TWI472519B - N-butylidenephthalide-containing pharmaceutical composition for treating liver injury and improving liver function - Google Patents
N-butylidenephthalide-containing pharmaceutical composition for treating liver injury and improving liver function Download PDFInfo
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本發明係關於正-亞丁基苯酞(n-butylidenephthalide)內於治療例如肝纖維化、肝硬化或肝發炎之肝損傷的用途，及用於改善肝功能的用途。The present invention relates to the use of n-butylidenephthalide for treating liver damage such as liver fibrosis, cirrhosis or hepatic inflammation, and for improving liver function.
肝病為華人之常見疾病，目前全球有3.5億人口感染慢性B型肝炎，台灣B型肝炎的帶原者約3、400萬人(15%~20%)，C型肝炎的帶原者約3、40萬(2%)，慢性肝病每年約造成6,000人死亡，肝癌死亡則達5,000人，全球肝炎市場和需求仍快速擴張中，在1930年代約有90%的處方藥或成藥，是屬於植物來源的藥品，但隨著科技進步，合成藥物也逐漸成為目前的常用藥物，但仍有許多問題有待克服，因此化學藥物、生物製劑、中藥衍伸成分，都已成為全球醫藥產業的重要趨勢，希望能在這拯救肝疾病的行列之中，扮演積極角色，渴望不管在肝保健與治療上，能為更多病患帶更多的福祉。慢性肝病及肝硬化近來總位於國人十大死因之列，至今很少藥物能治療或預防。Liver disease is a common disease among Chinese. At present, 350 million people worldwide are infected with chronic hepatitis B. The number of patients with hepatitis B in Taiwan is about 3 million (15% to 20%), and those with hepatitis C are about 3 400,000 (2%), chronic liver disease causes about 6,000 deaths per year, and liver cancer deaths reach 5,000. The global hepatitis market and demand are still expanding rapidly. In the 1930s, about 90% of prescription drugs or finished drugs belonged to plant sources. Drugs, but with the advancement of science and technology, synthetic drugs have gradually become the commonly used drugs, but there are still many problems to be overcome, so chemical drugs, biological agents, Chinese medicine derivatives have become an important trend in the global pharmaceutical industry, hope It can play an active role in the rescue of liver diseases, and is eager to bring more benefits to more patients regardless of liver health and treatment. Chronic liver disease and cirrhosis have always been among the top ten causes of death among Chinese people. So far, few drugs can be treated or prevented.
肝纖維化是由不同病因引起慢性肝損傷，啟動肝星狀細胞(HSC)，促進以膠原為主的細胞外基質(ECM)各成份合成增多，降解減少或代償不足，從而使ECM在肝臟異常沉積而引起的肝臟的纖維化病變。肝纖維化進一步發展，導致肝小葉結構改建，假小葉及結節形成，稱為肝硬化。肝硬化可以繼發或併發多種嚴重疾病。肝硬化不可逆轉，但在肝纖維化細胞分子學機制研究日益發展之下，其前期病理進程肝纖維化可能可應用化學藥物控制或逆轉，在抗肝纖維化或治療肝硬化之應用，非常重要。Liver fibrosis is caused by different causes of chronic liver injury, which initiates hepatic stellate cells (HSC), promotes the synthesis of collagen-based extracellular matrix (ECM) components, reduces degradation or undercompensation, and makes ECM abnormal in the liver. Fibrotic lesions of the liver caused by deposition. Further development of liver fibrosis leads to structural alteration of the hepatic lobule, formation of pseudolobules and nodules, called cirrhosis. Cirrhosis can be secondary or complicated by many serious diseases. Liver cirrhosis is irreversible, but in the development of molecular mechanisms of hepatic fibrosis cells, liver fibrosis may be controlled or reversed by chemical agents in the early pathological process. It is very important in the application of anti-fibrosis or cirrhosis. .
正-亞丁基苯酞(n-butylidenephthalide,n-BP)已知為當歸(Angelica sinensis)及川芎(Ligusticum chuanxiong)等植物中所含揮發油的主成分之一(Chao and Lin,Chinese Medicine,2011,6:29;Yan et al.,Drug Metabolism and Disposition,2008,36:400-408)，其具有如下所示結構One of the main component of volatile oil benzene phthalate, butylene (n-butylidenephthalide, n-BP ) is known as Angelica (Angelica sinensis) and Chuanxiong (Ligusticum chuanxiong) and the like contained in a plant (Chao and Lin, Chinese Medicine, 2011, - n 6:29; Yan et al., Drug Metabolism and Disposition, 2008, 36: 400-408), having the structure shown below
於先前文獻中已知，亞丁基苯酞具有多種生化功能，例如具有端粒酶活性抑制活性，而有用於作為抑制/治療腦瘤(TW I298259)；可與香茅醇組合，有用於抑制一氧化氮及/或***素E 2生成以及減輕發炎(TW I331033)；可經由抑制質子幫浦的活性而抑制胃酸分泌，有用於消化性潰瘍的治療(KR20050023998)；與1,3-雙(2-氯乙基)-1-亞硝基脲(BCNU)足合而有用於抑制人類肝腫瘤細胞的生長(Yu et al.,J Agric Food Chem,2010,58:1630-1638)；經由誘發Nur77受體的表現而有用於肝細胞腫瘤的治療(Chen et al.,2008,Mol Pharmacol,74:1046-1058)。It is known in the prior literature that butylene benzoquinone has various biochemical functions, such as telomerase activity inhibitory activity, and is useful as an inhibitory/therapeutic brain tumor (TW I298259); it can be combined with citronellol to inhibit one. Nitric oxide and/or prostaglandin E 2 production and alleviation of inflammation (TW I331033); inhibition of gastric acid secretion by inhibiting proton pump activity, treatment for peptic ulcer (KR20050023998); and 1,3-double (2 -Chloroethyl)-1-nitrosourea (BCNU) is sufficient for inhibiting the growth of human liver tumor cells (Yu et al., J Agric Food Chem, 2010, 58: 1630-1638); by inducing Nur77 The expression of the receptor is useful for the treatment of hepatocellular tumors (Chen et al., 2008, Mol Pharmacol, 74: 1046-1058).
然而，目前尚未有人提出亞丁基苯酞對於包括肝發炎、肝硬化、肝纖維等肝損傷具有治療效果，同時也未有證據顯示，亞丁基苯酞，對於肝損傷具有治療效果之外，也具有逆轉肝纖維化病理進程所導致之肝損傷的肝功能恢復效果。However, it has not been proposed that butylidene benzoquinone has therapeutic effects on liver damage including liver inflammation, cirrhosis, liver fibrosis, etc., and there is no evidence that butylene phenyl hydrazine has a therapeutic effect on liver damage, and also has Reversing the liver function recovery effect of liver damage caused by pathological process of liver fibrosis.
本發明係提供一種用於預防或改善肝損傷之醫藥組成物，其係包含治療有效量之式(I)所示之正-亞丁基苯酞(n-butylidenephthalide)化合物(Z型)或其E型、或E型與Z型的混合、或其等之醫藥上可接受鹽或酯作為有效成份：The present invention provides a pharmaceutical composition for preventing or ameliorating liver damage, which comprises a therapeutically effective amount of a n-butylidenephthalide compound (Z type) represented by formula (I) or an E thereof. Type, or a mixture of E and Z, or a pharmaceutically acceptable salt or ester thereof as an active ingredient:
以及醫藥上可接受之載劑。And a pharmaceutically acceptable carrier.
於本發明的一態樣中，該有效成份係化學合成者。於本發明的一態樣中該有效成份係由植物萃取而得者。於本發明的一態樣中，該醫藥組成物係當歸(Angelica sinensis)及川芎(Ligusticum chuanxiong)之萃取物。In one aspect of the invention, the active ingredient is a chemical synthesizer. In one aspect of the invention, the active ingredient is derived from a plant. In one aspect of the present invention, the pharmaceutical composition of Angelica (Angelica sinensis) and Chuanxiong (Ligusticum chuanxiong) of extract.
本發明亦提供一種使用前述式(I)所示之正-亞丁基苯酞化合物化合物(Z型)或其E型、或E型與Z型的混合、或其等之醫藥上可接受鹽或酯作為有效成份於製造用於治療肝損傷之醫藥品之用途。於本發明的一態樣中，該有效成份係化學合成者。於本發明的一態樣中，該有效成份係由植物萃取及分離而得者。The present invention also provides a mixture of the n-butylene benzoquinone compound (Z type) represented by the above formula (I) or its E form, or a mixture of E type and Z form, or a pharmaceutically acceptable salt thereof or The use of an ester as an active ingredient for the manufacture of a medicament for the treatment of liver damage. In one aspect of the invention, the active ingredient is a chemical synthesizer. In one aspect of the invention, the active ingredient is obtained by plant extraction and separation.
於本發明的一態樣中，該植物包括(但不限於)當歸(Angelica sinensis)及川芎(Ligusticum chuanxiong )。In one aspect of the present invention, the plant including (but not limited to) Angelica (Angelica sinensis) and Chuanxiong (Ligusticum chuanxiong).
於本發明的一態樣中，該肝損傷係肝纖維化、肝硬化或肝發炎。In one aspect of the invention, the liver injury is liver fibrosis, cirrhosis or liver inflammation.
於本發明的一態樣中，該醫藥組成物及醫藥品的投藥劑量為每天每公斤體重該有效成份8 mg至500 mg。In one aspect of the invention, the pharmaceutical composition and the pharmaceutical preparation are administered in an amount of from 8 mg to 500 mg per kg of body weight of the active ingredient per day.
於本發明的一態樣中，該肝損傷係由病毒、酒精或化學藥物引起。In one aspect of the invention, the liver injury is caused by a virus, alcohol or chemical agent.
於本發明的一態樣中，該醫藥組成物及醫藥品係經由口服、皮下、皮膚塗抹、靜脈注射、或一緩釋型式予以投藥。In one aspect of the invention, the pharmaceutical composition and the pharmaceutical product are administered orally, subcutaneously, dermally, intravenously, or in a sustained release form.
本發明亦提供一種改善肝功能之醫藥組成物，一種用於改善肝功能之醫藥組成物，其係包含治療有效量之前述式(I)所示之正-亞丁基苯酞(n-butylidenephthalide)化合物(Z型)或其E型、或E型與Z型的混合、或其等之醫藥上可接受鹽或酯作為有效成份。The present invention also provides a pharmaceutical composition for improving liver function, a pharmaceutical composition for improving liver function, which comprises a therapeutically effective amount of n-butylidenephthalide represented by the above formula (I). The compound (Z form) or its E form, or a mixture of the E form and the Z form, or a pharmaceutically acceptable salt or ester thereof, as an active ingredient.
本發明亦提供一種使用前述式(I)所示之正-亞丁基苯酞化合物化合物(Z型)或其E型、或E型與Z型的混合、或其等之醫藥上可接受鹽或酯作為有效成份於製造用於改善肝功能之醫藥品之用途。The present invention also provides a mixture of the n-butylene benzoquinone compound (Z type) represented by the above formula (I) or its E form, or a mixture of E type and Z form, or a pharmaceutically acceptable salt thereof or The use of an ester as an active ingredient for the manufacture of a medicament for improving liver function.
於本發明的一態樣中，該肝功能之改善包括製造白蛋白能力的改善及縮短凝血時間。In one aspect of the invention, the improvement in liver function includes an improvement in the ability to produce albumin and a shortened clotting time.
本發明將以下列實施例進一步具體說明，惟該等實施例之揭示不應視為任何限制本發明之意圖。The invention is further illustrated by the following examples, but the disclosure of the examples should not be construed as limiting the invention.
本發明所提供之醫藥組成物，其係包含治療有效量之式(I)的正-亞丁基苯酞化合物化合物(Z型)或其E型、或E型與Z型的混合、或其等之醫藥上可接受鹽或酯作為有效成份：The pharmaceutical composition provided by the present invention comprises a therapeutically effective amount of a n-butylene benzoquinone compound (Z type) of the formula (I) or an E form thereof, or a mixture of E type and Z type, or the like A pharmaceutically acceptable salt or ester as an active ingredient:
以及醫藥上可接受之載劑。And a pharmaceutically acceptable carrier.
醫藥上可接受之鹽類，只要為醫藥上可接受且為無毒性鹽者即可，並無特別限定，例如可為無機鹽類或有機鹽類。醫藥上可接受之酯，只要為醫藥上可接受且為無毒性酯即可，並無特別限定。於本發明之醫藥組成物中，可根據需要選擇正-亞丁基苯酞化合物的Z型或E型、或E型與Z型的混合、或其等之醫藥上可接受鹽或酯，並無特別限定。The pharmaceutically acceptable salt is not particularly limited as long as it is pharmaceutically acceptable and non-toxic, and may be, for example, an inorganic salt or an organic salt. The pharmaceutically acceptable ester is not particularly limited as long as it is pharmaceutically acceptable and is a non-toxic ester. In the pharmaceutical composition of the present invention, a Z-form or an E-form of a n-butylene benzoquinone compound, or a mixture of an E-form and a Z-form, or a pharmaceutically acceptable salt or ester thereof, may be selected as needed. Specially limited.
正-亞丁基苯酞可被化學合成。另外，正-亞丁基苯酞已知存在於當歸(Angelica sinensis)或川芎(Ligusticum chuanxiong)等藥用植物中，且其萃取方法已見於先前技術，如TW 200616656所揭示之當歸之丙酮萃取方法。以此項技術領域所習知之萃取方法，自植物萃取所得之萃取物也可使用作為本發明醫藥組成物中之有效成分。n-Butylene benzoquinone can be chemically synthesized. Further, n-butylidene benzoquinone is known to be present in medicinal plants such as Angelica sinensis or Ligusticum chuanxiong, and its extraction method has been found in the prior art, such as the Angelica extract method of Angelica sinensis disclosed in TW 200616656. The extract obtained by plant extraction can also be used as an active ingredient in the pharmaceutical composition of the present invention by an extraction method conventionally known in the art.
醫藥上可接受之載劑，為無毒性、惰性固體或半固體、稀釋劑、囊封裝物質、凝膠基劑或任何形式之配方佐劑，對於不同投藥方式，可利用習知方法將醫藥組成物配製成各種適當劑型(dosage form)。合適之醫藥可接受載劑為此項技術領域中所熟知。A pharmaceutically acceptable carrier is a non-toxic, inert solid or semi-solid, a diluent, a pouch, a gel base or a formulation adjuvant of any form. For different modes of administration, the pharmaceutical composition can be formed by conventional methods. The materials are formulated into a variety of suitable dosage forms. Suitable pharmaceutical acceptable carriers are well known in the art.
取決於投藥途徑，患者年齡、健康狀況、體重、欲治療之症狀等，醫藥組成物之投藥形式可為口服、皮下、皮膚塗抹、靜脈注射、或緩釋型式予以投藥。可根據習知醫藥組合技術與醫藥可接受載劑混合，其中該載劑取決於所欲投藥之製劑形式而可有寬廣範圍之形式。Depending on the route of administration, the patient's age, health status, weight, symptoms to be treated, etc., the pharmaceutical composition can be administered orally, subcutaneously, dermally, intravenously, or in a sustained release form. It may be admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical combination techniques, wherein the carrier may be in a wide range depending on the form of preparation to be administered.
此處所描述的概念將以下列實施例進一步說明之，該等實施例不限制申請專利範圍中所描述本發明之範疇。The concepts described herein are further illustrated by the following examples, which do not limit the scope of the invention described in the claims.
本發明以大鼠慢性肝損傷模式證實其可行性。The invention confirms the feasibility of the rat chronic liver injury mode.
[建立慢性肝纖維化模式以及評估肝臟功能][Building a pattern of chronic liver fibrosis and assessing liver function]
本實施例中係以自LASCO公司(台灣)獲得之102隻八週齡Wistar大鼠進行。所有步驟皆遵循道德準則規範且為國立東華大學之研究機構之動物照護與使用委員會所核准。In the present example, 102 8-week-old Wistar rats obtained from LASCO Corporation (Taiwan) were used. All steps are in accordance with ethical guidelines and approved by the Animal Care and Use Committee of the research institute of the National Dong Hwa University.
為了建立大鼠慢性肝損傷模式，將78隻成年雄性Wistar大鼠(320±20g)以每公斤體重5毫升硫代乙醯胺(TAA(Sigma-Aldrich))，每三日一次進行8周腹腔注射作為纖維化模式組。其餘24隻作為正常組(Normal組)，以一般生理鹽水取代TAA進行操作。於第4、6、8及10週結束時(亦即分別為第28、42、56及72日)，犧牲動物且收集心臟血液樣品。該等樣品以生化分析儀(Integra 800；Roche，Hollidton，MA，USA)測定肝臟功能指標，包括麩胺草醋酸轉胺酶(GOT)、麩胺丙酮酸轉胺酶(GPT)、血清白蛋白以及凝血酶原時間。In order to establish a rat model of chronic liver injury, 78 adult male Wistar rats (320 ± 20 g) were given 5 ml of thioacetamide per kg body weight (TAA (Sigma-Aldrich)), and the peritoneal cavity was performed once every three days for 8 weeks. Injection was performed as a group of fibrotic patterns. The remaining 24 were used as the normal group (Normal group), and the normal saline was used instead of TAA. At the end of Weeks 4, 6, 8, and 10 (i.e., Days 28, 42, 56, and 72, respectively), animals were sacrificed and heart blood samples were collected. These samples were tested for liver function using a biochemical analyzer (Integra 800; Roche, Hollidton, MA, USA), including glutamic acid transaminase (GOT), glutamine pyruvate transaminase (GPT), serum albumin. And prothrombin time.
本實施例使用之正-亞丁基苯酞(Alfa,cat. A10353)以橄欖油為溶劑配製成每毫升80毫克或每毫升500毫克之劑量進行下列實驗。纖維化模式組之大鼠於誘導第4週時，隨機分成以下四組：(1) BP 80 mg/kg治療組(共18隻)，於5週到8週TAA誘導期間以每天一次之頻率與每公斤體重80毫克正-亞丁基苯酞之劑量進行灌食治療；(2) BP 500 mg/kg治療組(共18隻)，於第5週到8週TAA誘導期間以每天一次之頻率與每公斤體重500毫克正-亞丁基苯酞之劑量進行灌食治療；(3)控制組(Control)(共18隻)，於第5週到8週TAA誘導期間以每天一次之頻率與每公斤體重1毫升橄欖油之劑量進行大鼠灌食；(4) TAA組(共24隻)，於第5週到8週TAA誘導期間不進行治療。N-butylene benzoquinone used in this example (Alfa , cat. A10353) The following experiment was carried out with olive oil as a solvent to prepare a dose of 80 mg per ml or 500 mg per ml. Rats in the fibrotic model group were randomly divided into the following four groups at the 4th week of induction: (1) BP 80 mg/kg treatment group (18 total), once daily during the TAA induction period from 5 weeks to 8 weeks With a dose of 80 mg of n-butylidene benzoquinone per kg of body weight; (2) BP 500 mg/kg treatment group (18 total), once every day during the 5th to 8th week of TAA induction With a dose of 500 mg of n-butylidene benzoquinone per kg of body weight for feeding; (3) Control group (control of 18), during the 5th to 8th week of TAA induction, once a day and every time Rats were fed with a dose of 1 ml of olive oil in kg; (4) TAA group (24 in total), and no treatment was performed during TAA induction from week 5 to week 8.
誘導期間，大鼠肝損傷程度可先初步以肉眼觀察其是否逐漸呈毛髮不佳、沉鬱、消瘦、口角染有暗紅色穢液並有下痢便等中毒特徵後，於第4、6、8、10週各犧牲6隻未治療之TAA組，檢測其血清中GOT、GPT、總膽紅素、白蛋白濃度、凝血酶原時間等肝功能指標，並由肝組織切片染色結果判定肝臟發炎與纖維化之情形。During the induction period, the degree of liver injury in rats can be firstly observed by the naked eye whether it is gradually showing poor hair, depression, weight loss, dark red sputum in the mouth and sputum and other poisoning characteristics, after 4, 6, and 8. At the 10th week, 6 untreated TAA groups were sacrificed, and liver function indexes such as GOT, GPT, total bilirubin, albumin concentration and prothrombin time were measured, and liver inflammation and fiber were determined by liver tissue section staining results. The situation.
自犧牲之大鼠取得肝臟後，將最大右葉肝割取厚約0.3公分組織塊固定於10%的中性福馬林(formalin)液中，再以石蠟包埋。包埋組織的連續3-μm切片以蘇木紫-伊紅染色法(hematoxylin and eosin)或梅生三色染色(Masson's trichrome)進行染色來辨識細胞型態及纖維化組織中的膠原蛋白堆積情形。肝纖維化為膠原蛋白過量累積於肝臟中，且因而膠原蛋白的累積為評估肝纖維化之重要指標。梅生三色染色法通常用於建定纖維化肝臟中膠原蛋白組織的增加。梅生三色染色法係將經染色之樣品於56℃置於Bouin’s溶液(Sigma-Aldrich)中1小時後，依序以下列溶液染色：Mayer's hematoxylin solution(Sigma-Aldrich) 5分鐘、Biebrich scarlet-acid fuchsin ssolution(Sigma-Aldrich) 15分鐘、phosphomolybdic acid-phosphotungstic acid(Sigma-Aldrich) 15分鐘以及aniline blue(Sigma-Aldrich) 5分鐘。After the liver was obtained from the sacrificed rat, the largest right lobe liver was cut into a thickness of about 0.3 cm and fixed in 10% neutral formalin solution, and then embedded in paraffin. Serial 3-μm sections of the embedded tissue were stained with hematoxylin and eosin or Masson's trichrome to identify collagen accumulation in cell type and fibrotic tissue. Liver fibrosis is an accumulation of collagen in the liver, and thus the accumulation of collagen is an important indicator for evaluating liver fibrosis. Meisheng trichrome staining is commonly used to establish an increase in collagen tissue in fibrotic liver. The Meisheng trichrome staining method stained samples were placed in Bouin's solution (Sigma-Aldrich) at 56 ° C for 1 hour, and then stained sequentially with the following solution: Mayer's hematoxylin solution (Sigma-Aldrich) for 5 minutes, Biebrich scarlet-acid Fuchsin ssolution (Sigma-Aldrich) for 15 minutes, phosphomolybdic acid-phosphotungstic acid (Sigma-Aldrich) for 15 minutes and aniline blue (Sigma-Aldrich) for 5 minutes.
控制組、BP 80 mg/Kg治療組及BP 500 mg/Kg治療組的大鼠於第6、8及10週結束時(亦即分別為第42、56及72日)，犧牲動物且收集心臟血液樣品。該等樣品以生化分析儀(Integra 800；Roche，Hollidton，MA，USA)測定肝臟功能指標，包括麩胺草醋酸轉胺酶(GOT)、麩胺丙酮酸轉胺酶(GPT)、血清白蛋白、總膽紅素以及凝血酶原時間。然後，自犧牲之大鼠所取得之肝臟組織亦進行組織學分析。Rats in the control group, BP 80 mg/Kg treatment group, and BP 500 mg/Kg treatment group at the end of weeks 6, 8 and 10 (ie, on days 42, 56, and 72, respectively), sacrificed animals and collected hearts. Blood sample. These samples were tested for liver function using a biochemical analyzer (Integra 800; Roche, Hollidton, MA, USA), including glutamic acid transaminase (GOT), glutamine pyruvate transaminase (GPT), serum albumin. , total bilirubin and prothrombin time. Then, the liver tissue obtained from the sacrificed rats was also subjected to histological analysis.
其結果示於第1圗及第2圖。第1a圗及第2a圖顯示於各種實驗組中大鼠肝臟之總體檢查結果。應注意者為肝臟的顏色與體積，是否有滲出、增厚或結塊的存在，以及肝臟表面為平滑或有結節。正常的肝臟為紅棕色且具有平滑表面，而由BP-80 mg/kg及500 mg/kg治療組可觀察到甚至較正常肝臟為佳的病理現象。但在受到損傷而有病理學上的變化時，肝臟變大且具有顆粒表面與僵硬的質感，如同控制組所觀察到者。The results are shown in Figures 1 and 2. Figures 1a and 2a show the results of the overall examination of the rat liver in various experimental groups. It should be noted that the color and volume of the liver, the presence of exudation, thickening or agglomeration, and the smooth or nodular surface of the liver. The normal liver was reddish brown with a smooth surface, and even better than normal liver pathology was observed in the BP-80 mg/kg and 500 mg/kg treatment groups. However, when there is a pathological change due to injury, the liver becomes large and has a grainy surface and a stiff texture, as observed by the control group.
受到損傷的肝臟組織的蘇木紫-伊紅染色切片顯示(第1b圖及第2b圖)，由TAA誘發肝纖維化大鼠的肝臟可觀察到組織空洞化、壞死與細胞核的降解。BP-80 mg/kg及500 mg/kg治療組的大鼠於第8週與第10週時明顯可見組織降解與空洞化的恢復，於控制組(橄欖油)仍顯示嚴重的發炎與壞死。The hematoxylin-eosin stained sections of the damaged liver tissue (Fig. 1b and Fig. 2b) showed that tissue hollowing, necrosis and nuclear degradation were observed in the liver of rats with liver fibrosis induced by TAA. Rats in the BP-80 mg/kg and 500 mg/kg treatment groups were significantly able to recover from tissue degradation and vacancies at weeks 8 and 10, and showed severe inflammation and necrosis in the control group (olive oil).
第1c圖及第2c圖的梅生三色染色解明了於肝纖維化大鼠中，膠原蛋白於肝臟之嚴重的累積。BP-80 mg/kg及500 mg/kg治療組於第8週及第10週時，可觀察到膠原蛋白的降解。BP-80 mg/kg及500 mg/kg治療組於第12週時幾乎無膠原蛋白的累積，而控制組仍顯示實質的膠原蛋白的累積。The three-color syrup staining of Figures 1c and 2c illustrates the severe accumulation of collagen in the liver in rats with liver fibrosis. Collagen degradation was observed in the BP-80 mg/kg and 500 mg/kg treatment groups at weeks 8 and 10. The BP-80 mg/kg and 500 mg/kg treatment groups had almost no collagen accumulation at week 12, while the control group still showed substantial collagen accumulation.
本發明係採用METAVUIIR scoring system為標準進行評估。其評分指標如下表所示：The present invention was evaluated using the METAVUIIR scoring system as a standard. The scoring indicators are shown in the following table:
其結果示於第3圖及第4圖。所有數據顯示為平均值與標準偏差。為了比較兩組之間不同處置的效果，數據以Student’s t-test分析。當p<0.05時認為有顯著性(*)。當p<0.01時認為有高顯著性(**)。當p<0.001時認為有極高顯著性(***)。其餘圖式之數據處理方式相同。The results are shown in Figures 3 and 4. All data are shown as mean and standard deviation. To compare the effects of different treatments between the two groups, the data was analyzed by Student's t-test. Significant (*) was considered when p < 0.05. High significance (**) was considered when p < 0.01. When p < 0.001, it was considered to be extremely significant (***). The rest of the schema is handled in the same way.
由第3圖及第4圖結果可知，TAA誘發後，大鼠的肝纖維化與發炎活性指標分數，明顯增高。而於BP-80 mg/kg及500 mg/kg治療組，於第8及10週的纖維化指數與發炎活性，顯著降低。控制組則仍維持與TAA組相當之纖維化指數與發炎活性。From the results of Fig. 3 and Fig. 4, it was found that the scores of liver fibrosis and inflammatory activity of rats were significantly increased after TAA induction. In the BP-80 mg/kg and 500 mg/kg treatment groups, the fibrosis index and inflammatory activity at 8 and 10 weeks were significantly reduced. The control group still maintained a fibrosis index and inflammatory activity comparable to the TAA group.
GOT(glutamic oxaloacetic transaminase麩胺草醋酸轉胺酶)及GPT(麩胺丙酮酸轉胺酶)為細胞酵素，當細胞發炎或變性時，就會從細胞被釋放到血液中。雖然GOT及GPT存在於心臟、肌肉等其他細胞中，但肝細胞中的GOT及GPT活性最高，所以，GOT及GPT在血液中的濃度可以作為肝細胞是否發炎或變性的指標。GOT (glutamic oxaloacetic transaminase) and GPT (glutamine pyruvate transaminase) are cytokines that are released from the cells into the bloodstream when the cells become inflamed or degenerated. Although GOT and GPT are present in other cells such as heart and muscle, the GOT and GPT activities in hepatocytes are the highest. Therefore, the concentration of GOT and GPT in the blood can be used as an indicator of whether the liver cells are inflamed or degenerated.
結果示於第5圖。由第5圖可知，在以TAA誘發大鼠慢性肝損傷4週後，GOT及GPT的濃度明顯上升(GOT由91.5±24.8 U/L增加至348.0±198.7 U/L，GPT由43.1±9.3 U/L增加至101.1±28.9 U/L)。第10週後，在BP-80 mg/kg及500 mg/kg治療組的GOT及GPT濃度均明顯下降。由此可知，正-亞丁基苯酞對於包括肝纖維化及肝發炎之肝損傷，具有顯著的功效。The results are shown in Figure 5. As can be seen from Fig. 5, the concentration of GOT and GPT increased significantly after 4 weeks of chronic liver injury induced by TAA (GOT increased from 91.5±24.8 U/L to 348.0±198.7 U/L, and GPT was 43.1±9.3 U. /L increased to 101.1 ± 28.9 U / L). After the 10th week, the concentrations of GOT and GPT in the BP-80 mg/kg and 500 mg/kg treatment groups were significantly decreased. From this, it can be seen that n-butylidene benzoquinone has a remarkable effect on liver damage including liver fibrosis and hepatic inflammation.
總膽紅素包括直接膽紅素和間接膽紅素。直接膽紅素亦稱為結合膽紅素，是經過肝臟處理後與葡萄糖醛酸結合的水溶性的膽紅素，間接膽紅素即非結合膽紅素，是細胞破壞後游離出的未被肝臟處理的非水溶性的膽紅素。總膽紅素增高，直接膽紅素、間接膽紅素也增高，常見於肝細胞性黃疸如急性黃疸型肝炎、重症肝炎、慢性活動性肝炎、肝硬化等。所以在臨床生化分析上，也將總膽紅素作為肝功能的指標之一。Total bilirubin includes direct bilirubin and indirect bilirubin. Direct bilirubin, also known as bilirubin, is a water-soluble bilirubin that binds to glucuronic acid after liver treatment. Indirect bilirubin, which is unconjugated bilirubin, is liberated after cell destruction. Liver-treated water-insoluble bilirubin. Increased total bilirubin, direct bilirubin, indirect bilirubin also increased, commonly seen in hepatic jaundice such as acute jaundice hepatitis, severe hepatitis, chronic active hepatitis, cirrhosis. Therefore, in clinical biochemical analysis, total bilirubin is also used as an indicator of liver function.
其結果示於第6圖。由第6圖可知，以TAA誘發大鼠慢性肝損傷4週後，相較於正常組，總膽紅素明顯升高。而在進行正-亞丁基苯酞的灌食後，BP-80 mg/kg及500 mg/kg治療組的總膽紅素相較於控制組，明顯降低。由此可知，正-亞丁基苯酞可明顯改善肝損傷之肝功能。The result is shown in Fig. 6. As can be seen from Fig. 6, after 4 weeks of chronic liver injury induced by TAA, total bilirubin was significantly increased compared with the normal group. After feeding with n-butylene benzoquinone, the total bilirubin in the BP-80 mg/kg and 500 mg/kg treatment groups was significantly lower than that in the control group. It can be seen that n-butylidene benzoquinone can significantly improve the liver function of liver injury.
白蛋白係於肝臟細胞中合成，而且因為其半衰期長達20天，則白蛋白的量明顯減少，被認為是起因於肝臟長時期的發炎或疾病。因此，臨床上也將白蛋白作為肝功能的指標之一。Albumin is synthesized in liver cells, and because its half-life is up to 20 days, the amount of albumin is significantly reduced, which is thought to be caused by inflammation or disease of the liver for a long period of time. Therefore, albumin is also clinically used as an indicator of liver function.
其結果示於第7圖。由第7圖的結果可知，白蛋白的濃度，以TAA誘發慢性肝損傷4週時，並無顯著變化的原因，應係白蛋白的半衰期為20天左右。而8週後，TAA組及控制組則可見顯著的減少(相對於正常組)。但以正-亞丁基苯酞灌食後，BP-80 mg/kg及500 mg/kg治療組的白蛋白的濃度不僅回復且達相當於正常組的水準，由此可知，正-亞丁基苯酞對於肝臟長時期損傷，亦有顯著改善功效。The result is shown in Fig. 7. From the results of Fig. 7, it can be seen that the concentration of albumin did not change significantly when TAA induced chronic liver injury for 4 weeks, and the half-life of albumin should be about 20 days. After 8 weeks, a significant reduction (relative to the normal group) was seen in the TAA group and the control group. However, after feeding with n-butylene benzoquinone, the concentrations of albumin in the BP-80 mg/kg and 500 mg/kg treatment groups not only recovered but reached the level equivalent to the normal group, which indicates that n-butylidene benzoquinone It also has a significant improvement in liver damage over a long period of time.
凝血酶原是凝血因子的前身。凝血因子中有一大部分是肝臟製造的，所以當肝的功能(特別是合成能力)不好時，除了白蛋白降低之外，凝血因子也將降低而造成凝血時間延長。測量此數值之意義在於了解體內凝血因子的量及功能，藉以測定肝臟功能是否正常。Prothrombin is the precursor to clotting factors. A large part of the clotting factor is made by the liver, so when the function of the liver (especially the ability to synthesize) is not good, in addition to the decrease in albumin, the clotting factor will also decrease and the clotting time will be prolonged. The significance of measuring this value is to understand the amount and function of coagulation factors in the body to determine whether the liver function is normal.
其結果示於第8圖。由該結果可知，以TAA誘發大鼠的肝損傷後，明顯導致凝血時間的延長(相較於正常組)。而以正-亞丁基苯酞灌食後，相較於控制組，BP-80 mg/kg及500 mg/kg治療組的凝血時間縮短，且恢復到與正常組相當的時間。由此可知，正-亞丁基苯酞對於肝損傷的改善，尤其是肝的再生能力，具有顯著的效果。The result is shown in Fig. 8. From the results, it was revealed that the liver injury induced by TAA significantly caused an increase in clotting time (compared to the normal group). After feeding with n-butylidene benzoquinone, the clotting time of the BP-80 mg/kg and 500 mg/kg treatment groups was shortened compared with the control group, and returned to the normal group for a comparable period of time. From this, it can be seen that n-butylidene benzoquinone has a remarkable effect on the improvement of liver damage, especially the ability of the liver to regenerate.
由上述研究結果顯示，相較於控制組，經口服正-亞丁基苯酞治療四週後，血清生化分析顯示治療組之GOT、GPT及總膽紅素皆較早恢復正常水平，其白蛋白及凝血酶原時間亦有明顯恢復，而其膠原蛋白堆積程度及肝發炎情形亦顯著改善；證明正-亞丁基苯酞幫助肝損傷之修復，並具有可治療及預防肝纖維化和肝硬化的潛力。From the above research results, serum biochemical analysis showed that the GOT, GPT and total bilirubin in the treatment group returned to normal levels earlier than the control group, after treatment with oral n-butylene benzoquinone, the albumin and Prothrombin time also recovered significantly, and the degree of collagen accumulation and liver inflammation were also significantly improved; it proved that n-butylene benzoquinone helps repair liver damage and has the potential to treat and prevent liver fibrosis and cirrhosis. .
根據本發明提供一種醫藥組成物，其係包含正-亞丁基苯酞作為活性成分，且可有效的改善包括肝發炎、肝纖維化及肝硬化等肝損傷，有用於作為肝損傷之預防及/或治療用途。According to the present invention, there is provided a pharmaceutical composition comprising n-butylidene benzoquinone as an active ingredient, and which can effectively improve liver damage including liver inflammation, liver fibrosis and cirrhosis, and is useful for prevention of liver damage and/or Or therapeutic use.
在上述說明中，已描述關於特定實施例之概念。然而，本領域中具有通常知識之技術人員應理解在不脫離下列申請專利範圍所提出之本發明範圍的情況下，可進行各種修改和變更。因此，應將本說明書視為說明性而非限制性，且意欲將所有這類修改涵括於本發明之範疇中。In the above description, the concept of a particular embodiment has been described. However, it will be understood by those skilled in the art that various modifications and changes can be made without departing from the scope of the invention as set forth in the appended claims. Accordingly, the description is to be considered as illustrative and not restrictive.
第1a圖為控制組(橄欖油投用組)與正-亞丁基苯酞(80 mg)對TAA誘發肝纖維化之總體觀察圖。Figure 1a shows the overall observation of TAA-induced liver fibrosis in the control group (orange oil application group) and n-butylene benzoquinone (80 mg).
第1b圖為控制組(橄欖油投用組)與正-亞丁基苯酞(80 mg)對TAA誘發肝纖維化之蘇木紫-伊紅染色圖。Figure 1b is a staining diagram of the hematoxylin-eosin staining of TAA-induced liver fibrosis in the control group (orange oil application group) and n-butylene benzoquinone (80 mg).
第1c圖為控制組(橄欖油投用組)與正-亞丁基苯酞(80 mg)對TAA誘發肝纖維化之梅生三色染色圖。Figure 1c shows the trichochrome staining of TAA-induced liver fibrosis in the control group (orange oil application group) and n-butylidene benzoquinone (80 mg).
第2a圖為控制組(橄欖油投用組)與正-亞丁基苯酞(500 mg)對TAA誘發肝纖維化之總體觀察圖。Figure 2a shows the overall observation of TAA-induced liver fibrosis in the control group (orange oil application group) and n-butylidene benzoquinone (500 mg).
第2b圖為控制組(橄欖油投用組)與正-亞丁基苯酞(500 mg)對TAA誘發肝纖維化之蘇木紫-伊紅染色圖。Figure 2b is a staining diagram of the hematoxylin-eosin staining of TAA-induced liver fibrosis in the control group (orange oil application group) and n-butylene benzoquinone (500 mg).
第2c圖為控制組(橄欖油投用組)與正-亞丁基苯酞(500 mg)對TAA誘發肝纖維化之梅生三色染色圖。Figure 2c shows the trichochromic staining of TAA-induced liver fibrosis in the control group (orange oil application group) and n-butylene benzoquinone (500 mg).
第3圖為各實驗組治療纖維化之肝纖維化指標分數圖。Figure 3 is a graph showing the scores of liver fibrosis indicators for treatment of fibrosis in each experimental group.
第4圖為各實驗組治療纖維化之肝發炎活性指標分數圖。Fig. 4 is a graph showing the scores of liver inflammatory activity indicators for treating fibrosis in each experimental group.
第5A圖為正-亞丁基苯酞對TAA誘發肝損傷之GOT變化之影響圖。Figure 5A is a graph showing the effect of n-butylidene benzoquinone on GOT changes in TAA-induced liver injury.
第5B圖為正-亞丁基苯酞對TAA誘發肝損傷之GOT變化之影響圖。Figure 5B is a graph showing the effect of n-butylidene benzoquinone on GOT changes in TAA-induced liver injury.
第6圖為正-亞丁基苯酞對TAA誘發肝損傷之總膽紅素之影響圖。Figure 6 is a graph showing the effect of n-butylene benzoquinone on total bilirubin induced by TAA in liver injury.
第7圖為正-亞丁基苯酞對TAA誘發肝損傷之白蛋白之影響圖。Figure 7 is a graph showing the effect of n-butylidene benzoquinone on albumin induced by TAA.
第8圖為正-亞丁基苯酞對TAA誘發肝損傷之凝血酶原時間之影響圖。Figure 8 is a graph showing the effect of n-butylidene benzoquinone on prothrombin time of TAA-induced liver injury.
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| WO2016015203A1 (en) * | 2014-07-28 | 2016-02-04 | 国立中兴大学 | Use of butenylphthalide, usage method thereof and method for preparing same into pharmaceutical composition |
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