TWI736023B - Urena lobate extract, preparation method and use thereof - Google Patents
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本發明關於一種野棉花萃取物，特別是一種野棉花水萃取物、製備方法及用途。The present invention relates to a wild cotton extract, in particular to a wild cotton water extract, preparation method and application.
嘌呤於體內的最終代謝產物為尿酸，經尿液或糞便排出體外，當尿酸生成過多或***不良時，過多的尿酸會累積於血液中，而形成高尿酸血症，國人的高尿酸血症盛行率偏高，無症狀的高尿酸血症患者無須服用藥物，透過飲食控制即可有效降低血液中的尿酸濃度，但若未積極控制尿酸濃度，尿酸濃度過高的時間越長，尿酸鈉鹽結晶可能會沈積於關節處而引起痛風關節炎，屆時則需要依賴藥物降低尿酸濃度，以緩解症狀。The final metabolite of purine in the body is uric acid, which is excreted in urine or feces. When uric acid is produced too much or poorly excreted, too much uric acid will accumulate in the blood to form hyperuricemia. Hyperuricemia is prevalent in Chinese The rate is high. Asymptomatic hyperuricemia patients do not need to take drugs. They can effectively reduce the uric acid concentration in the blood through diet control. However, if the uric acid concentration is not actively controlled, the longer the uric acid concentration will be, the longer the uric acid concentration will be, the sodium urate will crystallize It may deposit on joints and cause gouty arthritis. At that time, you need to rely on drugs to reduce the concentration of uric acid to relieve symptoms.
根據健保資料推估，每年約有20萬國人使用異嘌呤醇(Allopurinol)來治療痛風或痛風性關節炎，然而國人帶有HLA-B*5801基因的比例較高，可能在服用異嘌呤醇後產生不良反應，包含皮疹、皮膚癢、腹瀉及胃痛等，甚至發生嚴重過敏反應，例如史蒂芬強森症候群(Steven-Johnson Syndrome，SJS)或毒性表皮溶解症(Toxic Epidermal Necrolysis，TEN)，我國自實施藥害救濟制度以來，異嘌呤醇為申請藥害救濟給付的第一名藥物，因此亟需開發副作用小且安全性高的替代藥物，為痛風患者帶來更安全的照護。According to health insurance data, about 200,000 people in the country use allopurinol to treat gout or gouty arthritis each year. However, the proportion of people with HLA-B*5801 gene is higher, which may be after taking isopurinol. Adverse reactions, including skin rash, itching, diarrhea and stomach pain, and even severe allergic reactions, such as Steven-Johnson Syndrome (SJS) or Toxic Epidermal Necrolysis (TEN), are implemented in China Since the drug damage relief system, isopurinol has been the first drug to apply for drug damage relief benefits. Therefore, it is urgent to develop alternative drugs with small side effects and high safety to bring safer care to patients with gout.
本發明揭露一種野棉花萃取物之用途，其用以製備降低尿酸含量之一組成物。The present invention discloses the use of a wild cotton extract to prepare a composition for reducing the content of uric acid.
本發明另揭露一種野棉花萃取物之用途，其用以製備抑制黃嘌呤氧化酶活性之一組成物。The present invention also discloses the use of a wild cotton extract to prepare a composition that inhibits the activity of xanthine oxidase.
本發明另揭露一種野棉花萃取物之製備方法，其包含：一萃取步驟，將一野棉花植物置於一水中進行萃取，以取得一萃取液，其中該水的溫度介於60至100 °C之間；一過濾步驟，過濾該萃取液以取得一濾液；以及一乾燥步驟，乾燥該濾液，以取得一野棉花萃取物。The present invention also discloses a method for preparing a wild cotton extract, which comprises: an extraction step of placing a wild cotton plant in a water for extraction to obtain an extract, wherein the temperature of the water is between 60 and 100 °C Between; a filtration step, filtering the extract to obtain a filtrate; and a drying step, drying the filtrate to obtain a wild cotton extract.
本發明另揭露一種野棉花萃取物，其由上述之製備方法所製得。The present invention also discloses a wild cotton extract, which is prepared by the above-mentioned preparation method.
本發明之該野棉花萃取物可由水萃法或其他方式所製得，屬於副作用小且安全性高的天然萃取物，由於具有降低尿酸含量及抑制黃嘌呤氧化酶活性之功效，可製成不同形式的組成物取代已知痛風藥物，以更為安全的方式有效降低尿酸濃度，避免患者出現不良反應。The wild cotton extract of the present invention can be prepared by water extraction or other methods. It is a natural extract with low side effects and high safety. Due to the effect of reducing the content of uric acid and inhibiting the activity of xanthine oxidase, it can be prepared in different ways. The form of the composition replaces known gout drugs, effectively reducing the concentration of uric acid in a safer way, and avoiding adverse reactions in patients.
請參閱第1圖，本發明之一較佳實施例，一種野棉花萃取物之製備方法10包含下列步驟：「萃取步驟」11、「過濾步驟」12、「濃縮步驟」13及「乾燥步驟」14，藉由該製備方法10製得一野棉花水萃取物，其中野棉花(Urena lobata L.)為錦葵科(Malvaceae)野棉花屬(Urena)小灌木，又稱地桃花，分佈於台灣平地及低海拔區域，莖皮富含纖維，單葉互生，小枝及葉柄上有星狀毛，花瓣五枚，呈粉紅色或白色，果實為蒴果，表面具有鉤刺，可鉤附於動物上傳播繁殖。Please refer to Figure 1, a preferred embodiment of the present invention, a
於一萃取步驟11中，將一野棉花植物置於一水中進行萃取，以取得一萃取液，較佳地，該野棉花植物係以熱水萃取法進行萃取，其中該水的溫度介於60至100 °C之間，萃取時間介於60至120分鐘，該野棉花植物及該水的固液比(w/v)介於1：10至1：15之間，該萃取步驟11結束後進行一過濾步驟12，過濾該萃取液，將該野棉花植物自該萃取液中移除後取得一濾液，較佳地，於該過濾步驟12前，重複進行該萃取步驟11，收集所有萃取液後再進行過濾，接著進行一乾燥步驟14，乾燥該濾液以取得該野棉花萃取物，產率介於5至8 %之間，在本實施例中，該製備方法10另包含一濃縮步驟13，於該乾燥步驟14前濃縮該濾液，以減少該濾液體積，縮短乾燥時間。In an extraction step 11, a wild cotton plant is placed in a water for extraction to obtain an extract. Preferably, the wild cotton plant is extracted by a hot water extraction method, wherein the temperature of the water is 60 To 100 °C, the extraction time is 60 to 120 minutes, the solid-to-liquid ratio (w/v) of the wild cotton plant and the water is between 1:10 to 1:15, after the extraction step 11 is over Perform a
較佳地，該野棉花植物為全株植物的乾燥樣品，包含根、莖及葉，更佳地，另包含花及果實，但本發明不以此為限制，該野棉花植物可取自根、莖、葉、花及果實中至少一種或其組合的乾燥樣品。Preferably, the wild cotton plant is a dried sample of the whole plant, including roots, stems, and leaves, and more preferably, flowers and fruits, but the present invention is not limited to this. The wild cotton plant can be taken from roots. , Stems, leaves, flowers and fruits at least one or a combination of dried samples.
在本實施例中，將包含根、莖、葉、花及果實的野棉花全株植物裁碎後，放入60 °C烘箱乾燥三天，接著取200克的乾燥樣品，加入2500毫升的逆滲透水，煮沸後轉小火煮1小時，重複萃取兩次後混合，以紗布過濾萃取液後，取得1500毫升的濾液，以減壓濃縮法將濾液濃縮至120毫升，最後以真空冷凍乾燥法進行乾燥，取得12.5克乾燥粉末，並置於4 °C儲存，本發明不限制過濾、濃縮及乾燥方式，在其他實施例中，可選擇不同方式進行過濾、濃縮及乾燥。In this example, the whole wild cotton plant including roots, stems, leaves, flowers and fruits was cut into pieces and dried in an oven at 60 °C for three days. Then 200 grams of dried samples were taken and 2500 milliliters of reverse was added. Infiltrate the water, boil and simmer for 1 hour. Repeat the extraction twice and mix. After filtering the extract with gauze, 1500ml of filtrate is obtained. The filtrate is concentrated to 120ml by vacuum concentration method, and finally by vacuum freeze-drying method. After drying, 12.5 g of dry powder is obtained and stored at 4°C. The present invention does not limit the filtration, concentration, and drying methods. In other embodiments, different methods may be selected for filtration, concentration, and drying.
本發明之該野棉花萃取物係使用該水萃取該野棉花植物所製得，該野棉花萃取物可經由上述之該製備方法10所製得，或經由其他方法所製得，本發明不以此為限制。The wild cotton extract of the present invention is prepared by using the water to extract the wild cotton plant. The wild cotton extract can be prepared by the above-mentioned
為了確認該野棉花萃取物的功效，分別給予高尿酸血症實驗動物50.0 mg/kg、100.0 mg/kg及250.0 mg/kg的該野棉花萃取物，採集血液分離出血清後，測定血清中的尿酸(uric acid)濃度及黃嘌呤氧化酶(xanthine oxidase，XO)活性，其中黃嘌呤氧化酶為動物體內尿酸合成的重要酵素，催化次黃嘌呤(hypoxanthine)氧化成黃嘌呤(xanthine)，並催化黃嘌呤氧化成尿酸，當黃嘌呤氧化酶被抑制時，尿酸的合成路徑受阻，因此可降低體內的尿酸濃度，而試驗結果顯示該野棉花萃取物確實具有降低尿酸及黃嘌呤氧化酶含量的功效，因此可用以製備降低尿酸含量或抑制黃嘌呤氧化酶活性之一組成物，其中該組成物可為醫藥組成物、食品組成物或飲品組成物，例如藥物或健康食品。In order to confirm the efficacy of the wild cotton extract, 50.0 mg/kg, 100.0 mg/kg and 250.0 mg/kg of the wild cotton extract were given to experimental animals with hyperuricemia. The concentration of uric acid and the activity of xanthine oxidase (xanthine oxidase, XO). Xanthine oxidase is an important enzyme for the synthesis of uric acid in animals. It catalyzes the oxidation of hypoxanthine to xanthine and catalyzes Xanthine is oxidized to uric acid. When xanthine oxidase is inhibited, the synthesis path of uric acid is blocked, so the concentration of uric acid in the body can be reduced. The test results show that the wild cotton extract does have the effect of reducing the content of uric acid and xanthine oxidase. Therefore, it can be used to prepare a composition that reduces the content of uric acid or inhibits the activity of xanthine oxidase, wherein the composition can be a pharmaceutical composition, a food composition or a beverage composition, such as a medicine or a health food.
高尿酸血症動物實驗：Animal experiment of hyperuricemia:
進行實驗前，將4週齡大且體重介於22.0-24.0公克的ICR小鼠飼養於動物中心一週，動物中心具有自動空氣調節設備，室溫25.0±1.0 °C，相對濕度55.0±1.0 %，且明暗週期為12小時，以市售的一般動物飼料餵食實驗小鼠，並使其自由飲水，實驗小鼠的管理及照顧均遵循中華民國實驗動物學會出版之實驗動物飼養與管理指南。Before the experiment, 4 weeks old ICR mice weighing 22.0-24.0 grams were kept in the animal center for one week. The animal center has automatic air conditioning equipment, room temperature 25.0±1.0 °C, relative humidity 55.0±1.0%, The light-dark cycle is 12 hours. The experimental mice are fed with commercially available general animal feed and allowed to drink freely. The management and care of the experimental mice follow the laboratory animal breeding and management guidelines published by the Institute of Laboratory Animals of the Republic of China.
將實驗小鼠隨機分成6個組別(n=10)，分別為控制組、誘發組、正對照組、低劑量組、中劑量組及高劑量組，控制組小鼠不誘發急性高尿酸血症，僅餵以二次水(double distilled water，DDW)作為安慰劑，其餘組別皆給予尿酸酶抑制劑(Uricase inhibitor)，以誘發小鼠形成急性高尿酸血症，尿酸酶可催化尿酸氧化為5-羥基異尿酸，當尿酸酶受到抑制時，會增加血尿酸濃度而引起高尿酸血症，在本實施例中，係以腹腔注射方式給予250.0 mg/kg的氧嗪酸鉀(Potassium Oxonate，PO)懸濁液，以誘發小鼠形成急性高尿酸血症，其中懸濁液包含羧甲基纖維素鈉鹽(Carboxymethyl Cellulose Sodium salt, CMC-Na)、吐溫80(Tween-80)、氧嗪酸鉀及水。The experimental mice were randomly divided into 6 groups (n=10), namely the control group, the induction group, the positive control group, the low-dose group, the middle-dose group and the high-dose group. The control group mice did not induce acute hyperuricemia Only double distilled water (DDW) was given as a placebo, and the other groups were given uricase inhibitors to induce acute hyperuricemia in mice. Uricase can catalyze the oxidation of uric acid. It is 5-hydroxyisouric acid. When uricase is inhibited, it will increase the blood uric acid concentration and cause hyperuricemia. In this example, 250.0 mg/kg of Potassium Oxonate (Potassium Oxonate) was administered by intraperitoneal injection. , PO) suspension to induce acute hyperuricemia in mice, where the suspension contains Carboxymethyl Cellulose Sodium salt (CMC-Na), Tween-80, Potassium oxazine and water.
誘發組小鼠於誘發後1小時給予DDW作為安慰劑，並在給予安慰劑1小時後犧牲小鼠，正對照組小鼠於誘發後1小時給予5.0 mg/kg的異嘌呤醇(Allopurinol)，並在給予異嘌呤醇1小時後犧牲小鼠，異嘌呤醇是一種尿酸合成抑制劑，為常見的高尿酸血症藥物，用以抑制黃嘌呤氧化酶活性，避免嘌呤轉化為尿酸，因此可降低尿酸含量，低劑量組、中劑量組及高劑量組的小鼠分別口服灌食以DDW配製成的不同劑量野棉花樣品(50.0、100.0及250.0 mg/kg)，連續3天於早上及下午各別餵食野棉花樣品一次，並於第4天早上餵食野棉花樣品前1小時，誘發小鼠形成急性高尿酸血症，並在給予野棉花樣品1小時後犧牲小鼠，各組別小鼠犧牲後收集其血液，並將血液離心10分鐘(6000 rpm)以取得血清，儲存於-20 °C備用。Mice in the induction group were given DDW as a placebo one hour after the induction, and sacrificed one hour after the placebo was given. The mice in the positive control group were given 5.0 mg/kg allopurinol one hour after the induction. The mice were sacrificed 1 hour after the administration of isopurinol. Isopurinol is a uric acid synthesis inhibitor and a common hyperuricemia drug. It is used to inhibit xanthine oxidase activity and prevent the conversion of purines into uric acid, so it can reduce Uric acid content, mice in the low-dose group, middle-dose group and high-dose group were orally fed with different doses of wild cotton samples (50.0, 100.0 and 250.0 mg/kg) prepared with DDW for 3 consecutive days in the morning and afternoon The wild cotton samples were fed once, and 1 hour before the wild cotton samples were fed in the morning of the 4th day, the mice were induced to develop acute hyperuricemia, and the mice were sacrificed 1 hour after the wild cotton samples were given. The mice of each group After sacrifice, the blood was collected and centrifuged for 10 minutes (6000 rpm) to obtain serum, which was stored at -20 °C for later use.
血清尿酸濃度測定方法如下，將約20.0 μL血清加入含有1.0 mL試劑混合物的試管中，於37.0°C下放置5分鐘後，以546 nm波長進行吸光度測定，標準物為6.0 mg/dL尿酸標準物，其中血清尿酸濃度計算公式為： 血清尿酸濃度(mg/dL) = (標準物吸光度/樣品吸光度) x 6 The method for determining the serum uric acid concentration is as follows. Add about 20.0 μL of serum to a test tube containing 1.0 mL of the reagent mixture. After placing it at 37.0°C for 5 minutes, measure the absorbance at 546 nm. The standard is 6.0 mg/dL uric acid standard. , Wherein the calculation formula of serum uric acid concentration is: Serum uric acid concentration (mg/dL) = (standard absorbance/sample absorbance) x 6
請參閱第2圖，誘發組的尿酸含量比控制組增加了16.13 %，且誘發組尿酸含量為正對照組尿酸含量的35倍，表示本實驗成功誘發小鼠高尿酸血症實驗模型，而低劑量組、中劑量組及高劑量組之間呈現劑量依賴的現象，餵食的該野棉花萃取物濃度越高，則小鼠血中的尿酸含量越低，雖然該野棉花萃取物的抑制效果不如異嘌呤醇，但中劑量組及高劑量組的尿酸含量比誘發組減少了9.92 %及12.5 %，因此本發明之該野棉花萃取物確實可降低血清中的尿酸含量。Please refer to Figure 2. The uric acid content of the induced group increased by 16.13% compared to the control group, and the uric acid content of the induced group was 35 times that of the positive control group, indicating that the experiment successfully induced the experimental model of hyperuricemia in mice, but low There is a dose-dependent phenomenon among the dose group, the middle dose group and the high dose group. The higher the concentration of the wild cotton extract fed, the lower the uric acid content in the blood of the mice, although the inhibitory effect of the wild cotton extract is not as good as Isopurinol, but the uric acid content of the middle-dose group and the high-dose group were reduced by 9.92% and 12.5% compared with the induced group. Therefore, the wild cotton extract of the present invention can indeed reduce the uric acid content in the serum.
血清黃嘌呤氧化酶活性係根據Prajda和Weber學者於1975年提出的方法進行測定，將實驗小鼠的血清加入含有4 mM黃嘌呤的磷酸鹽緩衝液(50 mM，pH 7.5)中，於37.0°C下反應30分鐘，接著加入20 μL的100 %三氯乙酸(Trichloroacetic acid，TCA)終止反應，最後離心混合物20分鐘(3500 rpm)，測定上清液在292 nm波長下的吸光度，其中黃嘌呤氧化酶活性以微單位/每毫升(μU/mL)表示。Serum xanthine oxidase activity was measured according to the method proposed by Prajda and Weber scholars in 1975. The serum of experimental mice was added to phosphate buffer (50 mM, pH 7.5) containing 4 mM xanthine at 37.0° React for 30 minutes at C, then add 20 μL of 100% trichloroacetic acid (Trichloroacetic acid, TCA) to stop the reaction, and finally centrifuge the mixture for 20 minutes (3500 rpm), and measure the absorbance of the supernatant at a wavelength of 292 nm. Oxidase activity is expressed in microunits per milliliter (μU/mL).
請參閱第3圖，高劑量組之黃嘌呤氧化酶活性最低，比控制組降低了26.8%，比誘發組降低了19.86 %，甚至比正對照組降低了2.89 %，由此可知，本發明之該野棉花萃取物確實可降低黃嘌呤氧化酶活性，並且可推論本發明之該野棉花萃取物係透過抑制黃嘌呤氧化酶活性來降低尿酸含量。Please refer to Figure 3, the high-dose group has the lowest xanthine oxidase activity, which is 26.8% lower than the control group, 19.86% lower than the induced group, and even 2.89% lower than the positive control group. It can be seen that the present invention is The wild cotton extract can indeed reduce the xanthine oxidase activity, and it can be inferred that the wild cotton extract of the present invention reduces the uric acid content by inhibiting the xanthine oxidase activity.
28天餵食毒性試驗：28-day feeding toxicity test:
為了了解該野棉花萃取物是否對哺乳類動物產生毒性影響，根據健康食品管理法中的安全評估方法進行28天餵食毒性試驗，實驗動物為4週齡SD大鼠，飼養於動物中心一週後進行毒性試驗，動物中心具有自動空氣調節設備，室溫25.0±1.0 °C，相對濕度55.0±1.0 %，且明暗週期為12小時，以市售的一般動物飼料餵食實驗大鼠，並使其自由飲水，實驗大鼠的管理及照顧均遵循中華民國實驗動物學會出版之實驗動物飼養與管理指南。In order to understand whether the wild cotton extract has toxic effects on mammals, a 28-day feeding toxicity test was conducted according to the safety assessment method in the Health Food Management Law. The experimental animal was a 4-week-old SD rat. The toxicity was carried out after being kept in the animal center for one week. In the experiment, the animal center has automatic air conditioning equipment, the room temperature is 25.0±1.0 °C, the relative humidity is 55.0±1.0%, and the light-dark cycle is 12 hours. The experimental rats are fed with commercially available general animal feed and allowed to drink freely. The management and care of experimental rats follow the guidelines for breeding and management of experimental animals published by the Society of Laboratory Animals of the Republic of China.
將實驗大鼠隨機分成4個組別(n=20，雌雄各半)，分別為控制組、低劑量組(50.0 mg/kg)、中劑量組(100.0 mg/kg)及高劑量組(250.0 mg/kg)，控制組大鼠未餵食野棉花樣品，僅餵以DDW為安慰劑，低劑量組、中劑量組及高劑量組大鼠於每天相同時間以胃管餵食野棉花樣品一次，每次投予體積為10.0 mL/kg，連續餵食28天，每天觀察二次(間隔時間不得少於6小時)，並紀錄實驗動物的臨床症狀，第28天餵食結束後禁食一晚，隔日犧牲後抽血及採集臟器，檢驗項目包含血清、血液、尿液、眼睛、器官及組織病理切片。The experimental rats were randomly divided into 4 groups (n=20, male and female), which were the control group, the low-dose group (50.0 mg/kg), the medium-dose group (100.0 mg/kg), and the high-dose group (250.0 mg/kg). mg/kg). The rats in the control group were not fed with wild cotton samples, but only DDW was fed as placebo. The rats in the low-dose, middle-dose and high-dose groups were fed wild cotton samples by gastric tube at the same time every day. The volume of the single dose is 10.0 mL/kg, continuous feeding for 28 days, observation twice a day (interval should not be less than 6 hours), and record the clinical symptoms of the experimental animals, fasting for one night after feeding on the 28th day, sacrifice on the next day Afterwards, blood was drawn and organs were collected. The inspection items included serum, blood, urine, eyes, organs, and histopathological sections.
於連續28天餵食野棉花樣品的期間，每天觀察並紀錄每隻實驗大鼠是否發生昏眩、間歇抽序、遲鈍、角弓反張、疲軟、僵直、瞳孔放大/縮小、呼吸急促、咳血、鼻分泌物、下痢、尿失禁等症狀，結果所有實驗大鼠皆存活，活動力及生理反應一切正常，沒有觀察到任何明顯毒性症狀，也沒有顯示任何死亡狀況。During the period of feeding the wild cotton samples for 28 consecutive days, observe and record every day whether each experimental rat has dizziness, intermittent pumping, dullness, reflex, weakness, stiffness, pupil dilation/reduction, shortness of breath, hemoptysis, Symptoms such as nasal secretions, diarrhea, urinary incontinence, etc. As a result, all experimental rats were alive, with normal motility and physiological reactions, and no obvious symptoms of toxicity were observed, nor did they show any death.
血清分析包含肝功能相關指標的天門冬胺酸轉胺酶(Glutamic Oxaloacetic Transaminase，GOT)、丙胺酸轉胺酶(Glutamic Pyruvic Transaminase，GPT)、鹼性磷酸酶(Alkaline phosphate，ALP)、總蛋白(Total protein)、白蛋白(Albumin)、球蛋白(Globulin)、白蛋白/球蛋白比值(A/G ratio)、總膽紅素(Bilirubin-total)，以及腎臟、胰臟、血脂肪及血清電解質相關指標的血中尿素氮(Blood Urea Nitrogen，BUN)、肌酸酐(Creatinine)、血糖(Glucose AC)、三酸甘油酯(Triglyceride)、總膽固醇(Cholesterol-total)、乳酸脫氫酶(Lactic dehydrogenase，LDH)、澱粉酶(Amylase)、鈉(Na)、鉀(K)、氯(Cl)、鈣(Ca)、磷(P)等20種，血清分析結果顯示餵食野棉花樣品不會對實驗大鼠的肝功能造成明顯不良影響，但顯示野棉花樣品對實驗大鼠腎臟造成些微影響，然而根據器官病理檢驗結果，零星個體出現腎臟病變，推測應為個體差異影響統計數據結果，與餵食野棉花樣品無關。Serum analysis includes Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT), Alkaline phosphate (ALP), total protein ( Total protein, Albumin, Globulin, A/G ratio, Bilirubin-total, kidney, pancreas, blood fat and serum electrolyte Related indicators: Blood Urea Nitrogen (BUN), Creatinine, Glucose AC, Triglyceride, Cholesterol-total, Lactic dehydrogenase , LDH), Amylase (Amylase), Sodium (Na), Potassium (K), Chlorine (Cl), Calcium (Ca), Phosphorus (P), etc. 20 kinds, serum analysis results show that feeding wild cotton samples will not affect the experiment The liver function of the rats caused obvious adverse effects, but it showed that the wild cotton samples had a slight impact on the kidneys of the experimental rats. However, according to the results of organ pathological examinations, sporadic individuals had kidney disease. It is speculated that individual differences may affect the statistical data results. The cotton sample is irrelevant.
血液生化檢驗包含紅血球(RBC)、白血球(WBC)、血小板 (Platelet)、血紅素(Hemoglobin)、血比容(Hematocrit)、平均紅血球容積(MCV)、平均紅血球血紅素量(MCH)、平均紅血球血紅素濃度(MCHC)、帶狀嗜中性白血球(Neutrophils band)、節狀嗜中性白血球(Neutrophils segmented)、淋巴球(Lymphocytes)、單核球(Monocytes)、嗜酸性白血球(Eosinophils)、嗜鹼性白血球(Basophils)等14種，血液分析結果顯示野棉花樣品對於實驗大鼠具有增加血液中紅血球及血小板的效果，雖然白血球、平均紅血球容積及平均紅血球血紅素量略為降低，但整體而言，餵食野棉花樣品並無產生明顯毒性反應。Blood biochemical tests include red blood cells (RBC), white blood cells (WBC), platelets (Platelet), hemoglobin, hematocrit, average red blood cell volume (MCV), average red blood cell hemoglobin (MCH), average red blood cell Heme concentration (MCHC), banded neutrophils band, nodular neutrophils segmented, lymphocytes, monocytes, eosinophils, eosinophils 14 kinds of alkaline white blood cells (Basophils), blood analysis results show that wild cotton samples have the effect of increasing red blood cells and platelets in the blood of experimental rats. Although white blood cells, average red blood cell volume and average red blood cell hemoglobin content are slightly reduced, overall , Feeding wild cotton samples did not produce obvious toxic reactions.
尿液分析包含外觀(Appearance)、酸鹼值(pH)、比重(Specific gravity)、蛋白質(Protein)、葡萄糖(Glucose)、膽紅素(Bilirubin)、尿膽素原(Urobilinogen)、酮體(Ketone body)、亞硝酸鹽(Nitrite)、潛血(Occult blood)、紅血球(RBC)、白血球(WBC)、上皮細胞(Epithelia cell)、細菌(Bacteria)、三重磷酸鹽(Triple phosphate)及圓柱體(Cast)等16種，其中分別於實驗前後(餵藥前及犧牲前)採集尿液，測量結果顯示酸鹼值及比重出現統計差異，然而兩者皆會受到動物飲水量、尿液收集時間及保存溫度影響，應與餵食野棉花樣品無關，蛋白質、酮體、亞硝酸鹽及三重磷酸鹽於實驗前後皆有檢出，但未發現與野棉花樣品相關的差異，葡萄糖、膽紅素、尿膽素原、紅血球、白血球、上皮細胞及圓柱體於實驗前後皆為正常值或無檢出，並無明顯觀察到野棉花樣品對實驗大鼠造成的影響。Urine analysis includes Appearance, pH, Specific gravity, Protein, Glucose, Bilirubin, Urobilinogen, ketone body ( Ketone body), nitrite (Nitrite), occult blood (Occult blood), red blood cell (RBC), white blood cell (WBC), epithelia cell, bacteria (Bacteria), triple phosphate and cylinder ( Cast) and other 16 types. Among them, urine was collected before and after the experiment (before feeding and before sacrifice). The measurement results showed statistical differences in pH and specific gravity. The effect of storage temperature should not be related to the feeding of wild cotton samples. Protein, ketone bodies, nitrite and triple phosphate were detected before and after the experiment, but no differences related to wild cotton samples were found, such as glucose, bilirubin, and urine. Bilidinogen, erythrocytes, white blood cells, epithelial cells and cylinders were all normal or undetectable before and after the experiment, and no obvious effects of wild cotton samples on experimental rats were observed.
眼睛檢查係以肉眼檢驗實驗大鼠的眼睛外部，所有實驗大鼠的眼睛皆無發炎或流淚的異常現象發生，野棉花樣品沒有對實驗大鼠的眼睛造成任何不良反應。The eye examination is to visually inspect the outside of the eyes of the experimental rats. All the experimental rats have no abnormal phenomenon of inflammation or tearing of the eyes. The wild cotton samples did not cause any adverse reactions to the eyes of the experimental rats.
除了血清、血液、尿液及眼睛外部檢查外，亦針對控制組及高劑量組大鼠進行器官及組織病理分析，器官分析係測量大腦、心臟、肝臟、腎臟、腎上腺、脾臟、性腺(卵巢或睪丸)的絕對重量(g)及相對重量(%)，皆無統計差異，而組織病理分析係針對上述器官組織進行病理檢驗，各組別的大腦、心臟、脾臟、胸腺及卵巢並未發現明顯組織病理變化，而零星個體的肝臟、腎臟、腎上腺及睪丸出現病理變化，但經統計分析，比較控制組及高劑量組的病理變化積分後發現並無顯著差異，由於從高劑量組組織切片中未發現與野棉花樣品劑量成正相關的組織病理變化，因此無須進行低劑量組及中劑量組的組織切片判讀。In addition to external examinations of serum, blood, urine, and eyes, organ and histopathological analysis of rats in the control group and high-dose group was also performed. There is no statistical difference between the absolute weight (g) and relative weight (%) of testicles. Histopathological analysis is performed on the above-mentioned organs and tissues. No obvious tissues were found in the brain, heart, spleen, thymus and ovary of each group. Pathological changes, while the liver, kidney, adrenal glands and testicles of sporadic individuals have pathological changes. However, after statistical analysis, there is no significant difference between the pathological changes of the control group and the high-dose group. It was found that the histopathological changes were positively correlated with the dose of wild cotton samples, so there was no need to interpret the tissue sections of the low-dose group and the middle-dose group.
除了上述檢驗分析外，每週至少測量一次實驗大鼠之體重、飲水量、攝食量及食物利用率(Feed Efficiency)，其中食物利用率計算公式如下： 食物利用率(%) = [體重增加量(g)/總飼料攝取量(g)] x 100 經統計分析，實驗大鼠體重、飲水量、攝食量及食物利用率並無顯著差異，因此連續餵食野棉花樣品不會對實驗大鼠體重、飲水量、攝食量及食物利用率造成不良影響。 In addition to the above test and analysis, the weight, water intake, food intake and feed efficiency of the experimental rats are measured at least once a week. The formula for calculating the food efficiency is as follows: Food utilization rate (%) = [weight gain (g)/total feed intake (g)] x 100 According to statistical analysis, there is no significant difference in the weight, water intake, food intake and food utilization of experimental rats. Therefore, continuous feeding of wild cotton samples will not cause adverse effects on experimental rats' weight, water intake, food intake and food utilization.
28天餵食毒性試驗結果發現本發明之該野棉花萃取物不會對動物造成不良影響，因此該野棉花萃取物的沒有可觀察之不良影響值(No-Observed-Adverse-Effect Level，NOAEL)可設定為250.0 mg/kg/day，且根據高尿酸血症動物實驗結果確認該野棉花萃取物確實可降低尿酸含量及抑制黃嘌呤氧化酶活性，因此可使用250.0 mg/kg/day劑量的該野棉花萃取物治療高尿酸血症及其他因尿酸過高所引起的疾病，相較於異嘌呤醇，本發明之該野棉花萃取物具有副作用小及安全性高的優點，可避免患者服用異嘌呤醇後發生不良反應。The 28-day feeding toxicity test results found that the wild cotton extract of the present invention will not cause adverse effects on animals, so the wild cotton extract has no observable adverse effect value (No-Observed-Adverse-Effect Level, NOAEL). It is set to 250.0 mg/kg/day, and according to the results of animal experiments with hyperuricemia, it is confirmed that the wild cotton extract can indeed reduce uric acid content and inhibit xanthine oxidase activity. Therefore, 250.0 mg/kg/day of the wild cotton extract can be used. Cotton extract treats hyperuricemia and other diseases caused by high uric acid. Compared with isopurinol, the wild cotton extract of the present invention has the advantages of less side effects and high safety, and can prevent patients from taking isopurine Adverse reactions occur after alcohol.
本發明之該野棉花萃取物具有抑制黃嘌呤氧化酶活性之功效，因此除了治療高尿酸血症，亦可用以治療其他與黃嘌呤氧化酶相關的疾病，如心血管疾病、糖尿病、缺血再灌流傷害、腦瘤及肝炎等。The wild cotton extract of the present invention has the effect of inhibiting the activity of xanthine oxidase. Therefore, in addition to treating hyperuricemia, it can also be used to treat other diseases related to xanthine oxidase, such as cardiovascular disease, diabetes, and ischemia. Perfusion injury, brain tumor and hepatitis, etc.
本發明之保護範圍當視後附之申請專利範圍所界定者為準，任何熟知此項技藝者，在不脫離本發明之精神和範圍內所作之任何變化與修改，均屬於本發明之保護範圍。The scope of protection of the present invention shall be determined by the scope of the attached patent application. Anyone who is familiar with the art and makes any changes and modifications without departing from the spirit and scope of the present invention shall fall within the scope of protection of the present invention. .
10…野棉花萃取物之製備方法 11…萃取步驟
12…過濾步驟 13…濃縮步驟
14…乾燥步驟
10... Preparation method of wild cotton extract 11...
第1圖：依據本發明之一較佳實施例，一種野棉花萃取物之製備方法流程圖。 第2圖：實驗動物血清中尿酸含量之長條圖。 第3圖：實驗動物血清中黃嘌呤氧化酶含量之長條圖。 Figure 1: According to a preferred embodiment of the present invention, a flow chart of a preparation method of wild cotton extract. Figure 2: Bar graph of uric acid content in the serum of experimental animals. Figure 3: Bargraph of the content of xanthine oxidase in the serum of experimental animals.
10…野棉花萃取物之製備方法 11…萃取步驟
12…過濾步驟 13…濃縮步驟
14…乾燥步驟
10... Preparation method of wild cotton extract 11...
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