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研究生:唐偉智
研究生(外文):Wei-chih Tang
論文名稱:桑椹萃取物及組成方的生物活性之研究
論文名稱(外文):Screening of the bioactivity of mulberry extract and it’s mixture
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:製藥科技研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:87
中文關鍵詞:桑椹、刺五加、抗氧化活性、自由基清除能力、貧血
外文關鍵詞:mulberryfree radical scavenging abilityantioxidant activityanemia
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本研究利用桑的桑葉、桑椹、桑枝和桑白皮為材料,以水及95 %酒精萃取濃縮後,經冷凍乾燥後,評估其水萃與酒萃取物之抗氧化活性。並取用桑椹與刺五加以單方和複方進行貧血改善效力評估。
實驗結果顯示各桑樹的藥用部位的水萃取物之抗氧化能力大多優於酒精萃取物,桑椹水萃取物含有較高量的類黃酮與總酚以及花青素並具有最佳的自由基清除效力。桑椹水萃取物亦具有較佳緩解鹿角膠菜誘發小鼠發炎腫脹的效果。選用桑椹與刺五加等比例混合之組物灌食至以苯聯胺 (phenylhydrazine;phz)誘發貧血實驗大鼠,持續餵一週兩後分別採取各液檢測紅球數量、素及平均容積參。
所得結果發現,貧血大鼠食用桑椹後增加的表現最為明顯。在其病理組織切片中對於PHZ導脾臟受損以桑椹處理可達到最好效果。以上萃取物對在其K562細胞株實驗中顯現各物質並無抑制K562 細胞的生長。由結知桑椹確具有改善的力關鍵字: 桑椹、刺五加、抗氧化活性、自由基清除能力、貧血
In the present study, extract by 95% alcohol extract and water from the antioxidant
activities of leaves, berries, twigs, and cortex of Morus alba were clarified by free
radical scavenging test. The contents of flavonoids and the total phenolic compounds
in the targeted extract of M. alba were also determined. The targeted extract of M.
alba was further combined with the extract from Siberian Ginseng (Eleutherococcus
senticosus), at the appropriate ratio to compose the prescription used in the
amelioration of anemia.
It has been shown that the antioxidant capacity of the water extract of mulberry was
better than that from 95% alcohol extract. The water extract of mulberry contained the
highest levels of total phenol, flavonoid and anthocyanidin as well. The DPPH
radical scavenging ability in water extract of mulberry was higher than the others. The
water extract of mulberry produced the better effect to reduce the foot edema in
carrageenan-treated mice. The abnormal levels in blood including red blood cells,
white blood cells and hemoglobin, higher hematocrit mean corpuscular volume as
well as mean corpuscular hemoglobin and mean corpuscular hemoglobin
concentration were ameliorated in phenylhydrazine-treated rat (PHZ-rat) receiving an
oral administration of formula of mulberry and Siberian Ginseng at the equal ratio for
7 or 14 days. The pathology and hematoxylin staining in spleen of PHZ-rat revealed
that the increase of focal extramedullary hematopoiesis in splenomegaly with
erythroid precursors and hemosiderosis in the red pulp was reduced by mulberry and
Siberian Ginseng (1:1) in combination. In addition, the above extracts showed
non-cytotoxic to the K562 cell line.The results demonstrated that formula of mulberry
and Siberian Ginseng at the equal ratio pose the potential for anemic disorders
amelioration.
中文摘要…………………..…………………………………………………….Ⅰ
英文摘要..……………………………………………………………………….Ⅱ
誌謝...……………………………………………………......................…………III
目錄…………………………………………………………………..................……Ⅳ
表錄……………………………………………………………………….…………VII
圖錄……………………..............................................................................................IX
第壹章 研究背景…………………..............................................................................1
一、 桑之文獻回顧........................................………………………………………..1
(一)栽桑起源...........................................................................................................1
(二)桑樹種類型態...................................................................................................1
(三)桑樹常見藥用部位...........................................................................................2
(四)刺五加簡介.......................................................................................................6
二、貧血簡介...............................................................................................................7
(一)以西醫觀點定義貧血.......................................................................................7
(二)貧血的總類.......................................................................................................7
(三)脾臟的免疫功能.............................................................................................9
(四)以中醫觀點定義貧血.....................................................................................10
第貳章 總論研究動機與架構....................................................................................12
(一) 研究動機.......................................................................................................12
(二) 研究架構.......................................................................................................13
第叁章 材料與方法....................................................................................................14
(一) 實驗材料.......................................................................................................14
(二) 實驗步驟與方法...........................................................................................19
1.桑之萃取方式.............................................................................................19
2.刺五加之萃取方式.....................................................................................19
3.市售四物飲取得...………….……………………………………….……19
4.四物煎劑製作與萃取方式………….……………………………………19
5.抗氧化物質含量測定.................................................................................19
(1)總類黃酮化合物含量測定.....................................................................19
V
(2)總多酚類化合物含量測定.....................................................................19
6.花青素含量測定.........................................................................................20
7.急毒性試驗(acute toxicity)………………………………………….……21
8.K562 細胞毒性試驗測定..........................................................................22
(1)細胞活化...............................................................................................22
(2)細胞毒性(Cytotoxicity)測試.................................................................22
9.體外清除α,α-diphenyl-β-picrylhydrazyl(DPPH)自由基效力測定...22
10.桑抽取物對於鎮痛及抗發炎之影響.........................................................23
(1) Carrageenin 誘發足蹠腫脹實驗.................................................... ...23
(2) 細胞激素TNF-α 與IL-6 含量測定......................................................23
11.貧血試驗.....................................................................................................24
(1) 血液生化檢查.......................................................................................24
(2) Hematoxylin and eosin (H&;E)染色實驗方法.....................................25
12.統計方式....................................................................................................26
第肆章 結果 ..............................................................................................................27
1. 各藥材水萃與酒萃物的產率...................................................................27
2. 成分分析…………………………………………………...……………27
(1)類黃酮含量分析..…...………………………………………...……...27
(2)總酚含量分析……..…………………………………………….……27
(3)花青素含量分析……………..……………….....................................28
3. 毒性試驗……………………………………...…………………………28
(1) 急性毒性試驗……………………...………………………………..28
(2) 細胞毒性試驗……………………...………………………………..29
4.藥理活性分析.............................................................................................29
(1)體外清除DPPH 自由基的效力……....................................................29
(2)鹿角膠菜(Carrageenan)誘導發炎實驗.........................................29
(3)貧血改善試驗………………………………………………………...31
(I)血球變化值檢測分析值....................................................................31
a. 給予待測物連續七天後實驗動物血球變化檢測分析值……31
b. 給予待測物連續十四天後實驗動物血球變化檢測分析值….31
(II)凝血變化檢測分析值…...................................................................32
VI
a. 給予待測物連續七天後實驗動物凝血變化檢測分析值...........32
b. 給予待測物連續十四天後實驗動物凝血變化檢測分析值.......32
(III) 血糖與血脂生化檢測分析值.......................................................32
a. 給予待測物連續七天後實驗動物血糖與血脂生化檢測分析值
...………………………………………………………………..32
b. 給予待測物連續十四天後實驗動物血糖與血脂生化檢測分析
值...................................................................................................33
(IV) 肝腎生化檢測分析值...................................................................33
a. 給予待測物連續七天後實驗動物肝腎生化檢測分析值...........33
b. 給予待測物連續十四天後實驗動物肝腎生化檢測分析值.......33
(V) 電解質檢測分析值.......................................................................33
a. 給予待測物連續七天後實驗動物電解質檢測分析值...............33
b. 給予待測物連續十四天後實驗動物電解質檢測分析值...........33
(VI) 給予待測物連續十四天後實驗動物組織重量變化分分析值...34
(VII) 給予待測物連續十四天後實驗動物組織病理組織切片評估..34
第伍章 討論................................................................................................................35
參考文獻......................................................................................................................37
表錄
表一、各藥材水萃物與酒萃物產率…………………………………………………43
表二、臺灣產桑樹的葉、桑椹、桑枝、桑白皮等藥用部位的水或乙醇萃取物所含
總酚與類黃酮含量的變化..............................................................................44
表三、大陸產桑樹的葉、桑椹、桑枝、桑白皮等藥用部位所得水或乙醇萃取物所
含總酚與類黃酮含量的變化..........................................................................45
表四、以口服方式投予桑椹與刺五加粗萃物(5 g/kg)至BALB/cByJNarl 小鼠進行
急毒極限測試(acute limit test)所得之死亡情形…………………………46
表五、 臺灣產桑葉、桑椹、桑枝、桑白皮的萃取物的DPPH 自由基清除能力.........47
表六、 大陸產桑葉、桑椹、桑枝、桑白皮的萃取物的DPPH 自由基清除能力.........48
表七、連續七天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或是
桑椹與刺五加等比例混合之複合物後,大鼠血球變化檢測分析值............49
表八、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或
是桑椹與刺五加等比例混合之複合物後,大鼠血球變化檢測分析值....50
表九、連續七天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或
是桑椹與刺五加等比例混合之複合物後,大鼠的凝血變化值檢測分析值
...........................................................................................................................51
表十、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或
是桑椹與刺五加等比例混合之複合物後,大鼠的凝血變化值檢測分析
值......................................................................................................................52
表十一、連續七天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或
是桑椹與刺五加等比例混合之複合物後,大鼠血糖與血脂的表現
值…………………………………………………………………............53
表十二、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠血糖與血脂的表現
值..................................................................................................................54
表十三、連續七天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或
是桑椹與刺五加等比例混合之複合物後,大鼠肝臟與腎臟功能相關的生
化檢測值......................................................................................................55
表十四、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
VIII
或是桑椹與刺五加等比例混合之複合物後,大鼠肝臟與腎臟功能相關的
生化檢測值..................................................................................................56
表十五、連續七天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加或
是桑椹與刺五加等比例混合之複合物後,大鼠電解質檢測分析值......57
表十六、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠電解質檢測分析值....58
表十七、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠離體器官重量分析值........59
表十八、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠離體器官病理切片半定
量分析值......................................................................................................60
IX
圖錄
圖一、Kuromanin (Cyanidin-3-O-glucoside)的檢量線……………………….….….61
圖二、臺灣桑椹水萃物所含kuromanin (Cyanidin-3-O-glucoside chloride)的HPLC
層析圖譜…………………..…………………………...........…………….…62
圖三、Keracyanin (Cyanidin-3-rutinoside)的檢量線………………………….…..…63
圖四、(a)臺灣桑椹水萃物、(b)臺灣桑椹酒萃物所含keracyanin (cyanidine-
3- rut inoside) 的HPLC 層析圖譜…….……………….…...…..…64
圖五、Callistephin (Pelargonidin-3-O-glucoside)的檢量線………………………….65
圖六、臺灣桑椹水萃物所含callistephin(pelargonidin-3-O-glucoside)的HPLC 層析
圖譜…....…………………………………………………………..……...….66
圖七、市售四物飲、四物煎劑、桑椹、刺五加或是桑椹與刺五加等比例混合之複
合物對K562 細胞存活率的影響...................................................................67
圖八、以鹿角膠菜誘發小鼠足部腫脹程度............................................................68
圖九、以鹿角膠菜所誘發足部腫脹,抽血觀看發炎相關細胞激素TNF-α 分泌
生成量..............................................................................................................69
圖十、以鹿角膠菜誘發足部腫脹,抽血觀看發炎相關細胞激素IL-6 分泌生成
量......................................................................................................................70
圖十一、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠離體肝臟組織切片
圖. . .. . . . . . . . . . . . . . . . . . . . . . . . . .……………………………………………71
圖十二、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠離體腎臟組織切片
圖. . . . . . . . . . . . . .…………………………………. . . . . . . . . . . . . . . . . . . . . .72
圖十三、連續十四天餵食PHZ 誘導大鼠市售四物飲、四物煎劑、桑椹、刺五加
或是桑椹與刺五加等比例混合之複合物後,大鼠離體脾臟組織切片
圖…………………………………...……………………………………..73
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