KR20160086457A - Composition comprising alcohol extract of GamiBangkeehwangkeetang for preventing and treating a rheumatoid arthritis - Google Patents

Abstract

The present invention relates to a composition for preventing and treating rheumatoid arthritis, comprising an alcohol extract of Angelica gigas Nakai as an active ingredient, containing nine types of complex medicinal herbs of Astragalus membranaceus Bunge, Sinomenium acutum, Aconiti Tuber, Achyranthes bidentata Blume, Clematis chinensis Osbeck, Atractylodes macrocephala Koidzumi, Zingiberis rhizome, jujube, and Glycyrrhizae Radix. A flavor-added Sinomenium acutum medicinal herb broth comprises 11-15 wt% of Sinomenium acutum, 11-15 wt% of Aconiti Tuber, 11-15 wt% of Achyranthes bidentata Blume, 8-12 wt% of Clematis chinensis Osbeck, 8-12 wt% of Atractylodes macrocephala Koidzumi, 8-12 wt% of Zingiberis rhizome, 8-12 wt% of jujube, 3-6 wt% of Glycyrrhizae Radix, and the remaining of Astragalus membranaceus Bunge.

Description

[0001] The present invention relates to a composition for preventing and treating rheumatoid arthritis,

The present invention relates to a composition for the prevention and treatment of rheumatoid arthritis, which comprises the extract of a spore of Kami Bangui Hwanggi-tang. More specifically, the present invention relates to a composition for prevention and treatment of rheumatoid arthritis, The present invention relates to a composition for prevention and treatment of rheumatoid arthritis, which comprises an extract of a mugwort hanguigang containing a herbal medicine as an active ingredient.

"Bangui Hwanggi-tang" means "bengi" and "hwanggi" each 12g, white bran 8g, licorice 6g, ginger 3pages, It refers to a prescription of Oriental medicine prescribed as a mixture of two. According to Dong-bok-bok (醫)), the body is heavy and painful, hateful to the wind, sweating spontaneously and urine does not come out well, or the heat does not come down completely at the cold end, I continue to write this head and my whole body aches and urine does not come out well. Nephritis, dermatitis, myositis, arthritis, and urticaria. Also, I spend a lot of sweat while my body is running. It is said that the above medicine is eaten by dipping in water.

Korean Patent Registration No. 10-0735904 entitled " Purification composition containing herbal extracts ", and method for producing herbal extracts containing herb extracts using the herbal extracts) comprises a herbal composition containing at least 50% by weight of herbal extract powder as an essential ingredient X-containing tablet composition and a method for preparing the herbal extract-containing tablet, which is prepared by directly kneading the composition without adding water or an organic solvent. Which is blended with 1025% by weight of microcrystalline cellulose, 0.25.0% by weight of silicon dioxide and 110% by weight of pregelatinized starch, and by improving the fluidity, the titration and the disintegration power of the mixture, Tablets can be prepared by tabletting, and the tablets prepared by the above-mentioned preparation method can be produced by a conventional wet granulation method It is possible to produce the tablet at a size 0.50.6 times larger than that of the crude tablet. Therefore, it is possible to reduce the inconvenience of the patient's feeling due to the large size of the tablet upon taking the tablet, and it is possible to produce a high- It is possible to reduce the number and amount of the dose. In this patent document, "Bangui Hwanggi-tang is a recipe prescribed in the" Gyeonggi Gyeokjip ", which is a prescription for bangui, licorice, white 耆, 黄耆, ginger, (1) the lack of nutrients in the cells, so that the water that normally exists in the stroma (stroma) should be released to the skin more than necessary, and (2) the impurities to be discharged as kidneys are weak in the kidney function It can not be excreted in the kidneys and excreted into the skin and sweating is not restless. 3) It sweats much so that the body feels exhausted and gets a lot of cold. 4) It has excellent resistance to various inflammation symptoms due to weak resistance. For these symptoms, Bangui Hwanggi Tang enhances nutrition of the cells, strengthens the skin tension, strengthens the kidney function, strengthens the immune function by suppressing sweating , To alleviate divergence of body heat And promotes blood circulation. "

However, it has not been possible to provide a composition suitable for the prevention and treatment of rheumatoid arthritis, which contains a bamboo hankyang tang added with Kuman Bangui Hankitang, especially a spirulina extract of Kami Bangui Hankitang.

The present invention relates to a composition for the prevention and treatment of rheumatoid arthritis comprising, as an active ingredient, an alcoholic extract of Kami Bangui Higgigi Tang containing nine kinds of herbal medicines including Hwanggi, Bangui, The purpose is to provide.

The composition for the prevention and treatment of rheumatoid arthritis, which comprises the extract of a spore of Kami Bangui Hwanggi-tang according to the present invention, is characterized in that it contains a combination of nine kinds of herbal medicines including Hwanggi, Bangui, Cheondudo, And the extract of Hwanggi-tang as an active ingredient.

The Kamibugi Hwanggi-tang contains 11 to 15% by weight of an antiseptic, 11 to 15% by weight of a rainbow, 11 to 15% by weight of a mist, 8 to 12% By weight to 12% by weight, licorice by 3% by weight to 6% by weight, and balance as the remainder.

The alcoholic extract of Kami-Bangi Hwanggi-tang may be added to 1,000 to 1,400 ml of 70 to 90% alcohol based on 90 g of Kami Bangui Hwanggi-tang, extracted by reflux extraction, and concentrated by evaporation.

According to the present invention, there is provided a pharmaceutical composition for treating rheumatoid arthritis, which comprises, as an active ingredient, a spirit extract of Kami Bangui Hwanggi-tang containing 9 kinds of herbal medicines such as Hwanggi, Bangui, Cheondudo, The present invention provides a composition for preventing and treating rheumatoid arthritis.

1 is a flow chart showing a manufacturing process of a tissue.
FIG. 2 is a chromatogram showing the results of analysis of high performance liquid chromatography of Kamibuman-Hwanggi-tang (BHT) extract.
Figure 3 is a graph showing the effect of BHT extract on serum AST in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 4 is a graph showing the effect of BHT extract on serum ALT in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
5 is a graph showing the effect of BHT extract on creatinine of serum in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 6 is a graph showing the effect of BHT extract on serum BUN in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
7 is a graph showing the cytotoxicity test results of BHT extracts in RAW 264.7 cells.
Figure 8 is a graph showing DPPH free radical scavenging activity of BHT extract at various concentrations.
Figure 9 is a graph showing ABTS cation radical scavenging activity of BHT extract at various concentrations.
10 is a graph showing the effect of BHT extract on the production of reactive oxygen species in RAW 264.7 cells.
Figure 11 is a graph showing the effect of BHT extract on the production of LPS-induced nitric oxide (NO) in RAW 264.7 cells.
12 is a graph showing the effect of BHT extract on LPS-induced IL-1β production in RAW 264.7 cells.
Figure 13 is a graph showing the effect of BHT extract on LPS-induced IL-6 production in RAW 264.7 cells.
Figure 14 is a graph showing the effect of BHT extract on the production of LPS-induced IL-17 in RAW 264.7 cells.
Figure 15 is a graph showing the effect of BHT extract on LPS-induced IL-21 production in RAW 264.7 cells.
16 is a graph showing the effect of BHT extract on LPS-induced TNF-a production in RAW 264.7 cells.
Figure 17 is a graph showing the effect of BHT extract on the production of LPS-induced GM-CSF in RAW 264.7 cells.
18 is a graph showing the effect of BHT extract on the production of LPS-induced MCP-1 in RAW 264.7 cells.
19 is a graph showing the effect of BHT extract on serum IL-1 beta levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
20 is a graph showing the effect of BHT extract on serum IL-6 levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 21 is a graph showing the effect of BHT extract on serum IL-17 levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 22 is a graph showing the effect of BHT extract on serum IL-21 levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
23 is a graph showing the effect of BHT extract on serum TNF-a levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
24 is a graph showing the effect of BHT extract on serum GM-CSF levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
25 is a graph showing the effect of BHT extract on serum MCP-I levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
26 is a graph showing levels of IL-1 beta mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 27 is a graph showing levels of IL-6 mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
28 is a graph showing levels of IL-17 mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
29 is a graph showing levels of IL-21 mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 30 is a graph showing levels of TNF-a mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
31 is a graph showing the levels of GM-CSF mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
32 is a graph showing levels of MCP-1 mRNA in spleen from a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
33 is a graph showing the effect of BHT extract on serum IgM levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 34 is a graph showing the effect of BHT extract on serum IgG levels in a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
35 is a graph showing the effect of BHT extract on the level of WBC in the blood of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 36 is a graph showing the effect of BHT extract on the level of neutrophils in the blood of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 37 is a graph showing the effect of BHT extract on the level of lymphocytes in the blood of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 38 is a graph showing the effect of BHT extract on the level of mononuclear cells in the blood of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
39 is a graph showing the effect of BHT extract on the level of hs-CRP in the serum of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
FIG. 40 is a photograph showing a comparison between the control group and the experimental group of the collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 41 is a graph showing the effect of BHT extract on rheumatoid arthritis index score in collagen-induced DBA / 1 mice.
FIG. 42 is a photograph showing the effect of BHT extracts imaging cartilage degradation using fine tomography at the hind foot of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 43 is a graph showing the effect of BHT extract exhibiting reduced BV (bone volume) using micro tomography of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
Figure 44 is a graph showing the effect of BHT extracts showing reduced BS (bone surface area) / BV (bone volume) ratio using 3D tomography of a collagen-induced rheumatoid arthritis model of DBA / 1 mice.
45 is a photograph showing the results of histopathological examination analysis of the effects of BHT extract of collagen-induced rheumatoid arthritis model of DBA / 1 mice.

Hereinafter, the present invention will be described in detail with reference to specific examples.

The composition for the prevention and treatment of rheumatoid arthritis, which comprises the extract of a spore of Kami Bangui Hwanggi-tang according to the present invention, is characterized in that it contains a combination of nine kinds of herbal medicines including Hwanggi, Bangui, Cheondudo, And the extract of Hwanggi-tang as an active ingredient.

The Bengi ( Sinomenium : Acutum ( Thunb .) Rehder & EH Wilson ) is a medicinal product made by using vine stalks and rootstocks (Korea and Japan). China uses various kinds of bungi in each region, but officially called as Cheongpung, ( Sinomenium acutum var. Cinereum Rehder & EHWilson ) are used together. In Korea, Stephania tetrandra S. Moore , which is native to northern China, is used together, but it is different from Bangui and its composition is also different. The name Bangui is a medicinal substance that provides all the clues of luck and disaster, pleasure and anxiety as well as those who have gone through the prehistoric times. There is also a claim that the name is given because of the unique pattern of medicinal materials. The medicine of this medicine is very painful and cold. The efficacy is customary for the treatment of limb pain, limb pain, paralysis, guanwasa, edema, angelic disease, and revenge. Analgesic action, analgesic action, antipyretic action, antipyretic action, rhabdomyosynthesizing action, bronchial smooth muscle relaxation action, antibacterial and antitumor action have been reported. Bunggi is a circular or elliptical section with dark grayish brown side and vertical grooves and lump-shaped protrusions. The throat has a distinctive pattern because it is arranged radially with staggered conduit and dark brown radiating tissues staggered. In other names of Bangui, there are Harry, Rebun, and Seokhae. In Japan, Bangui is native, so it is called Waji. . Bugi is distributed in a wide variety of names according to the area of China, and it is called 'Cheongpung', 'Daechungmokyeong', 'Chungol Mountain', ' In addition, Bunbanggi (防 防 己) is known as Hanbanggi (漢 防 己), Sanggou, Sanso, Doji, Dongchung, Baekjok,

The above-mentioned cloud cover ( Aconitum carmichael Debeaux ) is a medicinal product made from root roots of the root of Odu (Korea, China). I call the root of the odu to be rich and call it the root. When Odu was first planted, I was called Root, and it was named Odu because it resembled the head of a crow. Growing up here is rich (附子), which means that the child is attached to the mother. This medicine is odorless, paralyzes the tip of the tongue, has a spicy flavor, is hot, and poisonous. It is used for dryness and rheumatism, arthritis, arthritis, swelling and sore symptoms, swelling and numbness, sore throat and sore throat, swelling and swelling. The pharmacological action of this drug has been reported to increase cardiac muscle contractility, increase blood pressure, antiinflammatory, analgesic, anti cold function, immune enhancement, adrenocorticotropic action and hypoglycemic action. Its appearance is uneven cone shape or spindle shape. Outer surface is grayish brown or blackish brown. On top there are traces of pine stalks, root and roots, and there is horizontal wrinkle pattern. The quality is hard and difficult to break. The cross-section of this medicine is powdery, the brow is brown, and the neck is milky white to grayish white. Other names are 卽 子, 喙 喙, and 烏頭. Due to symptoms and blood stasis caused by all kinds of windsongs and colds, it is possible to treat symptom and semi-passive affection, Heal the cold (cold), warm the book (臟腑), and the hard part of the cervical area is treated to cure the symptoms of abdominal pain (腹邪) by the cold (寒邪) is treated. Lee: It removes the frailness of the cold (寒 濕), passes the meridian well, defeats the cold (cold), and removes the mass of abdomen caused by the cold (寒邪). Wang Hogo (王 好 古): It treats the lack of humor in the human body and the winds that occur in the liver. Lee Ji-jin: It is said that the efficacy of helping Yangchi (气气) to defeat the scent of 阴寒 (气气) is like a rich man, but a little gentle.

The wooseul (牛膝) is in our country soemureup with amaranth (Achyranthes Acyranthes bidentata Blume and Achyranthes fauriei H. Lev. & Vaniot ) in Japan. It is the root of the japonica Nakai or 牛膝 ( Acyranthes bidentata Blume ). China recognizes only Acridanthes bidentata Blume . There was a doctor who treated old bone or muscle diseases, patients who had liver and kidney disease, and tried to find a true disciple who was his successor. He decided to stay at the disciples' house with a fleeting pattern and watch their attitude. As a result, only the youngest disciple treated this moneyless mentally. The lawmaker, who applauded him, told him the efficacy of herbal medicine and said, "It is medicine to treat diseases of the liver and kidney as well as bones and muscles, and save many people with this medicine." The disciples used this herb as a master 's will and became a name, and the appearance of the herb' s knuckles was like a cow 's knee and called' 牛 '. This medicine has little odor and is mucoid. The taste is slightly worn, and the quality is not balanced to any one side [苦 acid 平]. If you use raw whiskey, you can eliminate the ejaculation and boils, and if you grind it, you will see the liver and the gods and strengthen the muscles and skeleton. It removes eosinophilia, and it is used for physiological impoverishment, postpartum abdominal pain. It replenishes bone marrow, it is used for arthritis with good sound, and it treats the mouth and tongue rash caused by the diarrhea. The pharmacological effects of uterine stimulation, cholesterol lowering, diuretic action, hypoglycemic action, and liver function improvement have been reported. Appearance is a long, long, circumferential circular root, or a circular root with a side root. The root head is attached or removed with a little rhizome. The roots are usually rod-shaped or slightly curved, and the outer surface is yellow or yellowish brown with many vertical wrinkles and sparse ridges. Other names include 牛 莖, hundredfold, mountain), 節节 菜, and 鷄 膠 骨. In China, the wax is called the Henan Mountain Is regarded as a good product, and is called Hui 牛 knee.

The wiryeongseon (威靈仙) is Ranunculaceae euahri (Clematis of mandshurica Maximowicz) or other dongsok roots as medicine (Korea) made of closely related plants in China, euahri (Clematis mandshurica Maximowicz ), martial arts ( Clematis chinensis Osbeck . ), Fertilizer ( Clematis hexapetala Pall . ), And the root of Clematis armandii Franch . Japan uses the roots of euphoria, militia, and food. In ancient China, a good married couple lived and my husband leaned against a stone pillar from outside and became paralyzed as a paralyzed person for 10 years. At this time, a member of the council instructed her husband about herbal medicine, and said, "Your husband is a stroke, because of the wind and the humidity. She cut off the roots and called the water, cut it, and made it with the makgeolli to make it eaten. After drying, she put the powder into the vinegar and put it on the limbs. After a few months, her husband was recovered enough to walk. She asked the lawmaker for the name of the herb, and she immediately gave it a name because she did not have a name yet. She said, "Let's call it a spirit of medicine, because the nature of medicine is majestic and fresh." This medicine has no odor, and the taste is spicy and sour, and its quality is warm. The line removes customs and treats joint bending, limb paralysis, back pain, limb pain, muscle paralysis, bruises. It is used for the pain of hypertension and meridians. Pharmacological actions have been reported in hypotension, smooth muscle excitement, diuretic action, hypoglycemic action, pain relief, and antimicrobial action. Appearance is a chunk with uneven roots. It has a short stalk of woody top at the top, and many long roots. The outer surface of the rootstock is yellowish brown and fibrous, the outer surface of the root is brown or dark brown, has vertical wrinkles, is easy to bend, and the neck and skin tend to fall off. Other names include Nongsoo (老虎 鬚), Ryo (无 消), Yeonseon (霊 仙), Wenjin ship (仙 脚 威靈仙仙), Loseon (仙 仙) And Tomoki Ogori.

The white pearl is an extract of Atractylodes japonica Koidzumi) or baekchul (Atractylodes macrocephala roots and stems or jupi to remove the dried herbs (Korea), China baekchul (Atractylodes macrocephala Koidzumi) only writing in Japan sapju (Atractylodes japonica Koidzumi) and China baekchul (Atractylodes ovata) in Koidzumi) Lt; / RTI > It is said to be a letter patterned to the shape of the roots and branches of leaves. It is said to be similar to the nature of the leaves. It is also known as mountain 芥, and the leaves are similar to thistles. One beautiful old white crane was planted in the place where it was called Zamam, and it became an herb medicine of the place when it took care of it. Then on the 9th of the lunar month, on September 9th, she turned to be a beautiful woman wearing a chrysanthemum pattern and a white embroidered white skirt. She came down in a cloud and handed her a flower to a lady of a senator. On September 9 of the following year, the beauty appeared again, and the curious lady 's wife put her thread on the skirt of the beautiful woman and followed her back. At the end of the thread, anyone who looked at it seemed to be looking at a potion. The lady of the senator who is blinded by greed catches the bill which seems to be staying for a thousand years suddenly It is said that it is a great light suddenly, and the wife 's eyes are far away and the flower has disappeared. Even now, there is a village called Haksan in China, and I call it Baekje especially. This medicine has a peculiar odor, a little bit of taste, and if you chew it, it is viscous and its quality is warm [甘甘 溫]. Leek is a weak function of the stomach (脾胃), news, malaise, yellowish color of the face is dilute, dilute, good for diarrhea and stasis of the body is swollen and digestion can help to excrete the water. It is also used for boobs, coughs, clear sputum due to accumulation of water and wetness in the spleen. 脾气 (脾气) When sweating naturally on the skin due to weakness, pregnancy, vomiting use. It is also used for colds and limb pain with gastrointestinal disorders. Pharmacological increase of mice weight gain and endurance, increase phagocytic ability, promotion of cell immune function was reported. In addition, intestinal (intestinal) inhibition and control of excitement, anti-ulcer and liver function protection, immune function hyperactivity, vasodilation, diuretic and hypoglycemic action, anti-cancer action has been reported. Appearance is an uneven mass or an inconsistently curved circumference. Outer surface is pale yellowish yellow or pale yellowish white with grayish brown color and vagina is hard and not broken. The folded surface is fibrous, and yellowish brown discharge is scattered. There are cup-shaped small protrusions in various places. There are non-continuous vertical wrinkles and grooves, marks on the roots, stems on the upper ends, or spots of shoots. Other names are 백 白 마 마 마 마 마 마 가 부 부 부 부 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 가 (吃力 伽), mountain mountain (mountain), mountain (岳), and heaven (天).

The above-mentioned health means dry ginger, and in Korea, China and Japan, ginger ( Zingiber officinale Roscoe ) dried roots. In Japan, it is called dried ginger. In Korea, health powder is also medicinal. The Hindu Danang area of the East India is presumed to be the country of origin and it is said that it was cultivated 2,500 years ago in China. Now Sichuan province is known as the origin of ginger. In Korea, ginger cultivation was recorded for the first time in Goryeo (9th year of King Hyeongjong, 1018), and it was recorded that ginger was written as a royal slave. According to one theory, 1,300 years ago, a man named Shin Man Seok went to China to buy ginger and planted it in Bongdong, Wanju County, which is said to be the beginning of ginger cultivation in Korea. Ginger is described in the Chinese doctrine of 2,000 years ago and is used as medicines in almost all of the oriental medicine prescriptions. In life, it is widely used as a directional substance which fixes unpleasant odor and taste such as fishy smell. This medicine has a peculiar smell and the medicine is spicy and hot. Chest and abdomen are cold, there is pain in the stomach, the stomach is not digested, and it is effective for vomiting and diarrhea. It is also used when the belly is weak, the limbs are cold, the cold winds have seawater, the respiration is bad, and the belly is cold and diarrhea. Pharmacological effect stimulates gastric secretion, activates intestinal peristalsis, promotes digestion, vomiting, and stimulates the heart to stimulate blood pressure and promote blood circulation. It has anti-inflammatory, analgesic and acaricidal action. It is made up of unevenly split rootstock and its fragments. It is somewhat pressed, easily bent, bent ovate, long ovate, and gnarled on both sides. The crust is grayish yellow and the outer surface is grayish white with white powder. On the folded surface, secretions are scattered in dark brown small spots, and microscopically, they are filled with yellowish substances of essential oil. Health is also referred to as white ginkgo (白 薑) or ginkgo (薑 薑). Charcoal is used for hemostasis.

The jujube is the fruit of the jujube, also known as jujube or tree nectar. The surface is reddish brown, elliptical, 1.5 to 2.5 cm long, and has a sweet taste when cooked red. Fruit is not only reproductive, but also dried and dried after being harvested and used as cookie, cooking and medicine. Jujube is processed in daily life and used as jujube, jujube tea, jujube vinegar, jujube porridge. Honey juice as a processed product is popular in China, Japan and Europe. In one room, it is used as diuretic, tonic, and emollient. In Korea, Chungcheongbuk-do Boeun (恩恩) jujube is famous.

The licorice is licorice in Korea and JapanGlycyrrhiza uralensis Fischer), European licorice (Glycyrrhiza glabra L.) Or other roots and stem parts of plants, with or without skin. In China, licoriceGlycyrrhiza uralensis Fischer: Licorice), light and licorice (Glycyrrhiza glabra L.: Light 果 licorice), licorice licorice (Glycyrrhiza inflata Batal: 果 甘草) dried roots are used. Licorice is called ural licorice and curly licorice. It grows in Russia (Siberia), Iran, Afghanistan, Pakistan, China (Gansuk province, Shin Kangseong), Mongolia and grows in Korea. European licorice is distributed in Southern Europe, Central Asia and China. As a variant of European licorice, there are Russian licorice, Persian licorice, and Iran licorice, but not medicinal. An old lawmaker was rumored to be well - healed, and the patients were pushed back, but the visit was too frequent for the patient to wait. The wife of the senator worried about the patients who were waiting and found a pile of pudding which he accidentally tried to use as a firewood in the kitchen and tasted it. She used all herbs as medicines, and thought that this grass would work, and gave it to the patients. Later, when the clinic checked their symptoms, they found that each symptom had an effect. In addition, it harmonizes all medicines with the medicinal effect, and it is also called the national aged in the sense of the master of the country and the master of the king. Licorice smells unusual and has a sweet taste. Licorice harmonizes the toxicity of all medicines and makes the medicinal effect appear, regulates the fever and morale of the book, makes good communication of all blood vessels, and strengthens the muscles and bones. Pharmacological action is effective in detoxification, hepatitis, urticaria, dermatitis, eczema. Jinhae, genomes, muscle relaxation, diuretic, anti-inflammatory action and inhibits peptic ulcer. Licorice crust is reddish brown or dark brown with vertical wrinkles and occasionally shrubs, buds and scales. The outer skin of the peeled licorice is pale yellow and fibrous. This medicine is also called kuno (國 老), ichigo (草 草), mandarin (蜜 甘), wax (蜜 草), Yeongtong (靈 草), kelp (甛 草), 草 (草).

The Hwanggi (黄: Astragalus membranaceus BUNGE ) is one of the main medicines of the Kami Bangui Hwanggi Tang, and its tongues are grass (foundation), Dai (giant ginseng), Dai (grandchild), Dok (mind), cotton 黄, The main symptom is that it breaks the old boil, discharges the pus and reduces the pain, and it causes the skin diseases such as leprosy, hemorrhoids, fleas, And cures all infections in children by supplementing frail ones. Separate record: It has the effect of defeating the 瘀血 (ejaculation) between the cold and the fever in the womb of the woman and treating the weak and tired of the male, stopping the thirst, It treats diarrhea, encourages energy, and benefits 阴气 (阴气). Ⓒ 权 (cunning): Cough and weakness of the kidney (god) to weaken the ears of the symptoms of darkening and healing the pass, healing the boil on the back and complement the body. Nichuan Hokusaku (anecdote of capital): Helps ki and strengthens the blood (blood), strengthens the muscles (skeleton) and skin (lean muscle) (Infantile ejaculation), intestinal wind (long wind), blood plumage (blood plumage) It also treats all postpartum illness and treats menstrual disorders, coughs, sputum, head winds (fever), fever poisoning (open fever), and eye irritation (purpose). Zhang element (Zhang element): I am weak, I cure cold sweat, replenish lung 气 (discard), remove the lung fire (lung) and heartburn (deepening), strengthen the skin, help the stomach Removed the heat and removed all the meridians.王 好 古 (王 고子): 阴寒 经脉 (leprosy gyeongma) and the disease of the disease (amblyopia) due to 奇寒 发热 (寒热 热) (Reverse) and can not tolerate bowel symptoms were treated.

The Kamibugi Hwanggi hot water has 11 to 15% by weight, preferably 12 to 14% by weight, 11 to 15% by weight, preferably 12 to 14% by weight, and 11 to 15% by weight, Preferably 8 to 12% by weight, preferably 9 to 11% by weight, more preferably 8 to 12% by weight, preferably 8 to 12% %, Jujube 8 to 12 wt%, preferably 9 to 11 wt%, licorice 3 to 6 wt%, preferably 4 to 5 wt%, and balance as the balance.

The alcoholic extract of Kami-Bangi Hwanggi-tang is added to 1,000 to 1,400 ml, preferably 1,100 to 1,300 ml, of 70 to 90%, preferably 75 to 85% of alcohol, based on 90 g of Kamiubanguiragi-tang, , Followed by evaporation and concentration into powder.

The composition for wound healing, which comprises the extract of a spore of Kami-Bangi Hwanggi-tang, can be used to prevent and treat rheumatoid arthritis of an animal other than a human.

In the above description, "Kamibugi Hwanggi-tang" is described as a kind of a formulation of "tang". However, this is merely a name for convenience, and does not necessarily mean that it is used for extracting alcohol after preparation with hot water, It is to be understood that this means that a mixture of Bangui, Tianzhu, Wooze, Gujisi, Baicheng, Health, Jujube and Licorice is directly applied to the extraction of alcohol to produce the alcoholic extract.

Hereinafter, preferred embodiments and comparative examples of the present invention will be described.

The following examples are intended to illustrate the invention and should not be construed as limiting the scope of the invention.

Example

1. Materials

1) cells

The RAW 264.7 cells used in the experiments were purchased from Korean Cell Line Bank, Seoul, Korea.

2) Medicinal products

GamiBangkeehwangkeetang (hereafter referred to as " BHT ) was purchased from Omni Hub Co., Ltd., Daegu, Republic of Korea, and purchased from TBRC-RIC, Daejeon University, and used after selection. The contents and contents (one copy) are as follows (Table 1).

designation Pharmacognostic name Weight (g) Hwanggi Astragali Radix 14 Bangui Sinomenii Caulis et Rhizoma 12 Cloudiness Aconiti Tuber 12 The Achyranthis Radix 12 Command line Clematidis Radix 9 Boiling Atractylodis Rhizoma Alba 9 health Zingiberis Rhizoma 9 Jujube Zizyphi Fructus 9 licorice Glycyrrhizae Radix et Rhizoma 4 Sum 90

3) Animal and feed

Male DBA / 1 mice (16-22 g) at 5 weeks of age were supplied from Central Experimental Animals Co., Ltd., Seoul, Korea, and were fed with solid feed (Purina) and water at a temperature of 22 ± 2 ° C, Humidity, 55 ± 15%, 12 hours-12 hours (light-dark cycle). This experiment was conducted in accordance with the animal ethics code of the Animal Experimental Ethics Committee of the Great War of the Antarctic (Animal Use Ethics Committee Approval Number - DJUARB 2014-014).

4) Reagent

The reagents used were: isopropanol (Sigma Co., USA; gelred; Sigma); dulbecco's phosphate buffered saline (D-PBS; Welgene, Korea) Dulbecco's Modified Eagle's Medium (Gibco BRL Co., USA), fetal bovine serum (FBS; Invitrogen Co, USA) ), A lipopolysaccharide (LPS), a cell viability assay kit (Daeilab sevice) and a nitric oxide detection kit (Nitric Oxide Detection Kit) Materials Intron Biotechnology, dimethyl sulfoxide (DMSO), 1,1-diphenyl-2-picryl-hydrazyl (DPPH: 1,1 (Sigma), penicillin (Hyclone, Co.), streptomycin (hiclone in the United States of America), agarose (Fm) in the United States of America FMC Co.), (2,7) -dichlorodihydrofluorescin diacetate (DCFH-DA: (2,7) -dicyclodihydrofluorescin diacetate; Sigma in the United States of America), 2,2'-azinobis Benzothiazoline-6-sulfonic acid (ABTS: 2,2'-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid; sigma in the United States), formaldehyde (Sigma, USA), trypan blue (Sigma, USA), Immunization Grade Bovine Type II Collagen Solution (Chondrex Co., USA), incomplete prunate excipient Incomplete Freund's Adjuvant; Complete Freund's Adjuvant (Conrex in the United States); Ethanol (Merck Co., Germany; Mouse Cytokine Milliplex Map Immunoassay Kit IL-17AF ELISA Kit (e. G., EBioscience < / RTI > CO (USA)) was used for immunocytochemical analysis of the mouse cytokine milliplex map immunoassay kit (Millipore Co., Indomethacin, USA) ), IL-21 Enzyme Immunoassay Kit (United States Bioscience), MCP-1 Enzyme Immunoassay Kit (BioScience in the United States), GM-CSF Enzyme Mouse immunoglobulin M enzyme immunoassay kit (US Biotechnology), mouse immunoglobulin G enzyme immunoassay kit (United States Bioscience), total RNA A total RNA prep kit (Intronbio., Folin-Ciocalteu's phenol reagent, Merck, Germany, Gallic acid, Quercetin, (Sigma in the United States), sodium carbonate (Sigma in the United States), aluminum nitrate nonahydrate (Sigma in the United States), potassium acetate solution Standard solution of nitric acid (HNO ₃ ; Duksan, As, Pb, Hg, and Cd in the United States) is available from Escipi Science (Canada) SCP Science), water (Duksan in Korea), acetonitrile (Duksan in Korea), polin-cyocaluphenol reagent (Merck in Germany), gallic acid (Sigma in the United States), sodium carbonate Sigma) were used.

5) Devices

The equipment used was a rotary vacuum evaporator (BCo. B-480, Switzerland), a freeze dryer (EYELA Co. FDU-540, Japan), a CO ₂ incubator Materials such as Forma scientific Co., a clean bench (Vision scientific Co., Korea), autoclave (Sanyo Co., Japan, vortex mixer (Vision Scientific, Korea), a spectrophotometer (Shimadizu Co. in Japan, a centrifuge in the United States, a deep-freezer (Sanyo in Japan), a thermo- A thermocycler system (MWG Biotech Co., Germany; ice-maker; Vision Scientific, Korea), a plate shaker (Lab-Line Co., USA) ), Enzyme Immunoassay Reader (Becton Dickinson, Co., USA, Light Microscope, Carl Zeiss, Germany), an ELISA reader (Molecular Devices Co., USA, flow cytometer , An inductively coupled plasma spectrometer (ICP) (Shimatsu, Japan), a mercury analyzer (TELEDYNE, USA), Fusion-SL (Germany), Vilber Lourmat Leeman Labs), and high performance liquid chromatography (HPLC; Shimatsu, Japan).

2. Method

1) Sample extraction

1,200 ml of 80% alcohol (C ₂ H ₅ OH) was added to 90 g of BHT, refluxed for 3 hours, filtered and concentrated under reduced pressure in a vacuum rotary evaporator. The concentrated solution was lyophilized in a freeze dryer to obtain 4.6 g of powder. The powder was diluted with distilled water to the necessary concentration while being stored in an ultra-low temperature freezer (-80 ° C).

2) Heavy metal inspection

For the analysis of lead, arsenic and cadmium, 0.5 g of BHT extract was placed in a container for microwave sample pretreatment, and 10 of nitric acid was added. The vessel was placed in a hood to remove the generated gas and decomposed using a microwave sample pretreatment device. After the decomposition, the decomposed solution was filtered through a filter paper, placed in a volumetric flask, diluted with water to the appropriate concentration range of the standard solution, and used as a sample solution. Separately, 10 ml of nitric acid was put into a container for exclusive use in the microwave sample pretreatment apparatus and used as a blank test liquid by the same method as the preparation of the test liquid. A calibration curve was prepared using the prepared test solution, standard solution and blank test solution using an inductively coupled plasma spectrometer (ICP), calibrated with a blank test solution, and the test solution was measured. For mercury analysis, 50 mg of BHT extract was precisely weighed and measured using a mercury analyzer without special pretreatment.

3) HPLC analysis

30 mg of BHT extract was dissolved in 1 ml of 80% alcohol, filtered through a 0.45 μm membrane filter, and 20 μl of the filtrate was used as an HPLC sample. The HPLC was performed using a system controller (CBM-20A), a pump (LC-20AD), a column oven (CTO-20A) and a diode array detector (SPD-M20A) 18 (ZORBAX Eclipse Plus C18 (250, 5)) was used. (0% B), 5 to 40 minutes (30% B), 40 to 60 minutes (80%), and 10 minutes % B) and 60 to 80 minutes (100% B). The flow rate was 1.0 ml / min, the column temperature was maintained at 40 ° C, and the ultraviolet wavelength was set at 210 nm.

4) liver function test

Blood samples were collected from the serum by using the cardiac puncture method to measure the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum. Blood was solidified at room temperature for 30 minutes, centrifuged at 3,000 rpm for 15 minutes, and the serum was separated and analyzed by the Institute of Scientific Research, Seoul, Korea. AST and ALT activity were measured by a biochemical automatic analyzer using the principle of the JSCC UV method of the Japanese Society for Microbiological Resources.

5) New function tests

To measure creatinine and BUN (blood urea nitrogen) activity in the serum, blood was collected using the cardiac puncture method at the end of the experiment. Blood was solidified at room temperature for 30 minutes, centrifuged at 3,000 rpm for 15 minutes, and the serum was separated and analyzed by the Institute of Scientific Research, Seoul, Korea. The creatinine content was determined by the Creatinine Jaffe Method and the content of BUN by a biochemical automatic analyzer using the principle of Kinetic UV assay for urea / urea nitrogen.

6) Cytotoxicity measurement

RAW 264.7 cells were seeded at 2 × 10 ⁴ cells / well in a 96-well plate and cultured for 24 hours. Before the experiment, the culture medium was replaced with fresh culture medium, BHT was treated at a concentration of 1, 10, 100 (μg / ml), and cultured for another 24 hours. After culturing, 10 μl of a water-soluble tetrazolium salt solution (WST solution) was added and reacted in a cell incubator (37, 5% CO ₂ ) for 30 minutes. After the reaction, the change in absorbance at 450 nm was measured and the cell survival rate as a percentage was shown for the control group.

7) RAW 264.7 cell culture

The frozen RAW 264.7 cells were transferred to a 50 ml tube, and 9 ml of PBS was added to suspend the cells, followed by centrifugation at 1,200 rpm for 5 minutes to remove the supernatant. The cells were suspended in a tube containing 1 ml of DMEM medium consisting of 10% fetal bovine serum (FBS) and 1% penicillin. 9 ml of the medium was placed in a 100-mm dish, and the cells were suspended and cultured in a cell incubator (37 ° C, 5% CO ₂ ). The number of subcultures was made at least 5 times and the samples were adapted for 24 hours before treatment.

8) Antioxidant efficacy measurement

(1) Determination of total phenol content

The phenol content of the BHT extract was determined by the method of Gutfinger. 0.5 ml of 50% Folin-Ciocalteu's phenol reagent was added to 1 ml of the extracted sample solution, and the mixture was reacted at room temperature for 3 minutes. 1 ml of saturated Na ₂ CO ₃ solution and 7.5 ml of distilled water were added to the reaction solution, and the mixture was allowed to stand for 30 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken and absorbance was measured at 760 nm. The content of total phenol content was determined according to the calibration curve prepared using gallic acid as a standard material. GAE (gallic acid equivalent) / g was used as a measurement unit.

(2) Measurement of total flavonoid content

The flavonoid content of the BHT extract was measured by the method of Nieva Moreno et al. 10 ml of aluminum nitrate, 0.1 ml of 1M potassium acetate and 4.3 ml of 80% ethanol were added to 0.5 ml of a mixture of 0.1 ml of each sample and 0.9 ml of 80% ethanol, and the mixture was allowed to stand at room temperature for 40 minutes Absorbance was measured at 415 nm and the content was determined from a standard curve prepared using quercetin.

(3) Measurement of radical scavenging activity of 2,2-diphenyl-1-picrylhydrazide (DPPH)

Free radical scavenging activity test is a method using stable free radical DPPH. BHT extract was diluted to a final concentration of 1, 10, 100, 1,000 (㎍ / ㎖), and 150 ㎕ of 0.2 mM DPPH solution dissolved in ethanol and 100 ㎕ of BHT extract were mixed 37 Lt; 0 > C for 30 minutes. After the reaction, the absorbance was measured at 517 nm. The control solution of the sample solution was added with distilled water, and ethanol was added to the DPPH solution as a control. The DPPH free radical scavenging rate was calculated according to the following equation.

[Equation 1]

(4) Determination of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability

The ABTS assay was performed by adapting the previously reported method to a 96-well plate. The BHT extract was diluted to a final concentration of 1, 10, 100, 1,000 (ug / ml) and the ABTS solution was diluted with 7.4 mM ABTS (2,2'-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid) and 2.6 mM potassium persulphate were prepared and allowed to stand in a dark place for one day to form a cation (ABTS +). Then, the absorbance was measured at 734 nm and the absorbance value was 1.5 or less 150 μl of the diluted ABTS + solution and 5 μl of the BHT extract, respectively, were reacted at room temperature for 10 minutes, and the absorbance was measured at 734 nm. The antioxidative ability of the control group was determined by the percentage of the ABTS radical scavenging ability according to the following formula 2 using distilled water as a control.

[Equation 2]

(5) Production of reactive oxygen species (ROS) in cells

2 ', 7'-dichlorofluoresce diacetate (DCF-DA) was used to measure reactive oxygen species (ROS) in RAW 264.7 cells. RAW 264.7 cells were plated at 12 × 10 ⁵ cells / well in a 12-well plate and cultured for 24 hours. BHT extract was treated at a concentration of 1, 10, 100 (㎍ / ml), treated at a concentration of 1 μg / ml of LPS, and then incubated for 24 hours at 37 ° C in 5% CO ₂ Lt; / RTI > After incubation, the cells were centrifuged at 1,200 rpm for 5 minutes, and the collected cells were washed twice with cold PBS, added with DCF-DA to 10 μM, and kept at room temperature for 15 minutes. After staining, cold PBS was added and the mixture was centrifuged at 1,200 rpm for 5 minutes. Then, the supernatant was removed, and 400 μl of PBS was suspended. The fluorescence intensity was analyzed using a flow cytometer.

9) Anti-inflammatory efficacy measurement

(1) Nitric oxide (NO) measurement

NO concentration was determined by measuring the concentration of nitric oxide in the culture solution using a Griess Reagent System. RAW 264.7 cells were plated in 96 well plates at 2 × 10 ⁴ cells / well and cultured for 24 hours. BHT extracts were treated with 1, 10, 100 (㎍ / ㎖) and 1 ㎍ / ㎖ of LPS, respectively, for 24 hours. 50 쨉 l of N1 buffer was added to each well and reacted at room temperature for 10 minutes. Then 50 쨉 l of N2 buffer was added to each well and allowed to react for 10 minutes. After the reaction, the absorbance was measured at 540 nm. The NO concentration of the culture was determined using a standard curve for each concentration of nitrite standard.

(2) Measurement of cytokine and chemokine production

Intracellular inflammatory cytokines were cultured for 24 hours in RAW 264.7 cells / well in a 12-well plate at 2 × 10 ⁵ cells / well and replaced with fresh culture medium. BHT extract was added at a concentration of 1, 10, 100 (ig / ml) , Treated with a concentration of 1 μg / ml of LPS, and cultured in a cell culture incubator (37 ° C., 5% CO ₂ ) for another 24 hours. IL-17, IL-21, MCP-1 and GM-CSF were measured with an enzyme immunoassay (ELISA) assay, and the concentrations of IL-1β, IL-6 and TNF-α were measured using Luminex as a supernatant after centrifugation. Reader).

10) Induction of rheumatoid arthritis and sample treatment

Five-week-old DBA / 1 mice were stabilized for 2 weeks and then fed to a 7-week-old mouse. The bovine type 2 collagen solution at a concentration of 2 mg / ml was mixed with a full-fledged excipient (CFA) And 0.1 ml of the mixed solution (100 쨉 g of type II collagen) was slowly injected through the subcutaneous vein 1.5 to 3 cm below the base of the tail to induce the first induction. Two weeks after the first induction, it was induced by injecting one of the sole with the incomplete prunette excipient (IFA) instead of the complete prunate excipient. The experiment was divided into four groups, namely, a normal group and a control group to which distilled water was administered, a positive control group to which indomethacin (hereinafter referred to as "Indo") was administered, and an experimental group to which BHT was administered, (200 mg / kg). During the experiment, free diet was applied. The dose of BHT and Indo was calculated by taking a single dose per 60 kg of adult body weight and taking a sample from a single intake of 30 g of body weight.

11) Measurement of rheumatoid arthritis index (AI)

RA index (Rheumatoid Arthritis index score), clinical scores of 0 to 4 points (0 to 16 points) for each foot on each 4 paws of DBA / 1 mice after the 2nd injection, change in ankle thickness, incidence Were measured. Measurements were recorded weekly from week 9 to week 13 and the criteria were as follows (Table 2).

Rheumatoid arthritis index Inflammation-induced degree 0 No inflammation One A small degree of inflammation 2 Light swelling 3 Medium swelling 4 Severe swelling

12) Measurement of immune cells and hs-CRP in blood

After completion of the experiment, blood samples were collected from the DBA / 1 mice by cardiopulmonary resuscitation and then centrifuged at 6,500 rpm for 20 minutes to determine leukocyte, neutrophil, lymphocyte, and mononuclear cell contents and hs-CRP : high-sensitivity C-reactive protein) to a scientific research institute in Seoul.

13) Serum cytokine, chemokine and immunoglobulin measurement

After completion of the experiment, blood was collected by cardiac puncture under anesthesia with ethyl ether, and the serum was separated by centrifugation at 6,500 rpm for 20 minutes. IL-1β, IL-6 and TNF-α concentrations were measured using a custom-made 6-plex cytokine Milliplex panel as follows. 25 μl of 50-fold diluted serum was added to each well, and 25 μl of assay buffer, matrix buffer and antibody-immobilized beads were added to each well, followed by incubation at room temperature for 2 hours And then washed twice with a washing buffer. Then, 25 μl of detection antibody was added thereto, followed by cow reaction at room temperature for 1 hour. Streptavidin-Phycoerythrin (25 μl) was further added thereto, followed by reaction at room temperature for 30 minutes. And washed twice with buffer. After washing, 150 μl of PBS was added, shaking was performed for 5 minutes, and the measurement was performed using Luminex.

IL-17, IL-21, MCP-1 and GM-CSF were measured using an enzyme immunoassay kit as follows. 100 μl of coating buffer was dispensed in Microwell, allowed to stand overnight at 4 ° C, and then washed three times with washing buffer. Block buffer (200 μl per well) was added to each well, and the mixture was kept at room temperature for 1 hour, washed with washing buffer, and 100 μl of 50-fold diluted serum was dispensed. Respectively. Then 100 μl of the detection antibody was treated and washed for 1 hour. 100 μl of avidin-horseradish peroxidase (Avidin-HRP) was added thereto, reacted at room temperature for 30 minutes, and then washed seven times with washing buffer . After 100 μl of Substrate Solution was added, 50 μl of Stop Solution was added 15 minutes later and the absorbance was measured at 450 nm with an enzyme immunoassay reader.

Measurement of IgM and IgG production was carried out using an enzyme immunoassay kit as follows. Antibodies were diluted in coating buffer, coated on microwells and allowed to stand overnight at 4 < 0 > C. Each well was washed three times with washing buffer, and 100 쨉 l of 50-fold diluted serum was dispensed. This was reacted at room temperature for 1 hour, washed twice with washing buffer, treated with avidin-HRP conjugated antibody (100 μl), reacted at room temperature for 1 hour, and then washed again. 100 μl of TMB substrate was added to each well. After reacting in a dark place for 30 minutes, 50 μl of the stop solution was treated, and the absorbance was measured at 450 nm with an enzyme immunoassay reader.

14) Measurement of gene expression

(1) RNA extraction

After the end of the experiment, RT-PCR was performed to measure the rheumatoid arthritis-inducing factor expressed in the splenic tissue. The separated splenic tissue was frozen, the tissue was disrupted with a homogenizer, 1 ml of easy blue was added, and 200 μl of chloroform (CHCl ₃ ) was added and mixed again for 15 seconds. After centrifuging at 13,000 rpm using a 4 ° C centrifuge, about 400 μl of the supernatant was recovered, mixed with 400 μl of a binding buffer, and the solution was slowly poured into the column. After centrifugation at 13,000 rpm with a centrifuge at 4 ° C, wash buffer was added, centrifuged again, washed again with washing buffer, and 50 μl of elution buffer was added. Respectively.

(2) cDNA synthesis

RT (reverse transcription) reaction mixture (reaction buffer, dNTPs mixture, and ribonuclease inhibitor (RNase inhibitor), a stabilizer (stabilizer), oligo dT ₁₅ primer (oligo dT ₁₅ primer) of the reverse transcription premix kit (RT premix kit) ) Was added to distilled water treated with diethyl pyrocarbonate (DEPC) so as to have a final volume of 20 μl to 1 μg of total RNA. This 20 μl reaction mixture was mixed well, centrifuged at 2,000 rpm for 5 seconds, and reacted in a heating block at 45 ° C. for 60 minutes to synthesize a first strand cDNA. After incubation at 95 ° C for 5 minutes, M-MLV RT was inactivated and the synthesized cDNA was used for PCR.

(3) Measurement of gene expression in spleen tissue

RT-PCR was performed by adding each sample and primer to a mixture containing 250 mM dNTPs, RT buffer (10 mM Tris-HCl, pH 9.0, 30 mM KCl, 1.5 mM HCl ₂ ) in ₁ U / (primers) were added and polymerase chain reaction was performed. IL-1β is 2 min at 56 ° C, 10 min at 95 ° C, 15 sec at 60 ° C, 2 min at 60 ° C for IL-6, 15 min at 95 ° C, 15 sec at 60 ° C, 2 minutes at 95 ° C for 10 minutes, 60 ° C for 15 seconds, IL-21 at 53 ° C for 2 minutes, 95 ° C for 10 minutes, 60 ° C for 10 seconds, TNF- 10 min at 60 ° C, 10 min at 60 ° C, 2 min at 56 ° C, 10 min at 95 ° C, 15 sec at 60 ° C, 2 min at 55 ° C for GM-CSF, PCR was carried out at 35 to 40 cycles (cycles) for 15 seconds. The primers used were as follows (Table 3). After electrophoresis on 1% agarose gel, the presence of gene expression was photographed with ultraviolet light to identify bands for each group and RNA expression was shown.

primer F / R Base sequence rotation Annealing (℃) IL-1? F GTT GAC GGA CCC CAA AAG AT 40 56 R AAG GTC CAC GGG AAA GAC AC IL-6 F CCT ACC CCA ATT TCC AAT GC 35 60 R CGC ACT AGG TTT GCC GAG TA IL-17 F AAC CAA AAC CAG GGC ATT TC 35 57 R GAT TTC TTG CTG AAT GGC GA IL-21 F GAA AGC CTG TGG AAG TGC AA 35 53 R GGA AAG GAT GTG GGA GAG GA TNF-a F CTA CTC CTC AGA GCC CCC AG 35 58 R AGG CAA CCT GAC CAC TCT CC MCP-1 F GCT CAT AGC AGC CAC CTTCA 35 56 R GGG GTC AGC ACA GAT CTC CT GM-CSF F CCT TGA ACA TGA CAG CCA GC 35 55 R AGG GCT ATA CTG CCC TCC AA

15) Radiographic examination and structural parameter measurement

At the end of the 5-week experiment, the animals were sacrificed and the hind paws were separated for each group. After separation, the specimen of the foot was cut to the maximum extent before post-stiffness, and fixed in 10% formaldehyde solution. Micro-CT and Structural Parameter measurement were carried out at Raon Bio Co., . The microtomography was performed in 3D (3D) after the entire hind foot was taken. Structural parameters were measured on the basis of the middle toe (BV) and on the bone surface (BS) The rate of inflammation of the joints was calculated.

16) Observation of tissue staining

After the ankle was cut with anesthetized with ethyl ether (C ₂ H ₅ OC ₂ H ₅ ), the tissues extracted from each group were fixed in 10% neutral formalin for 48 hours. The tissues were washed for 12 hours in tap water flowing through the fixed tissues, The fixative solution was completely removed. For the dehydration of the tissue, dehydration was performed in order of increasing concentration from 60% to 100% alcohol, and xylene was subjected to a transparent process and then a paraffin block was prepared. The prepared block was cut to a thickness of 3 to 4 μm using a microtome, deparaffinized and functionalized, and stained with hematoxyline and eosin (H & E) staining and masson-trichrome ), Respectively, and observed and photographed on an optical microscope (Fig. 1).

3. Statistical processing

The results were statistically analyzed using the unpaired Student's t-test and ANOVA of SPSS 11.0. The significance was tested at p <0.05, p <0.01 and p <0.001.

4. Experimental results

(1) Heavy metal content

As a result of measuring the heavy metal content of BHT, cadmium was detected below the reference value, and lead, arsenic and mercury were not detected (Table 4).

lead arsenic cadmium Mercury Allowable density
(Mg / kg) 5 3 0.3 0.2 BHT Non-detection Non-detection 0.002 Non-detection

2. HPLC analysis

As a result of HPLC pattern analysis of BHT, peaks were observed at retention times of 9.00 min, 15.96 min, 18.80 min, 33.87 min and 42.45 min at 254 nm (Fig. 2). (ACE 5 C18, 250X4.6 mm), sample injection volume: 10 μl, mobile phase: water (A) / acrylonitrile (B ), Gradient elution: 0 to 5 minutes (0% B), 5 to 40 minutes (30% B), 40 to 60 minutes (80% flow rate: 1.0 ml / min, detector: PDA detector.

3. Effect on liver function

1) Aspartate aminotransferase (AST) content

The AST of liver function measurement was 125.4 ± 6.4 U / L in normal group, 146.7 ± 5.8 U / L in control group, 165.6 ± 7.1 U / L in positive control group and 133.8 ± 6.3 U / L, showing no difference compared to the normal group, and thus it was safe (FIG. 3). In Figure 3, the DBA / 1 mouse model was traced for 5 weeks after administration of BHT. Results are expressed as mean ± standard deviation.

2) alanine aminotransferase (ALT) content

The ALT was measured as 46.7 ± 3.3 U / L in the normal group, 43.3 ± 5.3 U / L in the control group, 63.3 ± 2.1 U / L in the positive control group, 47.2 ± 6.7 U / L, showing no difference compared to the normal group, and thus it was safe (FIG. 4). In Figure 4, the DBA / 1 mouse model was traced for 5 weeks after administration of BHT. Results are expressed as mean ± standard deviation.

4. Influence on renal function

1) Content of creatinine (Cr)

The creatinine level was 1.6 ± 0.3 (㎎ / ㎗) in the normal group, 1.2 ± 0.6 (㎎ / ㎗) in the control group, 1.6 ± 0.3 (㎎ / ㎗) in the positive control group, And 1.4 ± 0.7 (mg / dl), respectively, showing no difference compared to the normal group (FIG. 5). In Figure 5, the DBA / 1 mouse model was traced for 5 weeks after administration of BHT. Results are expressed as mean ± standard deviation.

2) BUN (Blood urea nitrogen) content

The mean BUN of the new functional test was 50.0 ± 2.1 mg / dl in the normal group, 46.7 ± 3.8 mg / dl in the control group, 43.3 ± 4.2 mg / dl in the positive control group, 43.3 ± 3.9 mg / ㎗, showing no difference compared to the normal group (Fig. 6). In Figure 6, the DBA / 1 mouse model was traced for 5 weeks after administration of BHT. Results are expressed as mean ± standard deviation.

5. Cytotoxicity to RAW 264.7 cells

Cell viability of RAW 264.7 cells was found to be 111.0%, 108.9% and 95.5% at the concentration of 1, 10, and 100 ㎍ / ㎖ of BHT extract, respectively, when the control group was 100.0% (Fig. 7). In Figure 7, each cell was treated with 1, 10 and 100 (占 퐂 / ml) BHT for 24 hours. Cytotoxicity was measured using MTT assay. Results are expressed as mean + standard deviation from three independent experiments.

6. Effect on antioxidant efficacy

1) Total phenol content

The total phenol content of the BHT extract was measured using gallic acid as a reference material, and the phenol content was 215.0 ± 4.4 mg / g as shown in Table 5.

Sample Total phenol content (mg / g extract) BHT 215.0 ± 4.4

2) Total flavonoid content

The total flavonoid content in the BHT extract was measured using quercetin as a standard substance, and the flavonoid content was 22.3 ± 1.4 mg / g as shown in Table 6.

Sample Total flavonoid content (mg / g extract) BHT 22.3 ± 1.4

3) Effect on DPPH radical scavenging ability

The DPPH elimination rate of BHT extract was 10.8% at 1 ㎍ / ㎖ concentration, 12.6% at 10 ㎍ / ㎖ concentration, 21.7% at 100 ㎍ / ㎖ concentration and 66.3% at 1,000 ㎍ / ㎖ concentration, To increase the radical scavenging ability (Fig. 8). In Fig. 8, the extract was incubated with DPPH solution for 30 minutes at 37 占 폚. The activity was determined by measuring the absorbance at 517 nm. Results are expressed as mean + standard deviation from three independent experiments.

4) Effect on ABTS radical scavenging ability

The ABTS scavenging activity of BHT extract was 9.7% at 1 ㎍ / ㎖ concentration, 10.1% at 10 ㎍ / ㎖ concentration, 20.9% at 100 ㎍ / ㎖ concentration and 70.1% at 1,000 ㎍ / To increase the radical scavenging ability (Fig. 9). In Fig. 9, the extracts were incubated with DPPH solution for 10 minutes at 37 占 폚. The activity was determined by measuring the absorbance at 732 nm. Results are expressed as mean + standard deviation from three independent experiments.

5) Effect on production of reactive oxygen species (ROS)

The inhibitory activities of BHT extracts were 19.3% in normal group, 92.2% in 1 ㎍ / ㎖, 90.0% in 100 ㎍ / ㎖, 100 ㎍ / ( P : 0.01, *: p < 0.05) at 10, 100 (ug / ml) concentration (FIG. 10). In Figure 10, each cell was treated with 1, 10 and 100 (占 퐂 / ml) BHT extract and LPS (1 占 퐂 / ml) for 24 hours. The ROS generation was analyzed by flow cytometry. Results were expressed as mean ± standard deviation from three independent experiments (significant results, **: p <0.01, *: p <0.05).

7. Effect on anti-inflammatory efficacy

1) Effect on production of NO (NO)

In the RAW 264.7 cells, the NO production of the BHT extract was 38.2% in the control group, 100.0% in the control group, 97.5% in the 1 μg / ㎖ concentration, 93.3% in the 10 μg / ( P <0.01, *: p <0.05) at a concentration of 10, 100 (㎍ / ㎖) compared to the control group. In Fig. 11, RAW 264.7 cells were treated with 1, 10 and 100 (占 퐂 / ml) BHT extract and LPS (1 占 퐂 / ml) for 24 hours. The amount of nitrogen oxide in the supernatant was measured using a grease reagent. Results were expressed as mean ± standard deviation from three independent experiments (significant results, **: p <0.01, *: p <0.05).

2) Effects on cytokines and chemokines

(1) Effect on IL-1β production amount

The amount of IL-1β produced by the BHT extract in RAW 264.7 cells was 28.6 ± 1.7 pg / ㎖ in the control group and 6.1 ± 2.3 pg / ㎖ in the normal group, and 28.3 ± 2.1 pg / ml at the concentration of 1 μg / , 26.6 ± 2.7 pg / ㎖ at 10 ㎍ / ㎖ and 20.7 ± 3.3 pg / ㎖ at 100 ㎍ / ㎖, respectively, and a significant decrease (*: p <0.05) was observed at the concentration of 100 ㎍ / (Fig. 12). In Fig. 12, RAW 264.7 cells were treated with BHT extracts at 1, 10 and 100 (占 퐂 / ml) in the presence of LPS (1 占 퐂 / ml) for 24 hours. Results were expressed as mean + standard deviation from three independent experiments.

(2) Effect on the amount of IL-6 produced

The amount of IL-6 produced by the BHT extract in RAW 264.7 cells was 1419.7 ± 182.5 pg / ml in the control group and 342.6 ± 168.9 pg / ml in the normal group, and 1355.7 ± 156.4 pg / ml at the concentration of 1 μg / , 1233.1 ± 188.6 pg / ml at a concentration of 10 μg / ml, and 1200.9 ± 103.8 pg / ml at a concentration of 100 μg / ml (FIG. 13). In Figure 13, RAW 264.7 cells were treated with BHT extracts at 1, 10, and 100 (占 퐂 / ml) in the presence of LPS (1 占 퐂 / ml) for 24 hours. Results were expressed as mean + standard deviation from three independent experiments.

(3) Effect on the amount of IL-17 produced

The amount of IL-17 produced in the RAW 264.7 cells was 20.7 ± 2.6 pg / ml in the control group and 4.4 ± 1.5 pg / ml in the normal group, and 20.5 ± 2.0 pg / ml at the concentration of 1 μg / , 19.3 ± 1.7 pg / ml at a concentration of 10 μg / ml, and 17.5 ± 1.4 pg / ml at a concentration of 100 μg / ml, showing a decrease at all concentrations compared to the control (FIG. 14). In Figure 14, RAW 264.7 cells were treated with BHT extracts at 1, 10, and 100 (ug / ml) in the presence of LPS (1 ug / ml) for 24 hours. Results were expressed as mean + standard deviation from three independent experiments.

(4) Effect on the amount of IL-21 produced

The amount of IL-21 produced in the RAW 264.7 cells was 0.7 ± 0.1 pg / ml in the control group and 0.3 ± 0.1 pg / ml in the normal group, and 0.2 ± 0.1 pg / ml at the concentration of 1 μg / , 0.3 ± 0.1 pg / ml at a concentration of 10 μg / ml, and 0.2 ± 0.1 pg / ml at a concentration of 100 μg / ml (FIG. 15). In Figure 15, RAW 264.7 cells were treated with BHT extracts at 1, 10, and 100 (ug / ml) in the presence of LPS (1 ug / ml) for 24 hours. Results were expressed as mean + standard deviation from three independent experiments.

(5) Effect on production of TNF-α

The amount of TNF-α produced in RAW 264.7 cells was 10096.9 ± 330.8 pg / ml in the control group, 3412.6 ± 66.7 pg / ml in the normal group, and 9477.1 ± 210.8 pg / ml at the concentration of 1 μg / , And 9504.9 ± 332.2 pg / ㎖ at the concentration of 10 ㎍ / ㎖, and 8544.3 ± 345.4 pg / ㎖ at the concentration of 100 ㎍ / ㎖, indicating a significant (*: p <0.05) reduction in the concentration of 100 ㎍ / (Fig. 16). In Fig. 16, RAW 264.7 cells were treated with BHT extracts at 1, 10 and 100 (占 퐂 / ml) in the presence of LPS (1 占 퐂 / ml) for 24 hours. Results were expressed as mean ± standard deviation from three independent experiments (significant results, *: p <0.05).

(6) Effect on production of GM-CSF

The amount of GM-CSF produced in the RAW 264.7 cells was 70.2 ± 6.6 pg / ㎖ in the control group and 23.9 ± 5.4 pg / ㎖ in the normal group, and 71.1 ± 5.7 pg / ml at the concentration of 1 ㎍ / , 66.6 ± 4.0 pg / ml at a concentration of 10 μg / ml, and 59.8 ± 4.1 pg / ml at a concentration of 100 μg / ml, and decreased at a concentration of 10, 100 (㎍ / ml) compared with the control group ). In Fig. 17, RAW 264.7 cells were treated with BHT extracts at 1, 10 and 100 (占 퐂 / ml) in the presence of LPS (1 占 퐂 / ml) for 24 hours. Results were expressed as mean + standard deviation from three independent experiments.

(7) Effect on the amount of MCP-1 produced

The amount of MCP-1 produced in RAW 264.7 cells was 507.7 ± 59.1 pg / ml in the control group and 203.7 ± 47.3 pg / ml in the normal group, and 450.6 ± 45.8 pg / ml at the concentration of 1 μg / , 448.9 ± 51.1 pg / ml at a concentration of 10 μg / ml, and 423.3 ± 40.4 pg / ml at a concentration of 100 μg / ml, showing a decrease at all concentrations compared to the control (FIG. In FIG. 18, RAW 264.7 cells were treated with BHT extracts at 1, 10 and 100 (占 퐂 / ml) in the presence of LPS (1 占 퐂 / ml) for 24 hours. Results were expressed as mean + standard deviation from three independent experiments.

8. Effect on serum cytokine and chemokine production

1) Effect on IL-1β production

The amount of IL-1β produced after the administration of BHT was 217.5 ± 74.1 pg / ㎖ in the normal group, 755.0 ± 68.4 pg / ㎖ in the control group, 472.9 ± 45.5 pg / ㎖ in the positive control group and 533.8 ± 50.9 pg / ml, indicating a significant decrease (**: p < 0.01) compared to the control group (Fig. 19). In Figure 19, the results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01).

2) Effect on the amount of IL-6 produced

The amount of IL-6 produced after the administration of BHT was 423.4 ± 88.8 pg / ㎖ in normal group, 675.9 ± 107.5 pg / ㎖ in control group, 373.1 ± 79.0 pg / ㎖ in positive control group and 375.3 ± 90.1 pg / ml, indicating a significant decrease (**: p <0.01, *: p <0.05) compared to the control group (Fig. 20). In Figure 20, results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01, *: p <0.05).

3) Effect on the amount of IL-17 produced

The amount of IL-17 produced after the administration of BHT was 9.7 ± 0.4 pg / ㎖ in the normal group, 13.4 ± 1.4 pg / ㎖ in the control group, 10.3 ± 0.3 pg / ㎖ in the positive control group and 10.2 ± 0.4 pg / ml, indicating a decrease compared to the control (Fig. 21). In Figure 21, the results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01, *: p <0.05).

4) Effect on the amount of IL-21 produced

The amount of IL-21 produced after the administration of BHT was 88.8 ± 10.4 pg / ㎖ in the normal group, 193.2 ± 13.9 pg / ㎖ in the control group, 101.5 ± 12.2 pg / ㎖ in the positive control group, 131.8 ± 15.3 pg / ml, showing a significant decrease (**: p <0.01, *: p <0.05) in comparison with the control group (FIG. 22). In Fig. 22, the results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01, *: p <0.05).

5) Effect on TNF-α production

The amount of TNF-α produced after the administration of BHT was 134.3 ± 31.7 pg / ㎖ in the normal group, 274.4 ± 37.2 pg / ㎖ in the control group, 204.8 ± 25.5 pg / ㎖ in the positive control group and 186.0 ± 30.8 pg / ml, indicating a significant (*: p < 0.05) reduction compared to the control (Fig. 23). In Fig. 23, the results are expressed as mean + standard deviation. Statistically significant values were calculated by Student's t-test comparing to the control (significant results, *: p <0.05).

6) Effect on GM-CSF production

The amount of GM-CSF produced after the administration of BHT was 90.5 ± 27.8 pg / ㎖ in the normal group, 288.9 ± 87.5 pg / ㎖ in the control group, 124.5 ± 21.9 pg / ㎖ in the positive control group and 198.0 ± 19.5 pg / ml, showing a significant decrease (**: p <0.01, *: p <0.05) compared to the control group (FIG. 24). In Fig. 24, the results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01, *: p <0.05).

7) Effect on production of MCP-1

The amount of MCP-1 produced after the administration of BHT was 437.8 ± 43.5 pg / ㎖ in the normal group, 1037.6 ± 134.5 pg / ㎖ in the control group, 661.1 ± 108.9 pg / ml in the positive control group and 603.5 ± 54.6 pg / ml, indicating a significant decrease (**: p <0.01, *: p <0.05) compared to the control group (FIG. 25). In Fig. 25, the results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01, *: p <0.05).

9. Effect on gene expression

1) IL-1? Gene expression

The expression of IL-1β gene after the administration of BHT was found to be 34.3% in the normal group, 48.2% in the normal group, and 54.3% in the BHT group when the control group was 100.0% (Fig. 26). In Fig. 26, the results are expressed as mean + standard deviation.

2) Expression of IL-6 gene

The expression of IL-6 gene after the administration of BHT was 34.3% in the normal group, 49.8% in the normal group, and 40.1% in the BHT group, respectively, when the control group was 100.0% (Fig. 27). In Figure 27, results are expressed as mean + standard deviation.

3) Expression of IL-17 gene

After the end of BHT administration, gene expression of IL-17 was 10.4% in the control group, 100.0% in the control group, 64.4% in the normal control group, and 70.5% in the BHT administration group, (Fig. 28). In Fig. 28, the results are expressed as mean + standard deviation.

4) Expression of IL-21 gene

The expression of IL-21 gene after the administration of BHT was found to be 35.6% in the normal group, 37.2% in the normal group, and 70.6% in the BHT-treated group when the control group was 100.0% (Fig. 29). In Figure 29, results are expressed as mean + standard deviation.

5) TNF-α gene expression

The expression of TNF-α gene after the administration of BHT was 21.9% in the normal group, 44.3% in the normal group, and 67.9% in the BHT-treated group, respectively, when the control group was 100.0% (Fig. 30). In Figure 30, results are expressed as mean + standard deviation.

6) Expression of GM-CSF gene

After the administration of BHT, the expression of GM-CSF gene was 44.3% in the normal group, 43.7% in the normal group, and 59.9% in the BHT group when the control group was 100.0% (Fig. 31). In Figure 31, results are expressed as mean + standard deviation.

7) Expression of MCP-1 gene

The expression of MCP-1 gene after the completion of BHT administration was 32.7% in the normal group, 57.3% in the normal group, and 60.2% in the BHT-treated group when the control group was 100.0% (Fig. 32). In Figure 32, results are expressed as mean + standard deviation.

10. Effect on serum immunoglobulin production

1) Effect on IgM production

The IgM production in the serum was 10.4 ± 5.9 pg / ㎖ in the normal group, 49.7 ± 9.3 pg / ㎖ in the control group, 18.1 ± 7.7 pg / ㎖ in the positive control group, 31.8 ± 5.4 pg / (**: p < 0.01, *: p < 0.05) compared to the control group (FIG. 33). In Figure 33, results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01, *: p <0.05).

2) Effect on IgG production

The amount of IgG produced after the administration of BHT was 54.4 ± 7.8 pg / ㎖ in the normal group, 127.3 ± 10.5 pg / ㎖ in the control group, 89.6 ± 8.8 pg / ㎖ in the positive control group, 95.7 ± 8.9 pg / Ml, indicating a significant decrease (**: p < 0.01) compared to the control (Fig. 34). In Figure 34, results are expressed as mean + standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, **: p <0.01).

11. Influence on Immune Cells in Blood

1) Effect on leukocyte production

The amount of leukocyte produced after the end of BHT administration was 2.3 (thousand) / ㎕ (Thous / ㎕) in normal group, 5.9 (thousand) / ㎕ in control group, 2.7 (thousand) / ㎕ in positive control group, ( P <0.01, *: p <0.05) compared to the control group (FIG. 35). In Figure 35, the results are expressed as mean ± standard deviation (significant results, **: p <0.01, *: p <0.05).

2) Effect on neutrophil production

The ratio of neutrophils to blood leukocyte counts after the administration of BHT was found to be 3.5% in the normal group, 8.2% in the control group, 5.4% in the positive control group and 6.4% in the BHT group and decreased compared to the control group 36). In Figure 36, results are expressed as mean + standard deviation.

3) Effect on lymphocyte production

The ratio of lymphocytes to blood leukocytes after the administration of BHT was 1.5% in the normal group, 3.9% in the control group, 1.4% in the positive control group and 1.7% in the BHT group, p < 0.05) (Fig. 37). In Figure 37, results are expressed as mean + standard deviation (significant results, *: p < 0.05).

4) Effect on Monocyte Production

The ratio of mononuclear cells to leukocytes in blood after the end of BHT administration was 0.3% in the normal group, 0.7% in the control group, 0.2% in the positive control group and 0.1% in the BHT group, *: p <0.001, **: p <0.01) (FIG. 38). In Figure 38, the results are expressed as mean ± standard deviation (significant results, ***: p <0.001, **: p <0.01).

12. Effect on serum hs-CRP

After the administration of BHT, the serum hs-CRP levels were 0.2 ± 0.1 mg / L in the normal group, 0.9 ± 0.1 mg / L in the control group, 0.2 ± 0.1 mg / L in the positive control group and 0.3 ± 0.1 mg / ℓ, indicating a significant decrease (**: p <0.01) compared to the control group (FIG. 39). In Figure 39, the results are expressed as mean ± standard deviation (significant results, **: p <0.01).

13. Gross observation and AI measurement of DBA / 1 mice

FIG. 40 is a photograph showing the degree of RA improvement at 9 weeks, 11 weeks, and 13 weeks in the positive control group and the BHT administration group during 5 weeks during which the experiment was performed. In the control group, paw, ankle, While the positive control group and the BHT-treated group were gradually decreased and showed a large decrease compared to the control group at 13th week (FIG. 40). These results were reflected in AI. The AI index was measured by sensory evaluation of the depth of RA after taking pictures every week from 9th to 13th week after inducing RA. The control group was 12.2 at 9th, 14.3 at 10th, 13.5 at 11th, 11.9 in the 12th week and 11.4 in the 13th week. The positive control group showed 12.1 in the 9th, 11.0 in the 10th, 8.7 in the 11th, 7.9 in the 12th, and 5.9 in the 13th, The BHT administration group showed a significant decrease (***: p <0.001) compared to the control group (12.0, 12.7, 11.11, 9.8, ). The rheumatoid arthritis index was defined as the sum of the individual scores scored as 0 (no arthritis), 1 (small degree of arthritis), 2 (mild swelling), 3 (moderate swelling) and 4 (severe swelling). Results are expressed as mean ± standard deviation. Statistically significant values were calculated by comparing Student's t-test with the control (significant results, ***: p <0.001).

14. Influence on joints

1) Effect on joint condition

After the end of the experiment, micro-tomography of the posterior foot and conversion to 3D showed that the degeneration of the mid- to upper-jaw joints and the fracture between the nodes were severe in the control group (FIG. 42A and B, ), And the fracture between the joints of the mid- and upper-jaw joints and the joints was relatively small in the BHT-treated group (the squares C and D in FIG. 42) in addition to the positive control group. In Figure 42, A is a group of normal mice, B is collagen-induced rheumatoid arthritis group, C is collagen-induced rheumatoid arthritis group treated with indomethacin, and D is collagen-induced rheumatoid arthritis group treated with BHT .

2) Effect on bone density

After the end of the experiment, the hind paw was analyzed by 3D CT (3D CT) and bone mineral density (BV) was calculated to be 0.36 (U ^ 3) in the normal group, (U ^ 3), positive control group had 0.37 (U ^ 3) and BHT treated group had 0.38 (U ^ 3), showing higher bone density values than the control group (FIG. 43). In Figure 43, results are expressed as mean + standard deviation.

3) Influence on joint inflammation

After the end of the experiment, the hindpaw was analyzed by 3-D micro-tomography and the ratio of bone to bone surface (BS) and bone volume was calculated. The degree of inflammation of the joint was measured as 17.10 (1 / U) (**: p <0.01) decreased compared to the control group, which was 26.9 (1 / U) in the control group, 17.0 (1 / U) in the positive control group and 18.0 44). In Figure 44, the results are expressed as mean + standard deviation. Statistically significant values were calculated by Student's t-test comparing to the control (significant results, *: p <0.05).

15. Impact on organizational change

After the end of the experiment, hematoxylin and eosin (H & E) and masson-trichrome (MT) staining were performed in the hindpaw tissues and the result was measured at 25x magnification (25x) Granulocytes, mononuclear cells, fibrocytes, subsynovial inflammation, and hyperplasia of the synovial cells caused cartilage and bone subsidence, whereas BHT-treated group Compared with the control group, immune cell infiltration, cartilage subsidence, and synovial cell damage were relatively decreased (FIG. 45). 45, A is a group of normal mice, B is collagen-induced rheumatoid arthritis group, C is collagen-induced rheumatoid arthritis group treated with indomethacin, and D is collagen-induced rheumatoid arthritis group treated with BHT . The sections were contrast dyed with hematoxylin and eosin, Mars-trichrome.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

(Without reference numerals)

Claims (4)

Which is characterized by containing as an active ingredient an extract of Kami Bangui Hwanggi-tang containing 9 kinds of herbal medicines such as Hwanggi, Bangui, Cheondudo, A composition for preventing and treating rheumatoid arthritis. The method according to claim 1,
11 to 15% by weight of Kamiibanguiranggi, 11 to 15% by weight of silkworm, 11 to 15% by weight of astaxanthin, 8 to 12% by weight of chewing gum, 8 to 12% To 12% by weight of liquorice, 3 to 6% by weight of licorice, and a residual amount of sulfur. The method according to claim 1,
Wherein the alcoholic extract of Kami-Bangi Hwanggi-tang is added to 70 to 90% of alcoholic beverage (1,000 to 1,400) on the basis of 90 g of Kami-Bangi Hwanggi-tang, extracted by reflux extraction and then evaporated and concentrated to powder. A composition for the prevention and treatment of rheumatoid arthritis, which comprises a extract of Hwanggi-tang's alcohol. A method for preventing and treating rheumatoid arthritis in an animal other than a human using the composition for the prevention and treatment of rheumatoid arthritis, which comprises the extract of a spore of Hwanggi-tang, according to any one of claims 1 to 3.

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