楊 帆,張亞梅
江西中醫(yī)藥大學 江西民族傳統(tǒng)藥現(xiàn)代科技與產(chǎn)業(yè)發(fā)展協(xié)同創(chuàng)新中心,南昌 330004
密花樫木(Dysoxylumdensiflorum)產(chǎn)于我國云南西雙版納(勐臘、景洪),生于海拔520~800 m的濕性熱帶季節(jié)性谷邊、河邊坡地的雨林中[1]?,F(xiàn)代研究表明,密花樫木中含有豐富的倍半萜、三萜、二萜、檸檬苦素類等化學成分,部分化合物具有良好的抗菌、抗腫瘤、鎮(zhèn)痛等多種藥理活性[2-5]。蛋白質酪氨酸磷酸酶(proteintyrosine phosphatase,PTPs)是一組催化蛋白質酪氨酸脫磷酸化的酶。蛋白質酪氨酸磷酸酶1B(proteintyrosine phosphatase 1B,PTP-1B)通過激活的胰島素受體和下游底物蛋白去磷酸化,在調節(jié)胰島素作用中發(fā)揮關鍵作用[6-7]。因此PIP-1B是胰島素信號轉導通路中的關鍵的負調節(jié)蛋白,PTP-1B抑制劑是開發(fā)抗糖尿病藥物的重要選擇。課題組前期發(fā)現(xiàn)楝科樫木屬植物多脈樫木中的二萜類成分表現(xiàn)出對PTP-1B抑制作用[8],同屬其他植物尚未見此活性研究報道。因此,為進一步探究密花樫木的活性成分,本實驗通過硅膠柱色譜、半制備色譜、制備色譜結合核磁共振波譜技術對密花樫木根提取物乙酸乙酯萃取部位進行研究,以期為抑制PTP-1B活性成分的發(fā)現(xiàn)提供依據(jù)。
Bruker Avance Ⅲ 600型核磁共振儀(德國Bruker公司);LC-3000型制備高效液相色譜儀(北京創(chuàng)新通恒科技有限公司);制備型色譜柱(250 mm × 21.2 mm,10 μm)(北京慧得易科技有限責任公司);半制備型色譜柱(Luna C-18,250 mm×10 mm,10 μm)(美國Phenomenex公司);硅膠G(100~200,200~300目,青島海洋化工廠);酶標儀(賽默飛世爾科技(中國)有限公司);重組人PTP-1B蛋白(Abcam公司);氯化鈉(國藥集團化學試劑有限公司);三羥甲基氨基甲烷Tris(Gen-view科技有限公司);硝基苯基磷酸二鈉(p-nitrophenyl phosphate,p-NPP)(上海麥克林生化科技有限公司);乙二胺四乙酸二鈉(ethylene diamine tetraacetic acid,EDTA)(北京索萊寶科技有限公司);二硫蘇糖醇(DL-dithiothreitol,DTT)(北京索萊寶科技有限公司);半制備及制備液相所用試劑為色譜純,其他均為分析純。
密花樫木根于2020年5月采自云南勐海,經(jīng)江西中醫(yī)藥大學江西民族傳統(tǒng)藥現(xiàn)代科技與產(chǎn)業(yè)發(fā)展協(xié)同創(chuàng)新中心張亞梅副教授鑒定為楝科蔥臭木屬植物密花樫木根(Dysoxylumdensiflorum)。憑證標本(2020-05)存放于江西中醫(yī)藥大學江西民族傳統(tǒng)藥現(xiàn)代科技與產(chǎn)業(yè)發(fā)展協(xié)同創(chuàng)新中心。
密花樫木根10 kg,粉碎,采用回流提取法用95%乙醇提取3次,每次1 h,合并提取液,過濾后減壓濃縮得到總浸膏2400 g,將總浸膏用乙酸乙酯-水(1∶1)萃取,減壓濃縮后得到乙酸乙酯萃取物491.35 g。取乙酸乙酯浸膏150 g用硅膠色譜柱(15 cm × 15 cm)分離,以石油醚-丙酮(10∶1、7∶1、4∶1、1∶1)和95%乙醇為洗脫劑,得到3個流分(MG1~MG3)。
MG1部位分別經(jīng)兩次制備HPLC(乙腈-水80∶20→95∶5、乙腈-水80∶20)得到MZ1a和MZ1b,MZ1a分別經(jīng)正向半制備HPLC(石油醚-丙酮8∶1)、半制備HPLC(乙腈-水80∶20)得到化合物19(4.8 mg,tR= 12 min)、20(1.1 mg,tR= 17 min)。MZ1b分別經(jīng)正向半制備HPLC(石油醚-丙酮8∶1)、半制備HPLC(乙腈-水70∶30)得到化合物18(1 mg,tR= 40 min)。
MG2部位分別經(jīng)兩次制備HPLC(乙腈-水60∶40→95∶5、乙腈-水50∶50)得到化合物10(91.7 mg,tR= 25 min)、22(30.3 mg,tR= 36 min)和MZ2a~MZ2f,MZ2a分別經(jīng)正向半制備HPLC(石油醚-丙酮6∶1)、半制備HPLC(乙腈-水60∶40→95∶5)得到化合物7(1 mg,tR= 29 min);MZ2b分別經(jīng)正向半制備HPLC(石油醚-丙酮8∶1)、半制備HPLC(乙腈-水60∶40→95∶5)得到化合物5(1 mg,tR= 13 min);MZ2c分別經(jīng)正向半制備HPLC(石油醚-丙酮8∶1)、半制備HPLC(乙腈-水60∶40→95∶5)得到化合物11(1 mg,tR= 18 min);MZ2d分別經(jīng)正向半制備HPLC(石油醚-丙酮10∶1)、半制備HPLC(乙腈-水60∶40)得到化合物12(5.1 mg,tR= 24 min)、14(7 mg,tR= 23 min)、15(9.2 mg,tR= 26 min)、21(4.5 mg,tR= 26 min);MZ2e分別經(jīng)正向半制備HPLC(石油醚-丙酮10∶1)、半制備HPLC(乙腈-水60∶40)得到化合物6(1 mg,tR= 32 min);MZ2f分別經(jīng)正向半制備HPLC(石油醚-丙酮10∶1)、半制備HPLC(乙腈-水60∶40)得到化合物8(45.1 mg,tR= 50 min)、9(2.4 mg,tR= 50 min)、13(1 mg,tR= 17 min)。
MG3部位分別經(jīng)兩次制備HPLC(乙腈-水50∶50→80∶20、乙腈-水30∶70→60∶40)得到MZ3a~MZ3c,MZ3a分別經(jīng)正向半制備HPLC(石油醚-丙酮3∶1)、半制備HPLC(乙腈-水40∶60)得到化合物17(3.3 mg,tR= 26 min);MZ3b分別經(jīng)正向半制備HPLC(石油醚-丙酮6∶1)、半制備HPLC(乙腈-水45∶55)得到化合物1(1 mg,tR= 17 min)、2(1 mg,tR= 19 min)、3(1.5 mg,tR= 22 min)、4(1 mg,tR= 29 min)、16(1 mg,tR= 34 min);MZ3c分別經(jīng)正向半制備HPLC(石油醚-丙酮3∶1)、半制備HPLC(乙腈-水55∶45)得到化合物23(11.4 mg,tR= 13 min)。
以對p-NPP作為底物,蘇拉明鈉為陽性對照,根據(jù)PTP-1B水解p-NPP 的磷酸基團而產(chǎn)生顏色反應來測定PTP-1B的活性。測試樣品用DMSO溶解,配制為質量濃度10 mg/mL的母液,取母液10 μL加緩沖液至質量濃度梯度為0、0.0125、0.025、0.05、0.1、0.2 mg/mL,在96孔板中加入緩沖液(180 μL緩沖體系組成為11 mmol/L Tris-HCl pH 7.5、55 mmol/L DTT、1.1 mmol/L EDTA、33 mmol/Lp-NPP),37 ℃再回溫5 min后加入p-NPP 50 μL,37 ℃再回溫5 min后加入樣品,酶20 μL,37 ℃孵育30 min后,加入mol/L NaOH 10 μL終止反應。以不含樣品的系統(tǒng)為空白對照,利用SpectraMax MD5酶標儀測定405 nm處吸光度(A)[9]。應用Graphpad軟件計算半抑制濃度(IC50)。
化合物1淡黃色膠狀;分子式為C20H34O2。1H NMR(600 MHz,CDCl3)δ:5.38(1H,t,J= 6.9 Hz,H-14),4.14(2H,d,J= 7.1 Hz,H-15),3.45(1H,d,J= 11.0 Hz,H-3),1.67(3H,s,H-16),1.04(3H,s,H-18),0.96(3H,s,H-19),0.84(3H,d,J= 6.8 Hz,H-20),0.82(3H,s,H-17);13C NMR(150 MHz,CDCl3)δ:25.2(C-1),27.4(C-2),76.3(C-3),34.1(C-4),136.3(C-5),25.5(C-6),27.1(C-7),33.3(C-8),40.5(C-9),131.7(C-10),39.8(C-11),34.5(C-12),140.9(C-13),122.7(C-14),59.4(C-15),16.0(C-16),16.5(C-17),24.0(C-18),20.1(C-19),21.1(C-20)。以上數(shù)據(jù)與文獻[10]報道基本一致,故鑒定化合物1為5(10),13E-halimadiene-3α,15-diol。
化合物2白色粉末;分子式為C20H34O2。1H NMR(600 MHz,CDCl3)δ:5.38(1H,tq,J= 6.9,1.3 Hz,H-14),4.83(2H,dd,J= 36.5,1.5 Hz,H-18),4.33(1H,t,J= 3.1 Hz,H-3),4.13(2H,d,J= 6.9 Hz,H-15),1.67(3H,d,J= 1.3 Hz,H-16),1.24(3H,s,H-20),0.81(3H,d,J= 6.4 Hz,H-17),0.76(3H,s,H-19);13C NMR(150 MHz,CDCl3)δ:16.5(C-1),27.0(C-2),74.6(C-3),160.8(C-4),39.7(C-5),38.1(C-6),32.8(C-7),36.7(C-8),39.1(C-9),48.5(C-10),36.4(C-11),34.6(C-12),140.6(C-13),122.8(C-14),59.4(C-15),16.5(C-16),16.0(C-17),109.4(C-18),22.9(C-19),18.2(C-20)。以上數(shù)據(jù)與文獻[11]報道基本一致,故鑒定化合物2為(-)-agbanindiol A。
化合物3白色結晶;分子式為C21H36O4。1H NMR(600 MHz,CDCl3)δ:5.67(1H,q,J= 1.2 Hz,H-14),3.68(3H,s,15-OCH3),3.58(1H,m,H-3),2.16(3H,d,J= 1.2 Hz,H-16),1.25(3H,s,H-18),1.12(3H,s,H-19),0.79(3H,d,J= 6.2 Hz,H-17),0.74(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:16.5(C-1),30.6(C-2),76.6(C-3),76.5(C-4),41.5(C-5),32.5(C-6),26.6(C-7),36.4(C-8),38.9(C-9),41.0(C-10),36.9(C-11),34.9(C-12),161.9(C-13),115.0(C-14),167.4(C-15),19.3(C-16),16.1(C-17),21.7(C-18),17.4(C-19),18.5(C-20),51.0(15-OCH3)。以上數(shù)據(jù)與文獻[12]報道基本一致,故鑒定化合物3為polylauioid H。
化合物4白色結晶;分子式為C20H34O2。1H NMR(600 MHz,CDCl3)δ:5.68(1H,d,J= 1.5 Hz,H-3),3.61(2H,m,H-15),1.85(3H,d,J= 1.3 Hz,H-18),1.08(3H,s,H-19),0.86(3H,d,J= 6.6 Hz,H-20),0.79(3H,m,H-16),0.76(3H,s,H-17);13C NMR(150 MHz,CDCl3)δ:35.5(C-1),200.7(C-2),172.7(C-3),125.3(C-4),39.5(C-5),34.8(C-6),26.8(C-7),35.7(C-8),38.4(C-9),45.5(C-10),29.9(C-11),34.7(C-12),29.3(C-13),39.7(C-14),60.7(C-15),19.6(C-16),15.6(C-17),18.2(C-18),18.9(C-19),17.9(C-20)。以上數(shù)據(jù)與文獻[13]報道基本一致,故鑒定化合物4為2-oxopopulifolic acid。
化合物5透明膠狀;分子式為C20H32O3。1H NMR(600 MHz,CDCl3)δ:5.89(1H,m,H-3),1.98(3H,d,J= 1.4 Hz,H-20),1.13(3H,s,H-19),0.91(9H,dt,J= 6.8,4.2 Hz,H-16,17,18);13C NMR(150 MHz,CDCl3)δ:48.3(C-1),199.0(C-2),128.9(C-3),159.6(C-4),74.6(C-5),34.2(C-6),40.3(C-7),25.3(C-8),35.9(C-9),40.1(C-10),33.5(C-11),48.1(C-12),210.7(C-13),52.7(C-14),24.9(C-15),22.9(C-16),23.0(C-17),17.2(C-18),21.4(C-19),19.7(C-20)。以上數(shù)據(jù)與文獻[14]報道基本一致,故鑒定化合物5為dysoxydenone C。
化合物6白色結晶;分子式為C21H32O3。1H NMR(600 MHz,CDCl3)δ:5.73(1H,m,H-3),5.62(1H,d,J= 1.4 Hz,H-13),3.67(3H,s,15-OCH3),1.88(3H,d,J= 1.3 Hz,H-16),1.87(3H,d,J= 1.4 Hz,H-18),1.11(3H,m,H-19),0.89(3H,d,J= 6.7 Hz,H-17),0.81(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:34.8(C-1),200.2(C-2),125.6(C-3),172.4(C-4),39.8(C-5),35.5(C-6),26.9(C-7),35.9(C-8),38.9(C-9),45.4(C-10),35.8(C-11),26.8(C-12),160.5(C-13),115.7(C-14),166.6(C-15),25.3(C-16),15.7(C-17),18.9(C-18),18.4(C-19),17.7(C-20),50.9(15-OCH3)。以上數(shù)據(jù)與文獻[15]報道基本一致,故鑒定化合物6為2-oxo-ent-cleroda-3,13Z-dien-15-oic acid。
化合物7透明膠狀;分子式為C20H32O2。1H NMR(600 MHz,CDCl3)δ:5.86(1H,m,H-14),5.71(1H,m,H-3),5.19(1H,dt,J= 17.3,1.8 Hz,H-15a),5.07(1H,dd,J= 10.8,1.8 Hz,H-15b),1.87(3H,d,J= 1.3 Hz,H-18),1.26(3H,s,H-16),1.10(3H,s,H-19),0.82(3H,d,J= 6.2 Hz,H-17),0.80(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:34.9(C-1),200.4(C-2),125.5(C-3),172.5(C-4),39.8(C-5),35.5(C-6),26.8(C-7),35.9(C-8),38.3(C-9),45.6(C-10),31.2(C-11),34.7(C-12),73.1(C-13),144.7(C-14),112.1(C-15),30.0(C-16),15.6(C-17),18.9(C-18),18.3(C-19),18.0(C-20)。以上數(shù)據(jù)與文獻[16]報道基本一致,故鑒定化合物7為nakamurol B。
化合物8白色粉末;分子式為C21H32O3。1H NMR(600 MHz,CDCl3)δ:5.68(1H,t,J= 1.2 Hz,H-3),5.61(1H,q,J= 1.3 Hz,H-14),3.63(3H,s,15-OCH3),2.10(3H,d,J= 1.2 Hz,H-16),1.85(3H,d,J= 1.2 Hz,H-18),1.07(3H,s,H-19),0.80(3H,d,J= 6.1 Hz,H-17),0.78(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:34.8(C-1),199.8(C-2),125.3(C-3),172.2(C-4),39.7(C-5),35.4(C-6),26.7(C-7),35.9(C-8),38.6(C-9),45.5(C-10),35.4(C-11),33.9(C-12),160.2(C-13),115.0(C-14),166.9(C-15),19.0(C-16),15.5(C-17),18.8(C-18),18.2(C-19),17.7(C-20),50.6(15-OCH3)。以上數(shù)據(jù)與文獻[17]報道基本一致,故鑒定化合物8為methyl (13E)-2-oxoneocleroda-3,13-dien-15-oate。
化合物9白色結晶;分子式為C22H34O3。1H NMR(600 MHz,CDCl3)δ:5.72(1H,q,J= 1.1 Hz,H-3),5.31(1H,td,J= 7.3,1.6 Hz,H-14),4.52(1H,m,H-15),2.04(3H,s,OAc),1.88(3H,s,H-18),1.74(3H,m,H-16),1.11(3H,s,H-19),0.85(3H,d,J= 6.3 Hz,H-17),0.80(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:36.3(C-1),200.4(C-2),125.9(C-3),172.7(C-4),40.2(C-5),35.4(C-6),27.2(C-7),36.5(C-8),39.2(C-9),45.9(C-10),35.9(C-11),25.5(C-12),143.5(C-13),119.2(C-14),61.1(C-15),24.1(C-16),16.1(C-17),19.3(C-18),18.7(C-19),18.2(C-20),21.5(CH3COO),171.4(CH3COO)。以上數(shù)據(jù)與文獻[18]報道基本一致,故鑒定化合物9為15-acetoxy-ent-3,13E-clerodadien-2-one。
化合物10白色結晶;分子式為C20H34O3。1H NMR(600 MHz,CD3OD)δ:5.34(1H,m,H-14),4.06(2H,d,J= 6.8 Hz,H-15),3.48(1H,q,J= 3.2 Hz,H-3),1.85(1H,dd,J= 12.4,2.0 Hz,H-10),1.67(3H,d,J= 1.3 Hz,H-16),1.19(3H,s,H-18),1.11(3H,s,H-19),0.79(3H,d,J= 5.9 Hz,H-17),0.74(3H,s,H-20);13C NMR(150 MHz,CD3OD)δ:17.6(C-1),31.0(C-2),76.8(C-3),77.3(C-4),42.5(C-5),33.5(C-6),27.9(C-7),37.3(C-8),39.7(C-9),41.7(C-10),38.4(C-11),34.1(C-12),140.7(C-13),124.2(C-14),59.4(C-15),16.6(C-16),16.5(C-17),21.3(C-18),17.9(C-19),19.0(C-20)。以上數(shù)據(jù)與文獻[17]報道基本一致,故鑒定化合物10為(3α,4β,13E)-neoclerod-13-ene-3,4,15-triol。
化合物11透明膠狀;分子式為C20H34O2。1H NMR(600 MHz,CDCl3)δ:5.89(1H,dd,J= 17.4,10.8 Hz,H-14),5.20(1H,dd,J= 17.4,1.2 Hz,H-15a),5.05(1H,dd,J= 10.8,1.2 Hz,H-15b),1.27(3H,s,H-16),1.04(3H,s,H-18),0.95(3H,s,H-19),0.82(6H,d,J= 6.4 Hz,H-17,20);13C NMR(150 MHz,CDCl3)δ:25.4(C-1),27.7(C-2),76.5(C-3),40.1(C-4),136.4(C-5),27.4(C-6),27.6(C-7),33.5(C-8),40.4(C-9),111.9(C-10),30.1(C-11),36.6(C-12),73.5(C-13),145.4(C-14),100.1(C-15),24.1(C-16),16.1(C-17),25.8(C-18),20.4(C-19),21.5(C-20)。以上數(shù)據(jù)與文獻[10]報道基本一致,故鑒定化合物11為5(10),14-halimadiene-3α,13ξ-diol。
化合物12白色結晶;分子式為C20H32O2。1H NMR(600 MHz,CDCl3)δ:5.87(1H,m,H-3),1.89(3H,d,J= 1.5 Hz,H-20),0.91(9H,m,H-12,17,18),0.84(3H,s,H-19);13C NMR(150 MHz,CDCl3)δ:54.6(C-1),199.4(C-2),127.0(C-3),163.3(C-4),47.9(C-5),26.1(C-6),43.5(C-7),24.5(C-8),40.2(C-9),37.8(C-10),33.8(C-11),16.9(C-12),48.0(C-13),210.9(C-14),52.6(C-15),24.7(C-16),22.8(C-17),22.7(C-18),17.2(C-19),22.1(C-20)。以上數(shù)據(jù)與文獻[19]報道基本一致,故鑒定化合物12為dysokusone A。
化合物13淡黃色膠狀;分子式為C18H28O2。1H NMR(600 MHz,CDCl3)δ:5.72(1H,s,H-3),2.12(3H,m,H-16),1.88(3H,d,J= 2.0 Hz,H-18),1.11(3H,d,J= 2.6 Hz,H-19),0.83(6H,m,H-17,20);13C NMR(150 MHz,CDCl3)δ:35.1(C-1),200.3(C-2),125.7(C-3),172.6(C-4),40.0(C-5),35.7(C-6),27.0(C-7),36.4(C-8),38.4(C-9),46.2(C-10),30.9(C-11),37.2(C-12),208.6(C-13),30.3(C-16),15.9(C-17),18.4(C-18),19.1(C-19),17.9(C-20)。以上數(shù)據(jù)與文獻[20]報道基本一致,故鑒定化合物13為14,15-dinorclerod-3-ene-2,13-dione。
化合物14透明膠狀;分子式為C20H32O3。1H NMR(600 MHz,CDCl3)δ:5.67(1H,s,H-14),2.93(1H,d,J= 1.8 Hz,H-3),2.16(3H,s,H-16),1.17(3H,s,H-18),1.04(3H,s,H-19),0.79(3H,d,J= 6.3 Hz,H-17),0.66(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:19.8(C-1),28.2(C-2),62.3(C-3),66.6(C-4),37.4(C-5),35.2(C-6),28.4(C-7),37.2(C-8),39.3(C-9),48.1(C-10),36.2(C-11),36.5(C-12),164.0(C-13),115.2(C-14),171.9(C-15),17.0(C-16),16.1(C-18),15.5(C-17),18.7(C-19),19.5(C-20)。以上數(shù)據(jù)與文獻[21]報道基本一致,故鑒定化合物14為3,4-epoxyclerodan-13E-en-15-oic acid。
化合物15透明膠狀;分子式為C20H30O2。1H NMR(600 MHz,CDCl3)δ:6.03(1H,s,H-13),5.87(1H,m,H-3),2.27(3H,s,H-12),2.12(1H,m,H-16),1.86(3H,d,J= 1.6 Hz,H-20),0.93(3H,s,H-17),0.92(3H,s,H-18),0.90(3H,s,H-19);13C NMR(150 MHz,CDCl3)δ:53.8(C-1),199.4(C-2),126.9(C-3),163.2(C-4),47.5(C-5),24.8(C-6),40.8(C-7),27.6(C-8),39.7(C-9),37.5(C-10),161.5(C-11),21.3(C-12),124.6(C-13),201.0(C-14),54.5(C-15),25.2(C-16),22.8(C-17),22.8(C-18),16.9(C-19),22.0(C-20)。以上數(shù)據(jù)與文獻[22]報道基本一致,故鑒定化合物15為dysokusone G。
化合物16透明膠狀;分子式為C22H38O4。1H NMR(600 MHz,CDCl3)δ:5.32(1H,t,J= 7.4 Hz,H-14),4.54(2H,m,H-15),3.58(1H,t,J= 2.9 Hz,H-3),2.04(3H,s,OAc),1.24(3H,s,H-18),1.12(3H,s,H-19),0.80(3H,d,J= 6.0 Hz,H-17),0.72(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:16.6(C-1),21.3(C-2),76.6(C-3),76.5(C-4),41.5(C-5),32.5(C-6),26.6(C-7),36.3(C-8),39.0(C-9),40.9(C-10),37.4(C-11),26.0(C-12),144.4(C-13),118.4(C-14),61.1(C-15),23.9(C-16),16.1(C-17),21.6(C-18),17.4(C-19),18.4(C-20),171.3(CH3COO),30.6(CH3COO)。以上數(shù)據(jù)與文獻[23]報道基本一致,故鑒定化合物16為15-acetyloxyl-3α,4β-dihydroxy-neoclerod-13Z-ene。
化合物17透明膠狀;分子式為C20H36O2。1H NMR(600 MHz,CDCl3)δ:5.40(1H,t,J= 7.0 Hz,H-14),4.14(2H,d,J= 6.9 Hz,H-15),1.68(3H,s,H-16),1.28(3H,s,H-18),1.02(3H,s,H-19),0.79(3H,d,J= 5.4 Hz,H-20),0.72(3H,s,H-17);13C NMR(150 MHz,CDCl3)δ:27.3(C-1),32.0(C-2),33.1(C-3),42.1(C-4),76.2(C-5),37.1(C-6),37.1(C-7),23.5(C-8),43.2(C-10),38.9(C-11),36.8(C-12)123.2(C-13),40.9(C-14),59.6(C-15),21.2(C-16),18.4(C-17),16.7(C-18),16.2(C-19),14.9(C-20)。以上數(shù)據(jù)與文獻[24]報道基本一致,故鑒定化合物17為[1α(E),2β,4aβ,8aα]-5-(decahydro-4a-hydroy-1,2,5,5-tetra-methyl-1-naphthalenyl)-3-methl-2-penten-1-ol。
化合物18淡黃色膠狀;分子式為C20H34O。1H NMR(600 MHz,CDCl3)δ:5.38(1H,tq,J= 6.9,1.3 Hz,H-14),5.17(1H,ddt,J= 4.2,2.7,1.2 Hz,H-3),4.12(2H,d,H-15),1.66(3H,m,H-16),1.57(3H,d,J= 3.1 Hz,H-20),0.98(3H,d,J= 0.7 Hz,H-19),0.79(3H,d,J= 6.4 Hz,H-17),0.70(3H,s,H-18);13C NMR(150 MHz,CDCl3)δ:36.8(C-1),27.0(C-2),120.5(C-3),144.6(C-4),38.3(C-5),36.9(C-6),27.6(C-7),36.4(C-8),38.7(C-9),46.5(C-10),18.4(C-11),32.9(C-12),140.9(C-13),123.0(C-14),59.5(C-15),16.6(C-16),18.5(C-17),18.1(C-18),20.0(C-19),16.1(C-20)。以上數(shù)據(jù)與文獻[25]報道基本一致,故鑒定化合物18為kolavenol。
化合物19無色結晶;分子式為C20H32O2。1H NMR(600 MHz,CDCl3)δ:5.71(1H,m,H-3),5.37(1H,tq,J= 6.9,1.3 Hz,H-14),4.12(2H,m,H-15),1.88(3H,d,J= 1.3 Hz,H-18),1.66(3H,m,H-16),1.11(3H,d,J= 0.7 Hz,H-19),0.83(3H,dd,J= 4.2,1.9 Hz,H-17),0.80(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:35.1(C-1),200.6(C-2),125.6(C-3),172.8(C-4),40.0(C-5),35.9(C-6),27.0(C-7),36.1(C-8),138.9(C-9),45.8(C-10),35.7(C-11),32.4(C-12),40.0(C-13),123.5(C-14),59.5(C-15),16.8(C-16),15.9(C-17),19.1(C-18),18.5(C-19),18.1(C-20)。以上數(shù)據(jù)與文獻[17]報道基本一致,故鑒定化合物19為(13E)-2-oxoneocleroda-3,13-dien-15-ol。
化合物20透明膠狀;分子式為C20H34O2。1H NMR(600 MHz,CDCl3)δ:5.42(1H,tq,J= 7.0,1.3 Hz,H-3),5.35(1H,dt,J= 4.6,1.5 Hz,H-14),4.15(1H,s,H-2),4.13(2H,d,J= 7.0 Hz,H-15),1.64(3H,s,H-16),0.94(3H,s,H-19),0.81(3H,d,J= 6.6 Hz,H-18),0.73(3H,s,H-17),0.73(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:27.6(C-1),65.5(C-2),150.1(C-3),122.2(C-4),38.5(C-5),36.4(C-6),36.6(C-7),36.4(C-8),38.9(C-9),41.0(C-10),28.1(C-11),32.4(C-12),141.2(C-13),122.9(C-14),59.6(C-15),16.7(C-16),16.0(C-17),18.2(C-18),18.7(C-19),18.5(C-20)。以上數(shù)據(jù)與文獻[26]報道基本一致,故鑒定化合物20為2β-hydroxykolavenol。
化合物21透明膠狀;分子式為C20H34O2。1H NMR(600 MHz,CDCl3)δ:5.39(1H,ddq,J= 7.0,5.5,1.3 Hz,H-14),4.14(2H,d,J= 6.9 Hz,H-15),2.92(1H,d,J= 1.9 Hz,H-3),1.67(3H,d,J= 1.3 Hz,H-16),1.17(3H,s,H-18),1.04(3H,s,H-19),0.78(3H,m,H-17),0.64(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:15.5(C-1),28.4(C-2),62.4(C-3),66.6(C-4),37.4(C-5),37.3(C-6),28.3(C-7),36.2(C-8),39.2(C-9),48.0(C-10),37.0(C-11),33.2(C-12),140.7(C-13),123.1(C-14),59.6(C-15),16.7(C-16),16.1(C-17),17.0(C-18),19.9(C-19),18.8(C-20)。以上數(shù)據(jù)與文獻[27]報道基本一致,故鑒定化合物21為ent-3β,4β-epoxyclerod-13E-en-15-ol。
化合物22透明膠狀;分子式為C22H36O3。1H NMR(600 MHz,CDCl3)δ:5.69(1H,p,J= 1.1 Hz,H-3),4.05(2H,m,H-15),2.02(3H,s,OAc),1.86(3H,d,J= 1.4 Hz,H-16),1.08(3H,m,H-18),0.87(3H,d,J= 6.5 Hz,H-20),0.79(3H,d,J= 6.2 Hz,H-17),0.77(3H,s,H-19);13C NMR(150 MHz,CDCl3)δ:35.6(C-1),200.5(C-2),125.6(C-3),171.3(C-4),39.9(C-5),35.0(C-6),27.0(C-7),35.6(C-8),38.6(C-9),45.7(C-10),34.7(C-11),30.3(C-12),29.5(C-13),35.9(C-14),62.9(C-15),19.4(C-16),15.8(C-17),18.5(C-18),19.1(C-19),18.1(C-20),172.7(CH3COO),21.1(CH3COO)。以上數(shù)據(jù)與文獻[13]報道基本一致,故鑒定化合物22為2-oxodihydrokolavenol acetate。
化合物23透明膠狀;分子式為C22H40O3。1H NMR(600 MHz,CDCl3)δ:5.41(1H,m,H-14),4.13(2H,d,J= 7.0 Hz,H-15),3.79(1H,t,J= 2.9 Hz,H-3),3.34(2H,2dq,J= 11.0,7.0 Hz,4-OCH2CH3),3.11(3H,d,J= 7.0 Hz,4-OCH2CH3),1.68(3H,s,H-16),1.15(3H,s,H-18),1.09(3H,s,H-19),0.76(3H,d,J= 6.3 Hz,H-17),0.70(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:16.2(C-1),30.7(C-2),72.0(C-3),79.4(C-4),42.4(C-5),32.0(C-6),26.9(C-7),36.1(C-8),38.6(C-9),39.5(C-10),37.3(C-11),33.2(C-12),141.5(C-13),123.0(C-14),59.6(C-15),16.3(C-16),16.6(C-17),14.5(C-18),17.7(C-19),18.5(C-20),57.0(4-OCH2CH3),16.3(4-OCH2CH3)。以上數(shù)據(jù)與文獻[17]報道基本一致,故鑒定化合物23為(3α,4β,13E)-4-ethoxyneoclerod-13-ene-3,15-diol。
本文對分離得到的23個化合物進行PTP-1B抑制活性篩選,結果見表1,與陽性藥蘇拉明鈉對比(抑制率62.31%,IC50值為134.81±8.36 μmol/L),化合物3、12、16、21表現(xiàn)出PTP-1B抑制活性,IC50值分別為31.25±0.64、0.30±0.56、0.64±0.51、78.50±0.59 μmol/L。
表1 化合物對PTP-1B的影響Table 1 Effects of the compounds on PTP-1B
本項研究從密花樫木根乙酸乙酯萃取物中共分離得到23個成分,并且進行PTP-1B抑制活性篩選,在此基礎上對構效關系進行初步探討?;衔?、16、21均為clerodane型,化合物12為prenyleudesmane型,且抑制效果最好,提示prenyleudesmane型二萜抑制活性強于clerodane型;化合物3、21均為E構型,化合物16為Z構型,而化合物16表現(xiàn)出較強的抑制活性,僅次于化合物12,提示Z型二萜類化合物抑制活性較強;化合物3、16結構中含有乙?;乙种苹钚跃^好,乙?;欠衽c抑制活性有關還需進一步探討。
二萜類成分在小分子天然PTP-1B抑制劑開發(fā)中發(fā)揮了不錯的潛力,樫木屬植物是二萜類成分的豐富來源[28],從該屬植物中挖掘天然來源的抗糖尿病候選藥物是值得進一步探索。