200324586 玖'發明說明 (碰:編明所屬之技術領域、先前技術、內容、實施方式及圖式簡單說明) 【發明所屬之技術領域】 一種可用來保護肝臟的氨基酸、維生素組合複方係由glydne(甘胺酸)、alanine(丙 胺酸）、taurine(牛磺酸）、vit C (維他命C)、vitE(維他命E)搭配而成 [先前技術] 根據衛生署統計，國人肝臟疾病的罹患率及死亡率一直居高不下，肝癌在10大 死因的排行榜不是第一就是第二，稱爲台灣的國病。有鑑於此保肝健康食品的開 發爲當務之急。 之前硏究護肝的配方往往都是用單一配方，並硏究機制，不過卻往往對於老鼠 餵食極大的量，例如本配方中的甘胺酸，之前的硏究就是混入飼料，老鼠以5% 的胃食量去進行實驗，如果換算成人往往就需要極大的餵食量，雖然甘氨酸目前 沒有安全劑量上的限制，不過吃過多的氨基酸往往不見得會有好的現象，且硏究 者往往就只給予一個配方一種功效，而肝病的產生許多原因，有可能細菌性或病 毒性肝損傷、化學性肝損傷、酒精性肝損傷，而沒有一個配方有對於各類的肝損 傷完全進行硏究，所以本實驗就是收集文獻上硏究的配方，由肝臟損傷的路徑， 去找尋最佳的搭配組合的配方，而成本及風味的考量於本實驗中也給予注意到， 希望可以找到一個組合配方，對於肝臟各方面的疾病都有改善的效果。 【內容】 本發明之獨特創意主要在l〇〇cc的產品中，添加glycine(甘胺酸)(0.2-25 % )、 alanine(丙胺酸)(0.1-20% )、taurine(牛磺酸)(0.1-20 % )、維他命 C(〇.〇〇l -5 % )、維 他命Ε(0·0001-1% )依每個單方對於肝臟的作用機制進行搭配，使此配方可以達到 完整的保肝效果。且經實驗發現，確實可以在老鼠急性肝炎 (D-galactosamine/lipopolysaccharide模式），和慢性肝損傷(CC14模式)下發揮護肝的 效果，如圖一，且由肝臟生化指標GOT及GPT可以發現， D-galactosamine/lipopolysaccharide 的誘發下可以使 GOT 和 GPT 降 85%以上，而在 慢性肝炎的模式下可以降50%以上，如圖二，除此之外對於喝酒前使用此素材也 可以於酒後明顯降低血液中的酒精濃度(可以於酒後0.5小時，血中酒精濃^降低 60%以上），如圖三，而使酒後老鼠的活動力不會因爲酒精的影響而醉倒。而配方 中可能最有效的物質glycine，根據之前文獻發現，此複方的添加明顯比之前文獻 進行實驗添加劑量降低10倍以上，而效果依然顯著。 此配方應用廣，可以添加於牛奶及茶飮料，如此可以降低茶飲料的溫味及降低 牛奶製品中糖類的添加量，所以除了可以改善風味更可增加護肝機能性。 “ 200^24586 [實施方式] 在100cc的產品中，添加甘胺酸（0.2-25 % )、丙胺酸（0.1-20% )、牛磺酸(0.1-20 % )、維 他命C(0.001 % )、維他命Ε(0·0001-1% )之配方，依下列之實驗，確認其具有顯著之保 肝效果· 一、實驗方法 1 ·急性肝炎的實驗 老鼠(ICR strain)依下表格一進行分組並預先餵食老鼠3天，誘發前一晚使老鼠 隔夜禁食(16小時），第四天一早灌食所要測試的素材，一小時後，於腹腔注射半 乳糖胺(400mg/Kg)加上Lipopolysaccharide(3//g/Kg)進行急性肝炎的誘發。觀察老 鼠存活現象並於誘發後第6.5小時進行眼窩採血測GOT及GPT護肝指標 表格一：急性肝炎實驗的分組情形 餵食老鼠的素材 灌食次數 (共4天） 每次灌食相當於 人 實驗 η値 C-water 只餵食水 1 ^/day 100cc 5隻 1 X 護肝口服液 1 勿day 100cc 5隻 Ganoderma Tsugae 雙X極品靈芝 1 ^/day lOOcc-4顆膠囊 4隻 2·慢性肝炎的實驗 老鼠(Wistar大鼠)依下面表格二進行分組，本次試驗以口服投予方式供藥，其 中四氯化碳(20 %溶於橄欖油；0.2 ml /100 g body weight)或橄欖油每週投予 兩次，固定於週六及週三早上8:00 - 9:00。無菌水、Silymarin或試驗物質每日 固定於13:00-14:00投予。並於於試驗期間（第1、3、6週結束時）及犧牲後 採血，進行血清生化値。犧牲後並測量肝臟蛋白質含量及各項抗氧化指標 (superoxide dismutase、glutathione、glutathione peroxidase 及 catalase ) 表格二：慢性肝炎實驗分組情形200324586 发明 'Invention description (touch: indicate the technical field to which it belongs, the prior art, content, embodiments and diagrams) [Technical field to which the invention belongs] An amino acid and vitamin combination compound that can be used to protect the liver is made by glycdne ( Glycine), alanine (alanine), taurine (taurine), vit C (vitamin C), vitE (vitamin E) [Previous technology] According to the statistics of the Department of Health, the incidence and death of liver disease in Chinese The rate has always been high, and liver cancer is ranked first or second in the top 10 causes of death, known as Taiwan ’s national disease. In view of this, the development of liver-protecting healthy foods is a top priority. In the past, the formula for studying liver protection often used a single formula and studied the mechanism, but it often fed a large amount of mice, such as the glycine in this formula. The previous study was to mix it into the feed. For the experiment of gastric appetite, if it is converted into an adult, a large feeding amount is often required. Although there is currently no limit on the safe dose of glycine, it is not always good to eat too much amino acid, and researchers often only give it. One formula has one effect, and there are many causes of liver disease. There may be bacterial or viral liver damage, chemical liver damage, and alcoholic liver damage. None of the formulas fully investigate all types of liver damage. The experiment is to collect research formulas in the literature, from the path of liver damage to find the best combination formula, and cost and flavor considerations are also noted in this experiment, hoping to find a combination formula for the liver All aspects of disease have an improvement effect. [Content] The unique idea of the present invention is mainly in the 100cc product, adding glycine (glycine) (0.2-25%), alanine (alanine) (0.1-20%), taurine (taurine) (0.1-20%), Vitamin C (0.001-5%), Vitamin E (0.0001-1%) according to each single action mechanism of the liver, so that this formula can achieve a complete protection Liver effect. And it was found through experiments that it can indeed play a protective effect on liver in rats with acute hepatitis (D-galactosamine / lipopolysaccharide mode) and chronic liver injury (CC14 mode), as shown in Figure 1, and can be found from the liver biochemical indicators GOT and GPT. D-galactosamine / lipopolysaccharide can reduce GOT and GPT by more than 85%, and in chronic hepatitis mode, it can reduce more than 50%, as shown in Figure 2. In addition, this material can also be used after drinking. Significantly reduce the alcohol concentration in the blood (can be reduced by more than 60% in 0.5 hours after drinking alcohol, as shown in Figure 3), so that the activity of the drunk rats will not be drunk due to the influence of alcohol. Glycine, which may be the most effective substance in the formula, is found in the previous literature that the addition of this compound is obviously more than 10 times lower than the experimental addition in the previous literature, and the effect is still significant. This formula is widely used and can be added to milk and tea condiments. This can reduce the warm taste of tea drinks and reduce the amount of sugars in milk products, so in addition to improving the flavor, it can also increase liver protection. "200 ^ 24586 [Embodiment] In 100cc products, add glycine (0.2-25%), alanine (0.1-20%), taurine (0.1-20%), vitamin C (0.001%) 1. The formula of vitamin E (0.0001-1%) is confirmed to have significant liver-protective effects according to the following experiments. I. Experimental method 1 Experimental mice with acute hepatitis (ICR strain) are grouped according to the following table and The rats were fed for 3 days in advance, and the rats were fasted overnight (16 hours) the night before, and the material to be tested was administered in the early morning on the fourth day. One hour later, galactosamine (400 mg / Kg) plus lipopolysaccharide ( 3 // g / Kg) to induce acute hepatitis. Observe the survival of the rats and perform eye socket blood collection for GOT and GPT liver protection indicators at 6.5 hours after induction. Table 1: Grouping of acute hepatitis experiments (Total 4 days) Each feeding is equivalent to human experiment η 値 C-water only feeds water 1 ^ / day 100cc 5 pieces 1 X Hugan oral liquid 1 Do not day 100cc 5 pieces Ganoderma Tsugae double X Super Ganoderma lucidum 1 ^ / day lOOcc-4 capsules 4 experimental mice with chronic hepatitis (Wistar rats ) Grouped according to the following Table 2. The test was administered by oral administration, in which carbon tetrachloride (20% in olive oil; 0.2 ml / 100 g body weight) or olive oil was administered twice a week. Fixed at 8:00-9:00 on Saturday and Wednesday morning. Sterile water, Silymarin or test substance is administered daily at 13: 00-14: 00. And during the test period (weeks 1, 3, and 6) At the end) and after sacrifice, blood was collected for serum biochemical tests. Liver protein content and various antioxidant indexes (superoxide dismutase, glutathione, glutathione peroxidase, and catalase) were measured after sacrifice. Table 2: Grouping of chronic hepatitis experiments
試驗組別 投予物質及劑量 實驗 n値 每次灌食相當 於人 Naive 薇橫油(0.2 mL/100g B.W.) /無菌水(10 mL/kg B.W.) 1 0隻 1 oocc Postive control 四氯化碳(0.2 mL/lOOg B.W.) /無菌水(10 mL/kg B.W.) 1 0隻 1 00CC Negative control 四氯化碳（0·2 mL/lOOg B.W·) / silymarin(200mg/kg B.W.) 10隻 1 00CC 200524586Substances and dosage experiments in the test group n 値 Each administration is equivalent to human Naive Weiheng oil (0.2 mL / 100g BW) / sterile water (10 mL / kg BW) 1 0 1 oocc Postive control carbon tetrachloride (0.2 mL / lOOg BW) / Sterile water (10 mL / kg BW) 1 0 100CC Negative control Carbon tetrachloride (0.2 mL / 100g BW ·) / silymarin (200mg / kg BW) 10 100CC 200524586
Recipe IX 四氯化碳(0.2 mL/100g B.W.)獄驗物質(1〇·4 miykg B.W.) 1 0隻 1 〇 0 c c Recipe 2X 四氯化碳(〇·2 mL/lOOg B.W.)獄驗物質(20·8 ml/kg B.W.) 1 0隻 ^〇Q^cc~ 3 ·解酒微動物模式 老鼠(ICR strain)分組情形如表格三，老鼠先行隔夜禁食，接著餵食依下表分組進 行灌食，半小時後，灌入適當濃度的酒精(25%的酒精〇·7 c c / 4 0 g體重），於 酒後0.5小時、2.5小時、4.5小時，進行眼窝採血。4°C冰存。血液進行GC儀器 的酒精濃度分析(血液要爲全血不凝固，EDTA72mg/mL取25//L進行抗凝血用） 表格三：解酒實驗分組情形 _ 餵食老鼠的素材 每次灌食相當於人 實驗 η値 C-water 只餵食水 100cc 4隻 Recipe 1 x 護肝口服液 100cc 2隻 Recipe 3X 護肝口服液 300cc 4隻 二、實驗結果 1 ·急性肝炎的實驗Recipe IX carbon tetrachloride (0.2 mL / 100g BW) jail test substance (10.4 miykg BW) 10 only 100 cc Recipe 2X carbon tetrachloride (0.2 mL / 100g BW) jail test substance ( (20 · 8 ml / kg BW) 1 0 ^ 〇Q ^ cc ~ 3 · The grouping of mice with hangover micro-animal model (ICR strain) is shown in Table 3. The rats were fasted overnight and then fed according to the following table for group feeding. After half an hour, an appropriate concentration of alcohol (25% alcohol 0.7 cc / 40 g body weight) was infused, and blood was collected from the eye socket at 0.5 hours, 2.5 hours, and 4.5 hours after drinking. Store at 4 ° C on ice. Analysis of alcohol concentration in blood by GC instrument (blood should be whole blood without coagulation, EDTA 72mg / mL should be taken at 25 // L for anticoagulation) Table 3: Grouping of hangover experiments Human experiment η 値 C-water only feed water 100cc 4 Recipes 1 x Hugan Oral Liquid 100cc 2 Recipe 3X Hugan Oral Liquid 300cc 4 only 2. Experimental results 1 · Experiment of acute hepatitis
此次實驗，乃經由建立動物篩選模式GalN/LPS的誘發，找到對老鼠最佳 的誘發劑量，在由參考之前硏究的文獻及不斷的動物試驗，找尋出氨基 酸及維他命最佳組合及比例，此設計出的配方經實驗確實可以明顯改善 由GalN+LPS所誘發的急性肝炎，經由採血測肝臟的的生化指標GOT及 GPT，可以下降達95%以上(圖一），肝臟生化指標的功效甚至比目前台灣 的健康食品通過護肝認證的靈芝有更佳的護肝效果，老鼠經由GalN/LPS 誘發，老鼠往往容易因爲LPS 的作用過於強烈而導致體內的免疫系統過於活化產生許多自由基及 Cytokine，而自行攻擊自己自身的細胞，造成老鼠死亡。當老鼠餵食護肝 口服液後，老鼠的存活率大大提升，經過實驗發現存活率可以由至餵食 水老鼠都死亡的情形，變成100%(圖二），可以看出護肝口服液可以於急 性肝損傷發揮極大的護肝保護效果。 2 ·慢断炎的實驗 慢性肝炎乃依標準的四氯化碳模式進行誘發，結果可以發現經過四氯化 200524586 碳的誘發下確實可以讓老鼠的生化指標GOT及GPT明顯升高(圖三），甚至達 數千，不過經由灌食護肝口服液，就可以於第3週以後明顯看到老鼠的護肝生 化指標下降，而其他和護肝有關的指標列於下表。綜合表格四及圖三，可以看 出護肝口服液可以在四氯化碳誘發下有明顯具有保護效果。 表格四：慢性肝炎實驗各組的數據 試驗組別 Albumin 第6週 Albumin 第8週 Spleen ύ Hydroxyproline 的量 肝蛋白質 總量 LP0的量 GSH的量 Postive control— silymarin 2.92+0.07 154土 0.09 2·16土 0.22 4558.6±339.1 150·1±4·7 4.52±0.31 L81±0.07 Negative control -water 2·98±0·08 2·3±0·1 2.63± 0.18 5194·4±172 141·8±4·1 5.35±0.29 1·64±0·05 Recipe IX 3.25±0.06 2·62土 0.08 2·05± 0.14 4329.3±166.5 163.0+3.9 3.79±0.37 1·66±0·09 Recipe 2X 3.33±0.04 3.64土 0.07 1·82土 0.15 3928.3±292.2 166.5±6.1 3.73±0.17 1.73±0.06 Naive 3.47+0.06 3.4±0.06 0·89土 0.04 1740.3±59.5 2243±4.3 3.32±0.22 1·49±0·2 3.解酒試驗動物模式 此實驗可以由老鼠血中的酒精可以看出，此配方確實可以在0.5小時內，阻 止老鼠體內酒精濃度的上昇(圖四），符合文獻推測的降低酒精在胃部的排空，_所 以酒精會先鎖在胃部慢慢釋出，所以血中所測的酒精濃度就很低，慢慢把酒經由 胃部釋出到血液中，所以老鼠也能漸漸適應酒精的濃度，所以老鼠的活動情形也 相對的好很多，反觀控制組(只喝水），酒精會立刻進入血液中，老鼠也可以在10 分鐘到半小時看到老鼠有酒醉的情形，不想動或爬行蹣跚的現象。由此結果可以 看出護肝口服液確實可以降低酒醉的現象，而血液中的酒精濃度降低，這樣進入 肝臟的酒精也會較少且較緩，這樣可以減輕肝臟的負擔，進而也可以保護酒精性 的肝損傷。 總結此配方所用的單方不僅價格便宜且都是安全無虞的單方，不會有萃取及製 程上的差異，加上不會有顏色及添加口感上的差異，所以應用範圍大。最重要是 對於肝臟有非常顯著的保護效果。 200,524586 【圖式簡單說明】 圖一、急性肝炎誘發下，護肝口服液在生化指標，可以看到明顯的降低 圖二、急性肝炎誘發下，護肝口服液在老鼠存活情形所看到的效果，本次申請的 專利配方可以提高老鼠存活率 圖三、慢性肝炎誘發下，護肝口服液可以明顯降低GOT (圖三之A)及GPT(圖 三之B)値且效果優於正控制組一silymarin 圖四、護肝口服液降低血中酒精濃度的效果 【指定代表圖】 圖一及圖二 續次頁（發明說明頁不敷使用時，請註記並使用續頁）In this experiment, GalN / LPS was used to establish the animal screening mode to find the best dose to induce mice. Before consulting the literature and continuous animal experiments, we found the optimal combination and ratio of amino acids and vitamins. The designed formula can indeed obviously improve the acute hepatitis induced by GalN + LPS. The blood biochemical indexes GOT and GPT can be reduced by more than 95% (Figure 1). The efficacy of liver biochemical indexes is even Ganoderma lucidum, which has passed liver protection certification in Taiwan, has better liver protection effect. Rats are induced by GalN / LPS. Rats are often prone to excessive activation of the body's immune system due to the action of LPS. Many free radicals and Cytokine , And attacked its own cells, causing the mouse to die. After the rats were fed Hugan Oral Liquid, the survival rate of the mice was greatly improved. It was found through experiments that the survival rate could be changed from 100% of the mice that fed water to death (Figure 2). It can be seen that Hugan Oral Liquid can be used for acute Liver injury exerts great liver protection effect. 2 · Chronic hepatitis is induced according to the standard carbon tetrachloride model. As a result, it can be found that the induction of carbon tetrachloride 200524586 carbon can indeed increase the biochemical indicators GOT and GPT in rats (Figure 3). Even up to several thousand, but after administering Hugan Oral Liquid, you can clearly see that the liver biochemical indexes of mice decrease after the third week, while other indexes related to liver protection are listed in the table below. Combining Table 4 and Figure 3, it can be seen that Hugan Oral Liquid can have obvious protective effects induced by carbon tetrachloride. Table 4: Data of each group of chronic hepatitis experiment. Test group Albumin Week 6 Albumin Week 8 Spleen pha Hydroxyproline Amount of total liver protein LP0 Amount of GSH A postive control—silymarin 2.92 + 0.07 154 soil 0.09 2 · 16 soil 0.22 4558.6 ± 339.1 150 · 1 ± 4 · 7 4.52 ± 0.31 L81 ± 0.07 Negative control -water 2 · 98 ± 0 · 08 2 · 3 ± 0 · 1 2.63 ± 0.18 5194 · 4 ± 172 141 · 8 ± 4 · 1 5.35 ± 0.29 1.64 ± 0.05 Recipe IX 3.25 ± 0.06 2.62 ± 0.08 2.05 ± 0.14 4329.3 ± 166.5 163.0 + 3.9 3.79 ± 0.37 1.66 ± 0 · 09 Recipe 2X 3.33 ± 0.04 3.64 ± 0.07 1 82 soil 0.15 3928.3 ± 292.2 166.5 ± 6.1 3.73 ± 0.17 1.73 ± 0.06 Naive 3.47 + 0.06 3.4 ± 0.06 0 89 soil 0.04 1740.3 ± 59.5 2243 ± 4.3 3.32 ± 0.22 1.49 ± 0 · 2 3. Mode This experiment can be seen from the alcohol in the blood of mice. This formula can indeed prevent the increase of alcohol concentration in mice within 0.5 hours (Figure 4), which is consistent with the literature's speculation to reduce alcohol emptying in the stomach. Alcohol is first released in the stomach and slowly released, so the alcohol concentration measured in the blood is very low. Released from the stomach into the blood, so the mouse can gradually adapt to the concentration of alcohol, so the mouse's activity situation is relatively much better. In contrast, in the control group (only drinking water), the alcohol will immediately enter the blood, and the mouse can also From 10 minutes to half an hour, the rats are drunk and do not want to move or crawl. From this result, it can be seen that Hugan Oral Liquid can indeed reduce drunkenness, and the concentration of alcohol in the blood is reduced, so that less and slower alcohol enters the liver, which can reduce the burden on the liver and further protect Alcoholic liver injury. In summary, the unilateral formula used in this formula is not only cheap but also safe and secure. There will be no differences in extraction and process, plus no differences in color and taste, so it has a wide range of applications. Most importantly, it has a very significant protective effect on the liver. 200,524586 [Simplified illustration of the figure] Figure 1. Acute hepatitis induced by Hugan Oral Liquid in biochemical indicators, you can see a significant decrease. Figure 2. Acute hepatitis induced by Hugan Oral Liquid in the survival of mice. The patented formulation of this application can improve the survival rate of mice. Figure 3. Under the induction of chronic hepatitis, Hugan Oral Liquid can significantly reduce GOT (Figure 3A) and GPT (Figure 3B). Control group one silymarin Figure four, the effect of Hugan oral solution on reducing alcohol concentration in blood [designated representative chart] Figures one and two on the next page (If the description page of the invention is insufficient, please note and use the continued page)