KR20110023133A - A mixed herb medicine extracts having osteoarthritis treatment activity - Google Patents

Abstract

PURPOSE: A composition containing herb mixture extract is provided to ensure antioxidation and to suppress osteoarthritis. CONSTITUTION: A herb mixture extract contains 20-30 weight% of Acanthopanax gracilistylus Smith, 20-30 weight% of Achyranthis japonica Nakai, 20-30 weight% of Eucommia ulmoides Oliv., and 20-30 weight% of Chinemys reevesil Gray as active ingredient. A pharmaceutical composition for treating and preventing osteoarthritis contains the herb mixture extract as an active ingredient. A health food composition for treating and preventing osteoarthritis contains the herb mixture extract as an active ingredient.

Description

A mixed herb medicine extracts having osteoarthritis treatment activity with osteoarthritis treatment activity

The present invention relates to herbal extracts of Ogapi, dew, head worm and ear plate having osteoarthritis therapeutic activity, more specifically, herbal extracts of Ogapi, dew, head worm and ear plate having osteoarthritis therapeutic activity and containing it as an active ingredient It relates to a pharmaceutical composition and nutraceutical composition for the treatment and prevention of osteoarthritis.

With the recent improvement in the standard of living and the development of life sciences and medical technology, the life expectancy of humans is prolonged, and the average life expectancy is likely to be 85 years or older within the next 20 years. Is expected to increase and become a full-fledged aging society. As the elderly population increases, patients with degenerative diseases such as dementia, arteriosclerosis, and arthritis are increasing, which has brought economic and mental difficulties to the family and the family, which is becoming a social problem.

Although the market for drugs and functional foods related to degenerative diseases is rapidly increasing due to the increase in the elderly population, the development of fundamental treatment or preventive medicines for degenerative diseases is still insufficient. Prevention and treatment of aging and related diseases are mainly focused on drugs and surgery, and there are few health functional foods in terms of prevention. Therefore, it is necessary to develop a concrete concept of aging prophylaxis which has functional and impact using food-based natural substances that have secured safety in the clinical aspect. It is increasing.

Due to the development of life sciences, functional food materials and products are being developed from medicinal and edible resources that have been used in the private sector, and are rapidly spreading to the industrial world. Especially as Korea enters an aging society, 80% and 75 years of age are over 55 years old. Most of the above cases are known to have a bone joint disease. Arthritis, a representative degenerative disease, is known to have at least 600,000 patients in Korea, and the most popular treatment for bone disease (arthritis, osteoporosis) has been sold worldwide (the global market condition for disease by disease, 2005).

Degenerative diseases such as arthritis are increasing with age and eating habits, and despite the development of various treatments, they still remain serious diseases due to aging population and Western eating habits. It is considered to be a major factor for lowering quality. The discovery of materials for improving degenerative diseases and the evaluation of the efficacy of bioactive substances for the development of functional foods and the establishment of functional foods can accelerate the commercialization and commercialization of more functional food candidates. These diseases are genetic factors and environmental factors such as dietary life. It is very important to study the functional material with biological activity contained in biological material because it can increase the immunity ability by continually ingesting the food with preventive effect.

Another problem for an aging society is that with the development of medical science, as social life improves, the entry of an aging society is accelerating, and attention to skin beauty is increasing due to aging. The causes related to aging have been confirmed by various factors such as stress, ultraviolet rays and free radicals, but there is no solution yet, and there is a tendency to increase the functionality by using various natural herbal materials except molding. To this end, there is a need for an approach to global commercialization through the development of functional materials, the development of delivery system materials to efficiently absorb these materials into the skin, and the development of ease of use and design.

According to data from the Korea Food and Drug Administration in 2008, health functional foods sold in Korea totaled 730.2 billion won in 2007, among which red ginseng products accounted for 45.2%, accounting for 45.2%, followed by aloe products (79.7 billion won) and vitamin / calcium supplements ( 77.7 billion won), ginseng products (35 billion won) and glucosamine-containing products (26.9 billion won). In particular, glucosamine-containing products are steadily sold to the elderly.

However, opinions are mixed between the medical community and related industries as to whether glucosamine is really effective in preventing and treating arthritis. Manufacturers claim that glucosamine promotes the production of proteins that make up cartilage, leading to cartilage health in middle-aged and menopausal women, but most doctors say that pain relief works to relieve joint pain, but there is no proven evidence of preventing or treating arthritis. .

Recently, researches to find functional foods related to arthritis and new natural products from natural products that have been used as traditional medicine or folk medicine have been in the spotlight around the world, especially in developed countries such as the United States, Europe and Japan. Currently, basic and clinical researches to separate and identify neuroprotective substances from natural plants and develop them into medicines are being conducted by research institutes in developed countries, and health functional foods and natural products from natural foods with relatively high stability and excellent effects. The interest of the world's leading pharmaceutical companies is increasing to promote development.

The domestic cosmetics industry, which is related to anti-aging and beauty, is a blue ocean industry that forms a market of 8 trillion won in 2007, and it is supporting the national industry by forming the largest market after the pharmaceutical industry among fine chemical products. In particular, the functional cosmetics market is growing at a high growth rate of more than 8% annually. Currently, more than 90% of functional cosmetic raw materials are dependent on overseas imports, and their import growth rate is accelerating. The development of new materials that can dominate the global cosmetics market with international competitiveness not only in the domestic market but also in the global market is a top priority.

In order to treat arthritis, basic methods such as rest and exercise and physical therapy are used, but medical medications are mainly used.In anti-inflammatory treatment of arthritis, intra-articular injections of non-steroidal anti-inflammatory drugs or glucocorticoids are used. Mainly used. There are antimalarial agents, ezthioprine, gold salt, cyclophosphamide, sulfasalazine, penicillin, and methotrexate, which are used as treatments for controlling joint diseases, but these compounds cause serious side effects and have a small effect.

The cause of arthritis in bone disease is not known with the exception of several diseases, such as purulent and inflammatory arthritis caused by bacterial infection. Recently, many studies have found that the disease-specific genes of congenital factors are found in patients with ankylosing spondylitis or rheumatoid arthritis. There is a bar.

Recently, the approach to inhibit inflammatory cytokines such as IL-6 and TNF-α has been attracting much attention in the treatment of inflammatory diseases such as rheumatoid arthritis. Antibody drugs (such as Reicade) or TNF receptors against TNF cytokines Protein drugs (eg, Enbrel) are currently on the market and are generating high sales.

New arthritis drugs for IL-6 inhibitors are also under development (2006), and prostaglandins that are distributed in organs or body fluids have various physiological effects such as uterine contractions, capillary dilation, gastric secretion suppression, and bronchial muscle contraction and relaxation. New mechanisms of anti-inflammatory drugs are being developed to raise prostaglandin D2 and prematurely terminate the inflammatory response.

The external factors of skin aging are ultraviolet rays, pollution, drinking, stress, and malnutrition. The basic research to delay skin aging caused by these external factors is skin moisturizing, protection, and vitality. In recent years, research has been conducted to develop new moisturizing agents and increase the moisturizing effect by intercellular lipids such as ceramide. Skin protection includes research on UV protection, research on the effective removal of free radicals such as harmful oxygen, and research on enhancing skin immune function.In the vitality level, natural blood or biotechnologies are used to promote blood circulation. Research is underway on substances that promote high skin metabolism.

Retinol, its stabilizing raw materials, and retinol derivatives are the main raw materials for wrinkle improvement, and natural extracts based on folk medicine or traditional medicine are being developed, but they are still weak and the structure of retinol is very unstable and fragile. In addition to retinol, retinol palmitate, and adenosine medicin A, which are known as raw materials by combining technology, various new materials are being promoted by each company as wrinkle-improving functional new materials, and the basic candidates of these functional materials are mainly It is derived from hospital dermatology, and a single substance using skin cell activation effect and collagen synthesis promotion is mainly developed and applied.

About 300 domestic patents related to degenerative arthritis have been applied, and patents related to anti-inflammatory patents of natural materials and compositions for treating arthritis including seaweed extract materials and glucosamine have been applied. In addition, about 120 joint patent applications have been filed by 2005.

Existing arthritis medicines have serious side effects and are less effective, and consumers' preference for functional foods with fewer side effects is increasing. Glucosamine-containing products that are steadily sold to the elderly have little side effects, but medical relief of joint pain Since there is no effective analgesic effect, but no prevention or treatment of arthritis has been proven, the exploration of new functional materials supported by efficacy will lead to an aging society, and the demand for bone disease improvement products will increase rapidly with the interest in joint diseases. Expected

In the present invention, to search for useful ingredients from traditional biological resources based on folk remedies that have been used for a long time without side effects, and to prepare data to develop more effective bone joint protection and bone disease prevention functional food, traditional herbal composition processed food Presenting examples of their transition into health functional foods, and improving their competitiveness of functional materials technology in Korea, which is about 50% compared to developed countries, and developing individual drug certification methods for useful ingredients of traditional materials, led to the present invention. It became.

It is therefore an object of the present invention to provide a mixed herbal extract effective for the treatment and prevention of arthritis.

Another object of the present invention is to provide a composition for treating and preventing arthritis, containing the mixed herbal extract as an active ingredient.

The object of the present invention as described above was achieved by selecting the organs of the herb, hyssop, worms and ear plates, and mixing them to produce a herbal mixture extract, and then confirm the articular treatment and prophylactic effect of the extract.

The present invention provides a herbal medicine mixed extract having therapeutic activity for osteoarthritis, which contains a staphylococcus, hyssop, head worm and ear plate as active ingredients.

In the present invention, "herbal herbal extract" or "herbal extract" refers to an extract prepared by extracting the herbal mixture consisting of the scabies, dew, worms and ear plates in the same way.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the medicine directly or indirectly by inherent pharmacological action. It means containing a main component).

In the present invention, the herbal mixture extract is preferably 20 to 30% by weight, 20 to 30% by weight, 20 to 30% by weight of larvae and 20 to 30% by weight of ear pads.

In another aspect, the present invention provides a pharmaceutical composition for the treatment and prevention of osteoarthritis, containing the mixed extract of the herbal medicines of Ogapi, dew, head worm and ear plate as an active ingredient.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Dosage forms of the pharmaceutical compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

In another aspect, the present invention provides a health functional food composition for improving osteoarthritis, containing the herbal extracts of the ogapi, Haedongcho, safflower seed and eoseongcho as an active ingredient.

"Health functional food" as defined in the present invention means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and "functional" means a human body It means the ingestion for the purpose of obtaining a useful effect in health use such as nutrient control or physiological action on the structure and function of.

In the present invention, to prepare a mixed extract using herbal medicines that are widely used in herbal medicine from ancient times, and to verify the efficacy of osteoarthritis prevention and treatment in osteoarthritis-induced animal models as well as inhibitory effects of inflammation-related factors through in vitro and cell experiments. The experiment was conducted.

13 experimental groups were obtained by obtaining SPF SD (Sprague-Dawley) rats as experimental animals. Osteoarthritis was induced by injecting MIA solution into the left knee cavity of all test groups except the normal control group, and the normal control group received the same amount of saline instead of the MIA solution. After confirmation of arthritis induction, normal control water and arthritis control group received normal drinking water for 1 week, 5 days a week, and 4 weeks from day 6, and the remaining test groups received oral administration of each single natural extract and mixture. Two positive controls were orally administered glucosamine and celecoxib, respectively.

Observations of animal body weight during the entire experimental period showed that the normal control group tended to be higher than the rest, but there was no significant difference. There was no significant difference between the arthritis control group and the rest of the test groups.

Weight load is an indirect indicator of the onset of MIA-induced osteoarthritis and the status of osteoarthritis. In the present study, the weight load was measured 3 days after the MIA injection (Day 4), and all of the osteoarthritis-induced groups were significantly lower than the normal control group. As a result of measuring weight load at weekly interval from day 4, day 11 showed significantly higher weight load in the MIX3 group which mixed Ogapi, hyssop, tofu, and old plate compared to the arthritis control group (OA group). And Celecoxib-treated PC (C) group were significantly higher than the arthritis control group (OA group). In addition, on day 25, the weight load of the MIX3 group was significantly higher than that of the negative control OA group. On day 32, MIX1 group, MIX2 group, MIX3 group, PC (G) group and PC (C) group who received Mix 1, Mix 2, Mix 3, Glucosamine and celecoxib were 12% and 11%, respectively, than OA group. , 14%, 17%, 14% and 23% were observed to increase significantly.

The expression level of TNF-alpha, a representative proinflammatory cytokine, was measured by ELISA to determine the changes in inflammation-related factors in plasma of all experimental groups. Overall, low levels of expression were observed, and there was no significant difference in comparison with the control group.

As a result of measuring ALT and AST contents to confirm liver damage of test substance, no significant difference was found. Also, the histopathological findings of liver tissue did not show any abnormal findings. Therefore, the extracts used in this experiment are considered to be harmless to living organisms.

The activity of antioxidant enzymes changes with or without oxidative stress. On the other hand, when the oxidative stress level of the living body is low, it was reported that the antioxidant activity of tissues and erythrocytes did not significantly change. Intracellular antioxidant enzymes include SOD, CAT, GSH-Px, and GR, which react directly with free radicals and peroxides to prevent oxidative damage to cells.

SOD is an enzyme that converts superoxide anion radicals into H ₂ O ₂ , which causes the formation of other free radicals and lipid peroxidation stages early in the process of generating free radicals. Erythrocyte SOD activity was higher in the MIX1, MIX2, MIX3, PC (G), and PC (C) groups treated with Mixture 1, Mixture 2, Mixture 3, Glucosamine, and celecoxib than in the OA group. Significantly increased compared to the control. The removal of H ₂ O ₂ produced by SOD is responsible for CAT and GSH-Px.

CAT is mainly present in peroxysomes and mitochondria and exhibits reductive activity in molecules smaller than lipid peroxides produced during lipid peroxidation. GSH-Px is distributed mainly in the cytoplasm and acts to reduce H ₂ O ₂ , but not in other cells. It is mainly used to remove toxic peroxides. CAT has catalytic activity for reducing H ₂ O ₂ produced by SOD to H ₂ O and peroxidation activity involved in oxidation of hydrogen donors such as methanol, ethanol, formman and phenol. Erythrocyte CAT activity was significantly increased in the MIX2 group, the PC (G) group, and the PC (C) group administered Mixed 2, Glucosamine and celecoxib.

By the action of GSH-Px, H ₂ O ₂ and reduced GSH become oxidized GSSG in water to generate water. GSH-Px also prevents peroxidative damage to tissues by catalyzing reactions that produce GSSG, alcohol and water from peroxides and GSH. Erythrocyte GSH-Px activity was significantly higher in MIX1 group, MIX2 group and PC (G) group treated with Ogapi, thawed bark, Emulsion, Mixed 1 and Glucosamine. Antioxidant activity was found to be significantly higher in the group treated with test substance than the control group of arthritis. It was also observed that antioxidant activity was significantly increased in MIX1 and MIX2 as well as in the single substance group.

For microscopic observation of articular cartilage, three stainings were performed: H & E, toluidine blue, and Azan staining. As a result of staining, the osteoarthritis-controlled arthritis control group showed severe degeneration of the articular cartilage, while compared to the arthritis control group, the MIX1 (ogapi + thawing skin + dew) group, the EU (tobacco) group, and the MIX2 (ogapi + thawing skin + safflower seed + Echocho) In the cartilage group and PC (G) (glucosamine) group, the exposure of the calcified zone was observed in some cartilage, but the exposure range was not wide, and the proliferation of cartilage cells to repair the cartilage damage was observed. The results of scoring histopathological findings on articular cartilage were similarly similar to the MIX1 (ogapi + thawing skin + ursula) group, the EU (tofu) group, the MIX2 (ogapi + thawing skin + safflower seed + estuary) group and PC (G) ( Glucosamine) group was observed to be significantly reduced than the arthritis control group.

In conclusion, after administration of the three mixtures, the administration of Ogapi, Tofu, MIX1 (Ogapi + Haedongpi + Wooslung), MIX2 (Ogapi + Haedongpi + Safflower + Eochocho) and MIX3 (Ogapi + Tooth + Bichung + Earpan) induced osteoarthritis One animal model has been shown to have the effect of inhibiting and alleviating the progression of osteoarthritis. In addition, antioxidant enzyme activity was significantly increased, and histopathological findings on articular cartilage were significantly higher than those of the control group. Histopathological observations of articular cartilage showed that the loss of cartilage substrates such as proteoglycans and collagen of articular cartilage was alleviated compared to the arthritis control group, and the weight load of these substance groups was significantly improved compared to the arthritis control group. Therefore, supplementation of MIX1 (ogapi + thaw skin + hyssop), MIX2 (ogapi + thaw skin + safflower + Eochocho) and MIX3 (ogapi + hyssop + nematode + gupan) seems to have an effect of inhibiting the progression of osteoarthritis and protecting bone joints. .

As described above, the herbal extracts of Ogapi, dew, head worm and ear plate according to the present invention are harmless to the living body, have excellent antioxidant activity, proliferate chondrocytes for repairing cartilage damage, inhibit the progression of osteoarthritis, There is an excellent effect of protecting the bone joints, the present invention is a very useful invention in the pharmaceutical and nutraceutical industry for the treatment and prevention of osteoarthritis.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1: Preparation of Herbal Mixture Extract

In this example, six herbal medicines (see Table 1), which are widely used as herbal medicines in oriental medicine and private prescription, were selected and used as test materials.

TABLE 1

Common name Scientific name part Supply source Two tooth Eucommia ulmoides Oliv. (EU) fell Domestic production Eoseongcho Houttuynia cordata Thunb. (HC) leaf Domestic production Ogapi Acanthopanax gracilistylus Smith (AG) fell Made in China Dew Achyranthis japonica Nakai (AJ) Root Domestic production Safflower Carthamus tinctorius L. (CT) Seed Domestic production Thawed blood Kalopanax pictus (KP) skin Made in China

Three herbal preparations were prepared by mixing as shown in Table 2 below to prepare herbal extracts for the treatment of arthritis by using the six single herbal samples.

TABLE 2

Herbal mixture (Abb.) Composition herbal MIX-1 (AKA) Ogapi (AG), thawed blood (KP), right dew (AJ) MIX-2 (AKCH) Ogapi (AG), Haedongpi (KP), Safflower (CT), Eoseongcho (HC) MIX-3 (AAEC) Ogapi (AG), Usel (AJ), Toothworm (EU), Ear plate (CR ^* )

^* Gwipan (Gray Chinemys reevesil; CR)

100 g of the herbal mixture of Table 2 was ultrasonically extracted twice at 40 ° C. for 3 hours using 1 L of ionized water, followed by filtration (Whatman No. 2). The filtrate was concentrated under reduced pressure, dissolved in 100 ml of ionized water, cooled, and the supernatant was concentrated under reduced pressure again to prepare a hot water extract.

An ethanol extract was prepared in the same manner as above except that 80% ethanol was used as the extraction solvent.

Example 3 Quantification of Functional Substances (Total Polyphenol Content and Total Flavonoid Content Determination)

Among the many phytochemicals present in plants, polyphenolic substances are widely distributed in various foods, and they can act as natural antioxidants. In particular, flavonoids, which are the main plant-based polyphenols, have been reported to have various physiological activities such as antioxidant, anticancer, hypertension suppression, caries prevention, and anti-inflammatory.

Therefore, the present embodiment is present in the ethanol extracts of M-1 (ogapi, haedongpi, wort), M-2 (ogapi, haedongpi, safflower seed, Eochocho), M-3 (ogapi, wedge, tofu, old plate) The total polyphenol content was measured using tannic acid as a reference material, and the total flavonoid content was determined using quercetin as a reference material.

The total polyphenol content was applied to the Folin-Denis method. Dissolve 1 mg of each sample in 1 mL of distilled water, add 2 mL of 2-fold dilute Folin reagent to 2 mL of 10-fold dilution, mix well, leave for 3 minutes, add 2 mL of 10% Na ₂ CO ₃ , and add 1 After the reaction time, the content was determined from a standard curve prepared by measuring absorbance at 700 nm using a UV / Visible spectrophotometer (UVIKON 922, Kontron, Italy). At this time, the standard curve using tannic acid was prepared by measuring the absorbance at 700 nm in the same manner as the final concentration of tannic acid to 0, 5, 25, 50, 75 μg / mL.

The total flavonoid content of the sample was measured using the method of Nieva Moreno et al. To 0.5 mL of a mixture of 0.1 mL of each sample and 0.9 mL of 80% ethanol, 0.1 mL of 10% aluminum nitrate, 1 M potassium acetate, and 4.3 mL of 80% ethanol were added to room temperature for 40 minutes, and the absorbance was measured at 415 nm. It was. The total flavonoid content was calculated from the standard curve prepared using quercetin.

As shown in Table 3 below, the total polyphenol content of M-2 ethanol extract was 58.98, 60.79 and 57.74 mg / g, respectively, showing a content of 50 mg / g or more. Flavonoid content showed similar tendency to polyphenol content, and M-2 showed higher content than other herbal extracts.

TABLE 3

sample Total polyphenols ^One
(mg / g) Total flavonoids ²
(mg / g) EU 42.18 ± 5.63 2.54 ± 3.05 HC 58.98 ± 7.83 36.86 ± 8.79 AG 23.25 ± 9.35 4.96 ± 0.48 AJ 5.97 ± 1.25 - CT 60.79 ± 10.90 19.42 ± 1.00 KP 31.60 ± 4.22 1.88 ± 0.34 M-1 31.32 ± 4.28 4.98 ± 0.70 M-2 57.74 ± 4.92 14.74 ± 3.81 M-3 28.13 ± 2.94 1.75 ± 0.21

Example 4 Measurement of Biological Activity

Example 4-1 Antioxidant Activity Measurement

The most characteristic mechanism of antioxidants is the reaction with free radicals. The radical scavenging action is used as a measure of antioxidant activity or inhibiting aging in the human body by donating electrons to active radicals. Radical scavenging activity of the herbal extracts was measured using DPPH and ABTS radicals, respectively. DPPH is a dark purple, relatively stable active radical that is reduced by antioxidants, aromatic amines, etc., to search for antioxidants, and ABTS is a 2,2'-azobis colored cationic radical. As a measure of antioxidant activity based on the reduction, ABTS radicals were scavenged by the antioxidant power of the extract, and the degree of removal of active radicals decolorized to blue-green was indicated by absorbance values. It is finished inside and can be measured in a short time.

The free radical scavenging activity of the samples was measured by reducing the DPPH, which is a stable radical.The samples were dissolved in 99% methanol and 800 μL of the diluted solution for each concentration, and 200 μL of 0.15 mM DPPH solution dissolved in methanol were added and left at room temperature for 30 minutes. Absorbance was then measured at 517 nm. The free radical scavenging activity of each sample was expressed as an IC ₅₀ value, which is the concentration of the sample required to reduce the absorbance of the control group without the sample to 1/2. At this time, BHA, a synthetic antioxidant, and L-ascorbic acid, a natural antioxidant, were used for the activity comparison.

Antioxidant activity was measured by ABTS + cation decolorization assay. 7 mM 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma Chemical Co., USA) and 2.45 mM potassium persulfate were mixed at the final concentration and left for 24 hours in a room temperature cow. ABTS + was formed and diluted with phosphate buffered saline (PBS, pH 7.4) to have an absorbance value of 0.70 (± 0.02) at 732 nm. 10 μL of the sample was added to 990 μL of the diluted solution, and the absorbance was measured after standing for exactly 1 minute. Scavenging activity of ABTS radicals is expressed as IC ₅₀ value.

As shown in Table 4, first, the RC ₅₀ value of the DPPH radical was 63.49 μg / mL of MIXII, similar to the result of the total polyphenol and flavonoid content, showing excellent DPPH radical scavenging ability in the extract, and the RC ₅₀ value of the ABTS radical was 41.61. μg / mL showed a similar trend with DPPH radical scavenging ability. As such, the radical scavenging activity of these extracts was lower than that of BHA and L-ascorbic acid (RC ₅₀ value = 2.43 ~ 3.90 μg / mL) used as a positive control group, but had significant activity. Similar to the report that the electron donating ability is an indicator of antioxidant activity against phenolic acid, flavonoids and other phenolic substances, the extracts of this study were found to be proportional to the results of polyphenol and flavonoid content.

TABLE 4

sample RC ₅₀ (μg / mL) ¹ DPPH RSA ABTS RSA MIX-1 122.16 ± 6.62 104.94 ± 5.51 MIX-2 63.49 ± 2.07 41.61 ± 1.31 MIX-3 150.76 ± 18.11 123.98 ± 0.56 BHA ² 2.43 ± 0.84 3.56 ± 1.09 L-AsA ³ 3.90 ± 1.12 3.48 ± 0.07

² t -Butylatedhydroxyanisole

³ L-ascorbic acid (L-AsA)

Example 4-2 Anti-inflammatory Activity Measurement

(1) Measurement of 5-LO and COX-2 Inhibitory Activity

5-LO inhibition efficacy was measured using a lipoxygenase inhibitor screening assay kit. After 10 μL of sample, 90 μL of 5-LO (220 units / mL) and 10 μL of 1 mM arachidonic acid were added and reacted at room temperature for 5 minutes. Then, 100 μL of chromogen was added and reacted at room temperature for 5 minutes, followed by ELISA. Absorbance was measured at 490 nm using an autoreader. At this time, NDGA (nordihydroguaiaretic acid) was used as a positive control group, and the inhibition rate was calculated according to the following equation.

% Inhibition = [(absorbance of control group-absorbance of experimental group) / absorbance of control group] × 100

COX-2 inhibitory efficacy was tested using a COX inhibitor screening assay kit. 10 μL of blood, 5 μL of enzyme, and 950 uL of buffer were added to 20 μL of sample. After reaction at 37 ° C, 10 μL of arachidonic acid was added and reacted at 37 ° C for 2 minutes to activate COX-2. After adding the color source of μL and reacted at 25 ℃ for 90 minutes, the absorbance was measured at 420nm using an ELISA autoreader. In this case, indomethacin, one of nonsteroidal anti-inflammatory drugs (NSAIDs), was used as a positive control group, and the inhibition rate was calculated according to the following equation.

% Inhibition = [(absorbance of control group-absorbance of experimental group) / absorbance of control group] × 100

Table 5 shows the results of measuring 5-LO and COX-2 inhibitory effects of the herbal mixture extract at 25 ㎍ / mL. As a result, all of the 5-LO inhibitory effect except the herbal extract extract III showed less than 50% inhibition, whereas the COX-2 inhibitory effect showed a high inhibitory effect of the herbal mixture II was 99.13%.

TABLE 5

sample 5-LO inhibition rate (%)  COX-2 inhibition rate (%) EU 21.53 ± 0.04 82.24 ± 1.06 HC 0.93 ± 0.91 77.06 ± 0.87 AG 36.23 ± 0.35 90.92 ± 0.09 AJ 22.11 ± 0.30 90.00 ± 0.13 CT 2.31 ± 2.05 85.08 ± 0.78 KP 15.97 ± 1.88 89.45 ± 0.89 MIX-1 45.60 ± 0.12 66.47 ± 0.76 MIX-2 32.64 ± 0.28 99.13 ± 3.37 MIX-3 52.43 ± 1.01 71.68 ± 5.65

(3) Verification of inhibitory effect on the generation of inflammatory markers

① Cell culture and statistical analysis

RAW 264.7 cells were distributed from Korean Cell Line Bank (KLCB) and 5% using DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS (fetal bovine serum) and 1% antibiotic (penicillin / streptomycin). Subculture was performed every 2 to 3 days in a 37 ℃ incubator containing CO ₂ . Cells were injected into 96 well plates (1 × 10 ⁵ cells / well), adhered to the cells, and 100 ng / mL of LPS (lipopolysaccharide) was added thereto, followed by 24 hours of incubation.

The quantitative analysis of herbal extracts and bioactive substances was shown as the average value after repeated measurements twice, and the physiological activity was measured as the average after three repeated measurements. The significance analysis of each section is omitted.

② Measurement of cell viability

LPS is a cell wall material of Gram-negative bacteria. Monocytes or macrophages are activated by trace LPS and produce and release various cytokines, arachidonic acid metabolites, free radicals, NO, and the like.

Cell viability was measured using the MTT assay. In other words, RAW 264.7 cells were dispensed in a 96 well plate at a concentration of 1 × 10 ⁵ cells / 200 μL, culture was removed for 24 hours, and the concentration-specific samples dissolved in 200 μL of DMEM medium to which LPS (100 ng / mL) was added. Was added to each well and incubated for 24 hours in a 5% CO ₂ incubator at 37 ℃. To this was added 20 μL of a MTT solution prepared at a concentration of 5 mg / mL with phosphate buffered saline (PBS, pH 7.4) and further incubated for 4 hours under the same culture conditions. The supernatant was removed, and 100 μL of DMSO was added to each well, followed by stirring for 10 minutes. The absorbance was measured at 550 nm. Cell viability was calculated according to the following equation.

% Cell viability = (S / C) × 100,

S: ΔO.D. of the sample. ; C: ΔO.D. of the control.

Cell survival is shown in FIG. 6 when the LPS-induced oxidative stress-induced ethanol extract was treated at 50 μg / mL in a concentration of 50 μg / mL. As a result, the cell viability was more than 90% in all samples. Therefore, NO inhibitory activity was measured at a concentration of 50 μg / mL, which is the concentration of the sample that does not toxic to cells.

③ Nitric oxide (NO) inhibitory activity

NO mediates important physiological functions such as blood vessel homeostasis and apoptosis-induced action under normal physiological conditions, but a large amount of NO kills normal cells and induces inflammation, resulting in acute or chronic inflammatory diseases (15). ). Therefore, effective NO secretion regulation is considered as a treatment for acute or chronic inflammatory diseases. This is one of the active oxygen, and recently examined the inhibitory effect of NO production known to play an important role in causing inflammation.

NO inhibition was measured by Green et al. RAW 264.7 cells were dispensed into 96 well plates at a concentration of 1 × 10 ⁵ cells / mL using DMEM medium, and then incubated for 24 hours by treating the test medium and fresh medium containing LPS (100 ng / mL) simultaneously. 100 μL of the cell culture supernatant and 100 μL of Griess reagent (1% sulfanilamide + 0.1% naphthylethylendiamine / 2.5% phosphoric acid) were mixed for 10 minutes in a 96-well plate and absorbed at 540 nm using an ELISA reader. Measured.

The result of measuring the NO production inhibitory effect in the form of NO ₂ ⁻ present in the cell culture solution by treating the herbal mixture extract at a concentration of 50 μg / mL in LPS-induced RAW 264.7 cells is shown in FIG. 7. As a result of the measurement, NO inhibitory effect was higher in the order of MIX-2MIX-1MIX-3.

④ Western blot analysis

Raw 264.7 cells (1.2 × 10 ⁶ cells / mL) were incubated 18 hours before, and each sample was treated with LPS (50 ng / mL) for 30 minutes and then incubated. After incubation, cells were washed with PBS (phosphate buffered saline) and eluted for 30 minutes by the addition of 100 μL. The cell eluate was centrifuged at 13,000 rpm for 15 minutes to remove debris. Protein concentration was quantified using a Bio-Rad Protein Assay Kit by standardizing BSA (bovine serum albumin). 15 μg of eluate was denatured by 10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane at 180 mA for 90 minutes. The blocking of the membrane was performed for 1 hour at room temperature with TTBS solution containing 5% skim milk. In order to measure the expression level of COX-2 and iNOS, anti-rabbit COX-2 and iNOS were diluted with TTBS solution containing 5% skim milk, respectively, as a primary antibody, reacted at room temperature, and washed at least 5 times with TTBS. . The secondary antibody was reacted with HRP (horseradish peroxidase) conjugated anti-rabbit antibody at room temperature for 1 hour and then washed 5 times with TTBS for 30 minutes. Next, the membrane was reacted with ECL western blot detection reagent (Amersham, USA) for 2 minutes and then subjected to X-ray film.

⑤ RNA isolation and RT-PCR

Raw 264.7 cells (1.2 × 10 ⁶ cells / mL) were incubated 18 hours before, and each sample was treated with LPS (50 ng / mL) for 30 minutes and then incubated for 12 hours. Total RNA extraction from the incubated cells was performed using TRI-reagent (MRC) and under RNase-free conditions. The reaction was stopped by heating 4 μg of total RNA with RT mixture [RNA buffer, oligo (dT) ₁₈ primer, dNTP], RNase inhibitor, and M-MuLV reverse transcriptase for 60 minutes at 42 ° C and then 5 minutes at 95 ° C.

Polymerase Chain Reaction (PCR) is a 2 μL cDNA, 1 μL of 5 'and 3' primer, 16.5 μL PCR mixture (10X buffer, 10 mM dNTP, 50% glycerol, 5 mg / mL) to amplify the gene from the synthesized cDNA. crezol red) and 0.2 μL Taq polymerase were mixed to perform PCR under the conditions of Table 5. The product produced by PCR was subjected to electrophoresis on 1% agarose gel and stained with ethidium bromide to identify specific bands.

TABLE 6

PCR conditions

Gene Primers Number of
cycles Amplified fragment
sizes COX-2 s 5'-ccg tgg tga atg tat gag ca-3 '
as 5'-cct cgc ttc tga tct gtc tt-3 ' 18 439 bp iNOS s 5′-atg gct tgc ccc tgg aag ttt ctc-3 ′
as 5′-cct ctg atg gtg cca tcg ggc atc-3 ′ 24 818 bp MCP-1 24 IL-1β 24 527 bp TNF-α 20 383 bp Actin 16 493 bp

⑥ PGE ₂  And LTB ₄ Production measurement

Prostagladin (PG) is known as a hormone that regulates the immune system. In particular, PGE2 (prostagladin E2) is an important factor that induces the inflammatory response is known to be produced by the expression of COX-2, the discovery of drugs that inhibit COX-2 expression and PGE ₂ production is arthritis, atopic skin diseases It is known to be very important for the improvement or treatment of immune diseases and cancer diseases accompanied with inflammatory reactions.

The production amount of PGE ₂ and LTB _{4 in} the cell culture was measured using an assay kit (enzyme immunoassay kit; Cayman Chemical Co., Ann Arbor, MI, USA). RAW 264.7 cells were dispensed into 96 well plates at a concentration of 1 × 10 ⁵ cells / mL using DMEM medium, and then incubated for 24 hours by treating the test medium and fresh medium containing LPS (100 ng / mL) simultaneously. At this time, the cell culture supernatant was taken to measure PGE ₂ and LTB ₄ .

As a result of measuring the effect of the mixed herbal extract on PGE ₂ production, the control group showed a concentration of about 136.34 pg / mL, whereas the LPS treatment group showed an increase of about 10 times to 1431.96 pg / mL. In addition, the LPS treatment group was treated with a mixture of herbal extract (10 μg / mL) and indomethacin (0.1 μg / mL) to significantly reduce PGE ₂ production in all groups by about 1.5 to 2 times. (See Figure 8).

As a result of measuring the effect of the herbal extracts on the production of LTB ₄ , the control group was found to have a concentration of about 8.98 pg / mL, whereas the LPS treatment group showed an increase of about 3.5 times to about 94.54 pg / mL. In addition, LTB ₄ production was significantly decreased in the LPS treatment group in all groups treated with herbal extract (10 μg / mL) and NDGA (0.1 μg / mL). In particular, the MIX-2 treated group showed the lowest yield of 57.10 pg / mL (see FIG. 9).

Example 5 Verification of Arthritis Improvement Efficacy Using Degenerative Arthritis-Induced Animal Experiment Model

Example 5-1 Animal Design

Experimental animals were obtained from 156 Biolinks of 156 SPF SD rat 6-week-old males. They were acclimated to solid feed for experimental animals for 1 week, and then divided into 13 groups by random method after weighing. Osteoarthritis was induced by injecting MIA (monosodium iodoacetate) solution into the left knee cavity of all test groups except group 1 (group 1), and normal control group was injected with the same amount of saline instead of MIA solution. Test substance was orally administered once a day from day 6, 5 days a week, and 4 weeks after confirmation of arthritis induction. Body weight and body weight were measured weekly during the administration of the test substance, and an autopsy was performed 4 weeks after the administration to confirm the functional effect of the test substance. Biochemical tests were performed on the blood obtained from the blood collection, and histopathological examination of the knee cartilage confirmed the efficacy of the test substance against osteoarthritis.

Example 5-2 Test System and Breeding Environment

(1) test system

① Species: SPF SD (Sprague-Dawley) rat

② Gender and age at acquisition and weight: 6 weeks old after male, 150 ~ 180g

③ Source: Korea Biolink

④ Date of Acquisition: April 23, 2009

⑤ Composition of test system: After measuring weight, it is composed by random method

⑥ Individual identification is indicated by the individual identification card notation for each breeding box, and the skin coloring method for each animal.

(2) Animal Breeding Environment

① Animal Room No.: Daegu Catholic University Biosafety Center SPF Laboratory Animal Room Ⅱ

② Humidity range: temperature 23 ± 3 ℃, relative humidity 50 ± 10%

③ Contrast cycle: Fluorescent lighting 12hr (08:00 to 20:00 off)

④ Roughness 150 ~ 300 Lux

⑤ Breeding box type (size): housed in polysulfone breeding box (260W × 420L × 150H mm) during purified quarantine, administration and observation period.

⑥ Animals per breeding box: 3 per cage

(3) feed

① Type: Solid feed for laboratory animals

② Source: Harlan Co., Ltd. (USA)

③ Feeding method: Free intake of radiation sterilized feed

④ Pollutant identification: Conducted in accordance with the SOP on the inspection of pollutants in feed and water of the Biosafety Center.

(4) water

① Type: drinking water

② Source: Free intake of ultraviolet sterilized ground water through water bottle

③ Analysis of pollutants: Refer to the data examined by Gyeongsangbuk-do Institute of Health and Environment according to the SOP on pollutants in feed and water of Biosafety Center.

Example 5-3 Establishment of Osteoarthritis Animal Model

(1) Osteoarthritis and Osteoarthritis Animal Model

Normal joints maintain proper function by the interaction of the shape of the joint surface, the biological adjustment of the mechanical elements of the joint structure, the structure of the supporting tissue, etc. The shape of the joint is an important factor in determining the degree of stress applied to the tissue. to be. Proper force exerted on the articular cartilage stimulates nourishment and chondrocytes from the synovial fluid to form an appropriate matrix, but an uneven overloaded joint causes functional changes in chondrocytes and partial necrosis and reactive proliferation of the cells. , Colonies of cells are formed, and the secretion of degrading enzymes such as collagenase and protease into the joint cavity increases arthritis. Causative factors of osteoarthritis include abrasion, trauma, aging or congenital abnormalities, but eventually, the secretion of degrading enzymes increases, causing osteoarthritis.

Animal models of osteoarthritis using surgery and chemicals have been introduced for the study of human osteoarthritis. The bones that make up the knee joint do not fit well together, causing frequent cranial crucite ligament, medial collateral ligament, and medial meniscus damage. In humans, rupture of the anterior cruciate knee ligament occurs best, and osteoarthritis is caused when the anterior cruciate knee ligament is left ruptured. Using these methods, various animals such as rats, horses, dogs, and rabbits have been used as osteoarthritis models. In general, surgical methods are caused by removing the cranial crucite ligament, medial collateral ligament cutting and medial meniscus. However, this surgical method has a long induction period and has a disadvantage in that forced exercise must be performed using a treadmill after surgery for effective induction. Therefore, there is a need for animal models that can cause osteoarthritis in a short time. Injecting chemicals such as collagenase or MIA into the knee joint cavity can cause osteoarthritis in a relatively short time. The knee joint is composed of collagen, proteoglycan and chondrocytes. Collagenase breaks down collagen and causes osteoarthritis. Intra-articular MIA injection causes chondrocyte necrosis in articular cartilage. MIA-induced arthritis in rodents is very similar to osteoarthritis in humans, such as proteoglycan matrix loss and joint failure. Therefore, this study induced osteoarthritis by injecting MIA solution into the rat's left knee joint cavity.

(2) Production of osteoarthritis animal model using MIA

After anesthetizing the animals adapted for one week to induce degenerative arthritis, sterilize the left knee joints of all test animals except the normal control group, and use MIA solution (monosodium iodoacetate 2mg in 30uL 0.9% sterile) in the knee cavity with a 0.3mL syringe (31G). ) 30ul was injected, and the normal control was injected with the same amount of saline instead of the MIA solution. In order to confirm the induction of arthritis, arthritis was induced by measuring the weight load on day 4, which is 3 days after MIA injection. Weight load is an indicator that can indirectly evaluate the presence and progression of degenerative arthritis.

Example 5-4 Test Group and Test Substance Administration

The experimental animals were 12 rats of 6 weeks old (150-180 g body weight) of SPF SD rats (Sprague-Dawley) rats. After adapting to the solid feed for experimental animals for 1 week, they were divided into 13 groups as shown in Table 6 by a random method after weighing.

<Table 7>

Composition of test group

Group No. Group ID
(Group name) induction of
osteoarthritis
(Induced osteoarthritis) treatment
(Test substance administration) One NC (Normal Control) Normal control - Tap water 2 OA (Osteoarthritis) Arthritis Control Left knee Tap water 3 AG Test group Left knee Ogapi 4 KP 〃 Left knee Thawed blood 5 AJ 〃 Left knee Dew 6 MIX-1 〃 Left knee Mixed 1 7 EU 〃 Left knee Tofu 8 CT 〃 Left knee Safflower seed 9 HC 〃 Left knee Eoseongcho 10 MIX-2 〃 Left knee Mixed 2 11 MIX-3 〃 Left knee Mixed 3 12 PC (G) (Positive Control) Positive control group Left knee Glucosamine 13 PC (C) (Positive Control) 〃 Left knee Celecoxib

The test substance in each group was administered orally natural extracts of each group orally as a clinical route for humans.

Test substance was administered orally once a day, 5 days a week, 4 weeks from day 6 after confirmation of arthritis induction. Normal control group and arthritis control group were fed with normal drinking water, and test groups from 3 to 11 were orally administered at a dose of 500 mg / kg / day.

Glucosamine (Ceun Kun Dang Health), which was used as a positive control, was also administered orally at 500 mg / kg / day, and another positive control, celecoxib (Korea Pfizer Pharmaceuticals), was set to 30 mg / kg / day considering the drug. By oral administration. Each test and positive control dose was set according to the results of previous studies.

Example 5-5 Observation and Inspection Items

(1) Clinical symptoms observation and organ weight comparison

Basic clinical symptoms such as weight, water intake, urine volume, activity, and behavioral abnormalities were observed for each experimental animal. In addition, after the end of the experiment, the animals were examined and the organ weight was measured.

(2) weight load measurement

Weight load is used as an indirect indicator to confirm the induction of osteoarthritis by MIA. Weight load is a measure of the degree to which the supporting ability of the osteoarthritis-induced leg is reduced. Previous studies have shown that the weight-bearing load of arthritis-induced animals is significantly reduced. Therefore, it is possible to confirm the induction of osteoarthritis through weight load, and to be measured every period, it can be an important indicator to compare the significant difference of osteoarthritis between test substances. In this experiment, weekly weight load was measured from day 4 using an incapacitance tester (Linton, UK), and the value was expressed as the ratio of left leg in which osteoarthritis was induced to the normal right leg (see FIG. 2).

(3) blood biochemical observation

The experimental animals were opened after ether anesthesia, and blood was collected from the abdominal vein to separate plasma. To determine the changes in inflammation-related factors in the blood, TNF-α, a representative inflammatory cytokine, was measured using an ELISA kit (R & D system, USA). In addition, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) contents were measured using an automated serum analyzer (Thermo Electron, USA) to confirm the hepatotoxicity of the test substance.

(4) Histopathological observation of liver tissue

In order to determine the hepatotoxicity of the test substance, a part of the liver tissue extracted at the time of animal sacrifice was fixed in 10% formaldehyde solution for 24 hours, and then exchanged twice with the same solution. Dehydrated with double ethanol and embedded in paraffin, which was cut into 4 μm thick, stained with H & E (hematoxylin-eosin), and observed at 200 times magnification of liver tissue cells under an optical microscope.

(5) Measurement of antioxidant enzyme activity in blood

① SOD (Superoxide dismutase): It was measured by Marklund's method (1974) using color development by automatic oxidation of pyrogallol in alkaline state. The enzyme activity unit was determined as the amount of protein that inhibits the automatic oxidation of the pyrogallol solution reacted without adding the enzyme solution by 50%.

② CAT9Catalase): Abei (1974) method, H ₂ O to the absorbance changes and the molar extinction coefficient of H ₂ O ₂ in the _two obtained, the concentration of H ₂ O ₂ and then decreased H ₂ O ₂ nmol / min / mg using the Enzyme activity was calculated by protein.

③ GSH-Px (Catalase): The method of Paglia and Valentine (1967) measured the extent of absorbance of NADPH at 340 nm when the oxidized glutathione was reduced by glutathione reductase and NADPH. It is expressed as the amount of enzyme that produces 1 nmol of oxidized NADPH for 1 minute.

(6) Observation of histopathological findings of articular cartilage

The femur and tibia were separated and fixed in 10% neutral formalin solution for 2 days, and then demineralized for 4 weeks in 10% EDTA solution. After the general tissue treatment process was embedded in pararaffin (pararaffin) to prepare a tissue section of 4㎛ (Fig. 3, 4)). H & E (Hematoxylin-Eosin) staining was used to observe tissue changes, and MT (Masson's Trichrome) staining was used to observe the expression of collagen fibers in the knee cartilage and knee cartilage through toluidine blue staining. The degree of proteoglycan loss was examined by light microscopy.

Table 10-12 shows the criteria for evaluating histopathological changes in articular cartilage. The grades (Table 8) according to the changes in the articular cartilage structure and the stages (Table 9) according to the extent of the damage were obtained. The total cartilage damage was scored.

This method was applied with a slight modification of the Mankin's score (see p. 8) method, which is widely used in osteoarthritis evaluation experiments. Mankin's score was used to evaluate the depth of injury but lacked an assessment of the extent of damage. osteoarthritic human hips.J Bone Joint Surg [Am] 53: 523-537, 1971).

<Table 8>

Grade Evaluation of Histopathology-Mankin's Score of Osteoarthritis-induced Cartilage

Category Grade  1. Structure
  a. Normal 0   b. Surface irregularity One   c. Pannus and surface irregularity 2   d. Clefts to transitional zone 3   e. Clefts to radial zone 4   f. Clefts to calcified zone 5   g. Complete disorganization 6  2. Cells
  a. Normal 0   b. Diffuse hypercellularity One   c. Cloning 2   d. Hypocellularity 3  3. Masson ’s Trichrome staining
  a. Normal 0   b. Slight reduction One   c. Moderate reduction 2   d. Severe reduction 3   eNo dye noted 4  4.Toluidine Blue staining
  a. Normal 0   b. Slight reduction One   c. Moderate reduction 2   d. Severe reduction 3   eNo dye noted 4  5. Tidemark integrity
  a. Intact 0   b. Crossed by blood vessels One

<Table 9>

Histopathological-Stage Assessment of Osteoarthritis-induced Cartilage

Stage
% Involvement (surface, area, volume) Stage 0 No osteocartilage activity area Stage 1 <10% Stage 2 10-25% Stage 3 25-50% Stage 4 > 50%

<Table 10>

Evaluation of Histopathology Score for Osteoarthritis-induced Cartilage Changes

Grade of

Structure Stage S1 S2 S3 S4 G0 0 0 0 0 G1 One 2 3 4 G2 2 4 6 8 G3 3 6 9 12 G4 4 8 12 16 G5 5 10 15 20 G6 6 12 18 24

Score = Grade × Stage (Score evaluation for cartilage structure change is calculated by multiplying grade score and stage score)

(7) Statistical Processing

All the results of this experiment were calculated using the SPSS package program (SPSS 12.0 for Windows, USA) which is one of the computer statistics program. The significance of the mean difference between the arthritis control group and the test group was tested by student's t-test. The significance level was P <0.05 and all measurements were expressed as mean ± SD.

Example 6 Arthritis Improvement Efficacy Verification Results Using a Degenerative Arthritis-Induced Animal Experiment Model

Example 6-1: Clinical symptoms and weight change

As a result of observing the weight change of the experimental group during the experimental period, body weight increased continuously in all experimental groups including the NC group throughout the experimental period. There was no significant difference. After MIA injection, the weight of other test groups except NC group was lower than that of NC group. No significant clinical symptoms were observed during the entire experimental period (FIG. 10).

Example 6-2: Effect on Weight Load

Weight load is an indirect indicator that can determine the presence of osteoarthritis and the status of osteoarthritis. In this experiment, the weight support rate of osteoarthritis-induced leg was lower than that of non-induced leg. In this experiment, the ratio of osteoarthritis-induced left leg to the right leg was shown. Existing studies show that osteoarthritis induces a greater degree of weight loss.

Three days after the osteoarthritis induction by MIA (day 4), the weight load was significantly decreased in all groups with osteoarthritis than in the normal control group. The results of measuring the weight load at weekly intervals from day 4 are shown in FIG. 11. On day 11, the MIX-3 group showed significantly higher weight load than the OA group, and on day 18, the MIX-3 and PC (C) groups were significantly higher than the OA group. In addition, on day 25, the body weight of the EU, CT, MIX-3, PC (G) and PC (C) groups was significantly higher than that of the negative control OA group. On day 32, MIX1 group, EU group, MIX-2 group, MIX-3 group, PC (G) group and PC (C) group were significantly increased than OA group.

Example 6-3: Effect on long-term weight

After confirming the induction of osteoarthritis, the organs were sacrificed for 4 weeks after the test substance was administered for 4 weeks from day 6, and there was no significant difference in liver tissue, heart, kidney, and spleen weight. .

Example 6-4: Results on TNF-alpha Content

The expression level of TNF-alpha, a representative proinflammatory cytokine, was measured by ELISA to determine the changes in inflammation-related factors in plasma. As a result, overall low level of expression was observed, and there was no significant difference between groups, but the expression level of the CT group to which safflower seed extract was administered tended to be the lowest (FIG. 13).

Example 6-5: Effect on the ALT and AST content of liver damage index

ALT and AST are indicators of hepatic damage that are sensitive to hepatocellular destruction. ALT and AST are present specifically in the liver, and AST is also present in the heart, skeletal muscle, and red blood cells, and is associated with myocardial infarction, skeletal muscle disease, and hemolytic disease. After comparing the ALT and AST contents after the end of the experiment, all groups did not show a significant difference, so the extracts used in this experiment were considered harmless (FIG. 14).

Example 6-6 6: Histopathological Observation of Liver Tissues

The histopathologic findings of liver by test substance in MIA-induced osteoarthritis model are as follows (FIG. 15). As a result of H & E staining to confirm the hepatotoxicity of the test substance, no abnormalities were found in all the test groups to which the test substance was administered, and in the OA group, the control group of the arthritis control group.

Example 6-7 Effect on Antioxidant Enzyme Activity

Antioxidant enzyme activity comparison results are shown in FIG. 16. SOD is an enzyme that converts superoxide anion radicals into H ₂ O ₂ , which causes the formation of other free radicals and lipid peroxidation stages early in the process of generating free radicals. In this experiment, SOD activity was higher than that of OA group, which was a control group of arthritis, KP group, MIX-1 group, MIX-1 group, MIX-1 group, MIX-2 group, MIX-3 group, glucosamine and celecoxib group. Group 2, MIX-3, PC (G), and PC (C) groups were significantly increased by 34%, 34%, 31%, 56%, 63%, 47%, and 50%, respectively, compared to the control group. .

CAT has catalytic activity for reducing H ₂ O ₂ produced by SOD to H ₂ O and peroxidation activity involved in oxidation of hydrogen donors such as methanol, ethanol, formic acid and phenol. Erythrocyte CAT activity was measured in AG group, AJ group, EU group, CT group, MIX-2 group, PC (G) group and PC (C group) administered Ogapi, hyssop, tofu, safflower seed, MIX-2, glucosamine and celecoxib. ) Significantly increased in the group (see FIG. 17).

By the action of GSH-Px, H ₂ O ₂ and reduced GSH become oxidized GSSG in water to generate water. In addition, GSH-Px catalyzes the reactions that produce GSSG, alcohol and water from peroxides and GSH to prevent peroxidative damage to tissues. Erythrocyte GSH-Px activity was measured in the group of AG, KP, AJ, MIX-1, CT, HC, MIX- treated with Ogapi, Haedongpi, Emulsion, MIX-1, Safflower, Eoseongcho, MIX-2 and Glucosamine. In group 2 and PC (G) group, 45%, 69%, 65%, 62%, 92%, 60%, 68%, 69% were significantly higher (see FIG. 18).

Example 6-8: Histopathological Change

In the NC group, the normal control group, the joint surface was smooth, the arrangement of chondrocytes and the matrix was balanced, and the cartilage substrates such as proteoglycan and collagen were abundant. In addition, no hypertrophy of chondron (cartilage cell including pericellular matrix; chondrocytes including pericellular matrix) or proliferative change of chondrocytes were observed (see FIG. 19). The cartilage matrix is composed of sulfide glycosaminoglycans, called proteoglycans, and polymers that are complexes of proteins, which appear blue when stained with toludine blue dyes. Therefore, when looking at the knee joint of the normal control group stained with toludine blue, it was possible to observe abundant proteoglycans stained with blue (see FIG. 19). In addition, the collagen fibers were abundant in the matrix of the articular cartilage, so that the collagen fibers dyed in blue on Messon's Trichrome stain could be confirmed (see FIG. 19).

In contrast, in the osteoarthritis-induced negative control group, severe degeneration of the articular cartilage was observed, and most of the cartilage was lost, severe exposure of the calcification zone and proliferation of peripheral soft tissues were observed. Reference).

In the AG, KP, AJ, CT, HC, MIX-3, and PC (C) groups, the calcification zone was exposed due to the loss of some cartilage. In some cartilage, cartilage degeneration was also observed in the negative control group, and the loss of cartilage substrate was obvious. In MIX-1, EU, MIX-2, and PC (G) groups, calcification zone exposure was observed in some cartilage, but the range of exposure was not wide, and proliferation of chondrocytes to repair cartilage damage was observed. It became. In addition, the loss of cartilage substrates such as proteoglycan and collagen was alleviated compared to the negative control group (see FIG. 19).

The results of scoring histopathological observations on the articular cartilage of each test group are as follows (FIG. 20). Compared to the OA group, which is a control group of arthritis, the KP group, AJ group, CT group, HC group, and MIX3 group showed a slightly lower degree of articular cartilage damage, but it is not a significant change. However, the AG group (ogapi), MIX1 group (mixed 1), EU group (tofu), MIX2 (mixed 2) group and glucosamine-treated PC (G) group showed significant differences from the negative control group and had less cartilage damage. Is observed. The PC (C) group administered celecoxib showed a slightly lower score compared to the OA group, but it was not considered a significant change.

1 is a schematic diagram illustrating an animal experiment schedule design.

Figure 2 is a diagram showing the incapacitance tester (a) and the actual measurement appearance (b) which is a weight load measurement instrument.

3 is a view showing the tissue cross-sectional direction and cross section.

4 shows the histological structure of articular cartilage.

5 shows an example of histopathological evaluation.

6 is a graph showing the effect of the herbal blend extract on RAW cell viability.

7 is a graph showing the inhibitory effect of the herbal blend extract on NO production in LPS-promoted RAW 264.7 cells.

8 is a graph showing the effect of herbal blend extract on LPS-induced PGE ₂ production in RAW 264.7 cells.

Figure 9 LPS-induced LTB ₄ in RAW 264.7 cells A graph showing the effect of herbal blend extract on production.

10 is a graph showing the weight change of each group during the entire test period.

11 is a graph showing the change in weight load of each group during the entire test period.

12 is a graph showing the effect of the herbal mixture extract on organ weight.

13 is a graph showing the results of measuring the amount of TNF-α expression in plasma.

14 is a graph showing the results of ALT and AST content measurement in plasma.

15 shows the results of morphological analysis of liver tissue.

Figure 16 is a diagram showing the results of antioxidant enzyme activity measurement.

17 and 18 are graphs showing the results of measuring antioxidant enzyme activity.

19 is a diagram showing the effect of the herbal extracts on osteoarthritis- H & E, TB, MT staining, respectively.

20 is a graph showing histopathological changes by test substances of osteoarthritis-induced lesions.

Claims (4)

Herbal mixed extract with therapeutic activity for osteoarthritis, containing Ogapi, hyssop, larvae and ear plate as active ingredients According to claim 1, wherein the herbal mixture extract is 20 to 30% by weight, 20 to 30% by weight of dew, 20 to 30% by weight of larvae and 20 to 30% by weight of ear packs. A pharmaceutical composition for the treatment and prevention of osteoarthritis, which contains a mixed extract of herbal medicines of Ogapi, hyssop, larvae and ear plate as an active ingredient. A health functional food composition for improving osteoarthritis, which contains a mixture of herbal extracts of Ogapi, hyssop, tofu and ear plate as an active ingredient.

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