TW201221129A

TW201221129A - Treatment of pain, inflammation and liver injury and hepatoprotection with ergosta-7,9(11),22E-trien-3 β -ol

Info

Publication number: TW201221129A
Application number: TW100109534A
Authority: TW
Inventors: Yueh-Hsiung Kuo; Guan-Jhong Huang
Original assignee: Univ China Medical
Priority date: 2010-11-26
Filing date: 2011-03-21
Publication date: 2012-06-01
Also published as: TWI409073B

Abstract

Disclosed herein is that ergosta-7, 9(11), 22E-trien-3 β -ol can be used in the treatment of pain, inflammation and liver injury and hepatoprotection.

Description

201221129 六、發明說明：【發明所屬之技術領域】本發明是有關於使用麥角甾-7,9(11)，22五-三烯-30-醇來治療疼痛（pain)、發炎（inflammation)與肝損傷（liver injury) 以及保護肝臟（hepatoprotection)。【先前技術】已知在好氧生物體（aerobic organisms)利用氧氣進行呼吸反應（respiration)的過程中會生成活性氧族（reactive oxygen species，ROS)[例如，過氧化物（peroxide)和過氧化氫 (hydrogen peroxide)]以及自由基（free radicals)[例如，經基自由基（hydroxyl radicals)和過氧化自由基（peroxy radicals)] ，而游離輻射（ionizing radiation)以及暴露於藥物或外來化合物（xenobiotics)[例如，四氣化碳（carbon tetrachloride, CC14)]亦會導致活性氧族與自由基的生成。活性氧族以及自由基非常地不穩定，它們會快速地與生物體内的基團或物質反應，進而導致細胞或組織的氧化性損傷（oxidative damage)，例如DNA損傷、多不飽和脂肪酸（polyunsaturated fatty acid)氧化[亦被稱為脂質過氧化（lipid peroxidation)]以及胺基酸氧化。一般而言，生物體内存在有由抗氧化酵素（antioxidant enzymes)所構成的交互作用網路（interacting network)來保護細胞或組織免於氧化性損傷，最為熟知的抗氧化酵素包括 :超氧化物歧化酶（suPer〇xide dismutase，SOD)(EC 1.15.1.1) 、過氧化氫酶（catalase, CAT)(EC 1·11·1_6)、麩胱甘肽過氧 201221129 化酶（glutathione peroxidase，GPx)(EC 1.11.1.9)以及麵胱甘肽還原酶（glutathione reductase，GSH reductase)(EC 1.8.1.7) 。然而，當活性氧族以及自由基的數量超過細胞或組織本身的抗氧化能力時，便會形成氧化性壓力（oxidative stress) 。現今氧化性壓力已被發現到在下列各種不同疾病的退化性或病理學過程（degenerative or pathological processes)中扮演一個重要的角色：老化（aging)、癌症（cancer)、發炎 (inflammation)以及肝損傷（liver injury)。發炎（inflammation)意指一種生物體對抗外部壓力刺激 (external stressful stimuli)或病原體（pathogen)的保護反應。在發炎的初期，受損傷的細胞或組織會釋放出大量的趨化激素（chemokines)，使得免疫系統中的多核白血球 (polynuclear leukocytes)[例如，嗜中性球（neutrophil)]以及單核球（monocytes)往受損傷處聚集[被稱為浸潤（infiltration)] 。同時，巨噬細胞會被活化並促使細胞膜上的卵磷脂 (phosphatidylcholine，PC)藉由填月旨酶 A2 (phospholipase A2) 被水解成花生四稀酸（arachidonic acid，AA)，花生四稀酸接著藉由環加氧酶-2 (cyclooxygenase-2，COX-2)而被代謝成大量的前列腺素（prostaglandin，PG)、前列環素（prostacyclin)以及凝血脂素A2 (thromboxane A2)來增強發炎反應。活化的巨嗤細胞也會藉由釋放腫瘤壞死因子-a (tumor necrosis factor-α，TNF-α)來促進其他巨噬細胞釋放出各種不同的前發炎性細胞激素（proinflammatory cytokine)[包括介白素-1 (interleukin-1，IL-1)、IL-6 以及 IL-8]，並且透過誘導性一 4 201221129 氧化氮合成酶（inducible nitric oxide synthase, iNOS)的作用來生成能夠舒張血管以及殺滅病原體的一氧化氮。此外，活性氧族以及自由基也被免疫系統用來殺滅病原體而成為發炎反應的重要成員。雖然目前已存在許多人工化合物可分別供用於改善氧化性壓力以及發炎，但這些人工化合物均會產生程度不等的副作用[諸如胃潰癌（gastric ulcer)以及胃炎（gastritis)]。如果能夠透過從藥性溫和且副作用較低的天然中草藥中找尋能夠改善氧化性壓力並且具有抗發炎以及止痛活性（antiinflammatory and analgesia activities)的化合物或萃取物，應可以提供一在使用上更為安全的藥物。樟芝又名“牛樟芝”）屬於無褶菌目（Aphyllophorales)、多孔菌科（Polyporaceae)、薄孔菌屬，主要生長在台灣高海拔的樟樹（Cz’/irtamowMm 上。在臺灣民俗醫學上，樟芝的子實體（fruiting body)被用於治療食品或藥物中毒（food or drug intoxication) 、腹濱（diarrhea)、腹痛（abdominal pain)、高血壓 (hypertension)、皮膚搔癢（skin itching)以及癌症（Peng C.C. et al. (2007), J. Ethnopharmacol., 109:93-103) o jl匕夕卜，樟芝的子實體亦被證實具有免疫調節、抗氧化以及肝臟保護效用（im.munomodulating，antioxidative, and hepatoprotective effects)(Shen Y.C. et al. (2004), Planta. Med., 70:310-314 ； Hsiao G. et al. (2003), J. Agricultural And Food Chemistry, 51:3302-3308) 〇 201221129 曾有文獻報導：樟芝的發酵培養液（fermented culture broth)具有對抗數種腫瘤細胞株的細胞毒性（cytotoxic activity )(Peng C.C. eia/· (2007)，同上述）；樟芝的發酵培養液的濾液（filtrate)具有對抗四氣化碳-誘發的肝毒性（CC14-induced hepatic toxicity)的肝臟保護效用以及抗氧化性質 (antioxidant properties)(Song T.Y. et al. (2003)， J. dgWcw/iwra/ Fooc? 51:1571-1577);樟芝的菌絲體（mycelia)具有抗發炎（anti-inflammation)與血管舒張 (vasorelaxation)的效用、對抗多種癌細胞株的細胞毒性以及抗-B 型肝炎病毒活性（anti-hepatitis B virus activity)(Shen Y.C. et al. (2004), FEMS Microbiology Letter, 231:137-143 ； Wang G.J. et al (2003), Life Science, 73:2769-2783 ； Yeh C.T. et al. (2009), Cancer Letter, 285:73-79 ； Lee I.H. et al. (2002), FEMS Microbiology Letter, 209:63-67) ° 在一個先前的研究中，發明人從樟芝的浸沉培養液 (submerged broth)中成功地分離出12種化合物，其中包括 11個已知化合物[亦即麥角甾-7,9(11)，22五-三烯-30-醇 (ergosta-7,9(ll),22£-trien-3p-ol)、過氧化麥角固醇 (ergosterol peroxide)、（4-羥基苯基）乙酸甲酯[methyl (4-hydroxyphenyl) acetate]、香草酸（vanillin)、4-經基苯甲酸· (4-hydroxybenzaldehyde)、十六酸（hexadecanoic acid)、5-曱氧基曱基 σ夫喃-2-曱酿（5-methoxymethylfuran-2-carbaldehyde) 、5-經基曱基吱喃-2-曱酸（5-hydroxymethylfuran-2-carbaldehyde) 、11-羥基十二内酯（ll-hydroxy-γ- 6 201221129 dodecalactone)、2-(2-經乙基）紛[2-(2-hydroxyethyl) phenol] 以及 12-經基十二酸甲醋（12-hydroxydodecanoic acid methyl ester)]，以及1個新穎化合物[亦即10·羥基-γ-十二内酯（1〇-hydroxy-y-dodecalactone)](Shao Y.Y. et al. (2008), Natural Product Research, 22:1151-1157) ° 為了暸解從樟芝的浸沉培養液中所分離出的化合物的生物活性，在一個隨後的研究中，發明人選用具有一如下面所示之化學式的麥角甾-7,9( 11 )，22五-三烯-3β-醇來進行活體外抗癌實驗：201221129 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to the use of ergosta-7,9(11),22penta-triene-30-ol for the treatment of pain and inflammation. With liver injury and liver protection (hepatoprotection). [Prior Art] It is known that reactive oxygen species (ROS) are formed during aerobic organisms using oxygen for respiration (for example, peroxide and peroxidation) Hydrogen peroxide] and free radicals [eg, hydroxyl radicals and peroxy radicals], and ionizing radiation and exposure to drugs or foreign compounds ( Xenobiotics) [eg, carbon tetrachloride (CC14)] also leads to the formation of reactive oxygen species and free radicals. Reactive oxygen species and free radicals are very unstable, and they react rapidly with groups or substances in the body, leading to oxidative damage to cells or tissues, such as DNA damage, polyunsaturated fatty acids (polyunsaturated). Fatty acid) [also known as lipid peroxidation] and amino acid oxidation. In general, there is an interacting network of antioxidant enzymes in the organism to protect cells or tissues from oxidative damage. The most well-known antioxidant enzymes include: superoxide. Dismutase xide dismutase (SOD) (EC 1.15.1.1), catalase (CAT) (EC 1·11·1_6), glutathione peroxidase (GPx) (EC 1.11.1.9) and glutathione reductase (GSH reductase) (EC 1.8.1.7). However, when the amount of reactive oxygen species and free radicals exceeds the antioxidant capacity of the cells or tissues themselves, oxidative stress is formed. Oxidative stress has now been found to play an important role in the degenerative or pathological processes of various diseases: aging, cancer, inflammation, and liver damage. (liver injury). Inflammation refers to the protective response of an organism against external stressful stimuli or pathogens. In the early stages of inflammation, damaged cells or tissues release large amounts of chemokines, making polynuclear leukocytes (eg, neutrophil) and mononuclear balls in the immune system (eg, nucleus) Monocytes) accumulate in the affected area [known as infiltration]. At the same time, macrophages are activated and promote phosphatidylcholine (PC) on the cell membrane to be hydrolyzed to arachidonic acid (AA) by alipase A2, followed by arachidonic acid It is metabolized into a large number of prostaglandin (PG), prostacyclin and thromboxane A2 by cyclooxygenase-2 (COX-2) to enhance the inflammatory response. . Activated giant scorpion cells also promote the release of various proinflammatory cytokine (including interleukin) by other macrophages by releasing tumor necrosis factor-a (TNF-α). Interleukin-1 (IL-1), IL-6 and IL-8], and through the action of inducible nitric oxide synthase (iNOS) to generate diastolic blood vessels and kill Eliminate the pathogen's nitric oxide. In addition, reactive oxygen species and free radicals are also used by the immune system to kill pathogens and become important members of the inflammatory response. Although many artificial compounds are currently available for improving oxidative stress and inflammation, respectively, these artificial compounds produce varying degrees of side effects [such as gastric ulcer and gastritis]. If it is possible to find compounds or extracts that can improve oxidative stress and have antiinflammatory and analgesia activities from natural herbal medicines with mild medicinal properties and low side effects, it should be possible to provide a safer use. drug. Anthracnium, also known as "Bagba lucidum", belongs to the genus Aphyllophorales, Polyporaceae, and the genus Phaeocystis. It is mainly grown on Taiwan's high-altitude eucalyptus (Cz'/irtamowMm. In Taiwanese folk medicine, The fruiting body of Antrodia camphorata is used to treat food or drug intoxication, diarrhea, abdominal pain, hypertension, skin itching, and cancer. (Peng CC et al. (2007), J. Ethnopharmacol., 109:93-103) o jl匕, the fruiting body of Antrodia camphorata has also been confirmed to have immunomodulatory, antioxidant and hepatic protective effects (im.munomodulating, Antioxidative, and hepatoprotective effects) (Shen YC et al. (2004), Planta. Med., 70:310-314; Hsiao G. et al. (2003), J. Agricultural And Food Chemistry, 51:3302-3308) 〇201221129 There have been reports in the literature that the fermented culture broth of 樟芝 has cytotoxic activity against several tumor cell lines (Peng CC eia/· (2007), same as above); fermentation of Antrodia camphorata The filtrate of the culture fluid has liver protection effect against anti-hemocarbon-induced hepatic toxicity and antioxidant properties (Song TY et al. (2003), J. dgWcw). /iwra/ Fooc? 51:1571-1577); Mycelia of Antrodia camphorata has anti-inflammation and vasorelaxation effects, cytotoxicity against various cancer cell lines, and anti-B Anti-hepatitis B virus activity (Shen YC et al. (2004), FEMS Microbiology Letter, 231: 137-143; Wang GJ et al (2003), Life Science, 73: 2769-2783; Yeh CT et al. (2009), Cancer Letter, 285:73-79; Lee IH et al. (2002), FEMS Microbiology Letter, 209:63-67) ° In a previous study, the inventor from Antrodia Twelve compounds were successfully isolated from submerged broth, including 11 known compounds [ie, ergosin-7,9(11),22penta-triene-30-ol (ergosta-7) , 9(ll), 22£-trien-3p-ol), ergosterol peroxide, (4-hydroxyphenyl) Methyl (4-hydroxyphenyl) acetate, vanillin, 4-hydroxybenzaldehyde, hexadecanoic acid, 5-nonyloxyindolyl -2- brewing (5-methoxymethylfuran-2-carbaldehyde), 5-hydroxymethylfuran-2-carbaldehyde, 11-hydroxydodelactone (ll-hydroxy-) Γ- 6 201221129 dodecalactone), 2-(2-ethylethyl) phenol], and 12-hydroxydodecanoic acid methyl ester, and 1 Novel compound [i.e., 1 〇-hydroxy-y-dodecalactone] (Shao YY et al. (2008), Natural Product Research, 22: 1151-1157) ° The biological activity of the compound isolated from the immersion culture solution of Antrodia camphorata. In a subsequent study, the inventors chose ergot -7,9(11),22-5-three having the chemical formula shown below. Alkenyl-3β-alcohol for in vitro anticancer experiments:

經由實驗結果證實：麥角甾-7,9(11)，22五-三烯-3β-醇具有抑制肝癌細胞生長的效用，它對於人類肝癌細胞株PLC/PRF/5 的 50%抑制濃度（5〇% inhibition concentration, IC5〇)為 20.5 pg/mL (未發表的數據）。經研究，發明人意外地發現麥角甾_7,9(ii),225-三烯-•3β-醇除了具有抑制肝癌細胞生長的效用之外，還具有止痛 (analgesia)、抗發炎（anti-inflammatory)以及改善肝損傷 (ameliGfating liver injury)的效用，因而被預期可供用於治療疼痛、發炎與肝損傷以及保護肝臟（hepatoprotection)。【發明内容】 201221129 發明概要於是’在第一個方面’本發明提供一種用於治療疼痛的藥學組成物’其包含有麥角甾_7,9(11)，22£-三烯_3(3_醇。在第二個方面，本發明提供一種用於治療發炎的藥學組成物’其包含有麥角甾-7,9(11)，22£-三烯-3β-醇。在第二個方面，本發明提供一種用於治療肝損傷的藥學組成物，其包含有麥角留_7,9(11)，22五-三烯_3β-醇。在第四個方面，本發明提供一種用於保護肝臟的組成物，其包含有麥角甾-7,9(11)，22五-三烯-30-醇。特徵與優點，在參照以本發明的上述以及其它目的、下的洋細說明與較佳實施例和隨文檢附的圖式後，將變得明顯。發明的詳細說明It was confirmed by experimental results that ergot 甾-7,9(11),22-trien-3-β-alcohol has the effect of inhibiting the growth of liver cancer cells, and its 50% inhibitory concentration on human hepatoma cell line PLC/PRF/5 ( 5〇% inhibition concentration, IC5〇) was 20.5 pg/mL (unpublished data). After research, the inventors unexpectedly discovered that ergoside _7,9(ii), 225-triene-•3β-alcohol has anti-inflammatory (analgesia) and anti-inflammatory (anti) in addition to the effect of inhibiting the growth of liver cancer cells. -inflammatory) and the effect of improving ameliGfating liver injury, and thus are expected to be useful for the treatment of pain, inflammation and liver damage, and protection of the liver (hepatoprotection). SUMMARY OF THE INVENTION 201221129 Summary of the Invention Thus, in a first aspect, the present invention provides a pharmaceutical composition for treating pain, which comprises ergot 甾7,9(11), 22£-triene_3 ( In a second aspect, the present invention provides a pharmaceutical composition for treating inflammation comprising 'ergost-7,9(11),22 £-triene-3β-ol. In one aspect, the present invention provides a pharmaceutical composition for treating liver damage comprising ergoline-7,9(11),22penta-triene-3β-ol. In a fourth aspect, the present invention provides A composition for protecting a liver comprising ergoside-7,9(11),22penta-triene-30-ol. Features and advantages, with reference to the above and other objects of the present invention The detailed description of the invention and the accompanying drawings will become apparent.

(comprises)”具有一對應的意義。除非另外有所定義在本文中所使用的所有技術性斑(comprises)" has a corresponding meaning. Unless otherwise defined, all technical spots used in this article

富然’本發明決不受到所描述的方法和材料之限制。 8 201221129 已知樟芝的菌絲體具有抗發炎與血管舒張的效用、對抗多種癌細胞株的細胞毒性以及抗_B型肝炎病毒活性，而樟芝的發酵培養物的濾液具有對抗四氣化碳_誘發的肝毒性的肝臟保護效用以及抗氧化性質。為了開發可用於治療疼痛以及發炎之藥物，發明人嘗試從樟芝的浸沉培養物中找出具有止痛以及抗發炎活性的有效活性成分。於是，發明人選用先前由樟芝的浸沉培養物中所分離出的麥角甾· 7,9(11)，22五-三烯_30_醇來進行活體内止痛試驗如咖〇 analgesia test)[包括乙酸·誘發的扭體試驗（阶以扣… induced writhing test)以及福馬林_誘發的腳掌舔吮試驗 (fcmnalin-induced paw Hcking㈣)]。乙酸誘發的扭體試驗是一種被用來評估一待測物是否具有周邊止痛效用 (peripheral analgesia effect)的方法，而福馬林誘發的腳掌舔吩試驗是一種透過將福馬林注射至小鼠腳掌來模擬人類臨床疼痛病況（human clinical pain _仙〇吵並分別在注射之後的第0至5分鐘的期間[被稱為早期（eariy _小可反映神經性疼痛（neur〇genic pain)]以及帛i5至4〇分鐘的期間[被稱為晚期（丨ate phase)，可反映發炎性疼痛 (infla細atory pain)]來觀察小鼠舔吹腳掌的時間，藉此而確認-待測物對於神經性疼痛以及發炎性疼痛是否具有止痛效用的方法。經由實驗結果證實：麥角⑴），现三渗 3β_醇具有減輕發炎性疼痛的效用，因而被預期在治療疼痛上具有極大潛力。於是，本發明揭示麥角^7,9⑴），22£_三烯β醇供應用於製傷—用來治療疼痛之醫藥品的用途。 201221129 因此，本發明提供一種用於治療疼痛的藥學組成物，其包含有麥角甾_7,9(11)，22五-三烯-3β-醇。如本文中所使用的，“治療（treating)”或“治療 (treatment)” 意指減少（reducing)、減輕（alleviating)、改善 (ameliorating)、緩解（relieving)' 或控制（controlling)—疾病 (disease)或障礙（disorder)的一或多個臨床徵兆（clinical sign) ，以及降低（lowering)、停止（stopping)或逆轉（reversing)— 正在被治療中的病況（condition)或症狀（symptom)之嚴重性 (severity)的進展（progression)。依據本發明的藥學組成物可利用熟習此藝者所詳知的技術而被製造成一適合於非經腸道地（parenterally)、局部地 (topically)或口服地（orally)投藥的劑型，這包括，但不限於，注射品（injection)[例如，無菌的水溶液或分散體 (dispersion)]、無菌的粉末、疑劑（tablet)、片劑（troche)、丸劑（pellet)、膠囊（capsule)以及類似之物》依據本發明的藥學組成物可以一選自於下列所構成的群組中的非經腸道途徑來投藥：腹膜内注射（intraperitoneal injection)、皮下注射（subcutaneous injection)、肌肉内注射 (intramuscular injection)、靜脈内注射（intravenous injection) 、舌下投藥（sublingual administration)以及穿皮投藥 (transdermal administration) ° 在本發明的一個較佳具體例中，該藥學組成物被製成適於以腹膜内注射被投藥的劑型。在本發明的另一個較佳具體例中，該藥學組成物被製 10 201221129 成適於口服投藥的劑型。依據本發明的藥學組成物可進一步包含有一被廣泛地使用於藥物製造技術之藥學上可接受的載劑。例如，該藥學上可接受的載劑可包含一或多種選自於下列的試劑：溶劑（solvent)、乳化劑（emulsifier)、懸浮劑（suspending agent) 、分解劑（decomposer)、黏結劑（binding agent)、賦形劑 (excipient)、安定劑（stabilizing agent)、螯合劑（chelating agent)、稀釋劑（diluent)、膠凝劑（gelling agent)、防腐劑 (preservative)、潤滑劑（lubricant)、吸收延遲劑（absorption delaying agent)、脂質體（liposome)以及類似之物。在本發明的一個較佳具體例中，該藥學組成物包含有一藥學上可接受的溶劑，而且該溶劑是下列的任一者：含糖溶液、含有醇[諸如乙醇、丙二醇（propanedipl)、甘油以及甘露糖醇（mannitol)等]的水性溶液、油[諸如花生油（peanut oil)、橄欖油（olive oil)、芝麻油（sesame oil)、篦麻油（castor oil)、棉花轩油（cottonseed oil)以及大立油（soybean oil)等]、甘油、有機溶劑以及脂質體（liposome)。本發明亦提供一種用於治療一帶有疼痛之個體的方法，其包括對該個體投藥以麥角甾-7,9(11),225-三烯-3β-醇。另外，發明人將麥角甾-7,9(11),22五-三烯-30-醇拿來進行鹿角菜膠-誘發的腳掌水腫試驗（carrageenan-induced paw edema test)，俾以評估麥角甾-7,9(11)，22五-三烯-3β-醇是否具有抗發炎活性（anti-inflammatory activity)而可有效地抑制鹿角菜膠注射所引起的急性發炎（acute inflammation)。經由 201221129 實驗結果證實：麥角H9(ll)，22【三烯_3β-醇具有抗發炎的效用。於是’本發明揭示供應用於製備一用來治療發炎的藥學組成物的用途。因此，本發明提供一種用於治療發炎的藥學組成物，其包含有麥角留-7,9(11)，22£-三烯-3β-醇。依據本發明，該藥學組成物的投藥途徑、劑型以及可供使用之藥學上可接受的載劑是如上面所述者。纟發明亦提供-種用於治療—帶有或被懷疑帶有發炎之個體的方法，其包括對該個體投藥以麥角留_7,9(11)，22五_ 三稀-3β-醇。在鹿角菜膠-誘發的腳掌水腫試驗中，發明人觀察到：在以鹿角菜膠注射誘發急性發炎之後，在小鼠肝臟中的3 種抗氧化酵素[包括超氧化物歧化酶（super〇xide dismmase， SOD)、過氧化氫酶（catalase，CAT)以及麵耽甘狀過氧化酶 (glutathione peroxidase，GPx)]的活性被提高，這顯示麥角留 -7,9(11)，22五-三稀-；^醇具有改善肝損傷的效用。於是，發明人選用經四氣化碳誘發化學性肝損傷（ccl4_induced chemical liver injury)的小鼠作為動物模型，俾以評估麥角留_7,9(11)，22五-三缔斗醇在改善肝損傷上的效用。經由實驗結果證實··麥角D，9⑴），2此三浠_3(3_醇可以顯著地改善四氣化碳對肝臟所造成的損傷。基於上述，麥M_7,9(11)，2此三烯斗醇被預期具有可用於治療肝損傷的高度潛力。於是，本發明揭示麥角留_ 7,9(11)，22五-三烯等醇供應用於製備一用來治療肝損傷之藥 12 201221129 學組成物的用途。因此’本發明提供一種用於治療肝損傷的藥學組成物，其包含有麥角留-7,9(11)，22五-三烯-3β-醇。如本文中所使用的，“肝損傷，，意指任何由一或多個外在或内在的因素（internal or external factors)及其組合直接或間接地造成肝臟在結構上或功能上的損傷，該等因素包括，但不限於：暴露於肝毒性化合物（hepat〇t〇xic e〇mp〇unds)或輻射（radiation)、機械性損傷（mechanical damage)、遺傳得病傾向（genetic predispositi〇n)、病毒感染（virai infecti〇ns)以及自體免疫疾病.（autoimmune diseases)。在本發明的一個較佳具體例中，該肝損傷是化學性肝損傷（chemical liver injury)、病毒性肝損傷⑽^ η· 或酒精性肝損傷（aleGhc)lie _…㈣。在本發明的一個較佳具體例中，該肝損傷是化學性肝損傷。在本發明、固更佳具體例中’該化學性肝損傷是四氣化碳(⑽^誘發的肝損傷。依據本發明，該藥學組成物的投藥途徑、劑型以及可供使用之藥學切接受的_是如上面所述者。傷之Γ = 一種^^療—帶有或被懷疑帶有肝損 7Qnn〇^ 法，其包括對該個體投藥以麥角留_ 7,9(11)，22 五-三婦♦醇。 ^ 基於麥角“，9⑴），挪三稀氯化碳對肝㈣造成的損傷•頁者地改善四 ⑴)，2此：婦貝傷本發明亦預期麥角留. —埽，，供應用於製備-用來保護肝臟之組成 13 201221129 物的用途。因此，本發明提供一種用於保護肝臟的組成物，其包含有麥角甾-7,9(11)，22五-三烯-30-醇。如本文中所使用的，“保護肝臟（hepatoprotection)”意指保護或預防肝臟以及相關聯的組織[包括，但不限於，肝靜脈（hepatic vein)以及門靜脈（portal vein)]免於損傷。依據本發明，該組成物被製造成有如一醫藥品或食品產品的形式。依據本發明，該醫藥品的投藥途徑、劑型以及可供使用之藥學上可接受的載劑是如上面所述者。依據本發明，該組成物可被當成食品添加物（food additive)，藉由習知方法於原料製備時被添加，或是於食品的製作過程中被添加，而與任一種可食性材料被配製成供人類與非人類動物攝食的食品產品。依據本發明，該食品產品的種類包括，但不限於：奶粉（milk powder)、飲料（beverages)、甜點（confectionery)、糖果（candies)、發酵食品（fermented foods)、烘培產品 (bakery products)、動物飼料（animal feeds)、健康食品 (health foods)以及腊食補充品（dietary supplements)。本發明亦提供一種用於保護一個體之肝臟的方法，其包括對該個體投藥以麥角甾-7,9(11),22五-三烯-3β-醇。【實施方式】較佳實施例之詳細說明本發明將就下面的實施例來做進一步說明，但應瞭解 14 201221129 的是，該等實施例僅是供例示說明用，而不應被解釋為本發明的實施上的限制。實施例實驗材料： 1 ·下面實施例中所使用的樟芝（Jcawp/ioraia)的浸沉培養物（submerged culture)的柬乾粉末（freeze-dried powder)是由葡萄王企業股份有限公司的生物工程中心 (Biotechnology Center of Grape King, Inc.)(台灣，中爐）所提供，生產批號為MZ-247。 2.下面實施例中所使用的雄性銘印控制區（imprinting control region, ICR)小鼠（6至8週大，體重約為18至25 g)是購自於樂斯科生物科技股份有限公司（BioLasco Taiwan Co.，Ltd)。所有的實驗動物被飼養於光照與黑暗各為12小時、室溫維持在22±1°C以及相對濕度維持在 55±5%的塑膠玻璃籠子（plexiglass cages)中，而且水分與飼料被充分地供給。在實驗之前，給予動物至少2週的期間去適應環境。有關實驗動物的一切實驗程序是依據國家衛生研究院（National Institutes of Health, NIH)的實驗動物飼養管理及使用規範（Guide for the Care and Use of Laboratory Animals)，並遵從國際疼痛研究協會 (International Association for the Study of Pain)的指導原則來進行。一般實驗方法： 1. 一般血清樣品（genera丨serum sample)的製備 15 201221129 將由實驗動物所採集到的血液在4°C下以3,000 rpm離心歷時10分鐘，所得到的血清被使用作為一般血清樣品並且被冷凍保存於-80°C下備用。 2. 組織萃取物樣品（tissue extract sample)的製備將由實驗動物所取得的組織以冷生理食鹽水（cold normal saline)予以洗滌後立刻轉移至等體積的冷生理食鹽水中，接著，在 4°C 下以一均質機（homogenizer)(Polytron PT-1200，Kinematica)予以均質處理歷時1分鐘，繼而在4°C下以12,000 xg離心所形成的均質物（homogenate)歷時5分鐘。之後，收取上清液，將所收取的上清液以一為10。/。（v/v) 的最終濃度（final concentration)加入至生理食鹽水中並均勻混合，藉此而得到組織萃取物樣品備用。 3. 統計學分析（statistical analysis) 在下面的實施例中，實驗數據是以平均值（mean)±平均值的標準誤差（standard error of the mean，SEM)來表示。各組間實驗數據的差異首先是藉由單因子變異數分析（one-way ANOVA)，繼而為雪費氏多重全距檢定（Scheffe’s multiple range test)來進行評估，若所得到的統計分析結果是p < 0.05 ，代表有統計學顯著性（statistical significance)。實施例1.源自於樟芝的浸沉培養物的麥角留-7,9(11)，22£-三稀-3p-醇[ergosta-7,9(ll)，22£"-trien-3p-ol】的純化源自於樟芝的浸沉培養物的麥角甾-7,9(11)，22五-三烯-3β-醇是參照 YI-YUAN SHAO ei a/. (2008)，/Voi/wci 16 201221129 22:1151-1 157當中所述的方法來進行純化。簡言之’秤取1.6 kg樟芝的浸沉培養物的凍乾粉末，在室溫下將之浸泡於16 L的曱醇（methanol)中並靜置歷時1天，繼而在真空下將甲醇蒸發而得到一殘餘物（residue)。此浸泡-靜置-蒸發步驟被重複進行3次，而得到一呈棕色之樟芝的浸沉培養物的曱醇萃取物（methanol extract)。將所得到的樟芝的浸沉培養物的曱醇萃取物懸浮於1 L滅菌水中，接著以水與乙酸乙醋（ethyl acetate, EtOAc)來進行分配分離（partition) 共計3次。所得到的乙酸乙醋分離部分（EtO Ac fraction)是使用一含有己烷（hexane)以及乙酸乙酯的混合溶液作為洗提液而在矽膠（silica gel)上被層析，繼而在使用10%乙酸乙酯（配於己烧中）作為移動相的高效能液相層析（high performance liquid chromatography, HPLC)中被進一步純化。上面所純化出的化合物的1H-NMR以及13C-NMR光譜是使用一 Bruker DMX-400光譜儀（spectrometer)而被摘測，所使用的溶劑為CDCI3，而化學位移（δ)是以ppm為單位。該化合物所測得的1H-NMR以及13C-NMR光譜數據被歸納如下： NMR (CDCI3, 400 MHz) δ ： 0.61 (s, 3 H, CH3), 0.80 (d, J =6.4 Hz, 3H, CH3), 0.B2 (d, J= 6.4Hz, 3H,CH3), 0.89 (d, J= 7.2Hz, 3H,CH3), 0.92 (s, 3H, CH3), 1.01 (d, 7=6.4 Hz, 3H,CH3), 3.61 (m, 1H, CH),5.14 (dd, 7=15.6, 7.2 Hz, 1H, CH), 5.20 (dd, J=\5.6, 8.0 Hz, 1H, CH),5.35 (m, 1H, CH), 5.55 (dd，J= 5.6, 2.0 Hz，CH)。 17 201221129 13C NMR (CDC13, 100 MHz) δ : 12.0, 16.3, 17.6，19.6, 20.0, 21.1，23.0, 28.3, 32.0, 33.1，37.0, 38.4, 39.1，40.4, 40.8, 42.8, 46.2， 54.6， 55.7， 70.5， 116.3， 119.6， 132.0， 135.6， 139.8, 141.3。依據所測得的光譜數據，該化合物被確認是一具有下列化學結構式的已知化合物[亦即麥角甾-7,9(11)，22五-三烯_ 3β-醇]:The invention is in no way limited by the methods and materials described. 8 201221129 It is known that the mycelium of Antrodia camphorata has anti-inflammatory and vasodilating effects, cytotoxicity against various cancer cell lines, and anti-B hepatitis virus activity, while the filtrate of the fermentation culture of Antrodia camphorata has anti-four gasification. Hepatoprotective effects of carbon-induced hepatotoxicity and antioxidant properties. In order to develop a drug which can be used for the treatment of pain and inflammation, the inventors attempted to find an effective active ingredient having analgesic and anti-inflammatory activity from a soaking culture of Antrodia camphorata. Therefore, the inventors selected the ergosterol 7, 7, (11), 22 penta-triene _30-ol previously isolated from the immersion culture of Antrodia camphorata for in vivo analgesic test such as curry analgesia test. [Including the acetic acid-induced writhing test and the fcmnalin-induced paw Hcking test (four)). The acetic acid-induced writhing test is a method used to assess whether a test substance has a peripheral analgesia effect, and the formalin-induced foot sputum test is a method of injecting formalin into the sole of a mouse. Simulating human clinical pain conditions (human clinical pain _ 〇〇并 and respectively during the 0 to 5 minutes after the injection [called early (eariy _ small can reflect neurouric pain (neur〇genic pain)] and 帛i5 During the period of 4 minutes [called 晚期ate phase, which can reflect inatory pain), the time of the mouse's paw is observed, thereby confirming that the analyte is neurologically Whether pain and inflammatory pain have an analgesic effect. It is confirmed by experimental results that ergot (1)), which has the effect of alleviating inflammatory pain, is expected to have great potential for treating pain. The invention discloses the use of ergot ^7,9(1)), 22 £-triene beta alcohol for the manufacture of a medicament for the treatment of pain. 201221129 Accordingly, the present invention provides a pharmaceutical composition for treating pain comprising ergoside _7,9(11),22 penta-triene-3β-ol. As used herein, "treating" or "treatment" means reducing, alleviating, ameliorating, relieving, or controllingling-disease ( One or more clinical signs of disease or disorder, and lowering, stopping, or reversing - the condition or symptom being treated (symptom) The progress of severity. The pharmaceutical composition according to the present invention can be manufactured into a dosage form suitable for parenterally, topically or orally, using techniques well known to those skilled in the art, including , but not limited to, an injection [eg, a sterile aqueous solution or dispersion], a sterile powder, a tablet, a troche, a pellet, a capsule, and the like Similarly, the pharmaceutical composition according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection. (intramuscular injection), intravenous injection, sublingual administration, and transdermal administration. In a preferred embodiment of the invention, the pharmaceutical composition is formulated to The dosage form to be administered is intraperitoneally injected. In another preferred embodiment of the invention, the pharmaceutical composition is formulated as a dosage form suitable for oral administration. The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, and binding agents. Agent), excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, Absorption delaying agents, liposomes, and the like. In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable solvent, and the solvent is any one of the following: a sugar-containing solution, containing an alcohol [such as ethanol, propanedipl, glycerin And an aqueous solution of mannitol, etc., oil [such as peanut oil, olive oil, sesame oil, castor oil, cottonseed oil, and Soybean oil, etc.], glycerin, organic solvents, and liposomes. The invention also provides a method for treating a pain-bearing individual comprising administering to the individual ergosta-7,9(11),225-triene-3β-ol. In addition, the inventors took ergoside-7,9(11),22pentatriene-30-ol for the carrageenan-induced paw edema test, and evaluated the wheat Whether or not keratin-7,9(11),22-trien-3-β-alcohol has anti-inflammatory activity can effectively inhibit acute inflammation caused by carrageenan injection. It was confirmed by the results of 201221129 that ergot H9(ll) and 22[triene_3β-alcohol have anti-inflammatory effects. Thus, the present invention discloses the use of a pharmaceutical composition for the preparation of a medicament for the treatment of inflammation. Accordingly, the present invention provides a pharmaceutical composition for treating inflammation comprising ergoline-7,9(11),22£-triene-3β-ol. In accordance with the present invention, the route of administration, dosage form, and pharmaceutically acceptable carrier for which the pharmaceutical composition is acceptable are as described above. The invention also provides a method for treating a subject with or suspected of having inflammation, which comprises administering to the individual ergoline _7,9(11),22 five-tris-3β-ol . In the carrageenan-induced paw edema test, the inventors observed: three antioxidant enzymes in the liver of mice after superinflammation induced by carrageenan injection [including superoxide dismutase (super〇xide) The activities of dismmase, SOD), catalase (CAT), and glutathione peroxidase (GPx) were increased, which showed ergot retention-7,9(11),22-5- Tris-l-alcohol has the utility of improving liver damage. Therefore, the inventors selected mice with ccl4_induced chemical liver injury as an animal model to evaluate ergot residues _7,9(11),22 five-triple alcohol Improve the effectiveness of liver damage. It was confirmed by experimental results that · ergot D, 9 (1)), 2 such three 浠 3 (3 - alcohol can significantly improve the damage caused by the four gasified carbon on the liver. Based on the above, Mai M_7, 9 (11), 2 This triterpene alcohol is expected to have a high potential for treating liver damage. Thus, the present invention discloses the supply of an alcohol such as ergot _ 7,9 (11), 22 penta-triene for the preparation of a liver for treating liver damage. Use of the composition 12 201221129 The present invention provides a pharmaceutical composition for treating liver damage comprising ergoline-7,9(11),22penta-triene-3β-ol. As used herein, "liver injury," means any structural or functional impairment of the liver caused directly or indirectly by one or more external or external factors and combinations thereof, Such factors include, but are not limited to, exposure to hepatotoxic compounds (hepat 〇t〇xic e〇mp〇unds) or radiation, mechanical damage, genetic predisposition 〇n, Viral infection (virai infecti〇ns) and autoimmune diseases. (au In a preferred embodiment of the invention, the liver injury is chemical liver injury, viral liver injury (10) η· or alcoholic liver injury (aleGhc) lie _ (4). In a preferred embodiment of the present invention, the liver injury is a chemical liver injury. In the present invention, in a more specific embodiment, the chemical liver injury is a carbonized carbon ((10)^ induced liver injury. In the present invention, the administration route, the dosage form, and the pharmaceutically acceptable form of the pharmaceutical composition are as described above. Injury Γ = a treatment - with or suspected of having liver damage 7Qnn 〇 ^ The method comprises administering to the individual ergots _ 7, 9 (11), 22 five-three women ♦ alcohol. ^ Based on ergot ", 9 (1)), the damage caused by the three diced carbon chloride to the liver (four) The page improves the four (1)), 2: the scallop injury. The present invention also contemplates the use of ergots, 埽, for the preparation of the composition 13 for use in the protection of the liver. Accordingly, the present invention provides a composition for protecting the liver comprising ergoside-7,9(11),22penta-triene-30-ol. As used herein, "hepatoprotection" means protecting or preventing the liver and associated tissues [including, but not limited to, hepatic veins and portal veins] from damage. According to the invention, the composition is manufactured in the form of a pharmaceutical or food product. In accordance with the present invention, the pharmaceutical administration route, dosage form, and pharmaceutically acceptable carrier for use are as described above. According to the present invention, the composition can be added as a food additive, added by a conventional method at the time of preparation of the raw material, or added during the production of the food, and matched with any edible material. Made into food products for human and non-human animals. According to the present invention, the types of food products include, but are not limited to, milk powder, beverages, confectionery, candies, fermented foods, bakery products. , animal feeds, health foods, and dietary supplements. The invention also provides a method for protecting a liver of a body comprising administering to the individual ergosta-7,9(11),22penta-triene-3β-ol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be further described with respect to the following embodiments, but it should be understood that the description of the embodiments is only for the purpose of illustration and should not be construed as Limitations on the implementation of the invention. EXAMPLES Experimental materials: 1 · The freeze-dried powder of the submerged culture of Jcawp/ioraia used in the following examples was bioengineered by Grape King Enterprise Co., Ltd. Provided by the Biotechnology Center of Grape King, Inc. (Taiwan, China), the batch number is MZ-247. 2. The male imprinting control region (ICR) mice used in the examples below (6 to 8 weeks old, weighing approximately 18 to 25 g) were purchased from Lesco Biotech Co., Ltd. (BioLasco Taiwan Co., Ltd). All experimental animals were housed in plexiglass cages with light and dark for 12 hours, room temperature maintained at 22 ± 1 ° C, and relative humidity maintained at 55 ± 5%, and the water and feed were adequately supply. Animals were given at least 2 weeks to adjust to the environment prior to the experiment. All experimental procedures for laboratory animals are based on the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and are in compliance with the International Association for Pain Research (International Association). The guiding principles for the Study of Pain). General Experimental Methods: 1. Preparation of a general serum sample (genera丨serum sample) 15 201221129 The blood collected from the experimental animals was centrifuged at 3,000 rpm for 10 minutes at 4 ° C, and the obtained serum was used as a general serum sample. It was stored frozen at -80 ° C for use. 2. Preparation of tissue extract sample The tissue obtained from the experimental animal was washed with cold normal saline and immediately transferred to an equal volume of cold physiological saline, followed by 4 ° C. The homogenization was carried out by homogenizer (Polytron PT-1200, Kinematica) for 1 minute, and then homogenate formed by centrifugation at 12,000 x g at 4 ° C for 5 minutes. Thereafter, the supernatant is collected, and the supernatant collected is taken as one. /. The final concentration of (v/v) was added to physiological saline and uniformly mixed, thereby obtaining a tissue extract sample for use. 3. Statistical analysis In the following examples, the experimental data is expressed as a mean error of the mean (SEM). The difference in experimental data between the groups was first evaluated by one-way ANOVA followed by Scheffe's multiple range test. If the statistical analysis obtained is p < 0.05 represents a statistical significance. Example 1. Ergots-7,9(11), 22 £-tris--3p-alcohol derived from the soaking culture of Antrodia camphorata [ergosta-7,9(ll),22£"-trien The purification of -3p-ol was derived from the ergoside culture of Antrodia camphorata, ergoside-7,9(11), and 22-trien-3-β-ol was referenced to YI-YUAN SHAO ei a/. (2008) , /Voi/wci 16 201221129 22:1151-1 157 the method described for purification. Briefly 'distilled lyophilized powder of 1.6 kg of immersion culture of Antrodia camphorata, soaked in 16 L of methanol at room temperature and allowed to stand for 1 day, then evaporate the methanol under vacuum And get a residue. This soaking-stabilizing-evaporating step was repeated 3 times to obtain a methanol extract of a immersion culture of brown anthraquinone. The sterol extract of the obtained immersion culture of Antrodia camphorata was suspended in 1 L of sterilized water, and then partitioned with water and ethyl acetate (EtOAc) for a total of 3 times. The obtained EtO Ac fraction was chromatographed on a silica gel using a mixed solution containing hexane and ethyl acetate as an eluent, followed by 10% in use. Ethyl acetate (in hexane) was further purified as a mobile phase in high performance liquid chromatography (HPLC). The 1H-NMR and 13C-NMR spectra of the above purified compound were extracted using a Bruker DMX-400 spectrometer using a solvent of CDCI3 and the chemical shift (δ) in ppm. The 1H-NMR and 13C-NMR spectral data of the compound were summarized as follows: NMR (CDCI3, 400 MHz) δ: 0.61 (s, 3 H, CH3), 0.80 (d, J = 6.4 Hz, 3H, CH3 ), 0.B2 (d, J= 6.4Hz, 3H, CH3), 0.89 (d, J= 7.2Hz, 3H, CH3), 0.92 (s, 3H, CH3), 1.01 (d, 7=6.4 Hz, 3H, CH3), 3.61 (m, 1H, CH), 5.14 (dd, 7=15.6, 7.2 Hz, 1H, CH), 5.20 (dd, J=\5.6, 8.0 Hz, 1H, CH), 5.35 (m , 1H, CH), 5.55 (dd, J = 5.6, 2.0 Hz, CH). 17 201221129 13C NMR (CDC13, 100 MHz) δ : 12.0, 16.3, 17.6, 19.6, 20.0, 21.1, 23.0, 28.3, 32.0, 33.1, 37.0, 38.4, 39.1, 40.4, 40.8, 42.8, 46.2, 54.6, 55.7, 70.5, 116.3, 119.6, 132.0, 135.6, 139.8, 141.3. Based on the measured spectral data, the compound was confirmed to be a known compound having the following chemical structural formula [i.e., ergosin-7,9(11),22penta-triene-3β-ol]:

實施例2·麥角甾-7,9(11),22五-三烯-3Ρ-醇的活體内止痛效用（i« vivo analgesia effect)的評估為了瞭解麥角甾-7,9(11)，22五-三烯-30_醇在活體内是否具有抑制疼痛的效用，依據上面實施例1所得到的麥角留_ 7,9(11)，22五-三烯-30-醇被拿來進行下面乙酸-誘發的扭體試驗（acetic acid-induced writhing test)以及福馬林-誘發的腳掌碌0允試驗（formalin-induced paw licking test)。 A、乙酸-誘發的扭體試驗將雄性ICR小鼠隨機分成5組（每組η = 6)，其中包括1 個病理對照組（pathological control group)、1個正對照組 (positive control group)以及3個實驗組（亦即，實驗組i、2 以及3)。病理對照組以及正對照組的小鼠分別經由腹膜内 18 201221129 注射（intraperitoneal injection)被投藥以生理食鹽水以及。引〇朵美洒辛（indomethacin，購自於Sigma)(配於生理食鹽水中，劑量為10 mg/kg)，而實驗組的小鼠經由腹膜内注射被投藥以麥角甾-7,9(11)，22£-三烯-3β-醇[配於羧曱基纖維素 (carboxymethyl cellulose，CMC)中]，其中實驗組 1 至 3 的劑量分別為1、5以及10 mg/kg。在投藥之後的第25分鐘，將 1% (v/v)乙酸溶液（Merck，Darmstadt, Germany)(劑量為 1〇 mL/kg)腹膜内注射至各組的小鼠體内。自乙酸注射起的$ 分鐘内’紀錄小鼠的扭體次數。所得到的實驗結果被顯示於圖1中。從圖1可見，與病理對照組相較之下，實驗組1至3 的扭體次數皆呈現下降的情形，同時會隨著麥角留_ 7,9(11)，22五-三烯-3β-醇劑量的增加而更趨於明顯。這個實驗結果顯示：麥角甾-7,9(11)，22£-三烯_30-醇以一劑量依賴性 (dose-dependent)的方式達到周邊止痛效用（peripherai analgesia effect) ° B、福馬林-誘發的腳掌舔吮試驗本實驗是依照上面A項中所述的方法來進行小鼠的分組以及投藥。在投藥之後的第3〇分鐘，將2Q此的5%福馬林(購自於Bi〇S〇urce International，Inc )注射至各組小鼠的右後腳掌的背側面（dorsal surfaee) ’接著在注射之後的第〇至 5分鐘的期間（亦即早期）以及第15至4〇八王4υ为鐘的期間（亦即晚期）紀錄小鼠㈣右後腳掌所花f的時間。所得到的實驗結果被顯示於圖2中。 19 201221129Example 2: Evaluation of the in vivo analgesic effect of ergoside-7,9(11),22-trien-3-indole-ol (i« vivo analgesia effect) In order to understand ergot -7,9(11) Whether the 22-trien-30-alcohol has a pain-inhibiting effect in vivo, according to the above-mentioned Example 1, the ergot residue _ 7,9 (11), 22 penta-trien-30-ol was taken The following acetic acid-induced writhing test and the formalin-induced paw licking test were performed. A. Acetic acid-induced writhing test Male ICR mice were randomly divided into 5 groups (n=6 per group), including 1 pathological control group, 1 positive control group, and 1 positive control group. Three experimental groups (ie, experimental groups i, 2, and 3). The pathological control group and the mouse of the positive control group were administered with physiological saline as well as intraperitoneal 18 201221129 injection (intraperitoneal injection). Indomethacin (purchased from Sigma) (in a physiological saline solution at a dose of 10 mg/kg), and the mice in the experimental group were administered with ergosin-7,9 via intraperitoneal injection. 11), 22 £-triene-3β-alcohol [in carboxymethyl cellulose (CMC)], wherein the doses of the experimental groups 1 to 3 were 1, 5, and 10 mg/kg, respectively. At the 25th minute after administration, 1% (v/v) acetic acid solution (Merck, Darmstadt, Germany) (dose of 1 〇 mL/kg) was intraperitoneally injected into each group of mice. The number of writhings in the mice was recorded within $ minutes from the injection of acetic acid. The experimental results obtained are shown in Fig. 1. As can be seen from Fig. 1, compared with the pathological control group, the number of writhings in the experimental groups 1 to 3 showed a decrease, and at the same time, the ergot leaves _ 7,9 (11), 22 penta-triene - The increase in the dose of 3β-alcohol is more pronounced. The results of this experiment showed that ergot -7,9(11), 22-trien- 30-alcohol reached the peripheral analgesia effect in a dose-dependent manner (peripherai analgesia effect) ° B, Fuma Lin-induced foot sputum test This experiment was conducted by grouping and administering mice according to the method described in item A above. At the 3rd minute after administration, 2% of this 5% fumarin (purchased from Bi〇S〇urce International, Inc.) was injected into the dorsal surfaee of the right hind paw of each group of mice. The period from the third to the fifth minute after the injection (i.e., early) and the period from the 15th to the fourth week of the eighth to fourth kings (i.e., late) recorded the time spent by the mouse (four) on the right hind paw. The experimental results obtained are shown in Figure 2. 19 201221129

綜合以上乙酸-誘發的扭體試驗以及福馬林_誘發的腳掌舔舐試驗的實驗結果，發明人認為··麥角甾= 烯-3β-醇是透過周邊作用（peripheral acti〇n)而在活體内達到抑制發炎性疼痛的效用。實施例3.麥角留_7,9(11)，22五·三烯_3p_醇的活體内抗發炎Combining the results of the above acetic acid-induced writhing test and the test results of the formalin-induced paw sputum test, the inventors believe that ergoside = ene-3β-alcohol is in a living body through a peripheral action (peripheral acti〇n) The effect of inhibiting inflammatory pain is achieved. Example 3. Anti-inflammatory in vivo of ergoline _7,9(11),22pentatriene_3p-alcohol

為了探討麥角留-7,9(11)，22仏三烯·3β_醇除了在活體内具有止痛效用之外，是否還具有抗發炎效用，依據上面實施例1所得到的麥角留_7,9(11)，22五-三烯，-醇被拿來進行下面的鹿角菜膠-誘發的腳掌水腫試驗（carrageenan_induced paw edema test)。實驗方法： A、鹿角菜膠-誘發的腳掌水腫試驗將雄性ICR小鼠隨機分成6組（每組η = 6)，其中包括J 個正常對照組（normal control group)、1個病理對照組、j個 20 201221129 正對照組以及3個實驗組（亦即’實驗組1、2以及3)。除了病理對照組以外，正常對照組以及正對照組的小鼠分別經由腹膜内注射被投藥以生理食鹽水以及吲哚美洒辛（劑量為 1 〇 mg/kg) ’而實驗組的小鼠經由腹膜内注射被投藥以麥角甾-7,9(11)，22五-三烯-3β-醇（配於CMC中），其中實驗組1至 3的劑罝分別為1、5以及10 mg/kg。在投藥之後的第3 0分鐘’除了正常對照組以外，將5 〇 μί的1/S鹿角菜膠懸浮液（鹿角菜膠是購自於sigma，配於生理食鹽水中）注射至其他各組小鼠的右後腳掌的疏面 (plantar surface)以誘發急性發炎（acute infiammati〇n)，而使仵小执的右後腳掌水腫。至於正常對照組是以生理食鹽水來代替鹿角菜膠懸浮液。分別在鹿角菜膠注射之前以及注射之後的第1、2、3、 4與5小時使用一體積測定儀（piethySm〇rneter)(Cat. No. 7159, Ugo Basile，Varese，Italy)來測量小鼠的右後腳掌的體積。小鼠右後腳掌的水腫體積是以小鼠右後腳掌在鹿角菜膠注射之後所測得的體積減去在鹿角菜膠注射之前所測得者的差值來表示。接著’藉由穿刺（puncture)而從各組小鼠的後大靜脈 (inferior vena cava)採集血液並依照上面“一般實驗方法，，的第1項「一般血清樣品的製備」當中所述的方法來進行一般血清樣品的製備，所得到的一般血清樣品被拿來進行下面B項至C項的分析。另外，將經採血的各組小鼠犧牲後分別取出肝臟以及 21 201221129 右後腳掌，其中肝臟被拿來進行下面D項的分析，而右後腳掌被拿來進行下面E至G項的分析。 B、血清亞琐酸鹽濃度（seruin nitrite concentration)的測定有關各組小鼠的血清一氧化氮濃度是藉由測量血清中的亞硝酸鹽（nitrite)濃度而被間接地決定，並且大體上是參In order to investigate whether ergott-7,9(11),22 quinone-3β-alcohol has an anti-inflammatory effect in addition to its analgesic effect in vivo, the ergot residue obtained according to Example 1 above _ 7,9(11),22-trienol,-ol was taken for the carrageenan_induced paw edema test. Experimental methods: A. Carrageenan-induced paw edema test Male ICR mice were randomly divided into 6 groups (n=6 per group), including J normal control group and 1 pathological control group. j 20 201221129 Positive control group and 3 experimental groups (ie, 'experimental groups 1, 2 and 3). In addition to the pathological control group, mice in the normal control group and the positive control group were administered with intraperitoneal injection of physiological saline and indomethacin (dose of 1 〇mg/kg), respectively. The intraperitoneal injection was administered with ergosta-7,9(11),22pentatriene-3β-ol (in CMC), and the doses of the experimental groups 1 to 3 were 1, 5, and 10 mg, respectively. /kg. At the 30th minute after administration, in addition to the normal control group, 5 〇μί of 1/S carrageenan suspension (carrageenan was purchased from sigma in physiological saline) was injected into other groups. The plantar surface of the right hind paw of the mouse induces acute inflammation (acute infiammati〇n), and the right hind paw of the sputum is edema. As for the normal control group, the sodium carrageenan suspension was replaced by physiological saline. Mice were measured using a volumetric analyzer (Catthy No. 7159, Ugo Basile, Varese, Italy) before and at 1, 2, 3, 4 and 5 hours after carrageenan injection. The volume of the right hind paw. The volume of edema in the right hind paw of the mouse is expressed as the difference between the volume measured by the right hind paw of the mouse after the injection of carrageenan minus the amount measured before the carrageenan injection. Then, 'puncture' was used to collect blood from the inferior vena cava of each group of mice and according to the method described in "General Experimental Methods, Item 1 "Preparation of General Serum Samples" above. To prepare a general serum sample, the resulting general serum samples were taken for analysis of items B to C below. In addition, each group of blood-collected mice was sacrificed and the liver was removed and 21 201221129 right hind paws, wherein the liver was taken for analysis of the following D items, and the right hind paw was taken for analysis of the following E to G items. B. Determination of serum sulphur concentration (seruin nitrite concentration) The serum nitric oxide concentration of each group of mice is determined indirectly by measuring the concentration of nitrite in the serum, and is substantially Reference

照 Recknagel R.O. ei α/. (1991)，//"epaioioxz.co/ogj； CRCPhoto Recknagel R.O. ei α/. (1991), ///quot;epaioioxz.co/ogj; CRC

Press，401-430當中所述的方法來進行。簡言之，取適量的上面A項所得到的一般血清樣品並以蒸餾水予以稀釋5倍 ’接著加入適量的硫酸鋅（zinc sulfate)至一為15 g/L的最終濃度並混合均勻，然後在室溫下以1〇,〇〇〇 xg離心歷時5分鐘。收取上清液，繼而以每井100 的體積將上清液分別加入至一微量培養盤的各井中。接著，對各井加入1〇〇 0 的革利士試劑（Griess reagent)[含有1°/。胺苯璜醯胺 (sulfanilamide)以及〇. 1% N-萘基乙烯二胺二氯化氫（N-naphthylethylenediamine dihydrochloride)，配於 2.5%多麟酸 (polyphosphoric acid)中]並在室溫下進行反應歷時1〇分鐘。之後，以一微量讀取儀（Molecular Devices, Orleans Drive, Sunnyvale，CA)來讀取各井在波長540 nm下的吸光值 (OD540)。將所得到的OD54〇數值分別根據預先以具有不同已知濃度的亞硝酸鈉（NaN02)相對於它們自身的〇D54〇數值所作出的一標準曲線而被換算成亞硝酸鹽濃度（μΜ)。 C、血清TNF-α濃度的測定有關各組小鼠的血清TNF-a濃度測定是使用 BioSourceTM TNF-a ELISA 套組（Cat. No· KMC3011， 22 201221129The method described in Press, 401-430 is carried out. Briefly, take an appropriate amount of the general serum sample obtained in item A above and dilute it 5 times with distilled water. Then add the appropriate amount of zinc sulfate to a final concentration of 15 g/L and mix well, then Centrifuge at 1 Torr, xg for 5 minutes at room temperature. The supernatant was collected, and the supernatant was separately added to each well of a microplate at a volume of 100 per well. Next, 1 〇〇 0 of Griess reagent was added to each well [containing 1 ° /. Sulfonamide and %. 1% N-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid] and react at room temperature for a period of time 1 minute. Thereafter, the absorbance (OD540) of each well at a wavelength of 540 nm was read with a micro reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). The obtained OD54 〇 values were converted into nitrite concentrations (μΜ) according to a standard curve previously prepared with sodium nitrite (NaN02) having different known concentrations relative to their own 〇D54 〇 values. C. Determination of serum TNF-α concentration The serum TNF-a concentration of each group of mice was determined using the BioSourceTM TNF-a ELISA kit (Cat. No. KMC3011, 22 201221129)

Invitrogen)來進行。各組所測得的吸光值分別根據預先以具有不同已知濃度的TNF-α標準品（Camarillo, CA)相對於它們自身的吸光值所作出的一標準曲線而被換算成TNF-α濃度 (pg/mL)。 D、小鼠肝臟組織内的抗氧化酵素活性的測定在本實驗中，發明人選用超氧化物歧化酶（superoxide dismutase, SOD)、過氧化氫酶（catalase, CAT)以及麩胱甘肽過氧化酶（glutathione peroxidase, GPx)作為抗氧化酵素活性的指標，俾以評估麥角甾-7,9(11),22五-三烯-3β-醇在急性發炎時是否能夠藉由提高抗氧化酵素活性來防止活性氧族 (ROS)以及自由基被大量地生成而減輕肝臟的氧化性損傷。首先，將上面Α項所得到的小鼠肝臟依照上面“一般實驗方法”的第2項「組織萃取物樣品的製備」當中所述的方法來進行組織萃取物樣品的製備，所得到的組織萃取物樣品被拿來分別進行下面的酵素活性測定。有關總超氧化物歧化酶（SOD)活性的測定是參照Flohe L. and Otting F. (1984)，105:93-104 當中所述的方法來進行。簡言之，取10 pL組織萃取物樣品並予以加入3 mL Tris-HCl缓衝液（pH 8·2)以及7 μΐ^五倍子酚 (pyrogallol)，在混合均勻後立刻於25t下使用分光光度計 (Beckman DU53 0)以一為1 5秒的時間間隔來讀取在波長325 nm下的吸光值歷時1分鐘。在本測定方法中，一單位的超氧化物歧化酶活性被定義為：五倍子酚的自氧化速率 (autooxidation rate)被降低達50%所需的SOD數量，所得到 23 201221129 的數值是以U/mg蛋白質來表示。有關總過氧化氫酶（CAT)活性的測定是參照Aebi H. (1984)，五π少mo/.，105:121-126當中所述的方法來進行。簡言之，取適量組織萃取物樣品並以50 mM磷酸鉀緩衝液予以稀釋1000倍，然後取2 mL經稀釋的組織萃取物樣品以及1 mL過氧化氫（30 mM)並混合均勻，之後立刻於 25°C下使用分光光度計以一為15秒的時間間隔來讀取在波長240 nm下的吸光值歷時1分鐘。在本測定方法中，一單位的過氧化氫酶活性被定義為：每分鐘所消耗的過氧化氫的奈莫耳數（nmol)，所得到的數值是以U/mg蛋白質來表示〇有關總麩胱甘肽過氧化酶（GPx)活性的測定是參照 Paglia E.D. and Valentine W.N. (1967), J. Lab. Clin. Med., 70:158-169當中所述的方法來進行。簡言之，取200 pL的麵胱甘肽還原酶（glutathione reductase，GR)(5 U/mL)、50 pL 的 GSH (40 mM)、620 pL 磷酸鉀緩衝液（0.25M，pH 7.4)以及 100 pL經磷酸溶液稀釋20倍的組織萃取物樣品並混合均勻。接著，依序加入新鮮配製的10 pL NADPH (20 mM)以及 20 μι 異丙苯過氧化氫（cumene hydroperoxide)(15 mM)，之後立刻於25°C下使用分光光度計以一為15秒的時間間隔來讀取在波長340 nm下的吸光值歷時1分鐘。在本測定方法中，一單位的麵胱甘肽過氧化酶活性被定義為：每分鐘所氧化的NADPH的奈莫耳數（nmol)，所得到的數值是以 U/mg蛋白質來表示。 24 201221129 E ' 小鼠腳掌組織内的丙二搭（malondialdehyde，MDA)濃度的測定丙二醒 (malondialdehyde，MDA)是多不飽和脂肪酸氧化的主要產物，丙二醛濃度可被用來評估氧化性損傷 (oxidative damage)的程度。有關丙二醛濃度的測定是參照 Tatum V.L. and Chow C.K. (1996), Free Radical Research, 25:133-139當中所述的方法來進行。簡言之，將各組由上面 A項所得到的其中1隻腳掌依照上面“一般實驗方法”的第2 項「組織萃取物樣品的製備」來進行組織萃取物樣品的製備，接著，取0.4 mL組織萃取物樣品，予以加入0.4 mL硫巴比妥酸試劑（thioarbituric acid reagent，TBA reagent)[含有 0.4%硫巴比妥酸以及 0.2% 丁基化經基甲苯（butylated hydroxytoluene, BHT)]並混合均勻，繼而置於90°C水浴中進行反應歷時45分鐘。待反應物冷卻至室溫後，予以加入等體積的η- 丁醇（n-butanol)並混合均勻，所形成的混合物以 3，000 i*pm離心歷時10分鐘。之後，收取一部分上清液，並使用BCA蛋白質分析套組（BCA Protein Assay Kit，Pierce)來測定上清液的蛋白質含量（protein content)，而其餘的上清液是使用以一 VERSAmax微量培養盤讀取儀（VERSAmax microplate reader, Molecular Devices)在波長 535 nm 下測量吸光值（OD535)。另一方面，具有不同已知濃度的1,1，3,3-四乙氧基丙烷 (1,1,3,3-tetraethoxypropane，TEP)溶液被拿來進行相同的實驗，並依據濃度及其相對應的吸光值（〇D535)而做出一標準 25 201221129 曲線（standard curve)。各組所測得的吸光值（OD535)是藉由該標準曲線而被換算成丙二醛濃度（nmol)，再除以對應的蛋白質含量，藉此而計算出每mg蛋白質所含有的丙二醛濃度 (nmol/mg 蛋白質）。 F、小鼠腳掌組織内的誘導性一氧化氮合成酶（inducible nitric oxide synthase, iNOS)以及環加氧酶-2 (eye丨ooxygenase-2，COX-2)表現量的西方墨點分析 (Western Blotting) 各組由上面A項所得到的1隻腳掌大體上是依照上面“ 一般實驗方法’’的第2項「組織萃取物樣品的製備」來進行組織萃取物樣品的製備，不同之處在於：使用一含有10 mM的3-[(3-膽醯胺丙基）二曱基銨基]-1-丙烷-磺酸酯{3-[(3-cholamidopropyl)dimethylammonio]-l-propane-sulfonate}、1 mM 苯甲基續醯氟（phenylmethylsulfonyl fluoride)、5 pg/mL 抑肽酶（aprotinin)、1 μΜ抑肽素（pepstatin)以及10 μΜ亮肽酶（leupeptin)的溶解緩衝液（lysis buffer)來代替生理食鹽水〇Invitrogen). The absorbance values measured for each group were converted to TNF-α concentrations according to a standard curve previously prepared with respect to their own absorbance values of TNF-α standards (Camarillo, CA) having different known concentrations. Pg/mL). D. Determination of Antioxidant Enzyme Activity in Mouse Liver Tissue In this experiment, the inventors selected superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidation. The enzyme (glutathione peroxidase, GPx) is used as an indicator of antioxidant enzyme activity to evaluate whether ergot 甾-7,9(11),22-trien-3-β-ol can increase antioxidant enzymes in acute inflammation. The activity prevents the active oxygen species (ROS) and free radicals from being formed in a large amount to reduce the oxidative damage of the liver. First, the liver of the mouse obtained in the above item is subjected to the preparation of the tissue extract sample according to the method described in the second item "Preparation of the tissue extract sample" of the "General Experimental Method" above, and the obtained tissue extract is obtained. The samples were taken for the following enzyme activity assays. The determination of total superoxide dismutase (SOD) activity is carried out by the method described in Flohe L. and Otting F. (1984), 105: 93-104. Briefly, 10 pL of tissue extract sample was taken and added to 3 mL of Tris-HCl buffer (pH 8·2) and 7 μL of pyrogallol, and the spectrophotometer was used at 25t immediately after mixing. Beckman DU53 0) The absorbance at a wavelength of 325 nm was read at a time interval of 1 5 seconds for 1 minute. In the present assay, one unit of superoxide dismutase activity is defined as the amount of SOD required to reduce the autooxidation rate of gallic phenol by 50%, and the value of 23 201221129 is U/ The mg protein is used to indicate. The measurement of total catalase (CAT) activity was carried out in accordance with the method described in Aebi H. (1984), five π less mo/., 105: 121-126. Briefly, take an appropriate amount of tissue extract sample and dilute it 1000 times with 50 mM potassium phosphate buffer, then take 2 mL of the diluted tissue extract sample and 1 mL of hydrogen peroxide (30 mM) and mix well, immediately after The absorbance at a wavelength of 240 nm was read at 25 ° C for 1 minute using a spectrophotometer at a time interval of 15 seconds. In the present assay, one unit of catalase activity is defined as the number of nanomoles (nmol) of hydrogen peroxide consumed per minute, and the values obtained are expressed in terms of U/mg protein. The measurement of glutathione peroxidase (GPx) activity was carried out by the method described in Paglia ED and Valentine WN (1967), J. Lab. Clin. Med., 70: 158-169. Briefly, 200 pL of glutathione reductase (GR) (5 U/mL), 50 pL of GSH (40 mM), 620 pL of potassium phosphate buffer (0.25 M, pH 7.4) and 100 pL of tissue extract sample diluted 20 times with phosphoric acid solution and mixed well. Next, freshly prepared 10 pL NADPH (20 mM) and 20 μM cumene hydroperoxide (15 mM) were added sequentially, followed by a spectrophotometer at 25 ° C for 15 seconds. The time interval was taken to read the absorbance at 340 nm for 1 minute. In the present assay, one unit of glutathione peroxidase activity is defined as the number of nanomolecules (nmol) of NADPH oxidized per minute, and the values obtained are expressed in U/mg protein. 24 201221129 E 'Determination of malondialdehyde (MDA) concentration in mouse foot tissue Malondialdehyde (MDA) is the main product of oxidation of polyunsaturated fatty acids, and malondialdehyde concentration can be used to evaluate oxidative properties. The extent of oxidative damage. The determination of the concentration of malondialdehyde was carried out in accordance with the method described in Tatum V.L. and Chow C.K. (1996), Free Radical Research, 25: 133-139. Briefly, each group of one of the soles obtained from item A above was prepared according to the second item "Preparation of tissue extract samples" in the "General Experimental Method" above, and then 0.4 was taken. mL tissue extract sample was added with 0.4 mL thioarbituric acid reagent (TBA reagent) [containing 0.4% thiobarbituric acid and 0.2% butylated hydroxytoluene (BHT)] The mixture was homogeneously mixed and then placed in a 90 ° C water bath for 45 minutes. After the reaction was cooled to room temperature, an equal volume of n-butanol was added and mixed well, and the resulting mixture was centrifuged at 3,000 i*pm for 10 minutes. Thereafter, a portion of the supernatant was collected and the BCA Protein Assay Kit (Pierce) was used to determine the protein content of the supernatant, while the remaining supernatant was used as a VERSAmax microplate. The reader (VERSAmax microplate reader, Molecular Devices) measured the absorbance (OD535) at a wavelength of 535 nm. On the other hand, solutions of 1,1,3,3-tetraethoxypropane (TEP) having different known concentrations were taken for the same experiment and based on the concentration and A standard 25 201221129 curve is made for the corresponding absorbance (〇D535). The absorbance (OD535) measured by each group is converted to the malondialdehyde concentration (nmol) by the standard curve, and divided by the corresponding protein content, thereby calculating the amount of C 2 per mg of protein. Aldehyde concentration (nmol/mg protein). Western blot analysis of F, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the foot tissue of mice (Western blot) Blotting) Each group of the soles obtained from item A above is generally prepared according to the second item "Preparation of tissue extract samples" in the "General Experimental Method" above, except that the sample is prepared. : using a 3-[(3-cholestyryl)diamidinosyl]-1-propane-sulfonate containing 10 mM {3-[(3-cholamidopropyl)dimethylammonio]-l-propane-sulfonate }, 1 mM phenylmethylsulfonyl fluoride, 5 pg/mL aprotinin, 1 μΜpeptin (peptatin), and 10 μl of leupeptin lysis buffer (lysis buffer) ) instead of physiological saline 〇

各組的組織萃取物樣品是使用迷你垂直式電泳槽（Bio-1^(1，1；8八）來進行308-?八0丑分析，繼而使用小鼠抗-丨1^08 單株抗體和小鼠抗-COX-2單株抗體（均是購自於Santa Cruz, CA)作為一次抗體（primary antibody)(以5%脫脂乳予以稀釋 500倍），以及綴合有辣根過氧化氫酶的山羊抗小鼠IgG抗體（購自於Sigma)作為二次抗體（secondary antibody)(以5% 脫脂乳予以稀釋2,000倍），並且使用Hyperfilm™以及ECL 26 201221129 試劑（皆是購自於Amersham International pic.)來進行西方墨點分析。最後，使用一凝膠成像系統（gel logic 1500, Kodak) 來進行照相。另外，以β-肌動蛋白（β-actin)作為内部對照組（internal control)並且使用抗 β-肌動蛋白抗體（anti β-actin antibody)(sc-47778)(Santa Cruz, CA)作為一次抗體（以 5%脫脂乳予以稀釋2000倍）以及綴合有辣根過氧化氫酶的山羊抗小鼠IgG抗體作為二次抗體（secondary antibody)(以5%脫脂乳予以稀釋1000倍）來進行相同的實驗。所得到的影像是使用柯達分子成像軟體（Kodak Molecular Imaging Software，版本 4.0.5)(Eastman Kodak Company，Rochester, NY)來進行分析，藉此蛋白質帶 (protein band)能夠被半-定量地計算出所對應的蛋白質表現量，蛋白質表現量接而以其所對應的β-肌動蛋白蛋白質表現量來予以標準化（normalized)，然後將病理對照組之經標準化的蛋白質表現量當作100%，藉此而計算出其他各組相對於病理對照組的蛋白質表現量百分比（％病理對照組）。 G、組織病理學檢驗（histopathological examination) 在室溫下將各組的其餘腳掌浸泡於一固定溶液[含有 1.85%甲酿（formaldehyde)以及1%乙酸]中，並予以固定歷時 1週。經固定的小鼠腳掌被進行乙醇脫水處理，接著以石蠟 (paraffin)(Sherwood Medical)予以包埋，並且進行切片而得到厚度為5 μιη的切片。經脫躐的切片是以蘇木精-伊紅 (hematoxylin-eosin)來進行染色，然後使用一光學顯微鏡 27 201221129 (ECLIPSE，TS100, Nikon)在⑽χ的放大倍率下來進行型態觀察並使用一數位相機__Elements D2 3〇,咖，Bu二 387)來拍照。，，m 另外，在一為400X的放大倍率下來進行嗜中性球的計數。有關嗜令性球的計數是由各組隨機地選出3_5個切片每個切片任選5個視野來進行。實驗數據是明有視野所計數出的嗜中性球數目的平均值來表示。結果： A、小鼠水腫腳掌的體積變化圖3顯示在鹿角菜膠-誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9〇 ^22五三烯_ 3β-醇，在鹿角菜膠注射之前（亦即，第〇小時）以及注射之後的第卜2、3、4與5小時制—體積測定儀來測量小鼠的右後腳掌的水腫體積變化。從圖3可見，與病理對照組相較之下，在鹿角菜膠注射之後的第1、2以及3小時，實驗組i至3的腳掌水腫體積沒有顯著的差異，然而在鹿角菜膠注射之後的第4以及5 小時’實驗組2以及3的腳掌水腫體積皆呈現下降的情形 ’同時會隨著麥角f7,9⑴)，挪三稀_3卜醇劑量的增加而更趨於明顯。這個實驗結果顯* :麥角^7,9(11)，22£-三稀_ 3β-醇具有改善腳掌水腫現象的效用。 Β、血清亞硝酸鹽濃度的測定圖4顯示在鹿角菜膠誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9⑴m五-三稀_ 28 201221129 3 β-醇’在鹿角菜膠注射之後的第傻扪弟5小時所測得的血清亞石肖酸鹽濃度。月從圖4可見，與病理對照組相較之下，實驗組】至3 的血清亞《鹽濃度皆呈現下降的情形，科會隨著麥角错-哪⑽厂三稀·3β_醇劑量的增加而更趨於明顯。特別地，實驗、组3 %血清亞靖酸鹽濃度幾乎相同於正對照組所具者。這個實驗結果顯示：麥肖留·7,9⑴），22五三稀勢醇以一劑量_依賴性的方式來抑制一氧化氮的生成。 C、血清TNF-α濃度的測定圖5顯示在鹿角菜膠-誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9(1丨），22五三烯— 3Ρ-醇，在鹿角菜膠注射之後的第5小時所測得的血清tnf· a濃度。從圖5可見，與病理對照組相較之下，實驗組2以及3 的血清TNF-a濃度明顯地被降低。這個實驗結果顯示：麥角甾-7,9(11)，22£-三烯-3β-醇具有降低血清TNF_a濃度的效用。 D、小鼠肝藏組織内的抗氧化酵素活性的測定有關小鼠肝臟組織内的超氧化物歧化酶、過氡化氮酶以及麵胱甘肽過氧化啤的活性測定結果被歸納於下面的表^ 中。 29 201221129 表1. f角，_7,9(11)，22£_三烯_3卜醇在鹿角菜膠_誘發的腳掌水腫試f ^對於小鼠肝臟内的超氧化物歧化酶（CAT)、過氧^氫酶（SOD)以及麩胱甘肽過氧化酶（GPx)活性的Tissue extract samples from each group were subjected to 308-?8 ugly analysis using a mini vertical electrophoresis tank (Bio-1^(1,1;8-8), followed by mouse anti-丨1^08 monoclonal antibody And mouse anti-COX-2 monoclonal antibody (both purchased from Santa Cruz, CA) as a primary antibody (diluted 500-fold with 5% skim milk), and conjugated with horseradish hydrogen peroxide Enzyme goat anti-mouse IgG antibody (purchased from Sigma) as a secondary antibody (diluted 2,000 times with 5% skim milk) and using HyperfilmTM and ECL 26 201221129 reagents (all purchased from Amersham) International pic.) for Western blot analysis. Finally, a gel imaging system (gel logic 1500, Kodak) was used for photography. In addition, β-actin (beta-actin) was used as an internal control (internal control). And using an anti-beta-actin antibody (sc-47778) (Santa Cruz, CA) as a primary antibody (diluted 2000 times with 5% skim milk) and conjugated with horseradish peroxidation Hydrogenase goat anti-mouse IgG antibody as secondary antibody (secondary a The same experiment was performed with ntibody) (1000 times diluted with 5% skim milk). The resulting image was obtained using Kodak Molecular Imaging Software (version 4.0.5) (Eastman Kodak Company, Rochester, NY). The analysis is performed so that the protein band can be semi-quantitatively calculated for the corresponding protein expression, and the protein expression is normalized by the corresponding β-actin protein expression. Then, the normalized protein expression of the pathological control group was taken as 100%, thereby calculating the percentage of protein expression of the other groups relative to the pathological control group (% pathological control group) G, histopathological examination (histopathological test) Examination) The remaining soles of each group were immersed in a fixing solution [containing 1.85% of formaldehyde and 1% acetic acid] at room temperature and fixed for 1 week. The fixed paws of the mice were subjected to ethanol dehydration. The treatment was followed by embedding with paraffin (Sherwood Medical) and sectioned to obtain a section having a thickness of 5 μm . The dislocated sections were stained with hematoxylin-eosin and then subjected to pattern observation using a light microscope 27 201221129 (ECLIPSE, TS100, Nikon) at a magnification of (10) 并 and using a digit. Camera __Elements D2 3 〇, coffee, Bu II 387) to take pictures. , ,m In addition, the neutrophil count is performed at a magnification of 400X. The count of the eugenic ball is randomly selected from each group of 3 to 5 slices, and each slice is selected to have 5 fields of view. The experimental data is expressed as the average of the number of neutrophils counted in the field of view. Results: A. Volume change of mouse edema soles Figure 3 shows that different doses of ergots were administered to mice by intraperitoneal injection before the carrageenan-induced paw edema test. _7,9〇^22 pentadiene _ 3β-alcohol, measuring the edema volume change of the right hind paw of mice before the carrageenan injection (ie, the third hour) and the second, third, fourth and fifth hour volumetric instruments after the injection . As can be seen from Fig. 3, there was no significant difference in the volume of edema of the soles of the experimental groups i to 3 at 1, 2, and 3 hours after the injection of carrageenan, but after the carrageenan injection. In the 4th and 5th hour of the experiment, the volume of the edema of the feet of the experimental groups 2 and 3 showed a decrease, and it became more obvious with the increase of the dose of ergot f7, 9 (1) and the trichome. The results of this experiment show that * ergot ^7,9 (11), 22 £-tris- 3β-alcohol has the effect of improving the edema of the sole of the foot. Determination of sputum and serum nitrite concentration Figure 4 shows that different doses of ergot are administered to mice by intraperitoneal injection prior to the carrageenan-induced paw edema test. _7,9(1)m5-three-dilution_ 28 201221129 3 β -Alcohol' serum sulphate concentration measured 5 hours after the carrageenan injection. The month can be seen from Figure 4, compared with the pathological control group, the experimental group] to 3 serum sub-"Salt concentration showed a decline, the department will follow the ergot wrong - which (10) plant three rare 3β-alcohol dose The increase is more obvious. In particular, the experimental, group 3% serum yttrium acid salt concentration was almost the same as that of the positive control group. The results of this experiment show that: Maishawu·7,9(1)), 22,5,3-divalent alcohol inhibits nitric oxide production in a dose-dependent manner. C. Determination of serum TNF-α concentration Figure 5 shows that mice were administered different doses of ergots via intraperitoneal injection before the carrageenan-induced paw edema test. _7,9(1丨),22五三Alkene-3-ol, serum tnf·a concentration measured at 5 hours after carrageenan injection. As can be seen from Fig. 5, the serum TNF-a concentrations of the experimental groups 2 and 3 were significantly lowered as compared with the pathological control group. The results of this experiment show that ergoside-7,9(11), 22-trien-3-β-alcohol has the effect of lowering the serum TNF_a concentration. D. Determination of the activity of antioxidant enzymes in the liver tissue of mice The results of the activity determination of superoxide dismutase, peroxidase and glutathione in the liver tissue of mice are summarized in the following. Table ^. 29 201221129 Table 1. f-angle, _7, 9 (11), 22 £ _ triene _ 3 phenol in carrageenan _ induced paw edema test f ^ for superoxide dismutase (CAT) in mouse liver , peroxylase (SOD) and glutathione peroxidase (GPx) activity

實驗組 3.81+0.06° 4.03+0.Q86 1-54+0.11 19.88+0.49 1.04+0.04 20,45i0.45c 1.27+0,14 ~ Λ -—-__·〜二wu 21.97±0.98ά 1 62+0 β < 0·01 ’與病理對照組相比較。 Ρ < 0.05，與病理對照組相比較。 GP^T X_U/mg蛋白皙、 2.18+0.16 0.48+0.08- 從表1可見，與正常對照組相較之下，病理對照組的超氧化物歧化酶、過氧化氫酶以及麵胱甘肽過氧化酶活性皆被降低。而與病理對照組相較之下，正對照組以及實驗組3小鼠的超氧化物歧化酶、過氧化氣酶以及麵耽甘狀過氧化酶活性均明顯地被提高W0.0D。這個實驗結果顯示 :麥角留-7,9(1 D，22【三稀_邛·醇具有提高小鼠肝臟内的超氧化物歧化酶、過氧化氫酶以及麵胱甘肽過氧化酶活性的效用。 Ε '小鼠腳掌組織内的丙二醛濃度的測定圖6顯示在鹿角菜膠-誘發的腳掌纟腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角甾-7,9(11) 22Ε =烯邛_醇’在鹿角菜膠注射之後的第5小時收取右後腳掌：織所測得的丙二醛濃度》從圖6可見，與病理對照組相較之下，實驗缸【至3 30 201221129 的丙一醛濃度皆呈現下降的情形，同時會隨著麥角甾_ 7,9(11)，22£-三烯_3卜醇劑量的增加而更趨於明顯。特別地，實驗、.且3的丙二醛濃度比正對照組所具者還低。這個實驗結果顯示：麥角甾-7,9(11)，22£_三烯_3p醇以一劑量·依賴性的方式來減輕水腫腳掌組織處的氧化性損傷。 F '小鼠腳掌組織内的iN〇S以及COX-2表現量的西方墨點分析圖7顯示在鹿角菜膠_誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予麥角留_7,9(11)，22五-三烯_3β_醇，在鹿角菜膠注射之後的第5小時收取右後腳掌組織進行西方墨點分析所測得的iNOS以及COX-2表現量結果。從圖7可見，與病理對照組（將病理對照組的蛋白質表現量當作100%)相較之下，正對組以及實驗組3小鼠的 _S以及C0X-2表現量皆明顯地呈現下降的情形，特別地 ’實驗組3的iNOS以及C0X_2表現量分別比正對照組所具者還低。這個實驗結果顯示：麥角甾—7,90 ^,22五三烯· 3β-醇具有降低水腫腳掌組織之iN〇s以及c〇x_2表現量的效用。 G、組織病理學檢驗圖8是一切片染色圖，其顯示在鹿角菜膠_誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投藥麥角留_ 7,9(11)，22五-三烯-3β-醇，並且在鹿角菜膠注射之後的第$小時所取得之右後腳掌切片的蘇木精伊紅染色結果。圖9是根據圖8的染色結果任選5個視野進行嗜中性球計數而得 31 201221129 到的結果。從圖8可見，與正常對照組（A區）相較之下，病理對照組（B區）的切片顯示帶有中等程度的脈管外紅血球細胞 (extravascular red blood cells)以及大量嗜中性球浸潤的出血 (hemorrhage)。與病理對照組相較之下，正對照組（c區）以及貫驗組3 (D區）的切片顯示在發炎反應上的降低。從圖9 可見’在正對照組以及實驗組3小鼠中，每個視野的平均嗜中性球數目皆明顯地被降低至4〇以下。特別地，在實驗組3 t ’每個視野的平均嗜中性球數目要比正對照組所具者還低。這個實驗結果顯示：麥角甾·7,9(11)，22五_三烯_3(3_ 醇具有降低嗜中性球浸潤以及減輕發炎的效用。綜合以上的實驗結果，發明人認為：麥角甾_ 7’9(11)，22五-二烯-3β-醇的抗發炎效用可能是經由提高肝臟内的超氧化物歧化酶、過氧化氫酶以及麩胱甘肽過氧化酶活性而透過抑制TNF-α與一氧化氮的表現來降低水腫腳掌組織中的丙二醛、iNOS以及C0X-2表現量。另外’發明人注意到，麥角甾_7,9(11)，22五-三烯-30-醇在鹿角菜膠-誘發的水腫腳掌試驗中可以提高超氧化物歧化酶、過氧化氫酶以及麩胱甘肽過氧化酶這3種抗氧化酵素的活性。發明人據此而認為：麥角甾_7,9(11)，22五_三烯_3卜醇具有改善肝損傷的效用，在製備供用於治療肝損傷的醫藥品以及保護肝臟之組成物上應有極大的潛力。因此，在下面的實施例中，發明人選用帶有以四氣化碳-誘發的化學陳肝損傷（CCl4-in(luced chemical liver injury)的小鼠作為動 32 201221129 物模型來命估麥角留22£-三烯_3p_醇在治療肝損傷以及保護肝臟上的效用。實施例4.麥角甾_7,9(11)，22五-三烯-3p_醇供用於治療化學性肝損傷的評估 A、以CCh誘發化學性肝損傷將雄f生ICR小鼠隨機分成6組（每組n = 6)，其中包括1 個正吊對照組、1個病理對照組、1個正對照組以及3個實驗組（亦即，實驗組卜2以及3)。正常對照組以及病理對照 .且的小鼠經由腹膜内注射被投藥以生理食鹽水（劑量為五5 mL/kg'，而正對照組以及實驗組的小鼠分別經由腹膜内注射被技藥以水飛莉（siIymarinX配於1% CMC _，劑量為25 g g)以及麥角留-7,9(11)，22五-三烯_3β-醇（配於i〇/0 CMC中 ’劑®分別為2·5、5與10 mg/kg)，每天注射1次，連續注射7天。在最後1次注射之後的第i小時，除了正常對照組以外’將配於橄欖油中的聽ccl4〇 5 mL/kg)以腹膜内注射的方式施打至其他各組的小鼠體内’俾以誘發化學性肝損傷。至於正常對照組是以生理食鹽水來代替如4。 y在C14 '主射之後的第24小時以乙醚（ethyl ether)將小鼠麻醉，蕤由空蜡由穿刺而從小鼠的頸動脈（carotid arteHes)採集企液。其中_部分的血液在代下以I·碎離心歷時3G分鐘，所得到的血清樣品被拿來進行下面b項的分析’而其餘的血液是依照上面“一般實驗方法，，的當中所述的方法來 33 201221129 品被拿來進行下面C項樣品的製備，所得到的一般血清樣至D項的分析。另外，將經採血的各組小鼠犧牲後取出肝臟的肝臟被分成兩個部分，直中_ 斤侍到Experimental group 3.81+0.06° 4.03+0.Q86 1-54+0.11 19.88+0.49 1.04+0.04 20,45i0.45c 1.27+0,14 ~ Λ -——-__·~2 wu 21.97±0.98ά 1 62+0 β < 0·01 ' is compared with the pathological control group. Ρ < 0.05, compared with the pathological control group. GP^T X_U/mg peptone, 2.18+0.16 0.48+0.08- It can be seen from Table 1 that the superoxide dismutase, catalase and glutathione of the pathological control group were compared with the normal control group. Oxidase activity is reduced. Compared with the pathological control group, the activities of superoxide dismutase, peroxidase and oxidative peroxidase in the positive control group and the experimental group 3 were significantly increased by W0.0D. The results of this experiment showed that ergot leaves -7,9 (1 D, 22 [three dilute 邛醇 alcohol has increased superoxide dismutase, catalase and caspase peroxidase activity in the liver of mice)效 'Determination of Malondialdehyde Concentration in Mouse Foot Tissues Figure 6 shows that mice were dosed with different doses of ergosta-7 via intraperitoneal injection prior to the carrageenan-induced paw edema test. 9(11) 22Ε = enoquinol-alcohol's charge of the right hind paw at the 5th hour after carrageenan injection: the concentration of malondialdehyde measured by weaving" can be seen from Figure 6, compared with the pathological control group. The concentration of propionaldehyde in the experimental tank [to 3 30 201221129 showed a decrease, and it became more obvious with the increase of the dose of ergoside _ 7,9 (11), 22 £-triene _3 phenol. In particular, the concentration of malondialdehyde in the experiment, and 3 was lower than that in the positive control group. The results of this experiment showed that: ergoside-7,9(11), 22£_triene_3p alcohol Dose-dependent manner to reduce oxidative damage in the edema and foot tissue. Western blotting of iN〇S and COX-2 in the foot tissue of F 'mouse Figure 7 shows that mice were administered ergoside _7,9(11),22 penta-triene_3β-ol via intraperitoneal injection prior to the carrageenan-induced paw edema test, after carrageenan injection At the 5th hour, the results of iNOS and COX-2 measured by Western blot analysis were collected from the right hind paw tissue. It can be seen from Fig. 7 that the pathological control group (the protein expression of the pathological control group is regarded as 100%) In contrast, both the _S and C0X-2 expressions of the positive and the experimental group 3 mice showed a significant decrease, especially the 'iNOS and C0X_2 expressions of the experimental group 3 were higher than those of the positive control group, respectively. The results of this experiment show that: ergoside-7,90^,22,5-triene-3β-alcohol has the effect of reducing the expression of iN〇s and c〇x_2 in edema of the foot of the foot. G, histopathological examination Figure 8 is a section staining diagram showing administration of ergoline _ 7,9(11), 22 penta-triene-3β-ol to mice via intraperitoneal injection prior to the carrageenan-induced paw edema test, And hematoxylin and eosin staining of the right hind paw slice taken at the first hour after the carrageenan injection Results Fig. 9 is the result of neutrophil count according to the staining result of Fig. 8 and the neutrophil count of 31 201221129. It can be seen from Fig. 8 that compared with the normal control group (area A), the pathological control is compared. Sections of group (B area) showed moderate extravascular red blood cells and a large amount of hemorrhage in the neutrophil infiltration. Compared with the pathological control group, the positive control group ( Sections of zone c) and group 3 (zone D) showed a decrease in inflammatory response. It can be seen from Fig. 9 that in the positive control group and the experimental group 3 mice, the average number of neutrophils per field of view was significantly reduced to less than 4 inches. In particular, the average number of neutrophils per field of view in the experimental group 3 t ' was lower than that of the positive control group. The results of this experiment show that: ergot 7·7,9(11), 22 pentatriene _3 (3_ alcohol has the effect of reducing neutrophil infiltration and reducing inflammation. Based on the above experimental results, the inventor believes that: The anti-inflammatory effect of keratin _ 7'9(11), 22-dien-3β-alcohol may be through the enhancement of superoxide dismutase, catalase and glutathione peroxidase activity in the liver. By inhibiting the expression of TNF-α and nitric oxide, the amount of malondialdehyde, iNOS and COX-2 in the edema of the plantar tissue was reduced. In addition, the inventor noticed that ergot _7,9(11),22 -Triene-30-ol can increase the activity of three antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, in the carrageenan-induced edema foot test. Therefore, it is believed that ergot 甾7,9(11),22penta-triene-3-butanol has the effect of improving liver damage, and should be prepared for the preparation of a drug for treating liver damage and protecting the liver. Great potential. Therefore, in the following examples, the inventors chose to carry a chemical with a carbonized gas induced by four gasifications. Injury (CCl4-in (luced chemical liver injury) mice as a model of the activity 32 201221129 to estimate the utility of ergot 22-triene _3p-alcohol in the treatment of liver damage and protection of the liver. Ergocarp 甾7,9(11),22penta-triene-3p-alcohol for the evaluation of chemical liver injury A. CH-induced chemical liver injury Randomized ICR mice were randomly divided into 6 groups ( n = 6) in each group, including 1 positive control group, 1 pathological control group, 1 positive control group, and 3 experimental groups (ie, experimental group 2 and 3). Normal control group and pathological control And the mice were administered with physiological saline (in a dose of five 5 mL/kg' via intraperitoneal injection, while the mice in the positive control group and the experimental group were intravenously injected with sicurone (siIymarinX). At 1% CMC _, the dose is 25 gg) and ergot -7,9 (11), 22 penta-triene _3β-alcohol (in the i〇 / 0 CMC 'agent ® respectively, 2. 5, 5 and 10 mg/kg), once a day, for 7 consecutive days. At the ith hour after the last injection, in addition to the normal control group, 'will be used in olive oil to listen to ccl4〇5 mL/kg) was administered intraperitoneally to mice in other groups to induce chemical liver injury. As for the normal control group, physiological saline was used instead of 4. For example, y is in C14 The mice were anesthetized with ethyl ether at the 24th hour afterwards, and the human body was collected from the carotid arteries (carotid arteHes) of the mice by puncture by empty wax. The blood of the _ part was centrifuged by I. At 3G minutes, the obtained serum sample was taken for the analysis of item b below' while the rest of the blood was taken according to the method described in the above "General Experimental Methods, 33 201221129" for the following sample C. Preparation, analysis of the resulting general serum samples to item D. In addition, the liver of each group of mice that have been sacrificed after sacrifice is divided into two parts, and the patient is directly

至^ ^分肝臟被拿來進行下面E 分析。刀析’而另—部分的肝臟被拿來進行下面r項的 B、血清丙胺酸轉胺酶⑷anine amin〇transferase，及天門冬胺酸轉胺酶（aspartate amin〇transfe⑽， AST)活性的測定 ’ 有關上面A項中所得到的血清樣品的丙胺酸轉胺酶以及天門冬胺酸轉胺酶活性測定是委由億立健醫事檢驗 (Easy-Check Clinical Laboratory)來代為進行。所得到的實驗結果被顯示於圖1 〇至1 1中。從圊10以及圖11可見，與正常對照組相較之下，病理對照組的血清丙胺酸轉胺酶以及天門冬胺酸轉胺酶活性在 CCU注射之後明顯地被升高。而與病理對照組相較之下，正對照組以及實驗組1至3的血清丙胺酸轉胺酶以及天門冬胺酸轉胺酶活性皆明顯地被降低，特別地，實驗組丨至3 會隨著麥角留-7,9(11 )，22£-三稀-3β·醇劑量的增加而更趨於明顯。這個實驗結杲顯示：麥角甾·7,9(11)，22五-三烯_3卜醇以一劑量-依賴性的方式來防止血清丙胺酸轉胺酶以及天門冬胺酸轉胺酶活性的升高。 C、血清亞硝酸鹽濃度的測定本實驗是依照上面實施例3的「Β、血清亞;ε肖酸鹽濃度 34 201221129 的測定I營φ從、+，u、的方法來進行血清亞硝酸鹽濃度的測定所知到的實驗結果被顯示於圖12中。了見，與正常對照組相較之下，病理對照組的血”鹽濃度被明顯地升高。而與病理對照組相較之 • '、丨至3的血清亞硝酸鹽濃度皆呈現下降的情形 ’同時會隨著麥角留_7,9⑴)，22五三烯，-醇劑量的增加而趨U肖別地，實驗組3的血清亞石肖酸鹽濃度比正對照組所具者還低。這個實驗結果顯示：麥角留· 7’9(11)，22五-二烯·3β_醇以一劑量_依賴性的方式來抑制一氧化氮的生成。 D、血清TNF-ct濃度的測定〜本實驗是依照上面實施例3的「c、血清TNF_a濃度的 :定」當中所述的方法來進行血清TNF_a濃度的測定。所得到的實驗結果被顯示於圖13中。從圖13可見，與正常對照組相較之下，病理對照組的血π TNF-a濃度被明顯地升高。而與病理對照組相較之下，實驗組1至3的血清TNF_a濃度皆呈現下降的情形，同時會隨著麥角留_7,9(1仏⑽·三婦_3β_醇劑量的增加而更趨於明顯。特別地’實驗組3的血清TNF_a濃度比正對照組所具者還低。這個實驗結果顯示：麥角崔_7,9(11)，22五-三烯_ 3β-醇以—劑量-依賴性的方式來降低血清TNF-a濃度。 E、小鼠肝臟組織内的抗氧化酵素活性的測定本實驗是依照上面實施例3的「D、小鼠肝臟組織内的抗氧化酵素活性的測定」當中所述的方法來進行超氧化物 35 201221129 每過氧化氫酶以及鼓脱甘狀過氧化酶的活性測定。所得到的實驗結果被顯示於圖14至16中。從圖14至ι6可見，在ecu注射之後，與正常照相齡夕 ΠΓ 一、、、您下，病理對照組的超氧化物歧化酶、過氧化氫酶以及麵耽甘肽過氧化酶活性皆被明顯地降低。而與病理對照組相較之下，實驗組1 1 3的超氧化物歧化酶、過氧化氫酶以及麵胱甘肽過氧化酶活性皆明顯地被提高，同時會隨著麥角甾-7,9(11)，22五-三烯-3戸-醇劑量的增加而更趨於明顯。特別地，實驗組3的超氧化物歧化酶、過氧化氫酶以及麩胱甘肽過氧化酶活性皆分別比正對照組所具者還高。這個實驗結果顯示：麥角留_7,9(11)，22五_三烯·3p•醇具有提高超氧化物歧化酶、過氧化氫酶以及麵胱甘肽過氧化酶活性的效用。 F、小鼠肝職組織内的丙二搭濃度的測定本實驗是依照上面實施例3的「E、小鼠腳掌組織内的丙一醛濃度的測定」當中所述的方法來進行丙二醛濃度的測定。所得到的實驗結果被顯示於圖17中。從圊17可見，與病理對照組相較之下，實驗組丨至3 的丙二醛濃度皆呈現下降的情形，同時會隨著麥角留_ 7’9(11)’22五-三烯-3β-醇劑量的增加而更趨於明顯。特別地，實驗組3的丙二醛濃度比正對照組所具者還低。這個實驗結果顯不：麥角甾-7,9(11)，22五-三烯_3β·醇以一劑量-依賴性的方式來減輕肝臟組織的氧化性損傷程度。 G、小鼠肝臟組織内的iNOS以及^❶尽之表現量的西方墨 36 201221129 點分析本實驗是依照上面實施例3的「F、小鼠腳掌組織内的 iNOS以及⑶X·2表現量的西方墨點分析」t中所述的方法來進行iNOS錢C0X_2表現量的分析。所得到的實驗結果被顯示於圖18中。從圖18可見，與正常對照組(將正常對照組的蛋白質表現量當作loo%)相較之下，病理對照組的iN〇s以及c〇x_2 表現量被明顯地升高。而與病理對照組相較之下，正對照組以及實驗組3的iNOS以及C〇X_2表現量皆明顯地呈現下降的情开），特別地，實驗組3的iNOS以及c〇X-2表現量皆分別比正對照組所具者還低。這個實驗結果顯示：對於▼有化學性肝損傷的小鼠而言，麥角甾7,90 ^,22五三烯_ 3β-醇具有降低肝臟組織之iN〇s以及c〇x_2表現量的效用〇 Η、小鼠肝臟組織内的麩胱甘肽濃度的測定首先’由上面Α項中所得到的各組的1隻小鼠腳掌大體上是依照上面“一般實驗方法，，的第2項「組織萃取物樣品的製備」來進行組織萃取物樣品的製備，不同之處在於：使用20〇111]^1'1^-11(：1(?117.2)來代替生理食鹽水。有關肝臟組織麩胱甘肽濃度的測定是參照Ellman，G.L. {\959)，Archives of Biochemistry and Biophysics,名2..Ί0-ΊΊ 售中所述的方法並稍作修改後來進行。簡言之，將720 pL的 Tris-HCl緩衝液（2〇〇 mM, pH 7.2)加入至組織萃取物樣品中 ’繼而予以加入16〇 pL的5%三氣乙酸（trichloroacetic acid， 37 201221129 TCA)並混合均勻，所得到的混合物在4。〇下以ι〇,〇〇〇離〜歷時5分鐘。取33〇上清液與66〇爾曼試劑 (Ellman’s reagent)混合均勻，之後，以—VERSAmax 微量培養盤讀取儀來讀取在波長4〇5 nm下的吸光值（〇D4Q5)。各組所測得的0〇4〇5數值分別根據預先以具有不同已知濃度的麩胱甘肽相對於它們自身的〇Ε>4〇5數值所作出的相關曲線而被換算成麩胱甘肽濃度（nm〇1)，再除以各組依據上面 F項所測得的蛋白質總量，藉此而計算出各組中每mg蛋白質所含有的麵胱甘肽濃度（nm〇l/mg蛋白質）。所得到的實驗結果被顯示於圖19中。從圖19可見，與正常對照組相較之下，病理對照組的 GSH濃度被明顯地降低。而與病理對照組相較之下，實驗組1至3小鼠的麩胱甘肽濃度皆明顯地被升高，同時會隨著麥角甾-7,9(11 )，22五-三稀-3β-醇劑量的增加而更趨於明顯。特別地，實驗組2至3的麩胱甘肽濃度比正對照組所具者還高。這個實驗結果顯示：麥角留_7,9(11)，22心三烯_3(3_ 醇以一劑量-依賴性的方式來提高肝臟組織的麵胱甘肽濃度而減輕氧化性壓力。 I、組織病理學檢驗本實驗是依照上面實施例3的「g、組織病理學檢驗」當中所述的方法來進行’不同之處在於：在一為的放大倍率下來進行觀察以及拍照。所拍下的結果被顯示於圖 20中。從圖20可見，在病理對照組（Β區）中，C(：l4可以誘發 38 201221129 包括增加之退化（increased degeneration)、壞死（necrosis)、肝炎（hepatitis)以及門三體炎（portal triaditis)的組織學變化。除了正常對照組以外，所有組別在中央小葉區 (centrolobular zone)皆展現出氣球化退化（baU〇〇ning degeneration)以及肝細胞壞死的現象。而與病理對照組相較之下，實驗組1至3小鼠在氣球化退化以及肝細胞壞死的程度較輕（D區〜F區）。這個實驗結果顯示：麥角留_ 7,9(11)，22£-三烯-30-醇具有改善化學性肝損傷的效用。綜合以上的實驗結果，發明人認為：麥角甾_ 7,9(11)，22£-三烯-3β-醇能夠抑制脂質過氧化、在肝臟内增高抗氧化酵超氧化物歧化酶、過氧化氫酶以及麩胱甘肽過氧化酶活性、降低TNF-cx與一氧化氮的血清濃度，以及降低誘導性一氧化氮合成酶（iNOS)與環加氧酶_2 (c〇x_2)的表現量，透過抗發炎效用保護肝臟免於化學性肝損傷，進而具有治療肝損傷以及保護肝臟的效用。於本說明書中被引述之所有專利和文獻以其整體被併入本案作為參考資料。若有所衝突時’本案詳細說明(包含界定在内）將佔上風。雖然本發明已參考上述特定的具體例被描述’明顯地在不背離本發明之範圍和精神之下可作出很多的修改和變化。因此意欲的是，本發明僅受如隨文檢附之中請專利範圍所示者之限制。【圖式簡單說明】圖i顯示在乙酸-誘發的扭體試驗之前經由腹膜内注射 39 201221129 對小鼠投予不同劑量的麥角甾-7,9(11)，22五-三烯-30-醇，並自乙酸注射起的5分鐘内所紀錄的小鼠扭體次數結果，其中病理對照組表示經乙酸誘發疼痛的小鼠；正對照組表示以10 mg/kg吲哚美洒辛來處理經乙酸誘發疼痛的小鼠；實驗組1至3分別表示以1、5與10 mg/kg麥角留_ 7’9(11)，22五-二稀-3β-醇來處理經乙酸誘發疼痛的小鼠；以及 *、“**’’與“***’’分別表示户<〇〇5、户<〇〇【與户<〇，與病理對照組相比較；圖2顯示在福馬林_誘發的腳掌舔舐試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角甾_7,9(11)，22£_三烯_3(3_ 醇，並分別在福馬林注射之後的第〇至5分鐘的期間（早期）以及第15至40分鐘的期間（晚期）所紀錄的小鼠舔舐右後腳旱的時間，其中病理對照組表示以生理食鹽水來處理經福馬林誘發疼痛的小鼠；正對照組表示以1〇 mg/kg吲哚美洒辛來處理經福馬林誘發疼痛的小鼠；實驗組丨至3分別表示以1、5與10 mg/kg麥角留_79⑴）22£三烯_3β醇來處理經福馬林誘發疼痛的小鼠；以及“*，，'“**，，與“***，，分別表示與^〇顧，與病理對照組相比較；圖3顯示在鹿角菜膠-誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9(1 三稀_ 3P-醇’在鹿角菜膠注射之前（第Q小時）以及注射之後的第i 2 3 4與5小時使用—體積測定儀來測量小鼠的右後腳掌的水腫體積變化，小鼠腳掌的水腫體積是以小鼠右後腳掌在鹿角菜膠懸浮液注射之後所測得的體積減去在鹿角菜 40 201221129 =洋液注射之前所測得者的差值來表示，其中病理對照 ^示以生理食鹽水來處理經鹿角轉誘發”水腫的小組表示以1〇㈣"1嗓美洒辛來處理經鹿角菜穋誘發腳旱水腫的小鼠；實驗組！至3分別表示以】、5與 g g麥角甾-7,9(11)，22五-三烯,_醇來處理經鹿角菜膠誘發腳掌水腫的小鼠；以及“*，，、““，，與“—分別表示〆 0.05 U.Oi與户以刪，與病理對照組相比較；圖4顯示在鹿角菜膠-誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9⑴乂三烯_ ^醇曲’在鹿角菜敎射之後的第5小時所測得的血清亞石肖辰度其中正常對照組表示以生理食鹽水來處理雄性 R】鼠，病理對照組表示以生理食鹽水來處理經鹿角菜膠誘’X腳掌水腫的小鼠正對照組表示以⑺mg/kg吲哚美洒辛來處理經鹿角菜膠誘發腳掌水腫的小鼠；實驗組1至3 分別表示以1、5與1〇 mg/kg麥角留_7 9(11) 22五三烯邓_ 醇來處理經鹿角菜膠誘發腳掌水腫的小鼠；“###，，表示尸〈 • 001與正常對照組相比較；以及“**，，與“***，，分別表示尸 <〇.〇1與/?< 0.001，與病理對照組相比較；圖5顯示在鹿角菜膠_誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9(11),22五_三烯_ 3 β醇’在鹿角菜膠注射之後的第5小時所測得的血清tnf-α '辰度’其中正常對照組表示以生理食鹽水來處理雄性ICR 小鼠，病理對照組表示以生理食鹽水來處理經鹿角菜膠誘發腳掌水腫的小鼠；正對照組表示以1〇 mg/kg吲哚美洒辛 41 201221129 來處理經鹿角菜膠誘發腳掌水腫的小鼠；實驗組1至3分別表不以1、5與10 mg/kg麥角甾·7,9(11)，22五三烯_3卜醇來處理經鹿角菜膠誘發腳掌水腫的小鼠；“###”表示夕〈 0.001，與正常對照組相比較；以及“**，，與“***，，分別表示户 <0.01與；?<0.001，與病理對照組相比較；圖6顯不在鹿角菜膠_誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9(11)，22£_三烯_ 3 β醇在鹿角菜膠注射之後的第5小時收取右後腳掌組織所測得的丙二醛濃度，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經鹿角菜膠誘發腳掌水腫的小鼠；正對照組表示以1〇 mg/kg吲哚美洒辛來處理經鹿角菜膠誘發腳掌水腫的小鼠；實驗組1至3分別表示以i、5與1〇 mg/kg麥角甾_ 7,9(11)，22五-三烯-3β_醇來處理經鹿角菜膠誘發腳掌水腫的小自· ‘‘###，，全一- 鼠’ 表不尸<0.001，與正常對照組相比較；以及“*，，、與分別表示〆0.05、Ρ<0_01 與 ρ<〇·〇〇ι，與病理對照組相比較；圖7顯示在鹿角菜膠_誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投予麥角留_7,9(11)，22五_三烯_3卜醇，在鹿角菜膠注射之後的第5小時收取右後腳掌組織進行西方墨點分析所測得的iNOS以及COX-2表現量結果，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經鹿角菜膠誘發腳掌水腫的小鼠；正對照組表示以10 mg/kg吲哚美洒辛來處理經鹿角菜 42 201221129 膠誘發腳草水腫的小鼠，貫驗組3表示以1 〇 mg/kg麥角甾_ 7,9(11)，22五·三烯-3β-醇來處理經鹿角菜膠誘發腳掌水腫的小鼠；“###”表示；? <0.001，與正常對照組相比較；以及“**”與分別表示户<〇.〇1與ρ<0·001，與病理對照組相比較圖8是一切片染色圖，其顯示在鹿角菜膠_誘發的腳掌水腫試驗之前經由腹膜内注射對小鼠投藥麥角甾- 7,9(11)，22五-三烯-30-醇，並且在鹿角菜膠注射之後的第5小時所取得之右後腳掌切片的蘇木精_伊紅染色結果，其中A 區表示以生理食鹽水來處理雄性ICR小鼠的正常對照組；B 區表示以生理食鹽水來處理經鹿角菜膠誘發腳掌水腫的小鼠的病理對照組；C區表示以1 〇 mg/kg吲哚美洒辛來處理經鹿角菜膠誘發腳掌水腫的小鼠的正對照組；以及D區表示以10 mg/kg麥角甾_7,9(11)，22五-三烯-3β-醇來處理經鹿角菜膠誘發腳掌水腫的小鼠的實驗組3 ; 圖9疋根據圖8的染色結果任選5個視野進行嗜中性球計數而得到的結果，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經鹿角菜膠誘發腳掌水腫的小鼠；正對照組表示以i 〇 mg/kg吲哚美洒辛來處理經鹿角菜膠誘發腳掌水腫的小鼠；實驗組3表示以1〇 mg/kg麥角留-7,9(11)，22£-三稀_3β_醇來處理經鹿角菜膠誘發腳掌水腫的小鼠；“###，，表示ρ<〇〇〇1 ，與正常對照組相比較；以及“**，，與“***，，分別表示〇〇1 與ρ< 0.001，與病理對照組相比較； 43 201221129 圖10顯示在以四氯化碳（ecu)誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角甾-7,9(11),22仏三烯-3β-醇’在ecu注射之後的第24小時所測得的血清酵素丙胺酸轉胺酶（alanine amin〇transferase，ALT)活性，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經C(：14誘發化學性肝損傷的小鼠，正對照組表示以25 mg/kg水飛薊來處理經CC14誘發化學性肝損傷的小鼠；實驗組丨至3表示分別以25、5 以及10 mg/kg麥角甾_7,9(11)，22五-三烯-3β-醇來處理經CC14 誘發化學性肝損傷的小鼠；“料“表示^<0.001，與正常對照組相比較；以及“*，，、“**，，與“***，，分別表示户< 0 05、 0.01與；?< 〇_〇〇1，與病理對照組相比較；圖11顯示在以CCU誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角甾-7,9(11)，22£-三烯-3爲-醇 ’在CCI4注射之後的第24小時所測得的血清天門冬胺酸轉胺酶（aspartate aminotransferase，AST)活性，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經CCU誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛薊來處理經CC14誘發化學性肝損傷的小鼠；實驗組1至3表示分別以2.5、5以及1〇 mg/kg麥角留_7,9(11)，22五-三烯-3β-醇來處理經CC14誘發化學性肝損傷的小鼠；“###”表示p < 0.001，與正常對照組相比較；以及“**，，與“***，，分別表示尸<〇.〇1與尸<0〇〇1，與病理對照組相比較； 44 201221129 圖12顯不在以CCU誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角留_79(11)，22心三烯_3卜醇，在CCU注射之後的第24小時所測得的血清亞硝酸鹽濃度 ’其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠，病理對照組表示以生理食鹽水來處理經CC14誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛薊來處理經 CCU誘發化學性肝損傷的小氣；實驗組丨至3表示分別以 2.5、 5以及1〇 mg/kg麥角留_7,9(11) 22五_三烯_3|3_醇來處理經CCU誘發化學性肝損傷的小鼠；“###，，表示户<〇〇〇1，與正常對A?、組相比較；以及“ * ”、“ * * ”與“ * * *，，分別表示尸〈 0.05、p< 〇.〇1與户^刪’與病理對照組相比較；圖13顯不在以cc丨*誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角甾_7,9(11)，22五三烯_3(3-醇 ’在CCl4注射之後的第24小時所測得的血清TNF-α濃度，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠 ’病理對照組表示以生理食鹽水來處理經Cci4誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛薊來處理經 CCU誘發化學性肝損傷的小鼠；實驗組1至3表示分別以 2.5、 5以及1〇 mg/kg麥角留_7,9(11)，22£•三烯_3卜醇來處理經CCU誘發化學性肝損傷的小鼠；“###’’表示户<〇〇〇i，與正常對照組相比較；以及“**’，與‘‘***，，分別表示户<〇〇1與户 <0.001，與病理對照組相比較；圖14顯示在以cci4誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角甾_7,9(1 ”刀仏三烯^卜醇 45 201221129 ，在CCU注射之後的第24小時收取肝臟組織所測得的超氧化物歧化酶活性，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠，·病理對照組表示以生理食鹽水來處理經 CC〗4誘發化學性肝損傷的小鼠；正對照組表示以水飛薊來處理經CC14誘發化學性肝損傷的小鼠；實驗組i 至3表示分別以2·5、5以及10 mg/kg麥角留_7 9(11) 22五_ 三烯-3β-醇來處理經CCU誘發化學性肝損傷的小鼠；“###” 表示/? < 〇.〇〇 1 ’與正常對照組相比較；以及“*，，、“**，，與分別表示p<0.05、p<0.01與〆〇 001，與病理對照組相比較；圖15顯示在以CC!4誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9〇 υ，22£三稀普醇在CCI4注射之後的第24小時收取肝臟組織所測得的過氧化氫酶活性’纟中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經誘發化學性肝損傷的小鼠；正對照組表示以25叫㈣水飛莉來處理經CC14誘發化學性肝損傷的小鼠；實驗組i至3 表示分別以2.5、5以及10 mg/kg麥角留_7 9⑴）22五三浠_ 邛-醇來處理經ecu誘發化學性肝損傷的小鼠；“###，，表示p < 0.001 ’與正常對照組相比較；以及“*，，、“**，，鱼‘‘*"，，八別表不〆〇·〇5、p<0.01與p<〇〇〇卜與病理對照組相比較；圖16顯示在以CCl4誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角[7,9(1 υ，以三稀·3ρ醇 46 201221129 ，在CCU注射之後的第24小時收取肝臟組織所測得的麩胱甘肽過氧化酶活性，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經CCU誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛莉來處理經CCl4誘發化學性肝損傷的小鼠；以及實驗組1至3表示分別以2.5、5以及1〇 mg/kg麥角留· 7,9(11)，22五-三稀-3β-醇來處理經ccu誘發化學性肝損傷的小鼠；“###”表示；^<0.00!，與正常對照組相比較；以及‘‘*，，、“"”與分別表示〆 0.05、p<〇〇1 與 ρ<〇〇〇ι，與病理對照組相比較；圖17顯不在以CCU誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角崔_7 9⑴），_三稀，·醇，在CCU注射之後的第24小時收取肝臟組織所測得的丙二醛濃度，其中正常對照組表示以生理食鹽水來處理雄性icr J既，病理對照組表不以生理食鹽水來處理經CC14誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛莉來處理經CCU誘發化學性肝損傷的小鼠；實驗組i至3表示分別以2.5、5以及10mg/kg麥角留_7,9(11)22£三稀_3卜醇來處理經cci4誘發化學性肝損傷的小鼠；“###，，表*户<〇〇〇1 ’與正常對照组相比較；以及“*，，、“**，，與“***，，分別表示户 <0·05、ρ<0.01與p<〇〇〇1，與病理對照組相比較；圖18顯示在以ccu誘發化學性肝損傷之前經由腹膜内注射對小鼠投予麥角留_7,9(11)，挪三稀_邱_醇，在响注後的第24小時收取肝臟組織經西方墨點分析所測得的 47 201221129 iNOS以及COX-2表現量分析結果，其中正常對照組表示以生理食鹽水來處理雄性ICR小鼠；病理對照組表示以生理食鹽水來處理經CCU誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛莉來處理經CC14誘發化學性肝損傷的小鼠，貫驗組3表示以10 mg/kg麥角留^9(^)22五三烯_ 3β-醇來處理經CCI4誘發化學性肝損傷的小鼠；“###，，表示夕 <0.001 ’與正常對照組相比較；以及“**，，與“***，，分別表示 Ρ< 0.01與ρ< 0.001 ’與病理對照組相比較；圖19顯示在以CCU誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角留_7,9(11},22五_三烯·3p_醇，在ecu注射之後的第24小時收取肝臟組織所測得的麩胱甘肽濃度，其中正常對照組表示以生理食鹽水來處理雄性 ICR小鼠；病理對照組表示以生理食鹽水來處理經誘發化學性肝損傷的小鼠；正對照組表示以25 mg/kg水飛薊來處理經（：(：14誘發化學性肝損傷的錢；實驗組丨至3表不分別以2·5、5以及1〇 mg/kg麥角留_7 9(11) 22五三烯_3p_ 醇來處理經CCU誘發化學性肝損傷的小鼠；“###”表示户〈 0.0 01 ’與正常對照組相比較；以及“ “ 斑“ * * *，，分別表不p< 0.05、ρ< 0·(Π與〇〇〇1，與病理對照組相比較 :以及圖20是一切片染色圖，其顯示在以CC14誘發化學性肝損傷之前經由腹膜内注射對小鼠投予不同劑量的麥角留_ 7’9(11)，22五-三稀_邱_醇，在ccl4注射之後的第24小時所取得之腳掌切片的蘇木精-伊紅染色結果，其中A區表示以生 48 201221129 理食鹽水來處理雄性ICR小鼠的正常對照組；b區表示以生理食鹽水來處理經CC14誘發化學性肝損傷之小鼠的病理對R?、組，C區表示以10 mL/kg °引α朵美洒辛來處理經cci4誘發化學性肝損傷之小鼠的正對照組；以及D至F區分別表示以2.5、5以及10 mg/kg麥角留_7,9(11)，22£-三烯-30-醇來處理經CCU誘發化學性肝損傷之小鼠的實驗組1至3。【主要元件符號說明】 (無） 49The liver was taken to ^^ for the following E analysis. Knife analysis 'and another part of the liver was taken for the following r terms B, serum alanine transaminase (4) anine amin〇transferase, and aspartate amin〇transfe (10), AST) activity assay The determination of alanine transaminase and aspartate transaminase activity in the serum samples obtained in item A above was carried out by the Easy-Check Clinical Laboratory. The experimental results obtained are shown in Figures 1 to 11. As can be seen from 圊10 and Fig. 11, the serum alanine transaminase and aspartate transaminase activities of the pathological control group were significantly increased after CCU injection as compared with the normal control group. Compared with the pathological control group, the serum alanine transaminase and aspartate transaminase activities in the positive control group and the experimental group 1 to 3 were significantly reduced, in particular, the experimental group 丨3 With the increase of the dosage of ergots -7,9 (11), 22 £-tris--3β-alcohol, it became more obvious. This experimental study showed that ergot 77,9(11),22penta-triene-3-butanol prevented serum alanine transaminase and aspartate transaminase in a dose-dependent manner. Increased activity. C. Determination of serum nitrite concentration In this experiment, serum nitrite was prepared according to the method of "Β, serum sub-; ε octate concentration 34 201221129 of the above Example 3, I φ, +, u, The experimental results obtained by the measurement of the concentration are shown in Fig. 12. It is seen that the blood salt concentration of the pathological control group was significantly increased as compared with the normal control group. Compared with the pathological control group, the serum nitrite concentration of ', 丨 to 3 decreased, and it increased with the dose of ergot _7,9(1)), 22,5-triene, and alcohol. In the other way, the serum sulphate concentration of the experimental group 3 was lower than that of the positive control group. The results of this experiment show that ergosporin 7'9(11), 22penta-diene-3β-alcohol inhibits the formation of nitric oxide in a dose-dependent manner. D. Measurement of Serum TNF-ct Concentration ~ In this experiment, the serum TNF_a concentration was measured in accordance with the method described in "c, serum TNF_a concentration:" in Example 3 above. The experimental results obtained are shown in Fig. 13. As can be seen from Fig. 13, the blood π TNF-a concentration of the pathological control group was significantly increased as compared with the normal control group. Compared with the pathological control group, the serum TNF_a concentration in the experimental group 1 to 3 decreased, and the dose of _7,9 (1仏(10)·三妇_3β_ alcohol increased with ergot. It is more obvious. In particular, the serum TNF_a concentration in experimental group 3 is lower than that in the positive control group. The results of this experiment show: ergot _7,9 (11), 22 penta-triene _ 3β- Alcohol reduces serum TNF-a concentration in a dose-dependent manner. E. Determination of Antioxidant Enzyme Activity in Mouse Liver Tissue This experiment is based on the "D, mouse liver tissue resistance" in Example 3 above. The method described in the "Measurement of Oxidase Activity" was carried out to measure the activity of superoxide 35 201221129 per catalase and typhoid peroxidase. The experimental results obtained are shown in Figures 14 to 16. It can be seen from Fig. 14 to Fig. 6 that after ecu injection, the activities of superoxide dismutase, catalase and glutathione peroxidase in the pathological control group are significantly different from those of the normal photographic age. The ground was lowered. Compared with the pathological control group, the experimental group was over 1 1 3 Oxide dismutase, catalase and caspase peroxidase activities were significantly increased, along with ergosta-7,9(11),22penta-triene-3戸-ol doses The increase was more obvious. In particular, the activities of superoxide dismutase, catalase and glutathione peroxidase in experimental group 3 were higher than those in the positive control group, respectively. : ergoline retention _7,9 (11), 22 penta-triene·3p• alcohol has the effect of increasing superoxide dismutase, catalase and caspase peroxidase activity. Measurement of the concentration of propylene glycol in the occupational tissue This experiment is to measure the concentration of malondialdehyde according to the method described in "E, measurement of the concentration of propionaldehyde in the paw tissue of the mouse" in Example 3 above. The experimental results obtained are shown in Fig. 17. As can be seen from 圊17, compared with the pathological control group, the concentration of malondialdehyde in the experimental group 丨3 was decreased, and it remained with ergot _ 7 The increase in the dose of '9(11)'22 penta-triene-3β-ol is more pronounced. In particular, the experimental group 3 The concentration was lower than that of the positive control group. The results of this experiment were not obvious: ergot 甾-7,9(11),22penta-triene_3β· alcohol reduced liver tissue in a dose-dependent manner. The degree of oxidative damage. G. The amount of iNOS in the liver tissue of mice and the amount of expression of Western ink 36 201221129 Point analysis This experiment is based on the above Example 3, "F, iNOS in mouse foot tissue and (3) X· 2 Western blot analysis of the amount of performance. The method described in t is used to analyze the amount of iNOS money C0X_2. The experimental results obtained are shown in Figure 18. As can be seen from Figure 18, compared with the normal control group (normal control) In comparison with the protein expression of the group as loo%, the expression of iN〇s and c〇x_2 in the pathological control group was significantly increased. Compared with the pathological control group, the expression levels of iNOS and C〇X_2 in the positive control group and the experimental group 3 were significantly decreased. In particular, the iNOS and c〇X-2 expression of the experimental group 3 were observed. The amount is lower than that of the positive control group. The results of this experiment show that for mice with chemical liver injury, ergoside 7,90 ^,22,5-triene _ 3β-alcohol has the effect of reducing the expression of iN〇s and c〇x_2 in liver tissue. Determination of the concentration of glutathione in the liver tissue of cockroaches and mice First of all, the mouse feet of each group obtained from the above items are generally in accordance with the above "general experimental method, item 2" Preparation of tissue extract samples to prepare tissue extract samples, except that: 20〇111]^1'1^-11 (:1 (?117.2) was used instead of physiological saline. The measurement of the concentration of glutathione was carried out by referring to the method described in Ellman, GL {\959), Archives of Biochemistry and Biophysics, name 2. Ί0-ΊΊ, and a slight modification. Briefly, 720 pL of Tris-HCl buffer (2 mM, pH 7.2) was added to the tissue extract sample, followed by the addition of 16 〇pL of 5% trichloroacetic acid (37 201221129 TCA). And mix well, the resulting mixture is at 4. 〇以〇〇〇〇〜〜历〜〜〜〜〜〜〜〜〜 The 33 上 supernatant was mixed well with Ellman's reagent, and then the absorbance at a wavelength of 4 〇 5 nm (〇D4Q5) was read with a -VERSAmax microplate reader. The values of 0〇4〇5 measured by each group were converted to glutosine according to the correlation curves previously obtained with values of glutathione having different known concentrations relative to their own 〇Ε>4〇5 values. The peptide concentration (nm〇1) was divided by the total amount of protein measured by each group according to the above item F, thereby calculating the concentration of glutathione per mg protein in each group (nm〇l/mg). protein). The experimental results obtained are shown in Fig. 19. As can be seen from Fig. 19, the GSH concentration of the pathological control group was significantly lowered as compared with the normal control group. Compared with the pathological control group, the glutathione concentration in the experimental group 1 to 3 mice was significantly increased, and along with the ergot -7,9 (11), 22 five-three rare The increase in the dose of -3β-alcohol is more pronounced. In particular, the glutathione concentrations of the experimental groups 2 to 3 were higher than those of the positive control group. The results of this experiment showed that ergot leaves _7,9(11), 22-triene _3 (3_ alcohol increased the concentration of glutathione in liver tissue in a dose-dependent manner to reduce oxidative stress. , Histopathological examination This experiment was carried out in accordance with the method described in "g, histopathological examination" of Example 3 above. 'The difference is that observation and photographing were performed at a magnification of one. The results are shown in Figure 20. As can be seen from Figure 20, in the pathological control group (Β), C(:l4 can induce 38 201221129 including increased degeneration, necrosis, hepatitis And the histological changes of portal triaditis. All groups except the normal control group showed ballooning degradation (baU〇〇ning degeneration) and hepatocyte necrosis in the central centrolobular zone. Compared with the pathological control group, the mice in the experimental group 1 to 3 had less degree of ballooning degradation and hepatocyte necrosis (D area ~ F area). The results of this experiment showed: ergot leaves _ 7,9 (11), 22 £-triene-30-alcohol has the effect of improving chemical liver damage. Based on the above experimental results, the inventors believe that: ergoside _ 7,9 (11), 22 £-triene-3β -Alcohol inhibits lipid peroxidation, increases antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase activity in the liver, decreases serum concentrations of TNF-cx and nitric oxide, and reduces induction The expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (c〇x_2) protect the liver from chemical liver damage through anti-inflammatory effects, which in turn has the effect of treating liver damage and protecting the liver. All patents and documents cited in this specification are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the the the the the the the the The invention has been described as being <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; [Simple diagram Figure i shows that mice were dosed with different doses of ergosta-7,9(11),22penta-triene-30-ol via intraperitoneal injection 39 201221129 prior to the acetic acid-induced writhing test. The number of writhing times recorded in the mice within 5 minutes from the injection of acetic acid, in which the pathological control group indicated mice induced pain by acetic acid; the positive control group showed that acetic acid induced pain was treated with 10 mg/kg 吲哚美辛辛Mice; experimental groups 1 to 3 represent mice treated with acetic acid-induced pain at 1, 5 and 10 mg/kg ergoside _ 7'9 (11), 22 penta-di- 3 -β-alcohol, respectively; And *, "**'' and "***'' respectively indicate that the household <〇〇5, household<〇〇[with household<〇, compared with the pathological control group; Figure 2 shows in Formalin_ Mice were administered different doses of ergorogane _7,9(11), 22 _triene _3 (3_ol, and the following after the formalin injection, via intraperitoneal injection before the induced foot sputum test. The time from the sputum to the 5-minute period (early period) and the 15th to 40-minute period (late period) recorded in the right hind paw of the mouse, in which the pathological control group indicated that the physiological diet Water was used to treat mice induced by formalin-induced pain; the positive control group showed that mice treated with formalin-induced pain were treated with 1 〇mg/kg 吲哚美辛辛; experimental groups 丨 to 3 were expressed as 1, 5 and 10 mg/kg ergot _79(1)) 22 £ triene _3β alcohol to treat mice induced by formalin-induced pain; and “*,,' “**,, and “***, respectively, and ^ Regardless, compared with the pathological control group; Figure 3 shows that mice were given different doses of ergots via intraperitoneal injection before the carrageenan-induced paw edema test. _7,9 (1 Tris _ 3P- The alcohol's edema volume change in the right hind paw of the mouse was measured before the carrageenan injection (Q hours) and at the i 2 3 4 and 5 hours after the injection - the volume of the edema of the mouse's foot was The volume measured after the injection of the right hind paw of the mouse in the suspension of carrageenan was subtracted from the difference measured in the carrageen 40 201221129 = before the injection of the liquid, wherein the pathological control showed physiological saline. The group that deals with edema induced by antlers is treated with 1〇(4)"1嗓美辛辛Lu carageenan induced edema in mice dry foot; experiment! To 3, respectively, to 5, gg ergot -7,9 (11), 22 penta-triene, _ alcohol to treat mice induced by carrageenan edema; and "*,,," ,, and "- respectively represent 〆0.05 U.Oi and households to delete, compared with the pathological control group; Figure 4 shows that different doses of mice were administered via intraperitoneal injection before the carrageenan-induced paw edema test. Ergoline retention _7,9(1) nintriene _ ^ alcohol ko's serum sulphate measured at the 5th hour after the staghorn squid shot. The normal control group indicated that male R was treated with physiological saline. The control group indicated that the mice in the control group treated with the carrageenan-induced X-foot edema in physiological saline were treated with (7) mg/kg 吲哚美辛辛 to treat mice with edema induced by carrageenan; experimental group 1 Up to 3 indicates that mice treated with carrageenan-induced foot edema were treated with 1,5 and 1〇mg/kg ergot _7 9(11) 22 pentaene Deng _ alcohol; "###,, The corpse < 001 is compared with the normal control group; and "**,, and "***, respectively, represent the corpse < 〇.〇1 and /?< 0.001, and The pathological control group was compared; Figure 5 shows that mice were administered different doses of ergots via intraperitoneal injection before the carrageenan-induced paw edema test. _7,9(11),22penta-triene_3 The β-alcohol's serum tnf-α 'end' measured at the 5th hour after carrageenan injection. The normal control group indicated that male ICR mice were treated with physiological saline, and the pathological control group showed physiological saline. The mice treated with carrageenan induced foot edema; the positive control group indicated that mice treated with carrageenan induced foot edema were treated with 1〇mg/kg 洒美辛辛41 201221129; experimental groups 1 to 3 respectively Mice treated with carrageenan-induced edema of the foot were treated with 1,5 and 10 mg/kg ergosin·7,9(11),22pentatriene_3 phenol; “###” means 夕<0.001 , compared with the normal control group; and "**,, and "***, respectively, represent households <0.01 and;?<0.001, compared with the pathological control group; Figure 6 is not in the carrageenan_induced Prior to the edema test of the foot, mice were dosed with different doses of ergots via intraperitoneal injection _7,9(11), 22 £ _ triene _ 3 β alcohol The concentration of malondialdehyde measured in the right hind paw tissue was collected at the 5th hour after the injection of carrageenan. The normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the treatment was performed with physiological saline. The mice in the carrageenan induced foot edema; the positive control group indicated that the mice infected with carrageenan induced foot edema were treated with 1〇mg/kg 吲哚美辛; the experimental groups 1 to 3 respectively represented i, 5 and 1〇mg/kg ergot 甾 7, 7, (11), 22 penta-triene-3β-alcohol to treat the small sacred edema induced by carrageenan. ''###,, all one-rat' The table was not 0.001, compared with the normal control group; and "*,,, and 〆0.05, Ρ<0_01 and ρ<〇·〇〇ι, respectively, compared with the pathological control group; Figure 7 shows the antlers The mice were given ergoline _7,9(11),22 pentatriene _3 phenol by intraperitoneal injection before the vegetable gum _ induced foot edema test, which was collected at the 5th hour after carrageenan injection. The results of iNOS and COX-2 measured by Western blot analysis were performed on the right hind paw tissue. Physiological saline was used to treat male ICR mice; the pathological control group indicated that mice treated with carrageenan-induced foot edema were treated with physiological saline; the positive control group was treated with 10 mg/kg of succulent 42 201221129 Glue-induced edema in mice, test group 3 indicated that the carrageenan-induced foot was treated with 1 〇mg/kg ergoside _ 7,9(11), 22 penta-triene-3β-ol Edema mice; "###" means; ? < 0.001, compared with the normal control group; and "**" and respectively represent households <〇.〇1 and ρ<0·001, and pathological control group Comparing Figure 8 is a section staining diagram showing the administration of ergosin- 7,9(11),22-triene-30- to mice by intraperitoneal injection prior to the carrageenan-induced paw edema test. Alcohol, and hematoxylin-eosin staining of the right hind paw slice obtained at the 5th hour after the carrageenan injection, wherein the A region represents a normal control group for treating male ICR mice with physiological saline; The area indicates the pathological control group of mice treated with carrageenan-induced foot edema by physiological saline; 1 〇mg/kg 洒美辛辛 to treat the positive control group of mice induced by carrageenan-induced edema; and D-zone means 10 mg/kg ergot _7,9(11),22- Trien-3β-alcohol to treat experimental group 3 of mice induced by carrageenan-induced edema of the foot; Figure 9疋 Results obtained by performing neutrophil counts according to the staining results of Figure 8 for 5 fields of view, wherein normal The control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that mice treated with carrageenan-induced foot edema were treated with physiological saline; the positive control group indicated that i 〇mg/kg 洒美辛辛To treat mice with edema induced by carrageenan; experimental group 3 showed that treatment with carrageenan was treated with 1〇mg/kg ergot -7,9(11), 22£-three dilute _3β-ol Mice with edema on the soles of the feet; "###," means ρ<〇〇〇1, compared with the normal control group; and "**,, with "***, respectively, 〇〇1 and ρ< 0.001, Compared with the pathological control group; 43 201221129 Figure 10 shows the small intraperitoneal injection before the chemical liver injury induced by carbon tetrachloride (ecu) Alanine amin〇transferase (alien amin〇transferase) measured at the 24th hour after ecu injection at different doses of ergosin-7,9(11),22仏triene-3β-alcohol ALT) activity, in which the normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that mice treated with C (:14 induced chemical liver injury) were treated with physiological saline, and the positive control group indicated 25 Mg/kg silymarin to treat mice with chemical liver injury induced by CC14; experimental group 丨 to 3 means ergodonta _7,9(11),22 five-three at 25, 5 and 10 mg/kg respectively Alkenyl-3β-alcohol to treat mice induced by CC14-induced chemical liver injury; “Material” means ^<0.001 compared with normal control group; and “*,,, “**,, and “*** , respectively, represent households < 0 05, 0.01 and ;? < 〇_〇〇1, compared with the pathological control group; Figure 11 shows that the mice were administered via intraperitoneal injection before the CCU induced chemical liver injury Serum aspartate transaminase measured at different doses of ergosin-7,9(11),22£-triene-3 as-alcohol' at 24 hours after CCI4 injection Aspartate aminotransferase (AST) activity, in which the normal control group indicated that the male ICR mice were treated with physiological saline; the pathological control group indicated that the mice with chemical liver injury induced by CCU were treated with physiological saline; The mice induced by CC14 induced chemical liver injury were treated with 25 mg/kg silymarin; the experimental groups 1 to 3 showed 6.5, 5 and 1 〇mg/kg ergots respectively _7,9(11),22 Penta-trien-3β-ol to treat mice induced by CC14-induced chemical liver injury; “###” means p < 0.001 compared with normal control group; and “**,, and “*** , respectively, representing corpse <〇.〇1 and corpse<0〇〇1, compared with pathological control group; 44 201221129 Figure 12 shows that mice were not administered by intraperitoneal injection before CCU induced chemical liver injury Different doses of ergot leaves _79 (11), 22 core triene _3 phenol, serum nitrite concentration measured at 24 hours after CCU injection 'where the normal control group indicated treatment with physiological saline Male ICR mice, pathological control group indicated treatment of chemical liver induced by CC14 with physiological saline Injured mice; the positive control group indicated that 25 mg/kg of milk thistle was used to treat small gas caused by CCU-induced chemical liver injury; the experimental group 丨 to 3 showed 2.5, 5 and 1 〇mg/kg ergots respectively _ 7,9(11) 22 penta-triene_3|3_ alcohol to treat mice induced by CCU induced chemical liver injury; "###,, indicates households < 〇〇〇 1, with normal pairs of A? Comparison with the group; and "*", "**" and "***, respectively, representing the corpse <0.05, p< 〇.〇1 and household^ deleting' compared with the pathological control group; Mice were administered different doses of ergot 甾7,9(11),22pentatriene_3 (3-alcohol' 24th after CCl4 injection by intraperitoneal injection before cc丨* induced chemical liver injury Serum TNF-α concentration measured in hours, in which the normal control group indicated that male ICR mice were treated with physiological saline. The pathological control group indicated that mice treated with Cci4-induced chemical liver injury were treated with physiological saline; The group indicated that mice treated with CCU-induced chemical liver injury were treated with 25 mg/kg of milk thistle; the experimental groups 1 to 3 showed _7,9 (11) at 2.5, 5 and 1 〇mg/kg ergot, respectively. ,2 2 £•Triene_3 phenol to treat mice induced by CCU-induced chemical liver injury; “###'' indicates household <〇〇〇i, compared with normal control group; and “**', And ''***, respectively, indicates that the household <〇〇1 and household<0.001, compared with the pathological control group; Figure 14 shows that the mice were administered via intraperitoneal injection before induced chemical liver injury with cci4 Different doses of ergot 甾7,9 (1 仏仏仏 45 45 45 201221129, the superoxide dismutase activity measured by liver tissue was collected at 24 hours after CCU injection, the normal control group indicated Male ICR mice were treated with physiological saline. The pathological control group showed that mice with chemical liver injury induced by CC 4 were treated with physiological saline. The positive control group indicated that the chemical resistance induced by CC14 was treated with milk thistle. Liver-injured mice; experimental groups i to 3 indicated that CCU-induced chemical liver was treated with 2.6, 5, and 10 mg/kg ergot _7 9(11) 22 pentatriene-3β-ol, respectively. Injured mice; "###" means /? < 〇.〇〇1 'Compared with the normal control group; and "*,,," **,, P<0.05, p<0.01 and 〆〇001, respectively, compared with the pathological control group; Figure 15 shows that mice were administered different doses of ergots via intraperitoneal injection prior to induction of chemical liver injury with CC!4. _7,9〇υ, 22 £ tri-dipropanol received catalase activity measured in liver tissue at 24 hours after CCI4 injection. 'The normal control group indicated that male ICR mice were treated with physiological saline. The pathological control group indicated that the mice with induced chemical liver injury were treated with physiological saline; the positive control group indicated that the mice induced by CC14 induced chemical liver injury were treated with 25 (4) Shui Fei Li; experimental groups i to 3 The mice treated with ecu-induced chemical liver injury were treated with 2.5, 5, and 10 mg/kg ergot _7 9(1)) 22 5.3 浠邛-alcohol respectively; "###, indicating p < 0.001 'Compared with the normal control group; and "*,,,",,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Comparison; Figure 16 shows that mice were administered via intraperitoneal injection prior to induction of chemical liver injury with CCl4 Different doses of ergot [7,9 (1 υ, to three dilute 3 ρ alcohol 46 201221129, glutathione peroxidase activity measured in liver tissue at 24 hours after CCU injection, of which normal control group The male ICR mice were treated with physiological saline; the pathological control group was treated with physiological saline to treat mice induced by CCU-induced chemical liver injury; the positive control group was treated with 25 mg/kg of water lily to induce CCl4-induced Chemical liver injury mice; and experimental groups 1 to 3 indicated that ccu was treated with 2.5, 5, and 1 〇mg/kg ergoside 7,9(11), 22 five-tris-3β-alcohol, respectively. Mice inducing chemical liver injury; "###" means; ^<0.00!, compared with the normal control group; and ''*,,, """ and 〆0.05, p< 1 compared with ρ<〇〇〇ι, compared with pathological control group; Figure 17 shows that mice were given different doses of ergot cucurbita via intraperitoneal injection before CCU induced chemical liver injury _7 9(1)), _3 Dilute, alcohol, the concentration of malondialdehyde measured by liver tissue at 24 hours after CCU injection, of which normal The photo group indicated that the male icr J was treated with physiological saline. The pathological control group did not treat the mice with chemical liver injury induced by CC14 with physiological saline; the positive control group indicated that it was treated with 25 mg/kg water lily. Mice with chemical liver injury induced by CCU; experimental groups i to 3 indicated that cci4-induced chemistry was treated with 2.5, 5, and 10 mg/kg ergot _7,9 (11) 22 £ tris- 3 octanol, respectively. Mice with liver injury; "###,, table * household < 〇〇〇 1 ' compared with the normal control group; and "*,,, **,, and "***, respectively, Households <0·05, ρ<0.01 and p<〇〇〇1, compared with pathological control group; Figure 18 shows that mice were administered ergots via intraperitoneal injection before ccu-induced chemical liver injury _ 7,9 (11), migrating three _ _ Qiu _ alcohol, in the 24th hour after the sounding, the liver tissue was measured by Western blot analysis of 47 201221129 iNOS and COX-2 performance analysis results, of which normal control The group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated that mice treated with CCU-induced chemical liver injury were treated with physiological saline. The positive control group indicated that mice treated with CC14-induced chemical liver injury were treated with 25 mg/kg of water lily. The test group 3 indicated that 10 mg/kg ergot leaves ^9(^)22 pentadiene _ 3β- Alcohol was used to treat mice induced by CCI4 to induce chemical liver injury; "###, indicating that eve <0.001' was compared with the normal control group; and "**,, with "***, respectively, Ρ< 0.01 vs. ρ < 0.001 ' compared with pathological control group; Figure 19 shows that mice were administered different doses of ergots via intraperitoneal injection before CCU induced chemical liver injury _7,9(11},22 _ Triene·3p-alcohol, the glutathione concentration measured by liver tissue was collected at 24 hours after ecu injection, wherein the normal control group indicated that male ICR mice were treated with physiological saline; the pathological control group indicated Physiological saline was used to treat mice with induced chemical liver injury; positive control group indicated treatment with 25 mg/kg silymarin (: (: 14 induced chemical liver damage; experimental group 丨 to 3) Treatment of chemical liver injury induced by CCU with 2·5, 5 and 1〇mg/kg ergot _7 9(11) 22 pentacene _3p_ alcohol Rat; "###" indicates that the household < 0.0 01 ' is compared with the normal control group; and " " spot " * * *, respectively, indicating p < 0.05, ρ < 0 · (Π and 〇〇〇 1, with Comparison of the pathological control group: and Figure 20 is a section staining diagram showing that mice were administered different doses of ergots via intraperitoneal injection before CC14 induced chemical liver injury _ 7'9 (11), 22 Five-three-dilute _ _ _ alcohol, the results of hematoxylin-eosin staining of the foot slice obtained at the 24th hour after ccl4 injection, in which the A area represents the treatment of male ICR mice with raw saline 2012 201221129 The normal control group; the b-zone indicates that the pathology of the mice induced by CC14-induced chemical liver injury was treated with physiological saline, and the group C was expressed at 10 mL/kg °. The positive control group of the mice that induced chemical liver injury; and the D to F areas indicated that _7,9(11), 22 £-triene-30-ol was added at 2.5, 5, and 10 mg/kg ergot, respectively. Experimental groups 1 to 3 of mice treated with CCU-induced chemical liver injury were treated. [Main component symbol description] (none) 49

Claims

201221129 VII. Patent application scope: 1. A pharmaceutical composition for treating pain, which comprises ergot Cui 7,9(11), 22 penta-triene-30-ol. 2. A pharmaceutical composition according to the scope of the patent application, which is a dosage form for oral administration. 3. A pharmaceutical composition as claimed in claim i, which is in the form of a non-zinc intestinal administration. 4. A ergosta-7,9(11),22-trien-3-ene alcohol supply for use in the preparation of a medicament for the treatment of pain. 5. The use of the fourth application of the patent scope wherein the pharmaceutical product is in a dosage form for oral administration. 6. The use of claim 4, wherein the pharmaceutical product is in a form for parenteral administration. 7. A pharmaceutical composition for treating inflammation comprising 'ergostine-7'9(11), 22penta-tris-3-ol-alcohol. 8. A pharmaceutical composition according to claim 7 of the patent application, which is in the form of a dosage form for oral administration. 9. A pharmaceutical composition as claimed in claim 7 which is in a form for parenteral administration. i 〇 — — — — 甾甾甾甾甾甾甾甾甾甾甾甾甾甾甾甾甾甾供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应供应The use of the pharmaceutical product is in the form of a dosage form for oral administration. 12. The use of the scope of the patent application, wherein the pharmaceutical product is a pharmaceutical composition for the treatment of liver damage, which comprises a pharmaceutical composition for parenteral administration of 5, 201221129, which comprises wheat Corner 甾·7,9(11), 22£-triene_3β_ alcohol. 14. The pharmaceutical composition of claim 13, wherein the liver injury is a chemical liver injury. 15. The pharmaceutical composition according to claim 14, wherein the chemical liver damage is caused by chemical vapor damage induced by carbon monoxide. 16. A pharmaceutical composition as claimed in claim 13 which is in the form of a pharmaceutical administration. , Service 17. The pharmaceutical composition of claim 13 is a dosage form for parenteral administration. 18. The use of ergots _7,9〇1), 22 £ triene _3 (3-alcohol supply for preparation - use of pharmaceuticals for the treatment of liver damage. 19. If the scope of application for patents is 18 The use of the pharmaceutical product is a dosage form for oral administration. 八20· The use of the 18th item of the patent application wherein the pharmaceutical product is in a form for parenteral administration. The composition for protecting the liver comprises ergot 7'9(11), 22 £; - triterpene _3β·ol. 22. The composition of claim 21, which can be manufactured as A form of a pharmaceutical product or a food product. 23. The composition of claim 22, wherein the pharmaceutical product is in a dosage form for oral administration. 24_ The composition of claim 22, wherein The pharmaceutical product is a dosage form for parenteral administration. 25. A ergoside-7,9(11),22penta-triene-3β-ol is supplied for the preparation of a liver for protection. The use of a pharmaceutical or food product. 26. For the use of the scope of claim 25, wherein the pharmaceutical product is for oral administration Dosage form. 27. The use Patent application range of item 25, wherein the pharmaceutical dosage form is for a parenteral administration. 52