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黃果茄果甾體成分及其體外抗炎活性

2023-10-30 06:12:00許振鵬郝智超王達(dá)圣匡海學(xué)楊炳友
中成藥 2023年10期
關(guān)鍵詞:黃果無定形分子式

劉 艷,孟 欣,許振鵬,潘 娟,管 偉,郝智超,王達(dá)圣,匡海學(xué),楊炳友

(黑龍江中醫(yī)藥大學(xué),教育部北藥基礎(chǔ)與應(yīng)用研究重點實驗室,黑龍江 哈爾濱 150040)

黃果茄為茄科茄屬植物,收錄于《中藥大辭典》 《中華本草》,傳統(tǒng)藥用部位為根、果實、種子,具有清熱利濕、消瘀止痛的功效。黃果茄作為阿育吠陀醫(yī)學(xué)中一種重要常用藥物,在印度、斯里蘭卡、巴基斯坦等國被廣泛用于咳嗽、哮喘,甚至是癌癥等疾病的治療[1-3],也可用于抗菌、抗感染、改善糖尿病癥狀等[4-5]。目前國內(nèi)外學(xué)者對其各方面藥理作用進(jìn)行了研究,然而對其化學(xué)成分的研究不足。課題組研究發(fā)現(xiàn),黃果茄果中化學(xué)成分復(fù)雜多樣,甾體類[6]、芳香類[7]、脂質(zhì)類[8]化合物是其主要成分,為進(jìn)一步開發(fā)利用黃果茄果,本研究對其正丁醇部分、乙酸乙酯部分、水部分分別進(jìn)行分離純化,共得到31 個化合物,并對其體外抗炎活性進(jìn)行檢測,為充分開發(fā)利用黃果茄果資源提供理論基礎(chǔ)。

1 材料

Waters e2695-2424-2998 型、515-2414 型高效液相色譜儀及XBridge C18色譜柱(4.6 mm×250 mm,5 μm; 19 mm×250 mm,10 μm)、SunFire C18色譜柱(4.6 mm× 250 mm,5 μm; 10 mm×250 mm,5 μm)、Atlantis? prep T3色譜柱(19 mm×250 mm,10 μm)(美國Waters 公司); LC-20AR/RID-20A 型半制備高效液相色譜儀(日本島津公司); Bruker-400/600 型超導(dǎo)核磁共振光譜儀(德國Bruker 公司); FDU-1200 型冷凍干燥機(jī)(日本EYELA 公司); 硅膠柱(青島海洋化工廠); ODS(日本YMC 公司); 反相MCI (日本三菱公司);薄層色譜硅膠60 F254板、Rp-18 硅膠板 (德國Merck 公司); AB-8 大孔樹脂(滄州寶恩吸附材料科技有限公司); 制備型薄層層析硅膠板(煙臺江友硅膠開發(fā)有限公司)。甲醇、乙腈為色譜純(德國Merck 公司); 氘代試劑(美國劍橋公司); 水為超純水[屈臣氏集團(tuán)(香港) 有限公司]。

RAW 264.7 細(xì)胞系 (武漢大學(xué)細(xì)胞保藏中心); 胎牛血清(以色列BI 公司); DMEM (美國Corning 公司); 脂多糖(美國Sigma 公司); 無水對氨基苯磺酸(天津市光復(fù)精細(xì)化工研究所);Nα-萘乙二胺(天津市傲然精細(xì)化工研究所)。吲哚美辛(天津天藥藥業(yè)股份有限公司)。

黃果茄果于2016 年3 月采收于廣西桂林,經(jīng)黑龍江中醫(yī)藥大學(xué)藥學(xué)院藥用植物學(xué)教研室樊銳鋒副教授鑒定為茄科茄屬植物黃果茄Solanum xanthocarpumSchrad.et Wendl.的果實,標(biāo)本(編號20160327) 保存于黑龍江中醫(yī)藥大學(xué)藥學(xué)院中藥化學(xué)實驗室。

2 提取與分離

取干燥粉碎的黃果茄果9.0 kg,6 倍量70%乙醇回流提取3 次,每次2 h,濾過,合并提取液,減壓回收溶劑,得提取物2 473.6 g,加水混懸均勻,依次用石油醚、乙酸乙酯、水飽和正丁醇萃取,減壓濃縮干燥,得石油醚部位膏狀物34.0 g、乙酸乙酯部位膏狀物150.1 g、正丁醇部位冷凍干燥粉末393.8 g、水部位冷凍干燥粉末1 605.0 g。

正丁醇部位冷凍干燥粉末(280.0 g) 經(jīng)硅膠柱分離,以二氯甲烷-甲醇(10 ∶1 ~0 ∶1) 梯度洗脫,經(jīng)TLC、HPLC 檢識合并,得到Fr.Ⅰ~Fr.Ⅶ。Fr.Ⅰ(7.7 g) 經(jīng)硅膠柱,以二氯甲烷-甲醇(1 ∶0~0 ∶1) 梯度洗脫,得到Fr.Ⅰ-1~Fr.Ⅰ-18。Fr.Ⅰ-8 經(jīng)TLC 檢識,以二氯甲烷-甲醇(20 ∶1) 為展開劑,得到化合物21 (2.2 mg)。Fr.Ⅱ(66.3 g)經(jīng)ODS 柱,以甲醇-水(1 ∶4 ~1 ∶0) 梯度洗脫,得到Fr.Ⅱ-1~Fr.Ⅱ-5。Fr.Ⅱ-2 (12.2 g) 經(jīng)硅膠柱分離,再經(jīng)制備HPLC,以80%甲醇分離純化,得到化合物9 (5.9 mg,tR=76.8 min),以82%甲醇分離純化,得到化合物10 (6.1 mg,tR=19.8 min)、11 (6.5 mg,tR=104.6 min)、12 (6.5 mg,tR=104.6 min)、13 (5.4 mg,tR=40.7 min)。Fr.Ⅲ(82.7 g) 經(jīng)ODS 柱,以甲醇-水(1 ∶4 ~1 ∶0)梯度洗脫,得到Fr.Ⅲ-1 ~Fr.Ⅲ-5。Fr.Ⅲ-4 (19.0 g) 經(jīng)ODS 柱,再經(jīng)制備HPLC,以68%甲醇分離純化,得到化合物3 (166.5 mg,tR=24.5 min),以52%甲醇分離純化,得到化合物22 (15.0 mg,tR=65.2 min),以62% 甲醇分離純化,得到化合物5 (15.2 mg,tR=142.1 min),以67%甲醇分離純化,得到化合物2 (76.3 mg,tR=20.4 min),以59%甲醇分離純化,得到化合物31 (22.5 mg,tR=40.3 min),以46%甲醇分離純化,得到化合物30(7.0 mg,tR=60.2 min)、21 (2.2 mg)。Fr.Ⅲ-5(21.3 g) 經(jīng)硅膠柱分離,再經(jīng)制備HPLC,以76%甲醇分離純化,得到化合物4 (7.8 mg,tR=20.7 min),以73% 甲醇分離純化,得到化合物6(4.8 mg,tR=20.8 min),以77% 甲醇分離純化,得到化合物1 (60.2 mg,tR=16.8 min)、7 (219.5 mg,tR=15.6 min),以66%甲醇分離純化,得到化合物8 (8.0 mg,tR=101.0 min),以甲醇分離純化,得到化合物24 (13.1 mg,tR=31.3 min)。Fr.Ⅳ(42.7 g) 經(jīng)ODS 柱,以甲醇-水(1 ∶9 ~1 ∶0)梯度洗脫,得到Fr.Ⅳ-1 ~Fr.Ⅳ-15。Fr.Ⅳ-10 經(jīng)硅膠柱分離,得到化合物15 (35.4 mg)、18 (16.8 mg); Fr.Ⅳ-11 經(jīng)硅膠柱分離,再經(jīng)制備HPLC,以甲醇-水-濃氨水(64 ∶36 ∶0.01) 分離純化,得到化合物16 (31.8 mg,tR=103.06 min),以甲醇-水-濃氨水(54 ∶46 ∶0.01) 分離純化,得到化合物17 (37.5 mg,tR=17.4 min)。Fr.Ⅳ-12 經(jīng)硅膠柱分離,再經(jīng)制備HPLC,以甲醇-水-濃氨水(72 ∶28 ∶0.01) 分離純化,得到化合物19 (24.5 mg,tR=65.0 min),以甲醇-水-濃氨水(80 ∶20 ∶0.01) 分離純化,得到化合物20 (6.3 mg,tR=53.2 min)。Fr.Ⅴ(38.5 g) 經(jīng)ODS 柱,以甲醇-水(1 ∶9~1 ∶0) 梯度洗脫,得到Fr.Ⅴ-1 ~Fr.Ⅴ-12。Fr.Ⅴ-7、Fr.Ⅴ-9 經(jīng)ODS 柱分離,經(jīng)制備HPLC,以73%甲醇分離純化,得到化合物14 (12.1 mg,tR=30.3 min) 以75%甲醇分離純化,得到化合物28 (2.9 mg,tR=18.2 min)。

乙酸乙酯部位膏狀物(100.0 g) 經(jīng)硅膠柱分離,以二氯甲烷-甲醇(1 ∶0 ~0 ∶1) 梯度洗脫,經(jīng)TLC、HPLC 檢識合并,濃縮干燥,得到Fr.A ~Fr.E。Fr.A (18.5 g) 經(jīng)硅膠柱,以聚乙烯-乙酸乙酯(1 ∶0 ~1 ∶0) 梯度洗脫,得到Fr.A-1 ~Fr.A-21。Fr.A-3 經(jīng)硅膠柱,以聚乙烯-乙酸乙酯(30 ∶1 ~1 ∶0) 梯度洗脫,得到化合物25 (9.8 mg)、26 (19.6 mg)。Fr.B (33.0 g) 經(jīng)硅膠柱,以二氯甲烷-甲醇(1 ∶0 ~0 ∶1) 梯度洗脫,得到Fr.B-1 ~Fr.B-5。Fr.B-3 經(jīng)硅膠柱,以聚乙烯-乙酸乙酯(20 ∶1~1 ∶0) 梯度洗脫,得到化合物22(15.5 mg)、27 (10.6 mg)。Fr.E (15.3 g) 經(jīng)ODS 柱,以甲醇-水(1 ∶9 ~1 ∶0) 梯度洗脫,得到Fr.E-1 ~Fr.E-43。Fr.E-37 經(jīng)制備HPLC,以93%甲醇分離純化,得到化合物23 (2.3 mg,tR=105.5 min)。

水部位冷凍干燥粉末(1 000.0 g) 溶于2.0 L水中,緩緩加入95% 乙醇8.0 L,充分?jǐn)嚢?,? ℃冷藏12 h,濾取上清液,濃縮后得提取物86.6 g,經(jīng)AB-8 大孔樹脂柱,依次用0、10%、20%、30%、40%、50%、60%、70%、80%、90%、100%甲醇洗脫,經(jīng)TLC、HPLC 檢識合并,得到Fr.1~Fr.4。Fr.4 (36.4 g) 經(jīng)MCI 柱,以甲醇-水(3 ∶7 ~1 ∶0) 梯度洗脫,得到Fr.4-1 ~Fr.4-31。Fr.4-14 經(jīng)制備HPLC,以62% 甲醇分離純化,得到化合物29 (2.5 mg,tR=30.4 min)。

3 結(jié)構(gòu)鑒定

化合物1: 白色無定形粉末,分子式C57H92O27,HR-ESI-MSm/z: 1 209.592 2 [M+H]+,1 226.619 4[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 5.29(1H,m,H-6),0.79 (3H,s,18-CH3),1.02(3H,s,19-CH3),1.06 (3H,d,J=6.9 Hz,21-CH3),1.38 (3H,s,27-CH3),1.75 (3H,d,J=6.2 Hz,Glc-H-6″),1.62 (3H,d,J=6.2 Hz,Glc-H-6?);13C-NMR (100 MHz,C5D5N)δ:37.5 (C-1),30.2 (C-2),78.1 (C-3),40.5(C-4),140.8 (C-5),121.9 (C-6),32.3 (C-7),31.7 (C-8),50.3 (C-9),37.2 (C-10),21.1 (C-11),39.0 (C-12),39.8 (C-13),56.5(C-14),32.2 (C-15),81.0 (C-16),62.7 (C-17),16.2 (C-18),19.4 (C-19),38.6 (C-20),15.2 ( C-21),120.3 ( C-22),33.1 ( C-23),34.0 (C-24),83.9 (C-25),77.5 (C-26),24.4(C-27),100.3 ( Glc-C-1′),78.5 ( Glc-C-2′),77.0 (Glc-C-3′),77.6 (Glc-C-4′),77.8 (Glc-C-5′),61.3 ( Glc-C-6′),102.1 ( Rha-C-1″),72.6 (Rha-C-2″),72.6 (Rha-C-3″),74.0 (Rha-C-4″),70.5 ( Rha-C-5″),18.5 ( Rha-C-6″),102.9 ( Rha-C-1?),72.6 ( Rha-C-2?),72.8(Rha-C-3?),74.2 ( Rha-C-4?),70.4 ( Rha-C-5?),18.7 ( Rha-C-6?),105.0 ( Glc-C-1″″),75.4 (Glc-C-2″″),78.6 (Glc-C-3″″),71.6 (Glc-C-4″″),78.0 ( Glc-C-5″″),62.9 ( Glc-C-6″″),105.5 ( Glc-C-1″?),75.2 ( Glc-C-2″?),78.6(Glc-C-3″?),71.8 ( Glc-C-4″?),78.4 ( Glc-C-5″?),62.5 (Glc-C-6″?)。以上數(shù)據(jù)與文獻(xiàn) [9]報道基本一致,故鑒定為multifidoside。

化合物2: 白色無定形粉末,分子式C45H72O17,HR-ESI-MSm/z: 885.485 6 [M +H]+,902.512 7[M+NH4]+。1H-NMR (400 MHz,CD3OD)δ: 0.81(3H,s,18-CH3),0.98 ( 3H,s,19-CH3),1.06 (3H,d,J=6.8 Hz,21-CH3),1.26 (3H,s,27-CH3);13C-NMR (100 MHz,CD3OD)δ:38.6 (C-1),30.8 (C-2),79.2 (C-3),39.4(C-4,20),142.0 (C-5),122.6 (C-6),32.8(C-7,8),51.7 (C-9),38.1 (C-10),22.0 (C-11),40.9 (C-12),41.6 (C-13),57.7 (C-14),33.2 (C-15),82.2 (C-16),63.2 (C-17),16.6(C-18),19.9 (C-19),15.1 (C-21),121.8 (C-22),33.6 (C-23),33.8 (C-24),85.6 (C-25),70.4 (C-26),24.3 (C-27),100.4 (Glc-C-1′),79.3 (Glc-C-2′),78.0 (Glc-C-3′),79.9 (Glc-C-4′),76.6 ( Glc-C-5′),62.0 ( Glc-C-6′),102.3 ( Rha-C-1″),72.1 ( Rha-C-2″),72.3( Rha-C-3″),73.7 ( Rha-C-4″),69.8 ( Rha-C-5″),17.9 (Rha-C-6″),103.0 (Rha-C-1?),72.2(Rha-C-2?),72.4 ( Rha-C-3?),73.9 ( Rha-C-4?),70.7 (Rha-C-5?),18.0 (Rha-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[10-11] 報道基本一致,故鑒定為nuatigenin-3-O-β-chacotriose。

化合物3: 白色無定形粉末,分子式C51H86O21,HR-ESI-MSm/z: 1 035.575 1 [M+H]+,1 052.602 0[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 5.32(1H,m,H-6),0.70 (3H,s,18-CH3),1.03(3H,s,19-CH3),1.16 (3H,d,J=6.5 Hz,21-CH3),1.02 (3H,m,27-CH3),4.82 (1H,d,J=7.6 Hz,Glc-H-1′),6.38 (1H,s,Rha-H-1″),1.75 (3H,d,J=6.1 Hz,Rha-H-6″),5.84(1H,s,Rha-H-1?),1.61 (3H,d,J=6.2 Hz,Rha-H-6?);13C-NMR ( 100 MHz,C5D5N )δ:37.6 (C-1),30.3 (C-2),78.2 (C-3),39.1(C-4),140.9 (C-5),122.1 (C-6),32.3 (C-7,8),50.5 (C-9),37.1 (C-10),21.4 (C-11),40.2 (C-12),42.4 (C-13),57.1 (C-14),24.7(C-15),28.3 (C-16),53.1 (C-17),12.1 (C-18),19.5 (C-19),41.6 (C-20),12.5 (C-21),73.1 (C-22),33.8 (C-23),31.3 (C-24),34.3(C-25),75.4 ( C-26),17.7 ( C-27),100.3(Glc-C-1′),78.0 (Glc-C-2′),78.3 (Glc-C-3′),78.7 (Glc-C-4′),77.0 (Glc-C-5′),61.4 (Glc-C-6′),102.1 ( Rha-C-1″),72.6 ( Rha-C-2″),72.8 (Rha-C-3″),74.0 (Rha-C-4″),69.6 (Rha-C-5″),18.7 ( Rha-C-6″),103.0 ( Rha-C-1?),72.6 ( Rha-C-2?),72.9 ( Rha-C-3?),74.2(Rha-C-4?),70.5 ( Rha-C-5?),18.6 ( Rha-C-6?),105.0 (Glc-C-1″″),75.3 (Glc-C-2″″),78.7( Glc-C-3″″),71.8 ( Glc-C-4″″),78.6 ( Glc-C-5″″),62.9 (Glc-C-6″″)。以上數(shù)據(jù)與文獻(xiàn)[12]報道基本一致,故鑒定為solaviaside A。

化合物4: 白色無定形粉末,分子式C45H72O16,HR-ESI-MSm/z: 869.490 7 [M +H]+,886.517 7[M+NH4]+。1H-NMR (400 MHz,CD3OD)δ: 5.38(1H,brd,J=3.6 Hz,H-6),0.83 (3H,s,18-CH3),1.04 (3H,s,19-CH3),0.96 (3H,d,J=7.1 Hz,21-CH3),0.80 (3H,d,J=6.6 Hz,27-CH3),4.50 (1H,d,J=7.8 Hz,Glc-H-1′),5.20 (1H,d,J= 1.4 Hz,Rha-H-1″),1.23(3H,d,J=6.8 Hz,Rha-H-6″),4.83 (1H,d,J=1.4 Hz,Rha-H-1?),1.28 (3H,d,J=6.5 Hz,Rha-H-6?);13C-NMR (100 MHz,CD3OD)δ: 38.6 (C-1),31.6 (C-2),79.3 (C-3),39.5(C-4),141.9 (C-5),122.6 (C-6),33.0 (C-7),32.7 (C-8),51.7 (C-9),38.0 (C-10),22.0 (C-11),41.0 (C-12),41.5 (C-13),57.7(C-14),33.2 (C-15),82.1 (C-16),64.0 (C-17),17.5 (C-18),19.9 (C-19),42.7 (C-20),15.1 ( C-21),110.3 ( C-22),32.8 ( C-23),30.7 (C-24),29.4 (C-25),68.1 (C-26),17.0(C-27),100.4 ( Glc-C-1′),79.2 ( Glc-C-2′),76.6 (Glc-C-3′),79.8 (Glc-C-4′),78.0 (Glc-C-5′),61.9 ( Glc-C-6′),102.3 ( Rha-C-1″),72.3 ( Rha-C-2″,3″),73.9 ( Rha-C-4″),69.8(Rha-C-5″),18.0 ( Rha-C-6″),103.0 ( Glc-C-1?),72.1 (Glc-C-2?),72.2 (Glc-C-3?),73.7(Glc-C-4?),70.6 (Glc-C-5?),17.8 (Glc-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[13] 報道基本一致,故鑒定為薯蕷皂苷。

化合物5: 白色無定形粉末,分子式C52H86O22,HR-ESI-MSm/z: 1 063.569 7 [M+H]+,1 080.597 0[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 5.32(1H,m,H-6),0.82 (3H,s,18-CH3),1.04(3H,s,19-CH3),1.07 (3H,d,J=7.5 Hz,21-CH3),1.04 (3H,s,27-CH3),3.35 (3H,s,28-OCH3),4.87 (1H,d,J=7.8 Hz,Glc-H-1′),6.41 (1H,s,Rha-H-1″),1.76 (3H,d,J=6.2 Hz,Rha-H-6″),5.87 (1H,s,Rha-H-1?),1.63 (3H,d,J=6.1 Hz,Rha-H-6?);13C-NMR (100 MHz,C5D5N)δ: 37.5 (C-1),30.2 (C-2),78.1 (C-3),39.0 (C-4),140.8(C-5),121.8 (C-6),32.2 (C-7),31.4 (C-8),50.1 (C-9),37.2 (C-10),20.8 (C-11),38.9(C-12),41.3 (C-13),55.5 (C-14),35.3 (C-15),119.0 (C-16),72.4 (C-17),13.8 (C-18),19.4 (C-19),34.2 (C-20),17.4 (C-21),87.5 (C-22),30.0 (C-23),32.0 (C-24),34.1(C-25),75.1 (C-26),17.7 (C-27),50.4 (C-28),100.4 (Glc-C-1′),77.8 (Glc-C-2′),78.6(Glc-C-3′),78.7 (Glc-C-4′),77.0 (Glc-C-5′),61.3 (Glc-C-6′),102.0 (Rha-C-1″),72.6 (Rha-C-2″),72.9 ( Rha-C-3″),74.2 ( Rha-C-4″),69.6 ( Rha-C-5″),18.7 ( Rha-C-6″),102.9(Rha-C-1?),72.6 ( Rha-C-2?),72.8 ( Rha-C-3?),74.0 (Rha-C-4?),70.4 (Rha-C-5?),18.5(Rha-C-6?),105.0 (Glc-C-1″″),75.3 (Glc-C-2″″),78.6 (Glc-C-3″″),71.8 (Glc-C-4″″),78.0(Glc-C-5″″),62.9 (Glc-C-6″″)。以上數(shù)據(jù)與文獻(xiàn)[12] 報道基本一致,故鑒定為solaviaside B。

化合物6: 白色無定形粉末,分子式C50H82O22,HR-ESI-MSm/z: 1 035.538 9 [M+H]+,1 052.566 1[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 5.29(1H,t,H-6),0.79 (3H,s,18-CH3),1.03(3H,s,19-CH3),1.17 (3H,d,J=6.9 Hz,21-CH3),0.98 (3H,d,J=6.4 Hz,27-CH3),4.99 (1H,d,J=7.6 Hz,Gal-H-1′),6.29 (1H,s,Rha-H-1″),1.68 (3H,d,J=6.2 Hz,Rha-H-6″),5.04 (1H,d,J=7.5 Hz,Xyl-H-1?),4.89(1H,d,J=7.6 Hz,Glc-H-1″″);13C-NMR (100 MHz,C5D5N)δ: 37.3 (C-1),30 (C-2),78.2(C-3),38.6 (C-4),140.7 (C-5),121.5 (C-6),32.0 (C-7),31.4 (C-8),50.1 (C-9),36.9 (C-10,23),20.8 (C-11),40.3 (C-12),40.6 (C-13,20),56.3 (C-14),32.1 (C-15),81.1 (C-16),64 (C-17),16.2 (C-18),19.2(C-19),16.0 (C-21),110.4 (C-22),28.0 (C-24),34.0 (C-25),74.8 (C-26),16.9 (C-27),100.3 (Gal-C-1′),76.3 (Gal-C-2′),84.8 (Gal-C-3′),70.1 (Gal-C-4′),75.0 (Gal-C-5′),62.0(Gal-C-6′),102 (Rha-C-1″),72.4 (Rha-C-2″),72.7 (Rha-C-3″),74.0 (Rha-C-4″),69.2 (Rha-C-5″),18.4 ( Rha-C-6″),106.6 ( Xyl-C-1?),74.5 (Xyl-C-2?),77.3 (Xyl-C-3?),70.8 (Xyl-C-4?),66.9 ( Xyl-C-5?),104.8 ( Glc-C-1″″),75.0 (Glc-C-2″″),78.3 (Glc-C-3″″),71.5 (Glc-C-4″″),78.4 (Glc-C-5″″),62.7 (Glc-C-6″″)。以上數(shù)據(jù)與文獻(xiàn) [14] 報道基本一致,故鑒定為indioside C。

化合物7: 白色無定形粉末,分子式C51H82O22,HR-ESI-MSm/z: 1 047.539 8 [M+H]+,1 064.566 5[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 5.29(1H,brd,J=4.5 Hz,H-6),0.78 (3H,s,18-CH3),1.02 (3H,s,19-CH3),1.06 (3H,d,J=6.9 Hz,21-CH3),1.38 (3H,s,27-CH3),4.96 (2H,d,J=7.8 Hz,Glc-H-1′,1″″),5.86(1H,s,Rha-H-1″),1.75 (3H,d,J=6.2 Hz,Rha-H-6″),6.40 ( 1H,s,Rha-H-1?),1.62(3H,d,J=6.2 Hz,Rha-H-6?);13C-NMR (100 MHz,C5D5N)δ: 37.5 ( C-1),30.2 ( C-2),78.1 (C-3),40.5 (C-4),140.8 (C-5),121.9(C-6),32.3 (C-7),31.6 (C-8),50.3 (C-9),37.1 (C-10),21.1 (C-11),39.0 (C-12),39.8(C-13),56.5 (C-14),32.2 (C-15),81.0 (C-16),62.7 (C-17),16.2 (C-18),19.4 (C-19),38.6 ( C-20),15.2 ( C-21),120.2 ( C-22),33.1 (C-23),34.0 (C-24),83.9 (C-25),77.5(C-26),24.4 (C-27),100.3 (Glc-C-1′),78.6(Glc-C-2′),77.0 (Glc-C-3′),77.8 (Glc-C-4′),78.0 (Glc-C-5′),61.3 (Glc-C-6′),102.1 (Rha-C-1″),72.9 ( Rha-C-2″),72.6 ( Rha-C-3″),74.2 (Rha-C-4″),69.6 (Rha-C-5″),18.7 (Rha-C-6″),102.9 (Rha-C-1?),72.6 (Rha-2?),72.8(Rha-C-3?),74.0 ( Rha-C-4?),70.4 ( Rha-C-5?),18.5 ( Rha-C-6?),105.5 ( Glc-C-1″″),75.4 (Glc-C-2″″),77.8 (Glc-C-3″″),71.6 (Glc-C-4″″),78.5 (Glc-C-5″″),62.5 (Glc-C-6″″)。以上數(shù)據(jù)與文獻(xiàn) [15] 報道基本一致,故鑒定為aculeatiside A。

化合物8: 白色無定形粉末,分子式C45H72O18,HR-ESI-MSm/z: 901.480 8 [M +H]+,918.507 7[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 5.29(1H,brd,J=4.5 Hz,H-6),0.78 (3H,s,18-CH3),1.02 (3H,s,19-CH3),1.06 (3H,d,J=6.9 Hz,21-CH3),1.38 (3H,s,27-CH3),4.96 (2H,d,J=7.8 Hz,Glc-H-1′,1?),5.86(1H,s,Rha-H-1″),1.75 (3H,d,J=6.2 Hz,Rha-H-6″);13C-NMR ( 100 MHz,C5D5N )δ:37.4 (C-1),30.2 (C-2),78.2 (C-3),39.3(C-4),140.8 (C-5),121.8 (C-6),32.2 (C-7,15),31.6 (C-8),50.2 (C-9),37.0 (C-10),21.1 (C-11),39.8 (C-12),40.5 (C-13),56.5(C-14),80.9 (C-16),62.5 (C-17),16.2 (C-18),19.4 (C-19),38.6 (C-20),15.2 (C-21),120.2 ( C-22),33.1 ( C-23),34.0 ( C-24),83.8 ( C-25),77.5 ( C-26),24.4 ( C-27),102.5 (Glc-C-1′),75.6 (Glc-C-2′),76.7 (Glc-C-3′),78.3 (Glc-C-4′),77.2 (Glc-C-5′),61.6(Glc-C-6′),102.7 ( Rha-C-1″),72.7 ( Rha-C-2″),72.8 (Rha-C-3″),74.0 (Rha-C-4″),70.4(Rha-C-5″),18.6 ( Rha-C-6″),105.5 ( Glc-C-1?),75.4 (Glc-C-2?),78.6 (Glc-C-3?,5?),71.6 (Glc-C-4?),62.7 (Glc-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[16] 報道基本一致,故鑒定為26-O- (β-Dglucopyranosyl) nuatigenin 3-O-α-L-rhamnopyranosyl-(1→4) -β-D-glucopyranoside。

化合物9: 白色無定形粉末,分子式C39H62O12,HR-ESI-MSm/z: 723.431 9 [M+H]+。1H-NMR (600 MHz,C5D5N)δ: 5.30 (1H,brs,H-6),0.82(3H,s,18-CH3),1.06 ( 3H,s,19-CH3),1.14 (3H,d,J=6.9 Hz,21-CH3),0.69 (3H,d,J=5.5 Hz,27-CH3),4.98 (1H,d,J=7.9 Hz,Gal-H-1′),4.41 (2H,d,J=5.9 Hz,Gal-H-6′),6.31 (1H,brs,Rha-H-1″),1.69 (3H,d,J=6.2 Hz,Rha-H-6″);13C-NMR (150 MHz,C5D5N)δ: 37.5 (C-1),30.2 (C-2),77.7 (C-3),38.9 (C-4),140.9 (C-5),121.6 (C-6),32.2 ( C-7,15),31.6 ( C-8),50.2 ( C-9),37.1 (C-10),21.0 (C-11),39.8 (C-12),40.4(C-13),56.6 (C-14),81.0 (C-16),62.8 (C-17),16.3 (C-18),19.4 (C-19),41.9 (C-20),15.0 ( C-21),109.2 ( C-22),31.8 ( C-23),29.2 (C-24),30.5 (C-25),66.8 (C-26),17.2(C-27),100.7 ( Gal-C-1′),75.9 ( Gal-C-2′),76.6 ( Gal-C-3′,5′),70.8 ( Gal-C-4′),62.3(Gal-C-6′),102.0 ( Rha-C-1″),72.5 ( Rha-C-2″),72.8 (Rha-C-3″),74.2 (Rha-C-4″),69.3(Rha-C-5″),18.5 (Rha-C-6″)。以上數(shù)據(jù)與文獻(xiàn)[17] 報道基本一致,故鑒定為smilacinoside A。

化合物10: 白色無定形粉末,分子式C39H62O13,HR-ESI-MSm/z: 739.426 8 [M+H]+。1H-NMR (600 MHz,C5D5N)δ: 5.32 (1H,brs,H-6),0.85(3H,s,18-CH3),0.91 ( 3H,s,19-CH3),1.18 (3H,d,J=6.8 Hz,21-CH3),1.22 (3H,s,27-CH3),4.96 (1H,d,J=7.7 Hz,Glc-H-1′),4.27 (1H,overlap,Glc-H-6′a),4.14 (1H,overlap,Glc-H-6′b),5.92 (1H,brs,Rha-H-1″),1.73 (3H,d,J=6.1 Hz,Rha-H-6″);13C-NMR(150 MHz,C5D5N)δ: 37.4 (C-1),30.1 (C-2),78.2 (C-3),39.2 (C-4),140.8 (C-5),121.7 (C-6),32.2 (C-7,15),31.6 (C-8),50.2 (C-9),37.0 (C-10),21.0 (C-11),39.8(C-12),40.4 (C-13),56.6 (C-14),81.3 (C-16),62.9 (C-17),16.3 (C-18),19.3 (C-19),41.9 ( C-20),15.1 ( C-21),109.5 ( C-22),27.8 (C-23),33.8 (C-24),65.9 (C-25),69.8(C-26),26.9 (C-27),102.4 (Glc-C-1′),75.5(Glc-C-2′),76.6 (Glc-C-3′),78.1 (Glc-C-4′),77.1 (Glc-C-5′),61.4 (Glc-C-6′),102.6 (Rha-C-1″),72.6 ( Rha-C-2″),72.8 ( Rha-C-3″),74.0 (Rha-C-4″),70.3 (Rha-C-5″),18.5 (Rha-C-6″)。以上數(shù)據(jù)與文獻(xiàn)[18] 報道基本一致,故鑒定為dioseptemloside H。

化合物11: 白色無定形粉末,分子式C39H62O12,HR-ESI-MSm/z: 723.431 8 [M+H]+。1H-NMR (600 MHz,C5D5N)δ: 0.82 (3H,s,18-CH3),0.91(3H,s,19-CH3),1.14 (3H,d,J=6.8 Hz,21-CH3),1.08 (3H,d,J=6.8 Hz,27-CH3),1.74 (3H,d,J=6.1 Hz,Rha-H-6″);13C-NMR(150 MHz,C5D5N)δ: 37.4 (C-1),30.1 (C-2),78.2 (C-3),39.2 (C-4),140.8 (C-5),121.7 (C-6),32.1 (C-7),31.6 (C-8),50.2(C-9),37.0 (C-10),21.0 (C-11),39.8 (C-12),40.4 (C-13),56.6 (C-14),32.2 (C-15,23),81.0 (C-16),62.8 (C-17),16.3 (C-18),19.3 ( C-19),41.9 ( C-20),15.0 ( C-21),109.2 ( C-22),29.2 ( C-24),30.5 ( C-25),66.8 (C-26),17.2 (C-27),102.4 (Glc-C-1′),75.5 (Glc-C-2′),76.6 (Glc-C-3′),78.1 (Glc-C-4′),77.1 ( Glc-C-5′),61.4 ( Glc-C-6′),102.6 ( Rha-C-1″),72.6 ( Rha-C-2″),72.8( Rha-C-3″),74.0 ( Rha-C-4″),70.3 ( Rha-C-5″),18.5 (Rha-C-6″)。以上數(shù)據(jù)與文獻(xiàn)[19] 報道基本一致,故鑒定為progenin Ⅱ。

化合物12: 白色無定形粉末,分子式C39H62O12,HR-ESI-MSm/z: 723.431 8 [M+H]+。與化合物11互為同分異構(gòu)體。1H-NMR (600 MHz,C5D5N)δ:0.83 ( 3H,s,18-CH3),1.06 ( 3H,s,19-CH3),1.13 (3H,d,J=5.8 Hz,21-CH3),0.69(3H,d,J=5.5 Hz,27-CH3),1.74 (3H,d,J= 6.1 Hz,Rha-H-6″);13C-NMR ( 150 MHz,C5D5N)δ: 16.2 (C-18),19.3 (C-19),42.4(C-20),14.8 (C-21),109.6 (C-22),26.3 (C-23),26.1 (C-24),27.5 (C-25),65.0 (C-26),16.2 (C-27)。其余核磁碳譜數(shù)據(jù)與化合物11 相同,推測兩者結(jié)構(gòu)差異在于C-25 位構(gòu)型不同,化合物11 的C-25 位為R構(gòu)型,當(dāng)C-25 位為S構(gòu)型時,由于甲基處于直立鍵,對F 環(huán)的碳原子產(chǎn)生屏蔽作用,C-23、C-24、C-26、C-27 位移值會向高場移動,特別是C-23 位由于γ斜式效應(yīng),其位移值將向高場移動超過5.0×10-6[10],從而判斷化合物12 的C-25 為S構(gòu)型。以上數(shù)據(jù)與文獻(xiàn)[20]報道基本一致,故鑒定為yamogenin-3-O- [α-L-rhamnopyranosyl- (1→4)] -β-D-glucopyranoside。

化合物13: 白色無定形粉末,分子式C34H56O9,HR-ESI-MSm/z: 626.426 8 [M +NH4]+。1H-NMR(600 MHz,C5D5N)δ: 5.38 (1H,t,H-6),0.83 ( 3H,s,18-CH3),1.03 ( 3H,s,19-CH3),1.19 (3H,d,J=6.9 Hz,21-CH3),1.00(3H,d,J=6.7 Hz,27-CH3),3.26 (3H,s,28-OCH3),4.85 (1H,d,J=7.8 Hz,Glc-H-1′);13C-NMR (150 MHz,C5D5N)δ: 37.7 (C-1),32.6 (C-2),71.2 (C-3),43.4 (C-4),141.9(C-5),121.0 (C-6),32.2 (C-7),31.6 (C-8),50.3 (C-9),37.0 (C-10),21.1 (C-11),39.8(C-12),40.8 (C-13),56.6 (C-14),32.1 (C-15),81.2 (C-16),64.1 (C-17),16.2 (C-18,21),19.5 (C-19),40.4 (C-20),112.6 (C-22),30.7 (C-23),28.1 (C-24),34.2 (C-25),75.2 ( C-26),17.1 ( C-27),47.2 ( C-28),105.0 (Glc-C-1′),75.2 (Glc-C-2′),78.6 (Glc-C-3′),71.7 (Glc-C-4′),78.5 (Glc-C-5′),62.8(Glc-C-6′)。NOESY 譜中發(fā)現(xiàn)H-1 (δH1.81) 和Hβ-19 (δH1.03) 相關(guān),可知H-1 (δH1.11) 為α構(gòu)型,又發(fā)現(xiàn)Hα-1 (δH1.11) 和H-3 (δH3.85)相關(guān),從而判斷3-OH 為β 構(gòu)型。C-26 位的2 個質(zhì)子信號δ3.60 (26-Ha)、δ3.96 (26-Hb) 化學(xué)位移差值Δab為0.36,判斷25 位為R構(gòu)型。以上數(shù)據(jù)與文獻(xiàn) [21-22] 報道基本一致,故鑒定為(25R) -furost-5-en-3β,26-diol-22α-methoxy -26-Oβ-D-glucopyranoside。

化合物14: 白色無定形粉末,分子式C52H86O22,HR-ESI-MSm/z: 1 063.569 7 [M+H]+,1 080.596 5[M+NH4]+。1H-NMR (600 MHz,C5D5N)δ: 5.31(1H,brs,H-6),0.81 (3H,s,18-CH3),1.04(3H,s,19-CH3),1.18 (3H,d,J=5.2 Hz,21-CH3),1.00 (3H,d,J=6.5 Hz,27-CH3),3.26 (3H,s,28-OCH3),4.91 (1H,overlap,Glc-H-1′),6.39 ( 1H,s,Rha-H-1″),1.76(3H,d,J=5.9 Hz,Rha-H-6″),5.85 (1H,s,Rha-H-1?),1.62 (3H,d,J=5.9 Hz,Rha-H-6?);13C-NMR (150 MHz,C5D5N)δ: 37.4 (C-1),30.1 (C-2),78.6 (C-3),38.9 (C-4),140.8 (C-5),121.7 (C-6),32.2 (C-7),31.6(C-8),50.2 (C-9),37.0 (C-10),21.0 (C-11),39.7 (C-12),40.7 (C-13),56.5 (C-14),32.1 (C-15),81.2 (C-16),64.1 (C-17),16.2(C-18,21),19.3 (C-19),40.4 (C-20),112.6(C-22),30.7 (C-23),28.1 (C-24),34.1 (C-25),75.1 (C-26),17.1 (C-27),47.2 (C-28),100.2 (Glc-C-1′),77.7 (Glc-C-2′),78.4 (Glc-C-3′),78.0 (Glc-C-4′),76.8 (Glc-C-5′),61.2(Glc-C-6′),102.0 ( Rha-C-1″),72.5 ( Rha-C-2″),72.7 (Rha-C-3″),74.0 (Rha-C-4″),69.4(Rha-C-5″),18.6 ( Rha-C-6″),102.8 ( Rha-C-1?),72.4 (Rha-C-2?),72.8 (Rha-C-3?),73.8(Rha-C-4?),70.3 ( Rha-C-5?),18.4 ( Rha-C-6?),104.9 (Glc-C-1″″),75.1 (Glc-C-2″″),78.6( Glc-C-3″″),71.7 ( Glc-C-4″″),77.9 ( Glc-C-5″″),62.8 (Glc-C-6″″)。以上數(shù)據(jù)與文獻(xiàn)[23]報道基本一致,故鑒定為methyl protodioscin。

化合物15: 白色無定形粉末,分子式C45H73NO15,HR-ESI-MSm/z: 868.506 8 [M +H]+。1H-NMR (400 MHz,C5D5N)δ: 0.85 (3H,s,18-CH3),1.04(3H,s,19-CH3),1.07 (3H,d,J=7.0 Hz,21-CH3),0.80 (3H,d,J=5.1 Hz,27-CH3),6.40 (1H,s,Rha-H-1″),1.62 (3H,d,J=6.2 Hz,Rha-H-6″),5.85 (1H,s,Rha-H-1?),1.75(3H,d,J=6.2 Hz,Rha-H-6?);13C-NMR (100 MHz,C5D5N)δ: 37.6 ( C-1),30.2 ( C-2),78.0 (C-3),39.0 (C-4),140.8 (C-5),121.9(C-6),32.6 (C-7),31.6 (C-8),50.4 (C-9),37.2 (C-10),21.2 (C-11),40.1 (C-12),40.7(C-13),56.7 (C-14),32.4 (C-15),78.9 (C-16),63.6 (C-17),16.6 (C-18),19.4 (C-19),41.7 (C-20),15.8 (C-21),98.4 (C-22),34.7(C-23),31.1 (C-24),31.7 (C-25),48.1 (C-26),19.8 ( C-27),100.3 ( Glc-C-1′),78.1(Glc-C-2′),77.8 (Glc-C-3′),78.6 (Glc-C-4′),77.0 (Glc-C-5′),61.3 (Glc-C-6′),102.9 (Rha-C-1″),72.6 ( Rha-C-2″),72.8 ( Rha-C-3″),74.0 (Rha-C-4″),70.4 (Rha-C-5″),18.5 (Rha-C-6″),102.1 ( Rha-C-1?),72.6 ( Rha-C-2?),72.9 ( Rha-C-3?),74.2 ( Rha-C-4?),69.6(Rha-C-5?),18.7 (Rha-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[24] 報道基本一致,故鑒定為澳洲茄邊堿。

化合物16: 白色無定形粉末,分子式C45H73NO15,HR-ESI-MSm/z: 868.506 6 [M +H]+。與化合物15 互為同分異構(gòu)體。1H-NMR (400 MHz,C5D5N)δ: 0.85 (3H,s,18-CH3),1.04 (3H,s,19-CH3),1.07 (3H,d,J=7.0 Hz,21-CH3),0.80(3H,d,J=6.3 Hz,27-CH3),6.41 (1H,s,Rha-H-1″),5.87 (1H,s,Rha-H-1?);13C-NMR(100 MHz,C5D5N)δ: 37.5 (C-1),30.2 (C-2),78.0 (C-3),39.0 (C-4),140.8 (C-5),121.9 (C-6),32.4 (C-7),31.6 (C-8),50.4(C-9),37.2 (C-10),21.2 (C-11),40.2 (C-12),40.7 (C-13),56.1 (C-14),33.2 (C-15),78.6 (C-16),62.2 (C-17),16.9 (C-18),19.4(C-19),43.0 (C-20),16.2 (C-21),99.4 (C-22),27.1 (C-23),29.3 (C-24),31.5 (C-25),50.6 (C-26),19.8 (C-27),100.3 (Glc-C-1′),78.1 (Glc-C-2′),77.8 (Glc-C-3′),78.7 (Glc-C-4′),77.0 ( Glc-C-5′),61.3 ( Glc-C-6′),102.1 ( Rha-C-1″),72.6 ( Rha-C-2″),72.9( Rha-C-3″),74.2 ( Rha-C-4″),69.6 ( Rha-C-5″),18.7 (Rha-C-6″),102.9 (Rha-C-1?),72.6(Rha-C-2?),72.8 ( Rha-C-3?),74.0 ( Rha-C-4?),70.4 (Rha-C-5?),18.5 (Rha-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[25] 報道基本一致,故鑒定為βslamarine,并且它與化合物15 的差異在于F 環(huán)構(gòu)型相反,27-CH3耦合常數(shù)有明顯差異。

化合物17: 白色無定形粉末,分子式C45H73NO15,HR-ESI-MSm/z: 868.506 7 [M+H]+。碳譜顯示該化合物C-16 位碳信號與化合物15 ~16 的差異最明顯,即在于C-16 位構(gòu)型不同。1H-NMR (400 MHz,C5D5N)δ: 0.81 (3H,s,18-CH3),1.02 (3H,s,19-CH3),1.04 (3H,d,J=7.0 Hz,21-CH3),0.75 (3H,d,J=6.1 Hz,27-CH3),1.61(3H,d,J=6.8 Hz,Rha-H-6″),1.73 (3H,d,J=6.8 Hz,Rha-H-6?);13C-NMR (100 MHz,C5D5N)δ: 37.5 (C-1),30.2 (C-2),78.0 (C-3),39.0 (C-4),140.8 (C-5),121.8 (C-6),32.2 ( C-7),31.7 ( C-8,25),50.2 ( C-9),37.1 (C-10),21.1 (C-11),39.5 (C-12),40.8(C-13),56.4 (C-14),32.5 (C-15),81.1 (C-16),62.8 (C-17),16.3 (C-18),19.4 (C-19),42.1 (C-20),15.7 (C-21),98.5 (C-22),33.7(C-23),29.6 (C-24),46.7 (C-26),19.1 (C-27),100.3 (Glc-C-1′),78.0 (Glc-C-2′),77.8(Glc-C-3′),78.6 (Glc-C-4′),77.0 (Glc-C-5′),61.3 (Glc-C-6′),102.9 (Rha-C-1″),72.6 (Rha-C-2″),72.8 ( Rha-C-3″),74.0 ( Rha-C-4″),70.4 ( Rha-C-5″),18.5 ( Rha-C-6″),102.1(Rha-C-1?),72.6 ( Rha-C-2?),72.9 ( Rha-C-3?),74.2 (Rha-C-4?),69.5 (Rha-C-5?),18.7(Rha-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[26] 報道基本一致,故鑒定為16-epi-solamargine,為化合物15 ~16的同分異構(gòu)體。

化合物18: 白色無定形粉末,分子式C45H73NO16,HR-ESI-MSm/z: 884.501 9 [M +H]+。1H-NMR(400 MHz,C5D5N)δ: 0.85 (3H,s,18-CH3),1.04 (3H,s,19-CH3),1.07 (3H,d,J=7.0 Hz,21-CH3),0.80 (3H,d,J=5.2 Hz,27-CH3),5.18 (1H,d,J=7.8 Hz,Gal-H-1′),6.27 (1H,s,Rha-H-1″),1.67 (3H,d,J=6.1 Hz,Rha-H-6″),4.92 (1H,d,J=8.0 Hz,Glc-H-1?);13C-NMR (100 MHz,C5D5N)δ: 37.6(C-1),30.2 (C-2),77.5 (C-3),38.8 (C-4),140.9 (C-5),121.8 (C-6),32.6 (C-7),32.4(C-8),50.3 (C-9),37.2 (C-10),21.2 (C-11),40.1 (C-12),40.7 (C-13),56.7 (C-14),31.7 (C-15),78.5 (C-16),63.6 (C-17),16.6(C-18),19.4 (C-19),41.7 (C-20),15.7 (C-21),98.4 (C-22),34.7 (C-23),31.1 (C-24),31.6 ( C-25),48.1 ( C-26),19.8 ( C-27),100.4 (Gal-C-1′),76.6 (Gal-C-2′),84.9 (Gal-C-3′),70.5 (Gal-C-4′),75.0 (Gal-C-5′),62.6(Gal-C-6′),102.3 ( Rha-C-1″),72.6 ( Rha-C-2″),72.9 (Rha-C-3″),74.2 (Rha-C-4″),69.5(Rha-C-5″),18.7 ( Rha-C-6″),105.9 ( Glc-C-1?),75.2 (Glc-C-2?),78.8 (Glc-C-3?),71.6(Glc-C-4?),78.3 (Glc-C-5?),62.6 (Glc-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[27] 報道基本一致,故鑒定為澳洲茄堿。

化合物19: 白色無定形粉末,分子式C39H63NO11,HR-ESI-MSm/z: 722.448 6 [M +H]+。1H-NMR(400 MHz,C5D5N)δ: 0.86 (3H,s,18-CH3),0.89 (3H,s,19-CH3),1.07 (3H,d,J=7.1 Hz,21-CH3),0.80 (3H,d,J=5.3 Hz,27-CH3),1.72 (3H,d,J=6.2 Hz,Rha-H-6″);13C-NMR (100 MHz,C5D5N)δ: 37.5 (C-1),30.2 (C-2),78.3 (C-3),39.3 (C-4),140.9(C-5),121.8 (C-6),32.6 (C-7),31.6 (C-8),50.3 (C-9),37.1 (C-10),21.2 (C-11),40.1(C-12),40.6 (C-13),56.7 (C-14),32.3 (C-15),78.8 (C-16),63.5 (C-17),16.6 (C-18),19.4 (C-19),41.6 (C-20),15.7 (C-21),98.4(C-22),34.7 (C-23),31.1 (C-24),31.6 (C-25),48.1 (C-26),19.8 (C-27),102.5 (Glc-C-1′),75.6 ( Glc-C-2′),78.4 ( Glc-C-3′),78.8(Glc-C-4′),76.7 (Glc-C-5′),61.5 (Glc-C-6′),102.7 ( Rha-C-1″),72.7 ( Rha-C-2″),72.9( Rha-C-3″),74.0 ( Rha-C-4″),70.4 ( Rha-C-5″),18.6 (Rha-C-6″)。以上數(shù)據(jù)與文獻(xiàn)[28] 報道基本一致,故鑒定為客西茄堿。

化合物20: 白色無定形粉末,分子式C27H43NO2,HR-ESI-MSm/z: 414.337 6 [M +H]+。1H-NMR(400 MHz,C5D5N)δ: 0.88 (3H,s,18-CH3),1.03 (3H,s,19-CH3),1.08 (3H,d,J=7.1 Hz,21-CH3),0.80 (3H,d,J=5.5 Hz,27-CH3);13C-NMR (100 MHz,C5D5N)δ: 37.8(C-1),32.4 (C-2),71.3 (C-3),43.5 (C-4),142.0 (C-5),121.1 (C-6),32.6 (C-7,15),31.8 (C-8),50.5 (C-9),37.0 (C-10),21.3(C-11),40.2 (C-12),40.7 (C-13),56.8 (C-14),78.9 (C-16),63.5 (C-17),16.6 (C-18),19.8 (C-19),41.6 (C-20),15.7 (C-21),98.4(C-22),34.6 (C-23),31.1 (C-24),31.6 (C-25),48.0 (C-26),19.6 (C-27)。以上數(shù)據(jù)與文獻(xiàn)[29] 報道基本一致,故鑒定為澳洲茄胺。

化合物21: 白色無定形粉末,分子式C29H50O,HR-ESI-MSm/z: 415.394 4 [M +H]+,432.421 1[M+NH4]+。1H-NMR (400 MHz,C5D5N)δ: 3.85(1H,m,H-3),5.42 (1H,brd,J=4.4 Hz,H-6),0.68 (3H,s,18-CH3),1.06 (3H,s,19-CH3),0.99 (3H,d,J=6.6 Hz,21-CH3),0.85(3H,overlap,26-CH3),0.87 (3H,overlap,27-CH3),0.88 (3H,overlap,29-CH3);13C-NMR(100 MHz,C5D5N)δ: 37.9 (C-1),32.7 (C-2),71.3 (C-3),43.6 (C-4),142.0 (C-5),121.3 (C-6),32.3 (C-7),32.2 (C-8),50.5(C-9),37.0 (C-10),21.4 (C-11),40.1 (C-12),42.6 (C-13),57.0 (C-14),24.6 (C-15),28.6 (C-16),56.3 (C-17),12.2 (C-18),20.0(C-19),36.5 (C-20),19.6 (C-21),34.3 (C-22),26.5 (C-23),46.1 (C-24),29.5 (C-25),19.1 (C-26),19.3 (C-27),23.4 (C-28),12.1(C-29)。以上數(shù)據(jù)與文獻(xiàn)[30] 報道基本一致,故鑒定為β-sitosterol。

化合物22: 白色無定形粉末,分子式C29H48O,HR-ESI-MSm/z413.378 4 [M+H]+。1H-NMR (600 MHz,CDCl3)δ: 3.52 (1H,m,H-3),5.35(1H,t,H-6),0.70 (3H,s,18-CH3),1.01(3H,s,19-CH3),1.02 (3H,d,J=6.7 Hz,21-CH3),5.15 (1H,dd,J=15.2,8.7 Hz,H-22),5.02 (1H,dd,J=15.2,8.7 Hz,H-23),0.83 (3H,d,J=6.8 Hz,26-CH3),0.79 (3H,d,J=6.5 Hz,27-CH3),0.85 (3H,t,J=7.4 Hz,29-CH3);13C-NMR (150 MHz,CDCl3)δ:37.4 (C-1),31.8 (C-2),72.0 (C-3),42.4(C-4),140.9 (C-5),121.9 (C-6),29.8 (C-7),32.0 (C-8,25),50.3 (C-9),36.7 (C-10),21.2 (C-11,26),39.8 (C-12),42.5 (C-13),57.0 (C-14),24.5 (C-15),29.1 (C-16),56.1 (C-17),12.2 (C-18),19.6 (C-19),40.6(C-20),21.4 (C-21),138.5 (C-22),129.4(C-23),51.4 (C-24),19.1 (C-27),25.6 (C-28),12.4 (C-29)。以上數(shù)據(jù)與文獻(xiàn)[31] 報道基本一致,故鑒定為豆甾醇。

化合物23: 白色顆粒狀固體,分子式C35H58O6,HR-ESI-MSm/z: 575.430 9 [M +H]+。1H-NMR(600 MHz,C5D5N)δ: 5.35 (1H,t,H-6),0.68 ( 3H,s,18-CH3),0.94 ( 3H,s,19-CH3),1.08 (3H,d,J=6.7 Hz,21-CH3),5.22(1H,dd,J=15.2,8.8 Hz,H-22),5.07 (1H,overlap,H-23),0.91 (3H,d,J=6.7 Hz,26-CH3),0.86 (3H,d,J=6.8 Hz,27-CH3),0.89(3H,t,J=7.4 Hz,29-CH3),5.07 (1H,d,J=7.8 Hz,Glc-H-1′),4.58 (1H,dd,J=11.8,2.0 Hz,Glc-H-6′a),4.43 (1H,dd,J=11.8,5.3 Hz,Glc-H-6′ b );13C-NMR ( 150 MHz,C5D5N)δ: 37.4 (C-1,10),30.2 (C-2),78.5(C-3),39.3 (C-4,12),140.9 (C-5),121.9(C-6),32.0 (C-7),32.1 (C-8),50.3 (C-9),19.4 (C-11,26),42.3 (C-13),56.9 (C-14),24.5 (C-15),29.3 (C-16,25),56.0 (C-17),12.5 (C-18),19.1 (C-19),40.7 (C-20),21.4(C-21),138.8 (C-22),129.4 (C-23),51.4(C-24),21.2 (C-27),25.7 (C-28),12.2 (C-29),102.5 (Glc-C-1′),75.3 (Glc-C-2′),78.6(Glc-C-3′),71.7 (Glc-C-4′),78.0 (Glc-C-5′),62.8 (Glc-C-6′)。以上數(shù)據(jù)與文獻(xiàn)[32] 報道基本 一 致,故 鑒 定 為 stigmasterol-3-O-β-Dglucopyranoside。

化合物24: 白色無定形粉末,分子式C35H60O6,HR-ESI-MSm/z: 577.446 7 [M +H]+。1H-NMR(600 MHz,C5D5N)δ: 5.35 (1H,t,H-6),0.67 ( 3H,s,18-CH3),0.94 ( 3H,s,19-CH3),1.00 (3H,d,J=6.1 Hz,21-CH3),0.87(3H,overlap,26-CH3),0.89 (3H,overlap,27-CH3),0.91 (3H,overlap,29-CH3),5.05 (1H,d,J=7.7 Hz,Glc-H-1′),4.56 (1H,brd,J=11.4 Hz,Glc-H-6′a),4.41 (1H,dd,J=11.4,5.3 Hz,Glc-H-6′ b );13C-NMR ( 150 MHz,C5D5N)δ: 37.4 (C-1),30.2 (C-2),78.4 (C-3),39.3 (C-4),140.9 (C-5),121.8 (C-6),32.1 (C-7),32.0 (C-8),50.3 (C-9),36.9(C-10),21.2 (C-11),39.9 (C-12),42.4 (C-13),56.8 (C-14),24.5 (C-15),28.5 (C-16),56.2 (C-17),12.1 (C-18),19.9 (C-19),36.3(C-20),19.4 (C-21),34.2 (C-22),26.4 (C-23),46.0 (C-24),29.4 (C-25),19.0 (C-26),19.2 ( C-27),23.4 ( C-28),11.9 ( C-29),102.5 (Glc-C-1′),75.3 (Glc-C-2′),78.5 (Glc-C-3′),71.6 (Glc-C-4′),78.1 (Glc-C-5′),62.8(Glc-C-6′)。以上數(shù)據(jù)與文獻(xiàn)[30] 報道基本一致,故鑒定為β-sitosterol β-D-glucopyranoside。

化合物25: 白色無定形粉末,分子式C31H54O,HR-ESI-MSm/z: 441.411 6 [M-H]-。1H-NMR(600 MHz,CDCl3)δ: 3.12 (1H,td,J=10.6,4.7 Hz,H-3),5.18 (1H,d,J=4.3 Hz,H-7);13C-NMR (150 MHz,CDCl3)δ: 37.2 (C-1),31.1 (C-2),76.4 (C-3),40.4 (C-4),46.8(C-5),26.8 (C-6),117.6 (C-7),139.3 (C-8),49.8 (C-9),35.0 (C-10),21.5 (C-11),39.7 (C-12),43.5 (C-13),55.1 (C-14),23.1(C-15),28.1 (C-16),56.2 (C-17),12.0 (C-18),14.3 (C-19),36.7 (C-20),19.0 (C-21),34.1 (C-22),26.4 (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),15.3 (C-30)。以上數(shù)據(jù)與文獻(xiàn)[33] 報道基本一致,故鑒定為24 (R) -ethyllophenol。

化合物26: 白色無定形粉末,分子式C37H54O4,HR-ESI-MSm/z: 563.409 8 [M +H]+。1H-NMR(600 MHz,CDCl3)δ: 0.62 (3H,s,18-CH3),0.93 (3H,s,19-CH3),0.95 (3H,d,J=6.7 Hz,21-CH3),3.73 ( 1H,m,H-22 ),0.81(3H,d,J=6.8 Hz,26-CH3),0.90 (3H,d,J=7.3 Hz,27-CH3),0.90 (3H,d,J=6.9 Hz,29-CH3),1.10 (3H,d,J=5.7 Hz,30-CH3),8.06 (2H,brd,J=7.4 Hz,C6H6-H-2′,6′),7.45 (2H,dd,J=7.4,7.4 Hz,C6H6-H-3′,5′),7.56 (1H,dd,J=7.4,7.4 Hz,C6H6-H-4′);13C-NMR (150 MHz,CDCl3)δ: 36.4 (C-1),26.3 (C-2),79.1 (C-3),31.9 (C-4),60.2 (C-5),200.3 (C-6),123.8 (C-7),161.1(C-8),51.2 (C-9),39.5 (C-10),21.9 (C-11),39.0 (C-12),45.2 (C-13),55.1 (C-14),22.7 (C-15),27.2 (C-16),53.3 (C-17),12.5(C-18),14.8 (C-19),42.8 (C-20),12.7 (C-21),71.2 (C-22),30.2 (C-23),41.6 (C-24),28.9 (C-25),17.8 (C-26),20.7 (C-27),23.8(C-28),12.0 ( C-29),17.6 ( C-30),130.7( C6H6-C-1′),129.7 ( C6H6-C-2′,6′),128.5( C6H6-C-3′,5′),133.0 ( C6H6-C-4′),166.6(7′-CO)。以上數(shù)據(jù)與文獻(xiàn)[34] 報道基本一致,故鑒定為carpesterol。

化合物27: 針狀結(jié)晶,分子式C36H52O4,HRESI-MSm/z: 549.394 5 [M +H]+。1H-NMR (600 MHz,CDCl3)δ: 4.69 (1H,td,J=10.5,4.5 Hz,H-3),0.62 (3H,s,18-CH3),0.93 (3H,s,19-CH3),0.95 (3H,d,J=6.7 Hz,21-CH3),3.76 (1H,m,22-H),0.84 (3H,d,J=6.6 Hz,26-CH3),0.85 (3H,d,J=6.7 Hz,27-CH3),0.89 (3H,d,J=6.6 Hz,28-CH3),1.10 (3H,d,J=5.8 Hz,29-CH3);13C-NMR(150 MHz,CDCl3)δ: 36.4 (C-1),26.3 (C-2),79.1 (C-3),31.9 (C-4),60.2 (C-5),200.4(C-6),123.8 (C-7),161.2 (C-8),51.2 (C-9),39.5 (C-10),21.9 (C-11),39.0 (C-12),45.2 (C-13),55.1 (C-14),22.7 (C-15),27.0(C-16),53.3 (C-17),12.5 (C-18),14.8 (C-19),42.6 (C-20),12.6 (C-21),70.9 (C-22),34.3 (C-23),34.7 (C-24),33.6 (C-25),15.2(C-26),18.5 (C-27),20.0 (C-28),17.6 (C-29),130.7 ( C6H6-C-1′),129.7 ( C6H6-C-2′,6′),128.5 ( C6H6-C-3′,5′),133.0 ( C6H6-C-4′),166.6 (CO-7′)。以上數(shù)據(jù)與文獻(xiàn)[35] 報道基本一致,核磁碳譜顯示它比化合物26 少1 個碳信號,由此推測缺少C-29 位甲基。在甲醇-二氯甲烷中得到了該化合物的單晶,通過Cu Kα X-射線晶體學(xué)分析進(jìn)一步證實了其結(jié)構(gòu),鑒定為norcarpesterol。

化合物28: 白色無定形粉末,分子式C39H60O15,HR-ESI-MSm/z: 769.402 8 [M +H]+。1H-NMR(600 MHz,C5D5N)δ: 5.34 (1H,brd,J=4.1 Hz,H-6),6.60 (1H,m,H-16),0.94 (3H,s,18-CH3),1.05 ( 3H,s,19-CH3),2.24(3H,s,21-CH3),4.95 (1H,overlap,Glc-H-1′),6.41 (1H,brs,Rha-H-1″),1.77 (3H,d,J=6.1 Hz,Rha-H-6″),5.86 (1H,brs,Rha-H-1?),1.64 (3H,d,J=6.1 Hz,Rha-H-6?);13C-NMR (150 MHz,C5D5N)δ: 37.3 (C-1),30.1 (C-2),78.0 (C-3),39.0 (C-4),141.2(C-5),121.5 (C-6),31.8 (C-7),30.3 (C-8),50.7 (C-9),37.1 (C-10),20.9 (C-11),35.1(C-12),46.2 (C-13),56.4 (C-14),32.2 (C-15),144.6 (C-16),155.2 (C-17),15.9 (C-18),19.2 (C-19),196.2 (C-20),27.0 (C-21),100.2 (Glc-C-1′),77.7 (Glc-C-2′),78.0(Glc-C-3′),78.6 (Glc-C-4′),76.9 (Glc-C-5′),61.2 (Glc-C-6′),102.0 (Rha-C-1″),72.5 (Rha-C-2″),72.8 ( Rha-C-3″),74.1 ( Rha-C-4″),69.5 ( Rha-C-5″),18.6 ( Rha-C-6″),102.9(Rha-C-1?),72.5 ( Rha-C-2?),72.7 ( Rha-C-3?),73.9 (Rha-C-4?),70.4 (Rha-C-5?),18.4(Rha-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[36] 報道基本一致,故鑒定為pregna-5,16-dien-3β-ol-20-one 3-Oα-L-rhamnopyranosyl- ( 1→2 ) - [α-Lrhamnopyranosyl- (1→4)] -β-D-glucopyranoside。

化合物29: 白色無定形粉末,分子式C39H60O16,HR-ESI-MSm/z: 785.396 4 [M +H]+。1H-NMR(600 MHz,C5D5N)δ: 5.34 (1H,brd,J=4.9 Hz,H-6),6.60 (1H,brs,H-16),0.93 (3H,s,18-CH3),1.06 ( 3H,s,19-CH3),2.24(3H,s,21-CH3),4.93 (1H,d,J=7.7 Hz,Gal-H-1′),6.30 ( 1H,brs,Rha-H-1″),1.69(3H,d,J=6.2 Hz,Rha-H-6″),5.20 (1H,d,J= 7.8 Hz,Glc-H-1?);13C-NMR (150 MHz,C5D5N)δ: 37.3 (C-1),30.0 (C-2),78.3 (C-3),38.7 (C-4),141.2 (C-5),121.4 (C-6),31.7 (C-7),30.2 (C-8),50.6 (C-9),37.0(C-10),20.8 (C-11),35.0 (C-12),46.2 (C-13),56.4 (C-14),32.2 (C-15),144.7 (C-16),155.1 (C-17),15.8 (C-18),19.2 (C-19),196.3 (C-20),27.0 (C-21),100.3 (Gal-C-1′),76.4 (Gal-C-2′),84.8 (Gal-C-3′),70.3(Gal-C-4′),74.8 (Gal-C-5′),62.5 (Gal-C-6′),102.1 ( Rha-C-1″),72.5 ( Rha-C-2″),72.8( Rha-C-3″),74.1 ( Rha-C-4″),69.3 ( Rha-C-5″),18.5 (Rha-C-6″),105.8 (Glc-C-1?),74.9(Glc-C-2?),78.4 (Glc-C-3?),71.5 (Glc-C-4?),77.4 (Glc-C-5?),62.4 (Glc-C-6?)。以上數(shù)據(jù)與文獻(xiàn)[12] 報道基本一致,故鑒定為3-O-α-Lrhamnopyranosyl- (1 →2) -O- [β-D-glucopyranosyl-(1→3)] -β-D-galactopyranosyl-pregna-5,16-dien-3β-ol-20-one。

化合物30: 白色無定形粉末,分子式C40H62O17,HR-ESI-MSm/z: 815.407 3 [M +H]+,832.434 0[M+NH4]+。1H-NMR (600 MHz,CD3OD)δ: 5.41(1H,brd,J=5.2 Hz,H-6),0.82 (3H,s,18-CH3),1.04 (3H,s,19-CH3),1.50 (3H,s,21-CH3),4.50 (1H,d,J=7.9 Hz,Glc-H-1′),5.21 (1H,brs,Rha-H-1″),1.26 (3H,d,J=6.2 Hz,Rha-H-6″),4.84 (1H,brs,Rha-H-1?),1.24 (3H,d,J=6.2 Hz,Rha-H-6?);13C-NMR(150 MHz,CD3OD)δ: 38.5 (C-1),30.7 (C-2),79.2 (C-3),39.5 (C-4),141.9 (C-5),122.4 (C-6),32.8 (C-7),32.2 (C-8),51.4(C-9),37.9 (C-10),21.2 (C-11),39.7 (C-12),41.3 (C-13),57.1 (C-14),32.8 (C-15),84.0 (C-16),64.9 (C-17),14.0 (C-18),19.8(C-19),75.4 (C-20),19.4 (C-21),180.1 (C-22),100.4 (Glc-C-1′),80.0 (Glc-C-2′),76.6(Glc-C-3′),79.3 (Glc-C-4′),78.0 (Glc-C-5′),62.0 (Glc-C-6′),102.3 (Rha-C-1″),72.4 (Rha-C-2″),72.2 ( Rha-C-3″),73.7 ( Rha-C-4″),69.8 ( Rha-C-5″),17.9 ( Rha-C-6″),103.0(Rha-C-1?),72.4 ( Rha-C-2?),72.2 ( Rha-C-3?),73.9 (Rha-C-4?),70.7 (Rha-C-5?),18.0(Rha-C-6?)。該化合物與化合物31 數(shù)據(jù)基本一致,僅在苷元E 環(huán)上差異較大,根據(jù)核磁譜圖判斷其結(jié)構(gòu)是后者20 位上的氫被羥基取代,以上數(shù)據(jù)與文獻(xiàn)[37] 報道基本一致,故鑒定為chonglouoside SL-7。

化合物31: 白色無定形粉末,分子式C40H62O16,HR-ESI-MSm/z: 799.412 8 [M +H]+,816.439 7[M+NH4]+。1H-NMR (600 MHz,CD3OD)δ: 5.41(1H,m,H-6),0.78 (3H,s,18-CH3),1.07(3H,s,19-CH3),1.31 (3H,d,J=7.6 Hz,21-CH3),5.22 ( 1H,brs,Glc-H-1′),4.86(1H,brs,Rha-H-1″),1.28 (3H,d,J=6.2 Hz,Rha-H-6″),4.52 (1H,d,J=7.9 Hz,Rha-H-1?),1.26 (3H,d,J=6.2 Hz,Rha-H-6?);13C-NMR (150 MHz,CD3OD)δ: 38.5 (C-1),30.7 (C-2),79.3 (C-3),39.5 (C-4),142.0(C-5),122.4 (C-6),33.0 (C-7),32.6 (C-8),51.7 (C-9),38.0 (C-10),21.5 (C-11),39.0(C-12),42.5 (C-13),55.8 (C-14),34.0 (C-15),84.8 (C-16),60.0 (C-17),14.0 (C-18),19.8 ( C-19),37.5 ( C-20),18.1 ( C-21),184.0 (C-22),100.8 (Glc-C-1′),78.2 (Glc-C-2′),78.5 ( Glc-C-3′),79.0 ( Glc-C-4′),77.4(Glc-C-5′),61.8 ( Glc-C-6′),102.5 ( Rha-C-1″),73.0 (Rha-C-2″),73.3 (Rha-C-3″),74.6( Rha-C-4″),70.0 ( Rha-C-5″),19.1 ( Rha-C-6″),103.4 (Rha-C-1?),73.0 (Rha-C-2?),73.2(Rha-C-3?),74.4 ( Rha-C-4?),70.9 ( Rha-C-5?),19.0 (Rha-C-6?)。以上數(shù)據(jù)與文獻(xiàn) [38]報道基本一致,故鑒定為dumoside。

4 體外抗炎活性研究

將RAW 264.7 細(xì)胞培養(yǎng)于含10% 胎牛血清、100 U/mL 青霉素/鏈霉素的DMEM 培養(yǎng)基中,對數(shù)生長期者接種于96 孔板中,細(xì)胞密度為1.0×104/孔,設(shè)置空白組、模型組、藥物組(各化合物),陽性組(吲哚美辛),每組3 個復(fù)孔,于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)過夜。次日,將5、10、20、30、50 μmol/L 待測藥物和終質(zhì)量濃度為1 μg/mL 的LPS 與細(xì)胞共同培養(yǎng)24 h,空白組加入DMEM 培養(yǎng)基,模型組加入含LPS 的培養(yǎng)基。取100 μL 培養(yǎng)上清,轉(zhuǎn)至新的96 孔板中,分別加入NO 檢測試劑Griess 試劑A 液、B 液(A 液為對氨基苯磺酸0.5 g,溶于150 mL 10% 稀醋酸中; B 液為N-α-萘乙二胺0.1 g,溶于20 mL 水中,10%稀醋酸稀釋至150 mL) 各50 μL,避光顯色30 min,在540 nm 波長處檢測光密度(OD) 計算IC50值,數(shù)據(jù)以(±s) 表示,結(jié)果見表1,可知化合物1~8、10、13、26、30~31 對NO 的抑制活性較高,IC50值在30~50 μmol/L 之間。

表1 各化合物抑制NO 的IC50值(±s,n=3)Tab.1 IC50 values of various compounds inhibiting NO (±s,n=3)

表1 各化合物抑制NO 的IC50值(±s,n=3)Tab.1 IC50 values of various compounds inhibiting NO (±s,n=3)

化合物IC50/(μmol·L-1)化合物IC50/(μmol·L-1)化合物IC50/(μmol·L-1)1 48.68±4.56 12—23>50 2 31.14±2.87 1333.74±3.21 24>50 3 41.38±4.63 14>50 25>50 4 42.15±4.34 15—2637.16±4.14 5 32.02±3.50 16—27—6 30.53±3.37 17—28>50 7 43.58±4.76 18—29>50 8 34.26±3.42 19—3045.16±4.62 9>50 20—3142.35±3.87 1046.42±4.10 21>50吲哚美辛2.24±0.26 11—22—

5 結(jié)論

本研究從黃果茄果70% 乙醇提取物中分離得到31 個化合物,包括14 個甾體皂苷、6 個甾體生物堿、7 個甾醇、2 個C21甾體、2 個C22甾體,其中化合物1、30 為首次從茄科植物中分離得到,2~8、16~17、31 為首次從該植物中分離得到,豐富了黃果茄果的化學(xué)成分,為之后相關(guān)開發(fā)應(yīng)用提供了基礎(chǔ)。同時,對各化合物體外抗炎活性進(jìn)行考察,發(fā)現(xiàn)21 個對LPS 誘導(dǎo)RAW 264.7 細(xì)胞的NO釋放有抑制效果。

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