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        多穗金粟蘭的化學(xué)成分及其抗炎活性研究

        2020-11-10 03:48:48黃偉明陳芳有卞玉婷張睿增劉定平雙鵬程羅永明
        關(guān)鍵詞:倍半萜分子式抗炎

        黃偉明,陳芳有,卞玉婷,張睿增,劉定平,雙鵬程,羅永明

        江西中醫(yī)藥大學(xué)藥學(xué)院,南昌 330004

        多穗金粟蘭(ChloranthusmultistachysPei)為金粟蘭科(Chloranthaceae)金粟蘭屬(ChloranthusSw.)植物,又名四塊瓦、大四塊瓦、四葉對(duì)、白毛七等,廣泛分布于我國(guó)南方地區(qū)。該植物常以全草入藥,其性味辛、苦、有小毒,具有祛濕散寒、活血止痛、散瘀解毒等功效,主治風(fēng)寒咳嗽、瘀血腫痛、腰腿痛、癤腫、皮膚瘙癢等病癥,是江西民間常用中草藥之一[1]。多穗金粟蘭的藥理學(xué)研究表明,其具有抗炎、抑菌、抗腫瘤等作用[2]。目前對(duì)于多穗金粟蘭的化學(xué)成分研究報(bào)道較少,只見(jiàn)少量的倍半萜類(lèi)、酰胺、甾醇等類(lèi)型化合物的研究報(bào)道[3],致使其藥效物質(zhì)基礎(chǔ)不清楚,嚴(yán)重影響了該藥的研究與開(kāi)發(fā)。因此,對(duì)該植物進(jìn)行系統(tǒng)的化學(xué)成分研究與藥理活性篩選十分必要。本課題組在前期的活性篩選中發(fā)現(xiàn),多穗金粟蘭的二氯甲烷部位有較好的抗炎活性。為進(jìn)一步闡明其抗炎活性的有效活性成分,為其臨床合理用藥提供科學(xué)依據(jù),我們對(duì)多穗金粟蘭的二氯甲烷部物的化學(xué)成分進(jìn)行了較深入的研究,并對(duì)部分化合物進(jìn)行了抗炎活性研究,以期為多穗金粟蘭的進(jìn)一步開(kāi)發(fā)和利用提供一定的參考依據(jù)。

        1 材料與方法

        1.1 儀器與材料

        Bruker AVANCE III HD 600MHz型核磁共振波譜儀(Bruker,瑞士);AB SCIEX Triple TOF5600+型高分辨飛行時(shí)間質(zhì)譜聯(lián)用儀(AB SCIEX,美國(guó));RAW264.7細(xì)胞株(中科院上海生命科學(xué)研究院);脂多糖(LPS);Griess試劑(碧云天公司);酶標(biāo)儀(BioTek公司,Synergy 4 MLFPTAD);氨基胍(Aminoguanidine);胰蛋白酶(Gibco);離心機(jī)(Beckman公司,AllegraX-15R);培養(yǎng)皿(Corning)、培養(yǎng)瓶(Corning)、48孔板(Corning);Waters 2695-2998型高效液相(Waters,美國(guó));Waters 515 制備型液相(Waters,美國(guó));葡聚糖凝膠Sephadex LH-20(Amersham Pharmacia Biotech,美國(guó));YMC-Pack ODS-A制備色譜柱(250 mm×20 mm,5 μm,YMC,日本);Buchi中壓制備液相色譜儀(Buchi,瑞士);YMC ODS反相色譜填料(50 μm,YMC,日本);PRP 512 A樹(shù)脂(75~100 μm,北京聚福樹(shù)脂廠(chǎng));AB-104N型分析天平(METTLER TOLEDO,瑞士);薄層硅膠板GF254(青島海洋化工廠(chǎng));柱色譜用硅膠(100~200,200~300目)青島海洋化工廠(chǎng);純凈水(哇哈哈,杭州);甲醇(星可,上海)為色譜純,其他所用試劑均為分析純(西隴科學(xué)股份有限公司)。

        多穗金粟蘭全草于2018年11月采自江西井岡山,由江西中醫(yī)藥大學(xué)藥學(xué)院鄧可眾副教授鑒定為金粟蘭科金粟蘭屬多穗金粟蘭(ChloranthusmultistachysPei)的全草。憑證標(biāo)本(20181128)保存于江西中醫(yī)藥大學(xué)藥學(xué)院中藥化學(xué)科研室。

        1.2 提取與分離

        多穗金粟蘭全草20 kg,切碎后加4倍量的95%乙醇提取2次,合并提取液,減壓濃縮至無(wú)醇味,得浸膏1 100 g。取部分總浸膏經(jīng)硅藻土柱色譜,依次用石油醚、二氯甲烷、乙酸乙酯、甲醇洗脫,各洗脫液分別減壓濃縮后得石油醚部位130 g、二氯甲烷部位375 g、乙酸乙酯部位30 g、甲醇部位300 g。

        二氯甲烷部位浸膏經(jīng)PRP 512 A樹(shù)脂柱色譜分離(梯度依次為30%、50%、70%、95%乙醇)得到4個(gè)組分A~D。其中B組分(100 g)經(jīng)硅膠(100~200目)柱色譜分離,以石油醚∶乙酸乙酯(20∶1→0∶1)為流動(dòng)相梯度洗脫,經(jīng)薄層色譜合樣后得到10個(gè)組分Fr1~Fr10。Fr8(8 g)通過(guò)中壓ODS柱色譜(甲醇-水)、葡聚糖凝膠Sephadex LH-20柱色譜(甲醇)和重結(jié)晶分離純化得到化合物5(20 mg)、11(20 mg)、14(9 mg)、15(8 mg)。Fr9(14.9 g)經(jīng)硅膠柱色譜分離(二氯甲烷-甲醇,50∶1→0∶1)、中壓ODS柱色譜(甲醇-水)和制備液相柱色譜(甲醇-水)分離純化得到化合物9(10 mg)、10(11 mg)、21(2 mg)、22(4 mg);其中C組分(107 g)經(jīng)硅膠(100~200目)柱色譜分離,以石油醚∶乙酸乙酯(30∶1→0∶1)為流動(dòng)相梯度洗脫,經(jīng)薄層色譜合樣后得到8個(gè)組分Fr1~Fr8。Fr4(18 g)通過(guò)硅膠柱色譜分離(二氯甲烷-甲醇,50∶1→0∶1)、中壓ODS柱色譜(甲醇-水)、葡聚糖凝膠Sephadex LH-20柱色譜(甲醇)和重結(jié)晶分離純化得到化合物1(2 mg)、6(15 mg)、7(14 mg)、12(13 mg)、13(10 mg)、16(11 mg)、17(12 mg)、18(4 mg)、19(3 mg)、20(4 mg);其中D組分(30 g)經(jīng)硅膠(100~200目)柱色譜分離,以石油醚∶乙酸乙酯(50∶1→0∶1)為流動(dòng)相梯度洗脫,經(jīng)薄層色譜合樣后得到5個(gè)組分Fr1~Fr5。Fr4(4.2 g)通過(guò)葡聚糖凝膠Sephadex LH-20柱色譜(甲醇)、薄層制備色譜和重結(jié)晶得到化合物2(3 mg)、3(3 mg)、4(3 mg)、8(9 mg)。

        1.3 抗炎活性篩選方法

        本實(shí)驗(yàn)采用脂多糖(LPS)誘導(dǎo)的RAW264.7細(xì)胞炎癥篩選模型,對(duì)分離得率較高的化合物(5~17)進(jìn)行了抗炎活性檢測(cè),采用Griess試劑法測(cè)定其中產(chǎn)生一氧化氮(NO)的含量[4]以評(píng)價(jià)化合物的抗炎活性。取對(duì)數(shù)生長(zhǎng)期的RAW264.7細(xì)胞經(jīng)胰蛋白酶消化后重懸得到1×106個(gè)/mL的細(xì)胞懸液,鋪板于48孔板中,每孔200 μL,在37 ℃、5% CO2培養(yǎng)箱中常規(guī)培養(yǎng)12 h。再用10 μmol/L的單體化合物預(yù)處理1 h,再加入1 μg/mL LPS(終濃度)進(jìn)行刺激,同時(shí)設(shè)置空白對(duì)照組(培養(yǎng)液)、非樣品組(LPS+培養(yǎng)液)、陽(yáng)性對(duì)照組(氨基胍+LPS+培養(yǎng)液),繼續(xù)培養(yǎng)持續(xù)刺激18 h。每組實(shí)驗(yàn)在相同條件下設(shè)置3個(gè)復(fù)孔。取100 μL細(xì)胞培養(yǎng)液,加入等量Griess試劑(含A B液分別50 μL,碧云天公司),經(jīng)搖床避光充分混勻15 min后,用酶標(biāo)儀于570 nm波長(zhǎng)下測(cè)定各孔OD值,計(jì)算化合物的NO生成抑制率[5]。

        2 實(shí)驗(yàn)結(jié)果

        2.1 結(jié)構(gòu)鑒定

        化合物1白色無(wú)定型粉末;HR-ESI-MS:m/z383.146 2 [M+Na]+(calcd for C20H24O6Na,383.146 5),分子式為C20H24O6;1H NMR(600 MHz,CD3OD)δ:6.94(1H,d,J=1.8 Hz,H-2),6.75(1H,d,J=8.1 Hz,H-5),6.82(1H,dd,J=8.1,2.1 Hz,H-6),5.48(1H,d,J=6.3 Hz,H-7),3.82(1H,m,H-8),3.75(1H,dd,J=11.1,7.2 Hz,H-9a),3.46(1H,q,J=6.3 Hz,H-9b),6.72(2H,s,H-2′,6′),2.62(2H,dd,J=8.7,6.9 Hz,H-7′),1.80(2H,m,H-8′),3.56(2H,t,J=6.5 Hz,H-9′),3.81(3H,s,3-OCH3),3.85(3H,s,3′-OCH3);13C NMR(150 MHz,CD3OD)δ:134.8(C-1),110.5(C-2),149.1(C-3),147.5(C-4),116.1(C-5),119.7(C-6),89.0(C-7),55.5(C-8),65.0(C-9),136.9(C-1′),114.0(C-2′),145.2(C-3′),147.5(C-4′),129.9(C-5′),117.9(C-6′),32.9(C-7′),35.8(C-8′),62.2(C-9′),56.3(3-OCH3),56.7(3′-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[6]對(duì)照基本一致,故鑒定化合物1為(7S,8R)-dihydrodehydrodiconiferyl alcohol。

        化合物2無(wú)色針狀結(jié)晶(甲醇);HR-ESI-MS:m/z429.372 2 [M+H]+(calcd for C29H49O2,429.372 7),分子式為C29H48O2;1H NMR(600 MHz,CDCl3)δ:3.68(1H,m,H-3),5.69(1H,br s,H-6),2.45(3H,m),2.24(1H,ddd,J=12.6,10.8,2.4 Hz),2.09~1.02(17H,m,包括1.19(3H,H-19)),0.69(3H,s,H-18),0.93(3H,d,J=6.6 Hz,H-21),0.83(3H,d,J=6.6 Hz,H-26),0.81(3H,d,J=6.6 Hz,H-27),0.85(3H,d,J=7.2 Hz,H-29);13C NMR(150 MHz,CDCl3)δ:36.5(C-1),31.3(C-2),70.7(C-3),41.9(C-4),165.2(C-5),126.3(C-6),202.5(C-7),45.6(C-8),50.1(C-9),38.8(C-10),21.4(C-11),38.4(C-12),43.2(C-13),50.1(C-14),26.5(C-15),28.7(C-16),54.8(C-17),12.1(C-18),17.5(C-19),36.2(C-20),19.1(C-21),34.1(C-22),26.2(C-23),45.9(C-24),29.2(C-25),19.2(C-26),20.0(C-27),23.2(C-28),12.1(C-29)。以上數(shù)據(jù)與文獻(xiàn)[7]對(duì)照基本一致,故鑒定化合物2為7-ketositosterol。

        化合物3無(wú)色針狀結(jié)晶(甲醇);HR-ESI-MS:m/z429.372 4 [M+H]+(calcd for C29H49O2,429.372 7),分子式為C29H48O2;1H NMR(600 MHz,CDCl3)δ:5.82(1H,d,J=0.9 Hz,H-4),4.35(1H,m,H-6),2.03(1H,m,H-7),0.74(3H,s,H-18),1.38(3H,s,H-19),0.92(3H,d,J=6.6 Hz,H-21),1.70(1H,m,H-25),0.81(3H,m,H-26),0.84(3H,m,H-27),0.85(3H,d,J=7.5 Hz,H-29);13C NMR(150 MHz,CDCl3)δ:37.2(C-1),34.4(C-2),200.6(C-3),126.5(C-4),168.6(C-5),73.4(C-6),38.7(C-7),29.9(C-8),53.7(C-9),38.1(C-10),21.1(C-11),39.7(C-12),42.6(C-13),56.2(C-14),24.3(C-15),28.3(C-16),56.0(C-17),12.2(C-18),19.7(C-19),36.3(C-20),18.9(C-21),34.0(C-22),26.7(C-23),46.0(C-24),29.3(C-25),20.0(C-26),19.2(C-27),23.2(C-28),12.1(C-29)。以上數(shù)據(jù)與文獻(xiàn)[8]對(duì)照基本一致,故鑒定化合物3為6β-羥基豆甾-4-烯-3-酮。

        化合物4無(wú)色針狀結(jié)晶(甲醇);HR-ESI-MS:m/z429.372 4 [M+H]+(calcd for C29H49O2,429.372 7),分子式為C29H48O2;1H NMR(600 MHz,CDCl3)δ:6.17(1H,d,J=1.8 Hz,H-4),4.33(1H,ddd,J=12.3,5.7,1.8 Hz,H-6),0.71(3H,s,H-18),1.18(3H,s,H-19),0.92(3H,d,J=6.6 Hz,H-21),0.83(3H,d,J=6.9 Hz,H-26),0.81(3H,d,J=6.9 Hz,H-27),0.85(3H,d,J=7.5 Hz,H-29);13C NMR(150 MHz,CDCl3)δ:36.4(C-1),34.3(C-2),199.6(C-3),119.8(C-4),171.7(C-5),68.9(C-6),41.6(C-7),34.0(C-8),53.9(C-9),39.2(C-10),21.2(C-11),39.6(C-12),42.6(C-13),55.7(C-14),24.3(C-15),28.3(C-16),56.1(C-17),12.1(C-18),18.4(C-19),36.2(C-20),18.8(C-21),34.0(C-22),26.2(C-23),45.9(C-24),29.3(C-25),20.0(C-26),19.2(C-27),23.2(C-28),12.1(C-29)。以上數(shù)據(jù)與文獻(xiàn)[9]對(duì)照基本一致,故鑒定化合物4為6α-羥基豆甾-4-烯-3-酮。

        化合物5白色粉末;HR-ESI-MS:m/z267.159 7 [M+H]+(calcd for C15H23O4,267.159 6),分子式為C15H22O4;1H NMR(600 MHz,CD3OD)δ:1.11(1H,m,H-1a),1.51(1H,m,H-1b),1.39(1H,m,H-2a),1.93(1H,m,H-2b),1.42(1H,m,H-3a),1.70(1H,m,H-3b),1.09(1H,t,J=3.5 Hz,H-5),2.44(1H,m,H-6a),2.82(1H,dd,J=13.2,2.7 Hz,H-6b),1.35(1H,d,J=8.4 Hz,H-9a),2.06(1H,d,J=13.2 Hz,H-9b),1.79(3H,d,J=1.5 Hz,H-13),1.32(3H,d,J=3.9 Hz,H-14),1.24(3H,s,H-15);13C NMR(150 MHz,CD3OD)δ:42.6(C-1),18.7(C-2),41.7(C-3),72.3(C-4),55.8(C-5),23.1(C-6),164.4(C-7),105.8(C-8),54.9(C-9),36.1(C-10),121.9(C-11),174.6(C-12),7.9(C-13),19.7(C-14),30.4(C-15)。以上數(shù)據(jù)與文獻(xiàn)[10]對(duì)比基本一致,故鑒定化合物5為4β,8β-二羥基-5α(H)-桉葉-7(11)-烯-8,12-內(nèi)酯。

        化合物6白色塊狀結(jié)晶(甲醇);HR-ESI-MS:m/z307.263 4 [M+H]+(calcd for C20H35O2,307.263 1),分子式為C20H34O2;1H NMR(600 MHz,CDCl3)δ:1.05(1H,m,H-1a),1.78(1H,m,H-1b),1.48(2H,m,H-2),1.02(1H,m,H-3a),1.72(1H,m,H-3b),1.22(1H,m,H-5),1.29(1H,m,H-6a),1.82(1H,m,H-6b),2.38(1H,ddd,J=12.6,4.2,2.4 Hz,H-7a),1.93(1H,td,J=12.6,5.1 Hz,H-7b),1.54(1H,m,H-9),1.36(1H,m,H-11a),1.52(1H,m,H-11b),1.24(1H,m,H-12a),1.76(1H,m,H-12b),5.92(1H,m,H-14),5.21(1H,dd,J=17.4,1.2 Hz,H-15a),5.06(1H,m,H-15b),1.28(3H,s,H-16),4.81(1H,q,J=1.5 Hz,H-17a),4.47(1H,q,J=1.5 Hz,H-17b),0.96(3H,s,H-18),3.73(1H,d,J=10.8 Hz,H-19a),3.36(1H,dd,J=10.8,1.2 Hz,H-19b),0.63(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:39.1(C-1),19.1(C-2),35.5(C-3),39.0(C-4),56.4(C-5),24.5(C-6),38.7(C-7),148.3(C-8),57.4(C-9),39.8(C-10),17.9(C-11),41.4(C-12),73.8(C-13),145.2(C-14),111.8(C-15),27.2(C-16),106.7(C-17),28.2(C-18),65.1(C-19),15.4(C-20)。以上數(shù)據(jù)與文獻(xiàn)[11]對(duì)照基本一致,故鑒定化合物6為13-epitorulosol。

        化合物7白色粉末;HR-ESI-MS:m/z305.210 6 [M+H]+(calcd for C19H29O3,305.211 1),分子式為C19H28O3;1H NMR(600 MHz,CDCl3)δ:1.15(1H,m,H-1a),1.82(1H,m,H-1b),1.54(1H,m,H-2a),2.17(1H,m,H-3a),1.37(1H,dd,J=12.0,2.7 Hz,H-5),1.99(1H,m,H-6a),2.42(1H,dt,J=9.9,2.5 Hz,H-7a),1.90(1H,m,H-9),2.56(1H,m,H-11a),6.42(1H,ddd,J=7.4,5.9,1.5 Hz,H-12a),9.32(1H,s,H-14),1.74(3H,d,J=1.2 Hz,H-16a),4.38(1H,s,H-17a),4.85(1H,br s,H-17b),1.25(3H,s,H-18),0.67(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:39.4(C-1),20.0(C-2),37.9(C-3),44.3(C-4),56.2(C-5),25.8(C-6),38.4(C-7),147.7(C-8),55.9(C-9),40.5(C-10),24.6(C-11),156.4(C-12),139.0(C-13),195.5(C-14),9.5(C-16),108.0(C-17),29.1(C-18),184.1(C-19),13.0(C-20)。以上數(shù)據(jù)與文獻(xiàn)[12]對(duì)照基本一致,故鑒定化合物7為15-nor-14-oxolabda-8(17),12E-dien-19-oic acid。

        化合物8無(wú)色油狀;HR-ESI-MS:m/z263.127 4 [M+H]+(calcd for C15H19O4,263.127 7),分子式為C15H18O4;1H NMR(600 MHz,CDCl3)δ:2.45(1H,ddd,J=15.3,4.5,3.0 Hz,H-2a),2.73(1H,td,J=15.3,6.0 Hz,H-2b),1.91(1H,td,J=14.4,4.5 Hz,H-3a),2.03(1H,ddd,J=13.5,6.0,3.0 Hz,H-3b),2.89(1H,s,H-5),3.07(1H,d,J=17.7 Hz,H-9a),2.95(1H,d,J=17.7 Hz,H-9b),7.12(1H,s,H-12),2.20(3H,d,J=1.2 Hz,H-13),1.73(3H,s,H-14),1.27(3H,s,H-15);13C NMR(150 MHz,CDCl3)δ:211.1(C-1),34.8(C-2),39.0(C-3),70.4(C-4),62.2(C-5),195.5(C-6),119.4(C-7),165.9(C-8),35.7(C-9),51.2(C-10),119.1(C-11),140.3(C-12),9.1(C-13),23.9(C-14),20.5(C-15)。以上數(shù)據(jù)與文獻(xiàn)[13]對(duì)照基本一致,故鑒定化合物8為4a-hydroxy-8,12-epoxyeudesma-7,11-diene-1,6-dione。

        化合物9黃色油狀;HR-ESI-MS:m/z731.228 3 [M-H]-(calcd for C39H39O14,731.229 4),分子式為C39H40O14;1H NMR(600 MHz,CDCl3)δ:2.28(1H,d,J=5.7 Hz,H-1),1.22(1H,d,J=5.1 Hz,H-2a),1.02(1H,q,J=8.1 Hz,H-2b),1.78(H,m,H-3),1.81(3H,s,H-13),1.16(3H,s,H-14),2.68(1H,m,H-15a),1.75(1H,t,J=12.3 Hz,H-15b),1.55(1H,d,J=9.3 Hz,H-1′),1.26(1H,m,H-2′a),0.63(1H,q,J=7.8 Hz,H-2′b),1.46(1H,s,H-3′),2.23(1H,dd,J=12.6,6.6 Hz,H-5′),3.04(1H,dd,J=18.3,12.6 Hz,H-6′a),2.39(1H,dd,J=18.3,6.6 Hz,H-6′b),2.60(1H,td,J=10.8,5.7 Hz,H-9′),5.40(1H,d,J=12.0 Hz,H-13′a),4.51(1H,d,J=12.0 Hz,H-13′b),0.95(3H,s,H-14′),4.32(1H,d,J=11.4 Hz,H-15′a),4.03(1H,d,J=11.4 Hz,H-15′b),6.57(1H,t,J=5.7 Hz,H-3′′),4.75(1H,dd,J=14.7,5.4 Hz,H-4′′a),4.63(1H,dd,J=14.7,6.0 Hz,H-4′′b),1.86(3H,s,H-5′′),2.79(1H,m,H-2′′′a),2.52(1H,m,H-2′′′b),2.71(1H,m,H-3′′′a),2.48(1H,m,H-3′′′b);13C NMR(150 MHz,CDCl3)δ:24.4(C-1),9.5(C-2),30.3(C-3),77.6(C-4),160.9(C-5),122.8(C-6),148.0(C-7),94.3(C-8),200.3(C-9),57.1(C-10),129.2(C-11),170.4(C-12),11.5(C-13),21.1(C-14),40.2(C-15),26.8(C-1′),10.1(C-2′),29.4(C-3′),77.4(C-4′),55.1(C-5′),24.1(C-6′),173.7(C-7′),85.7(C-8′),52.1(C-9′),45.2(C-10′),123.9(C-11′),171.3(C-12′),53.5(C-13′),24.4(C-14′),74.3(C-15′),168.0(C-1′′),129.5(C-2′′),136.6(C-3′′),61.9(C-4′′),13.0(C-5′′),172.3(C-1′′′),29.0(C-2′′′),29.0(C-3′′′),172.3(C-4′′′)。以上數(shù)據(jù)與文獻(xiàn)[14]對(duì)照基本一致,故鑒定化合物9為金粟蘭素 A。

        化合物10黃色油狀;HR-ESI-MS:m/z639.256 1 [M+Na]+(calcd for C36H40O9Na,639.256 4),分子式為C36H40O9;1H NMR(600 MHz,CDCl3)δ:2.18(1H,dt,J=8.7,4.8 Hz,H-1),1.19(1H,m,H-2a),0.63(1H,m,H-2b),1.96(1H,ddd,J=8.4,5.4,3.0 Hz,H-3),3.95(1H,s,H-9),1.36(3H,s,H-13),1.16(3H,s,H-14),2.96(1H,dd,J=18.0,7.2 Hz,H-15a),2.31(1H,dd,J=18.0,9.6 Hz,H-15b),1.66(1H,m,H-1′),0.66(1H,m,H-2′a),1.22(1H,m,H-2′b),1.66(1H,m,H-3′),1.65(1H,m,H-5′),2.46(1H,d,J=11.7 Hz,H-6′a),1.71(1H,d,J=14.1 Hz,H-6′b),2.65(1H,dd,J=9.6,7.2 Hz,H-9′),5.52(1H,s,H-13′a),6.21(1H,s,H-13′b),0.95(3H,s,H-14′),4.24(1H,d,J=11.1 Hz,H-15′a),4.19(1H,d,J=11.1 Hz,H-15′b),6.86(1H,m,H-2′′),1.82(3H,s,H-4′′),1.83(3H,s,H-5′′),3.45(3H,s,12-OCH3);13C NMR(150 MHz,CDCl3)δ:28.5(C-1),15.8(C-2),26.6(C-3),151.3(C-4),135.4(C-5),151.4(C-6),129.7(C-7),197.1(C-8),83.2(C-9),57.6(C-10),65.3(C-11),172.0(C-12),18.9(C-13),14.9(C-14),29.3(C-15),26.6(C-1′),10.3(C-2′),29.8(C-3′),79.0(C-4′),55.7(C-5′),27.8(C-6′),59.1(C-7′),95.6(C-8′),52.2(C-9′),43.0(C-10′),145.9(C-11′),168.4(C-12′),123.1(C-13′),24.0(C-14′),69.6(C-15′),167.9(C-1′′),128.2(C-2′′),138.7(C-3′′),14.7(C-4″),12.3(C-5″),52.2(12-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[15]對(duì)照基本一致,故鑒定化合物10為fortunilide K。

        化合物11淡黃色無(wú)定型粉末;HR-ESI-MS:m/z285.109 8 [M+Na]+(calcd for C15H18O4Na,285.109 7),分子式為C15H18O4;1H NMR(600 MHz,DMSO-d6)δ:4.87(1H,m,H-1),2.15(1H,m,H-2a),2.02(1H,m,H-2b),2.68(1H,m,H-3a),2.03(1H,m,H-3b),6.30(1H,s,H-5),2.70(1H,d,J=12.6 Hz,H-9a),2.39(1H,d,J=12.6 Hz,H-9b),1.91(3H,s,H-13),1.85(3H,d,J=1.5 Hz,H-14),1.58(3H,s,H-15);13C NMR(150 MHz,DMSO-d6)δ:127.8(C-1),25.5(C-2),29.4(C-3),147.9(C-4),129.0(C-5),190.8(C-6),154.7(C-7),109.3(C-8),48.2(C-9),135.4(C-10),135.4(C-11),170.2(C-12),9.9(C-13),24.5(C-14),17.8(C-15)。以上數(shù)據(jù)與文獻(xiàn)[16]對(duì)照基本一致,故鑒定化合物11為(1E,4Z)-8-hydroxy-6-oxogermacra-1(10),4,7(11)-trieno-12,8-1actone。

        化合物12白色粉末;HR-ESI-MS:m/z359.218 8 [M+Na]+(calcd for C20H32O4Na,359.219 2),分子式為C20H32O4;1H NMR(600 MHz,CD3OD)δ:1.16(1H,m,H-1a),1.88(1H,m,H-1b),1.50(1H,m,H-2a),1.86(1H,m,H-2b),1.07(1H,td,J=13.2,4.0 Hz,H-3a),2.13(1H,m,H-3b),1.36(1H,dd,J=12.0,3.0 Hz,H-5),1.94(2H,m,H-6),1.85(1H,m,H-7a),2.38(1H,m,H-7b),1.74(1H,m,H-9),2.00(1H,m,H-11a),2.32(1H,m,H-11b),5.38(1H,t,J=6.6 Hz,H-12),3.94(1H,dd,J=7.2,5.1 Hz,H-14),3.45(2H,m,H-15),1.61(3H,d,J=1.5 Hz,H-16),4.47(1H,s,H-17a),4.82(1H,s,H-17b),1.19(3H,s,H-18),0.66(3H,s,H-20);13C NMR(150 MHz,CD3OD)δ:40.6(C-1),21.2(C-2),39.4(C-3),45.4(C-4),57.5(C-5),27.4(C-6),39.7(C-7),149.6(C-8),57.9(C-9),41.4(C-10),23.6(C-11),129.3(C-12),135.1(C-13),79.1(C-14),65.9(C-15),12.3(C-16),107.9(C-17),29.6(C-18),181.3(C-19),13.4(C-20)。以上數(shù)據(jù)與文獻(xiàn)[17]對(duì)照基本一致,故鑒定化合物12為henrilabdane B。

        化合物13紅色塊狀結(jié)晶(甲醇);HR-ESI-MS:m/z345.133 3 [M+H]+(calcd for C19H21O6,345.133 2),分子式為C19H20O6;1H NMR(600 MHz,CDCl3)δ:7.56(2H,m,H-2,6),6.93(2H,m,H-3,5),6.01(1H,s,H-5′),7.77(1H,d,J=15.6 Hz,H-a),7.81(1H,d,J=15.6 Hz,H-b),3.84(3H,s,4-OCH3),3.86(3H,s,3′-OCH3),3.96(6H,d,J=0.9 Hz,4′,6′-OCH3);13C NMR(150 MHz,CDCl3)δ:128.3(C-1),130.3(C-2,6),161.6(C-4),114.5(C-3,5),107.1(C-1′),158.3(C-2′),131.0(C-3′),158.6(C-4′),87.2(C-5′),159.5(C-6′),125.2(C-a),143.0(C-b),193.3(C=O),55.6,56.1,56.2,60.9(OCH3)。以上數(shù)據(jù)與文獻(xiàn)[18]對(duì)照基本一致,故鑒定化合物13為2′-羥基-4,3′,4′,6′-四甲氧基查爾酮。

        化合物14白色粉末;HR-ESI-MS:m/z265.143 0 [M+H]+(calcd for C15H21O4,265.143 4),分子式為C15H20O4;1H NMR(600 MHz,CD3OD)δ:3.69(1H,m,H-1),1.82(1H,m,H-2a),1.76(1H,m,H-2b),1.63(2H,m,H-3),2.56(1H,s,H-5),3.09(1H,d,J=17.1 Hz,H-9a),2.72(1H,dt,J=17.1,0.9 Hz,H-9b),7.08(1H,s,H-12),2.19(3H,d,J=1.5 Hz,H-13),1.48(3H,s,H-14),1.02(3H,s,H-15);13C NMR(150 MHz,CD3OD)δ:78.3(C-1),28.5(C-2),38.3(C-3),71.2(C-4),62.1(C-5),197.3(C-6),119.4(C-7),166.4(C-8),40.0(C-9),44.7(C-10),139.7(C-11),119.2(C-12),9.2(C-13),24.6(C-14),14.7(C-15)。以上數(shù)據(jù)與文獻(xiàn)[3]對(duì)照基本一致,故鑒定化合物14為curcolonol。

        化合物15無(wú)色透明結(jié)晶(甲醇);HR-ESI-MS:m/z285.109 9 [M+Na]+(calcd for C15H18O4Na,285.109 7),分子式為C15H18O4;1H NMR(600 MHz,CDCl3)δ:2.91(1H,br d,J=11.1 Hz,H-1),2.22(1H,br d,J=14.1 Hz,H-2a),1.53(1H,m,H-2b),2.40(1H,br d,J= 14.1 Hz,H-3a),1.48(1H,m,H-3b),3.76(1H,s,H-5),2.80(1H,d,J=17.1 Hz,H-9a),3.67(1H,d,J=17.1 Hz,H-9b),7.08(1H,s,H-12),2.16(3H,s,H-13),1.31(3H,s,H-14),1.14(3H,s,H-15);13C NMR(150 MHz,CDCl3)δ:70.0(C-1),24.5(C-2),36.9(C-3),64.9(C-4),64.7(C-5),192.3(C-6),123.4(C-7),159.2(C-8),39.9(C-9),59.1(C-10),124.4(C-11),140.0(C-12),10.9(C-13),15.6(C-14),17.0(C-15)。以上數(shù)據(jù)與文獻(xiàn)[3]對(duì)照基本一致,故鑒定化合物15為zederone epoxide。

        化合物16黃色油狀;HR-ESI-MS:m/z755.267 1 [M+Na]+(calcd for C40H44O13Na,755.267 4),分子式為C40H44O13;1H NMR(600 MHz,CDCl3)δ:2.05(1H,m,H-1),1.00(1H,m,H-2a),0.32(1H,m,H-2b),1.87(1H,m,H-3),3.95(1H,J=3.9 Hz,H-6),3.87(1H,s,H-9),1.94(3H,d,J=0.6 Hz,H-13),1.03(3H,s,H-14),2.80(1H,m,H-15a),2.58(1H,m,H-15b),1.60(1H,m,H-1′),0.73(1H,m,H-2′a),1.35(1H,m,H-2′b),1.39(1H,m,H-3′),1.85(1H,m,H-5′),2.50(1H,m,H-6′a),2.71(1H,m,H-6′b),1.84(1H,m,H-9′),4.53(1H,d,J=12.0 Hz,H-13′a),5.09(1H,m,H-13′b),0.82(3H,s,H-14′),3.63(1H,d,J=11.7 Hz,H-15′a),4.62(1H,m,H-15′b),6.61(1H,ddq,J=6.6,5.1,1.2 Hz,H-c),4.63(1H,d,J=12.0 Hz,H-da),5.07(1H,m,H-db),1.92(3H,d,J=1.2 Hz,H-e),2.47(1H,m,H-ga),2.89(1H,m,H-gb),2.67(1H,m,H-ha),2.77(1H,m,H-hb),3.71(3Н,S,12-OCH3);13C NMR(150 MHz,CDCl3)δ:26.1(С-1),16.1(С-2),24.9(C-3),142.6(C-4),132.2(C-5),41.2(C-6),131.5(C-7),200.9(C-8),80.1(C-9),51.1(C-10),147.6(C-11),170.3(C-12),20.3(C-13),15.4(C-14),25.5(C-15),25.7(C-1′),11.8(C-2′),27.9(C-3′),77.2(C-4′),61.4(C-5′),23.5(C-6′),174.7(C-7′),93.4(C-8′),55.6(C-9′),45.1(C-10′),123.5(C-11′),171.7(C-12′),54.5(C-13′),26.2(C-14′),72.1(C-15′),167.2(C-a),129.3(C-b),135.6(C-c),61.4(C-d),13.1(C-e),171.7(C-f),28.8(C-g),29.3(C-h),172.1(C-i),52.5(12-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[19]對(duì)照基本一致,故鑒定化合物16為銀線(xiàn)草醇 B。

        化合物17黃色油狀;HR-ESI-MS:m/z549.246 5 [M+H]+(calcd for C32H37O8,549.248 3),分子式為C32H36O8;1H NMR(600 MHz,CDCl3)δ:1.83(1H,m,H-1,1′),0.87(1H,m,H-2,2′a),0.21(1H,q,J=3.9 Hz,H-2,2′b),1.96(1H,d,J=7.2 Hz,H-3,3′),7.16(1H,s,H-6,6′),3.80(1H,s,H-9,9′),1.50(3H,s,H-13,13′),0.99(3H,s,H-14,14′),2.95(1H,d,J=13.5 Hz,H-15,15′a),2.60(1H,d,J=13.5 Hz,H-15,15′b),3.64(3H,s,12,12′-OCH3);13C NMR(150 MHz,CDCl3)δ:24.8(C-1,1′),14.1(C-2,2′),28.1(C-3,3′),147.5(C-4,4′),136.0(C-5,5′),138.5(C-6,6′),137.4(C-7,7′),199.2(C-8,8′),81.1(C-9,9′),58.6(C-10,10′),47.9(C-11,11′),175.6(C-12,12′),28.7(C-13,13′),16.1(C-14,14′),38.4(C-15,15′),52.2(12,12′-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[19]對(duì)照基本一致,故鑒定化合物17為環(huán)銀線(xiàn)草醇 A。

        化合物18白色無(wú)定型粉末;HR-ESI-MS:m/z337.237 3 [M + H]+(calcd for C20H33O4,337.237 3),分子式為C20H32O4;1H NMR(600 MHz,CD3OD)δ:1.19(3H,s,H-1a),1.79(1H,m,H-1b),1.49(1H,m,H-2a),1.88(1H,m,H-2b),1.08(1H,td,J=13.5,4.2 Hz,H-3a),2.06(1H,m,H-3b),1.40(1H,d,J=12.3,Hz,H-5),2.01(1H,m,6a),1.91(1H,m,H-6b),1.97(1H,m,7a),2.41(1H,m,H-7b),2.13(1H,m,H-9),1.52(1H,m,H-11a),1.62(1H,m,H-11b),3.97(1H,d,J=10.2 Hz,H-12),5.52(1H,t,J=6.6 Hz,H-14),4.11(2H,d,J=6.6 Hz,H-15),1.66(3H,s,H-16),4.49(1H,d,J=1.8 Hz,H-17a),4.86(1H,s,H-17b),1.20(3H,s,H-18),0.61(3H,s,H-20);13C NMR(150 MHz,CD3OD)δ:40.3(C-1),21.1(C-2),39.4(C-3),45.2(C-4),57.6(C-5),27.6(C-6),39.9(C-7),150.3(C-8),53.2(C-9),41.2(C-10),31.5(C-11),75.9(C-12),142.4(C-13),124.7(C-14),59.2(C-15),12.1(C-16),107.0(C-17),29.6(C-18),181.3(C-19),13.5(C-20)。以上數(shù)據(jù)與文獻(xiàn)[17]對(duì)照基本一致,故鑒定化合物18為henrilabdane A。

        化合物19黃色油狀;HR-ESI-MS:m/z315.196 1 [M-H]-(calcd for C20H27O3,315.196 0),分子式為C20H28O3;1H NMR(600 MHz,CDCl3)δ:1.87(1H,m,H-1a),1.26(H,s,H-1b),1.90(1H,m,H-2a),1.54(1H,m,H-2b),2.17(1H,m,H-3a),1.10(1H,td,J=13.5,4.2 Hz,H-3b),1.43(1H,m,H-5),2.00(1H,m,H-6a),1.89(1H,m,H-6b),2.36(1H,m,H-7a),1.92(1H,m,H-7b),2.30(1H,m,H-9),2.74(1H,dd,J=15.3,3.3 Hz,H-11a),2.63(1H,dd,J=15.3,10.4 Hz,H-11b),6.11(1H,d,J=1.8 Hz,H-14),7.17(1H,d,J=1.8 Hz,H-15),1.96(3H,d,J=0.6 Hz,H-16),4.79(1H,d,J=1.5 Hz,H-17a),4.59(1H,s,H-17b),1.26(3H,s,H-18),0.70(3H,s,H-20);13C NMR(150 MHz,CDCl3)δ:39.2(C-1),20.1(C-2),38.1(C-3),44.3(C-4),56.3(C-5),26.0(C-6),38.6(C-7),148.1(C-8),53.8(C-9),40.5(C-10),21.8(C-11),150.8(C-12),113.5(C-13),113.0(C-14),139.5(C-15),10.3(C-16),107.1(C-17),29.2(C-18),182.9(C-19),12.8(C-20)。以上數(shù)據(jù)與文獻(xiàn)[20]對(duì)照基本一致,故鑒定化合物19為12-(3-methyl-furan)-labd-8(17)-en-19-oic acid。

        化合物20無(wú)色無(wú)定型粉末;HR-ESI-MS:m/z307.226 4 [M+H]+(calcd for C19H31O3,307.226 7),分子式為C19H30O3;1H NMR(600 MHz,CD3OD)δ:1.04(1H,td,J=12.6 ,5.4 Hz,H-1a),1.70(1H,m,H-1b),1.52(2H,m,H-2),1.25(2H,m,H-3),1.32(1H,m,H-5),1.45(1H,d,J=5.1 Hz,H-6a),1.32(1H,d,J=1.5 Hz,H-6b),2.26(1H,m,H-7a), 2.11(1H,m,H-7b),1.80(1H,m,H-9),1.66(2H,m,H-11),1.76(1H,m,H-12a),1.67(2H,m,H-12b),5.53(1H,s,H-14),1.22(3H,s,H-17),3.34(1H,d,J=10.8 Hz,H-18a),2.99(1H,d,J=10.8 Hz,H-18b),0.78(3H,s,H-19),0.85(3H,s,H-20);13C NMR(150 MHz,CD3OD)δ:40.1(C-1),19.4(C-2),36.5(C-3),38.8(C-4),48.5(C-5),23.3(C-6),36.6(C-7),139.7(C-8),49.6(C-9),39.1(C-10),19.6(C-11),32.8(C-12),43.8(C-13),126.2(C-14),181.6(C-15,16),25.2(C-17),72.0(C-18),18.5(C-19),16.1(C-20)。以上數(shù)據(jù)與文獻(xiàn)[21]對(duì)照基本一致,故鑒定化合物20為ent-8(9)-pimarene-20-hydroxy-16-nor-15-oic acid。

        化合物21黃色油狀;HR-ESI-MS:m/z314.138 8 [M+H]+(calcd for C18H19NO4,314.138 6),分子式為C18H18NO4;1H NMR(600 MHz,CD3OD)δ:7.10(1H,d,J=1.8 Hz,H-2),6.78(1H,d,J=8.1 Hz,H-5),7.01(1H,dd,J=8.1,2.1 Hz,H-6),7.42(1H,d,J=15.6 Hz,H-7),6.39(1H,d,J=15.6 Hz,H-8),7.05(1H,d,J=2.1 Hz,H-2′),6.71(1H,d,J=2.1 Hz,H-3′),6.70(1H,d,J=2.1 Hz,H-5′),7.04(1H,d,J=2.1 Hz,H-6′),2.74(2H,t,J=7.5 Hz,H-7′),3.45(2H,t,J=7.5 Hz,H-8′),3.87(3H,s,3-OCH3);13C NMR(150 MHz,CD3OD)δ:128.1(C-1),111.5(C-2),150.0(C-3),149.3(C-4),116.5(C-5),123.2(C-6),142.1(C-7),118.6(C-8),169.2(C-9),131.3(C-1′),130.7(C-2′),116.3(C-3′),157.0(C-4′),116.2(C-5′),130.7(C-6′),35.8(C-7′),42.6(C-8′),56.4(3-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[22]對(duì)照基本一致,故鑒定化合物21為反式-N-阿魏酰酪胺。

        化合物22白色粉末;HR-ESI-MS:m/z625.254 0 [M+H]+(calcd for C36H37N2O8,625.254 4),分子式為C36H36N2O8;1H NMR(600 MHz,DMSO-d6)δ:7.13(1H,s,H-2),6.89(1H,s,H-6),7.35(1H,d,J=15.6 Hz,H-7),6.46(1H,d,J=15.6 Hz,H-8),7.02(2H,m,H-2′,6′),6.68(2H,d,J=3.9 Hz,H-3′,5′),2.66(2H,d,J=8.1 Hz,H-7′),3.33(2H,m,H-8′),6.99(2H,d,J=8.4 Hz,H-2′′,6′′),6.67(2H,d,J=3.9 Hz,H-3′′,5′′),2.64(2H,d,J=7.5 Hz,H-7′′),3.32(2H,m,H-8′′),6.89(1H,s,H-2′′′),6.78(1H,d,J=8.1 Hz,H-5′′′),6.71(1H,dd,J=8.1,2.1 Hz,H-6′′′),5.88(1H,d,J=8.1 Hz,H-7′′′),4.21(1H,d,J=8.1 Hz,H-8′′′),3.83(3H,s,3-OCH3),3.75(3H,s,3′′′-OCH3),9.21(1H,s,4-OH),9.18(1H,s,4′′-OH),9.15(1H,s,4′′′-OH),8.06(1H,t,J=5.7 Hz,9′-NH),8.39(1H,t,J=5.7 Hz,9′′-NH);13C NMR(150 MHz,DMSO-d6)δ:128.6(C-1),111.7(C-2),144.1(C-3),148.7(C-4),128.5(C-5),115.9(C-6),138.7(C-7),119.7(C-8),129.6(C-1′),129.6(C-2′,6′),115.1(C-3′,5′),155.6(C-4′),34.4(C-7′),40.9(C-8′),165.2(C-9′),129.3(C-1′′),129.5(C-2′′,6′′),115.2(C-3′′,5′′),155.7(C-4′′),34.2(C-7′′),40.7(C-8′′),169.5(C-9′′),130.6(C-1′′′),110.4(C-2′′′),147.7(C-3′′′),146.9(C-4′′′),115.5(C-5′′′),118.8(C-6′′′),87.7(C-7′′′),55.9(C-8′′′),55.8(3-OCH3),55.7(3′′′-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[23]對(duì)照基本一致,故鑒定化合物22為grossamide。

        2.2 抗炎活性篩選結(jié)果

        本實(shí)驗(yàn)采用脂多糖(LPS)誘導(dǎo)的RAW264.7細(xì)胞炎癥篩選模型,以氨基胍為陽(yáng)性對(duì)照,對(duì)多穗金粟蘭中分離得率較高的13個(gè)化合物(5~17)進(jìn)行了抗炎活性檢測(cè)。結(jié)果顯示,當(dāng)化合物濃度為10 μmol/L時(shí)化合物5、8、9、10、11、13、14、15、16、17具有一定的NO生成抑制作用,表現(xiàn)出一定的抗炎活性,其中化合物5、10、11和13有較好的NO生成抑制作用,表現(xiàn)出中等的抗炎活性,結(jié)果見(jiàn)表1。

        表1 化合物5~17的抗炎活性大小Table 1 The anti-inflammatory activity of compounds 5-17

        3 結(jié)論

        本實(shí)驗(yàn)從多穗金粟蘭具有抗炎活性的二氯甲烷部位中分離得到22個(gè)化合物,包括9個(gè)倍半萜類(lèi)(5、8~11、14~17)、6個(gè)二萜(6、7、12、18~20)、2個(gè)酰胺(21、22),3個(gè)甾醇(2~4)、1個(gè)查爾酮(13)、1個(gè)木脂素(1),其中化合物2、4、21、22為首次從金粟蘭屬中分離得到,化合物1~10、18~22為首次從多穗金粟蘭中分離得到。并采用脂多糖(LPS)誘導(dǎo)的小鼠巨噬細(xì)胞RAW264.7釋放一氧化氮(NO)免疫炎癥細(xì)胞模型,對(duì)多穗金粟蘭中得率較高的13個(gè)化合物(5~17)進(jìn)行了抗炎活性測(cè)定,結(jié)果表明:9個(gè)倍半萜類(lèi)化合物和查爾酮(化合物13)都有一定的抗炎活性,其中化合物5、10、11、13表現(xiàn)出較好的抗炎活性。通過(guò)比較倍半萜化合物(5、8、11、14、15)的抗炎活性大小,可以發(fā)現(xiàn)倍半萜內(nèi)酯化合物(5、11)的抗炎活性明顯優(yōu)于其他倍半萜化合物(8、14、15),并且所分離的化合物中倍半萜內(nèi)酯的得率也是最高的,據(jù)此初步推測(cè)倍半萜內(nèi)酯類(lèi)化合物可能是多穗金粟蘭抗炎的主要活性成分。且此前也有大量文獻(xiàn)表明倍半萜內(nèi)酯類(lèi)化合物具有較好的抗腫瘤、強(qiáng)心等藥理活性[24],因此,應(yīng)更加注重多穗金粟蘭中倍半萜內(nèi)酯類(lèi)化學(xué)成分及其藥理活性的研究。本文進(jìn)一步豐富了多穗金粟蘭植物的研究?jī)?nèi)容,同時(shí)也為抗炎活性較好的先導(dǎo)化合物的發(fā)現(xiàn)、創(chuàng)新藥物的研發(fā),以及該藥用植物資源的開(kāi)發(fā)和利用提供了一定的參考依據(jù)。

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