一種含葡萄子萃取物配方之營養品’其主要係以牛石黃酸、 beta-胡蘿蔔素、葡萄子萃取物中之前花青素等混合加工，取其 具改善慢性肝炎的效果，另添加適量維生素£、維生素C等抗氧 化食品，以期達到更減輕慢性肝炎造成之傷害者。 六、英文新型摘要： 七、指定代表圓： (一） 本案指定代表圖為··第（j )圖 (二) 本代表圖之元件符號簡單說明： A-四氯化碳中毒 · 母 B-Silymarin 治療 C-前花青素配方治療 D-正常值 八、新型說明： 【發明之領域】 特別 私明疋有關於含葡萄子萃取物配方之營養品，， 者。種可有奴知性肝炎所造成傷害及症狀之營養品 【發明之背景】 硬化長Hi肝炎會導致肝臟纖維化，嚴重者，會造成肝 ’月之G〇T和GPT值昇高；自由基及其傷害 正< -MiA，脂質過氧化在肝臟纖維化的過程佔重要的角色。 慢性肝炎最終其血清白蛋白、肝臟蛋白質含量減少，脾臟 重量增加，肝臟脂質過氧化、及膠原蛋白含量增加。 已知四氯化碳所引起的肝炎、肝纖維化與其產生自由 基及脂質過氧化有密切的關係。因此一些抗氧化劑如維生 素E、C能改善四氯化碳誘發的慢性肝炎，最近的文獻也 指出胡蘿蔔素有改善慢性肝炎的效果。 牛磺酸(Taurine)是一種必須氨基酸，是體内抗氧化 劑之一（endogenous ant i oxi dant)。很多疾病與缺 taurine有關，包含肝功能不足，臨床也用於 改善肝功能。最近的研究顯示taurine對四氯化碳誘發急 性肝炎及肝細胞損傷有保護作用。對酒精所引起的脂肪肝 和脂質過氧化也有改善作用。 紅葡萄酒中所含的前花青素(Pr〇anthr〇cyanidins)具 有相當強的抗氧化作用，有益人類健康可用於預防多種疾 病，尤其對心A管疾病。目前國外已普遍的將葡萄軒抽出 物應用在健康食品。 有鑑於此，發明人乃針對上述食品應用於改善慢性肝 义造成傷害方面加以研究改進，終於有本發明產生。 【發明之綜合說明】 本叙明曰在共一種含葡萄子萃取物配方之營養品， 其主要係斜猶、beta—娜蔔素、前花青素等混合， 1274551A nutrient containing grape seed extract formula, which is mainly prepared by mixing bovine ascorbate, beta-carotene, and anthocyanin in grape seed extract, and has the effect of improving chronic hepatitis, and adding appropriate amount Antioxidant foods such as vitamins £ and vitamin C are expected to reduce the damage caused by chronic hepatitis. Sixth, the new type of abstract in English: VII. Designated representative circle: (1) The representative representative of the case is as follows: (j) (2) The symbolic symbol of the representative figure is simple: A-carbon tetrachloride poisoning · mother B- Silymarin treatment of C-proanthocyanidin formula treatment D-normal value eight, new description: [field of invention] special private diet with nutritional formula containing grapefruit extract,,. Nutritional products that can cause harm and symptoms caused by ignorant hepatitis. [Background of the invention] Hardening of Hi hepatitis can lead to liver fibrosis. In severe cases, the liver's G〇T and GPT values increase; free radicals and Its damage is positive - -MiA, lipid peroxidation plays an important role in the process of liver fibrosis. Chronic hepatitis eventually reduces serum albumin, liver protein content, spleen weight, liver lipid peroxidation, and collagen content. Hepatitis and liver fibrosis caused by carbon tetrachloride are known to be closely related to the production of free radicals and lipid peroxidation. Therefore, some antioxidants such as vitamins E and C can improve carbon tetrachloride-induced chronic hepatitis. Recent literature also points out that carotene has an effect of improving chronic hepatitis. Taurine is an essential amino acid and one of the endogenous ant i oxi dant. Many diseases are associated with a lack of taurine, including inadequate liver function, and are also used clinically to improve liver function. Recent studies have shown that taurine has a protective effect on carbon tetrachloride-induced acute hepatitis and hepatocyte damage. It also has an effect on fatty liver and lipid peroxidation caused by alcohol. The proanthocyanidins (Pr〇anthr〇cyanidins) contained in red wine have a strong anti-oxidation effect, which is beneficial to human health and can be used to prevent various diseases, especially for heart A tube disease. At present, grape stalk extracts have been widely used in health foods abroad. In view of this, the inventors have made research and improvement on the application of the above-mentioned foods to improve the damage caused by chronic liver, and finally the present invention has been produced. [Comprehensive Description of the Invention] This article describes a nutrient containing grape seed extract formula, which is mainly mixed with oblique, beta-nabin, proanthocyanidins, etc., 1274551
If? H ί 利用其具改善慢性肝炎的特性，夯添知雄維生素 c、玉米澱粉等食品，以期達到減輕慢性肝炎造成之傷害， 此為本發明之主要目的。 【較佳具體實施例之描述】 一、食品内容··（較佳重量比之組合成份) a.牛磺酸：50-90%。 b.維生素E : 1.8-2%。 c·維生素C: 8_8.25%。 d· beta-胡蘿蔔素：〇.5也55%。 e.前花青素·· 6-25%。 f·玉米澱粉或其他賦形劑：0-9.7%。 今以下列之比例作實驗之： a·牛磺酸：約70%。 b· b·維生素E :約1.8%。 c·維生素C :約8%。 d·前花青素：約1〇〇/。。 e· beta胡蘿蔔素：約〇. 5〇/。。 f·玉米殿粉或其他賦形劑：約9· 7〇/〇。 二、實驗評估·· 1.材料與方法 a·試驗物質配製： 上述組成之食品内容粉末狀，以0.5% CMC分別配 補充丨成濃度為10、50及100mg/ml之懸浮液，經口投予 、投予體積為每100克大鼠體重投與lml毫升。 對照藥物silymarin也同樣懸浮於〇·5% CMC，配 製濃度為60mg/ml。 b·劑量計算： 以大鼠200公克與70公斤人的表面積比值為〇· 〇18 為依據，若人體的建議劑量為每天1g，依據實 驗動物與人體表面積比等效劑量換算比率計算， 大鼠每天的劑量為90 mg/kg。選用劑量為每天投 予〇· ：1、0· 5、1· 0 g/kg，分別為人體建議劑量的 1.1、5· 5、Π· 1倍。控制組大鼠給予同體積的 〇· 5% CMC溶液。對照藥物siiymarin (sigma)使 用劑量為0· 6 g/kg。 2.實驗環境： 使用雄性Wistar大鼠，購自國科會國家實驗動物繁 殖及研究中心，飼養於中國醫藥學院醫學系動物室 。動物室設定溫度為22± 2 °C，光照為12小時亮、 12小時暗（早上7點亮，下午7點暗）。動物經兩 週適應後，篩選出健康大鼠使用。飼料使用福壽牌 老鼠飼料，飲水經逆滲透處理。 在試驗物質投予前一天，大鼠依體重大小，分成適 當重量群，進行隨機分組，除正常控制組7隻外， 其餘5組每組動物12隻。 3·試驗設計： 大鼠先投予試驗物質7天，而後每週兩次經口投予 四氯化碳溶液，每週第一次投予時，大鼠先絕食一 晚，第二次投予時不絕食。四氯化碳投予時間為早 上8點。試驗物質每天投予，時間為下午1點。於 四氣化碳投予後滿一、三、六、八週時，大氣在乙 醚麻醉下由尾動脈抽血，供血清生化值檢定，採血 當天，在採血之後才投予試驗物質。第八週採血時 ，大鼠也同時犧牲，迅速取下肝臟及脾臟，以冰冷 生理g鹽水洗淨後，吸乾水分，稱重。最大葉肝臟 切下1/4相同部位，浸於10%中性福馬林溶液，供 病理切片使用，其餘肝臟分裝3袋，儲存於一8(rc 備用。 每週稱體重一次，做為當週投予試驗物質的體重依 據。四氯化碳溶於橄欖油配製成2〇%的溶液，每次 投予量為0· 5 ml/rat。 (本試驗設計與衛生署公告的方法财不同，特別 加以說明如下：四氯化碳投予的方式可以s c·、 1· Ρ·、ι· m·或ρ· 〇·，基於經驗，四氯化碳溶於撤揽 油不易由s’a給藥’ ；ί·ρ·時部分肝臟會與四氣化碳 直接作用’且死亡率高，ρ 〇·四氯化碳經吸收後作 用到肝臟’ _較-致，城亡輪低。因此在本 試驗使用Ρ.α的方法。至於每週第—次投予先絕食 -晚’第二次投予不絕食，其目的在於控制產生的 肝損傷不會太嚴；t，也稍於太輕。試驗物質每天 投予，不因投予四氯化碳而中斷，乃因大鼠已中毒 ，為求試驗物質的有效，轉試驗物f—定的血中 濃度是必要的。採金時在投予試驗物之前，若在藥 物投予之後2小時採血，時間上較難掌控一致。） 4·檢驗測定 a·血清生化學檢驗 血液取得後，靜置待其凝固後，以47〇〇 rpm離心 15分鐘，取血清供生化檢驗用。第一、三、六週 只進行GOT及GPT檢驗，第八週最後犧牲時，除檢 驗GOT、GPT外，也測定血清白蛋白（aibumin)含 里。檢驗使用市售檢驗試劑（Roche)，以血清生 化自動分析儀測定(COBAS MIRA)。 b·血液凝血時間（pr〇thrombin time)檢驗 弟八週大鼠犧牲時取得的血液，部分與sod citrate(5%)以9:1混合，供血液血凝時間檢驗。 c·肝臟組織glutathione(GSH)含量測定 組織GSH 的測定依據Sedlak and Lindsay(1968) 的方法，使用5,5’-dithiobis-(2-nitrobenzoic acid)呈色，在412nm測吸光度。以//mole/g wet weight表示之。 d·肝臟組織lipid peroxidation測定 組織的脂質過氧化測定依據Ottawa et al(1979) 的方法，使用2_thiobarbituric acid呈色，在 532nm測吸光度。脂質過氧化的程度#njn〇le malondialdehyde/g wet weight表示之。 e·肝臟組織膠原蛋白（hydroxyprol ine)含量測定 Hydroxy proline的測定參照Neuman and Logan (1950)的方法。肝臟乾組織水解後加H2〇2氧化， 再以p-dimethylaminobenzoaldehyde呈色，於 540 nm測吸光值。Hydroxyprol ine 量以 mg/g tissue表示之。 f·肝臟組織 superoxide dismutase (SOD)、 catalase 、 glutathione peroxidase (GSH-Px) 活性測定If? H ί is used for improving the characteristics of chronic hepatitis, adding foods such as vitamin c and corn starch to reduce the damage caused by chronic hepatitis. This is the main purpose of the present invention. [Description of Preferred Embodiments] 1. Food Content·(Better Weight Ratio Combination) a. Taurine: 50-90%. b. Vitamin E: 1.8-2%. c· Vitamin C: 8_8.25%. d· beta-carotene: 〇.5 is also 55%. e. Proanthocyanidins · 6-25%. f·corn starch or other excipients: 0-9.7%. The experiment is carried out in the following proportions: a. Taurine: about 70%. b· b·Vitamin E: about 1.8%. c· Vitamin C: about 8%. d·proanthocyanidins: about 1〇〇/. . e· beta carotene: about 〇. 5〇/. . f· corn house powder or other excipients: about 9·7〇/〇. II. Experimental evaluation·· 1.Materials and methods a·Test substance preparation: The food content of the above composition is powdered and supplemented with 0.5% CMC to form a suspension with a concentration of 10, 50 and 100 mg/ml. The volume of administration and administration was 1 ml ml per 100 g of rat body weight. The control drug silymarin was also suspended in 〇·5% CMC at a concentration of 60 mg/ml. b. Dose calculation: Based on the ratio of the surface area of 200 grams of rats to 70 kg, 建议·〇18, if the recommended dose of the human body is 1g per day, based on the equivalent dose conversion ratio of experimental animals to human body surface area, rats The daily dose is 90 mg/kg. The dosage to be administered is 每天·: 1,0·5, 1·0 g/kg per day, which is the recommended dose of 1.1, 5·5, Π·1 times for the human body. The control group rats were given the same volume of 〇·5% CMC solution. The control drug siiymarin (sigma) was administered at a dose of 0.6 g/kg. 2. Experimental environment: Male Wistar rats were purchased from the National Experimental Animal Breeding and Research Center of the National Science Council and kept in the Animal Department of the Medical College of China Medical College. The animal room was set at a temperature of 22 ± 2 °C, and the light was illuminated for 12 hours and dark for 12 hours (7 in the morning and 7 in the afternoon). After two weeks of adaptation, the animals were screened for healthy rats. The feed uses Fushou brand mouse feed, and the drinking water is treated by reverse osmosis. One day before the administration of the test substances, the rats were divided into appropriate weight groups according to their body weight and randomly divided into groups, except for the normal control group of 7 rats, and the remaining 5 groups of 12 animals each. 3. Experimental design: The rats were first administered with the test substance for 7 days, then the oral administration of carbon tetrachloride solution twice a week. When the first dose was given every week, the rats were hunger for one night and the second time. Not hunger strikes. The carbon tetrachloride was administered at 8 o'clock in the morning. The test substance was administered daily at 1 pm. At the first, third, sixth, and eighth weeks after the administration of the four gasification carbons, the atmosphere was drawn from the tail artery by diethyl ether anesthesia for serum biochemical value verification. On the day of blood collection, the test substance was administered after blood collection. At the time of blood collection in the eighth week, the rats were also sacrificed at the same time. The liver and spleen were quickly removed, washed with ice-cold physiological g saline, and the water was absorbed and weighed. The largest leaf liver was cut into 1/4 of the same site, immersed in 10% neutral formalin solution for pathological section, and the remaining liver was divided into 3 bags and stored at 8 (rc spare). Weigh once a week, as The weight of the test substance is administered in a week. The carbon tetrachloride is dissolved in olive oil to prepare a 2% solution, and the dosage is 0.5 ml/rat. (The design of this experiment and the method announced by the Department of Health Different, especially explained as follows: the method of carbon tetrachloride can be given sc·, 1· Ρ·, ι· m· or ρ· 〇·, based on experience, carbon tetrachloride dissolved in the withdrawal oil is not easy by s' a administration '; ί·ρ· part of the liver will directly interact with the four gasified carbon' and the mortality rate is high, ρ 〇 · carbon tetrachloride is absorbed into the liver after absorption _ _ _, the city is low. Therefore, in this test, the method of Ρ.α is used. As for the first-weekly hunger strike-night, the second dose is not hunger strike, the purpose is to control the liver damage not too strict; t, also slightly Too light. The test substance is administered daily and is not interrupted by the administration of carbon tetrachloride. It is because the rat has been poisoned and is effective for the test substance. It is necessary to transfer the blood concentration of the test substance f. In the case of gold mining, if the blood is collected 2 hours after the administration of the drug, it is difficult to control the time in time.) 4. Test determination a· serum After the biochemical test blood was obtained, it was allowed to stand for coagulation, and then centrifuged at 47 rpm for 15 minutes to take serum for biochemical tests. Only GOT and GPT tests were performed in the first, third and sixth weeks, and the last sacrifice in the eighth week. In addition to GOT and GPT, serum albumin (aibumin) was also determined. The test was performed using a commercially available test reagent (Roche) and measured by a serum biochemical analyzer (COBAS MIRA) b. Blood clotting time (pr〇thrombin) Time) The blood obtained from the sacrifice of the eight-week rat was partially mixed with sod citrate (5%) for 9:1 for blood hemagglutination time test. c. Determination of tissue GSH by liver tissue glutathione (GSH) The method of Sedlak and Lindsay (1968) uses 5,5'-dithiobis-(2-nitrobenzoic acid) coloration to measure absorbance at 412 nm, expressed as //mole/g wet weight. d· Liver tissue lipid peroxidation assay tissue Lipid peroxidation assay The absorbance was measured at 532 nm according to the method of Ottawa et al (1979) using 2_thiobarbituric acid. The degree of lipid peroxidation is expressed by #njn〇le malondialdehyde/g wet weight. e. Determination of hydroxyprol ine content The determination of Hydroxy proline is based on the method of Neuman and Logan (1950). The liver tissue was hydrolyzed and then H2〇2 was oxidized, and then p-dimethylaminobenzoaldehyde was colored, and the absorbance was measured at 540 nm. The amount of Hydroxyprol ine is expressed in mg/g tissue. f· Liver tissue superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) activity assay
i. SOD 組織之前處理依據Xia et al (1995)的方法。 活性的測定依據Marklund and Marklund (1974)的方法。S0D活性定義為抑制 Pyrogallel自動氧化還原反應速率5〇%所需酵 素的量為一個單位(U)，以u/mg protein表示 之。蛋白質測定依具Lowry et al. (1951)的方 法。 ii. Glutathione peroxidase (GSH-Px) 肝組織之前處理依據Xia et al.(1995)的方法。 GSH-Px活性測定依據Hafeman et al. (1974)的方 法’測定時分為酵素反應與非酵素反應兩部份。 GSH-Px活性單位表示為： U=LogA[GSH]E-LogA[GSH]NE/1000 ΔΤ ^ △ [GSH]E :為酵素反應減少GSH的濃度， △ [GSH]NE :表示非酵素反應減少GSH的濃度。 肝組織的GSH-Ρχ活性以u/mg protein表示之。 iii. Catalasei. The SOD organization was previously processed according to the method of Xia et al (1995). The activity was determined according to the method of Marklund and Marklund (1974). S0D activity is defined as the amount of enzyme required to inhibit Pyrogallel's automatic redox reaction rate of 5 〇 % as one unit (U) expressed as u/mg protein. Protein assays are based on the method of Lowry et al. (1951). Ii. Glutathione peroxidase (GSH-Px) Liver tissue was previously treated according to the method of Xia et al. (1995). The GSH-Px activity assay was divided into two parts, an enzyme reaction and a non-enzyme reaction, according to the method of Hafeman et al. (1974). The GSH-Px activity unit is expressed as: U=LogA[GSH]E-LogA[GSH]NE/1000 ΔΤ ^ △ [GSH]E : Reduces the concentration of GSH for the enzyme reaction, △ [GSH]NE : indicates a decrease in non-enzyme reaction The concentration of GSH. The GSH-Ρχ activity of liver tissue is expressed in u/mg protein. Iii. Catalase
Catalase活性測定依據Aebi(1984)的方法。其 活性定義以K (一級反應之速率常數，min) 為一個單位（U)，以u/mg protein表示之。 K的計算方法如右： K=(2. 3/t2-tl)(l〇gAl/A2) ，其中Al:為tl=0秒時之吸光值，A2:為T2=25 秒時之吸光值。 g·病理檢驗 肝臟組織經福馬林固定後，進行臘包埋及切片製 作，使用兩種染色法，一為一般的Η·Ε· stain 1274551 成' i、編· (HematoxyHn and eosin stain)，另一種為膠 原蛋白的特殊染色即Masson，s stain。病理學比 較依照衛生署公告的護肝功能評估辦法，將肝臟 的損傷或纖維化分為四個等級，加以評分。為了 使病變的評分客觀病理切片的製作及判讀皆委託 臺灣養豬科學研究所病理生物系進行。 5·統計分析 除了病理判讀數據外，其它實驗所得數據均以單尾 變異數分析（one-way analysis of variance)，並 進行Dunnet測試，以p值小於0·05認為有顯著意義 。病理判讀依據結果以Kruskall Wallis η變異數分 析，而後進行Mann-Whitney U test檢定，以ρ值小 於0· 05認為有顯著意義。 三、結果 在為期九週的投予試驗物質過程中，正常控制組7隹 皆存活很好，四氯化碳組及給藥組各12隻，其中少 數大鼠對四氣化碳過於敏感而導致死亡或非常的瘦弱 ，剃除這些的數據，每組可用數據為1〇隻。 1·體重變化 四氯化碳組投予六週後大鼠平均體重明顯低於控制 組。試驗物質或silymarin處理之大氣對體重沒有 影響。但大氣犧牲時四氣化碳組及組有 10 1274551 祕、' U.i, ίΜ」嚴重腹水出現，若考慮脫水重量，經投藥之大鼠（ 0· 5、1· 0 g/kg)其體重或有可能較四氯化碳組高 2·對血清GOT、GPT值的影響 四氯化碳組投予之後第一、三、六及八週大鼠血清 GOT、GPT值均顯著高於控制組。於第一週，試驗物 - 質三個劑量組明顯降低GOT、GPT值。於第三、第六 — 及苐八週’试驗物質其用量依存性減少GOT、GPT值 。Silymarin處理之大鼠，僅降低第一週的G〇T、 GPm 〇 3·對血清albumin、及血液pr〇thrombin time的影響 四氯化碳組大鼠最後的血清a 1 bumin值較控制組低 ，而prothrombin time較控制組長。試驗物質高 劑里處理之大鼠可以增加血清以]^!!^]!值。試驗物 質對prothrombin time的縮短作用，雖無統計上的 ‘ 思義’但具有縮短的傾向。Silymarin處理之大鼠 對這些皆沒有影響。 4·對肝臟和脾臟重量及腹水隻數的影響 四氯化碳組大鼠最後的肝臟重量與控制組比較沒有 明顯差異，試驗物質高劑量可明顯增加肝臟重量。 四氯化碳組大鼠最後的脾臟重量明顯較控制組高。 試驗物質高劑量可減輕脾臟重量。大鼠最後犧牲時 ，四氯化碳組多數大鼠出現腹水，試驗物質可以減 11 1274551 彻彳' ^ I少腹水出現的隻數。Si lymarin處理之大鼠對這些 .如〜^*^一一 皆沒有影響。 5·對肝臟glutathione、lipid peroxidation、 protein、和hydroxyproline含量的影響 四氣化碳誘發大鼠慢性肝炎’導致肝臟的protein 含量降低。lipid peroxidation、hydroxyproline - 值上升，對glutathione含量沒有影響。試驗物質 可增加蛋白質含量，及減少hydroxyproline含量。 高劑量也可以降低肝臟lipid peroxidation程度。 試驗物質對glutathione含量沒有影響。Si lymar in 對這些皆沒有影響。 6·對肝臟S〇D、Catalase及GSH-Px活性的影響 四氯化碳誘發大鼠慢性肝炎，使肝臟抗氧化三種酵 素S 0 D、Catalase及GSH-Px的活性明顯低於控制 ^ 組，試驗物質對S 0 D、GSH-Px的活性沒有影響， 試驗物質（0· 5g/kg)可增加Catalase活性，但不具 用量依存性，Si lymar in對這三種酵素的活性皆沒 有影響。 7·病理變化 四氯化碳誘發大鼠慢性肝炎，以HE染色可以明顯 看出月曰肪/儿積及組織變性的情形。以Mass〇n染色， 在中央靜脈區及門脈區明顯出現結節纖維化之情形 12 1274551 。實驗物質之處理有意義的改善這些病變，Catalase activity was determined according to the method of Aebi (1984). Its activity is defined by K (rate constant of the first order reaction, min) as a unit (U) expressed as u/mg protein. The calculation method of K is as follows: K=(2. 3/t2-tl)(l〇gAl/A2), where Al: is the absorbance at tl=0 sec, and A2: absorbance at T2=25 sec. . g. Pathological examination After liver tissue was fixed by formalin, it was embedded in wax and sliced. Two kinds of staining methods were used, one was general Η·Ε· stain 1274551 into 'i, 编· (HematoxyHn and eosin stain), another One is the special staining of collagen, Masson, s stain. Pathological comparisons The liver damage or fibrosis was classified into four grades according to the Department of Health's liver function assessment method. In order to make the scores of the objective pathological sections of the lesions, the interpretation and interpretation were entrusted to the Department of Pathology, Taiwan Institute of Pig Research. 5. Statistical analysis In addition to the pathological interpretation data, the data obtained from other experiments were analyzed by one-way analysis of variance, and the Dunnet test was performed. The p-value was less than 0.05 and considered to be significant. The pathological interpretation results were analyzed by Kruskall Wallis η variability, and then the Mann-Whitney U test was performed. The ρ value was less than 0.05 and considered to be significant. 3. Results During the nine-week trial of the test substances, the normal control group survived very well. There were 12 carbon tetrachloride groups and 12 drug-administered groups, and a few of them were too sensitive to four-carbonized carbon. Leading to death or very thin, shaving these data, the available data for each group is 1〇. 1. Change in body weight The average body weight of rats in the carbon tetrachloride group was significantly lower than that in the control group after six weeks of administration. The test substance or the atmosphere treated with silymarin had no effect on body weight. However, when the atmosphere is sacrificed, the four gasified carbon groups and groups have 10 1274551 secret, 'Ui, ίΜ' severe ascites. If the dehydration weight is considered, the weight of the administered rats (0.5·1·0 g/kg) or It may be higher than the carbon tetrachloride group. 2. Effects on serum GOT and GPT values The serum GOT and GPT values of the rats in the first, third, sixth and eighth weeks after the administration of the carbon tetrachloride group were significantly higher than those in the control group. In the first week, the test substance-quality three dose groups significantly reduced the GOT and GPT values. In the third, sixth, and eighth weeks, the dose dependence of the test substances decreased the GOT and GPT values. In Silymarin-treated rats, only the first week of G〇T, GPm 〇3, serum albumin, and blood pr〇thrombin time were reduced. The final serum a 1 bumin value of the rats in the carbon tetrachloride group was lower than that of the control group. , and the prothrombin time is longer than the control group. Rats treated with high test substances can increase the serum to the value of ^^!!^]! The shortening effect of the test substance on the prothrombin time has a tendency to shorten although there is no statistical ‘thinking’. Rats treated with Silymarin had no effect on these. 4. Effects on liver and spleen weight and ascites only The final liver weight of the rats in the carbon tetrachloride group was not significantly different from that of the control group. The high dose of the test substance significantly increased the liver weight. The final spleen weight of the rats in the carbon tetrachloride group was significantly higher than that in the control group. High doses of test substance can reduce the weight of the spleen. At the final sacrifice of the rats, ascites occurred in most rats in the carbon tetrachloride group, and the test substance could be reduced by 11 1274551. Rats treated with Si lymarin had no effect on these, such as ~^*^. 5. Effects on liver glutathione, lipid peroxidation, protein, and hydroxyproline content Four-gasified carbon-induced chronic hepatitis in rats resulted in a decrease in protein content in the liver. The lipid peroxidation and hydroxyproline values increased, and had no effect on the glutathione content. The test substance increases the protein content and reduces the hydroxyproline content. High doses can also reduce the degree of liver lipid peroxidation. The test substance had no effect on the glutathione content. Si lymar in has no effect on these. 6. Effects on liver S〇D, Catalase and GSH-Px activity Carbon tetrachloride induced chronic hepatitis in rats, which significantly inhibited the activity of three antioxidant enzymes S 0 D, Catalase and GSH-Px in the liver. The test substance had no effect on the activity of S 0 D and GSH-Px. The test substance (0.5 g/kg) increased the activity of Catalase, but it was not dependent on the amount. Si lymar in had no effect on the activity of these three enzymes. 7. Pathological changes Carbon tetrachloride induced chronic hepatitis in rats. The HE staining can clearly show the situation of lunar pericarposis/infancy and tissue degeneration. Stained with Mass〇n, nodular fibrosis occurred in the central venous and portal areas 12 1274551 . The treatment of the experimental substance significantly improves these lesions,
Silymarin對這些沒有減輕作用。 曰由上述咸驗可知’由人的建議用量推算出試驗劑 里之4驗物貝’能明顯降低肝炎指標G〇T、GpT 值及肝纖維化的指標，如：血清蛋白減少、腹水出現 、脾腫大及肝臟膠原蛋白的增加。 肝臟受到傷害’使血清中之G〇T、GpT活性上昇 ，是最常見崎臟損傷生化指標，其中該GpT較有專一性， 而GOT亦存在於心臟、腎臟、骨絡肌、腦部。本織以四氣 化碳造成肝臟損傷，血清中G 〇 T、G p τ的活㈣顯上升， 而試驗物質能降低血清中之G〇T、听活性，顯示其能減 輕四氯化碳對肝臟造成之損傷。 GOT、GPT只能靜態的反應出賴最近受到的傷 ^雖是肝臟所有傷害之指標，但其無法估算肝臟還殘留 夕少能力。血液的凝固時間反應出肝臟合成凝血因子之能 力’月中之蛋白主要來自肝臟的合成，因此在慢性肝 二二長’血清白蛋白值下降’在本試驗四氯化 钱射变性肝炎，最後也出現血清白蛋白值下降，及血液 壯％間延長之情形，試驗物質能提昇血清中之白蛋白 值’有縮短凝血時間之傾向 慢性肝炎的肝魏減退。-料四减破誘發 肝臟蛛傷時會啟動再生的功能，因此肝臟重量增 13 ^274551 丄屬但若嚴重時肝臟會萎输 料肝” 擔讀喊誘發大鼠Silymarin has no mitigating effect on these.曰 From the above-mentioned salt test, it can be seen that 'the recommended amount of the human being used to calculate the 4 test specimens in the test agent' can significantly reduce the indicators of hepatitis G, T, GpT and liver fibrosis, such as: decreased serum protein, ascites, Splenomegaly and an increase in liver collagen. The liver is damaged. The activity of G〇T and GpT in serum is increased. It is the most common biochemical indicator of sagging injury. The GpT is more specific, and GOT is also present in the heart, kidney, skeletal muscle and brain. This weaving caused liver damage with four carbonized carbon, and the activity of G 〇T and G p τ in serum increased (4), while the test substance could reduce G〇T and auditory activity in serum, indicating that it can alleviate carbon tetrachloride. Damage caused by the liver. GOT and GPT can only react statically to the recent injuries. Although it is an indicator of all liver damage, it cannot estimate the ability of the liver to remain. The clotting time of the blood reflects the ability of the liver to synthesize clotting factors. The protein in the month is mainly derived from the synthesis of the liver, so the serum albumin value decreases in the chronic liver two-long 'in this test. When the serum albumin value decreases and the blood is prolonged, the test substance can increase the albumin value in the serum, which has a tendency to shorten the clotting time, and the liver Wei declines in chronic hepatitis. - The fourth dose is reduced. When the liver is injured, the liver will start to regenerate. Therefore, the liver weight increases by 13 ^ 274551. However, if it is severe, the liver will lose the liver.
㈣撕·量與㈣_較雖沒有差異，四氯化 碳組的肝臟明顯萎縮或明顯腫大，顯示最後誘導的狀況是 肝臟由腫捕絲縮_段，顺物f缺频重量靜 四氯化碳組，靖_賴胸含量，㈣示試驗 物質能增加肝臟再生能力。肝臟纖維化，使血液進入肝臟 党到阻力’引起門脈高壓，連帶影響到脾臟的也流，會使 脾臟禮大，在本試驗吨化碳婦大紐性肝炎，最^ 出現脾腫大讀形，試驗物質歧善_大之_，顯示 其有減輕肝臟纖維化之作用。(4) The amount of tearing and (4) _ Although there is no difference, the liver of the carbon tetrachloride group is obviously atrophied or obviously swollen, indicating that the last induced condition is that the liver is swollen by the swollen _ segment, the f The carbon group, Jing _ _ chest content, (four) shows that the test substance can increase liver regeneration capacity. Liver fibrosis, the blood into the liver party to the resistance 'caused portal hypertension, and the spleen affects the spleen, which will make the spleen ritual. In this trial, the ton of carbonized women with hepatitis, the most splenomegaly The test substance is _ _ _ _, which shows that it has the effect of reducing liver fibrosis.
慢性肝炎會引起肝臟纖維化，即結締組織增生，結締 組織主要由膠原蛋白構成，是膠原蛋白特 有的成分，測定Hydr〇xpr〇line的量可以反應膠原蛋白的 置’可用絲撕臟麟化的程度，在本試驗四氯化碳誘 叙大鼠丨艾性肝炎，其肝臟Hydroxproline含量明顯增加， 試驗物質能使Hydroxproline含量減少，顯示可減緩肝臟 纖維化的作用，此作用在組織病理檢驗得到進一步證 實。 已知四氯化碳造成肝臟纖維化，與自由基的傷害造成 lipid proxidation有關，在本試驗中，最後肝臟Hpid proxidation明顯增加，·本發明能降低lipid poxidation 的程度’顯示其護肝作用與減少自由基的傷害有密切關 14 12745¾ 在本試驗中，最後自由基與三種酵素S 〇 D、GSH-Px 及Catalase的活性明顯下降，試驗物質對此三種酵素沒有 改善作用，試驗物質亦不能增加肝臟内的抗氧化劑 glutathione含量，由這些結果推測，試驗物質的作用並 不是經由加強抗氧化酵素或抗氧化;劑glutathi〇ne的作， 主要還是其本身的抗氧化作用。 對照藥物silymarin依照衛生署公告的方法使用劑量 馨 為200mg/kg，本研究室先前的實驗以此劑量進行沒有得到 正面的結果。此次以600mg/kg進行，除了降低第一週的 GOT、GPT值外，對其餘的指標仍然沒有正面的結果。一些 臨床文獻指出Silymarin對慢性肝炎及肝硬化無效。動物 實驗方面，雖有對慢性四氣化碳肝損傷有效的結果，但也 有無效的結果。 為證實本發明之功效，請參閱第一及第二圖，其中第一圖所馨 示者為前花青素配方對肝臟傷害保護功能2SG〇t 值之比較，第二圖則為SGPT值比較，八週重複餵食，其中d為正 系值，A為四氯化碳中毒，b為經3办_^治療者，c為經前花 青素配方治療者，縱座標為週數，由上列二圖可知，前花青素配 方治療能使每週重複餵食大鼠四氯化碳致使SG0T及SGPT功能指 數幵尚現象彳于以改善，且改善之程度較法定治肝藥為 佳0 15 1274551 二二一第二圖為剷钯青素配方對四氯化碳傷害誘發之慢性肝炎血 清白蛋白含量之影響，而血清白蛋白之降低能導致水腫，其中d 為正常值，A為四氯化碳對肝之損傷，3為前花青素配方治療， 劑量效應為g/kg/day，C之劑量分別為〇·!、〇 5及1〇 (由左至 右），B之silymarin之則為〇· 6,由圖中明顯可知前花青素配方對 肝損傷之血清白蛋白有增加制，且其增加之幅度與配方劑量 成正相關，而法定治肝-silymarin則無明顯之功效。 第四圖前花青素配方對四氯化碳傷害引起之肝臟蛋白質的 影響，其蛋白質含量mg/g Tissue,劑量單位為g/kg/day其中〇為 正常質，A為四氯化碳之肝損傷，c為前花青素配方治療，分別 為〇·卜〇· 5及1· 〇 (由左至右），b為〇· Rsi 1γ·ίη，由圖中明 顯可知如^匕青素配方對因四氯化碳引起肝損傷致降低白蛋質含 量有增加_，且其增加之幅度触㈣彳量成正侧，而法定治 肝藥silymarin則無明顯之功效。 第五圖為前花青素配方對四氯化碳傷害肝臟引起脂肪過氧麝 化(LPO)作用之影響，lp〇之單位細m〇ie/mg pr〇tein ，其中D為正常值，A為四氯化碳肝損傷，C為前花青素配方治 療，其劑量為1· 〇G/KG/DAY，B為silymarin治療，由圖中可知，前 花青素配方能降低四氯化碳肝損傷引起之脂肪過氧化作用，但 silymarin則無此功效。 、 第六圖為前花青素配方對四氯化碳傷害肝臟引起大鼠之大 鼠隻數之f彡響，以每組十隻大u正常值為〇, a為四氯化碳 16Chronic hepatitis can cause liver fibrosis, that is, connective tissue hyperplasia. Connective tissue is mainly composed of collagen. It is a unique component of collagen. The amount of Hydr〇xpr〇line can be measured to reflect the presence of collagen. To the extent that in this test, carbon tetrachloride was used to induce sputum hepatitis in rats, the content of Hydroxproline in the liver was significantly increased, and the test substance could reduce the content of Hydroxproline, which showed that it can slow down the liver fibrosis. This effect was further confirmed by histopathological examination. Confirmed. It is known that carbon tetrachloride causes liver fibrosis, which is related to lipid proxidation caused by free radical damage. In this test, the liver Hpid proxidation is obviously increased. · The present invention can reduce the degree of lipid poxidation', showing its liver protection and reduction The damage of free radicals is closely related. 14 127453⁄4 In this test, the activity of free radicals and three enzymes S 〇D, GSH-Px and Catalase decreased significantly. The test substance did not improve the three enzymes, and the test substance could not increase the liver. The content of the antioxidant glutathione, from these results, speculated that the role of the test substance is not through the strengthening of antioxidant enzymes or antioxidants; glutathi〇ne, mainly its own antioxidant effect. The control drug silymarin was administered at a dose of 200 mg/kg according to the method published by the Department of Health. Previous experiments in this laboratory did not yield positive results at this dose. This time, at 600mg/kg, in addition to lowering the GOT and GPT values in the first week, there are still no positive results for the remaining indicators. Some clinical literature indicates that Silymarin is ineffective for chronic hepatitis and cirrhosis. In animal experiments, although there are effective results for chronic four-gasified carbon-hepatic injury, there are also ineffective results. To confirm the efficacy of the present invention, please refer to the first and second figures, wherein the first figure shows the comparison of the 2SG〇t values of the liver damage protection function of the proanthocyanidin formula, and the second figure compares the SGPT values. Repeated feeding for 8 weeks, where d is a positive value, A is carbon tetrachloride poisoning, b is treated by 3 _^, c is a pre-anthocyanidin formula, and the ordinate is the number of weeks. As shown in the second figure, the proanthocyanidin formula treatment can repeatedly feed the rats with carbon tetrachloride every week, which causes the SG0T and SGPT function index to be improved, and the degree of improvement is better than that of the legal liver medicine. 1274551 22:21 The second figure shows the effect of shovel palladium on the serum albumin content of chronic hepatitis induced by carbon tetrachloride injury, and the decrease of serum albumin can cause edema, where d is normal and A is tetrachloro Carbon damage to the liver, 3 for the treatment of proanthocyanidins, the dose effect is g / kg / day, the dose of C is 〇 ·!, 〇 5 and 1 〇 (from left to right), B of silymarin Then it is 〇·6, it is obvious from the figure that the proanthocyanidin formula has an increase in serum albumin for liver damage. And it increases the magnitude of dose formulations positive correlation, while the legal Liver -silymarin no significant effect. The fourth figure shows the effect of anthocyanin formula on liver protein caused by carbon tetrachloride injury. Its protein content is mg/g Tissue, the dosage unit is g/kg/day, where 〇 is normal and A is carbon tetrachloride. For liver injury, c is treated with anterior anthocyanin formula, which are 〇·卜〇·5 and 1· 〇 (from left to right), and b is 〇·Rsi 1γ·ίη, as is clearly seen from the figure. The formula has an increase in the content of white egg caused by liver damage caused by carbon tetrachloride, and the increase is in the range of (4) sputum into the positive side, while the legal liver medicine silymarin has no obvious effect. The fifth picture shows the effect of proanthocyanidin formula on the effect of carbon tetrachloride on the liver caused by fat peroxidation (LPO). The unit of lp〇 is fine m〇ie/mg pr〇tein, where D is normal, A For carbon tetrachloride liver injury, C is a proanthocyanidin formula, the dose is 1 · 〇 G / KG / DAY, B is silymarin treatment, as shown in the figure, the proanthocyanidin formula can reduce carbon tetrachloride Fat peroxidation caused by liver damage, but silymarin has no such effect. The sixth picture shows the number of rats in the rat induced by carbon tetrachloride in the anterior anthocyanin formula. The normal value of each group of ten large u is 〇, a is carbon tetrachloride.
之肝損傷’ C為前花！·素治療，其舰械_，分別為1、 0. 5及1.0 (由左至右）’ ^為如卿也治療由圖中可知前花 青素配方糾低四減韻剌起之產錢柄錢隻數功^ 但 silymarin則無。 由上所述可知，本發明之含葡萄子萃取物配方之營養 品確實具有降低血清GPT、GOT值、增加血清白蛋白 值、減輕脾麵大、增加賴蛋㈣含量、降低肝臟纖維 化及脂質過氧化程度之功效，確已具有產業上之利用性、 新顆性及進步性。 【圖式簡單說明】 第-圖為前花青素配核si lymaHn對賴傷#保護功能 之SG0T值之比較 第二圖為前花青素配方與silymarin對肝臟傷害保護功能 之SGPT值之比較 第三圖為前花青素配方對四氯化碳傷害誘發之慢性肝炎血 清白蛋白含量之影響 第四圖前花青素配方對四氣化碳傷害引起之肝臟蛋白質的 影響 第五圖為前花青素配方對四氯化碳傷害肝臟引起脂肪過氧 化作用之影響 第/、圖為前花青素配方對四氯化碳傷害肝臟引起大鼠之大 鼠隻數之影響 17 1274551 【主要元件符號酬】 A-四氯化碳中毒 B-Silymarin 治療 c-前花青素配方治療 D -正常值 18Liver damage 'C is the front flower! ·Sulf treatment, its _, _, 1, 0.5 and 1.0 (from left to right) ' ^ for the treatment of Ruqing also from the figure can be seen that the anterior anthocyanin formula to correct the four reduction rhyme The money is only counted ^ but the silymarin is not. It can be seen from the above that the nutrient containing grape seed extract formula of the present invention has the effects of lowering serum GPT, GOT value, increasing serum albumin value, reducing spleen surface, increasing lysed egg (4) content, reducing liver fibrosis and lipid. The effect of the degree of peroxidation has indeed been industrially utilized, new and progressive. [Simplified illustration] The first picture shows the comparison of the SG0T values of the protective function of the anterior anthocyanin nucleus si lymaHn. The second picture shows the comparison of the SGPT values of the anterior anthocyanin formula and silymarin on the liver injury protection function. The third picture shows the effect of proanthocyanidin formula on serum albumin content of chronic hepatitis induced by carbon tetrachloride injury. The fourth picture shows the effect of anthocyanin formula on liver protein caused by four gasification carbon damage. The effect of anthocyanin formula on the peroxidation of liver caused by carbon tetrachloride damage in the liver / / The picture shows the effect of proanthocyanidin formula on the number of rats in rats caused by carbon tetrachloride injury in the liver 17 1274551 [Main components Symbolic rewards A-carbon tetrachloride poisoning B-Silymarin treatment c-proanthocyanidin formula treatment D - normal value 18