TWI515004B - Novel biphenyl compounds extracted from Magnolia officinalis and their bioactive components and their preparation methods - Google Patents
Novel biphenyl compounds extracted from Magnolia officinalis and their bioactive components and their preparation methods Download PDFInfo
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本發明係有關於一種厚朴萃取出新聯苯化合物及生物活性成分及製備方法;更詳而言之,係指一種從厚朴分析出三種新聯苯化合物:5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),5,5'-diallyl-2-(allyloxy)biphenyl-2'-ol(2)及5,5'-diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2'-ol(3)的分析系統。另外, 抗發炎活性試驗之結果顯示5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),magnolol(4),honokiol(5),randainal(7),magnaldehyde D(8),及randaiol(9)對於fMLP/CB所誘導超氧陰離子(O2 ‧-)的產生,具有良好的抑制活性(IC50 4.42μg/mL)。另外,5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),magnolol(4),randainal(7),magnaldehyde D(8),及randaiol(9)對於fMLP/CB所誘導彈性蛋白酶(elastase)的釋放,具有良好的抑制作用(IC50 2.35μg/mL)。 The present invention relates to a magnified extraction of a novel biphenyl compound and a biologically active ingredient and a preparation method thereof; more specifically, a novel biphenyl compound is analyzed from Magnolia: 5 ' -allyl-5-( 1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) and 5,5 ' -diallyl-2-(3- Analysis system for methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ). In addition, the results of the anti-inflammatory activity test showed 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),honokiol( 5 ),randainal( 7 ),magnaldehyde D ( 8 ), and randaiol( 9 ) have good inhibitory activity on the production of superoxide anion (O 2 ‧- ) induced by fMLP/CB (IC 50 ) 4.42 μg/mL). In addition, 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),randainal( 7 ),magnaldehyde D( 8 ), and randaiol( 9 ) for fMLP/ The release of elastase induced by CB has a good inhibitory effect (IC 50 2.35 μg/mL).
厚朴屬於落葉喬木,分布於長江流域及中國南部,厚朴的樹皮又稱厚樸(Houpo),在中藥藥材屬於珍貴中藥材和珍稀保護植物,歷代本草中有記載,苦辛、溫中下元、燥濕化痰、散滿消積、降逆平喘、制菌利尿,主要用來治療胃腸不適,咳嗽哮喘,血栓性中風,焦慮,精神不濟;厚朴具有抑制胃潰瘍、抗過敏、細胞毒性、肌肉鬆弛、抗氧化、血管鬆弛、神經營養、抗真菌等作用。 Magnolia is a deciduous tree distributed in the Yangtze River basin and southern China. The bark of Magnolia is also known as Houpo. It is a precious Chinese herbal medicine and rare and protected plant in Chinese herbal medicines. It has been recorded in the past genus, bitter and warm. Yuan, dampness, phlegm, phlegm, phlegm, phlegm and diuresis, mainly used to treat gastrointestinal discomfort, cough, asthma, thrombotic stroke, anxiety, lack of spirit; Magnolia has inhibition of gastric ulcer, anti-allergy, cells Toxicity, muscle relaxation, anti-oxidation, vascular relaxation, neurotrophic, anti-fungal, etc.
本案發明人從事醫藥相關研究多年,對於醫藥相關知識有相當程度的了解,由前述分析得知,厚朴相當珍貴且材料成本高、治療效果廣泛,本發明人認為高價值的藥材治療效果應不止於此,所以針對厚朴研究是否還有新的治療功能。 The inventor of the present invention has been engaged in medical related research for many years, and has a considerable degree of understanding of medical related knowledge. From the above analysis, it is known that magnolia is quite precious, the material cost is high, and the therapeutic effect is wide. The inventors believe that the therapeutic effect of high-value medicinal materials should be more than that. Therefore, there is still a new therapeutic function for the study of Magnolia.
有鑑於此,本案發明人遂依其多年從事相關領域之研發經驗,針對前述進行深入研究,並依前述尋找新的萃取化合物方法,歷經長時間的努力研究與多次測試,終於完成本發明。 In view of this, the inventor of the present invention has carried out in-depth research on the above-mentioned research and development experience in the related fields, and has searched for a new method for extracting compounds according to the foregoing, and has completed the present invention after a long period of hard research and multiple tests.
本發明之主要目的係在提供一種厚朴萃取出新聯苯化合物及生物活性成分及製備方法,並藉由各種實驗數據分析得到的三種新聯苯化合物及具有抗發炎活性之化合物。 The main object of the present invention is to provide a novel biphenyl compound and a biologically active ingredient and a preparation method thereof, and to analyze three kinds of novel biphenyl compounds and compounds having anti-inflammatory activity by various experimental data.
A.利用本發明所述之萃取、層析、純化及結構解析步驟,發現三個新化合物:5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),5,5'-diallyl-2-(allyloxy)biphenyl-2'-ol(2)及5,5'-diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2'-ol(3)。 A. Using the extraction, chromatography, purification and structural resolution steps described in the present invention, three new compounds were found: 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),5 , 5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) and 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ).
B.三個新化合物之實驗數據如下:5'-Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1):colorless oil;UV(MeOH)λmax(log ε)209(4.55),255(3.82),295(3.90)nm;IR(neat)υmax 3521(OH),1639,1431,990,916(-CH=CH2),1607,1488,818(1,2,4-trisubstituted benzene)cm-1;1H NMR(CDCl3,400MHz):δ 3.34(2H,d,J=6.8Hz,H-7'),5.05(1H,ddt,J=10.4,1.6,1.6Hz,H-9'),5.08(1H,ddt,J=17.2,1.6,1.6Hz,H-9'),5.12(1H,br s,D2O exchangeable,OH),5.55(1H,ddd,J=10.4,0.8,0.8Hz,H-3"),5.70(1H,ddd,J=17.2,0.8,0.8Hz,H-3"),5.96(1H,ddd,J=17.2,10.4,6.8Hz,H-8'),6.09(1H,ddd,J=17.2,10.4,6.0Hz,H-2"),6.48(1H,br d,J=6.0Hz,H-1"),6.89(1H,d,J=8.0Hz,H-3'),6.90(1H,dd,J=8.0,2.0Hz,H-4),6.90(1H,d,J=2.0Hz,H-6),6.91(1H,d,J=8.0Hz,H-3),7.02(1H,d,J=2.0Hz,H-6'),7.05(1H,dd,J=8.0,2.0Hz,H-4');13C NMR(CDCl3,100MHz):δ 39.4(C-7'),108.8(C-4),109.5(C-6), 110.7(C-1"),115.7(C-3'),115.7(C-9'),122.1(C-3"),122.2(C-3),127.6(C-1'),129.0(C-4'),130.2(C-6'),130.7(C-1),132.0(C-2"),132.2(C-5'),137.7(C-8'),147.3(C-5),148.4(C-2),150.7(C-2');ESIMS m/z 321[M+Na]+;HRESIMS m/z 321.1107[M+Na]+(calcd for C18H18O4Na,321.1103). B. Experimental data for three new compounds are as follows: 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ):colorless oil;UV(MeOH)λ max (log ε)209( 4.55), 255 (3.82), 295 (3.90) nm; IR (neat) υ max 3521 (OH), 1639, 1431, 990, 916 (-CH=CH 2 ), 1607, 1488, 818 ( 1 , 2 , 4- Trisubstituted benzene) cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 3.34 (2H, d, J = 6.8 Hz, H-7 ' ), 5.05 (1H, ddt, J = 10.4, 1.6, 1.6 Hz, H-9 ' ), 5.08 (1H, ddt, J = 17.2, 1.6, 1.6 Hz, H-9 ' ), 5.12 (1H, br s, D 2 O exchangeable, OH), 5.55 (1H, ddd, J = 10.4, 0.8, 0.8 Hz, H-3 " ), 5.70 (1H, ddd, J = 17.2, 0.8, 0.8 Hz, H-3 " ), 5.96 (1H, ddd, J = 11.2, 10.4, 6.8 Hz, H -8 ' ), 6.09 (1H, ddd, J = 17.2, 10.4, 6.0 Hz, H-2 " ), 6.48 (1H, br d, J = 6.0 Hz, H-1 " ), 6.89 (1H, d, J = 8.0 Hz, H-3 ' ), 6.90 (1H, dd, J = 8.0, 2.0 Hz, H-4), 6.90 (1H, d, J = 2.0 Hz, H-6), 6.91 (1H, d , J = 8.0 Hz, H-3), 7.02 (1H, d, J = 2.0 Hz, H-6 ' ), 7.05 (1H, dd, J = 8.0, 2.0 Hz, H-4 ' ); 13 C NMR (CDCl 3 , 100MHz): δ 39.4 (C-7 ' ), 108.8 (C-4), 109.5 (C-6), 110.7 (C-1 " ), 115.7 (C-3 ' ), 115.7 (C- 9 ' ), 122.1 (C-3 " ), 122.2 (C-3), 127.6 (C-1 ' ), 129.0 (C-4 ' ), 130.2 (C-6 ' ), 130.7 (C-1) , 132.0 (C-2 " ), 132.2 (C-5 ' ), 137.7 (C-8 ' ), 147.3 (C-5), 148.4 (C-2), 150.7 (C-2 ' ); ESIMS m/ z 321[M+Na] + ;HRESIMS m/z 321.1107[M+Na] + (calcd for C 18 H 18 O 4 Na, 321.1103).
5,5'-Diallyl-2-(allyloxy)biphenyl-2'-ol(2):yellowish oil;UV(MeOH)λmax(log ε)205(4.72),286(3.91)nm;IR(neat)υmax 3407(OH),1640,1418,995,916(-CH=CH2),1608,1496,819(1,2,4-trisubstituted benzene)cm-1;1H NMR(CDCl3,400MHz):δ 3.41(2H,br d,J=5.2Hz,H-7'),3.43(2H,br d,J=5.6Hz,H-7),4.60(2H,ddd,J=5.2,1.6,1.6Hz,H-1"),5.10(1H,ddt,J=10.4,1.6,1.6Hz,H-9'),5.11(1H,ddt,J=10.4,1.6,1.2Hz,H-9),5.13(1H,ddt,J=17.2,1.6,1.6Hz,H-9'),5.13(1H,ddt,J=17.2,1.6,1.2Hz,H-9),5.27(1H,ddt,J=10.4,1.6,1.6Hz,H-3"),5.35(1H,ddt,J=17.2,1.6,1.6Hz,H-3"),5.99(1H,ddd,J=17.2,10.4,5.2Hz,H-2"),6.01(1H,ddd,J=17.2,10.4,5.2Hz,H-8'),6.04(1H,ddd,J=17.2,10.4,5.6Hz,H-8),6.42(1H,br s,D2O exchangeable,OH-2'),7.00(1H,d,J=8.0Hz,H-3),7.01(1H,d,J=8.0Hz,H-3'),7.12(1H,d,J=2.0Hz,H-6'),7.15(1H,dd,J=8.0,2.0Hz,H-4'),7.20(1H,dd,J=8.0,2.4Hz,H-4),7.21(1H,d,J=2.4Hz,H-6);13C NMR(CDCl3,100MHz):δ 39.3(C-7),39.4(C-7'),70.2(C-1"),113.6(C-3),115.5(C-9'),115.8(C-9),117.6(C-3'),118.2(C-3"),126.3(C-1'),127.8(C-1),129.0(C-4),129.2(C-4'),131.2 (C-6'),132.3(C-2"),132.6(C-5),132.6(C-6),134.1(C-5'),137.3(C-8),137.8(C-8'),152.0(C-2'),152.8(C-2);ESIMS m/z 329[M+Na]+;HRESIMS m/z 329.1515[M+Na]+(calcd for C21H22O2Na,329.1517). 5,5 ' -Diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ):yellowish oil;UV(MeOH)λ max (log ε)205(4.72),286(3.91)nm;IR(neat) υ max 3407(OH), 1640, 1418, 995, 916 (-CH=CH 2 ), 1608, 1496, 819 (1, 2 , 4-trisubstituted benzene) cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 3.41 (2H, br d, J = 5.2 Hz, H-7 ' ), 3.43 (2H, br d, J = 5.6 Hz, H-7), 4.60 (2H, ddd, J = 5.2, 1.6, 1.6 Hz, H-1 " ), 5.10 (1H, ddt, J = 10.4, 1.6, 1.6 Hz, H-9 ' ), 5.11 (1H, ddt, J = 10.4, 1.6, 1.2 Hz, H-9), 5.13 (1H , ddt, J =17.2, 1.6, 1.6 Hz, H-9 ' ), 5.13 (1H, ddt, J = 17.2, 1.6, 1.2 Hz, H-9), 5.27 (1H, ddt, J = 10.4, 1.6, 1.6Hz, H-3 "), 5.35 (1H, ddt, J = 17.2,1.6,1.6Hz, H-3"), 5.99 (1H, ddd, J = 17.2,10.4,5.2Hz, H-2 ") , 6.01 (1H, ddd, J = 11.2, 10.4, 5.2 Hz, H-8 ' ), 6.04 (1H, ddd, J = 17.2, 10.4, 5.6 Hz, H-8), 6.42 (1H, br s, D 2 O exchangeable, OH-2 ' ), 7.00 (1H, d, J = 8.0 Hz, H-3), 7.01 (1H, d, J = 8.0 Hz, H-3 ' ), 7.12 (1H, d, J =2.0 Hz, H-6 ' ), 7.15 (1H, dd, J = 8.0, 2.0 Hz, H-4 ' ), 7.20 (1H, dd, J = 8.0, 2.4 Hz, H-4), 7.21 (1H , d, J = 2.4 Hz, H-6); 13 C NMR (CDCl 3 , 100 MHz): δ 39.3 (C-7), 39.4 (C-7 ' ), 70.2 (C-1 " ), 113.6 (C -3), 115.5 (C-9 ' ), 115.8 (C-9), 117.6 (C-3 ' ), 118.2 (C-3 " ), 126.3 (C-1 ' ), 127.8 (C-1), 129.0 (C-4), 129.2 (C-4 ' ), 131.2 (C-6 ' ), 132.3 (C-2 " ), 132.6 (C-5), 132.6 (C-6), 134.1 (C-5 ' ), 137.3 (C-8), 137.8 (C -8 ' ), 152.0(C-2 ' ), 152.8(C-2); ESIMS m/z 329[M+Na] + ;HRESIMS m/z 329.1515[M+Na] + (calcd for C 21 H 22 O 2 Na, 329.1517).
5,5'-Diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2'-ol(3):colorless oil;UV(MeOH)λmax(log ε)206(4.73),287(3.89)nm;IR(neat)υmax 3380(OH),1674(-C=C(Me)2),1640,1414,993,914(-CH=CH2),1607,1496,822(1,2,4-trisubstituted benzene)cm-1;1H NMR(CDCl3,600MHz):δ 1.64(1H,d,J=1.2Hz,H-5"),1.74(1H,d,J=1.2Hz,H-4"),3.39(2H,br d,J=7.2Hz,H-7'),3.40(2H,br d,J=7.2Hz,H-7),4.57(1H,d,J=6.6Hz,H-1"),5.06(1H,ddd,J=10.4,1.6,1.6Hz,H-9'),5.08(1H,ddt,J=10.4,1.6,1.6Hz,H-9),5.10(1H,ddt,J=17.2,1.6,1.6Hz,H-9),5.10(1H,ddt,J=17.2,1.6,1.6Hz,H-9'),5.40(1H,tqq,J=6.6,1.2,1.2Hz,H-2"),5.98(1H,ddd,J=17.2,10.4,7.2Hz,H-8'),6.00(1H,ddd,J=17.2,10.4,7.2Hz,H-8),6.70(1H,s,D2O exchangeable,OH-2'),6.97(1H,d,J=8.0Hz,H-3'),6.99(1H,d,J=8.4Hz,H-3),7.09(1H,d,J=1.8Hz,H-6'),7.11(1H,dd,J=8.0,1.8Hz,H-4'),7.17(1H,d,J=2.4Hz,H-6),7.18(1H,dd,J=8.4,2.4Hz,H-4);13C NMR(CDCl3,150MHz):δ 18.1(C-5"),25.7(C-4"),39.4(C-7),39.5(C-7'),66.7(C-1"),114.1(C-3),115.4(C-9'),115.8(C-9),117.9(C-3'),118.8(C-2"),126.7(C-1'),128.3(C-1),128.9(C-4),129.2(C-4'),131.2(C-6'),132.3(C-5),132.7(C-6),134.1(C-5'),137.4(C-8),137.9(C-8'),139.0(C-3"),152.3(C-2'),152.9(C-2);ESIMS m/z 357 [M+Na]+;HRESIMS m/z 357.1828[M+Na]+(calcd for C23H26O2Na,357.1830). 5,5 ' -Diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ):colorless oil;UV(MeOH)λ max (log ε)206(4.73),287(3.89) Nm;IR(neat)υ max 3380(OH),1674(-C=C(Me) 2 ),1640,1414,993,914 (-CH=CH 2 ),1607,1496,822 (1,2,4- Trisubstituted benzene) cm -1 ; 1 H NMR (CDCl 3 , 600 MHz): δ 1.64 (1H, d, J = 1.2 Hz, H-5 " ), 1.74 (1H, d, J = 1.2 Hz, H-4 " ), 3.39 (2H, br d, J = 7.2 Hz, H-7 ' ), 3.40 (2H, br d, J = 7.2 Hz, H-7), 4.57 (1H, d, J = 6.6 Hz, H- 1 " ), 5.06 (1H, ddd, J = 10.4, 1.6, 1.6 Hz, H-9 ' ), 5.08 (1H, ddt, J = 10.4, 1.6, 1.6 Hz, H-9), 5.10 (1H, ddt , J =17.2, 1.6, 1.6 Hz, H-9), 5.10 (1H, ddt, J = 17.2, 1.6, 1.6 Hz, H-9 ' ), 5.40 (1H, tqq, J = 6.6, 1.2, 1.2 Hz , H-2 " ), 5.98 (1H, ddd, J = 11.2, 10.4, 7.2 Hz, H-8 ' ), 6.00 (1H, ddd, J = 11.2, 10.4, 7.2 Hz, H-8), 6.70 ( 1H, s, D 2 O exchangeable, OH-2 ' ), 6.97 (1H, d, J = 8.0 Hz, H-3 ' ), 6.99 (1H, d, J = 8.4 Hz, H-3), 7.09 ( 1H,d, J =1.8 Hz, H-6 ' ), 7.11 (1H, dd, J = 8.0, 1.8 Hz, H-4 ' ), 7.17 (1H, d, J = 2.4 Hz, H-6), 7.18 (1H, dd, J = 8.4, 2.4 Hz, H-4); 13 C NMR (CDCl 3 , 150 MHz): δ 18.1 (C-5 " ), 25.7 (C-4 " ), 39.4 (C-7) ), 39.5 (C-7 ' ), 66.7 (C-1 " ), 114.1 (C-3) , 115.4 (C-9 ' ), 115.8 (C-9), 117.9 (C-3 ' ), 118.8 (C-2 " ), 126.7 (C-1 ' ), 128.3 (C-1), 128.9 (C -4), 129.2 (C-4 ' ), 131.2 (C-6 ' ), 132.3 (C-5), 132.7 (C-6), 134.1 (C-5 ' ), 137.4 (C-8), 137.9 (C-8 ' ), 139.0 (C-3 " ), 152.3 (C-2 ' ), 152.9 (C-2); ESIMS m/z 357 [M+Na] + ; HRESIMS m/z 357.1828 [M+ Na] + (calcd for C 23 H 26 O 2 Na, 357.1830).
C.厚朴分離之化合物,藉由抑制formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B(fMLP/CB)所誘導人類嗜中性白血球產生超氧陰離子(O2 ‧-)及彈性蛋白酶(elastase)之效果,來評估其抗發炎活性。抗發炎活性試驗之結果顯示5'-allyl-5-(1-hydroxyallyloxy)-biphenyl-2,2'-diol(1),magnolol(4),honokiol(5),randainal(7),magnaldehyde D(8)及randaiol(9)對於fMLP/CB所誘導超氧陰離子(O2 ‧-)的產生,具有良好的抑制活性(IC50 4.42μg/mL)。另外,5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),magnolol(4),randainal(7),magnaldehyde D(8),及randaiol(9)對於fMLP/CB所誘導彈性蛋白酶(elastase)的釋放,具有良好的抑制作用(IC50 2.35μg/mL)。 C. Magnolia-separated compound, which produces superoxide anion (O 2 ‧- ) by inhibiting human neutrophils induced by foryl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB) The effect of elastase was evaluated to evaluate its anti-inflammatory activity. The results of the anti-inflammatory activity test showed 5 ' -allyl-5-(1-hydroxyallyloxy)-biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),honokiol( 5 ),randainal( 7 ),magnaldehyde D( 8 ) and randaiol( 9 ) have good inhibitory activity on the production of superoxide anion (O 2 ‧- ) induced by fMLP/CB (IC 50 ) 4.42 μg/mL). In addition, 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),randainal( 7 ),magnaldehyde D( 8 ), and randaiol( 9 ) for fMLP/ The release of elastase induced by CB has a good inhibitory effect (IC 50 2.35 μg/mL).
D.在所有分離化合物中,Randainal(7)及randaiol(9)為最有效的化合物,其對於抑制fMLP/CB所誘導彈性蛋白酶的釋放及超氧陰離子的產生之IC50值,分別為1.21±0.12及0.39±0.06μg/mL。此兩種化合物具有研究開發為抗發炎藥之潛力。 D. Among all the isolated compounds, Randainal ( 7 ) and randaiol ( 9 ) are the most effective compounds, and the IC 50 values for inhibiting the release of elastase induced by fMLP/CB and the production of superoxide anion are 1.21 ± 0.12 and 0.39 ± 0.06 μg/mL. These two compounds have the potential to be researched and developed as anti-inflammatory drugs.
為期許本發明之特徵及結構能夠有更詳盡之了解,請配合參閱附件一、附件二;附件一係為本發明之化合物結構圖,附件二係為本發明之化合物儀器分析結果數據圖。 In order to have a more detailed understanding of the features and structures of the present invention, please refer to Annex I and Annex II; Annex I is a structural diagram of the compound of the present invention, and Annex II is a data chart of the analysis results of the compound of the present invention.
本發明所述之厚朴萃取出新聯苯化合物的製備方法,其步驟如下: The method for preparing a novel biphenyl compound is extracted from the magnolia of the present invention, and the steps are as follows:
A.將厚朴樹皮切片陰乾後共得5.0kg,以甲醇冷浸抽取三次,所得之抽取液經減壓濃縮後,得到MeOH浸膏510g,接著以EtOAc:H2O(1:1,v/v)進行分配,得到EtOAc層抽出液,經減壓濃縮後得到EtOAc層抽出物(Fr.A,118g)。再將H2O層抽出液與n-BuOH(1:1,v/v)再進行分配,得到n-BuOH層抽出物(Fr.B,177g)及H2O層抽出物(Fr.C,206g)。 A. The dried bark bark was dried and dried to obtain a total of 5.0 kg, which was extracted three times with methanol, and the obtained extract was concentrated under reduced pressure to give MeOH (d) 510 g, followed by EtOAc:H 2 O (1:1, v /v) EtOAc (EtOAc) (EtOAc) The H 2 O layer extract was further distributed with n -BuOH (1:1, v/v) to obtain n -BuOH layer extract (Fr. B, 177 g) and H 2 O layer extract (Fr. C). , 206g).
B.取自步驟A之EtOAc層抽出物(Fr.A,118g)經管柱層析法分離(silica gel,70-230mesh,4.9kg),由n-hexane開始沖湜,漸次增加EtOAc及MeOH以提高其極性,共得到10個分劃:Fr.A1(2L,n-hexane),A2(7L,n-hexane/EtOAc,30:1),A3(7L,n-hexane/EtOAc,20:1),A4(6L,n-hexane/EtOAc,10:1),A5(5L,n-hexane/EtOAc,5:1),A6(6L,n-hexane/EtOAc,3:1),A7(5L,n-hexane/EtOAc,2:1),A8(6L,n-hexane/EtOAc,1:1),A9(6L,EtOAc),A10(6L,MeOH)。 B. The EtOAc layer extract (Fr. A, 118 g) from step A was separated by column chromatography (silica gel, 70-230 mesh, 4.9 kg), starting with n- hexane, gradually increasing EtOAc and MeOH. To increase its polarity, a total of 10 fractions were obtained: Fr.A1 (2L, n- hexane), A2 (7L, n- hexane/EtOAc, 30:1), A3 (7L, n- hexane/EtOAc, 20:1 ), A4 (6L, n- hexane/EtOAc, 10:1), A5 (5L, n- hexane/EtOAc, 5:1), A6 (6L, n- hexane/EtOAc, 3:1), A7 (5L) , n- hexane/EtOAc, 2:1), A8 (6L, n- hexane/EtOAc, 1:1), A9 (6L,EtOAc), A10 (6L, MeOH).
C.取自步驟B之Fraction A2(11.2g)經管柱層析法分離(silica gel,230-400mesh,505g),以n-hexane/acetone(40:1)進行沖湜,得到16個分劃(each 1.0L,Fr.A2-1~A2-16)。取Fr.A2-3(185mg)以preparative TLC(silica gel,CHCl3/EtOAc,60:1)精製純化,得到(-)-monoterpenylmagnolol(6)(20.5mg,R f =0.90)。Fr A2-4(150mg)經preparative TLC(silica gel,CHCl3/EtOAc,60:1)精製純化,得到randaiol(9)(3.8mg,R f =0.55)。Fr A2-6(165mg)經preparative TLC(silica gel,n-hexane/acetone,5:1)精製純化,得到 5,5'-diallyl-2-(allyloxy)biphenyl-2'-ol(2)(4.2mg,R f =0.55)。取A2-9(185mg)以preparative TLC(silica gel,n-hexane/acetone,10:1)精製純化,得到5,5'-diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2'-ol(3)(4.5mg)(R f =0.60)。取A2-11(145mg)以preparative TLC(silica gel,n-hexane/acetone,5:1)精製純化,得到5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1)(3.7mg)(R f =0.36)。 C. Fraction A2 (11.2 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 505 g), and washed with n- hexane/acetone (40:1) to obtain 16 partitions. (each 1.0L, Fr.A2-1~A2-16). Fr. A2-3 (185 mg) was purified by preparative TLC (silica gel, CHCl 3 /EtOAc, 60:1) to give (-)-monoterpenylmagnolol ( 6 ) (20.5 mg, Rf = 0.90). Fr A2-4 (150 mg) was purified by preparative TLC (silica gel, CHCl 3 /EtOAc, 60:1) to afford randaiol ( 9 ) (3.8 mg, Rf = 0.55). Fr A2-6 (165 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 5:1) to give 5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 )( 4.2 mg, R f = 0.55). A2-9 (185 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 10:1) to give 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol (3) (4.5mg) ( R f = 0.60). Take A2-11 (145mg) (silica gel n -hexane / acetone,, 5: 1) preparative TLC purification to give 5 '-allyl-5- (1- hydroxyallyloxy) biphenyl-2,2' -diol (1 ) (3.7 mg) ( R f = 0.36).
D.取自步驟B之Fraction A3(10.5g)經管柱層析法分離(silica gel,230-400mesh,475g),以n-hexane/acetone(3:1)進行沖湜,得到13個分劃(each 1.2L,Fr.A3-1~A3-13)。取Fr.A3-2(148mg)以preparative TLC(silica gel,n-hexane/acetone,6:1)精製純化,得到混合物β-sitostenone(10)及stigmasta-4,22-dien-3-one(11)(6.7mg)(R f =0.58)。取A3-4(235mg)以MeOH洗滌過濾後,以MeOH再結晶得到混合物β-sitosterol(12)及stigmasterol(13)(55mg)。Fr A3-6(272mg)以n-hexane洗滌過濾後,以CH2Cl2再結晶得到magnolol(4)(95mg)。 D. Fraction A3 (10.5g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 475 g), and washed with n- hexane/acetone (3:1) to obtain 13 partitions. (each 1.2L, Fr.A3-1~A3-13). Fr.A3-2 (148 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 6:1) to obtain a mixture of β-sitostenone ( 10 ) and stigmasta-4, 22-dien-3-one ( 11 ) (6.7 mg) ( R f = 0.58). A3-4 (235 mg) was washed with MeOH and filtered and then recrystallised from MeOH to give a mixture of β-sitosterol ( 12 ) and stigmasterol ( 13 ) (55 mg). Fr A3-6 (272 mg) was filtered with n- hexane elution and then recrystallized from CH 2 Cl 2 to give magnolol ( 4 ) (95 mg).
E.取自步驟B之Fraction A4(9.5g)經管柱層析法分離(silica gel,230-400mesh,420g),以n-hexane/acetone(3:1)進行沖湜,得到11個分劃(each 1.0L,Fr.A4-1~A4-11)。取Fr.A4-5(145mg)以preparative TLC(silica gel,CHCl3/MeOH,25:1)精製純化,得到randainal(7)(4.6mg)(R f =0.26)。取Fr.A4-6(138mg)以preparative TLC(silica gel,CHCl3/MeOH,25:1)精製純化,得到magnaldehyde D (8)(3.8mg)(R f =0.24)。取Fr.A4-7(220mg)以preparative TLC(silica gel,n-hexane/acetone,3:1)精製純化,得到honokiol(5)(90mg)(R f =0.36)。 E. Fraction A4 (9.5 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 420 g), and washed with n- hexane/acetone (3:1) to obtain 11 fractions. (each 1.0L, Fr.A4-1~A4-11). Take Fr.A4-5 (145mg) preparative TLC to (silica gel, CHCl 3 / MeOH , 25: 1) was purified to give randainal (7) (4.6mg) ( R f = 0.26). Take Fr.A4-6 (138mg) to preparative TLC (silica gel, CHCl 3 / MeOH, 25: 1) was purified to give magnaldehyde D (8) (3.8mg) (R f = 0.24). Take Fr.A4-7 (220mg) to preparative TLC (silica gel, n -hexane / acetone, 3: 1) was purified to give honokiol (5) (90mg) ( R f = 0.36).
F.取自步驟B之Fraction A7(9.8g)經管柱層析法分離(silica gel,230-400mesh,440g),以n-hexane/acetone(2:1)進行沖湜,得到14個分劃(each 900mL,Fr.A7-1~A7-14)。取Fr.A7-5(190mg)以preparative TLC(silica gel,n-hexane/acetone,3:1)精製純化,得到混合物3β-hydroxystigmast-5-en-7-one(14及3β-hydroxystigmasta-5,22-dien-7-one(15)(6.3mg)。 F. Fraction A7 (9.8 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 440 g), and washed with n- hexane/acetone (2:1) to obtain 14 partitions. (each 900mL, Fr.A7-1~A7-14). Fr.A7-5 (190 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 3:1) to give a mixture of 3β-hydroxystigmast-5-en-7-one ( 14 and 3β-hydroxystigmasta-5). , 22-dien-7-one ( 15 ) (6.3 mg).
將上述步驟之純化所得化合物進行抗發炎活性檢測,如下列步驟所示: The purified compound obtained in the above step was tested for anti-inflammatory activity as shown in the following steps:
(A)臺灣澤蘭地上部所分離之化合物 (A) Compounds isolated from the upper part of Zeeland, Taiwan
(B)human neutrophils (B)human neutrophils
(C)HBSS:Hank’s buffered saline solution (C)HBSS: Hank’s buffered saline solution
(D)fMLP:formyl-L-methionyl-L-leucyl-L-phenylalanine (D)fMLP: formyl-L-methionyl-L-leucyl-L-phenylalanine
(E)CB:cytochchalasin B (E)CB: cytochchalasin B
(F)SOD:superoxide dismutase (F)SOD: superoxide dismutase
(G)ferricytochrome c (G)ferricytochrome c
自年約20-30歲的健康捐血者(作息正常且禁服藥物二週以上),以真空 無菌採血管於手肘靜脈採血,約30-80ml。利用Ficoll gradient離心方法,將嗜中性白血球分離,方法如下:25℃,650g,男性全血離心10min,女性全血則離心8min,去除上清液,將下層血球的部份和3% dextran溶液以等體積混合,於室溫下靜置30min。將含豐富嗜中性白血球的上層覆蓋於等體積Histopaque-1077溶液的50ml離心管中,於20℃下,400g,離心35min,取pellet。利用低張溶液溶血的方法將殘存的紅血球脹破。最後,於4℃下,離心,去除上清液,將分離出的嗜中性白血球懸浮於冰浴的Hank’s buffered saline solution(HBSS)中。 Healthy blood donors from about 20-30 years old (normal work and banned drugs for more than two weeks), vacuum The sterile blood collection tube collects blood in the elbow vein, about 30-80 ml. The neutrophils were separated by Ficoll gradient centrifugation method as follows: 25 ° C, 650 g, male whole blood was centrifuged for 10 min, female whole blood was centrifuged for 8 min, the supernatant was removed, and the lower layer of blood cells and 3% dextran solution were removed. Mix in an equal volume and let stand at room temperature for 30 min. The upper layer containing abundant neutrophils was covered in a 50 ml centrifuge tube of an equal volume of Histopaque-1077 solution, and centrifuged at 400 g for 20 min at 20 ° C to obtain a pellet. The remaining red blood cells are broken by a method of hemolysis of a low-tension solution. Finally, the supernatant was removed by centrifugation at 4 ° C, and the separated neutrophils were suspended in Hank's buffered saline solution (HBSS) in an ice bath.
將含有0.5mg/ml ferricytochrome c、1mM CaCl2及1mM MgCl2的嗜中性白血球的懸浮液(6×105cells/ml),預熱5分鐘使其達37℃,在加入待測藥物作用5分鐘後,再加入fMLP(0.1μM)/cyto-chalasin B(CB,1μg/ml)反應10分鐘。使用紫外光分光光度計,於波長550nm下測量其吸光值。實驗中評估嗜中性白血球所釋出超氧自由基的量(extinction coefficient 21.1/mM/cm)可以經由superoxide dismutase(SOD,100U/ml)抑制ferricytochrome c還原,計算得知。 A suspension of neutrophils (6 × 10 5 cells / ml) containing 0.5 mg / ml of ferricytochrome c , 1 mM CaCl 2 and 1 mM MgCl 2 was preheated for 5 minutes to reach 37 ° C, and the drug to be tested was added. After 5 minutes, fMLP (0.1 μM)/cyto-chalasin B (CB, 1 μg/ml) was further added for 10 minutes. The absorbance was measured at a wavelength of 550 nm using an ultraviolet spectrophotometer. In the experiment, the amount of superoxide radical released by neutrophils (extinction coefficient 21.1/mM/cm) was evaluated by superoxide dismutase (SOD, 100 U/ml) to inhibit ferricytochrome c reduction.
△A550=extinction coefficient×1×△C △A 550 =extinction coefficient×1×△C
△C=△A550/(21.1/mM/cm×1cm) △C=△A 550 /(21.1/mM/cm×1cm)
△C=△A550×47.4nmol/ml △C=△A 550 × 47.4nmol/ml
其中為△A550待測樣品的吸光值減去SOD抑制組之吸光值,△C為待測檢品的O2 ‧-產量。 Among them, the absorbance of the sample of ΔA 550 to be tested is subtracted from the absorbance of the SOD inhibition group, and ΔC is the O 2 ‧ yield of the sample to be tested.
嗜中性白血球的懸浮液含Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide(Ms-Ala-Ala-Pro-Val-pNA),預熱5分鐘使達37℃,加入待測藥物作用5分鐘後,再加入fMLP/CB反應10分鐘。使用紫外光分光光度計,於波長405nm下測量其吸光值。 The suspension of neutrophils contains Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (Ms-Ala-Ala-Pro-Val-pNA), preheated for 5 minutes to reach 37 ° C, and added to the drug to be tested 5 After a minute, the fMLP/CB reaction was further added for 10 minutes. The absorbance was measured at a wavelength of 405 nm using an ultraviolet spectrophotometer.
以fMLP/CB(formyl-L-methionyl-L-leucyl-L-phenylalanine/cyto-chalasin B)來誘導嗜中性白血球(neutrophil)產生超氧陰離子(superoxide anion)及彈性蛋白酶(elastase),而超氧陰離子的產生及彈性蛋白酶的釋放與發炎性疾病的發生有關。因此,藉由對fMLP/CB誘導超氧陰離子產生及彈性蛋白酶釋放的抑制效果,可以用來評估分離化合物之抗發炎活性。 Using fMLP/CB (formyl-L-methionyl-L-leucyl-L-phenylalanine/cyto-chalasin B) to induce neutrophil to produce superoxide anion and elastase, and super The production of oxygen anions and the release of elastase are associated with the development of inflammatory diseases. Therefore, the anti-inflammatory activity of the isolated compound can be evaluated by the inhibitory effect of fMLP/CB on induction of superoxide anion production and elastase release.
實驗結果以平均值±標準差(SEM)(n=4)來呈現,且使用『Student’s t test』做比較,probability為0.05或以下被認為具統計學意義。 The experimental results were presented as mean ± standard deviation (SEM) (n = 4), and compared using "Student's t test", a probability of 0.05 or less was considered statistically significant.
對木蘭科植物厚朴樹皮進行化學成分之研究,目前由厚朴樹皮共分離得到3個為新聯苯化合物:5'-allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),5,5'-diallyl-2-(allyloxy)biphenyl-2'-ol(2),及5,5'-diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2'-ol(3),以及12個已知化合物(4-15)(Fig.1)(附件一)。上述化合物經各種實驗數據分析(包括2D-NMR:NOESY,COSY,HMBC,HSQC...等)以及比對相關文獻上之數據,而確認其結構。 The chemical constituents of magnolia bark were studied. At present, three new biphenyl compounds were isolated from the thick bark: 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ), and 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol ( 3 ), and 12 known compounds ( 4-15 ) (Fig. 1) (Annex I). The above compounds were analyzed by various experimental data (including 2D-NMR: NOESY, COSY, HMBC, HSQC, etc.) and the data in the related literature were confirmed.
三個為新聯苯化合物(1-3)以UV, IR,ESI-MS,HR-ESI-MS,NMR(1H-NMR,13C-NMR,DEPT,COSY,NOESY,HSQC,HMBC....),.....等各種實驗資料,決定其化學結構。十二個已知化合物(4-15),包括4個neolignans:magnolol(4),honokiol(5),(-)-monoterpenylmagnolol(6),randainal(7);2個norlignans:magnaldehyde D(8)及randaiol(9);以及6個steroids:a mixture of β-sitostenone(10)and stigmasta-4,22-dien-3-one(11),a mixture of β-sitosterol(12)and stigmasterol(13)及a mixture of 3β-hydroxystigmast-5-en-7-one(14)and 3β-hydroxystigmasta-5,22-dien-7-one(15),經與文獻資料比對其1H-NMR、IR、UV、MS等數據,結果均一致而確定其結構。 Three are new biphenyl compounds ( 1-3 ) with UV, IR, ESI-MS, HR-ESI-MS, NMR ( 1 H-NMR, 13 C-NMR, DEPT, COSY, NOESY, HSQC, HMBC.. ..), ..... and other experimental materials determine the chemical structure. Twelve known compounds ( 4-15 ), including 4 neolignans: magnolol ( 4 ), honiciol ( 5 ), (-)-monoterpenylmagnolol ( 6 ), randainal ( 7 ); 2 norlignans: magnaldehyde D ( 8 ) And randaiol( 9 ); and 6 steroids: a mixture of β-sitostenone( 10 ) and stigmasta-4,22-dien-3-one( 11 ), a mixture of β-sitosterol( 12 ) and stigmasterol( 13 ) And a mixture of 3β-hydroxystigmast-5-en-7-one( 14 ) and 3β-hydroxystigmasta-5,22-dien-7-one( 15 ), compared with the literature, 1 H-NMR, IR, UV, MS and other data, the results are consistent and determine its structure.
發炎,是人體正常的防禦反應,為了對抗外來病原菌而產生的保護機制,在局部呈現紅、熱、腫、痛等徵狀之現象。白血球在對抗外來病原菌時,會使用高氧化力的自由基,不但會殺死外來病原菌,同時周遭的正常組織或細胞也常被波及。正常細胞的DNA一旦受損,基因就會發生突變,影響細胞的生長與分化。若在重要的基因中產生突變,形成惡性腫瘤的機會就大增。由於慢性發炎是經年累月地刺激正常細胞,染色體的變異也一直在累積,最後就導致癌症,或者免疫系統也可能因為長期的負荷,讓許多慢性退化性疾病開始產生。 Inflammation is the normal defense response of the human body. In order to combat the foreign pathogens, the protective mechanism is characterized by red, hot, swollen, painful symptoms. When white blood cells fight against foreign pathogens, high-oxidation free radicals are used, which not only kills foreign pathogens, but also the surrounding normal tissues or cells are often affected. Once the DNA of a normal cell is damaged, the gene will mutate and affect the growth and differentiation of the cell. If mutations are made in important genes, the chances of developing malignant tumors increase. Since chronic inflammation is the stimulation of normal cells over the years, chromosome variation has been accumulating, eventually leading to cancer, or the immune system may also cause many chronic degenerative diseases to start due to long-term load.
研究指出約有百分之三十的癌症與慢性發炎或慢性感染有關;尤其是心肌梗塞、糖尿病、阿茲海默氏症、癌症、過敏性及自體免疫疾病等,現在有愈來愈多的證據顯示都與慢性發炎息息相關。舉例來說,有些心臟病發作的人,其實本身的膽固醇並不高,血管壁上慢性發炎所造成的粥狀硬化 塊剝落,啟動凝血機制,阻塞冠狀動脈才是心肌梗塞的原因;肝的慢性發炎變成肝癌,就是免疫系統的攻擊所造成的;子宮頸癌也是體內為了對抗人類乳突病毒的發炎反應所引起的;胃液逆流造成食道發炎也容易產生食道癌。因此,理論上如果能夠阻斷發炎反應,就可以明顯地抑制癌細胞。 Research indicates that about 30% of cancers are associated with chronic inflammation or chronic infections; especially myocardial infarction, diabetes, Alzheimer's disease, cancer, allergic and autoimmune diseases, and more and more The evidence shows that it is closely related to chronic inflammation. For example, some people with heart attacks actually have low cholesterol and atherosclerosis caused by chronic inflammation on the blood vessel wall. Block flaking, initiation of coagulation mechanism, occlusion of coronary artery is the cause of myocardial infarction; chronic inflammation of the liver becomes liver cancer, which is caused by the attack of the immune system; cervical cancer is also caused by the inflammatory reaction against human papillomavirus in vivo. Gastrointestinal reflux causes esophageal inflammation and is also prone to esophageal cancer. Therefore, in theory, if the inflammatory response can be blocked, the cancer cells can be significantly inhibited.
當人類嗜中性白血球(neutrophil)受到刺激活化後,會釋放出超氧陰離子(superoxide anion,O2 ‧-)、過氧化氫(hydrogen peroxide)及氫氧自由基(hydroxyl radical)等活性氧類[(reactive oxygen species(ROS)],以及會釋放出彈性蛋白酶(elastase),β-glucuronidase,lysozyme,PAF等媒介物質,而上述物質均和發炎性疾病有關如:類風濕關節炎、腎絲球腎炎、皮膚病、局部缺血性再灌流損傷、心肌梗塞、慢性肺阻塞疾病及氣喘。抑制嗜中性白血球過度或不適當活化被認為可以改善這些發炎性疾病。因此新的治療與預防發炎性疾病的藥物之開發,一直是醫藥界研究探討的熱門話題。由過去天然物所分離有關抗發炎活性成分之研究,顯示flavonoids、terpenoids及lignans等成分具有良好的抗發炎活性,因此由天然物經由抗發炎試驗之體外篩選,選擇抗發炎活性較『佳』之植物進行成分分離與藥理評估,對於開發新的抗發炎藥物是可行之路。 When human neutrophils are stimulated and activated, superoxide anion (O 2 ‧- ), hydrogen peroxide, and hydroxyl radicals are released. [(Reactive oxygen species (ROS)], and release of elastase (elastase), β-glucuronidase, lysozyme, PAF and other media, and these substances are related to inflammatory diseases such as: rheumatoid arthritis, kidney skein Nephritis, skin disease, ischemic reperfusion injury, myocardial infarction, chronic lung obstruction and asthma. Inhibition of neutrophilic leukemia or inappropriate activation is thought to improve these inflammatory diseases. Therefore, new treatment and prevention of inflammation The development of drugs for diseases has been a hot topic in the research of the pharmaceutical industry. Studies on anti-inflammatory active ingredients isolated from natural materials in the past have shown that flavonoids, terpenoids and lignans have good anti-inflammatory activity, so they are In vitro screening of anti-inflammatory tests, selection of plants with better anti-inflammatory activity than "good" for component separation and pharmacological evaluation , Developing new anti-inflammatory drugs is a viable path.
厚朴樹皮分離之化合物,藉由抑制formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B(fMLP/CB)所誘導人類嗜中性白血球產生超氧陰離子(O2 ‧-)及彈性蛋白酶(elastase)之效果,以評估其抗發炎活性。抗發炎活性之數據列於Table 1。Diphenyleneiodonium及phenylmethylsulfonyl fluoride分別當作[O2 ‧-產生]及[elastase釋放]之positive control。對於抗發炎試驗之結果,可歸納出以下七點結論: A compound isolated from the bark of bark, which produces superoxide anion (O 2 ‧- ) and elasticity by inhibiting formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB) in human neutrophils The effect of protease (elastase) to assess its anti-inflammatory activity. Anti-inflammatory activity data is listed in Table 1. Diphenyleneiodonium and phenylmethylsulfonyl fluoride were treated as positive controls for [O 2 ‧- production] and [elastase release], respectively. For the results of the anti-inflammatory test, the following seven conclusions can be summarized:
(a)5'-Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),magnolol(4),honokiol(5),randainal(7),magnaldehyde D(8),及Randaiol(9)對於fMLP/CB所誘導超氧陰離子(O2 ‧-)的產生,具有良好的抑制活性(IC50 4.42μg/mL)。 (a) 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),honokiol( 5 ),randainal( 7 ),magnaldehyde D( 8 ), and Randaiol ( 9 ) Good inhibitory activity (IC 50 ) for the production of superoxide anion (O 2 ‧- ) induced by fMLP/CB 4.42 μg/mL).
(b)5'-Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(1),magnolol(4),randainal(7),magnaldehyde D(8),及randaiol(9)對於fMLP/CB所誘導彈性蛋白酶(elastase)的釋放,具有良好的抑制作用(IC50 2.35μg/mL)。 (b) 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),randainal( 7 ),magnaldehyde D( 8 ), and randaiol( 9 ) for fMLP /CB induces the release of elastase, which has a good inhibitory effect (IC 50 2.35 μg/mL).
(c)5,5'-Diallyl-2-(allyloxy)biphenyl-2'-ol(2)(10μg/ml)在cytochalasin B(CB)存在下,會誘導人類嗜中性白血球釋放彈性蛋白酶。在不添加fMLP/CB條件下,5'-Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2'-diol(3)(10μg/ml)會獨自誘導人類嗜中性白血球釋放超氧陰離子(O2 ‧-)及彈性蛋白酶(elastase)。在不添加fMLP/CB條件下,Honokiol(5)(10μg/ml)會獨自誘導人類嗜中性白血球釋放彈性蛋白酶(elastase)。 (c) 5,5 ' -Diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) (10 μg/ml) induces the release of elastase from human neutrophils in the presence of cytochalasin B (CB). 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 3 ) (10 μg/ml) alone induces the release of superoxide anion from human neutrophils without the addition of fMLP/CB ( O 2 ‧- ) and elastase (elastase). Without the addition of fMLP/CB, Honokiol( 5 ) (10 μg/ml) alone induced the release of elastase from human neutrophils.
(d)在5'-allylbiphenyl-2,2'-diol類似化合物(1,4,6-9)中,化合物1,4及7-9,對於fMLP/CB所誘導超氧陰離子的產生及彈性蛋白酶的釋放,具有顯著的抑制活性。對於抑制超氧陰離子的產生方面,其活性強度順序為9(with 5-hydroxy group)>4(with 5-allyl group)>7(with(E)-3-oxoprop-1-enyl group at C-5)>8(with 5-formyl group)>1(with 1-hydroxyallyloxy group at C-5)。對於抑制彈性蛋白酶的釋放方面,其活性強度順序為7(with(E)-3-oxoprop-1-enyl group at C-5)>1(with 1-hydroxyallyloxy group at C-5)≒9(with 5-hydroxy group)>4(with 5-allyl group)>8(with 5-formyl group)。相較之下,化合物6(with a(1S,6R)-6-isopropyl-3-methylcyclohex-2-enyl group at C-3' and an allyl group at C-5)則不具活 性。 (d) In the 5 ' -allylbiphenyl-2,2 ' -diol analogs ( 1 , 4 , 6-9 ), compounds 1 , 4 and 7-9 , for the production and elasticity of superoxide anion induced by fMLP/CB The release of protease has significant inhibitory activity. For inhibition of superoxide anion production, the order of activity intensity is 9 (with 5-hydroxy group) > 4 (with 5-allyl group) > 7 (with( E )-3-oxoprop-1-enyl group at C- 5)> 8 (with 5-formyl group) > 1 (with 1-hydroxyallyloxy group at C-5). For inhibition of elastase release, the order of activity intensity is 7 (with( E )-3-oxoprop-1-enyl group at C-5)> 1 (with 1-hydroxyallyloxy group at C-5)≒ 9 (with 5-hydroxy group)> 4 (with 5-allyl group)> 8 (with 5-formyl group). In contrast, compound 6 (with a(1 S ,6 R )-6-isopropyl-3-methylcyclohex-2-enyl group at C-3 ' and an allyl group at C-5) is not active.
(e)Honokiol(5)(C-4位接OH)對於抑制超氧陰離子的產生,比其類似化合物magnolol(4)(C-2位接OH)更有效。 (e) Honokiol ( 5 ) (C-4 at OH) is more effective at inhibiting the production of superoxide anion than its similar compound magnolol( 4 ) (C-2 at OH).
(f)在所有分離化合物中,Randainal(7)及randaiol(9)為最有效的化合物,其對於抑制fMLP/CB所誘導彈性蛋白酶的釋放及超氧陰離子的產生之IC50值,分別為1.21±0.12及0.39±0.06μg/mL。 (f) Among all the isolated compounds, Randainal ( 7 ) and randaiol ( 9 ) were the most potent compounds, and their IC 50 values for inhibiting the release of elastase induced by fMLP/CB and the production of superoxide anion were 1.21, respectively. ±0.12 and 0.39±0.06 μg/mL.
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