TWI642433B - Use of composition of active ingredients in organic extract ofandrographispaniculatain kidney protection - Google Patents
Use of composition of active ingredients in organic extract ofandrographispaniculatain kidney protection Download PDFInfo
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Abstract
本發明係關於一種穿心蓮(Andrographis paniculata,AP)之有機溶劑萃取 物用於保護糖尿病患者腎臟功能之用途,特徵在於該穿心蓮有機溶劑萃取物係藉由將穿心蓮之乾燥莖之乙醇粗萃取物以乙醇、正己烷、正丁醇與乙酸乙酯等有機溶劑進行萃取、濃縮及乾燥而得,且包含具有藥理活性的成分(active ingredient,簡稱APai),主要包含230-400mg/g穿心蓮內酯(Andrographolides,簡稱A)、50-90mg/g新穿心蓮內酯(Neoandrographolide,簡稱NA)及20-50mg/g去氧去氫穿心蓮內酯(14-deoxy-11,12-dehydrogen-andrographolide,簡稱DDA)。 The invention relates to an organic solvent extract of Andrographis paniculata (AP) for protecting kidney function of a diabetic patient, characterized in that the extract of Andrographis paniculata organic solvent is obtained by using ethanol as a crude extract of dried stem of Andrographis paniculata An organic solvent such as n-hexane, n-butanol or ethyl acetate is extracted, concentrated and dried, and contains an active ingredient (APai), which mainly contains 230-400 mg/g andrographolide (Andrographolides). , abbreviated as A), 50-90mg / g neoandrographolide (NA) and 20-50mg / g dehydrogenated andrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, abbreviated as DDA).
Description
本發明係關於穿心蓮有機溶劑萃取物之具有降血糖活性成分組成物用於保護腎臟功能之新用途。更特別地,本發明係關於主要包含穿心蓮內酯、新穿心蓮內酯及去氧去氫穿心蓮內酯之穿心蓮有機溶劑萃取物活性成分(active ingredient,簡稱APai)組成物於保護糖尿病患者腎臟功能之應用。 The present invention relates to a novel use of a hypoglycemic active ingredient composition of an extract of Andrographis paniculata organic solvent for protecting kidney function. More particularly, the present invention relates to an active ingredient (APai) composition of andrographolide organic solvent extract mainly comprising andrographolide, neoandrographolide and deoxyhydroandrographolide for protecting kidney function in diabetic patients. application.
穿心蓮(Andrographis paniculata)是爵床科多年生草本植物。莖細長而深綠色,稜角處有縱溝和翼膜。莖的橫截面四方形。葉是披針形,無毛。中國、印度、東南亞和臺灣等地有廣泛栽培。氣輕微、味道極苦且持久。在馬來西亞,穿心蓮被稱為「Hempedu Bumi」,即是“地球膽汁”之意,也是傳統醫藥中味道最苦的植物之一。在中醫藥的五行學說中認為苦入心,而穿心蓮只要你含入一小片它的葉子,馬上可以感受到,那種刻骨銘心的苦,像是直入你的心中,故名『穿心蓮』。 Andrographis paniculata is a perennial herb of the genus. The stem is slender and dark green with longitudinal grooves and wing membranes at the corners. The stem has a square cross section. The leaves are lanceolate and glabrous. China, India, Southeast Asia and Taiwan are widely cultivated. It is mild, very bitter and long lasting. In Malaysia, Andrographis paniculata is called “Hempedu Bumi”, which means “earth bile” and one of the most bitter plants in traditional medicine. In the five elements of Chinese medicine, I think that bitterness comes into my heart, and if you wear a small piece of its leaves, you can feel it immediately. The kind of unforgettable bitterness seems to go straight into your heart, hence the name "through the heart."
穿心蓮的藥用部位是乾燥莖與葉子,穿心蓮葉含二萜內酯化合物:去氧穿心蓮內酯(Deoxyandrographolide)0.1%以上,穿心蓮內酯(Andrographolide)1.5%以上,新穿心蓮內酯(Neoandrographolide)0.2%以上,及高穿心蓮內酯 (Homoandrographolide),潘尼內酯(Panicolide)。還含有穿心蓮烷(Andrographan),穿心蓮酮(Andrographon),穿心蓮甾醇(Andrographosterin),β-谷甾醇-D-葡萄糖甙(β-sitosterol-D-glucoside)等。根部除了含有穿心蓮內酯之外,還含有5-羥基-7,8,2",3"-四甲氧基黃酮(Mono-o-methylwithtin)、5-羥基-7,8,2"-三甲氧基黃酮(Andrographin)、5,2"-二羥基-7,8-二甲氧基黃酮(Panicolin)、芹菜素-7,4"-二甲醚(Apigenin-7,4"-dimethyl ether)、a1-谷甾醇和KH2PO4等。全草尚含l4-去氧-11-氧化穿心蓮內酯醋(14-Deoxy-ll-oxoandrographolide)、14-去氧-11,12-二去氫穿心蓮內酯(14-Deoxy-ll,12-didehydroandrographolide)。另據初步分析,還含有甾醇皂甙、糖類及縮合鞣質等酚類物質。又從葉、嫩枝、胚軸、根部和胚芽所得的癒合組織,經培養分離,可得到三種倍半萜內酯化合物:欖核蓮內酯A、B和C(Pa-niculides A、B、C)。 The medicinal part of Andrographis paniculata is dry stems and leaves, and the penicidopsis leaves contain diterpene lactone compounds: Deoxyandrographolide (0.1%), andrographolide (Andrographolide) 1.5% or more, Neoandrographolide (Neoandrographolide) ) 0.2% or more, and high andrographolide (Homoandrographolide), panicolide (Panicolide). Also included are Andrographan, Andrographon, Andrographosterin, β-sitosterol-D-glucoside, and the like. In addition to the andrographolide, the root also contains 5-hydroxy-7,8,2",3"-tetramethoxylated flavonoids (Mono-o-methylwithtin), 5-hydroxy-7,8,2"-trimethyl Andrographin, 5,2"-dihydroxy-7,8-dimethoxyflavone (Panicolin), apigenin-7,4"-dimethyl ether (Apigenin-7,4"-dimethyl ether) , a1-sitosterol and KH 2 PO 4 and the like. Whole grass still contains l4-deoxy-ll-oxoandrographolide, 14-deoxy-11,12-didehydroandrographolide (14-Deoxy-ll,12- Didehydroandrographolide). According to preliminary analysis, it also contains phenolic substances such as sterol saponins, sugars and condensed tannins. The healing tissue obtained from leaves, shoots, hypocotyls, roots and germs can be isolated and cultured to obtain three sesquiterpene lactone compounds: lanolinide A, B and C (Pa-niculides A, B, C).
穿心蓮水萃取物(APHE)及穿心蓮內酯(Andrographolides,簡稱A)已被發表具有降血糖、降血脂、抗菌、抗發炎及抗癌等功能。其中穿心蓮內酯已知具有祛熱解毒,消炎止痛的功效,對細菌性、病毒性上呼吸道感染(急、慢性扁桃腺炎及咽喉腫痛)及細菌性痢疾、胃腸炎有特殊療效(Md.Sanower Hossain等人,The Scientific World Journal 2014:1-28,2014)。中華民國專利I355933已描述一種作為腫瘤壞死因子(TNF-α)拮抗劑之穿心蓮內酯及其藥學組成物。另外,已有利用穿心蓮內酯作為活性成分,製作一以治癒傷口及/或美白皮膚的醫藥組合物(中華民國專利201524532)。 Andrographis water extract (AP HE ) and andrographolide (Andrographolides ( A) have been published to have functions such as lowering blood sugar, lowering blood fat, antibacterial, anti-inflammatory and anti-cancer. Among them, andrographolide is known to have antipyretic, anti-inflammatory and analgesic effects, and has special effects on bacterial, viral upper respiratory tract infections (urgent, chronic tonsillitis and sore throat) and bacterial dysentery and gastroenteritis (Md. Sanower Hossain et al., The Scientific World Journal 2014: 1-28, 2014). The Republic of China Patent No. I355933 has described an andrographolide which is a tumor necrosis factor (TNF-α) antagonist and a pharmaceutical composition thereof. Further, a pharmaceutical composition for healing wounds and/or whitening skin has been prepared by using andrographolide as an active ingredient (Republic of China Patent 201524532).
糖尿病(Diabetes mellitus,簡稱DM)為一種人體將葡萄糖(糖類)轉換成能量的方式出現變化的疾病,其特徵在於伴隨著脂肪和蛋白質代謝紊亂的高血糖症。根據行政院衛生署資料統計,台灣糖尿病人口已逾一百四十萬, 其中超過三成以上的糖尿病患者有腎功能衰退的問題,台大醫院代謝內分泌科主任黃天祥醫師表示,長期的高血糖會傷害體內的血管,並進一步侵犯到佈滿微血管的器官如腎臟、眼睛、神經系統及大血管系統如心臟,因此一發現有糖尿病就必須注意腎病變徵兆,以免一旦腎臟功能受損,就很難再恢復回來。 Diabetes mellitus (DM) is a disease in which the body changes glucose (sugar) into energy, and is characterized by hyperglycemia accompanied by disorders of fat and protein metabolism. According to the statistics of the Health Department of the Executive Yuan, the population of diabetes in Taiwan has exceeded 1.4 million. More than 30% of diabetic patients have problems with renal function decline. Dr. Huang Tianxiang, director of the Department of Metabolism and Endocrinology, National Taiwan University Hospital, said that long-term hyperglycemia can damage blood vessels in the body and further invade organs such as kidneys, eyes and nervous system. And the large vascular system such as the heart, so once you find diabetes, you must pay attention to signs of kidney disease, so that once the kidney function is impaired, it is difficult to recover.
本發明係以穿心蓮乾燥莖中所含之具有降血糖活性的有機溶劑萃取物為目標,同時研究其對於糖尿病患者腎臟病變的防治。本發明之穿心蓮乾燥莖有機萃取物經分析,其中具有降血糖藥理活性的成分(active ingredient,APai)包含特定含量之指標化合物:穿心蓮內酯(Andrographolides,簡稱A)、新穿心蓮內酯(Neoandrographolide,簡稱NA)、及去氧去氫穿心蓮內酯(14-deoxy-11,12-dehydrogen-andrographolide,簡稱DDA)。 The invention aims at the prevention and treatment of renal lesions in diabetic patients by taking the organic solvent extract with hypoglycemic activity contained in the dried stem of Andrographis paniculata. The organic extract of the dried stem of Andrographis paniculata L. of the present invention is analyzed, wherein an active ingredient (APai) having a hypoglycemic pharmacological activity comprises a specific content of an indicator compound: Andrographolides (A) and Neoandrographolide (Neoandrographolide, Referred to as NA), and deoxygenated and dehydro-andrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, abbreviated as DDA).
於是,本發明之一方面係關於,一種穿心蓮有機溶劑萃取物用於製備保護糖尿病患者腎臟功能之組成物的用途,所述之穿心蓮有機溶劑萃取物主要包含230-400mg/g穿心蓮內酯(Andrographolides,簡稱A)、50-90mg/g新穿心蓮內酯(Neoandrographolide,簡稱NA)及20-50mg/g去氧去氫穿心蓮內酯(14-deoxy-11,12-dehydrogen-andrographolide,簡稱DDA)。於本發明之一較佳具體實施態樣,所述之穿心蓮有機溶劑萃取物主要包含180-380mg/g穿心蓮內酯、40-85mg/g新穿心蓮內酯及16-45mg/g去氧去氫穿心蓮內酯。 Accordingly, one aspect of the present invention relates to the use of an extract of Andrographis paniculata organic solvent for preparing a composition for protecting kidney function in a diabetic patient, wherein the extract of Andrographis paniculata organic solvent mainly comprises 230-400 mg/g andrographolide (Andrographolides) , abbreviated as A), 50-90mg / g neoandrographolide (NA) and 20-50mg / g dehydrogenated andrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, abbreviated as DDA). In a preferred embodiment of the present invention, the extract of Andrographis paniculata organic solvent mainly comprises 180-380 mg/g andrographolide, 40-85 mg/g of new andrographolide, and 16-45 mg/g of deoxygenated dehydrogenation. Andrographolide.
於本發明之一些具體實施態樣,所述之穿心蓮有機溶劑萃取物包含80-99%之活性成分(APai)。於本發明之一項具體實施態樣,所述之穿心蓮有機溶劑萃取物包含95-99%之活性成分(APai)。 In some embodiments of the invention, the Andrographis paniculata organic solvent extract comprises 80-99% active ingredient (APai). In a specific embodiment of the present invention, the Andrographis paniculata organic solvent extract comprises 95-99% active ingredient (APai).
於本發明之一些具體實施態樣,所述之穿心蓮有機溶劑萃取物活 性成分(APai)包含230-450mg/g穿心蓮內酯(Andrographolides,簡稱A)、50-90mg/g新穿心蓮內酯(Neoandrographolide,簡稱NA)及20-60mg/g去氧去氫穿心蓮內酯(14-deoxy-11,12-dehydrogen-andrographolide,簡稱DDA)。於本發明之一項較佳具體實施態樣,所述之穿心蓮有機溶劑萃取物活性成分(APai)包含230-280mg/g穿心蓮內酯、50-70mg/g新穿心蓮內酯及20-40mg/g去氧去氫穿心蓮內酯。 In some embodiments of the present invention, the Andrographis paniculata organic solvent extract active ingredient (APai) comprises 230-450 mg/g andrographolide (Andrographolides, abbreviated as A), 50-90 mg/g of new andrographolide (Neoandrographolide) , abbreviated as NA) and 20-60 mg / g dehydrogenated andrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, abbreviated as DDA). In a preferred embodiment of the present invention, the Andrographis paniculata organic solvent extract active ingredient (APai) comprises 230-280 mg/g andrographolide, 50-70 mg/g of new andrographolide and 20-40 mg/ g deoxygenated dehydroandrographolide.
於本發明之一些具體實施態樣,所述之穿心蓮有機溶劑萃取物包含99-100%穿心蓮有機溶劑萃取物活性成分(APai),其中所述之活性成分(APai)包含25%-40%穿心蓮內酯、5%-9%新穿心蓮內酯及2%-5%去氧去氫穿心蓮內酯。於本發明之一項具體實施態樣,所述之穿心蓮有機溶劑萃取物包含25-30%穿心蓮內酯、6-8%新穿心蓮內酯及3-4%去氧去氫穿心蓮內酯。 In some embodiments of the present invention, the Andrographis paniculata organic solvent extract comprises 99-100% Andrographis paniculata organic solvent extract active ingredient (APai), wherein the active ingredient (APai) comprises 25%-40% Andrographis paniculata Lactone, 5%-9% new andrographolide and 2%-5% deoxygenated andrographolide. In a specific embodiment of the present invention, the Andrographis paniculata organic solvent extract comprises 25-30% andrographolide, 6-8% new andrographolide, and 3-4% deoxyhydroandrographolide.
本發明之另一方面,係關於一種用於保護糖尿病患者腎臟功能之組成物,包含一具有降血糖藥理活性之穿心蓮有機溶劑萃取物,其特徵在於所述之有機溶劑萃取物包含23%-40%穿心蓮內酯、5%-9%新穿心蓮內酯及2%-5%去氧去氫穿心蓮內酯。 Another aspect of the invention relates to a composition for protecting kidney function of a diabetic patient, comprising an andrographis organic solvent extract having hypoglycemic pharmacological activity, characterized in that the organic solvent extract comprises 23%-40 % andrographolide, 5%-9% new andrographolide and 2%-5% deoxygenated andrographolide.
於本發明之一些具體實施態樣,所述之組成物為一醫藥組成物。於本發明之其他具體實施態樣,所述之所述之組成物為一保健食品組成物。 In some embodiments of the invention, the composition is a pharmaceutical composition. In other specific embodiments of the invention, the composition is a health food composition.
本發明之另一方面,係關於一種用於防止及調控糖尿病患者腎臟病變之醫藥組成物,包含所述之穿心蓮有機溶劑萃取物及醫藥上可接受的載體、稀釋劑或賦形劑。 Another aspect of the invention relates to a pharmaceutical composition for preventing and regulating kidney disease in a diabetic patient, comprising the extract of Andrographis paniculata organic solvent and a pharmaceutically acceptable carrier, diluent or excipient.
圖1為混合標準物溶液測得之多重層析圖譜。 Figure 1 is a multiple chromatogram of the mixed standard solution.
圖2係顯示各種劑量(E20,E40,E80mg/kg)之本發明穿心蓮有機溶劑萃取物APai對於菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠的飲水量(A)及飲食量(B)的影響。 Figure 2 is a graph showing the amount of water (A) and the amount of food consumed by the penicillin-streptomycin-induced type II diabetic mice in various doses (E20, E40, E80 mg/kg) of the Andrographis paniculata organic solvent extract APai. (B) The impact.
圖3係顯示各種劑量(E20,E40,E80mg/kg)之本發明穿心蓮有機溶劑萃取物APai對於菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠之口服葡萄糖耐受性試驗的影響。 Figure 3 is a graph showing the effects of various doses (E20, E40, E80 mg/kg) of the pericardium organic solvent extract APai on the oral glucose tolerance test induced by nicotinamide-streptomycin in type II diabetic mice. .
圖4係顯示各種劑量(E20,E40,E80mg/kg)之本發明穿心蓮有機溶劑萃取物APai對於菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠在28-天重複口服投藥之血糖值的影響。 Figure 4 is a graph showing the various doses (E20, E40, E80 mg/kg) of the pericardium organic solvent extract APai of the present invention for niacinamide-streptomycin-induced type II diabetic mice to repeat oral administration of blood glucose at 28-days. The impact of the value.
圖5係顯示各種劑量(E20,E40,E80mg/kg)之本發明穿心蓮有機溶劑萃取物APai對於菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠在28-天重複口服投藥之HbA1c值的影響。 Figure 5 is a diagram showing the various doses (E20, E40, E80 mg/kg) of the orthodontic organic solvent extract APai of the present invention for nicotinicinamide-streptomycin-induced type II diabetic mice in a 28-day repeated oral administration of HbA1c The impact of the value.
圖6係顯示正常組Sham(A)、STZ誘導第II型糖尿病小鼠組(B)、STZ誘導第II型糖尿病小鼠組以APai-10(E10mg/kg)(C)、以APai-20(E20mg/kg)(D)、以APai-40(E40mg/kg)(E)及以STZ+APai-80(E80mg/kg)(F)等各分組,在28-天中重複口服投藥處理之小鼠腎臟組織切片放大倍率為400x的蘇木精-伊紅染色結果。 Figure 6 shows the normal group Sham (A), STZ induced type II diabetic mice (B), and STZ induced type II diabetic mice with APai-10 (E10 mg/kg) (C), with APai-20 (E20mg/kg) (D), APai-40 (E40mg/kg) (E) and STZ+APai-80 (E80mg/kg) (F), etc., repeated oral administration in 28-day The mouse kidney tissue section was magnified with a 400x hematoxylin-eosin staining result.
於本說明書中所稱的“穿心蓮有機溶劑萃取物”係指,穿心蓮乾燥莖之乙醇粗萃取物進一步以乙醇、正己烷、正丁醇與乙酸乙酯等有機溶劑進行萃取、濃縮及乾燥而得者。經由活性測試及分析,本發明之穿心蓮有機溶劑萃取物包含具有降血糖活性成分(active ingredient,APai)。 The "andrographis organic solvent extract" as referred to in the present specification means that the crude ethanol extract of the dried stem of Andrographis paniculata is further extracted, concentrated and dried by an organic solvent such as ethanol, n-hexane, n-butanol or ethyl acetate. By. Through the activity test and analysis, the Andrographis paniculata organic solvent extract of the present invention comprises an active ingredient (APai).
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明,而該實施範例僅作為輔助說明,並非用於限制本發明之範圍。 The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.
實施例一、穿心蓮有機溶劑萃取物之製備Example 1 Preparation of Andrographis Organic Solvent Extract
將穿心蓮乾燥莖(4.0kg)切碎,並於迴流下以60升乙醇萃取兩次。將組合之萃取物於減壓下進行濃縮,而得到乾燥的粗萃取物(產率8.75%)。將前述之乙醇粗萃取物依序使用下列有機溶劑及萃取條件:乙醇(95%)在室溫下萃取36-72小時,重複5-7次、正己烷(100%)在室溫下萃取12-36小時,重複4-7次、正丁醇(100%)在室溫下萃取12-48小時,重複7-10次、乙酸乙酯(100%)在室溫下萃取12-36小時,重複7-10次,透過系統分離法進行連續萃取與分離,之後將所得之有機萃取物利用減壓濃縮機及烘箱進行濃縮與乾燥,取得萃取物粉末,即本發明之穿心蓮有機溶劑萃取物活性成分(APai)。 The dried stem of Andrographis paniculata (4.0 kg) was chopped and extracted twice with 60 liters of ethanol under reflux. The combined extracts were concentrated under reduced pressure to give a dried crude extract (yield: 8.75%). The foregoing crude ethanol extract is sequentially subjected to the following organic solvents and extraction conditions: ethanol (95%) is extracted at room temperature for 36-72 hours, repeated 5-7 times, and n-hexane (100%) is extracted at room temperature. -36 hours, repeat 4-7 times, n-butanol (100%) was extracted at room temperature for 12-48 hours, repeated 7-10 times, and ethyl acetate (100%) was extracted at room temperature for 12-36 hours. Repeating 7-10 times, continuous extraction and separation by system separation method, and then concentrating and drying the obtained organic extract by a vacuum concentrator and an oven to obtain an extract powder, that is, the activity of the organic solvent extract of the present invention Ingredients (APai).
實施例二、穿心蓮有機溶劑萃取物之活性成分(active ingredient,APai)組成物的指標化合物含量分析Example 2: Analysis of the content of the indicator compound of the active ingredient (APai) composition of the extract of the organic solvent of Andrographis paniculata
進一步利用二溶劑-HPLC分析系統,測定本發明穿心蓮有機溶劑萃取物活性成分組成物(APai)之主要組成物質的含量,溶劑A為100%乙腈,溶劑B為0.25%甲酸,分析條件如下述: Further, the content of the main constituent substance of the active ingredient composition (APai) of the organic solvent extract of the present invention is determined by a two-solvent-HPLC analysis system, the solvent A is 100% acetonitrile, and the solvent B is 0.25% formic acid, and the analysis conditions are as follows:
1. 移動相(Mobil phase) Mobile phase (Mobil phase)
起始條件為31%溶劑B,0-12分鐘改變成35%溶劑B,12-23分鐘改變成35%溶劑B,23-28分鐘改變成31%溶劑B,28-38分鐘改變成31%溶劑B。使用Shimadzu Class VPTM軟體進行整合與校正。透過高峰面積與線性回歸進行評估。 The starting conditions were 31% solvent B, 0-12 minutes changed to 35% solvent B, 12-23 minutes changed to 35% solvent B, 23-28 minutes changed to 31% solvent B, 28-38 minutes changed to 31% Solvent B. Using Shimadzu Class VP TM software integration correction. Evaluate through peak area and linear regression.
2. 管柱:LiChrospher® C18,5μm,(250 X 4.6mm,5μm) 2. Column: LiChrospher ® C18, 5 μ m, (250 X 4.6mm, 5μm)
3. 流速:1mL/min 3. Flow rate: 1mL/min
4. UV偵測:210,232 and 254nm,使用SPD-M10AVP photodiode array detector(Japan) 4. UV detection: 210, 232 and 254 nm, using SPD-M10AVP photodiode array detector (Japan)
5. 注射體積:20μL 5. Injection volume: 20 μ L
接著進行檢量線的製作,定量活性成分組合物中A,NA及DDA之含量。藉由配置標準物工作溶液(10-300μg/mL)及內在標準物工作溶液,來訂出檢量曲線(Calibration curve)數據。透過繪製標準物對內在標準物之高峰面積比例圖,來建構檢量曲線。準確度以日內(intra-day)及日間(inter-day)RSD表示。標準物之日內及日間RSD是分別於同一天及連續三天進行分析得到,每次實驗包含三重複組。檢出極限(LOD)係測定為對應於信號對雜訊比為3:1之標準物含量。定量極限(LOQ)係測定為能夠以可接受之精確度及正確度,在具有信號對雜訊比為至少10之實驗條件下得到的最低標準物濃度。 Next, the calibration curve was prepared to quantify the contents of A, NA and DDA in the active ingredient composition. Calibration curve data was prepared by configuring a standard working solution (10-300 μg/mL) and an internal standard working solution. The calibration curve is constructed by plotting the standard against the peak area ratio of the intrinsic standard. Accuracy is expressed in intra-day and inter-day RSD. The intra- and inter-day RSD of the standard was obtained on the same day and three consecutive days, and each experiment contained three replicates. The limit of detection (LOD) was determined to correspond to a standard to the signal to noise ratio of 3:1. The limit of quantitation (LOQ) is determined as the minimum standard concentration that can be obtained with acceptable accuracy and accuracy under experimental conditions with a signal to noise ratio of at least 10.
圖1所示為混合標準物溶液於210、232及254nm下測得之重疊層析圖譜圖中標示穿心蓮內酯(A)、新穿心蓮內酯(NA)及去氧去氫穿心蓮內酯(DDA)之吸收高峰位置。A、NA及DDA標準物之滯留時間(retention time)分別為6.13、11.83及18.68min,而卡巴氮平(內在標準物)之滯留時間為10.03mA、NA及DDA標準物係於232、210及210nm呈現最大吸光值。因此,A標準物的線性度、精確度及回收率係於232nm下進行計算;而NA及DDA標準物的線性度、精確度及回收率係於210nm下進行計算。此等選擇的吸光值亦應用於卡巴氮平(內在標準物)之吸光值。 Figure 1 shows the overlapping chromatograms of the mixed standard solutions at 210, 232 and 254 nm indicating andrographolide (A), neoandrographolide (NA) and deoxyhydroandrographolide (DDA). The absorption peak position. The retention times of A, NA and DDA standards were 6.13, 11.83 and 18.68 min, respectively, while the retention time of carbazapine (intrinsic standard) was 10.03 mA, NA and DDA standards were at 232, 210 and 210 nm exhibits a maximum absorbance value. Therefore, the linearity, accuracy, and recovery of the A standard were calculated at 232 nm; the linearity, accuracy, and recovery of the NA and DDA standards were calculated at 210 nm. The absorbance values of these choices are also applied to the absorbance of kabazapine (intrinsic standard).
經由注射八種濃度(10-300μg/20μL)之含有A、NA及DDA的標準物工作溶液,來評估標準曲線之線性度。將高峰面積與濃度進行最小平方線性回歸分析,以計算校準方程式和相關判定係數。單獨A標準物於濃度範圍20-300μg/20μL內獲得的線性度為0.9990±0.0003,或者以A標準物對內在標準物之鋒面積比與相關判定係數獲得的線性度為0.9948±0.0009。單獨NA標準物於 濃度範圍20-300μg/20μL內獲得的線性度為0.9986±0.0002,或者以NA標準物對內在標準物之鋒面積比與相關判定係數獲得的線性度為0.9939±0.0012。單獨DDA標準物於濃度範圍20-300μg/20μL內獲得的線性度為0.9956±0.0020,或者以DDA標準物對內在標準物之鋒面積比與相關判定係數獲得的線性度為0.9988±0.0004。 The linearity of the standard curve was evaluated by injecting eight concentrations (10-300 μg / 20 μL) of the standard working solution containing A, NA and DDA. A least squares linear regression analysis was performed on the peak area and concentration to calculate the calibration equation and the associated decision coefficients. The linearity of the individual A standard obtained in the concentration range of 20-300 μg/20 μL was 0.9990±0.0003, or the linearity obtained by the A standard to the intrinsic standard front area ratio and the correlation coefficient was 0.9948±0.0009. Individual NA standard The linearity obtained in the concentration range of 20-300 μg/20 μL was 0.9986±0.0002, or the linearity obtained by the NA standard to the intrinsic standard front area ratio and the correlation coefficient was 0.9939±0.0012. The linearity of the DDA standard obtained in the concentration range of 20-300 μg/20 μL was 0.9956±0.0020, or the linearity obtained by the DDA standard to the intrinsic standard front area ratio and the correlation coefficient was 0.9988±0.0004.
中間精確度可呈現出實驗室偏差,以日內及日間偏差表示。於同一天(日內)及於不同天(日間)進行A、NA及DDA標準物之八種濃度(10-300μg/20μL)判定,並以相對標準偏差百分比(%RSD)或變異係數表示(CV)。結果顯示,對於A標準物之日內及日間分析的%RSD係落在0.54-9.21範圍內;對於NA標準物之日內及日間分析的%RSD係落在0.54-8.03範圍內;而對於DDA標準物之日內及日間分析的%RSD係落在1.00-14.97範圍內。 Intermediate accuracy can present laboratory deviations expressed as intraday and interday deviations. On the same day (day) and eight kinds of concentrations for A, NA, and the standard DDA (10-300 μ g / 20 μ L ) determined at different days (day), and to percent relative standard deviation (% RSD) or a variant The coefficient is expressed in (CV). The results show that the %RSD for the intra- and inter-day analysis of the A standard falls within the range of 0.54-9.21; the %RSD for the intra- and inter-day analysis of the NA standard falls within the range of 0.54-8.03; and for the DDA standard The %RSD analyzed during the day and during the day falls within the range of 1.00-14.97.
該方法的準確性是從回收率研究確定。預分析樣品中摻入了三種不同濃度的A、NA及DDA標準物,然後將混合物以建議的方法進行分析。結果得知,A標準物之回收率範圍介於96.93-100.23%,而計算A標準物之平均回收百分比值為98.41±1.68%。NA標準物之回收率範圍介於97.03-102.14%,而計算NA標準物之平均回收百分比值為99.64±2.55%。DDA標準物之回收率範圍介於97.09-105.23%,而計算DDA標準物之平均回收百分比值為100.92±4.40%。 The accuracy of the method was determined from the recovery study. The pre-analytical samples were spiked with three different concentrations of A, NA and DDA standards and the mixture was analyzed in the recommended manner. As a result, the recovery rate of the A standard ranged from 96.93 to 100.23%, and the average recovery percentage of the calculated A standard was 98.41±1.68%. The recovery of NA standards ranged from 97.03 to 102.14%, while the average recovery percentage of calculated NA standards was 99.64 ± 2.55%. The recovery of the DDA standard ranged from 97.09 to 105.23%, while the average recovery percentage of the calculated DDA standard was 100.92 ± 4.40%.
藉由比對由光二極體列陣偵測儀得到之標準物與樣本的高峰光譜及純度,確定相對應於存在APai中之A、NA與DDA的高峰。圖1顯示本發明穿心蓮有機溶劑萃取物活性組成於210、232及254nm下的HPLC多重層析圖譜。存在APai中A、NA及DDA之含量計算結果如下表1所示。因此,穿心蓮內酯(A)含量為235.10±1.64mg/g;新穿心蓮內酯(NA)含量為52.31±4.62mg/g;去氧去氫穿心蓮內酯(DDA)含量為22.68±1.13mg/g。本實例提供之分析數值,會因所採 收之穿心蓮來源、產地及產季而有所變化,但經由以下的藥理活性實驗證明,本發明萃取方法所得之穿心蓮有機溶劑萃取物活性成分具有降血糖功效。 The peaks corresponding to the A, NA and DDA in the presence of APai are determined by comparing the peak spectra and purity of the standards and samples obtained by the photodiode array detector. 1 shows an HPLC multiple chromatogram of the active composition of the organic extract of Andrographis paniculata at 210, 232 and 254 nm. The calculation results of the contents of A, NA and DDA in the presence of APai are shown in Table 1 below. Therefore, the content of andrographolide (A) was 235.10±1.64 mg/g; the content of new andrographolide (NA) was 52.31±4.62 mg/g; the content of deoxyhydroandrographolide (DDA) was 22.68±1.13 mg/ g. The analytical values provided in this example will be The source, origin and season of the andrographis paniculata vary, but the pharmacological activity test proves that the active ingredient of the andrographis paniculata organic solvent extract obtained by the extraction method of the invention has hypoglycemic effect.
實施例三、穿心蓮有機溶劑萃取物活性成分組成物對於鏈佐黴素STZ-誘導之第II型糖尿病鼠之腎臟保護功效評估Example 3: Evaluation of the protective effect of the active ingredient composition of Andrographis paniculata organic solvent extract on streptozotocin STZ-induced type II diabetic rats
雄性ICR小鼠(19-21g)係購自台灣BioLASCO Co.,Ltd。小鼠餵養於環球科技大學之動物中心,溫度調控於22±2℃,相對濕度55±5%且於實驗前一周施予12小時光照/12小時黑暗週期。對於第II型糖尿病鼠之誘導,係將菸鹼醯胺(Sigma,Saint Louis,MO,USA)食鹽水溶液(210mg/kg b.w.)以腹膜內注射入小鼠,經15分鐘後將於使用前溶解於檸檬酸鹽緩衝液(pH 4.5)之鏈佐黴素(STZ)(Sigma,180mg/kg b.w.,i.p.注射)投藥給該小鼠。對照組係接受該二種載劑者。於實驗期間,每週一次記錄實驗動物之食物攝取量及體重。本發明穿心蓮有機溶劑萃取物活性成分組成物APai之投藥量為:E10組:APai 10mg/kg;E20組:APai 20mg/kg;E40組:APai 40mg/kg;E80組:APai 80mg/kg。其中為如前述實施例一所製備得之穿心蓮有機溶劑萃取物活性成分組成物,經Tween 20溶解後調整成0.5%甲基纖維素(CMC)溶液。 Male ICR mice (19-21 g) were purchased from BioLASCO Co., Ltd., Taiwan. Mice were fed an animal center at the University of Science and Technology of the United States, with a temperature regulation of 22 ± 2 ° C, a relative humidity of 55 ± 5% and a 12-hour light / 12-hour dark cycle one week prior to the experiment. For the induction of type II diabetic rats, nicotinic acid amide (Sigma, Saint Louis, MO, USA) aqueous saline solution (210 mg/kg bw) was intraperitoneally injected into mice and dissolved after 15 minutes. The mice were administered with citrate buffer (pH 4.5) of streptozotocin (STZ) (Sigma, 180 mg/kg bw, ip injection). The control group received the two carriers. The food intake and body weight of the experimental animals were recorded once a week during the experiment. The dosage of APai of the active ingredient composition of the organic solvent extract of the present invention is: E10 group: APai 10 mg/kg; E20 group: APai 20 mg/kg; E40 group: APai 40 mg/kg; E80 group: APai 80 mg/kg. Among them, the active ingredient composition of Andrographis paniculata organic solvent extract prepared as in the above Example 1 was dissolved in Tween 20 and adjusted to a 0.5% methylcellulose (CMC) solution.
臨床觀察Clinical Observation
每天至少進行兩次觀察動物之身體狀況,每次觀察的時間間隔不少於6小時,以確定動物的健康或死亡狀態。每天至少一次觀察試驗動物之臨 床病徵,記錄試驗動物所表現出的毒性作用,包括該等毒性作用的開始時間及過程。試驗結束後,將存活的小鼠犧牲;之後進行屍體解剖,採取血液樣本進行血清生化分析。 Observe the physical condition of the animal at least twice a day, with an interval of not less than 6 hours per observation to determine the health or death of the animal. Observe the presence of the test animal at least once a day Bed symptoms, recording the toxic effects exhibited by the test animals, including the onset time and course of such toxic effects. At the end of the experiment, the surviving mice were sacrificed; then autopsy was performed and blood samples were taken for serum biochemical analysis.
由臨床觀察之結果顯示,本發明之穿心蓮有機溶劑萃取物活性成分組成物APai(E10、E20、E40、E80mg/kg)可降低因菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠的小鼠的體重增加,但之間無劑量依存性(圖2)。 As a result of clinical observation, the active ingredient composition of the Andrographis paniculata organic solvent extract of the present invention APai (E10, E20, E40, E80 mg/kg) can reduce the type II diabetic mice induced by nicotinamide-streptomycin The mice gained weight but there was no dose dependency between them (Figure 2).
血清肌酸酐(Creatinine)分析Serum creatinine (Creatinine) analysis
肌酸酐(creatinine)又稱肌酐,是肌酸和磷酸肌酸代謝的終產物,它主要由肌肉中磷酸肌酸的非酶促反應生成。肌酸酐的產生量一定,而且不會在過濾後被腎小管重吸收。因此,它的排泄量反映血液中的肌酸酐含量和腎小球的過濾速率,也就是腎臟的機能。當腎發生嚴重病變時,肌酸酐排泄受阻,致使血液中肌酸酐含量升高。於是透過血液中肌酸酐的測定,可以知道腎功能是否有嚴重障礙,肌酸酐的值越高則顯示腎臟的障礙越大。 Creatinine, also known as creatinine, is the end product of creatine and creatine phosphate metabolism, which is mainly produced by the non-enzymatic reaction of creatine phosphate in muscle. The amount of creatinine produced is constant and will not be reabsorbed by the renal tubules after filtration. Therefore, its excretion reflects the creatinine content in the blood and the filtration rate of the glomerulus, which is the function of the kidney. When the kidney develops severe lesions, creatinine excretion is blocked, resulting in an increase in creatinine levels in the blood. Therefore, it is possible to know whether there is a serious disorder of renal function by measuring the creatinine in the blood, and the higher the value of creatinine, the greater the obstacle of the kidney.
本試驗以Randox套裝組進行分析(Randox Laboratories Ltd,Crumlin,UK)。取50μL血清加入500μL反應試劑(35mmol/L Picric acid,0.32mol/L Sodium Hydroxide)混合均勻後在37℃下反應,使用分光光度計於490nm檢測其吸光值,於30秒後讀取值為A1,2分鐘後讀取值為A2,計算方式為A2-A1之數值。鹼性溶液中的Creatinine與picric acid反應後會產生有色的複合物,最終以複合物之數值來澰測Creatinine之濃度含量。請提供詳細的實驗方法,包括使用的試劑、來源、檢測條件和評量標準等。 This test was performed in the Randox kit (Randox Laboratories Ltd, Crumlin, UK). Take 50 μ L of serum was added 500 μ L reaction reagent (35mmol / L Picric acid, 0.32mol / L Sodium Hydroxide) After the reaction was mixed at 37 ℃, using a spectrophotometer to detect the absorbance 490nm, read at 30 seconds The value is A1. After 2 minutes, the value is A2, and the calculation method is the value of A2-A1. The Creatinine in alkaline solution reacts with picric acid to produce a colored complex, and finally the value of the complex is used to measure the concentration of Creatinine. Please provide detailed experimental methods, including reagents used, sources, test conditions and evaluation criteria.
圖3係顯示各種劑量(E20,E40,E80mg/kg)之本發明穿心蓮有機溶劑萃取物APai對於菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠之口服葡萄糖耐受性試驗的影響,各數值以平均值±SEM表示。##P<0.01係相較於正常動物組(Sham組)。* P<0.05,** P<0.01,*** P<0.001係相較於STZ誘導處理組。之結果顯示, STZ明顯升高血清肌酸酐(Serum Creatinine)值,與正常組(Sham)比較時,有顯著的差異。投藥本發明之APai(E20、E40及E80mg/kg)可降低Nicotinamide-streptozotocin所誘導第II型糖尿病小鼠血清中肌酸酐升高的現象,其中以E40、E80mg/kg連續投藥28天的效果最佳。 Figure 3 is a graph showing the effects of various doses (E20, E40, E80 mg/kg) of the pericardium organic solvent extract APai on the oral glucose tolerance test induced by nicotinamide-streptomycin in type II diabetic mice. The values are expressed as mean ± SEM. ## P<0.01 is compared to the normal animal group (Sham group). *P<0.05, **P<0.01, ***P<0.001 compared to the STZ induction treatment group. The results showed that STZ significantly increased serum creatinine (Serum Creatinine) values, which were significantly different when compared with the normal group (Sham). Administration of APai (E20, E40 and E80mg/kg) of the present invention can reduce the increase of serum creatinine in mice with type II diabetes induced by nicotinamide-streptozotocin, and the effect of continuous administration for 28 days at E40 and E80 mg/kg is the most effective. good.
尿酸分析(Uric acid assay)Uric acid assay
尿酸的排出,除了少部分由腸道排出,其餘大部分皆由腎臟排出。腎臟功能不好的人,尿酸代謝的能力也相對變差,導致血中尿酸升高。因此,在常規腎功能的檢查通常也包括血尿酸分析。 Excretion of uric acid, except for a small part of it is excreted by the intestines, most of which are excreted by the kidneys. People with poor kidney function have a relatively poor ability to metabolize uric acid, leading to an increase in uric acid in the blood. Therefore, examination of routine renal function usually also includes blood uric acid analysis.
本試驗以Randox套裝組進行分析(Randox Laboratories Ltd,Crumlin,UK)。取1mL血清與10mL蒸餾水稀釋後取20μL加入1000μL反應試劑(200mmol/L HEPES buffer pH 7.55,0.25mmol/L 4-Aminophenazone,4.0mmol/L 3,5 DCHBS,200U/L Uricase,1000U/L Peroxidase)混合均勻後在37℃下反應5分鐘,使用分光光度計於520nm檢測quinoneimine之吸光值。 This test was performed in the Randox kit (Randox Laboratories Ltd, Crumlin, UK). Take 1mL 10mL serum taken after dilution with distilled water is added 20 μ L 1000 μ L reaction reagents (200mmol / L HEPES buffer pH 7.55,0.25mmol / L 4-Aminophenazone, 4.0mmol / L 3,5 DCHBS, 200U / L Uricase, 1000U /L Peroxidase) After mixing uniformly, the reaction was carried out at 37 ° C for 5 minutes, and the absorbance of quinoneimine was measured at 520 nm using a spectrophotometer.
尿酸(Uric acid)的生化值反應式如下:
於圖4中各數值以平均值±SEM表示。##P<0.01,##P<0.001係相較於正常動物組(Sham組)。* P<0.05,** P<0.01,*** P<0.001係相較於STZ誘導處理組。之結果顯示,STZ明顯升高HbA1c,與Sham比較時,有顯著的差異。投藥本發明之APai(E40,E80mg/kg),可降低菸鹼醯胺-鏈佐黴素所誘導第II型糖尿病小鼠血清中尿酸升高之現象,其中以E40mg/kg連續投藥28天的效果最佳。 The values in Figure 4 are expressed as mean ± SEM. ## P<0.01, ## P<0.001 is compared to the normal animal group (Sham group). *P<0.05, **P<0.01, ***P<0.001 compared to the STZ induction treatment group. The results showed that STZ significantly increased HbA1c, and there was a significant difference when compared with Sham. Administration of APai (E40, E80 mg/kg) of the present invention can reduce the increase of uric acid in serum of type II diabetic mice induced by nicotinamide-streptavidin, wherein E40 mg/kg is continuously administered for 28 days. The best results.
尿素分析(Urea assay)Urea assay
尿素是腎臟過濾新陳代謝後產生的廢物。正常成年人檢驗得出的數值應介乎2.1至7.1mmol/L,若腎臟功能出現問題,廢物無法適當排出,尿素值便會升高。因此,尿素氮和血肌酐同是腎功能的兩個最重要的指標,尿素氮數值的高與低反映著腎臟功能的好與壞。 Urea is the waste produced by the kidneys after filtering metabolism. The value obtained by normal adult testing should range from 2.1 to 7.1 mmol/L. If there is a problem with kidney function, the waste cannot be properly discharged and the urea value will increase. Therefore, both urea nitrogen and serum creatinine are the two most important indicators of renal function. The high and low values of urea nitrogen reflect the good or bad function of the kidney.
本試驗以Randox套裝組進行分析(Randox Laboratories Ltd,Crumlin,UK)。取5μL血清加入500μL反應試劑(150mmol/L Tris buffer pH 7.6,10U/ml Urease,2U/mL GLDH,0.26mmol/L NADH,3mmol/L Adenosine-5-diphosphate,14mmol/L α-oxoglutarate)混合均勻後在37℃下反應,於1,2,3分鐘使用分光光度計於340nm檢測其吸光值。 This test was performed in the Randox kit (Randox Laboratories Ltd, Crumlin, UK). Take 5 μ L of serum was added 500 μ L reaction reagents (150mmol / L Tris buffer pH 7.6,10U / ml Urease, 2U / mL GLDH, 0.26mmol / L NADH, 3mmol / L Adenosine-5-diphosphate, 14mmol / L α- Oxoglutarate) was uniformly mixed and reacted at 37 ° C, and its absorbance was measured at 340 nm using a spectrophotometer at 1, 2, and 3 minutes.
尿素的生化值反應式
圖5為檢測各種劑量(E20,E40,E80mg/kg)之本發明穿心蓮有機溶劑萃取物APai對於菸鹼醯胺-鏈佐黴素誘導第II型糖尿病小鼠在28-天重複口服投藥之HbA1c值的影響,其中各數值以平均值±SEM表示。##P<0.01係相較於正常動物組(Sham組);*P<0.05,**P<0.01係相較於STZ誘導處理組。結果顯示,STZ明顯升高尿素值,與正常對照組(Sham)比較時,有顯著的差異。而投藥本發明之APai(E40,E80mg/kg),可降低Nicotinamide-streptozotocin所誘導第II型糖尿病小鼠血清中尿素值升高的現象。 Fig. 5 is a view showing the HbA1c of the orthodontic organic solvent extract APai of the present invention for detecting various doses (E20, E40, E80 mg/kg) for niacinamide-streptomycin-induced type II diabetic mice in a 28-day repeated oral administration. The effect of the values, where each value is expressed as mean ± SEM. ## P<0.01 was compared with the normal animal group (Sham group); *P<0.05, **P<0.01 was compared with the STZ induction treatment group. The results showed that STZ significantly increased the urea value, which was significantly different from the normal control group (Sham). The administration of the APai (E40, E80 mg/kg) of the present invention can reduce the increase in the urea value in the serum of type II diabetic mice induced by Nicotinamide-streptozotocin.
腎臟組織病理學的觀察(Histopathological observation)Histopathological observation of the kidney (Histopathological observation)
割取腎臟,割取1公分見方的腎組織,放入10%的中性福馬林中, 做進一步的蘇木紫-伊紅染色法(Hematoxylin-Eosin stain病理染色。在進行組織病理學的比較時,可以Shackelford et al.(2002)(Toxicologic Pathology 30:93-96,2002)的半定量方法進行評估。損傷程度是嚴重性區分為五級:1=最輕度(minimal)(<1%);2=輕度(slight)(1-25%);3=中度(moderate)(26-50%);4=中度/嚴重(moderate/severe)(51-75%);5=嚴重/極嚴重(severe/high)(76-100%)。 The kidneys were cut, and 1 cm of kidney tissue was cut and placed in 10% neutral formalin for further hematoxylin-Eosin stain staining. Comparison of histopathology was performed. The semi-quantitative method of Shackelford et al. (2002) ( Toxicologic Pathology 30: 93-96, 2002) can be evaluated. The degree of damage is classified into five grades: 1 = minimal (<1%) 2;slight (1-25%); 3=moderate (26-50%); 4=moderate/severe (51-75%); 5= Severe/high (76-100%).
結果如圖6所示,STZ誘導的糖尿病小鼠之腎臟組織出現發炎性損傷,以本發明之穿心蓮有機溶劑萃取物APai(E20、E40、E80mg/kg)處理,沒有發現發炎等損傷,顯示本發明之穿心蓮有機溶劑萃取物APai能改善由STZ誘導的糖尿病小鼠之腎臟組織損傷現象。 As a result, as shown in Fig. 6, the kidney tissue of STZ-induced diabetic mice showed inflammatory damage, and the treatment was carried out with the organic solvent extract APai (E20, E40, E80 mg/kg) of the present invention, and no damage such as inflammation was observed. The inventive andrographis organic solvent extract APai can improve the renal tissue damage phenomenon of STZ-induced diabetic mice.
綜合以上之實驗結果,本發明得自穿心蓮有機溶劑萃取物之具有降血糖活性成分組成物(APai),具有減輕由菸鹼醯胺-鏈佐黴素所誘導第II型糖尿病的腎功能減退的病理現象,尤其能有效降低糖尿病鼠之血中尿酸值,以80mg/kg之劑量所達到的效果最為顯著,甚至可回復至與正常小鼠組一樣的數值,故本發明之穿心蓮降血糖活性成分組成物不僅能防止糖尿病患者腎臟功能受損,且可預防糖尿病患者之尿酸性腎病變(urate nephropathy)發生。因此,本發明之穿心蓮有機溶劑萃取物活性成分組成物可用於研發製造天然腎臟保護藥物或保健食品。 Based on the above experimental results, the present invention is derived from a hypoglycemic active ingredient composition (APai) of an extract of Andrographis paniculata organic solvent, which has reduced renal dysfunction induced by nicotinamide-streptinomycin. The pathological phenomenon can effectively reduce the uric acid value in the blood of diabetic rats, and the effect achieved by the dose of 80 mg/kg is the most significant, and even returns to the same value as the normal mouse group, so the hypoglycemic active ingredient of the present invention The composition not only prevents kidney function damage in diabetic patients, but also prevents the occurrence of urate nephropathy in diabetic patients. Therefore, the active ingredient composition of the Andrographis paniculata organic solvent extract of the present invention can be used for research and development of a natural kidney protecting drug or a health food.
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