on the upper limit of the content of oleanolinic acid, but if the oleanolinic acid is contained in excess of a certain level, the oleanolinic acid does not increase any more and is not preferable in terms of manufacturing and technical reasons. If it is about 10.0 weight% or less, a preferable effect can be obtained.

On the other hand, the content of isoferulic acid, which is another indicator substance in the herbal composition of the present invention, when the content is 0.1% by weight or more, preferably 0.1 to 0.5% by weight, the desired drug effect can be obtained. Herbal compositions with less than 0.1% by weight of isoferulic acid result in relatively low joint cartilage tissue inhibitory activity and joint tissue protective activity. In addition, in the present invention, there is no particular limitation on the upper limit of the content of isoferulic acid, but if it contains more than a certain level of isoferulic acid, it does not increase any more, and is preferable in terms of technical and economical preparation. Since it does not, about 0.5 weight% or less can obtain a preferable effect.

Oleanolinic acid and isoferulic acid selected by the present invention as indicators are important components of the herbal composition obtained in the present invention, and when they are contained in a certain content range, they can express a stronger drug effect by expressing a synergistic effect. In addition, as an active ingredient of the herbal composition obtained in the present invention, the action of other components other than the above components cannot be excluded.

According to the present invention, the herbal extract is

1) After reflux extraction with water or alcohol solution of 10-15 times the weight of the medicinal herb, it is filtered, and the residue is added with 7-12 times water or alcohol solution of the weight of the mixed herbal medicine, and then filtered by heating. Mix and filter,

2) The filtrate obtained in 1) was separated into layers by the same amount of lower alcohol or nonpolar solvent, and then concentrated under reduced pressure at 60 to 70 ° C.,

3) It is obtained by azeotropically concentrating with water of 50-100 times the total amount of the extract obtained in 2), homogeneous suspension with the same amount of water, and freeze-drying to prepare a powder extract.

Examples of the lower alcohol solvent used herein include isopropanol, propanol or butanol, and nonpolar solvents include ethyl acetate, dichloromethane, chloroform, carbon tetrachloride and methyl ethyl ketone. It is preferable in view of activity and extraction efficiency.

The present invention also includes a method of using a herbal composition containing the powder extract prepared as an active ingredient as a joint protectant.

As described above, the present invention is intended to have an active ingredient having excellent joint protection effect from the herbal composition mainly based on the gastric gland, and the gastric gland used in the present invention is collected in the fall.

The gastric gland collected at this time is extracted by the step of extracting with water or ethanol aqueous solution as well as the method of extracting hot water, which has been used as a conventional common bath, and separating the filtrate obtained by the same amount of lower alcohol and nonpolar solvent.

As described above, water or an alcoholic solution is added to the gastrointestinal medicinal plant and extracted under reflux for 2 to 5 hours. At this time, the amount of the aqueous or alcoholic solution is preferably 10 to 15 times the weight of the herbal ingredient. Then, the filtrate is collected by filtration, and the residue is added with an aqueous solution of water or alcohol of 7-12 times the weight of the crude ingredient, and then warmed again for 2 to 5 hours, filtered and mixed with the previous filtrate to increase the extraction efficiency. If the amount of water is too small, the stirring becomes difficult and the solubility of the extract is low, and the extraction efficiency is lowered. If the amount is too large, the amount of the lower alcohol and the nonpolar solvent used in the next purification step increases, which is not economical, causing problems in handling. Can be.

In the present invention, the method of re-extracting after the first extraction is adopted. Even in the case of mass production of the herbal extracts, even though the filtration is effective, the loss occurs because the water content of the herbal medicine itself is high, so the extraction efficiency is reduced only by the first extraction. This is to prevent this. In addition, as a result of verifying the extraction efficiency of each step, it was found that about 80 to 90% of the total extraction amount is extracted by the second extraction, and the third or more multistage extraction is not economical.

As described above, the extract obtained by extracting with water or a mixed solution of water and alcohol in the first and second stages is filtered and concentrated, and then purified impurities, such as unnecessary proteins, polysaccharides and fatty acids contained in the filtrate, in the present invention The impurities are purified by performing layer separation two to four times with an equal amount of lower alcohol or a non-polar solvent to obtain a solvent fraction. In this case, butyl alcohol, propyl alcohol or isopropyl alcohol is used as the lower alcohol, and the nonpolar solvent is ethyl acetate, dichloromethane, chloroform, carbon tetrachloride and methyl ethyl ketone, and the amount of lower alcohol or nonpolar solvent is less than that of the filtrate. In this case, the fine particles are formed by unnecessary components such as fatty acids, so that the separation of the layers is not smooth and the extraction content of the active ingredient is lowered.

The lower alcohol or nonpolar solvent fraction obtained after layer separation is concentrated under reduced pressure at 60 to 70 ° C. to remove the solvent remaining in the sample. The extract obtained after concentration is azeotropically concentrated two to three times with water of 25 to 50 times the total amount of the extract, and again suspended homogeneously by adding the same amount of water. The reason for azeotropic concentration with water at the time of concentrated drying is to effectively control the content of the remaining lower alcohol in order to use the obtained herbal extract as a pharmaceutical raw material.

The extract thus obtained is lyophilized to obtain a powdery extract. The extract is excellent in inhibiting articular tissue degrading enzymes and protecting joints. Thus, the herbal medicine using the extract is useful as a joint protecting agent.

It contains the powder extract obtained in the present invention, and is formulated by a conventional manufacturing method to prepare tablets, capsules, injections, and the like, of which lactose, microcrystalline cellulose, magnesium stearate, and the like, which are used as bases in the manufacture of tablets, are combined with the present invention. The use of extract in a ratio of 2: 1 can produce tablets that are active in treating arthritis.

In the preparation of such pharmaceuticals, herbal extracts may be used as such, but may be mixed with pharmaceutically acceptable carriers, excipients, diluents, and the like as powders, granules, capsules, or injections. Manufacturing is possible. In addition, the herbal extract according to the present invention has been used for food and medicinal use since ancient times, and there is no particular restriction on its dosage, body absorption, weight, age, sex, health status of the patient, administration time, administration method, excretion Rate, the severity of the disease, and the like. In general, the herbal extract is preferably administered about 0.1 to 10 mg per 1 kg body weight. Therefore, the composition containing the active ingredient of the present invention is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way according to the judgment of the expert and the needs of the individual to monitor or observe the administration of the drug as needed Specialized medications can be used or administered at regular intervals.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited by the examples.

Example 1 Preparation of Gastric Extract

After mixing evenly 250 g of the small stomach line about 3.0 cm, 2 liters of water was added and stirred well for 5 hours while stirring well. The filtrate was collected and collected, and the residue was added with 2 L of water and extracted by reheating for 3 hours. The filtrates were combined and concentrated to 1 L. The same amount of saturated n-butyl alcohol was added thereto, and the layers were separated three times. Then, only n-butyl alcohol was collected and concentrated under reduced pressure until the herbal extract was dried at 65 ° C. Most of n-butyl alcohol and water were evaporated, 0.1 L of water was added, the azeotropic concentration was repeated, and then repeated twice. Finally, the same amount of distilled water was added and suspended, followed by freeze-drying to obtain a powder gasoline extract.

The oleanolinic acid content of the herbal extract extracted and purified by the above method was 4.5% (w / w) and the isoferulic acid content was 0.2% (w / w).

Example 2 Preparation of Gastric Extract

After mixing evenly 250 g of a small stomach line about 3.0 cm, 2 L of 30% ethanol was added thereto, followed by extraction under reflux with stirring for 5 hours. The filtrate was collected and collected, and the residue was added to 2 L of 30% ethanol, and refluxed for 3 hours, and the filtrates were combined and concentrated to 1 L. The same amount of saturated n-butyl alcohol was added thereto, and the layers were separated three times. Then, only n-butyl alcohol was collected and concentrated under reduced pressure until the herbal extract was dried at 65 ° C. Most of n-butyl alcohol and water were evaporated, 0.1 L of water was added, the azeotropic concentration was repeated, and then repeated twice. Finally, the same amount of distilled water was added and suspended, followed by freeze-drying to obtain a powder gasoline extract.

The oleanolinic acid content of the herbal extract extracted and purified by the above method was 8.7% (w / w), and the isoferulic acid content was 0.4% (w / w).

In addition, the chromatogram analyzed by the method by HPLC is shown in Figure 1, wherein the HPLC analysis conditions are as follows.

1) Solvent:

division 0 min 60 minutes 70 minutes 80 minutes 0.1 NH ₃ PO ₄ 10% 60% 50% 50% Acetonitrile 90% 40% 50% 50%

2) Column: J'sphere (YMC) ODS-H80 (250 × 4.6 mm I.D., 4 μm)

3) flow rate: 1.0 ml / min

4) Detector: UV 254 nm

Comparative Example 1 Preparation of the Gastric Extract

After mixing evenly 250 g of the small stomach line about 3.0 cm, 2 liters of water was added and stirred well for 5 hours while stirring well. The filtrate was collected and collected, and the residue was added with 2 L of water and extracted by reheating for 3 hours. The filtrates were combined and concentrated to 1 L. Finally, the same amount of distilled water was added and suspended, followed by freeze-drying to obtain a powdered gasoline extract.

The oleanolinic acid content of the herbal extract extracted and purified by the above method was 0.2% (w / w), and the isoferulic acid content was 0.02% (w / w).

Comparative Example 2 Preparation of the Gastric Extract

After mixing evenly 250 g of a small stomach line about 3.0 cm, 2 L of 30% ethanol was added thereto, followed by extraction under reflux with stirring for 5 hours. The filtrate was collected and collected, and the residue was added to 2 L of 30% ethanol, and refluxed for 3 hours, and the filtrates were combined and concentrated to 1 L. Finally, the same amount of distilled water was added and suspended, followed by freeze-drying to obtain a powdered gasoline extract.

The oleanolinic acid content of the herbal extract extracted and purified by the above method was 0.8% (w / w), and the isoferulic acid content was 0.05% (w / w).

Experimental Example 1: Proteoglycan degradation inhibition test

In order to compare the joint tissue protective activity of the extracts prepared in Examples 1 and 2 and Comparative Examples 1 and 2, proteoglycans among the main components of cartilage tissue under conditions for culturing rabbit joint tissues and promoting cartilage degradation The degradation inhibition activity test of) was performed, and the results are shown in Table 1 below.

Experimental Method

Cartilage tissue was isolated from rabbit joints of 5 weeks old, and cultured at 55 ° C. in culture vessels set at 37 ° C., and then the extracts prepared according to Examples 1 and 2 and Comparative Examples 1 to 3 were added and interleukin one alpha ( Interleukin 1α, IL-1α) was added at a concentration of 5 ng / ml per cartilaginous tissue, followed by 60 hours of incubation, causing degradation of proteoglycans in the constituents of cartilage tissue, resulting in the degradation of proteoglycans in the culture medium. ) Was quantified by absorbance at 525 nm to the extent to which the 1,9-dimethylmethylene blue dye (1,9-dimethylmethylene blue dye) was developed.

division Test concentration (mg / ml) Glycosaminoglycan production inhibition rate (%) Example 1 1.0 87 0.3 65 0.1 57 0.03 39 Example 2 1.0 90 0.3 69 0.1 54 0.03 40 Comparative Example 1 1.0 66 0.3 45 0.1 34 0.03 22 Comparative Example 2 1.0 68 0.3 48 0.1 36 0.03 21

As shown in Table 1, the extract prepared as in the present invention can be seen that the degree of inhibition of proteoglycan degradation of the joint tissue is large.

Experimental Example 2: Arthritis Treatment Effect Test

In order to compare the joint tissue protective activity of the extracts prepared in Examples 1 and 2 and Comparative Examples 1 and 2, collagenase (Collagenase) is injected into the rabbit joint cavity to damage the joint tissues and thus tissues similar to human arthritis Rabbit model experiments showing pathological findings were performed, and the results are shown in Table 2 below.

Experimental Method

1 mg of collagenase per 1 kg was injected into 2.0-2.5 Kg rabbit joint cavity on the 1st and 4th day of the experiment to cause damage to the joint tissue similar to arthritis. Examples 1 to 2 and Comparative Examples 1 to 1 The extract prepared by 3 was orally administered for 4 weeks at 200 mg per 1 kg of rabbit (5 per group). Four weeks after the induction of arthritis, the rabbits were dissected to separate the joint tissues, demineralized, stained with safranin-O, and the joint tissues were observed by cartilage and synovial tissue under a microscope. Histopathological lesions were scored (no lesions: 0, fine lesions: 1, positive lesion findings: 2, severe lesions: 3, very severe lesions: 4).

division Cartilage tissue ¹⁾ Synovial tissue ²⁾ Total ³⁾ Control group (no treatment group) 11.9 10.7 22.6 Example 1 6.8 6.8 13.6 Example 2 6.4 6.4 12.8 Comparative Example 1 7.6 7.7 15.3 Comparative Example 2 7.2 7.3 14.5 1) Cartilage tissue (24 points of complete arthritis): Loss of superficial layer, cartilage erosion, destruction and damage of fibrotic cells 2) Synovial tissue (24 cases of complete arthritis): Hyperplasia of inner layer, hypertrophy, cell infiltration, proliferation of granulation tissue 3) Total (48 points of complete arthritis)

As shown in Table 2, it can be seen that the extract prepared as described in the present invention has an excellent degree of alleviating lesion intensification in histopathology by protecting joint tissue in the arthritis model induced by collagenase.

Preparation Example 1 Preparation of Tablet

Using the herbal extract of the present invention, the tablet for oral administration was prepared by using the wet granulation method and the dry granulation method with the following composition.

[Furtherance]

Herbal extract 200 mg, light silicic acid anhydrous 10 mg, magnesium stearate 2 mg, microcrystalline cellulose 50 mg, starch glycolate 25 mg, corn starch 113 mg, ethanol anhydride.

Preparation Example 2 Preparation of Ointment

The herbal extract of the present invention was used to prepare an ointment with the following composition.

[Furtherance]

Herbal extract 5 g, cetyl palmitate 20 g, cetanol 40 g, stearyl alcohol 40 g, myristan isopropyl 80 g, monostearic acid sorbitan 20 g, polysorbate 60 g, paraoxybenzoic acid propyl 1 g, para 1 g of methyl oxyanate, phosphoric acid and purified water

Preparation Example 3 Preparation of Injection

Using the herbal extract of the present invention was prepared an injection with the following composition.

[Furtherance]

Herbal extract 100 mg, mannitol 180 mg, sodium dihydrogen phosphate 25 mg, water for injection 2974 mg

Preparation Example 4 Preparation of Transdermal Agent

The herbal extract of the present invention was used to prepare a transdermal drug in the following manner.

[Composition 1]

Herb extract 0.4 g, sodium polyacrylate 1.3 g, glycerin 3.6 g, aluminum hydroxide 0.04 g, methyl paraben 0.2 g, water 14 g.

[Composition 2]

0.8 g of herbal extracts, 1.6 g of propylene glycol, 0.8 g of liquid paraffin, 0.4 g of isopropyl myristate, 16.4 g of gel bar 1430.

Experimental Example 3: Toxicity Test

Toxicity test was performed on the herbal extract of the present invention as follows. Specifically, herbal extracts were dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to the mice (10 mice per group) at 1 g / kg and observed for 7 days, but no rats died.

As described above, the herbal extract containing the active ingredient extracted from the gastric glands is useful as a joint protecting agent by showing cartilage inhibitory action and joint protection action.

Claims (6)

The herbaceous herb extract is an active ingredient, characterized in that the herbal composition for protection of arthrosis, characterized in that the oleanolinic acid and more than 2.0% by weight and isoferulic acid in the total extract as an active ingredient. The herbal composition for protecting joints according to claim 1, wherein the oleanolinic acid is present in an amount of 2.0 to 10.0% by weight and isoperulic acid in an amount of 0.1 to 0.5% by weight. Extracting the gastric gland with water or alcoholic aqueous solution, layering the filtrate obtained in the extraction step with the same amount of lower alcohol or non-polar solvent, concentrating under reduced pressure, azeotropically concentrating the extract obtained in the concentration step with water and freeze-drying Method of producing a herbal extract, characterized in that consisting of the steps of preparing a powder extract. According to claim 3, wherein the extraction step is reflux extracted with 10-15 times water or lower alcoholic aqueous solution of the gasoline, and then the residue obtained is 7 to 12 times water or lower alcoholic aqueous solution of the mixed drug weight The method of manufacturing a herbal extract, characterized in that the addition of the filtered, and filtered by the method of filtering again by adding the reflux extraction filtrate. The herbal composition selected from claims 1 to 2 is characterized in that it is contained as an active ingredient of joint protection and arthritis treatment. The joint protector according to claim 5, wherein the joint protector is an oral tablet, an oral capsule, an ointment, an injection or a transdermal drug.