李曉波,張高菊,張廣輝,楊生超,姜薇薇,c*

(云南農(nóng)業(yè)大學,a.云南省藥用植物生物學重點實驗室; b.西南中藥材種質(zhì)創(chuàng)新與利用國家地方聯(lián)合工程研究中心; c.理學院,昆明 650201)

姜狀三七(Panax zingiberensis)為五加科(Araliaceae)人參屬植物，根莖入藥，主要用于治療跌打損傷、勞虛咳嗽、外傷出血及貧血等癥,也作三七代用品。國外主要分布于尼泊爾中部、不丹高海拔地區(qū)、緬甸東枝、撣邦等地[1–4]，國內(nèi)分布于云南東南部的馬關(guān)、硯山等地。野生資源分布狹窄及過度采挖，導致該物種于1997年被國際自然保護聯(lián)盟列為瀕危種。由于材料的難以采集導致關(guān)于該植物的研究較少。目前只從該植物中分離鑒定了三萜皂苷類化合物不到10 個，有人參二醇型、人參三醇型和齊墩果烷型，且其生物活性的研究也鮮見報道[5–7]。本課題組在前期研究[8]的基礎上對其化學成分進行了系統(tǒng)研究，從中分離鑒定了9 個皂苷類化合物，為尋找活性物質(zhì)提供了化學基礎，也為姜狀三七植物資源的合理開發(fā)和利用奠定理論基礎。

1 材料和方法

1.1 材料

材料購自云南省普洱市鎮(zhèn)沅縣九甲鎮(zhèn)三臺村林下經(jīng)濟種植示范基地，經(jīng)云南農(nóng)業(yè)大學張廣輝教授鑒定為五加科人參屬植物姜狀三七(Panax zingiberensis)，標本存放于云南農(nóng)業(yè)大學西南中藥材種質(zhì)創(chuàng)新與利用國家地方聯(lián)合工程研究中心。

1.2 儀器和試劑

API Qstar Pulsa LC/TOF 質(zhì)譜儀，Bruker AM-400、DRX-500 和AVANCE III-600 MHz 超導核磁共振儀(瑞士Bruker 公司)，TMS 為內(nèi)標，化學位移δ用ppm 表示，耦合常數(shù)J用Hz 表示；中藥高速粉碎機(浙江永歷制藥機械有限公司)；旋轉(zhuǎn)蒸發(fā)儀(東京理化儀器有限公司)；超聲波清洗機(上海聲源超聲波儀器有限責任公司)；柱層析硅膠和硅膠G薄層板(青島海洋化工廠)；AB-8 (大孔樹脂天津波鴻樹脂科技有限公司)；反相硅膠板和柱層析用RP-18 (德國Merk 公司)；Sephadex LH-20 (Amer- sham Pharmacia Biotech 公司)；顯色劑(5%硫酸乙醇溶液),甲醇、氯仿、丙酮和乙酸乙酯為工業(yè)級重蒸后使用,正丁醇、冰乙酸為分析純，乙腈為色譜純,水為哇哈哈純凈水。

1.3 提取和分離

姜狀三七根莖粗粉2.8 kg，加8 倍量70%甲醇溶液常溫浸泡3 d，超聲提取3 次，提取時間分別為2、1.5和1.5 h，合并提取液回收甲醇至無醇味,藥液分次過已預處理好的AB-8 大孔樹脂柱(2 kg)，以蒸餾水洗脫至Molish 反應為陰性，再以80%甲醇(共8 L)洗脫，收集甲醇洗脫液，回收甲醇，濃縮干燥得姜狀三七總皂苷(440 g)。

將總皂苷吸附于1.0 kg 硅膠上進行柱層析，流動相以氯仿-甲醇-水(10∶1∶0～0∶1∶1)梯度洗脫,收集洗脫液，每份0.5 L，共收集292 份，薄層鑒別合并洗脫液得Fr1～Fr8 組分。組分Fr 4 (150 g)上硅膠柱色譜，用氯仿-甲醇-水溶劑系統(tǒng)進行梯度洗脫，得到3 個餾分Fr 4.1～Fr 4.3。Fr 4.1 (20 g)經(jīng)硅膠柱色譜，以氯仿-甲醇-水(9∶1∶0.2～7∶3∶0.5)梯度洗脫，再以Sephadex LH-20 (甲醇)純化，得到化合物1 (2 mg)、2 (8 mg)和3 (246 mg)。Fr 4.2 (50 g)經(jīng)反復硅膠柱層析(氯仿-乙酸乙酯-甲醇-水；氯仿-甲醇-水)梯度洗脫，得到化合物4 (6 mg)、5 (10 mg)、6 (10 mg)和7 (2 g)。Fr 4.3 (30 g)過Rp-18 柱色譜,以水-甲醇(9∶1～0∶1)梯度洗脫，再以Sephadex LH- 20 (甲醇)純化，得到化合物8 (6 mg)和9 (10 mg) (圖1)。

圖1 化合物1～9 的結(jié)構(gòu)Fig.1 Structures of compounds 1-9

1.4 結(jié)構(gòu)鑒定

化合物1白色結(jié)晶，ESI-MSm/z: 693 [M +Na]+;1H NMR (400 MHz,CD3OD):δ4.34 (1H,d,J=7.8 Hz,H′-6),1.76,1.32,1.12,1.09,1.00,0.99,0.94(each 3H,s,CH3×7);13C NMR (125 MHz,CD3OD):δ40.5 (C-1),27.5 (C-2),79.1 (C-3),40.4 (C-4),61.8(C-5),79.9 (C-6),45.3 (C-7),41.8 (C-8),50.9 (C-9),40.2 (C-10),32.0 (C-11),71.7 (C-12),49.5 (C-13),52.5 (C-14),31.9 (C-15),27.4 (C-16),52.5 (C-17),17.9 (C-18),17.5 (C-19),74.2 (C-20),22.6 (C-21),40.4 (C-22),26.4 (C-23),91.2 (C-24),146.2 (C-25),113.9 (C-26),17.8 (C-27),31.4 (C-28),16.8 (C-29),17.3 (C-30),105.5 (C-1′),75.5 (C-2′),80.9 (C-3′),72.0 (C-4′),77.7 (C-5′),62.9 (C-6′)。以上數(shù)據(jù)與文獻[9]報道基本一致，故鑒定為人參皂苷SL1。

化合物2白色粉末,ESI-MSm/z: 661 [M +Na]+;1H NMR (400 MHz,CD3OD):δ4.38 (1H,d,J=7.8 Hz,H-1′),1.70,1.64,1.34,1.17,1.11,1.02,1.01,0.96 (each 3H,s,CH3×8);13C NMR (100 MHz,CD3OD):δ40.2 (C-1),27.6 (C-2),79.9 (C-3),40.5(C-4),61.8 (C-5),77.6 (C-6),45.3 (C-7),41.8 (C-8),50.9 (C-9),40.4 (C-10),31.9 (C-11),71.7 (C-12),48.5(C-13),52.5 (C-14),31.9 (C-15),27.4 (C-16),55.1(C-17),17.8 (C-18),17.9 (C-19),74.5 (C-20),26.6(C-21),36.3 (C-22),23.3 (C-23),126.2 (C-24),132.1(C-25),26.0 (C-26),17.6 (C-27),31.4 (C-28),16.2(C-29),17.1 (C-30),105.5 (C-1′),75.5 (C-2′),81.8 (C-3′),71.7 (C-4′),79.0 (C-5′),62.9 (C-6′)。以上數(shù)據(jù)與文獻[10]報道基本一致，故鑒定為人參皂苷Rh1。

化合物3白色粉末,ESI-MSm/z: 653 [M -H]–;1H NMR (400 MHz,C5D5N):δ5.07 (1H,d,J=7.8 Hz,H-1′),2.06,1.63,1.56,1.55,1.39,1.26,1.06,0.80 (each 3H,s,CH3×8);13C NMR (100 MHz,C5D5N):δ39.7 (C-1),28.0 (C-2),78.6 (C-3),40.1(C-4),61.5 (C-5),80.1 (C-6),45.2 (C-7),41.1 (C-8),50.2 (C-9),39.4 (C-10),32.2 (C-11),71.1 (C-12),48.5(C-13),51.7 (C-14),31.2 (C-15),26.8 (C-16),54.2(C-17),17.4 (C-18),17.8 (C-19),73.3 (C-20),27.7(C-21),137.6 (C-22),127.4 (C-23),40.4 (C-24),81.3(C-25),25.2 (C-26),25.3 (C-27),31.8 (C-28),16.4(C-29),16.8 (C-30),106.0 (C-1′),75.5 (C-2′),79.7(C-3′),71.8 (C-4′),78.3 (C-5′),63.1 (C-6′)。以上數(shù)據(jù)與文獻[11]報道基本一致，故鑒定為三七皂苷R8。

化合物4白色粉末,ESI-MSm/z: 793 [M -H]–;1H NMR (400 MHz,CD3OD):δ6.53 (1H,d,J=7.6 Hz,H-1′),4.82 (1H,d,J= 7.2 Hz,H-1″),1.15,1.04,0.94,0.93,0.90,0.83,0.796,0.80 (each 3H,s,CH3×7);13C NMR (100 MHz,CD3OD):δ40.2 (C-1),26.4 (C-2),90.8 (C-3),40.0 (C-4),57.0 (C-5),19.5(C-6),33.5 (C-7),40.8 (C-8),49.0 (C-9),37.8 (C-10),24.7 (C-11),123.8 (C-12),144.8 (C-13),43.0 (C-14),28.7 (C-15),24.1 (C-16),48.0 (C-17),42.9 (C-18),47.4 (C-19),31.6 (C-20),35.0 (C-21),34.1 (C-22),28.6 (C-23),17.0 (C-24),16.1 (C-25),17.8 (C-26),26.4 (C-27),178.0 (C-28),33.5 (C-29),24.0 (C-30),106.7 (3-Glc,C-1′),75.5 (C-2′),78.0 (C-3′),73.7 (C-4′),75.5 (C-5′),176.6 (C-6′),95.7 (28-Glc,C-1″),73.9(C-2″),78.6 (C-3″),71.1 (C-4″),78.7 (C-5″),62.5(C-6″)。以上數(shù)據(jù)與文獻[12]報道基本一致，故鑒定為竹節(jié)參皂苷IVa。

化合物5白色粉末,ESI-MSm/z: 653 [M -H]–;1H NMR (400 MHz,C5D5N):δ5.03 (1H,d,J=7.8 Hz,H-1′),2.06,1.68,1.60,1.52,1.31,1.19,1.03,0.80 (each 3H,s,CH3×7);13C NMR (100 MHz,C5D5N):δ39.5 (C-1),28.1 (C-2),78.7 (C-3),40.5(C-4),61.6 (C-5),78.3 (C-6),45.1 (C-7),41.2 (C-8),50.2 (C-9),39.7 (C-10),32.2 (C-11),70.7 (C-12),48.9(C-13),52.4 (C-14),31.8 (C-15),28.0 (C-16),52.1(C-17),17.6 (C-18),17.9 (C-19),78.1 (C-20),26.4(C-21),27.8 (C-22),25.9 (C-23),74.5 (C-24),78.7(C-25),23.3 (C-26),30.1 (C-27),31.8 (C-28),16.4(C-29),17.3 (C-30),106.1 (3-Glc,C-1′),75.7 (C-2′),80.3 (C-3′),71.9 (C-4′),79.7 (C-5′),63.1 (C-6′)。以上數(shù)據(jù)與文獻[13]報道基本一致，故鑒定為越南人參皂苷R10。

化合物6白色粉末,ESI-MSm/z: 799 [M -H]–;1H NMR (500 MHz,CD3OD):δ5.15 (1H,d,J=7.8 Hz,H-1″),4.98 (1H,d,J= 7.8 Hz,H-1′),2.03,1.58,1.56,1.56,1.55,1.11,0.98,0.75 (each 3H,s,CH3×8);13C NMR (125 MHz,CD3OD):δ39.5 (C-1),28.0(C-2),78.7 (C-3),40.5 (C-4),61.5 (C-5),78.3 (C-6),45.2 (C-7),41.2 (C-8),50.1 (C-9),39.7 (C-10),31.0(C-11),70.4 (C-12),49.2 (C-13),51.5 (C-14),30.8(C-15),26.7 (C-16),51.7 (C-17),17.7 (C-18),17.7(C-19),83.4 (C-20),22.5 (C-21),36.1 (C-22),23.4(C-23),126.0 (C-24),131.1 (C-25),17.9 (C-26),25.9(C-27),31.9 (C-28),17.2 (C-29),16.2 (C-30),106.1(3-Glc,C-1′),75.5 (C-2′),80.3 (C-3′),71.8 (C-4′),80.0 (C-5′),63.1 (C-6′),98.3 (20-Glc,C-1″),75.2(C-2″),79.3 (C-3″),71.6 (C-4″),78.4 (C-5″),62.8 (C-6″)。以上數(shù)據(jù)與文獻[14]報道基本一致，鑒定為人參皂苷Rg1。

化合物7白色粉末,ESI-MSm/z: 955 [M -H]–;1H NMR (400 MHz,CD3OD):δ5.37 (1H,d,J=8.0 Hz,H-12),1.15,1.04,0.94,0.93,0.90,0.83,0.79(each 3H,s,CH3×7);13C NMR (100 MHz,CD3OD):δ39.8 (C-1),27.0 (C-2),90.8 (C-3),40.3 (C-4),57.0(C-5),19.0 (C-6),34.0 (C-7),40.7 (C-8),48.4 (C-9),37.5 (C-10),23.9 (C-11),123.8 (C-12),144.8 (C-13),41.9 (C-14),28.9 (C-15),23.5 (C-16),47.5 (C-17),42.2 (C-18),46.4 (C-19),32.0 (C-20),33.5 (C-21),33.5 (C-22),28.5 (C-23),17.7 (C-24),16.0 (C-25),17.7 (C-26),26.3 (C-27),178.1 (C-28),33.5 (C-29),24.0 (C-30),109.4 (3-Glc A,C-1′),73.9 (C-2′),86.4(C-3′),71.1 (C-4′),76.7 (C-5′),176.2 (C-6′),106.8(20-Glc,C-1″),75.4 (C-2″),76.6 (C-3″),76.5 (C-4″),78.7 (C-5″),63.2 (C-6″),95.7 (28-Glc,C-1?),73.9 (C-2?),82.9 (C-3?),71.1 (C-4?),78.7 (C-5?),62.4 (C-6?)。以上數(shù)據(jù)與文獻[12,15]報道基本一致,鑒定為菠棱皂苷A 28-O-葡萄糖苷。

化合物8白色粉末,ESI-MSm/z: 617 [M -H]–;1H NMR (400 MHz,C5D5N):δ6.32 (1H,d,J=6.5 Hz,H-1′),1.23,1.22,1.12,1.02,0.90,0.89,0.85(each 3H,s,CH3×7);13C NMR (100 MHz,C5D5N):δ39.1 (C-1),28.4 (C-2),78.3 (C-3),40.4 (C-4),55.9(C-5),18.9 (C-6),33.3 (C-7),39.5 (C-8),48.2 (C-9),39.7 (C-10),23.9 (C-11),123.0 (C-12),144.3 (C-13),41.9 (C-14),28.9 (C-15),23.5 (C-16),47.1 (C-17),42.2 (C-18),46.3 (C-19),30.9 (C-20),34.1 (C-21),32.6 (C-22),30.9 (C-23),15.7 (C-24),16.7 (C-25),17.6 (C-26),26.2 (C-27),176.7 (C-28),23.8 (C-29),33.2 (C-30),95.9 (28-Glc,C-1′),79.5 (C-2′),79.0 (C-3′),71.2 (C-4′),74.2 (C-5′),62.3 (C-6′)。以上數(shù)據(jù)與文獻[16]報道數(shù)據(jù)一致,故鑒定為齊墩果酸28-O-β-D-葡萄糖苷。

化合物9白色粉末,ESI-MSm/z: 793 [M -H]–；1H NMR (500 MHz,C5D5N):δ5.33 (1H,d,J=7.7 Hz,H-1″),4.91 (1H,d,J= 7.3 Hz,H-1′),1.24,1.22,1.03,0.95,0.92,0.91,0.66 (each 3H,s,CH3×7);13C NMR (125 MHz,C5D5N):δ38.6 (C-1),26.6 (C-2),89.3 (C-3),39.7 (C-4),55.8 (C-5),18.5 (C-6),33.4(C-7),39.6 (C-8),48.0 (C-9),36.9 (C-10),23.7 (C-11),122.6 (C-12),144.9 (C-13),42.0 (C-14),28.3(C-15),23.8 (C-16),46.7 (C-17),42.1 (C-18),46.5 (C-19),31.0 (C-20),34.2 (C-21),33.2 (C-22),28.2 (C-23),15.5 (C-24),16.8 (C-25),17.4 (C-26),26.2 (C-27),180.3 (C-28),23.8 (C-29),33.2 (C-30),106.0 (3-GluUA C-1′),82.8 (C-2′),78.5 (C-3′),73.7 (C-4′),77.6 (C-5′),174.9 (C-6′),105.3 (C-1″),77.9 (C-2″),78.5 (C-3″),71.6 (C-4″),77.2 (C-5″),62.6 (C-6″)。以上數(shù)據(jù)與文獻[5]報道一致，故鑒定為姜狀三七苷R1。

2 結(jié)果和討論

對姜狀三七根莖中的化學成分進行了研究,從70%甲醇提取物中分離鑒定了9 個皂苷類化合物,分別是人參皂苷SL1(1)、人參皂苷Rh1(2)、三七皂苷R8(3)、竹節(jié)參皂苷IVa (4)、越南人參皂苷R10(5)、人參皂苷Rg1(6)、菠棱皂苷A 28-O-葡萄糖苷 (7)、齊墩果酸28-O-β-D-葡萄糖苷 (8)和姜狀三七苷R1(9)?；衔?、3、5、7 和8 為首次從姜狀三七中分離得到?；衔?～3 和6 為原人參三醇型皂苷,化合物4、7～9 為齊墩果烷型皂苷，化合物5 為奧克梯隆醇型人參皂苷，此類型皂苷為首次從姜狀三七中得到。

人參皂苷Rg1(6)是中藥人參、三七等人參屬藥材的主要活性成分之一，可通過抗氧化作用維持細胞內(nèi)Ca2+的穩(wěn)態(tài)達到保護心肌細胞的作用[17]; 對多種動物中樞神經(jīng)系統(tǒng)疾病模型具有治療作用，主要是通過NMDA 受體、MAPK、MEK- ERK1/2-PI3K、Ca2+-CaM-CaMK II 等信號通路進行調(diào)控，作用于皮層、海馬、紋狀體、下丘腦,影響神經(jīng)遞質(zhì)的合成與釋放，促進神經(jīng)細胞生長、神經(jīng)突觸的發(fā)生，保護神經(jīng)元，促進神經(jīng)干細胞分化等[18]; 還可明顯抑制人白血病TF-1 細胞的增殖并促進其凋亡，輔助T細胞免疫活性增加，對小鼠免疫性肝損傷也有一定的保護作用[19]。此外,人參皂苷Rg1還有抗腫瘤,降血糖、降血脂，抗骨質(zhì)疏松和抗炎等作用[20]。

竹節(jié)參苷Ⅳa (4)也具有多種藥理活性，尤其是其卓越的心腦血管保護作用，降糖、降脂作用，抗炎、抗凝血作用以及特異性的腫瘤細胞增殖抑制作用等，相關(guān)作用機制及靶點主要包括激活SIRT1/ERK1/2/Homer1a 途徑，增強機體清除氧自由基的能力、降低心肌細胞膜脂質(zhì)過氧化的程度，上調(diào)PTEN、下調(diào)NF-κB 的表達，調(diào)節(jié)Ca2+、K+、Na+等離子的跨膜轉(zhuǎn)運，促進GSK-3β的磷酸化，抑制PKC的磷酸化，抑制Wnt/β-catenin 信號通路，抑制炎癥因子表達，抑制血小板聚集，誘導腫瘤細胞周期停滯及凋亡，導致病毒直接失活或抑制子代病毒釋放[21]。越南人參皂苷R10(5)為奧克梯隆醇型皂苷,是一類側(cè)鏈具有呋喃環(huán)的四環(huán)三萜類皂苷，主要存在于人參屬植物西洋參(P.quinquefolius)、竹節(jié)參(P.japonicus)、狹葉竹節(jié)參(P.bipinnatifidusvar.angustifolius)、珠子參(P.japonicusvar.major)、喜馬拉雅假人參(P.pseudo-ginsengsubsp.himalaicus)中，葫蘆科(Cucurbitaceae)和樺木屬(Betula)植物中也發(fā)現(xiàn)了多種奧克梯隆型皂苷。奧克梯隆型皂苷在這些植物中雖然含量甚微，但具有較強的生物活性，包括保護心肌損傷、增強神經(jīng)元活性、抗腫瘤等[22–23]。