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蒲黄化学成分研究

2015-02-12李锋涛陶伟伟杨念云李勇军段金廒

天然产物研究与开发 2015年9期
关键词:香蒲分子式柱层析

陈 毓 ,李锋涛,,陶伟伟,杨念云,李勇军,陈 巍,段金廒*

1江苏农牧科技职业学院,泰州 225300;2 南京中医药大学 江苏省中药资源产业化过程协同创新中心中药资源产业化与方剂创新药物国家地方联合工程研究中心,南京 210023

蒲黄为香蒲科植物水烛香蒲Typha angustifolia L.、东方香蒲Typha orientalis Presl 或同属植物的干燥花粉。具有止血、化瘀、通淋的功效。蒲黄的化学成分研究始于20 世纪20年代,至今从香蒲属植物中分离得到的化学成分有黄酮、烷烃、甾类、有机酸、多糖、挥发油等。研究发现,蒲黄的乙酸乙酯和正丁醇部位可明显缩短小鼠凝血时间[1];乙酸乙酯部位对家兔血小板聚集有明显的抑制作用,对小鼠和大鼠体内血栓形成也有明显抑制作用,并对血管内皮有明显的保护作用[2]。在药效学研究的基础上,我们开展了蒲黄化学成分的研究,对其乙酸乙酯和正丁醇部位进行了系统分离,以期为阐明其功效物质基础奠定基础。

1 仪器与材料

JASCO P-1020 旋光仪(日本Jasco 公司);IR100型红外光谱仪(美国Thermo 公司);Bruker Avance AV500/300 型核磁共振仪(德国Bruker 公司);SynaptTMQ-TOF 型质谱仪(美国Waters 公司);薄层层析和柱层析用硅胶(青岛海洋化工厂产品);其余试剂均为分析纯。

蒲黄购于南京药业股份有限公司中药饮片厂(产地:江苏,批号:20080923),经南京中医药大学段金廒教授鉴定为香蒲科植物水烛香蒲Typha angustifolia L.的花粉。

2 提取与分离

取乙酸乙酯部位浸膏,经硅胶柱层析分离,依次用石油醚-乙酸乙酯(2∶1→1∶2→0∶1)和乙酸乙酯-甲醇(10∶1)梯度洗脱,得到4 个组分(Fr.1~ Fr.4)。Fr.1 有晶体析出,重结晶后得到化合物1(500 mg)。Fr.1 经硅胶柱层析,石油醚-乙酸乙酯梯度洗脱,得到化合物25(18 mg)、10(35 mg)以及6 个组分(Fr.1-1~Fr.1-6)。Fr.1-1 和Fr.1-4 经硅胶柱层析分别得到化合物23(12 mg)、17(110 mg)和21(30 mg)、22(26 mg);Fr.1-2 和Fr.1-3 分别乙酸乙酯重结晶,得到化合物18(20 mg)和20(23 mg)。Fr.2 硅胶柱层析,石油醚-乙酸乙酯梯度洗脱,得到化合物24(14 mg)、19(10 mg)以及5 个组分(Fr.2-1~Fr.2-5)。Fr.2-1 经硅胶柱层析和葡聚糖凝胶柱层析,得到化合物9(8mg);Fr.2-3 和Fr.2-5 分别经乙酸乙酯重结晶得到化合物26(35mg)和11(16 mg);Fr.2-4 经硅胶柱层析,乙酸乙酯重结晶得到化合物12(9 mg)。Fr.3 经硅胶柱层析,石油醚-乙酸乙酯-甲醇梯度洗脱,得到化合物16(25mg)。

取正丁醇部位浸膏,经硅胶柱层析分离,依次用乙酸乙酯-甲醇(10∶1→5∶2→2∶1)梯度洗脱,得到3个组分(Fr.5~Fr.7)。Fr.6 经硅胶柱层析分离,乙酸乙酯-甲醇梯度洗脱,得到化合物13(15 mg)以及3 个组分(Fr.6-1~Fr.6-3)。Fr.6-1 经硅胶柱层析以及聚酰胺柱层析,得到化合物5(70 mg)及1 个组分,该组分进一步聚酰胺柱层析,得到化合物6(90 mg)、2(16mg)以及2 个组分,此2 个组分经制备HPLC 分离,分别得到化合物4(12 mg)、8(16 mg)和3(12mg)、7(8 mg)。Fr.6-2 经硅胶柱层析得到化合物14(35 mg)和15(30mg)。

3 结构鉴定

化合物1 白色粉末(EtOAc);ESI-MS m/z:271[M-H]-,分子式C15H12O5;UV λmax288;1H NMR(DMSO-d6,300 MHz)δ:12.16(1H,s,-OH),10.77(1H,s,-OH),9.58(1H,s,-OH),7.32(2H,d,J=8.43 Hz,H-2',6'),6.81(2H,d,J=8.40 Hz,H-3',5'),5.90(1H,s,H-6,8),5.41(1H,dd,J=12.8,2.73 Hz,H-2),3.25(1H,dd,J=12.81,17.01 Hz,H-3a),2.68(1H,dd,J=2.91,17.01 Hz,H-3b);13C NMR(DMSO-d6,75 MHz)δ:78.5(C-2),42.1(C-3),196.5(C-4),163.8(C-5),95.9(C-6),167.0(C-7),95.2(C-8),163.2(C-9),101.9(C-10),129.3(C-1'),128.4 (C-2',C-6'),115.3 (C-3',C-5'),158.2(C-4')。以上数据与文献[3,4]报道一致,鉴定为柚皮素。

化合物2 黄色粉末(EtOAc);ESI-MS m/z:623[M-H]-,分子式C28H32O16;UV λmax254,354;1H NMR (CD3OD,300 MHz)δ:7.93(1H,d,J=1.83 Hz,H-2'),7.60(1H,dd,J=1.83,8.43 Hz,H-6'),6.89(1H,d,J=8.43 Hz,H-5'),6.38(1H,s,H-8),6.19(1H,s,H-6),5.22(1H,d,J=7.50 Hz,H-1''),4.52(3H,s,1'''),3.93(3H,s,-OCH3),1.09(3H,d,J=6.21 Hz,H-6''');13C NMR(CD3OD,75 MHz)δ:159.3(C-2),135.9(C-3),179.7(C-4),163.4(C-5),100.5(C-6),166.7(C-7),95.4(C-8),158.9(C-9),106.1(C-10),123.4(C-1'),115.1(C-2'),151.3(C-3'),148.8(C-4'),116.6(C-5'),124.5 (C-6'),102.9(C-1''),76.4(C-2''),78.6(C-3''),72.8(C-4''),77.8(C-5''),69.0(C-6''),104.9(C-1'''),72.1(C-2'''),72.5(C-3'''),74.3(C-4'''),70.2(C-5'''),18.3(C-6'''),57.2(-OCH3)。以上数据与文献[5]报道一致,鉴定为异鼠李素-3-O-芸香糖苷(水仙苷)。

化合物3 黄色粉末(EtOAc);ESI-MS m/z:609[M-H]-,分子式C27H30O16;UV λmax255,355;1H NMR (CD3OD,300 MHz)δ:7.61(1H,d,J=2.19 Hz,H-2'),7.59(1H,d,J=7.50 Hz,H-6'),6.86(1H,d,J=8.61 Hz,H-5'),6.35(1H,S,H-8),6.16(1H,d,J=2.01 Hz,H-6),5.73(1H,d,J=7.68 Hz,H-1''),5.21(H,s,1'''),0.96(3H,d,J=6.21 Hz,6''');13C NMR(CD3OD,75 MHz)δ:158.9(C-2),135.0(C-3),179.8(C-4),100.2(C-6),166.2(C-7),95.0(C-8),158.9(C-9),106.4(C-10),123.7(C-1'),116.4(C-2'),150.1(C-3'),146.5(C-4'),117.7(C-5'),123.9(C-6'),100.8(C-1''),80.6(C-2''),78.7(C-3''),72.8(C-4''),79.4(C-5''),63.0(C-6''),103.1 (C-1'''),72.2 (C-2'''),72.8 (C-3'''),74.5(C-4'''),70.4(C-5'''),17.9(C-6''')。以上数据与文献[6]报道一致,鉴定为槲皮素-3-O-新橙皮糖苷。

化合物4 黄色粉末(EtOAc);ESI-MS m/z:755[M-H]-,分子式C33H40O20;UV λmax255,353;1H NMR (CD3OD,300 MHz)δ:7.60(1H,d,H-2'),7.58(1H,d,H-6'),6.86(1H,d,J=9.15 Hz,H-5'),6.35(1H,d,J=2.01 Hz,H-8),6.16(1H,d,J=2.01 Hz,H-6),5.58(1H,d,J=7.50 Hz,H-1''),5.22(1H,s,1'''),4.50(1H,S,1''''),1.07(3H,d,J=6.03 Hz,6''''),1.00(3H,d,J=6.21 Hz,6''');13C NMR(CD3OD,75 MHz)δ:159.4(C-2),134.9(C-3),179.7(C-4),163.5(C-5),100.2(C-6),166.0(C-7),95.1(C-8),158.9(C-9),106.4(C-10),123.9(C-1'),116.5(C-2'),149.9(C-3'),146.3(C-4'),117.9(C-5'),124.0(C-6'),100.9(C-1''),80.5(C-2''),79.3(C-3''),74.3(C-4''),77.5(C-5''),68.7(C-6''),102.7(C-1'''),72.8(C-2'''),72.5(C-3'''),74.3(C-4'''),70.2(C-5'''),18.2(C-6'''),103.1(C-1''''),72.7(C-2''''),72.3(C-3''''),74.3(C-4''''),70.4(C-5''''),18.0(C-6'''')。以上数据与文献[7]报道一致,鉴定为槲皮素-3-O-(2G-α-L-鼠李糖基)-芸香糖苷。

化合物5 黄色针晶(EtOAc);ESI-MS m/z:623[M-H]-,分子式C28H32O16;UV λmax254,354;1H NMR(DMSO-d6,500 MHz)δ:7.95(1H,d,J=2.20 Hz,H-2'),7.50(1H,dd,J=8.55,2.15 Hz,H-6'),6.91(1H,d,J=8.55 Hz,H-5'),6.43(1H,d,J=2.15 Hz,H-8),6.20(1H,d,J=2.15 Hz,H-6),5.76(1H,d,J=7.65 Hz,H-1''),5.03(H,S,1'''),0.66(3H,d,J=6.10 Hz,6''')。以上数据与文献[8]报道一致,经与异鼠李素-3-O-新橙皮糖对照品薄层比较,Rf值及显色行为一致。与异鼠李素-3-O-新橙皮糖对照品HPLC 图谱比较,保留时间及吸收波长均一致。鉴定为异鼠李素-3-O-新橙皮糖苷。

(1)合规性风险。建议学校首先实施必要的遴选程序,并经学校党委常委会等集体决策程序执行“三重一大”决策程序明确酒店的经营管理单位;其次,该模式涉及学校房产对外出租出借行为,相关租金需进行资产评估协商确定,协议期限不得超过5年,并履行相应报批报备手续。

化合物6 黄色针晶(EtOAc);ESI-MS m/z:769[M-H]-,分子式C34H42O20;UV λmax254,354;1H NMR(CD3OD,500 MHz)δ:7.92(1H,d,J=1.85 Hz,H-2'),7.56(1H,dd,J=8.55,2.10 Hz,H-6'),6.90(1H,d,J=8.55 Hz,H-5'),6.38(1H,d,J=2.15 Hz,H-8),6.18(1H,d,J=2.15 Hz,H-6),5.71(1H,d,J=7.56 Hz,H-1''),5.19(H,s,1'''),4.53(H,S,1''''),1.05(3H,d,J=6.10 Hz,6''''),0.92(3H,d,J=6.10 Hz,6''')。以上数据与文献[9]报道一致,经与香蒲新苷对照品薄层比较,Rf值及显色行为一致。与香蒲新苷对照品HPLC 图谱比较,保留时间及吸收波长均一致。鉴定为香蒲新苷。

化合物7 黄色粉末(EtOAc);ESI-MS m/z:593[M-H]-,分子式C27H30O15;UV λmax265,348;1H NMR (DMSO-d6,500 MHz)δ:8.0(1H,d,J=8.85 Hz,H-2'),6.88(1H,d,J=8.85 Hz,H-3'),8.0(1H,d,J=8.85 Hz,,H-6'),6.88(H,d,J=8.85 Hz,H-5'),6.42(1H,d,J=2.15 Hz,H-8),6.20(1H,d,J=2.15 Hz,H-6),5.6 (1H,d,J=7.60 Hz,H-1''),5.07(H,s,1'''),0.77(3H,d,J=6.15 Hz,6''')。以上数据与文献[10]报道一致,鉴定为山柰酚-3-O-新橙皮糖苷。

化合物8 黄色粉末(EtOAc);ESI-MS m/z:739[M-H]-,分子式C33H40O19;UV λmax265,348;1H NMR (CD3OD,500 MHz)δ:8.03(1H,d,J=8.85 Hz,H-2'),6.94 (1H,d,J=8.85 Hz,H-3'),8.03(1H,d,J=8.85 Hz,,H-6'),6.94(1H,d,J=8.80 Hz,H-5'),6.44(1H,d,J=2.15 Hz,H-8),6.29(1H,d,J=2.15 Hz,H-6),5.70(1H,d,J=7.65 Hz,H-1''),5.18(H,s,1'''),4.53(H,s,1''''),1.05(3H,d,J=6.10 Hz,6''''),0.91(3H,d,J=6.10 Hz,6''')。以上数据与文献[7]报道一致,鉴定为山柰酚-3-O-(2G-α-L-鼠李糖基)-芸香糖苷。

化合物9 无色针晶(Pet/ EtOAc);ESI-MS m/z:427[M-H]-,分子式C28H44O3;1H NMR (CDCl3,500 MHz)δ:6.50(1H,d,J=8.55 Hz,H-7),6.24(1H,d,J=8.55 Hz,H-6),5.23(1H,dd,J=7.30,13.25 Hz,H-22),5.15(1H,dd,J=8.55,13.25 Hz,H-23),3.97(1H,m,H-23),0.88(3H,s,H-18),0.82(3H,s,H-19),1.00(3H,d,H-21),0.91(3H,d,H-28),0.83(3H,d,H-27),0.82(3H,d,H-26);13C NMR (CDCl3,125 MHz)δ:34.7(C-1),30.5(C-2),66.4(C-3),36.9(C-4),82.1(C-5),135.4(C-6),130.1(C-7),79.4(C-8),51.1(C-9),36.9(C-10),20.6(C-11),39.3(C-12),44.5(C-13),51.7(C-14),23.4(C-15),28.6(C-16),56.2(C-17),12.8(C-18),18.2(C-19),39.7(C-20),20.9(C-21),135.2(C-22),132.3(C-23),42.7(C-24),33.0(C-25),19.6(C-26),19.9(C-27),17.5(C-28)。以上数据与文献[11]报道一致,鉴定为5α,8α-epidioxyergosta-6,22-dien-3β-ol。

化合物10 无色针晶(EtOAc);ESI-MS m/z:427[M-H]-,分子式C29H48O2;1H NMR (CDCl3,300 MHz)δ:0.96(3H,s,H-19),0.93(3H,d,J=6.39 Hz,H-21),0.85(3H,t,J=7.32 Hz,H-29),0.84(3H,d,J=6.42 Hz,H-26),0.82(3H,d,J=6.79 Hz,H-27),0.70(3H,s,H-18);13C NMR (CDCl3,75 MHz)δ:38.1(C-1),39.4(C-2),209.0(C-3),37.0(C-4),57.5(C-5),211.1(C-6),46.6(C-7),37.4(C-8),53.5(C-9),41.2(C-10),21.7(C-11),38.0(C-12),43.0(C-13),56.0(C-14),24.0(C-15),28.0(C-16),56.6(C-17),12.5(C-18),12.0(C-19),36.0(C-20),18.7(C-21),33.8(C-22),26.1(C-23),45.8(C-24),29.2(C-25),19.8(C-26),19.0(C-27),23.0(C-28),12.0(C-29)。以上数据与文献[12]报道一致,鉴定为stigmastan-3,6-dione。

化合物11 白色粉末(EtOAc);ESI-MS m/z:837[M+Na]+,分子式C51H90O7;1H NMR (CDCl3,500 MHz)δ:5.37(1H,m,H-6),4.37(1H,d,J=7.95 Hz,H-1'),2.36(3H,t,J=7.30 Hz,H-2''),1.00(3H,s,H-19),0.68(3H,s,H-18);13C NMR(CDCl3,125 MHz)δ:37.3(C-1),29.7(C-2),79.5(C-3),38.9(C-4),140.3(C-5),122.8(C-6),31.9(C-7),31.9(C-8),50.2(C-9),36.7(C-10),21.0(C-11),39.8(C-12),42.3(C-13),56.8(C-14),24.3(C-15),28.2(C-16),56.1(C-17),12.0(C-18),19.0(C-19),36.2(C-20),18.9(C-21),34.0(C-22),26.2(C-23),45.9(C-24),29.3(C-25),19.3(C-26),19.8(C-27),23.1(C-28),11.8(C-29),101.2(C-1'),73.6(C-2'),76.0(C-3'),70.1(C-4'),74.0(C-5'),63.2(C-6'),174.7(C-1'')。参照文献[13]和质谱数据,鉴定为胡萝卜苷-6'-棕榈酸酯。

化合物12 白色蜡状(EtOAc);ESI-MS m/z:893[M+Na]+,分子式C55H98O7;1H NMR (CDCl3,500 MHz)δ:5.36(1H,m,H-6),4.37(1H,d,J=7.95 Hz,H-1'),2.35(3H,t,J=7.35 Hz,H-2''),1.00(3H,s,H-19),0.68(3H,s,H-18);13C NMR(CDCl3,125 MHz)δ:37.3(C-1),29.7(C-2),79.5(C-3),38.9(C-4),140.3(C-5),122.1(C-6),31.9(C-7),31.9(C-8),50.2(C-9),36.7(C-10),21.1(C-11),39.8(C-12),42.3(C-13),56.8(C-14),24.3(C-15),28.2(C-16),56.1(C-17),12.0(C-18),19.0(C-19),36.1(C-20),18.8(C-21),34.0(C-22),26.2(C-23),45.9(C-24),29.3(C-25),19.3(C-26),19.8(C-27),23.1(C-28),11.8(C-29),101.2(C-1'),73.6(C-2'),76.0(C-3'),70.1(C-4'),74.0(C-5'),63.2(C-6'),174.7(C-1'')。参照文献[14]和质谱数据,鉴定为胡萝卜苷-6'-二十烷酸酯。

化合物13 无色颗粒(EtOAc);ESI-MS m/z:159[M+H]+,分子式C4H6N4O3;1H NMR (DMSOd6,500 MHz)δ:10.51(1H,s,H-1),8.03(H,s,H-3),6.88(H,s,H-6),5.76(1H,s,-NH2),5.24(s,H-5);13C NMR (DMSO-d6,125 MHz)δ:173.7(C-5),157.5(C-7),156.9(C-2),62.5(C-4)。以上数据与文献[15]报道一致,鉴定为尿囊素。

化合物14 白色粉末(EtOH);ESI-MS m/z:136[M+H]+,分子式C5H5N5;1H NMR (DMSO-d6,300 MHz)δ:12.78(1H,s,NH),8.12(1H,s,H-2),8.10(1H,s,H-8),7.10(2H,s,NH2);13C NMR (DMSOd6,75 MHz)δ:152.6(C-2),151.5(C-4),155.5(C-6),139.5(C-8)。参照文献[16]和质谱数据,鉴定为6-氨基嘌呤。

化合物15 白色粉末(EtOH);ESI-MS m/z:137[M+H]+,分子式C5H4N4O;1H NMR (DMSO-d6,300 MHz)δ:12.49(1H,s,NH),8.17(1H,s,H-8),7.97(1H,s,H-2);13C NMR (DMSO-d6,75 MHz)δ:144.8(C-2),153.4(C-4),119.4(C-5),155.6(C-6),140.4(C-8)。参照文献[16]和质谱数据,鉴定为次黄嘌呤。

化合物16 黄色粉末(EtOAc);ESI-MS m/z:113[M +H]+,分子式C4H4N2O2;1H NMR (DMSO-d6,300 MHz)δ:10.99(1H,s,NH),10.80(1H,s,NH),7.38(1H,d,J=7.68 Hz,H-6),5.45(1H,d,J=7.68 Hz,H-5)。以上数据与文献[17,18]报道一致,鉴定为尿嘧啶。

化合物17 白色蜡状(Pet);ESI-MS m/z:307[M +Na]+,分子式C18H36O2;1H NMR (DMSO-d6,500 MHz)δ:11.65(1H,s,COOH),2.18(2H,t,J=7.35 Hz,H-2),1.26~1.51(30H,overlapped),0.87(3H,t,J=6.70 Hz,H-18)。参照文献[19]和质谱数据,鉴定为硬脂酸。

化合物18 白色蜡状(Pet);ESI-MS m/z:223[M +Na]+,分子式C12H24O2;1H NMR (DMSO-d6,300 MHz)δ:11.92(1H,s,COOH),2.19(2H,t,J=6.95 Hz,H-2),1.26-1.49(18H,overlapped),0.86(3H,t,H-12)。鉴定为十二烷酸。

化合物19 无色针晶(EtOAc);ESI-MS m/z:167[M-H]-,分子式C8H8O4;1H NMR (CDCl3,500 MHz)δ:7.72(1H,dd,J=8.25,1.80 Hz,H-6),7.59(1H,d,J=1.80 Hz,H-2),6.98(1H,d,J=8.25 Hz,H-5),3.97 (3H,s,-OCH3)。以上数据与文献[20]报道一致,鉴定为香草酸。

化合物20 白色粉末(EtOAc);ESI-MS m/z:439[M-H]-,475[M+Cl]-,分子式C29H60O2;1H NMR (CD3OD,500 MHz)δ:3.80(2H,m,H-6,8),1.25~1.55(50H,m),0.89(6H,J=7.05 Hz,H-1,29);ESI-MS/MS m/z:421 [M-H2O]-,403 [M-2H2O]-,367[C24H47O2]-,323[C22H43O]-。以上数据与文献[21]报道一致,鉴定为二十九烷二醇-6,8。

化合物21 白色粉末(EtOAc);ESI-MS m/z:439[M-H]-,475[M+Cl]-,分子式C29H60O2;1H NMR (CD3OD,500 MHz)δ:3.51(2H,m,H-6,10),1.25-1.59(50H,m),0.89(6H,J=9.75 Hz,H-1,29);ESI-MS/MS m/z:421 [M-H2O]-,403 [M-2H2O]-,367[C24H47O2]-,295[C20H39O]-。以上数据与文献[21]报道一致,鉴定为二十九烷二醇-6,10。

化合物22 白色粉末(EtOAc);ESI-MS m/z:439[M-H]-,475[M+Cl]-,分子式C29H60O2;1H NMR (CD3OD,500 MHz)δ:3.52(2H,m,H-6,21),1.25~1.55(50H,m),0.89(6H,J=7.0 Hz,H-1,29);ESI-MS/MS m/z:421 [M-H2O]-,403 [M-2H2O]-,367[C24H47O2]-,295[C21H41O2]-。以上数据与文献[21]报道一致,鉴定为二十九烷二醇-6,21。

化合物23 白色粉末(EtOAc);ESI-MS m/z:381[M-H]-,分子式C26H54O;1H NMR (CD3OD,500 MHz)δ:3.52(2H,t,7.0 Hz,H-1),1.25-1.55(48H,m),0.89(3H,J=7.1 Hz,H-25)。以上数据与文献[22]报道一致,鉴定为二十六烷醇-1。

化合物24 白色粉末(EtOAc);ESI-MS m/z:241[M-H]-,分子式C16H34O;1H NMR (CD3OD,500 MHz)δ:3.58(2H,t,6.6 Hz,H-1),1.28-1.50(26H,m),0.89(3H,J=6.5 Hz,H-16)。以上数据与文献[23]报道一致,鉴定为十六烷醇-1。

化合物25 白色粉末(EtOAc);ESI-MS m/z:353[M+H]+,分子式C25H52;1H NMR (CD3OD,300 MHz) δ:0.88 (6H,t,2CH3),1.28 (46H,23CH2)。以上数据与文献[24]报道一致,鉴定为二十五烷。

化合物26 白色粉末(EtOAc);ESI-MS m/z:353[M+Na]+,分子式C19H38O4;1H NMR (CD3OD,500 MHz)δ:4.14(1H,dd,J=4.60,11.3 Hz,H-1a'),4.04(1H,dd,J=6.10,11.3 Hz,H-1b'),3.80(H,m,H-2'),3.54(2H,m,H-3'),2.33(2H,t,J=7.35,H-2),0.89(3H,t,J=6.70,H-19)。以上数据与文献[25,26]报道一致,鉴定为单棕榈酸甘油酯。

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