JP2009534374A - Anti-inflammatory or analgesic composition comprising white flower serpentine extract extract containing ursolic acid and oleanolic acid - Google Patents

Abstract

  An anti-inflammatory or analgesic composition comprising a purified white flower serpentine grass extract containing ursolic acid and oleanolic acid is disclosed. The ursolic acid and oleanolic acid content of the white flower serpentine extract is 80 to 95% by weight, and the ursolic acid: oleanolic acid ratio is 1: 0.05 to 0.6. .

Description

  The present invention relates to an anti-inflammatory or analgesic composition comprising a purified extract of Oldenlandia diffusa containing ursolic acid and oleanolic acid.

  Rheumatoid arthritis, which is a typical intractable inflammatory disease, is an autoimmune inflammatory disease induced by an abnormal immune reaction caused by the infiltration of inflammatory cells in the joint space, resulting in severe pain, joint deformation, and loss of function. It is a feature. For this reason, the principle of arthritis treatment used in the clinic aims to suppress pain and inflammation and minimize loss of joint function. Non-steroidal (NSAIDs) anti-inflammatory drugs such as aspirin and low-dose oral steroidal immunosuppressants such as methotrexate and imran are used as primary drug treatments for arthritis. I have an injection.

  However, although these drugs are known to have excellent anti-inflammatory effects and are frequently used in clinical practice, their use must be limited due to severe side effects (van Ede et al., Semin Arthritis Rheum., 27: 277, 1998; Peng S & Duggan, Expert. Opin. Drug Saf., 4: 157, 2005). For this reason, medical interest and research for effectively treating arthritis are still ongoing, and a huge budget is being spent on the development of new drugs with excellent therapeutic effects.

  In order to eliminate the above-mentioned side effects, arthritis treatments and therapeutic agents using natural products have been developed and applied actively in the clinic (Darshan & Doreswamy, Phytother. Res., 18: 343). , 2004). The therapeutic agents using some natural products and extracts thereof have been proven to have a strong anti-inflammatory effect and have the advantage of being extremely safe due to few side effects.

  On the other hand, the white flower serpentine grass is an annual plant (Hedyotis lindleyanna var. Hirsuta HARA or Oldenlandia diffusa L. ROXB.) That belongs to the Rubiaceae family. The branches are split and stand diagonally, with some white hair consisting of many cells (Yoshida. Y. et al., Int. J. Immunopharmacol., 19: 359, 1997). White flower serpentine grass is cold, but not poisonous, sweet and bitter, and the meridians that act are transgastric, trans large intestine, trans small intestine, etc. active blood circulation against stasis), various inflammations such as lung fever cough, tonsillitis, sore throat, appendicitis, diarrhea, jaundice, adnexitis, arthritis, digestive cancer, liver cancer It is used for various cancers such as lung cancer, lymph cancer and throat cancer (Tong, IH et al., Phytochemistry, 25: 1988, 1986).

  Of the natural product extracts, ursolic acid is a pentacyclic triterpenoid compound extracted from various plants, and its efficacy and mechanism in treating arthritis are known (Miceli, N. et al. ., J. Ethnopharmacol ,, 97: 261, 2005; Park et al., Phytother. Res., 18: 930, 2004).

  Existing anti-arthritis drugs aiming to minimize the loss of joint function by suppressing pain and inflammation in the affected area have excellent anti-inflammatory properties, but have serious side effects such as induction of diseases of the gastrointestinal tract There is a problem. In addition, there is a problem that arthritis treatments and therapeutic agents using natural products are inferior in anti-inflammatory effect compared to conventional arthritis therapeutic agents. For this reason, in the present field, the development of an anti-inflammatory and analgesic composition that is safe and effective for the human body is eagerly desired.

  Therefore, the present inventors have made an intensive effort to develop a composition for suppressing inflammation and analgesia, which has an excellent anti-inflammatory effect and analgesic effect, and has no risk of side effects such as induction of diseases of the gastrointestinal tract. As a result, it was confirmed that a composition containing ursolic acid and oleanolic acid extracted from natural white serpentine grass has anti-inflammatory and analgesic effects, and the present invention has been completed.

Detailed Description of the Invention

《Technical issues》
In one aspect, the present invention relates to an anti-inflammatory or analgesic pharmaceutical composition comprising a purified white flower serpentine extract containing ursolic acid and oleanolic acid.

  In another aspect, the present invention relates to an anti-inflammatory or analgesic health functional food containing a purified extract of white flower serpentine grass containing ursolic acid and oleanolic acid and a food additive that is foodically acceptable.

Technical solution

  In yet another aspect, the present invention provides (a) after crushing white serpentine grass, and then from the group consisting of lower alcohol, water, lower organic acid, lower alcohol ester, lower ketone, halogenated hydrocarbon, and mixtures thereof. Extracting with a selected solvent and concentrating; and (b) adding the same amount of ethyl acetate to the obtained concentrate, then concentrating after fractionating twice, and (c) obtaining the obtained Ethyl acetate: acetone: water was added to the concentrate at a ratio of 4.25 to 4.75: 4.25 to 4.75: 0.5 to 1.5 and dissolved at 30 to 60 ° C. Then, storing at 1 to 6 ° C. to form a precipitate, and (d) recovering an extract purified product containing ursolic acid and oleanolic acid from the precipitate, and ursolic acid and The method of manufacturing a antiinflammatory or blossom Hebishita grass purified extract of analgesic containing oleanolic acid.

  Other features and implementations of the invention will become even more apparent from the following detailed description and claims.

The HPLC analysis result of the white flower serpentine extract extraction refinement | purification by this invention is shown. It is the graph which collected the exudate in the sac after inducing acute inflammation, measured the number of leukocytes which migrated, and analyzed it as an index of inflammation. After centrifugation of the air sac (air-pouch) exudate is a graph showing the amount of PGE ₂ was measured by collecting the supernatant. It is a graph which measures and shows the edema of the right side (A) and the left side (B), respectively. It is the photograph which X-ray-photographed the right ankle of the mouse | mouth after 21 days passed since arthritis was induced. It is a graph which shows the result of having analyzed the effect of the white flower serpentine grass extract and refinement | purification (OE) with respect to the swelling of the soft tissue by arthritis, and the new bone growth through X-ray imaging of the foot part of the animal. After a lapse of 21 days since the induced arthritis is a graph showing the measured concentration of plasma PGE ₂ each group of blood were collected. It is a graph which shows the result of having evaluated the influence which the administration of the extract refinement | purification product of this invention gave to the sensitivity with respect to a physical irritation | stimulation after arthritis was induced | guided | derived, Comprising: Shows analgesic effect on left foot. It is the graph which evaluated the influence which white flower serpentine grass extract refinement | purification product (OE) exerted on the sensitivity with respect to the heat stimulus induced after arthritis was induced | guided | derived, Comprising: It shows thermal stimulus sensitivity. It is the photograph which shows the expression level of Fos protein which is a neuron | cell to leave in pain, A is what administered the excipient | filler, B is what administered ibuprofen, and C is the white flower snake of this invention. Tongue extract (OE) was administered. Graph showing the number of Fos protein-containing neurons in the right spinal cord due to chronic pain in the right foot that primarily induced arthritis (A) and the number of Fos-containing neurons in the left spinal cord explaining the metastasis of arthritis to the left foot (B) It is. It is the graph which measured the weight increase rate of the experimental group by which the white flower serpentine grass extract refinement | purification material (OE) of this invention was processed.

  The white flower serpentine extract and purified product according to the present invention can be produced through the following steps: (a) After crushing white flower serpentine grass, lower alcohol, water, lower organic acid, lower alcohol ester, lower ketone Extracting with a solvent selected from the group consisting of a halogenated hydrocarbon and a mixture thereof, and then concentrating; and (b) adding the same amount of ethyl acetate to the obtained concentrate, followed by fractionating twice. And (c) adding ethyl acetate: acetone: water to the concentrate obtained in a ratio of 4.25 to 4.75: 4.25 to 4.75: 0.5 to 1.5. And adding at a temperature of 30 to 60 ° C., storing at 1 to 6 ° C. to form a precipitate, and (d) purifying an extract containing ursolic acid and oleanolic acid from the precipitate. Collect Step.

  As the lower alcohol, methanol, ethanol, propanol, butanol and the like can be used, and as the lower organic acid, acetic acid, propionic acid, succinic acid, adipic acid and the like can be used. The lower alcohol ester may be methyl acetate or ethyl acetate, and the lower ketone may be acetone or methyl ethyl ketone. As the extraction solvent according to the present invention, it is most preferable to use an 80% aqueous methanol solution or water.

  The method for producing a purified extract of white flower serpentine according to the present invention is characterized in that a compound excellent in anti-inflammatory or analgesic effect can be obtained from white flower serpentine in a high yield by a simple method.

The extract of purified white flower serpentine according to the present invention exhibits anti-inflammatory and analgesic effects in an air sac acute inflammation model derived from zymosan and a chronic arthritis experimental animal model using Freund's adjuvant. The air sac inflammation model derived from zymosan has been widely used for the evaluation of efficacy against arthritis and related mechanism research by showing pathological symptoms similar to various inflammatory reactions shown in arthritic patients. The present inventors conducted an air sac experiment in advance in the present invention, and verified the anti-inflammatory effect of the white flower serpentine extract and purified product with various doses. As a result, from the 25 mg / kg dose treatment group, leukocyte migration in the air sac was effectively suppressed, and the maximum anti-inflammatory effect could be observed at a dose of 50 mg / kg. In addition, the PGE ₂ concentration of exudates in the air sac, which is known to increase due to an inflammatory reaction, was effectively suppressed at a dose of 50 mg / kg.

In chronic arthritis experiments, it was observed that administration of the composition according to the present invention showed the same degree of anti-inflammatory and analgesic efficacy as ibuprofen used as a positive control group. In particular, as a result of X-ray image analysis, it was observed that the new bone growth inhibitory effect was extremely excellent, and that it showed an excellent analgesic effect even in thermal hyperalgesia to physical and thermal stimuli. It can also be seen that the number of Fos protein-containing neurons expressed in the spinal cord during peripheral pain also decreases sharply compared to the negative control group (excipient). In particular, the concentration of PGE ₂ in plasma that increased in the chronic arthritis group was suppressed to the same level as that of the ibuprofen treatment group, which was a positive control group, by the purified white flower serpentine extract of the present invention.

  In addition, in order to determine whether or not the composition of the present invention can be used as a therapeutic agent, experiments on physiology and side effects were conducted. As a result, the administration of the purified extract of white flower serpentine grass containing ursolic acid and oleanolic acid according to the present invention is different from ibuprofen in that there is no decrease in body weight gain rate, decrease in feed intake rate, and digestive absorption disorder. I was able to observe. In the gastric damage experiment, the ursolic acid and oleanolic acid groups did not show a statistically different degree of gastric damage from the excipient group, whereas the ibuprofen group showed significant damage to the gastric mucosa compared to the control group. It can be seen that it increases.

  Summarizing the above results, the white serpentine extract-purified product containing ursolic acid and oleanolic acid according to the present invention has the same effect as ibuprofen, a NSAID drug, but has side effects on the gastrointestinal tract. It can be seen that the primary safety is greatly reduced.

  Therefore, the present invention provides an anti-inflammatory or analgesic pharmaceutical composition comprising a white flower serpentine extract-purified product containing ursolic acid and oleanolic acid.

  In the anti-inflammatory or analgesic pharmaceutical composition according to the present invention, the ursolic acid and oleanolic acid content of the purified white serpentine grass extract is 80 to 95% by weight, and the ursolic acid: oleanolic acid is used. The ratio is 1: 0.05 to 0.6. A white flower serpentine extract and purified product having the above-mentioned content ratio shows an even better effect in anti-inflammatory or analgesia than those using ursolic acid or oleanolic acid alone, and this is because ursolic acid and oleanolic acid work together It is recognized that this is due to the synergistic effect shown.

  The pharmaceutical composition according to the present invention includes birch, bamboo shoots, bamboo shoots, Paecilomyces japonica, cordyceps Militaris, yamabushitake, salmon, agaricus, bekkotake, maitake, moth, aritake ment, It can further contain one or more herbal extracts extracted from the group consisting of shiitake, himematsutake, narake, agaric, matsutake, and fungus extract, and consists of an antihistamine, anti-inflammatory agent, anticancer agent, and antibiotic. It can be formulated together with any one or more drugs selected from the group.

  The antihistamine of the present invention is a drug that antagonizes the action of histamine, which is one cause of allergic diseases, and the composition of the present invention makes it possible to block the possibility of inducing allergic diseases. Refers to a drug for sedating inflammation, and when the anti-inflammatory analgesic is further added to the composition of the present invention, the anti-inflammatory effect can be maximized. The anticancer agent is a general term for chemotherapeutic agents used for the treatment of malignant tumors, and the antibiotic is a substance that suppresses or kills the growth of other microorganisms in a small amount by a metabolite produced by the microorganism. Say. When the substance is further added to the composition of the present invention, effects such as an antiallergic effect and an anticancer effect can be expected in addition to the anti-inflammatory or analgesic effect of the present invention.

  The pharmaceutical dosage forms of the compositions of the invention can be used in the form of their pharmaceutically acceptable salts, or can be used alone or in combination with other pharmaceutically active compounds. Not only can it be used as a suitable set.

  The anti-inflammatory and analgesic pharmaceutical compositions according to the present invention are prepared by oral methods such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and It can be used in the form of a sterile injection solution. Carriers, excipients and diluents that can be included in the composition containing anti-inflammatory and analgesic composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulating, it can be prepared using diluents or excipients such as fillers, fillers, binders, wetting agents, disintegrating agents, surfactants and the like that are usually used. Examples of solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one or more excipients such as starch, calcium, etc. It can be prepared by mixing carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solution-resistant agents, emulsions, syrups, etc., but various excipients in addition to water and liquid paraffin, which are frequently used simple diluents. For example, wetting agents, sweeteners, fragrances, preservatives and the like can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a suppository base, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

  A suitable dose of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for a suitable effect, the composition of the present invention should be administered at 0.0001 to 100 mg / kg / day, preferably at 0.001 to 100 mg / kg / day. Administration may be performed once per day, or may be divided into several times. The dosage does not limit the scope of the present invention in any way.

  The anti-inflammatory and analgesic composition of the present invention can be administered to mammals such as rats, mice, domestic animals and humans through various routes. Any mode of administration is predictable, but can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura or intracranial injection.

  In another aspect, the present invention provides an anti-inflammatory or analgesic health functional food containing a purified extract of white flower serpentine grass containing ursolic acid and oleanolic acid and a food additive that is pharmaceutically acceptable.

  In the anti-inflammatory or analgesic health functional food according to the present invention, the ursolic acid and oleanolic acid content of the purified white serpentine grass extract is 80 to 95% by weight, and the ursolic acid: oleanolic acid is used. The ratio is 1: 0.05 to 0.6.

  The health functional foods according to the present invention include birch, bamboo shoots, bamboo shoots, cordyceps, sanagitake, yamabushitake, bamboo shoots, agaricus, bekko mushrooms, maitake, bamboo shoots, mushrooms, oyster mushrooms, shiitake mushrooms, matsutake mushrooms, It is characterized by further containing one or more kinds of herbal extracts extracted and purified selected from the group consisting of purified products.

  The anti-inflammatory or analgesic composition of the present invention can be used as a main component of food or an additive and auxiliary agent in the production of functional foods and health supplements.

  In this specification, the “functional food” means a food whose functionality is improved by adding the composition of the present invention to the general food. Functionality can be broadly classified into physical properties and physiological functions. When the composition of the present invention is added to general foods, the physical properties and physiological functions of general foods are improved. Is defined as “functional food”.

  For example, the anti-inflammatory or analgesic health functional food can be produced by using the anti-inflammatory or analgesic function of the composition of the present invention, and a functionally enhanced food, a cancer prevention beverage, and the like can be produced. Note that the composition of the present invention can also be applied to the production of dietary therapy or dietary supplements for arthritic patients.

  In addition to the above, the composition of the present invention comprises various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectinic acid and Contains food additives such as its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonates used in carbonated beverages be able to. In addition to these, the compositions of the present invention can contain pulp for the production of natural fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01 to 20 parts by weight per 100 parts by weight of the anti-inflammatory and analgesic composition of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are merely for explaining the present invention in more detail, and it is usual in the art that the scope of the present invention is not limited to these examples by the gist of the present invention. It is clear to those who have knowledge of.

Example 1: Manufacture of purified extract of white flower serpentine grass (OE) containing ursolic acid and oleanolic acid 10 kg of 80% methanol (MeOH) solution was added to 1 kg of white flower serpentine grass over 24 hours at room temperature. Extracted twice. The extract was concentrated 20 times in a vacuum concentrator, the same amount of ethyl acetate was added, and fractionation was repeated twice. After maximally concentrating the ethyl acetate fraction, the solution was dissolved by adding a solution of ethyl acetate: acetone: water = 4.5: 4.5: 1 to the concentrate at 40 to 50 ° C. Precipitation was confirmed by storage at a low temperature (4 ° C.). Centrifugation gave a white serpentine grass extract and purified product (OE) containing ursolic acid and oleanolic acid (purity: 89.9%, ursolic acid: oleanolic acid = 1: 0.37).

The purified white flower serpentine grass extract containing ursolic acid and oleanolic acid was analyzed by HPLC. The analysis conditions are moving bed acetonitrile: water (in 1.25% H ₃ PO ₄ ) = 86: 14 at a detector 206 nm = 86: 14, flow rate 0.5 mL / min, column is Phenomenex Luna C18, 250 * 4.60 mm. is there. FIG. 1 shows the results of HPLC analysis of a white flower serpentine extract and purified product according to the present invention. 2 mg of white flower serpentine extract and purified product are weighed and analyzed by adding 1 mL of 100% methanol. .

  As a result of HPLC analysis, peaks of oleanolic acid and ursolic acid appeared at 15.95 minutes and 16.3833 minutes, respectively.

Example 2: Anti-inflammatory and analgesic efficacy in air sac acute inflammation experimental animal model 2-1: Preparation of experimental animal model For verification of anti-inflammatory effect using air sac model, ICR mice (25 g, Hanrim experimental animal) were used. The light intensity was adjusted every 12 hours, the temperature and humidity were kept constant, and feed and water were frequently supplied. In order to induce acute inflammation in the mouse, an air sac is formed on the back of the mouse to form an artificial independent space, and then 0.1% zymosan (yeast cell wall) is injected in a volume of 500 μL to induce acute inflammation. Triggered.

2-2: Evaluation of anti-inflammatory effect (1) Evaluation of the number of leukocytes migrating to the air sac 70 ICR mice (25 g, Hanrim experimental animal) were selected and administered with the drugs listed in Table 1 below. 10 animals received vehicle only, 10 animals received ibuprofen and the remaining 50 animals (12.5 mg / kg, 25 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg; 10 animals each) Was administered with the white serpentine grass extract purified product (OE) containing ursolic acid and oleanolic acid prepared in Example 1.

  The same amount of excipient, ibuprofen, and white flower serpentine extract (OE) were administered in a volume of 50 μL / 10 g using a sonde, and 1 hour after the administration of the drug, After forming an artificially independent space, 0.1% zymosan (yeast cell wall) was injected in a volume of 500 μL to induce acute inflammation. After inducing acute inflammation, exudates in the sac were collected and the number of leukocytes migrated was measured and analyzed as an index of inflammation (FIG. 2). As a result, as shown in FIG. 2, as a result of the anti-inflammatory effect evaluation experiment using the white flower serpentine extract purified product (OE) of the present invention, the white flower serpentine grass extract purified product (OE) 25 mg / kg was used as an excipient. Compared to the control group, it was observed that the migration of inflammatory cells in the air sac was suppressed, and an excellent anti-inflammatory effect could be observed. In particular, the present invention shows the maximum anti-inflammatory effect at 50 mg / kg of white flower serpentine extract (OE), which is similar to the anti-inflammatory effect observed in the drug control group ibuprofen treatment (100 mg / kg). It was.

(2) Measurement of PGE ₂ amount in air sac exudates 10 mice administered vehicle, 10 mice administered ibuprofen, and white serpentine grass extract purified product (OE) of the present invention (50 mg / kg) After centrifuging the exudate extracted from the above-mentioned 2-2-1 of 10 mice administered with the supernatant, the supernatant was collected and the amount of PGE ₂ was measured (FIG. 3). As a result, as shown in FIG. 3, the oral administration group of white flower serpentine grass extract purified product (OE) 50 mg / kg of the present invention had a higher release amount of PGE ₂ than ibuprofen. It can be observed that the amount of PGE ₂ released is significantly reduced compared to the group.

  When summarizing the results of the above (1) and (2), the anti-inflammatory effect induced by the purified extract of white flower serpentine grass (OE) of the present invention is due to suppression of cyclooxygenase activity which plays an important role in acute inflammatory reaction I was able to confirm. Furthermore, 50 mg / kg white flower serpentine grass extract purified product (OE) was applied to the anti-arthritis efficacy evaluation experiment using the chronic arthritis experimental animal carried out as follows.

Example 3: Anti-inflammatory and analgesic efficacy in an experimental animal model of chronic arthritis 3-1: Preparation of an experimental animal model For verification of anti-inflammatory efficacy using a chronic arthritis model, Sprague-Dawley rats (180 g, Seoul National University Experimental Animal Research) ), The light intensity was adjusted every 12 hours, the temperature and humidity were kept constant, and feed and water were supplied freely.

  Twenty-four Sprague-Dawley rats were selected and administered with the drugs listed in Table 2. 8 animals received vehicle only, 8 animals received ibuprofen, and the remaining 8 animals received white snake tongue extract extract (OE) containing 50 mg / kg of ursolic acid and oleanolic acid of the present invention. did.

  The same amount of excipient, ibuprofen, ursolic acid and oleanolic acid extract of the present invention (OE) was administered in a volume of 50 μL / 10 g using a sonde. One hour after the administration of the drug, an air sac is formed to form an artificial independent space, and then Freund's adjuvant prepared for inducing arthritis (Mycobacterium butyricum is used as mineral oil). 50 μL each was injected into the sole of the rat.

3-2: Anti-inflammatory evaluation The drug was administered from the 12th day to the 21st day after inducing arthritis. The following items were measured as indices for pain and inflammation for arthritis, and the baseline value (day 0) was measured before inducing arthritis. The experiment was terminated 21 days after arthritis induction.

(1) Measurement of ankle edema After induction of chronic arthritis by injecting Freund's adjuvant, ankle edema could be observed in the right foot due to primary inflammatory reaction after about 3 days (FIG. 4 (A)). ). After about 12 days from the induction of arthritis, ankle edema was also observed from the left foot due to a secondary inflammatory reaction caused by a systemic immune reaction (FIG. 4B). As a result of verifying the anti-inflammatory effect by orally administering the white flower serpentine extract extract (OE) of the present invention from the 12th day after induction of arthritis, as shown in FIG. 4, the white flower serpentine extract extract (OE) of the present invention was obtained. ) The 50 mg / kg administration group was evaluated to effectively suppress ankle edema in both the primary inflammation and the metastasized left foot. Moreover, since the inhibitory effect of the ankle edema of the white flower serpentine grass extract purified product (OE) of the present invention shows an effect similar to that of the ibuprofen administration group which is a drug control group, it has an extremely excellent anti-inflammatory effect. It was evaluated.

  FIG. 5 is an X-ray photograph of the right ankle of the mouse 21 days after induction of arthritis. The ibuprofen 100 mg / kg administration group (B) and the white flower serpentine extract extract (OE) 50 mg / kg of the present invention were administered. It can be seen that the group (C) has a significantly high ability to suppress ankle edema. FIG. 6 is a graph showing the results of analyzing the effect of drugs on soft tissue swelling and new bone growth due to arthritis through X-ray imaging after X-ray imaging of an animal's foot as described above.

  As a result, as shown in FIG. 6, in the case of the right ankle of the experimental animal group to which the white flower serpentine grass extract purified product (OE) of the present invention was orally administered, soft tissue swelling and new bone growth induced by arthritis It can be seen that is significantly suppressed. In addition, it can be seen that the new bone growth of the left foot induced by the secondary inflammatory reaction is also reduced by the administration of the white flower serpentine extract extract (OE) of the present invention. In particular, the white serpentine grass extract and purified product (OE) of the present invention has an extremely excellent effect on the growth of new bone, and is similar to the inhibitory effect observed in the ibuprofen treatment group, which is a drug control group. .

(2) Measurement of PGE ₂ concentration in plasma As a result of collecting blood in each group and measuring the concentration of plasma PGE ₂ 21 days after arthritis induction, as shown in FIG. In the group to which the white flower serpentine extract extract (OE) containing ursolic acid and oleanolic acid according to the present invention was administered, the PGE ₂ concentration was significantly reduced. This result is similar to the inhibitory effect observed in the ibuprofen treatment group, which is a positive control group, and thus it can be re-verified that the composition of the present invention has an excellent anti-inflammatory effect. It was.

3-3: Analgesic evaluation (1) Mechanical threshold test After arthritis is induced, administration of the white serpentine grass extract purified product (OE) of the present invention affects the sensitivity of mechanical and physical stimuli. The impact was evaluated. The hyperalgesic response to mechanical stimuli is determined by using the analgesic effect measuring device (LE7356, manufactured by LETICA, Germany) by the mechanical force (g) according to the grade applied until the avoidance reaction or the pain complaint every 5 minutes. The average value was applied with the measured experimental value (Randal Celit method). The mechanical pain threshold in normal rats was 140-160 g. As a result of the experiment, the administration group of white flower serpentine extract (OE) showed an analgesic effect excellent in pain induced by physical stimulation after 21 days from the induction of arthritis in both the right foot and the left foot. (FIG. 8). In addition, as shown in FIG. 8A, in the case of the right foot, an analgesic effect superior to ibuprofen, which is a positive control drug, could be observed. Summarizing the above results, the white serpentine extract extract (OE) containing ursolic acid and oleanolic acid of the present invention has an excellent analgesic effect on pain caused by physical irritation when arthritis is induced. You can see that

(2) Thermal threshold test After arthritis was induced, the effect of white serpentine grass extract purified product (OE) on the sensitivity to the induced thermal stimulus was evaluated. The Hargreaves method was used to measure hyperalgesia to thermal stimuli in laboratory animals (Hergreaves et al., 1988). Rats were allowed to sit in a plastic chamber above the glass surface for 5 minutes and then focused and stimulated underneath the glass surface in contact with the sole by radiant heat. The evacuation latent time (sec), which is the time taken to show an avoidance reaction, is measured through a photosensitive cell connected to digital time. The intensity of light was adjusted to show an avoidance response in 9 to 10 seconds in normal rats. The experimental interval was 5 minutes, and the average value was applied after measuring twice. As a result of the experiment after induction of arthritis, the treated group of white serpentine grass extract (OE) containing ursolic acid and oleanolic acid of the present invention compared to the negative control group (vehicle) showed arthritis in both the right and left legs. It was observed that the increase in sensitivity to thermal stimuli sometimes observed was significantly suppressed (FIG. 9). From the results as described above, it can be seen that the white serpentine grass extract and purified product (OE) of the present invention has the same analgesic effect as ibuprofen, which is a positive control group, for heat-sensitive sensitive symptoms caused by arthritis.

(3) Numeric variation search of Fos protein in spinal cord In order to examine the pain-reducing effect of white flower serpentine extract (OE) containing ursolic acid and oleanolic acid of the present invention, it is a nerve cell that is put in pain The degree of expression of Fos protein was examined. The experimental method is as follows. After the experiment was completed (day 21), the animals were inhaled by anesthesia with 5% isoflurane for immunohistochemical staining of c-Fos protein in the spinal cord, perfused from calcium-free tyrode solution through the heart, and then Lunar's fixative Perfusion fixation was performed with (4% paraformaldehyde, 0.2% picric acid, 0.1 M phosphate buffer solution, pH 6.9, TPBS). Immediately after perfusion, the spinal cord was separated and postfixed in the same fixative for 4 hours. And cryoprotection was performed with 30% sucrose (sucrose in PBS, pH 7.4) over 48 hours at refrigerated temperature. The spinal cord was rapidly frozen with siphon gas, and then the L3-L5 spinal cord portion was cut into a 40-m-thick section using a cryostat (manufactured by Microm, Germany). Thereafter, the plate was washed 6 times with TPBS for 5 minutes, quenched with 0.3% H ₂ O ₂ / TPBS to eliminate the activity of endogenous peroxidase, and washed with 1% goat serum / 0.3% Triton X-100. The reaction was allowed to take place at room temperature for 1 hour to block nonspecific reactions (pre-blocking process). The floating section was reacted with a polyclonal rabbit c-Fos antibody (Calbiochem, 1: 10,000) at 4 ° C. for 24 hours and then washed 6 times with TPBS for 5 minutes, and this section was goatanti-RbIgG which is a secondary antibody. (Vector, 1% NGS / Triton X100 / TPBS 1: 200 dilution) and reacted at room temperature for 1 hour. Thereafter, the plate was washed with TPBS 6 times for 5 minutes, and subjected to a streptavidin (avidin-biotin reaction, 0.3% Tritons X / TPBS 1: 200 dilution) reaction at room temperature for 1 hour, and then with TPBS for 5 minutes. Washed 6 times. C-Fos expression was verified through the 3-3 diamino-benzidine (Sigma Chemicals, USA) reaction. Thereafter, the tissue slice was washed 6 times for 5 minutes each with TPBS, and the tissue section was subjected to a dehydration process using alcohol, followed by a clarification process using xylene. After the arthritis was induced, as a result of evaluating the effect of the white flower serpentine extract (OE) on the expression of Fos protein in the spinal cord, as shown in FIG. 10, compared with the negative control group (A), ibuprofen ( It can be seen that a small amount of Fos protein is observed in B) and the white serpentine grass extract purified product (OE) administration group (C) of the present invention. In addition, as shown in FIGS. 10 and 11, in the negative control group (vehicle), FLI in the right spinal cord caused by chronic pain in the right foot that primarily induced arthritis (Fos-like immune reaction, Fos protein expression) The number of nerve cells and the number of FLI neurons in the left spinal cord that explain the metastasis to the left foot of arthritis are superficial dorsal horn (SDH), spinal nuclei (nucleus proprius, NP), neck of dorsal horn, It can be seen that both increase rapidly at (NECK) sites (Kwon et al., Pain, 90: 271, 2001; Kwon et al., Life Sciences, 71: 191, 2002). In contrast, the white flower serpentine extract and purified product (OE) of the present invention has a shallow layer (SDH), spinal cord proper nucleus (NP), and neck (NECK) sites in both the right and left spinal cords. Decreased almost at the NP site in the left spinal cord than in the right side where arthritis was induced (FIG. 11).

  From the results as described above, the white flower serpentine grass extract purified product (OE) of the present invention not only temporarily reduces pain stimulation due to arthritis, but also neural pain into the spinal cord caused by metastasis of arthritis to the left foot. It can be seen that the stimulation is effectively suppressed. In addition, the inhibitory effect on the activity of nerve cells related to such pain is similar to that of the ibuprofen treatment group which is a positive control group. This is because the extract of purified white serpentine grass (OE) of the present invention has an excellent analgesic effect. It turns out that it is the result of reconfirming that it is.

3-4: Evaluation of side effects (1) Body weight measurement In order to confirm whether or not the white flower serpentine grass extract purified product (OE) of the present invention shows side effects on physiological phenomena, from the injection of adjuvant to the end of the experiment The change in body weight was measured every three days to conduct an experiment on the weight gain rate. As a result, as shown in FIG. 12, in the negative control group (excipient), it was observed that the weight gain rate rapidly decreased starting from day 9 after the induction of arthritis. . This suggests that the amount of feed intake decreases as arthritis becomes severe, and even if the feed is ingested, the decrease in the rate of increase in feed due to disease appears. In addition, the ibuprofen-treated group also showed a significant weight loss on the 21st day after inducing arthritis compared to the 9th day after inducing arthritis. It is considered to be caused by gastrointestinal disorders due to decreased activity of endogenous cyclooxygenase (Whittle, Fundam. Clin. Pharmacol., 17: 301, 2003). In contrast, the weight gain rate of the white flower serpentine extract purified product (OE) treatment group of the present invention does not show a statistically significant difference from the 9th day until the 21st day of arthritis induction, It turns out that the weight of the 9th day is maintained.

  From the above results, it can be seen that the white serpentine grass extract and purified product (OE) of the present invention has no side effects on basic physiological phenomena such as weight gain compared to ibuprofen showing similar anti-inflammatory activity.

(2) Gastric ulcer formation test Further, in order to evaluate the influence of the purified white snake tongue extract (OE) containing ursolic acid and oleanolic acid of the present invention on the gastrointestinal tract, the experiment was completed on the 21st day. Rats were necropsied and observed for gastric damage (Table 3). The frequency of lesions was measured by the number of animals with gastric lesions per group (n = 8). The steps of the degree of gastric mucosal lesion were divided into the following. 1 point for a punctate bleeding lesion, 2 points if the lesion site is smaller than 1 mm, 3 points if the lesion site is 1 to 2 mm, 4 points if the lesion site is 2 to 4 mm, and 4 mm for the lesion site Was also 5 (Meeroff et al., 1975). The total lesion score was obtained by adding the lesion scores according to the degree of damage.

  As a result, as shown in Table 3, ibuprofen, which is already known to induce gastrointestinal disorders, showed a considerably higher value of gastric damage when compared to the excipient group, The group treated with the purified extract of white serpentine grass (OE) containing ursolic acid and oleanolic acid according to the present invention was not able to observe a statistically significant difference from the excipient group. When the above results are summarized, it can be seen that the composition of the present invention has low efficacy in the gastrointestinal tract while exhibiting an effect similar to that of a drug already used in clinical practice.

  As described above in detail, the anti-inflammatory and analgesic composition of the present invention is a natural substance-derived substance and exhibits the same anti-inflammatory and analgesic efficacy as ibuprofen, which is a synthetic substance, but also induces gastrointestinal diseases. Therefore, it is safe to administer over a long period of time to patients who should be treated continuously. In addition, the method for producing a white flower serpentine extract and purified product according to the present invention has an advantage that a compound excellent in anti-inflammatory or analgesic effect can be obtained from white flower serpentine grass in a high yield by a simple method. In addition, the white flower serpentine extract-purified product containing ursolic acid and oleanolic acid at a certain ratio according to the present invention shows a synergistic effect by the joint action of both compounds, and ursolic acid or oleanolic acid is used alone. It is even better in anti-inflammatory or analgesic effect than it does.

  For this reason, the composition of this invention can be effectively used as a health functional food and medicine for anti-inflammatory and analgesic, such as arthritis.

  While specific portions of the subject matter of the present invention have been described in detail above, such specific descriptions are merely preferred embodiments for those having ordinary knowledge in the art, and thus the scope of the present invention. It will be clear that is not limited. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (13)

  A pharmaceutical composition for anti-inflammatory, comprising a white flower serpentine extract and purified product containing ursolic acid and oleanolic acid.   A pharmaceutical composition for analgesia comprising a white flower serpentine extract extract containing ursolic acid and oleanolic acid.   The ursolic acid and oleanolic acid content of the white flower serpentine extract and purified product is 80 to 95% by weight, The pharmaceutical composition according to claim 1 or 2.   The pharmaceutical composition according to claim 1 or 2, wherein the ratio of ursolic acid: oleanolic acid is 1: 0.05 to 0.6.   Bamboo shoot, bamboo shoots, bamboo shoots, cordyceps, sanagitake, yamabushitake, bamboo shoots, agaricus, mushrooms, maitake, bamboo shoots, oyster mushrooms, oyster mushrooms, shiitake mushrooms, dairy mushrooms, agaric mushrooms, and mushrooms The pharmaceutical composition according to claim 1 or 2, further comprising one or more kinds of herbal extracts extracted and purified.   The pharmaceutical composition according to claim 1 or 2, which is formulated together with one or more drugs selected from the group consisting of antihistamines, anti-inflammatory analgesics, anticancer drugs, and antibiotics.   A health functional food for anti-inflammation, comprising a white flower serpentine extract and purified product containing ursolic acid and oleanolic acid, and a food additive that is foodically acceptable.   A health functional food for analgesia comprising a white flower serpentine extract extract containing ursolic acid and oleanolic acid, and a food additive that is foodically acceptable.   The health functional food according to claim 7 or 8, wherein a content of ursolic acid and oleanolic acid in the white flower serpentine extract is 80 to 95% by weight.   The health functional food according to claim 7 or 8, wherein the ratio of ursolic acid: oleanolic acid is 1: 0.05 to 0.6.   Bamboo shoot, bamboo shoots, bamboo shoots, cordyceps, sanagitake, yamabushitake, bamboo shoots, agaricus, mushrooms, maitake, bamboo shoots, oyster mushrooms, oyster mushrooms, shiitake mushrooms, dairy mushrooms, agaric mushrooms, and mushrooms The health functional food according to claim 7 or 8, further comprising one or more kinds of herbal extract and purified product. A method for producing an anti-inflammatory or analgesic white serpentine extract extract containing ursolic acid and oleanolic acid, comprising the following steps:
(A) After crushing white flower serpentine grass, it is extracted with a solvent selected from the group consisting of lower alcohol, water, lower organic acid, lower alcohol ester, lower ketone, halogenated hydrocarbon, and mixtures thereof, and then concentrated. Step;
(B) adding the same amount of ethyl acetate to the obtained concentrate, then fractionating twice and then concentrating;
(C) Ethyl acetate: acetone: water was added to the obtained concentrate at a ratio of 4.25 to 4.75: 4.25 to 4.75: 0.5 to 1.5, and 30 to A step of dissolving at 60 ° C. and then storing at 1 to 6 ° C. to form a precipitate; and (d) a step of recovering an extract purified product containing ursolic acid and oleanolic acid from the precipitate.   The method according to claim 12, wherein the solvent is an 80% aqueous methanol solution or water.