201004634 七、指定代表圖： (一） 本案指定代表圖為：第（$ 、 (二) 本代表圖之元件符號簡單說明: 無 八、本案若有彳b學式時’請揭示最麟示發明特徵的化學式. 無 九、發明說明： 【發明所屬之技術領域】 本發明乃一有關含有牛樟芝成份的外用藥及供經絡穴位 注射治療的用藥，經由這兩種劑型及用藥途徑，可用來預防或 治療皮膚癌、皮膚炎、青春痘、微血管異常增生、關節炎、及 各種腫瘤、癌症。。 【先前技術】 牛樟芝加如胤>舰<3)為一種台灣特有的兹菌，為 多孔菌科薄孔屬（也之成員，在天然環境中， 201004634 匕八寄生在口灣特有的牛棒木（匚如瓜洲浙⑽灸Hay.) 腐化之髓部，成長相當緩慢，因此天然的牛樟芝相當稀少。台 灣原住民將它用來做撕、解毒，治獨、職的良藥。近年 來也發現口服牛樟芝對肝癌、肺癌、胃癌、膀胱癌、血癌、肝 炎、肝硬化等有相當的幫助。由於牛樟芝無毒性，又有保肝、 抗氧化、抗癌等功能，現多以保健食品販賣。目前牛樟芝的使 用為口服，雖有文獻提到靜脈注射，仍未有實際應用。但牛樟 之極苦，且口服的生体可利用率（bi〇availability)也相當 低，因此並非一理想的給藥方式。而靜脈注射會使藥物遍佈全 身，谷易引發不可預期的副作用，而且藥物經全身血液稀釋 後，有效k度降低，也無法使成份聚集到病灶位置因此藥界 積極的在研究標的給藥的方法。本發明即針對這些缺失，提供 解決的方法。 外用藥乃將藥物直接塗抹到皮膚上，使用簡便，可直接 作用到表皮病變，直接接觸，有效濃度高，收效快，且進入其 他器官的藥量也較少、，可能的副作用相對的也較少。正常的皮 膚通常表皮細胞排列較敏密，外物不易進入，但有病變的皮膚 則細胞排列較鬆散、粗Μ，甚至形成賴，因此藥物較容易進 入，發揮治療功效。本發明即利用此一特徵，使用含牛樟芝成 份的藥物來治療皮膚腫瘤、皮膚癌、皮敍、f春癌、微血管 異常增生、襲炎等各難病，收戦想不到的功效。 201004634 穴位注射則是利用人體的經絡系統，來進行標的給藥。 人體主要有12條經脈及奇經八脈，其分佈涵蓋全身，通達内 臟及表皮。每條經脈有特定的循行路徑及涵蓋範圍。分佈在經 脈上的穴位，在臨床上也各有其特定功能。這些經絡除了掌管 人体氣血的流動平衡外，深人的研究經脈也有傳送物 質的功能。以放射性物質注入穴位後，即可觀察到放射性物質 沿著穴位所屬經脈傳送放射性物質，並進入臟腑，傳送路徑與 經脈相當-致，非-般的擴散作用，更非經由血管系統傳送。 而且經脈祕有”氣人赌，，的特徵，即舰適#的穴位後，氣 會優先傳到病灶位置。利用經絡傳導的這種特徵，我們可以將 藥物有效的傳送到有病的位置。所需的藥物劑量低於一般口 服、皮下注射、肌肉注射或靜脈注射，而錄效快，可能的副 作用較小。因此本發明之另一目的，即利用經脈傳輸的這些特 ❹ 徵嗜如斜毅雜㈣物，經由穴健射，發揮標的給 藥及經穴刺激的雙重作用，達到預防及治療各種癌症、腔瘤、 微血管異常增生、關節炎、肝炎或肝硬化等功用。 【發明内容】 將天然牛料或採職細酵、@ _酵或縣騎所得人工 培養的牛樟芝ϋ絲體烘乾、磨粉、過篩網後，可以溶劑將有效 成份萃取出來。溶劑與牛樟芝的比例可以是10比卜或視需 6 201004634 要而調整。溶劑可以是酒精、醋酸乙酯、正戊己烷、甲醇、四 氟甲燒、二氯甲烧或其它具有類似性質的溶劑，其中，以酒精 較佳，酒精又以含有5%〜70%水份者更佳。牛樟芝有效成份相 當複雜，且含量隨菌種及培養條件而有相當的差異，要判定分 離物中是否含有效成份，除了常用的物理、化學、LC_MASS、 HPLC等分析方法外’也可以細胞存活、生長分析做為一指標。 下面實施例癌細胞生長抑制分析或微血管增生抑制分析方法 可做參考。 粗萃取分離物可以進一步純化，例如薄膜方離法、分子篩 透析法、液體色層（liquid chromatography)分離法、凝膠渗 透色層（gel permeation chromatography)分離法、離心法、 沈氣法、離子父換樹脂分離法，砍膠(siHca跃1)分離法、 HPLC、結晶法等皆可用來做成份的純化。然而純化不一定帶來 更高的活性或更好的經濟效益。採用多種成份並存的雞尾酒 (cocktail)組方’常可收到各成份功效加乘的效果。 分離或純化出來的牛樟芝成份可進一步濃縮或乾燥，然後 溶入藥理上可接受的溶劑，如水、乙醇、甘油、丙二醇、聚乙 二醇、油酸乙酯、笨甲酸苄酯、二曱基乙酰胺、N-CP-經基乙 基、乳跌胺、肉立蔻異丙基酯等，或這些溶劑的組合物。增溶 劑’如Tween-80、胆酸等也可適量使用，以增加溶解度。 7 201004634 牛樟芝分離物可以做成乳液劑型、凝膠劑型、軟膏劑型或 乳膏劑型以供外用，其濃度以l~5〇〇Ug/g較佳。乳液劑型可以 使用適1的聚醋酸乙烯酯、聚乙烯吡咯烷酮等。凝膠劑型可使 用適量果膠、西黃蓍膠、海藻膠、黃原膠、瓊脂、甲基纖維素、 叛曱基纖維素、竣丙基甲基纖維素、或卡波普(carb〇p〇1)等做 成凝膠劑。軟膏劑型可添加適量凡士林、蜂蠟、矽酮等做成軟 膏劑。乳膏劑麵可利用油、水混合做成乳膏劑，也可添加適 量的界面活性劑以改善乳膏的安定性。 除了可做成外用藥外，牛樟芝分離物也可以做成穴位注射 用藥。可溶人_上可接受的溶劑，如水、乙醇、甘油、丙二 醇聚乙一醇、油酸乙g旨、苯甲酸节醋、二甲基乙醜HCP— 經基乙基、魏胺、肉轉異丙基自旨等，或這些溶綱組合物。 增溶劑’如T_-80、胆酸等也可適量使用，以增加溶解度。 -般注射_的顧方法可用來處理牛樟芝穴位注射用藥，使 其成為無菌、安全的注射用藥。 牛樟芝分雜可，也可離域份賴雞尾酒組 合物，可㈣功效加成的效果。例如牛樟芝與紅麴共用，對癌 細胞的抑做果可大為增加。其他如葛根、夏鮮、苦參、天 花粉、草河東、漢防己、鵪胆子、豬原青、猫爪草、農吉利、 白頭翁、黃連、黃柏、大黃、薑、料、當歸、川g、減、 蒼求、丁公藤、丹參、錢、自爾、山錄、甘草、山楂、 8 201004634 白花蛇舌草、半枝蓮、威靈仙、桃仁、三棱、莪朮、丹参、乳 香、沒藥、冰片、紅花、漏蘆、金銀花、蒲公英、紫花地丁、 連輕、敗醬草、仙鹤草、土茯苓、桔梗、川貝母、皂角刺、生 南星、生草烏、雷公藤、生半夏、砒霜、柴胡、鱼腥草、紫草、 或板藍根等皆可選用。這些藥物有些含有抗癌成份，有些有抗 發炎功能，有些有活血通絡的功能，有些有止痛功能，有些則 有補氣的作用，這些都有助於提高整體療效，可根据病情加以 1 選用。 此外，乙醜膽驗、輔酶A，細胞色素〇、三鱗酸腺苷等有 增強經脈感傳的作用，可提高牛樟芝穴位注射用藥的功能。白 藥產醇(resveratrol)、黃_(£^〇11〇丨(18)、異黃酮 (isoflavonoids)、多酚(polyphenols)、木酚素（lignans)及其衍 生物則具有抗氧化及抗癌功能，可與牛樟芝合併使用，使功效 ，加乘。其他如 Is〇thiOCyanates、j|〇nacolin K、維他命 Βι、維他 命心、維他命c、NADH、抗生素等亦可與牛樟芝合併使用，以 提高牛樟芝的功效。 穴位注射用藥在進行穴位注射前，可先進行小劑量皮下注 射，確定患者沒有藥物過敏的問題，再進行穴位注射。穴位的 選取則可根据病症及穴位特性來取穴施治。其原則以循經選穴 為主，即在病变之經絡上選取穴位。也可以選用阿是穴或經外 奇穴。一般可選取2〜4個穴位，各穴位可輪流施打，每個穴位 9 201004634 =4 8號針觀射卜2 ml賴量。對年老者及身體虛弱者， 母-人劑置可酌減。注射頻率可卜2日一次或ι週一次，視病情 而增減。 癌細胞生長抑制分析 將癌細胞培養在含 Dulbecco modified Eagle Medium ( )1 /oPeniciUin-strptomycin，2mM L-glutamine 及 1 〇% 月σ牛血q的疋全培養基中，在攝氏π度，含^二氧化碳的怪 /m匣濕培養箱中培養至長滿培養皿後，移走培養基，以含〇. 2⑽201004634 VII. Designated representative map: (1) The representative representative of the case is: ($, (2) A brief description of the symbol of the representative figure: No. 8. If there is a 学b study in this case, please reveal the most invented invention. The chemical formula of the characteristic. Nine, the invention description: [Technical field of the invention] The present invention relates to an external medicine containing an ingredient of Antrodia camphorata and a medicine for injection and treatment of acupoints through acupuncture points, and the two dosage forms and the route of administration can be used for prevention or Treatment of skin cancer, dermatitis, acne, microvascular abnormalities, arthritis, and various tumors and cancers. [Prior Art] Astragalus membranaceus plus 胤; ship<3) is a Taiwan-specific strain of polyporus Branch of the genus (also a member, in the natural environment, 201004634 匕 寄生 寄生 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特 特It is quite rare. Taiwan's aborigines used it as a good medicine for tearing, detoxification, and treatment of independence and occupation. In recent years, oral administration of Antrodia camphorata has been found for liver cancer, lung cancer, stomach cancer, bladder cancer, blood cancer, and liver. Inflammation, cirrhosis, etc. are quite helpful. Because of the non-toxicity of Antrodia camphorata, and the functions of liver protection, anti-oxidation, anti-cancer, etc., it is now mostly sold as health food. At present, the use of Antrodia camphorata is oral, although there are references to intravenous injection. There is still no practical application. However, the burdock is extremely bitter, and the oral bioavailability (bi〇availability) is also quite low, so it is not an ideal mode of administration. Intravenous injection will cause the drug to spread throughout the body, and the valley may cause unpredictable The side effects, as well as the effective k-degree reduction of the drug after systemic blood dilution, can not cause the components to gather to the location of the lesion, so the pharmaceutical industry is actively studying the method of drug administration. The present invention provides a solution to these defects. The medicine is applied directly to the skin, and is easy to use, and can directly act on epidermal lesions, direct contact, high effective concentration, quick effect, and less dose into other organs, and relatively less side effects. Normal skin usually has denser epidermal cells, and foreign objects are not easy to enter, but the skin with lesions is arranged more. Dispersed, rough, and even formed, so the drug is easier to enter, play a therapeutic effect. The present invention uses this feature to treat skin tumors, skin cancer, skin, f spring cancer, microvascular abnormalities using drugs containing anthraquinone ingredients Hyperplasia, assault, and other difficult diseases, the unexpected effect. 201004634 Acupoint injection is the use of the body's meridian system to carry out the standard drug administration. The human body mainly has 12 meridians and odd eight veins, its distribution covers the whole body, Tongda Dirty and epidermis. Each meridian has a specific path and coverage. The acupoints distributed in the meridians also have their specific functions in the clinic. These meridians not only control the flow balance of human blood, but also the study of deep humans. The meridians also have the function of transmitting substances. After the radioactive substances are injected into the acupuncture points, the radioactive substances can be observed to transport the radioactive substances along the meridians of the acupoints, and enter the organs, and the transmission path and the meridians are equivalent, non-general diffusion, and more Transmitted via the vascular system. Moreover, the meridians have the characteristics of "gas gambling," and the characteristics of the ship's acupoints, the gas will be preferentially transmitted to the lesion location. Using this feature of meridian conduction, we can effectively deliver the drug to the diseased position. The required dose of the drug is lower than that of general oral, subcutaneous, intramuscular or intravenous injection, and the recording effect is fast, and the possible side effects are small. Therefore, another object of the present invention is to use the special characteristics of the meridian transmission. Yi Mis (four), through the point of the shot, to play the dual role of the target administration and acupoint stimulation, to prevent and treat various cancers, luminal tumors, microvascular abnormalities, arthritis, hepatitis or cirrhosis. After drying, grinding, and sifting the natural bovine or the fermented broiled broiled broiled broiled broiled broiled broiled broiled broiled broiled broiled broiled broiled broiled broth 10 Bib or as needed 6 201004634 Adjust. Solvent can be alcohol, ethyl acetate, n-pentane, methanol, tetrafluoromethane, dichloromethane or other similar properties The agent is preferably alcohol, and the alcohol is more preferably 5% to 70% water. The active ingredient of Antrodia camphorata is quite complicated, and the content varies considerably depending on the strain and the culture condition, and it is determined whether the isolate is Including active ingredients, in addition to the commonly used physical, chemical, LC_MASS, HPLC and other analytical methods, 'cell survival and growth analysis can also be used as an indicator. The following examples of cancer cell growth inhibition analysis or microvascular proliferation inhibition analysis methods can be used as a reference. The extraction isolate can be further purified, for example, membrane separation method, molecular sieve dialysis method, liquid chromatography separation method, gel permeation chromatography separation method, centrifugation method, gassing method, ion father resin separation Method, chopping (siHca leap 1) separation method, HPLC, crystallization, etc. can be used to purify the ingredients. However, purification does not necessarily lead to higher activity or better economic benefits. Cocktails with multiple ingredients (cocktail) The group side can often receive the effect of adding and multiplying the components. The isolated or purified ingredient of the antrodia camphorata can be further Shrink or dry, and then dissolved in a pharmacologically acceptable solvent, such as water, ethanol, glycerin, propylene glycol, polyethylene glycol, ethyl oleate, benzyl benzoate, dimercaptoacetamide, N-CP-based A base, a lactose amine, a isopropyl isopropyl ester, or the like, or a combination of these solvents. A solubilizing agent such as Tween-80, cholic acid, etc. may also be used in an appropriate amount to increase the solubility. 7 201004634 Astragalus membranaceus isolate can be made The emulsion dosage form, the gel dosage form, the ointment dosage form or the cream dosage form is for external use, and the concentration thereof is preferably from 1 to 5 〇〇 Ug/g. The emulsion dosage form may be a suitable polyvinyl acetate, polyvinylpyrrolidone or the like. The dosage form can be used with appropriate amount of pectin, tragacanth, seaweed, xanthan gum, agar, methyl cellulose, cytotoxic cellulose, propyl propyl methyl cellulose, or carbop (1). ) to make a gel. The ointment dosage form may be added with an appropriate amount of petroleum jelly, beeswax, anthrone or the like to form a soft paste. The cream surface can be mixed with oil or water to form a cream, or an appropriate amount of surfactant can be added to improve the stability of the cream. In addition to being used as a topical drug, the extract of Antrodia camphorata can also be used as acupoint injection. Soluble human _ acceptable solvent, such as water, ethanol, glycerin, propylene glycol, polyethylene glycol, oleic acid, benzoic acid vinegar, dimethyl uranium HCP - trans-ethyl, sulphate, meat The propyl group or the like, or these solute compositions. Solubilizers such as T_-80, cholic acid and the like can also be used in an appropriate amount to increase the solubility. The general injection method can be used to treat acupoint injections of Astragalus membranaceus, making it a sterile and safe injectable drug. The burdock can be mixed, and it can also be used as a cross-linking cocktail. For example, the combination of Antrodia camphorata and Rhododendron can greatly increase the inhibitory effect on cancer cells. Others such as Pueraria, Xia Xian, Sophora flavescens, Tianhua powder, Caohedong, Hanfangji, 鹌胆子, 猪原青, Cat's claw grass, Nongjili, Pulsatilla, Coptis, Phellodendron, Rhubarb, Ginger, Chinese, Angelica, Sichuan g, Reduction, Cangqiu, Dinggongteng, Danshen, Qian, Zier, Shanlu, Licorice, Hawthorn, 8 201004634 Hedyotis diffusa, Scutellaria barbata, Clematis, peach kernel, Sanling, Qishu, Salvia, frankincense, myrrh, Borneol, safflower, staghorn, honeysuckle, dandelion, purple diced, even light, succulent, sage, earthworm, platycodon, Fritillaria, saponin, raw southern star, raw grass, tripterygium, raw Pinellia, arsenic, Bupleurum, Houttuynia, comfrey, or radix is available. Some of these drugs contain anti-cancer ingredients, some have anti-inflammatory functions, some have the function of promoting blood circulation and collaterals, some have analgesic function, and some have qi-enhancing effects, which all contribute to the overall efficacy, and can be selected according to the condition. . In addition, B ugly test, coenzyme A, cytochrome sputum, adenosine triphosphate, etc. have the effect of enhancing the meridian transmission, which can improve the function of acupoint injection. Resveratrol, yellow _(£^〇11〇丨(18), isoflavoneoids, polyphenols, lignans and their derivatives have antioxidant and anti-cancer functions. It can be combined with burdock to enhance the efficacy and addition. Others such as Is〇thiOCyanates, j|〇nacolin K, vitamin Βι, vitamins, vitamin c, NADH, antibiotics, etc. can also be combined with burdock to improve the efficacy of burdock Before acupoint injection, acupoint injection can be performed by subcutaneous injection at a small dose to confirm that the patient has no drug allergy problem, and then acupoint injection. The selection of acupoints can be based on the symptoms and acupoint characteristics. According to the selection of acupoints, the acupoints are selected on the meridians of the lesions. Aya or Jingqi acupoints can also be used. Generally, 2 to 4 acupoints can be selected, and each acupoint can be applied in turn, each acupoint 9 201004634 = 4 No. 8 needle observation 2 ml amount. For the elderly and the weak, the mother-human agent can be reduced. The frequency of injection can be 2 or once every 1 week, depending on the condition. Suppress The cancer cells were cultured in a total medium containing Dulbecco modified Eagle Medium ( )1 /oPeniciUin-strptomycin, 2 mM L-glutamine and 1 〇% σ 牛 blood q at π °C, containing CO 2 After cultivating in a dampness incubator to a full dish, remove the medium to contain 〇. 2(10)
Trypsin-0. 53 mM EDTA之溶液稍加沖洗，再加入2〜3 mlTrypsin-0. 53 mM EDTA solution is slightly rinsed, then add 2~3 ml
Tiypsin-EDTA溶液，靜置約5〜15分鐘，待細胞分離後，加入 適1上述完全培養基，並計算細胞含量，使每ml含有約1〇, 〇〇〇 個細胞。將細胞放到96 well plate培養，並依試驗所需，加 入5以1的各種藥物，每一個培養條件5重覆。細胞經隔夜培 養後’取出以PBS緩衝溶液沖洗2次，加入MTT 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide 試劑’在攝氏37度下靜置2小時後，吸掉溶液，加入acidic isopropanol，兩小時後測其560nm之吸光強度，並與標準曲 線對照、換算細胞之存活率。圖1顯示牛樟芝分離物對肝癌細 胞有很強的抑制力，圖2顯示牛樟芝分離物物對肝癌細胞抑制 力隨劑量增加而增強，有dose-response的相關性。圖3顯示 201004634 牛樟之分離物對人及動物癌細胞皆有很強的抑制力，而對人類 正常骨綱葉幹細酬毒性相當低，@此牛樟芝可以有效的殺 死癌細胞，而不會有明顯副作帛。此與一般化療帛藥對正常細 胞亦有高毒性不同，因此牛樟芝用來做抗癌藥物是相當合適 的。 牛樟芝可單獨使用以抗癌，亦可與其他藥草、化合物做成組合 物，以收功效加乘的效果。圖4為牛樟芝與紅麴分離物合用 (1.1)的結果，其效果遠大於各自單獨使用。 牛樟芝段木培養 將至少1公分厚之牛樟段木浸泡在PDB培養基中，隔夜， 以高壓滅g峨氏m度滅g丨小時，冷卻後，將牛樟芝菌種接 種到木頭上，放人攝氏25度培養箱巾培養。待紅褐色牛樟芝長滿 木頭表面即可收成。 牛樟芝固態培養 將牛樟木屑、小麥、PDA，以2〇: 5:1混合，經攝氏121度， 高屢滅菌40分鐘。冷卻後，加入牛樟芝菌種，並放到攝氏四度 培養箱中培養。待紅褐色牛樟芝長滿後即可收成。 牛樟芝液態培養 將PDB培養基在500ml三角搖瓶中經攝氏121度，高壓滅 201004634 菌20分鐘。冷卻後’加入牛樟芝菌種，並放到攝氏25度迴旋振 盪培養箱中培養’ 120rpm。約2週即可收成。 牛樟芝分離物製備 將牛樟芝菌絲體烘乾、磨粉、過篩峨以6〇%酒精萃取， 隔夜’以3咖_ — ’取上清液，織賴濃縮、乾燥。再視 須要’溶入丙二醇或其他合適的溶劑中。 外敷動物試驗 10隻8週大之C57BL/6雄氣前腿皮下注射1〇5 Β1_癌 細胞，待腫瘤長至約8 mm3 (腫瘤體積以v=q.52xDi2x])2, ^為 腫瘤短邊直徑，D2為腫瘤長邊餘)後開始每天塗藥一次，每 次約2〇mg乳液。老鼠隨機分兩組，一組為空白對照組，所用 乳液不含牛樟芝成份。另一組為實驗組，所用乳液含牛掉芝分 離物（1〇〇⑽g)，每週記錄腫瘤大小2次。圖5為老鼠腫瘤 體積的變化平均值。顯科樟芝乳液可有效抑麵瘤的發展。 外敷人體試驗 一成年男性臉部長一黑褐色腫瘤，顏色不均勻，形狀不規 則，直徑約1公分，經診斷為黑色素癌瘤。每天塗含牛樟芝分 離物軟膏(1GG她），—週後可看出軸__，約兩週 12 201004634 後腫瘤消失，皮膚恢復平整。 穴位注射動物試驗 將VX2癌細胞打入3隻紐西蘭白兔New Zealand white rabbits (3.0-3.5 kg)之後腿，兩週後，將兔麻醉取出腫瘤並 切碎(0.1-0.2 cm3 )。另外，將12隻白兔剃毛，麻醉，切開腹 φ 腔，使肝腑暴露出來。用21號針將腫瘤組織接種到12隻白兔 之肝臟以祕肝癌。將12隻兔子分成敝，—㈣實驗組， 每天在太沖、曲泉兩穴位注射〇.3ml含牛樟芝分離物〇5“ g/ml)的穴位注射液’另一組則進行肌肉注射ο】的丙二醇 溶液。12天後，將兔子犧牲，查肝臟腫瘤。結果控制組肝腫 瘤平均直徑1. 5±0. 5公分，而實驗组則皆在〇· 5公分以内或無 腫瘤。The Tiypsin-EDTA solution was allowed to stand for about 5 to 15 minutes. After the cells were separated, the above complete medium was added, and the cell content was calculated so that each cell contained about 1 〇 of cells. The cells were cultured on 96 well plates, and 5 to 1 of each drug was added as required for the test, and each culture condition was repeated 5. After the cells were cultured overnight, 'take out 2 times with PBS buffer solution, add MTT 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide reagent' to stand at 37 ° C After 2 hours, the solution was aspirated, acidic isopropanol was added, and the absorbance at 560 nm was measured two hours later, and compared with the standard curve, the survival rate of the cells was converted. Figure 1 shows that the extract of Antrodia camphorata has a strong inhibitory effect on liver cancer cells. Figure 2 shows that the inhibitory effect of the isolates of Antrodia camphorata on liver cancer cells is enhanced with increasing dose, and there is a correlation of dose-response. Figure 3 shows that the 201004634 burdock isolate has strong inhibition on human and animal cancer cells, and the toxicity of human normal skeletal leaves is quite low. @此牛樟芝 can effectively kill cancer cells without Obviously a vice. This is different from the general chemotherapy and expectorant to normal cells, so it is quite suitable for the use of Antrodia camphorata for anticancer drugs. Antrodia camphorata can be used alone to fight cancer, and can also be combined with other herbs and compounds to achieve the effect of multiplying effects. Fig. 4 shows the results of the combination of the extracts of Antrodia camphorata and the red peony (1.1), which are much more effective than the respective use. The burdock section of the burdock is immersed in the PDB medium at least 1 cm thick of the burdock wood. After overnight, the cockroach is sterilized by high pressure. After cooling, the burdock strain is inoculated on the wood and placed in the Celsius. 25 degree culture box towel culture. Wait until the reddish-brown burdock is covered with wood. Solid culture of burdock burdock Wood, burdock, wheat, PDA, mixed with 2〇: 5:1, 121 ° C, high sterilization for 40 minutes. After cooling, the strain of Antrodia camphorata was added and cultured in a four degree Celsius incubator. Wait until the reddish brown burdock is full and ready to harvest. Liquid culture of Antrodia camphorata PDB medium was irradiated at 121 °C in a 500 ml triangular shake flask, and the bacteria were deactivated for 20 minutes at 201004634. After cooling, the strain of Antrodia camphorata was added and cultured in a 25-degree-rotating shaker incubator at '120 rpm. It will be harvested in about 2 weeks. Preparation of isolates of Antrodia camphorata The mycelium of Antrodia camphorata was dried, ground, sieved and extracted with 6〇% alcohol. The supernatant was taken overnight by 3 coffee _ ‘, woven and concentrated. Re-required to be dissolved in propylene glycol or other suitable solvent. Externally applied animal test 10 8 weeks old C57BL/6 male anterior leg subcutaneous injection of 1〇5 Β1_ cancer cells, until the tumor grows to about 8 mm3 (tumor volume with v=q.52xDi2x)) 2, ^ is short tumor The side diameter, D2 is the long side of the tumor), and then apply once a day, about 2 〇mg of emulsion each time. The rats were randomly divided into two groups, one of which was a blank control group, and the emulsion used did not contain the ingredient of Antrodia camphorata. The other group was the experimental group, and the emulsion used contained bovine officin fraction (1 〇〇 (10) g), and the tumor size was recorded twice a week. Figure 5 shows the mean change in tumor volume in mice. Xianke Anzhizhi emulsion can effectively inhibit the development of facial tumors. External application of human body test A male male face with a dark brown tumor, uneven color, irregular shape, about 1 cm in diameter, diagnosed as melanoma. Apply the ointment ointment ointment (1GG her) every day. After the week, the axis __ can be seen. After about two weeks 12 201004634, the tumor disappears and the skin recovers smoothly. Acupoint injection animal test VX2 cancer cells were injected into 3 New Zealand white rabbits (3.0-3.5 kg) legs. Two weeks later, the rabbits were anesthetized to remove the tumor and chopped (0.1-0.2 cm3). In addition, 12 white rabbits were shaved, anesthetized, and the abdomen φ cavity was cut to expose the liver sputum. Tumor tissue was inoculated into the liver of 12 white rabbits with a 21 gauge needle to secrete liver cancer. 12 rabbits were divided into sputum, - (4) experimental group, daily injection of 3. 3 ml of acupoint injection containing burdock isolate 〇 5" g / ml) at the two points of Tai Chong and Qu Quan 'the other group was intramuscularly injected ο The propylene glycol solution. After 12 days, the rabbit was sacrificed and the liver tumor was examined. Results The average diameter of the liver tumor in the control group was 1.5 ± 0.5 cm, while the experimental group was within 5 cm or no tumor.
穴位注射人體試驗 女病患72歲’峰斷為㈣部位胃癌第三期，無法 =，注射足三里、公孫、中胺、梁門，每天取繼位，各 /主射1. 2毫升牛樟芝分離物， ^ /曰為一療程，中間休息3天， 、，坐4療程後，已可進食 、iu&MRl檢查，腫瘤已近消失。 微血管增生抑制分析 13 201004634 將含有 VEGF (75 ng/ml; Pepro Tech，Inc.，Rocky Hill, NJ)、bFGF (300 ng/ml; Pepro Tech, Inc.)及測試藥品的 Matrigel (250 μΐ; Becton Dickinson, Bedford, MA)皮下 /主射入去胸腺的老鼠(BALB/c-^i/Zhi/)。Matrigel打入皮下後 隨即凝成一塊狀物。7天後，將老鼠犧牲掉’取出凝膠，以福 馬林(formalin)固定，以蠟包埋、切片（5-μιη)、染色 (Hematoxylin and Eosin)，然後以 video imaging system 加以 計數侵入Matrigel之微血管内皮細胞數目，每一切片取5〇個 視域（fields，20倍放大），並與未加藥的空白Matrigel相 比較’計算藥物對微血管細胞增生的抑制率。圖6顯示牛棒芝 分離物可有效抑制微血管增生。因此牛樟芝分離物適合於治療 微血管異常增生相關疾病，如癌、踵瘤、黃斑部病變、糖尿病 視網膜病變、風濕性關節炎、牛皮癬等。 以上所述實施例之揭示僅為本案可能的實施方式，其目的 在使人更容易瞭解本發明的内容。前面的說明已充份指出，本 發明絕不只限於此節的實施方式。因此不能以本節的内容來限 縮本發明的範圍。 【圖式簡單說明】 圖1·各種牛樟芝分離物對人皮膚黑色素癌細胞抑制率 圖2.牛樟芝分離物不同劑量對人皮膚黑色素癌細胞抑制率 圖3.牛樟芝分離物對人、老鼠、兔癌細胞及人骨髓間葉幹細 201004634 胞抑制率比較 圖4牛樟芝及紅麴分離混合物對人皮膚黑色素癌細胞抑制率 圖5.牛樟芝外用藥對老鼠皮膚黑色素癌腫瘤之抑制效果 圖6.牛樟芝分離物不同劑量對微血管增生抑制率 【主要元件符號說明】 ❹ 無 ❹ 15Acupoint injection human test female patient 72 years old 'peak breakage (four) part of the third stage of gastric cancer, can not =, injection Zusanli, Gongsun, Zhongamine, Liangmen, take the next day, each / main shot 1. 2 ml of Antrodia camphorata separation Things, ^ / 曰 for a course of treatment, rest for 3 days in the middle, ,, after 4 courses of treatment, can already eat, iu & MRl check, the tumor has disappeared. Microangiogenesis inhibition assay 13 201004634 Matrigel (250 μΐ; Becton) containing VEGF (75 ng/ml; Pepro Tech, Inc., Rocky Hill, NJ), bFGF (300 ng/ml; Pepro Tech, Inc.) and test drug Dickinson, Bedford, MA) Subcutaneous/primarily injected into the thymus mouse (BALB/c-^i/Zhi/). After Matrigel hit the skin, it condensed into a piece. After 7 days, the rats were sacrificed 'to remove the gel, fixed with formalin, embedded in wax, sliced (5-μιη), stained (Hematoxylin and Eosin), and then counted by video imaging system to invade Matrigel The number of microvascular endothelial cells, 5 sections per field (fields, 20-fold magnification), and the rate of inhibition of microvascular proliferation was calculated by comparison with unmedicated blank Matrigel. Figure 6 shows that the extract of Bovine Ganoderma lucidum can effectively inhibit microvascular proliferation. Therefore, the extract of Antrodia camphorata is suitable for the treatment of diseases associated with microvascular abnormalities such as cancer, neoplasms, macular degeneration, diabetic retinopathy, rheumatoid arthritis, psoriasis and the like. The disclosure of the above-described embodiments is only a possible embodiment of the present invention, and its purpose is to make it easier for a person to understand the contents of the present invention. The foregoing description has fully pointed out that the present invention is by no means limited to the embodiments of this section. Therefore, the scope of the present invention cannot be limited by the contents of this section. [Simplified illustration] Figure 1. Inhibition rate of various burdock isolates on human skin melanocytes. Figure 2. Inhibition rate of different doses of Antrodia camphorata isolates on human skin melanocytes. Figure 3. Burdock officin isolates on human, mouse, rabbit cancer Cell and human bone marrow mesothelium 201004634 Comparison of cell inhibition rateFig.4 Inhibition rate of human skin melanoma cells by the mixture of Antrodia camphorata and Rhizoma OxideFig. 5. Inhibition effect of external application of Antrodia camphorata on melanoma of mouse skinFig. 6. Selenium officinalis isolate Different doses of microvessel proliferation inhibition rate [main component symbol description] ❹ no ❹ 15