201242604 六、發明說明： 【發明所屬之技術領域】 本發明關於一種用於抗B型肝炎病毒之醫藥組合物’該組合物包 含有效劑量之樟芝萃取物質。該萃取物質包含抗病毒成份4-乙酿樟芝 醌醇B。 【先前技術】 B型肝炎是世界上最為常見的傳染性肝病，它是由B型肝炎病毒 (HBV)引起，這一病毒侵害肝臟。通常大多數成人都能夠抵抗β型肝炎 病毒，並能復原，但是也有許多成人無法抵抗Β型肝炎病毒，也包括 大多數受到傳染的嬰兒和兒童，他們被診斷為Β型肝炎的慢性病毒帶 原者，病毒可在其企液和肝臟很長時間，且他們可能將病毒傳染給他 人。目前全球有一十億人(將近有三分之一人口）已受到Β型肝炎病毒 傳染，許多人成功抵抗病毒並疾癒，但是也有四億人口無法抵抗病毒 而變成慢性攜帶病毒者’在所有族裔中，亞裔患Β型肝炎的比率最高； .美國亞太減慢性Β肝炎的比率多達15%，其他美國人只佔Q.挪，在 美國大概有-百五十萬的B型肝炎病毒攜帶者，亞太裔佔了—半以上。 目前治療B型肝炎病毒的方法中，干擾素注射劑可以抑制B肝病 毒的複製’降低肝臟發炎程度，可惜副作用大，療效有限，因此使用 之機會並不多。近年來’ 口服抗B浙炎鱗雜陸續綱發出來， 201242604 最早上市的為干安能Lamivudine(商品名Zeffix)，可以抑制病毒的複 製降低肝臟發炎’但缺點是停藥後《復發，甚至產生抗藥性；另一 種口服抗B肝藥物則是干適能Adefovir dipiv〇xil (商品名此卿ra)， 與干安能毅差不多’但較不會發生抗雜，對干絲無效之案例也 有效，但藥價貴許多，而且有潛伏的腎毒性，通常當第二線使用。因 此，開發新的抗病«物是目«界普遍之目標。 擇芝，學名為 Antrodia cmjAorata (Zang & Sm) 4 I， Ryvarden & Τ·Τ. Chang’又有一麵為輪滿ci•卿咖，屬於 台灣國寶級的珍貴藥用真菌，經常被應養生保健_途，一般民 間對於棒芝的稱呼有_、牛_、牛樟芝或是紅樟芝，《的稱呼 尚有樟益、雜、樟《、牛_、牛樟芝、紅樟、紅料......等名 稱，是台灣特有種的益菌類，全世界只生長在台灣山區海拔彻〜i咖 公尺間的牛樟樹幹雜〜材_或枯關狀讀木體暗潮濕之 表面’非常的珍貴且稀少，因此又被稱為「台灣森林中的寳石」。掉芝 為口灣特有藥’傳統民俗療法為保肝治癌之高價格藥方^傳統上 使用經驗敘述有關樟芝成份中含有的多_可活化細胞、改善體質， 增進人體⑽免疫調節，其中另—主要有效成份—三細化合物，應 用於抗癌/抗發炎及贿魏性產^，耗在市場摘廣為認可，因 而樟芝有藥用真菌之王的美譽。 【發明内容】 本發明關於一種抗β型肝炎病毒之醫藥組合物，其包含有效劑量之 201242604 樟芝萃取物’牛樟芝萃取物含有抗病毒成份4-乙醯樟芝酿；醇b (4-acety卜antroquinonol)，其結構式如下圖所示：201242604 VI. Description of the Invention: [Technical Field] The present invention relates to a pharmaceutical composition for anti-hepatitis B virus. The composition comprises an effective dose of an extract of Antrodia camphorata. The extract contains an antiviral component, 4-ethyl chrysanthemum sterol B. [Prior Art] Hepatitis B is the most common infectious liver disease in the world. It is caused by hepatitis B virus (HBV), which invades the liver. Most adults are generally resistant to the hepatitis B virus and can recover, but many adults are unable to fight the hepatitis B virus, including most infected infants and children, who are diagnosed with chronic hepatitis with chronic hepatitis. Viruses can be in their fluids and liver for a long time, and they may transmit the virus to others. At present, one billion people (nearly one-third of the population) have been infected by the hepatitis C virus. Many people have successfully resisted the virus and recovered, but there are also 400 million people who cannot resist the virus and become chronic carriers. Among ethnic groups, Asians have the highest rate of sputum hepatitis; US Asia Pacific reduces the rate of chronic sputum hepatitis by as much as 15%, other Americans only account for Q. Norwegian, and there are approximately -500,000 hepatitis B in the United States. The carriers of the virus, Asian-Pacific people accounted for more than half. In the current method for treating hepatitis B virus, interferon injection can inhibit the replication of hepatitis B virus, which reduces the degree of inflammation of the liver. Unfortunately, the side effects are large and the curative effect is limited, so there is not much chance of use. In recent years, the oral anti-B-Zhe-Yin-Zhan dynasty has been issued, and the earliest listed in 201242604 is Ganivudine (trade name Zeffix), which can inhibit the replication of the virus and reduce the inflammation of the liver. 'But the shortcoming is that after relapse, the recurrence or even the occurrence Drug resistance; another oral anti-B liver drug is dry adefovir dipiv〇xil (trade name this Qing ra), similar to Gan An Neng Yi, but less anti-heterogeneous, effective in cases of dry silk is also effective However, the drug is much more expensive and has latent nephrotoxicity, usually used as a second line. Therefore, the development of new disease resistance is a common goal in the world. Choizhi, the scientific name is Antrodia cmjAorata (Zang & Sm) 4 I, Ryvarden & Τ·Τ. Chang' has a side full of ci•qing coffee, a precious medicinal fungus belonging to the national treasure of Taiwan, often Health care _ way, the general folks for the name of the barley _, cattle _, burdock or red 樟 ,, "the name is still good, miscellaneous, 樟", cattle _, burdock, red carp, red material.. .... and other names, are the endemic species of endemic species in Taiwan. The world only grows in the mountainous area of Taiwan, and the burdock trunks of the yak yam ~ _ _ or the closed surface of the dark and moist surface of the wood Very precious and rare, it is also known as the "jewel in the forest of Taiwan."芝芝 is a special medicine for mouth Bay. Traditional folklore therapy is a high-priced prescription for liver cancer treatment. Traditionally, experience has been used to describe the multiple _ activating cells contained in the ingredients of Antrodia sinensis, improving the body and promoting the immune regulation of the human body (10). - The main active ingredient - three fine compounds, used in anti-cancer / anti-inflammatory and bribery products, is recognized by the market, so Apocynum has the reputation of the king of medicinal fungi. SUMMARY OF THE INVENTION The present invention relates to a pharmaceutical composition for anti-beta hepatitis virus, which comprises an effective dose of 201242604 Antrodia camphorata extract, Antrodia camphorata extract containing an antiviral component, 4-acetone, and an alcohol b (4-acety) Bu antroquinonol), its structural formula is shown below:
結構式(I) 本發明中所稱之樟芝萃取物’其包括但不限於樟芝菌絲體萃取 物'樟芝子實體萃取物，以及樟芝發酵液。 另本發明中所稱抗病毒成分「4-乙醯樟芝醌醇B」，其英文名稱為 「4-acetyl-antroquinonol」，如結構式（I)所示。 在本發明中所稱病毒包括但不限於野生型B型肝炎病毒、抗干安能 (Lamivudine-Resistant)B型肝炎突變病毒、腸病毒以及A型流感病 毒，在本發明之較佳實施例中，野生型B型肝炎病毒以及抗干安能 (Lamivudine-Resistant)B型肝炎突變病毒，有效濃度分別為 0· 17188pg/ml 至0.39063pg/na，較佳0. 97656pg/ml 至0.58594pg/nU， 最佳 0. 78125pg/ml ;以及 4. 6875pg/ml 至 1. 5625pg/ml，較佳 3. 9063pg/ml 至2. 3438pg/ml，最佳3.125pg/ml。 本發明之抗B型肝病毒之醫藥組合物，進一步包含一醫藥上可接受 之佐劑、載體或賦型劑。適當的藥物載體可包含惰性成分，該惰性成 分不會與本發明之化合物發生實質反應。用於非口服施藥的適當藥物 201242604 載體包含’例如’無菌水、生理食鹽水、抑菌食鹽水（包含約〇· 9% 苯基醇的食鹽水）、填酸緩衝液食鹽水、漢克溶液、林格式乳糖液及其 他相似載體。躲口服雜，可依則知製程，將其製成粉末狀、顆 粒狀、錠狀、液狀、膠狀或膏狀。本發明之醫藥組合物可以食品、飲 品、藥品、試劑或營養補充品之形式提供，或是藉由靜脈、肌肉、上 皮、腹腔、吸入、口含、口服、經皮等途徑施用。本發明中所述之“有 效劑量”，係指該化合物劑量在提供給一對象後能獲得有益的結果， 或者，該化合物的劑量能在體内或體外獲得預期的活性。以本發明為 例，受B型肝炎病毒感染之細胞表面抗原抑制強度在2〇%_35%為輕度抑 制(+);35%-50%為中度抑制(++) ; 50%-65%為強度抑制(+++);大於65%為 非常強度抑制(++++)。 本發明以體外細胞培養模式，利用酵素連結免疫吸附分析法 (Enzyme-Linked Immunosorbent Assay)，針對 B 型肝炎病毒野生型細 胞株(MS-G2 cell line)及干安能(Lamivudine)抗藥性突變株(JJ33 cell line)測定樟芝萃取物4_乙醯樟芝醌醇b對人類B型肝炎病毒表面抗原 及e抗原之作用。試驗結果顯示：4-乙醯樟芝醌醇b對B型肝炎病毒 野生型細胞株(MS-G2 cell line)及干安能抗藥性突變株(M33 cell line)，無細胞生長抑制作用下，對表面抗原具強度(+H)抑制作用，濃 度分別為0. 78125 yg/ml及3.125 pg/ml;對干安能抗藥性突變株QJ33 cell line)之e抗原具中度(H)抑制作用，濃度為3.125 pg/ml。 201242604 【實施方式】 細胞株及細胞培養 本發明使用兩種人類肝癌細胞株，一為MS-G2，此係哈佛大學Dr. Max Essex.之實驗室所建立(Sureau，C. et al.，1986. Production of hepatitis Bvirus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. CW/47: 37-47)，乃利用肝癌細胞株 HepG2 經轉 染(transfection)帶有完整HBV DNA (^)及新黴素抗性(neomycin resistance)基因的質體後，以G418篩選，再經由系列稀釋得到的細胞株， 命名為MS-G2。由於此細胞株有完整的HBVDNA的嵌入(integrated)， 且已形成·一個穩疋的細胞株，故可持續南量的分泌B型肝炎病毒表面抗 原(HBsAg)、e抗原(HBeAg)及病毒顆粒體(42 nm丹氏顆粒〇3116 particles and 22 nm次病毒顆粒subviral particles)，本計劃將以此作為b 型肝炎病毒野生型模式，進行抗病毒作用及分子生物層次之探討。另一 為本實驗室執行國科會計劃，所建立的干安能（又稱3TC)抗藥性突變株 之穩定轉殖細胞株，亦是利用肝癌細胞株HepG2經轉染帶有耐藥基因 位點(YMDD)突變之HBV DNA及抗潮黴素基因(hygr〇mycin resistance gene)的質體後，以潮黴素篩選，再經由系列稀釋得到的細胞株，命名為 M33，以此作為B型肝炎病毒抗干安能突變型模式，進行抗病毒作用及 分子生物層次之探討。 實施例一: 7 201242604 樟芝菌絲體3公斤以95%乙醇10公升加熱回流萃取四次，萃取液 經過濾後濃縮，減壓乾燥得乙醇萃取物384公克，將乙醇萃取物懸浮於 水以等量乙酸乙酯進行分配(partition)劃分，乙酸乙酯層經減壓濃縮可得 乙酸乙酯層劃分部157.57公克及水層劃分部159.51公克。 上述乙酸乙酯層劃分部157.57公克以矽膠管柱（10 cm i.d X 30 cm) 進行層析，依次以正己烷正己烷_乙酸乙酯（10: 1 2+10: 3+10: 44 10: 5->1: 1 2, v/v)+乙酸乙酯->甲醇每種比例各10L進行沖 提’每1L收集成一劃分部。其中正己烧-乙酸乙酯(1〇: 4)沖提之劃分部 56-63 (3.015 g)，以逆相製備級管柱 Tosoh ODS-80TS (21.5 mm X 300 mm, 10μηι)進行層析，以 H20-CH3CN (20:80)為移動相，流速 10 ml/min 進行層析’以265nm為檢測波長，管柱控溫40oC，可獲得4-乙醯樟芝 醌醇 B (131 mg)。 實施例二： 在24孔槽的細胞培養皿(petri-dish)，種入MS-G2細胞2xl05細胞 /ml，或M33細胞1.5 x10s細胞/mi，待隔夜後細胞充分附著，更新培養 基，同時給予不同濃度之4-乙醯樟芝醌醇b，每個濃度三重覆。給藥處 理48小時後’收集上層培養液，進行抗病毒活性測定，並同時以MTT (3-[4, 5-二甲噻嗤-2-基]2, 5-聯苯基-四唑漠）測試，留在24孔槽内的 細胞存活情形及藥物是否毒害及抑制細胞生長。MTT是一種黃色的染 劑’會被活細胞所吸收並經由粒線體中的破珀酸去氫酶(succinate 201242604 dehydrogenase)還原成藍紫色的曱臢(formazan)’可用來檢測細胞之存活與 生長變化，在24孔槽的細胞培養皿中，加入1 mg/miMXX溶液，於37 C下作用4小時，吸除上清液，加入1〇〇 giDMSO將藍紫色的結晶析出. 最後’以分光比色計自動酵素免疫分析儀(ELISAreader)在54〇啦測定吸 光值，反映存活細胞的多寡，用以作為藥物是否具細胞毒性(cyt〇t〇xici^) 之指標。 實施例三： 利用酵素連結紐靖分躲(Enzyme-Linked Assay> ELISA)，使用抗人類B猜炎病毒表面抗原之單株抗體及抗人類b型 肝炎病毒如/e抗狀多源紐轉錢疫_觸〔普錢份有限公 司，新竹縣’台灣〕’利用抗體—抗原—抗體酵素接合體之三明治複合 體，以四甲基聯苯胺(TMB)呈色劑呈色之，再以分光比色計臟故 00型自動酵素免疫分析儀在·聰測定之，所得的吸光值(〇d 反戯抗原❹寡。伽龍_吸光值當⑽，依以下公式計算 其抑制百分比（Inhibition %): 對照組吸光值-給藥組吸光值 X 100% =抑制百分比（Inhibition %) 對照組吸光值 201242604 抑制百分比(Inhibitionpercentage)在2〇一35%為輕度抑制(+)，抑制百 分比在35〜50%為中度抑制(++)，抑制百分比在5〇一65%為強度抑制 (+++)’抑制百分比在65%以上為非常強度抑制(++++)。 實施例四： 如下表-及表二所不，B型肝炎赫野生型細胞雜s_G2eeU⑽) 五個測試濃度（12· 5、6. 25、3.125、1. 5625 及 0. 78125 pg/ml)，12. 5、 6. 25、3.125及1. 5625 具有細胞生長抑制作用（抑制百分比%> 20%)，0.78125 無細胞生長抑制作用，對表面抗原具強度⑽抑 制作用’其抑制百分比為61. 3 ± 1.8 (圖1)。 表一 4-乙醯樟芝醌醇B在野生型HBV細胞株(MS_G2)中抗以及广 -HBeAg的效果 處理48 h pg/ml HBsAg HBeAg 一· MTT assay (抑制率！!〇 (抑制率%) (抑制率%) Mean ± S. E. Mean ± S. £ Mean 土 S. E. 控制組 0 . 0 —_ 0 DMS0 2.5 μΐ/ml 0.9 ± 1.6 aNU -2.9 ±1.1 201242604 4-乙醯樟芝醌 醇B 12.5 μ^ηύ (12.5 ppm) 6.25 μ^ιηΐ (6.25 ppm) 3.125μ§/ιη1 (3.125 ppm) 1.5625μ^πι1 0_78125μ_ ND ND ND ND 61.3 ± 1.8 ND 49.9 ±2.5 38.4 ±1.8 33.9 ± 1.0 30.0 ±3.9 16.7 ±2.3 蛇床子素 80 μΜ 52.0 ± 1.7 ND -13.7 土 0.8 (From獨活） 1. 實驗數值為單一實驗三重複結果 2. 肋:無偵測結果 3. DMSO組為溶劑控制組 4. 蛇床子素(〇sthole)為正對照組 抑制百分比(Inhibitionpercentage):介於20%_35%為輕度抑制(+) 介於35% - 50%為中度抑制(++) 介於50% - 65%為強度抑制(+++) 大於65%為非常強度抑制(++++) 表二野生型HBV細胞株(MS-G2) 處理 HBsAg HBeAg 4-乙醯樟芝醌醇Β +++ ND 一 (高濃度具細胞毒性） 蛇床子素（80 μΜ) +++ ND -- (此濃度無細胞毒性） 11 201242604 實施例五： 如下表3及表4所示’干安能抗藥性突變株_從腿e)八侧試 漢度(100、50、25、12.5、6.25、3.125、1 5625 β b25 及0.78125 pg/ml)，100、 50、25、12.5及6.25 具有細胞生長抑制作用(抑制百分比分 〇鳩、L5625及3.125 μ§/ηι1無細胞生長抑制作用，對表面抗原具輕 度⑴至強度(叫抑制作用，其抑制百分比分別為248土〇8、％ 7± i 〇 及56.6 ± 1.5 ’ 1. 5625及3.125 pg/ml對e抗原具輕度(+)至中度㈣抑制 作用，其抑制百分比分別為28.1±〇.9及45.1±2·〇(圖2)。 表三·4-乙醢樟芝轉Β在抗干安能之HBV突變細胞株(Μ33)中的抗_ηβ心 以及抗-HBeAg的效果 pg/ml HBsAg (抑制率%) Mean 土 S. E. Mean 土 S. E. MTT assay (抑制率%) Mean + S. E.Structural Formula (I) The Antrodia camphorata extract referred to in the present invention includes, but is not limited to, the Antrodia camphorata mycelium extract, the Antrodia camphorata fruit extract, and the Antrodia camphorata fermentation broth. Further, the antiviral component "4-ethyloxazol B" as referred to in the present invention has the English name "4-acetyl-antroquinonol" as shown in the structural formula (I). The viruses referred to in the present invention include, but are not limited to, wild type hepatitis B virus, Lamivudine-Resistant hepatitis B mutant virus, enterovirus, and influenza A virus, in a preferred embodiment of the present invention. , wild-type hepatitis B virus and Lamivudine-Resistant hepatitis B mutant virus, the effective concentration is 0. 17188pg/ml to 0.39063pg/na, preferably 0. 97656pg/ml to 0.58594pg/nU , the best is 0. 78125pg / ml; and 4. 6875pg / ml to 1. 5625pg / ml, preferably 3. 9063pg / ml to 2. 3438pg / ml, the best 3.125pg / ml. The pharmaceutical composition for anti-B hepatic virus of the present invention further comprises a pharmaceutically acceptable adjuvant, carrier or excipient. Suitable pharmaceutical carriers may contain inert ingredients which do not substantially react with the compounds of the present invention. Appropriate drug for non-oral administration 201242604 The carrier contains 'for example' sterile water, physiological saline, bacteriostatic saline (salt containing about 9% phenyl alcohol), acid buffer saline, Hank Solution, forest format lactose solution and other similar carriers. To avoid oral miscellaneous, it can be made into powder, granule, ingot, liquid, gel or paste according to the process. The pharmaceutical composition of the present invention may be provided in the form of a food, a drink, a drug, a reagent or a nutritional supplement, or may be administered by intravenous, intramuscular, epithelial, intraperitoneal, inhalation, buccal, oral, transdermal or the like. As used herein, "effective dose" means that the dose of the compound provides a beneficial result upon administration to a subject, or that the dose of the compound is such that it achieves the desired activity in vivo or in vitro. Taking the present invention as an example, the cell surface antigen inhibition intensity of the hepatitis B virus-infected cell is mildly suppressed (+) at 2% to 35%; 35% to 50% is moderately suppressed (++); 50%-65 % is intensity inhibition (+++); greater than 65% is very strength inhibition (++++). The present invention utilizes an enzyme-linked immunosorbent Assay in an in vitro cell culture mode to target a hepatitis B virus wild-type cell line (MS-G2 cell line) and a dry-sensitive (Lamivudine) resistant mutant strain. (JJ33 cell line) The effect of Antrodia camphorata extract 4_acetaminophen b on human hepatitis B virus surface antigen and e antigen was determined. The results showed that 4-ethylglycolide b inhibited the cell growth inhibition of the hepatitis B virus wild-type cell line (MS-G2 cell line) and the dry cell-resistant mutant strain (M33 cell line). The surface antigen has a strength (+H) inhibitory effect at concentrations of 0.778125 yg/ml and 3.125 pg/ml, respectively; moderate (H) inhibition of the e antigen of the QJ33 cell line) The concentration is 3.125 pg/ml. 201242604 [Embodiment] Cell line and cell culture The present invention uses two human liver cancer cell lines, one of which is MS-G2, which was established by the laboratory of Dr. Max Essex. of Harvard University (Sureau, C. et al., 1986). Production of hepatitis Bvirus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. CW/47: 37-47), which is transfected with hepatoma cell line HepG2 with intact HBV DNA (^) and After the plastid of the neomycin resistance gene, the cell strain obtained by G418 screening and serial dilution was named MS-G2. Since this cell line has integrated HBV DNA and has formed a stable cell line, it can continuously secrete hepatitis B virus surface antigen (HBsAg), e antigen (HBeAg) and virus particles. The body (42 nm dendritic particles 116 3116 particles and 22 nm subviral particles), this program will use this as a wild type of hepatitis B virus model for antiviral effects and molecular biological levels. The other is the stable transgenic cell line of the anti-drug mutant strain of Gananeng (also known as 3TC) established by the National Institute of Science and Technology. It is also transfected with the drug-resistant gene using HepG2 cell line. After the point (YMDD) mutant HBV DNA and the hygromycin resistance gene plastid, the cell line selected by hygromycin and then serially diluted was named M33, which was used as type B. Hepatitis virus anti-drying ability mutant mode, antiviral effect and molecular biological level discussion. Example 1: 7 201242604 3 kg of Antrodia camphorata mycelium was heated and refluxed four times with 10 liters of 95% ethanol. The extract was concentrated by filtration, dried under reduced pressure to give 384 g of ethanol extract, and the ethanol extract was suspended in water. An equivalent amount of ethyl acetate was partitioned, and the ethyl acetate layer was concentrated under reduced pressure to obtain 157.57 g of an ethyl acetate layer partition and 159.51 g of a water layer partition. 157.57 g of the above-mentioned ethyl acetate layer partitioning portion was subjected to chromatography on a silica gel column (10 cm id X 30 cm), followed by n-hexane-hexane-ethyl acetate (10:1 2+10:3+10:44 10: 5->1: 1 2, v/v) + ethyl acetate-> Methanol was eluted in 10 L of each ratio and collected into a partition every 1 L. The fraction 56-63 (3.015 g) which was extracted from the ethyl acetate-ethyl acetate (1〇: 4) was subjected to chromatography in the reverse phase preparative column Tosoh ODS-80TS (21.5 mm X 300 mm, 10 μηι). H20-CH3CN (20:80) was used as the mobile phase, and the flow rate was 10 ml/min for chromatography. With 265 nm as the detection wavelength and the column temperature was controlled at 40 °C, 4-acetylglycolate B (131 mg) was obtained. Example 2: In a 24-well cell culture dish (petri-dish), seeded MS-G2 cells 2xl05 cells/ml, or M33 cells 1.5 x 10s cells/mi. After overnight, the cells were fully adhered, the medium was updated, and the medium was given. Different concentrations of 4-ethylglycol sterol b, each concentration is triple-coated. After 48 hours of administration, the supernatant medium was collected for antiviral activity assay and simultaneously with MTT (3-[4, 5-dimethylthiazin-2-yl]2, 5-biphenyl-tetrazole Tested, cell survival in the 24-well tank and whether the drug is toxic and inhibits cell growth. MTT is a yellow dye that is absorbed by living cells and reduced to blue-purple guanidine (formazan) via succinate 201242604 dehydrogenase in mitochondria to detect cell survival and Growth changes, in a 24-well cell culture dish, add 1 mg / miMXX solution, act at 37 C for 4 hours, aspirate the supernatant, add 1 〇〇 giDMSO to precipitate blue-violet crystals. Finally 'to split the light The colorimeter auto-enzyme immunoassay analyzer (ELISA reader) measures the absorbance at 54 , to reflect the amount of viable cells used as an indicator of whether the drug is cytotoxic (cyt〇t〇xici^). Example 3: Enzyme-Linked Assay> (ELISA), using monoclonal antibodies against human B-guessitis surface antigen and anti-human hepatitis B virus such as /e anti-multiple Epidemic _ Touch [Pu Qian Co., Ltd., Hsinchu County 'Taiwan Province'' uses a sandwich-complex of antibody-antigen-antibody enzyme conjugate, which is colored with tetramethylbenzidine (TMB), and then split ratio Color meter dirty type 00 automatic enzyme immunoassay analyzer measured by · Cong, the resulting absorbance value (〇d anti-playing antigen oligo. Gaillon _ absorbance value (10), according to the following formula to calculate its percent inhibition (Inhibition %): Absorbance value of the control group - absorbance value of the administration group X 100% = percentage of inhibition (Inhibition %) Control group absorbance value 201242604 Inhibition percentage (inhibition percentage) at 2〇 to 35% for mild inhibition (+), inhibition percentage at 35~50 % is moderate inhibition (++), the percentage of inhibition is between 5 and 65% for intensity inhibition (+++), and the percent inhibition is above 65% for very strength inhibition (++++). Example 4: - and Table 2, B hepatitis B wild type cell miscellaneous s_G2eeU (10) Test concentrations (12·5, 6.25, 3.125, 1.525, and 0.78125 pg/ml), 12.5, 6.25, 3.125, and 1.565 have cell growth inhibition (% inhibition %) 20%), 0.78125 has no cell growth inhibition, and has an inhibitory effect on the surface antigen (10). The percent inhibition is 61.3 ± 1.8 (Fig. 1). Table 1 4-Acetylsterol B in wild-type HBV cell line (MS_G2) and HL-HBeAg treatment 48 h pg/ml HBsAg HBeAg-MTT assay (inhibition rate!!〇 (% inhibition rate) (% inhibition rate) Mean ± SE Mean ± S. £ Mean Soil SE control group 0 . 0 —_ 0 DMS0 2.5 μΐ/ml 0.9 ± 1.6 aNU -2.9 ±1.1 201242604 4-Acetylsteryl B 12.5 μ ^ηύ (12.5 ppm) 6.25 μ^ιηΐ (6.25 ppm) 3.125μ§/ιη1 (3.125 ppm) 1.5625μ^πι1 0_78125μ_ ND ND ND ND 61.3 ± 1.8 ND 49.9 ±2.5 38.4 ±1.8 33.9 ± 1.0 30.0 ±3.9 16.7 ± 2.3 osthole 80 μΜ 52.0 ± 1.7 ND -13.7 soil 0.8 (From alone) 1. The experimental value is a single experiment three repeated results 2. Rib: no detection result 3. DMSO group is solvent control group 4. osthole (〇sthole) is the percentage of inhibition in the positive control group (Inhibitionpercentage): between 20%_35% for mild inhibition (+) between 35% - 50% for moderate inhibition (++) between 50% - 65% Intensity inhibition (+++) greater than 65% for very strong inhibition (++++) Table 2 Wild-type HBV cell line (MS-G2) Treatment of HBsAg HBeAg 4-Acetylsterol Β +++ ND (high concentration cytotoxicity) osthole (80 μΜ) +++ ND -- (no cytotoxicity at this concentration) 11 201242604 Example 5: Stem-resistant mutants as shown in Tables 3 and 4 below From the leg e) eight sides of the test (100, 50, 25, 12.5, 6.25, 3.125, 1 5625 β b25 and 0.78125 pg / ml), 100, 50, 25, 12.5 and 6.25 have cell growth inhibition (% inhibition Tiller, L5625 and 3.125 μ§/ηι1 have no cell growth inhibition, and have mild (1) to intensity (called inhibition) on surface antigens, with inhibition percentages of 248 〇8, % 7± i 〇 and 56.6 ± 1.5, respectively. ' 1. 5625 and 3.125 pg/ml had mild (+) to moderate (four) inhibitory effects on e antigen, with inhibition percentages of 28.1 ± 〇. 9 and 45.1 ± 2 · 〇, respectively (Figure 2). Table 3. The effect of anti-ββ heart and anti-HBeAg in the anti-dry energy HBV mutant cell line (Μ33) pg/ml HBsAg (inhibition rate %) Mean soil SE Mean soil SE MTT assay (% inhibition) Mean + SE
4-乙醯樟 芝酿1醇B 100 μ^ιηΐ (100 ppm) 50 μ^ιηΐ (50 ppm) 25 μ^ιηΐ (25 ppm) 12·5 pg/ml (12.5 ppm) 6.25 μ^ιηΐ (6.25 ppm) 3·125 pg/ml 1.5625 μ^ιηΐ 0.78125 μ^ιηΐ 處理48 h4-Ethyl sinensis 1 alcohol B 100 μ^ιηΐ (100 ppm) 50 μ^ιηΐ (50 ppm) 25 μ^ιηΐ (25 ppm) 12·5 pg/ml (12.5 ppm) 6.25 μ^ιηΐ (6.25 Ppm) 3·125 pg/ml 1.5625 μ^ιηΐ 0.78125 μ^ιηΐ 48 h
ND ND ND ND ND 56.6 ± 1.5 35.7 ±1.0 24.8 ± 0.8 HBeAg (抑制率%)ND ND ND ND ND 56.6 ± 1.5 35.7 ±1.0 24.8 ± 0.8 HBeAg (inhibition rate %)
ND ND ND ND ND 45.1 ±2.0 28.1 ±0.9 -3.7 ± 7.9 94.1 ±2.0 63.6 ±1.1 41.8 ±3.5 36.7 士 2.8 22.5 ±3.9 19.9 ± 0.6 15.8 ± 1.0 10.1 ±1.6 12 201242604 蛇床子素 80 μΜ 50.8 ± 1.5. 12.8 ±0.6 -0.3 ±2.8 (From 獨 活） 1. 實驗數值為單一實驗三重複結果 2. ND:無偵測結果 3. DMSO組為溶劑控制組 4. 蛇床子素為正對照組 抑制百分比(Inhibition percentage) ··介於20% - 35%為輕度抑制(+) 介於35% - 50%為中度抑制(++) 介於50% - 65°/❶為強度抑制(+++) 大於65%為非常強度抑制(++++) 表四.抗干安能HBV突變細胞株(M33) 處理 HBsAg HBeAg 4-乙醯樟芝醌醇β +++ ++ (高濃度具細胞毒性） (向濃度具細胞毒性） 蛇床子素(80 Μ) +++ - _ (此濃度無細胞毒性） (此濃度無細胞毒性） 13 201242604 【圖式簡單說明】 圖一顯示4-乙醯樟芝醌醇B在野生型B型肝炎病毒細胞株(MS-G2)中抗 HBsAg的效果。 圖二顯示4-乙醯樟芝醌醇B在抗干安能之突變B型肝炎病毒細胞株 (M33)中抗HBsAg以及HBeAg的效果。 【主要元件符號說明】 無 14ND ND ND ND ND 45.1 ±2.0 28.1 ±0.9 -3.7 ± 7.9 94.1 ±2.0 63.6 ±1.1 41.8 ±3.5 36.7 ±2.8 22.5 ±3.9 19.9 ± 0.6 15.8 ± 1.0 10.1 ±1.6 12 201242604 Osthole 80 μΜ 50.8 ± 1.5. 12.8 ±0.6 -0.3 ±2.8 (From Independence) 1. The experimental value is the single experiment three repeated results 2. ND: no detection result 3. The DMSO group is the solvent control group 4. The osthole is the positive control group inhibition percentage ( Inhibition percentage) ·· between 20% - 35% for mild inhibition (+) between 35% - 50% for moderate inhibition (++) between 50% - 65°/❶ for strength suppression (+++ ) greater than 65% for very strong inhibition (++++) Table 4. Anti-drying HBV mutant cell line (M33) Treatment of HBsAg HBeAg 4-Acetylsterol β +++ ++ (high concentration with cells) Toxicity) (cytotoxicity to concentration) Osthole (80 Μ) +++ - _ (This concentration is non-cytotoxic) (This concentration is not cytotoxic) 13 201242604 [Simple diagram of the diagram] Figure 1 shows 4-acetamidine The effect of indomethacin B against HBsAg in wild-type hepatitis B virus cell line (MS-G2). Figure 2 shows the effect of 4-ethylglycolyl B on anti-HBsAg and HBeAg in a mutant hepatitis B virus cell line (M33) resistant to dryness. [Main component symbol description] None 14