第五章 女貞子藥理活性研究之萃取及劃分
第二節 清除 DPPH 藥理活性測試
a、配製 1mM DPPH 甲醇溶液
b、取 100λ之待測樣品，加入 100λDPPH 溶液，室溫下避光靜置 5 分鐘 c、使用光度計檢測 517nm 之吸光值
d、計算清除率
【各劃分層之活性測試】
利用清除DPPH進行實驗測定。如表所示，有氯仿層(LLC)及氯仿層的第10 個fraction(LLC-10)、第11個fraction(LLC-11)、第14個fraction(LLC-14)、和 水層(LLW)有良好的活性。利用此結果，再進一步作細部分離，取得有效化合物。
ITEM α-glucosidase DPPH Positive control 25.0% (Acarbose) 30.0%(Vit C)
LLC 46.2% 43.1 %
LLW 4.8% 58.8 %
LLC-1 13.6% -
LLC-2 9.1% -
LLC-3 23.5% -
LLC-4 29.5% -
LLC-5 34.9% -
LLC-6 98.4% -
LLC-7 94.4% -
LLC-8 53.6% -
LLC-9 19.0% -
LLC-10 - 10.2%
LLC-11 - 7.7%
LLC-12 17.6% -
LLC-13 16.7% -
LLC-14 13.0% 43.8%
LLC-15 10.0%
表 各劃分層藥理活性測試數據
第六章 結論
女貞子氯仿層萃取物經矽膠管柱層析與TLC片檢測合併後，得到15個劃分 層。將這15個劃分層，分別進行α-glucosidase抑制活性篩選、清除DPPH自由 基活性測試。測試結果:第4、5、6、7、8劃分層有良好的α-glucosidase抑制活 性;第10、11、14劃分層有不錯的DPPH自由基清除活性。
再將對α-glucosidase抑制活性較好的第3、4、5、6、7、8劃分層先做細 部的純化分離。共得到9個化合物，經結構鑑定後與文獻比對確認，分別為五環 三萜類- oleanolic acid(100)、3-acetyl oleanolic acid(102)、ursolic acid(99)、ursolic acid long-chain fatty acid ester(106)、lupeol(107)、
lupeol long-chain fatty acid ester(108)。及其它類-β-sitosterol(109)、
mannitol(27)及aliphatic alcohol(110)。
附錄一 植物的萃取與分離
將購自藥廠的女貞子果實1公斤以甲醇萃取5次，由甲醇萃取液經減壓濃縮 除去溶劑後，得到初抽物(LLM)199.36克，再依序以氯仿和水進行分配萃取，得 到氯仿層萃取物(CHCL3 Layer)101.8克及水層萃取物(H2O Layer) 56.98 克。其 中於水層萃取物發現有沉澱物反覆以再結晶法處理後生成一針狀結晶，與文獻比 對無誤後確認為mannitol(27)(0.51g)。
再利用α-glucosidase抑制活性測定篩選適當的分配萃取層，選定氯仿層 萃取物(α-glucosidase抑制率為46.2%; DPPH抑制率為43.1%)進行細部分離。以 矽膠管柱層析分離，氯仿與甲醇(CM=29：1）為沖提液，再加入甲醇漸增極性沖 得到一白色粉末為lupeol long-chain fatty acid ester(108)(0.39g)。
第4 個劃分層，以矽膠管柱層析分離，氯仿為沖提液，經收集濃縮得到各劃分層，
再經以TLC 片檢測合併後得到 7 個劃分層。其中 4-2 劃分層經再結晶法得到一 白色粉末為 lupeol(107)(13.89g)，為首次於女貞屬中發現之化合物。4-3 劃分 層，以矽膠管柱層析分離，正己烷與乙酸乙酯(NE=4:1)為沖提液，再加入乙酸乙
酯漸增極性沖提，經收集濃縮得到各劃分層，再經以TLC 片檢測合併後得到 5
個劃分層。其中4-3-1 劃分層經再結晶法得到一白色粉末為 aliphatic alcohol(110)(0.69g)。4-5-1 劃分層經再結晶法得到一白色粉末為 3-acetyl oleanolic acid(102)(0.30g)。
第5 個劃分層，以矽膠管柱層析分離，氯仿為沖提液，經收集濃縮得到各劃
分層，再經以TLC 片檢測合併後得到 6 個劃分層。其中 5-2 劃分層經再結晶法 得到一透明片狀結晶為β-sitosterol(109)(0.58g)。5-5 分層經再結晶法得到一白色 粉末為ursolic acid long-chain fatty acid ester(106)(0.38g)。
第6 個劃分層，先以多次再結晶法、反覆抽氣過濾，得到一淡黃色粉末。
剩餘母液以矽膠管柱層析分離，氯仿與甲醇(CM=29：1）為沖提液，經收集濃縮 得到各劃分層，再經以TLC 片檢測合併後得到 12 個劃分層。其中 6-7 劃分層經 再結晶法得到一透明片狀結晶，為 oleanolic acid(100)(0.39g)。
第8個劃分層，先以多次再結晶法、反覆抽氣過濾，得到一白色粉末。剩餘 母液以矽膠管柱層析分離，氯仿與丙酮(CA=4：1）為沖提液，經收集濃縮得到 各劃分層，再經以TLC片檢測合併後得到6個劃分層。其中8-2劃分層經再結晶法 得到一白色粉末為ursolic acid(99)(0.69g)。
附錄二 光譜數據 oleanolic acid(100) Colorless powder MP:
Formula:C30H48O3
IR(KBr)νmax :1456、1558、1686、2938cm-1
1H-NMR（CDCl3, 300 MHz）δ：
0.75,0.77,0.91,0.92,0.95,0.98,1.13(7XCH3) 2.80(1H, d, J=9.4 Hz, H-18)
3.19(1H, dd, J=4.8,11.2 Hz, H-3) 5.24(1H, br, H-12)
3-acetyl oleanolic acid(102) Colorless powder
MP:
Formula: C32H50O4
IR(KBr)νmax :1245、1365、1463、1696、1704、2945 cm-1
1H-NMR（CDCl3, 300 MHz）δ： Colorless powder MP:
Formula:C30H48O3
IR(KBr)νmax :1461、1690、2928、3580 cm-1
1H-NMR（CDCl3, 400 MHz）δ：
δ0.71,0.74, 0.78(3H,d , J=4.1 Hz, H29),0.79, 0.85(3H, d , J=4.4 Hz, H-30),0.93,0.99(7XCH3)
2.17(1H, d, J=10.9 Hz, H-18) 3.21(1H, brd, J=10.7 Hz, H-3)
ursolic acid long-chain fatty acid ester(106) Colorless powder
MP:
IR(KBr)νmax :1215、1462、1702、2848、2916、3355cm-1
1H-NMR（CDCl3, 300 MHz）δ：
lupeol(107) Colorless powder MP:
Formula: C30H50O
IR(KBr)νmax : 1379、1456、2849、2916、3355cm-1
1H-NMR（CDCl3, 300 MHz）δ：
0.76, 0.78, 0.82, 0.94, 0.96, 1.02, 1.63(3H, s, H-30)(7XCH3) 2.35(1H, td,J=5.6, 11.2 Hz, H-19)
3.16(1H, dd,J=5.2, 10.9 Hz, H-3) 4.56(1H, s, H-29b)
4.60(1H, s, H-29a)
lupeol long-chain fatty acid ester(108) Colorless powder
MP:
IR(KBr)νmax : 1174、1380、1456、1732、2853、2925cm-1
1H-NMR（CDCl3, 400 MHz）δ：
0.76,0.80,0.84,0.95,0.97,1.05,1.67(7XCH3) 1.24(br)
2.26(2H, t, J=7.4 Hz)
2.33(1H, td,J=5.9, 16.7 Hz, H-19) 4.44(1H, dd, J=5.7, 10.7 Hz H-3) 4.56(1H, s, H-29b)
4.67(1H, s, H-29a)
β-sitosterol(109) Colorless powder MP:
Formula:C29H56O
IR(KBr)νmax : 1379、1456、1637、2849、2916、3355 cm-1
1H-NMR（CDCl3, 400 MHz）δ：
Colorless needle MP: aliphatic alcohol(110)
Colorless powder MP:
Formula:CH3(CH2)nCH2OH
IR(KBr)νmax : 1215、1462、2848、2916 cm-1
1H-NMR（CDCl3, 400 MHz）δ：
0.86(3H, t, J=7.0 Hz) 1.25(br)
1.53(4H, m)
3.62(2H, t, J=6.6 Hz)
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