馬文杰 蘇志維 馬仲輝
摘 要:? 全緣葉紫珠(Callicarpa integerrima)具有祛風(fēng)散結(jié)和治風(fēng)濕瘰疬的功效,但目前對其化學(xué)成分的報道較少。為探究全緣葉紫珠根、莖的化學(xué)成分,該研究利用硅膠柱層析色譜、Sephadex LH-20葡聚糖凝膠柱層析色譜、ODS反相硅膠柱層析色譜以及高效液相色譜等現(xiàn)代分離方法對全緣葉紫珠根和莖的95%乙醇提取物進(jìn)行系統(tǒng)的分離純化,并運用NMR和ESI-MS等現(xiàn)代波譜技術(shù)對化合物進(jìn)行結(jié)構(gòu)鑒定。結(jié)果表明:從全緣葉紫珠根和莖的95%乙醇提取物中共鑒定了15個化合物分別為豆甾烷-4-烯-3-酮 (1)、(24R)-5α-豆甾烷-3,6-二酮 (2)、2′-羥基-4′-甲氧基二氫查爾酮 (3)、α-香樹脂醇 (4)、 β-谷甾醇 (5)、熊果酸 (6)、對羥基間甲氧基苯甲酸 (7)、4-羥基吡啶 (8)、對羥基苯甲酸 (9)、連翹酯苷B (10)、nepetifosides D (11)、異毛蕊花苷 (12)、毛蕊花苷 (13)、pedicularioside M (14)、 β-甲氧基連翹酯苷B (15)。除化合物4-6、12和13外,其他化合物均為首次從全緣葉紫珠植物中分離得到,其中化合物1、2、3、8、11和14為首次從紫珠屬植物中分離得到。該研究結(jié)果豐富了全緣葉紫珠植物的化合物庫,為該藥用植物的進(jìn)一步開發(fā)利用奠定了科學(xué)基礎(chǔ)。
關(guān)鍵詞: 紫珠屬, 全緣葉紫珠, 化學(xué)成分, 苯乙醇苷類, 三萜類
中圖分類號:? Q946文獻(xiàn)標(biāo)識碼:? A文章編號:? 1000-3142(2023)06-1135-10
Chemical constituents of Callicarpa integerrima
MA Wenjie1,2, SU Zhiwei2*, MA Zhonghui1
( 1. College of Agriculture, Guangxi University / Guangxi Key Laboratory of Sugarcane Biology / State Key Laboratory for Conservation and Utilization
of Subtropical Agro-Bioresources / Traditional Chinese Herbal Medicine Resources and Agriculturalization Research Institute, Guangxi University,
Nanning 530004, China; 2. Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, China )
Abstract:? Callicarpa integerrima has very good effects in emoving blood stasis and resolving static blood and treatment of rheumatism evil. However, there are few reports on its chemical constituents. In order to explore the chemical constituents from the roots and stems of C. integerrima, the 95% ethanol extracts of C. integerrima roots and stems were isolated and purified by diverse column chromatography, such as silica gel, Sephadex LH-20 gel column, ODS column chromatography, and preparative HPLC. In addition, these compounds were identified on the basis of NMR, ESI-MS as well as other modern spectral techniques. The results were as follows: A total of 15 compounds were isolated from the 95% ethanol extracts of the roots and stems of C. integerrima, which were identified as stigmast-4-en-3-one (1), (24R) -5α-stigmastane-3,6-dione (2), 2′-hydroxy-4′-methoxydihydrochalcone (3), α-amyrin (4), β-sitosterol (5), ursolic acid (6), 4-hydroxy-3-methoxy-benzoic acid (7), 4-pyridinol (8), p-hydroxybenzoic acid (9), forsythoside B (10), nepetifosides D (11), isoacteoside (12), acteoside (13), pedicularioside M (14), β-methoxy forsythoside B (15). All compounds, except for compounds 4-6, 12 and 13, were isolated from C. integerrima for the first time. Furthermore, compounds 1, 2, 3, 8, 11 and 14 were isolated from the genus of Callicarpa for the first time. Therefore, the results of this research has enriched the compound library and afford a scientific foundation for the further rational use of C. integerrima.
Key words: Callicarpa, Callicarpa integerrima, chemical constituents, phenylethanoids, triterpenoids
紫珠屬植物具有悠久的藥用歷史,首載于《本草拾遺》,民間常用于止血和解毒,該屬植物的化學(xué)成分主要有萜類、苯乙醇苷類、黃酮類,具有止血、抗菌、抗炎、改善記憶力等藥理作用(占麗麗等,2020)。目前,《中國藥典》(2020)收錄了4種紫珠屬植物,即廣東紫珠、裸花紫珠、大葉紫珠、杜虹花。以這4種紫珠屬植物為主要藥效成分研發(fā)的臨床藥物已得到了廣泛應(yīng)用,如以廣東紫珠為主要成分的抗宮炎膠囊和抗宮炎片,以裸花紫珠為主要成分的裸花紫珠片、裸花紫珠抑菌凝膠、裸花紫珠顆粒、裸花紫珠膠囊等,以大葉紫珠為主要成分的紫地寧血散和三七血傷凝膠囊,以杜虹花為主要成分的11號止血粉、痔炎消顆粒和婦炎靈膠囊等(莫霞和李瑤,2019;鄔秋萍等,2019)。由此可見,紫珠屬植物資源的開發(fā)利用具有廣闊的發(fā)展空間。
全緣葉紫珠(Callicarpa integerrima)為唇形科紫珠屬罕見的藤本型植物,系我國特有種(王雪芬等,1986),常生長于海拔200~700 m的山坡或谷地林中,主要分布于廣西、廣東、江西、福建等?。▍^(qū))(《中國植物志》編輯委員會,1982)。全緣葉紫珠的根和葉入藥,具有祛風(fēng)散結(jié)、治風(fēng)濕瘰疬的作用(柴玲等,2010)。目前已從全緣葉紫珠中發(fā)現(xiàn)二萜類、三萜類、黃酮類、揮發(fā)油等化學(xué)成分(王雪芬等,1986;柴玲等,2010;祝晨蔯等,2012;Di et al., 2021)。數(shù)十年來,國內(nèi)外的研究重點集中在該屬小喬木型、灌木型植物,如裸花紫珠、廣東紫珠和杜虹花等個別種類的化學(xué)及藥理活性研究,而對該屬罕見的藤本型植物全緣葉紫珠的化學(xué)成分研究卻很少報道,且其藥效物質(zhì)基礎(chǔ)仍不明確。全緣葉紫珠作為我國南方民間常用藥,明確其化學(xué)成分是其用藥安全和資源開發(fā)的根本前提。因此,為了更好地了解和應(yīng)用全緣葉紫珠這一藥用植物,闡明其化學(xué)成分組成和藥理藥效基礎(chǔ),進(jìn)而深入開發(fā)利用這一特色資源,該研究從全緣葉紫珠的根、莖提取物中共鑒定了15個化合物(圖1),分別鑒定為豆甾烷-4-烯-3-酮 (1)、(24R) -5α-豆甾烷-3,6-二酮 (2)、2′-羥基-4′-甲氧基二氫查爾酮 (3)、α-香樹脂醇 (4)、β-谷甾醇 (5)、熊果酸 (6)、對羥基間甲氧基苯甲酸 (7)、4-羥基吡啶 (8)、對羥基苯甲酸 (9)、連翹酯苷B (10)、nepetifosides D (11)、異毛蕊花苷 (12)、毛蕊花苷 (13)、 pedicularioside M (14)、 β-甲氧基連翹酯苷B (15)。除化合物4-6、12、13外,其他化合物均為首次從全緣葉紫珠植物中分離得到,其中化合物1、2、3、8、11、14為首次從紫珠屬植物中分離得到。這些化合物的發(fā)現(xiàn),豐富了紫珠屬植物的化合物庫,為進(jìn)一步系統(tǒng)地探究全緣葉紫珠的藥理活性以及作用機制奠定了基礎(chǔ),也為更加合理地開發(fā)和利用紫珠屬藥用植物資源提供了科學(xué)依據(jù)。
1 儀器與材料
DHG-9240電熱恒溫鼓風(fēng)干燥箱(上海精宏實驗設(shè)備有限公司);SHB-ⅢA循環(huán)水式真空泵(鞏義市予華儀器有限責(zé)任公司);AR224CN(上海電子分析天平奧豪斯儀器有限公司);CX-1000A粉碎機(上海市晟喜制藥機械有限公司);3510E-DTH超聲波清洗機(美國必能信公司);KQ-800DE數(shù)控超聲波清洗器(昆山市超聲儀器有限公司);AVIII HD 600 和AVIII 500 型核磁共振波譜儀(瑞士Bruker 公司);島津 LC-2030C 3D Plus高效液相色譜儀(日本島津公司);數(shù)顯恒溫水浴鍋 HH-S4(常州金壇良友儀器有限公司);鄭州長城DLSB-10/20低溫冷卻循環(huán)泵(鄭州長城科工貿(mào)有限公司);上海EYELA OSB-2200小型旋轉(zhuǎn)蒸發(fā)儀(上海愛朗儀器有限公司);WFH-203B暗箱式紫外分析儀(杭州齊威儀器有限公司);MCI GEL CHP20P樹脂填料(日本三菱化學(xué)公司);ODS反相色譜填料 C18 MB100-40/75 (富士化學(xué)有限公司);分析型 HPLC 色譜柱為YMC-Pack ODS-A 色譜柱(250 mm×4.6 mm, 5 μm);半制備型 HPLC 色譜柱為 YMC-Pack ODS-A 色譜柱(250 mm×10 mm, 5 μm);優(yōu)普UPH-II-20T純水機(南京優(yōu)普儀器設(shè)備有限公司);柱色譜硅膠(100~200目、200~300 目,煙臺江友硅膠開發(fā)有限公司);GF254薄層層析硅膠板(青島譜科分離材料有限公司);顯色劑為10%硫酸乙醇溶液,浸濕后加熱顯色;聚酰胺(浙江省臺州市路橋四甲生化塑料廠);Sephadex LH-20葡聚糖凝膠(40~70 μm, GE healthcare)。
石油醚、正丁醇購于天津富宇精細(xì)化工有限公司,乙酸乙酯購于天津大茂化學(xué)試劑廠,甲醇、二氯甲烷、氯仿購于上海泰坦化學(xué)有限公司,上述所用試劑均為分析純級別;氘代試劑購于Cambridge Isotope Laboratories, Inc.;色譜純乙腈、甲醇均購于上海星可高純?nèi)軇┯邢薰尽?/p>
本實驗所用全緣葉紫珠植物樣品于2019年4月采自廣東省廣州市從化區(qū)溫泉鎮(zhèn)溫泉派出所附近,經(jīng)廣西大學(xué)農(nóng)學(xué)院馬仲輝副教授鑒定為唇形科紫珠屬植物全緣葉紫珠 (Callicarpa integerrima)。憑證標(biāo)本(采集號:20190426)保存于廣西大學(xué)農(nóng)學(xué)院植物標(biāo)本室 (GAUA)。
2 提取分離
將陰干的全緣葉紫珠根0.55 kg、莖9.78 kg分別粉碎后,用95%乙醇室溫浸泡1周后各提取3次,提取液經(jīng)減壓濃縮得根部醇提物(41.66 g)、莖部醇提物(448.50 g)。將莖部醇提物(448.50 g)加熱水混懸后,依次用石油醚、乙酸乙酯和正丁醇進(jìn)行萃取,減壓濃縮后得到石油醚部位(46.26 g)、乙酸乙酯部位(72.90 g)和正丁醇部位(106.56 g)。
取根部乙醇提取物(41.66 g)經(jīng)聚酰胺柱層析,用甲醇-水(0∶100 ~ 30∶70 ~ 50∶50 ~ 70∶30 ~ 100∶0)梯度洗脫,得到11個流分(Fr.1 ~ Fr.11)。流分 Fr.9(1.00 g)經(jīng)Sephadex LH-20柱層析(氯仿∶甲醇=1∶1)純化,在氯仿甲醇混合液中重結(jié)晶,得到化合物1(7.10 mg)和化合物2(11.70 mg),經(jīng)薄層層析硅膠板進(jìn)行制備,得到化合物3(2.60 mg)。
取莖部石油醚萃取物(46.26 g)經(jīng)硅膠柱層析,用石油醚-二氯甲烷(98∶2 ~ 5∶5)梯度洗脫,得到15個流分(Fr.1 ~ Fr.15)。流分Fr.10(5.69 g)經(jīng)Sephadex LH-20柱層析(氯仿∶甲醇=1∶1)、硅膠柱層析(石油醚∶二氯甲烷= 19∶1),ODS反相硅膠柱 (90%甲醇水)反復(fù)純化,得到化合物4(21.20 mg),在氯仿甲醇混合液中重結(jié)晶,得到化合物5(10.11 mg)。Fr.14(6.80 g)經(jīng)MCI柱層析(85%甲醇水)和硅膠柱層析(石油醚∶二氯甲烷∶甲醇= 50∶99∶1)純化,得到化合物6(11.40 mg)。
取莖部乙酸乙酯萃取物(72.90 g)經(jīng)硅膠柱層析,二氯甲烷-甲醇(98∶2 ~ 5∶5)梯度洗脫,得到13個流分(Fr.1 ~ Fr.13)。Fr.5(3.64 g)經(jīng)Sephadex LH-20柱層析(氯仿∶甲醇=1∶1)純化,再經(jīng)半制備HPLC,18%乙腈水等度制備,流速2 mL·min-1,得到化合物7(10.00 mg, tR=19 min)和化合物8(4.50 mg, tR=27 min)。Fr.6(2.56 g)經(jīng)Sephadex LH-20柱層析(氯仿∶甲醇=1∶1)純化,再經(jīng)半制備HPLC,20%乙腈水(含0.1% 甲酸)等度制備,流速2 mL·min-1,得到化合物9(5.22 mg, tR=15 min)。Fr.12(56.11 g)經(jīng)硅膠柱層析(二氯甲烷-甲醇=95∶5 ~ 5∶5),中壓ODS反相柱層析(乙腈-水=15% ~ 100%),Sephadex LH-20柱層析(氯仿∶甲醇=1∶1),反復(fù)純化,得到化合物10 ( 69.90 mg )。再經(jīng)半制備HPLC,21%乙腈水(含0.1%甲酸)等度制備,流速2 mL·min-1,得到化合物11(7.04 mg, tR=52 min);34%甲醇水等度制備,流速2 mL·min-1,得到化合物12(40.00 mg, tR=31 min);35%甲醇水等度制備,流速2 mL·min-1,得到化合物13(94.00 mg, tR=20 min)和化合物14(13.00 mg, tR=27 min);15%乙腈水(含0.1%甲酸)等度制備,流速2 mL·min-1,得到化合物15(10.62 mg, tR=36 min)。
3 結(jié)構(gòu)鑒定
化合物1 白色針晶狀結(jié)晶,分子式為C29H48O。ESI-MS m/z: 413.37? [ M + H ]+。1H-NMR (600 MHz, CDCl3) δH:? 5.74 (1H, s, H-4), 0.92 (3H, s, H-19), 0.90 (3H, d, J = 7.1 Hz, H-21), 0.86 (3H, t, J = 8.1 Hz, H-29), 0.85 (3H, d, J = 7.1 Hz, H-26), 0.82 (3H, d, J = 6.6 Hz, H-27), 0.70 (3H, s, H-18)。13C-NMR (150 MHz, CDCl3) δC: 198.8 (C-3), 171.6 (C-5), 123.9 (C-4), 56.1 (C-17), 55.9 (C-14), 53.8 (C-9), 45.9 (C-24), 42.3 (C-13), 39.8 (C-10, C-12), 38.7 (C-8), 36.1 (C-20), 35.6 (C-1), 33.9 (C-2), 33.8 (C-22), 32.9 (C-6), 32.0 (C-7), 29.1 (C-25), 28.2 (C-16), 26.0 (C-23), 24.2 (C-15), 23.0 (C-28), 21.0 (C-11), 19.9 (C-27), 19.1 (C-26), 18.8 (C-21), 17.6 (C-19), 12.1 (C-18/29)。以上數(shù)據(jù)與文獻(xiàn)(Abdelhameed et al., 2020)報道的豆甾烷-4-烯-3-酮 (stigmast-4-en-3-one) 的數(shù)據(jù)基本一致,故鑒定化合物1為豆甾烷-4-烯-3-酮 (stigmast-4-en-3-one)。
化合物2 白色針晶狀結(jié)晶,分子式為C29H48O2。ESI-MS m/z: 429.37 [ M + H ]+。1H-NMR (600 MHz, CDCl3) δH:? 0.95 (3H, s, H-18), 0.92 (3H, d, J = 7.1 Hz, H-21), 0.86 (3H, t, J = 8.1 Hz, H-29), 0.83 (3H, d, J = 6.9 Hz, H-26), 0.81 (3H, d, J = 6.4 Hz, H-27), 0.70 (3H, s, H-19)。13C-NMR (150 MHz, CDCl3) δC:? 211.4 (C-6), 209.1 (C-3), 57.5 (C-5), 56.6 (C-17), 56.0 (C-14), 53.5 (C-9), 46.6 (C-7), 45.8 (C-24), 43.0 (C-13), 41.2 (C-10), 39.4 (C-2), 38.1 (C-1), 38.0 (C-8), 37.4 (C-12), 37.0 (C-4), 36.0 (C-20), 33.9 (C-22), 29.1 (C-25), 28.0 (C-16), 26.0 (C-23), 24.0 (C-15), 23.0 (C-28), 21.7 (C-11), 19.9 (C-26), 19.1 (C-27), 18.6 (C-21), 12.7 (C-18), 12.1 (C-19/29)。以上數(shù)據(jù)與文獻(xiàn)(李小軍等,2014)報道的(24R)-5α-豆甾烷-3,6-二酮[(24R)-5α-stigmastane-3,6-dione]的數(shù)據(jù)基本一致,故鑒定化合物2為 (24R)-5α-豆甾烷-3,6-二酮[(24R)-5α-stigmastane-3,6-dione]。
化合物3 白色無定形粉末,分子式為C16H16O3。ESI-MS m/z: 257.11? [ M + H ]+。1H-NMR (600 MHz, CD3OD) δH: 7.53 (1H, d, J=8.5 Hz, H-6′), 7.22 (5H, m, H-2/3/4/5/6), 6.36 (1H, d, J = 1.9 Hz, H-3′), 6.40 (1H, dd, J = 8.5, 2.1 Hz, H-5′), 3.83 (3H, s, H-4′), 3.22 (2H, t, J = 7.6 Hz, H-α), 2.90 (2H, t, J = 7.6 Hz, H-β)。13C-NMR (150 MHz, CD3OD) δC: 201.8 (C=O), 164.8 (C-4′), 163.0 (C-2′), 143.1 (C-1), 133.6 (C-6′), 129.5 (C-2/3/5/6), 127.0 (C-4), 113.3 (C-1′), 108.9 (C-3′), 99.9 (C-5′), 56.0 (4′-OCH3), 46.2 (C-α), 32.2 (C-β)。以上數(shù)據(jù)與文獻(xiàn)(Edyta et al., 2017)報道的2′-羥基-4′-甲氧基二氫查爾酮(2′-hydroxy-4′-methoxydihydrochalcone)的數(shù)據(jù)基本一致,故鑒定化合物3為2′-羥基-4′-甲氧基二氫查爾酮(2′-hydroxy-4′-methoxydihydrochalcone)。
化合物4 白色針狀結(jié)晶,分子式為C30H50O。ESI-MS m/z: 427.39 [M + H]+。 1H-NMR (600 MHz, CDCl3) δH: 5.13 (1H, t, J = 3.7 Hz, H-12), 3.22 (1H, dd, J = 11.3, 4.8 Hz, H-3), 1.07 (3H, s, H-27), 1.01 (3H, s, H-26), 1.00 (3H, s, H-23), 0.95 (3H, d, J = 0.8 Hz, H-25), 0.91 (3H, s, H-30), 0.87 (3H, s, H-28), 0.80 (3H, s, H-24), 0.74 (3H, d, J = 11.8 Hz, H-29)。 13C-NMR (150 MHz, CDCl3) δC: 139.6 (C-13), 124.4 (C-12), 79.1 (C-3), 59.1 (C-18), 55.2 (C-5), 47.7 (C-9), 42.1 (C-14), 41.5 (C-22), 40.0 (C-8), 39.7 (C-19), 39.6 (C-20), 38.8 (C-1), 38.8 (C-4), 36.9 (C-10), 33.8 (C-17), 33.0 (C-7), 31.3 (C-21), 28.8 (C-15), 28.1 (C-23), 28.1 (C-28), 27.3 (C-2), 26.6 (C-16), 23.4 (C-11), 23.3 (C-27), 21.4 (C-29), 18.4 (C-6), 17.5 (C-30), 16.9 (C-26), 15.7 (C-25), 15.6 (C-24)。以上數(shù)據(jù)與文獻(xiàn)(莫青胡等,2020)報道的α-香樹脂醇(α-amyrin)的數(shù)據(jù)基本一致,故鑒定化合物4為α-香樹脂醇(α-amyrin)。
化合物5 白色粉末,分子式為C29H50O。ESI-MS m/z: 415.39? [M + H]+。 1H-NMR (600 MHz, CDCl3) δH: 5.35 (1H, m, H-6), 3.52 (1H, m, H-3), 1.01 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.84 (3H, d, J = 2.9 Hz, H-26), 0.83 (3H, m, H-29), 0.81 (3H, m, H-27), 0.69 (3H, s, H-18)。 13C-NMR (150 MHz, CDCl3) δC: 140.8 (C-5), 121.7 (C-6), 71.8 (C-3), 56.8 (C-14), 56.1 (C-17), 50.1 (C-9), 45.9 (C-24), 42.3 (C-4), 42.3 (C-13), 39.8 (C-12), 37.3 (C-1), 36.5 (C-10), 36.2 (C-20), 34.0 (C-22), 31.9 (C-7), 31.9 (C-8), 31.9 (C-2), 29.2 (C-25), 28.3 (C-16), 26.0 (C-23), 24.3 (C-15), 23.0 (C-28), 21.1 (C-11), 19.8 (C-26), 19.4 (C-19), 19.0 (C-27), 18.8 (C-21), 12.0 (C-29), 11.9 (C-18)。以上數(shù)據(jù)與文獻(xiàn)(查顯進(jìn)等,2021)中報道的β-谷甾醇(β-sitosterol)的數(shù)據(jù)基本一致,故鑒定化合物5為β-谷甾醇(β-sitosterol)。
化合物6 白色粉末,分子式為C30H48O3。ESI-MS m/z: 457.36? [M + H]+。1H-NMR (600 MHz, CD3OD) δH: 5.26 (1H, t, J = 3.7 Hz, H-12), 3.29 (1H, dd, J = 3.3, 1.6 Hz, H-3), 1.32 (3H, m, H-27), 1.30 (3H, s, H-29), 1.27 (3H, s, H-24), 1.16 (3H, s, H-23), 0.90 (3H, m, H-30), 0.83 (3H, s, H-25), 0.75 (3H, s, H-26)。13C-NMR (150 MHz, DMSO) δC: 179.3 (C-28), 138.9 (C-13), 127.1 (C-12), 78.2 (C-3), 55.9 (C-5), 53.6 (C-18), 48.1 (C-17), 48.1 (C-9), 42.6 (C-14), 40.1 (C-8), 39.6 (C-1), 39.5 (C-4), 39.4 (C-19), 39.2 (C-20), 37.5 (C-22), 37.4 (C-10), 33.7 (C-7), 31.1 (C-21), 28.8 (C-23), 28.8 (C-2), 28.2 (C-15), 25 (C-16), 24 (C-11), 23.7 (C-27), 21.4 (C-30), 18.8 (C-6), 17.5 (C-26), 17.5 (C-29), 16.5 (C-24), 15.7 (C-25)。以上數(shù)據(jù)與文獻(xiàn)(陳雪林等,2020)報道的熊果酸(ursolic acid)的數(shù)據(jù)基本一致,故鑒定化合物6為熊果酸(ursolic acid)。
化合物7 白色粉末,分子式為C8H8O4。ESI-MS m/z: 169.04? [M + H]+。 1H-NMR (600 MHz, CD3OD) δH: 7.56 (1H, d, J = 1.8 Hz, H-2), 7.55 (1H, d, J = 2.0 Hz, H-6), 6.86~6.81 (1H, m, H-5), 3.89 (3H, s, 8-OCH3)。 13C-NMR (150 MHz, CD3OD) δC: 167.6 (C-7), 152.7 (C-4), 148.8 (C-3), 125.3 (C-6), 123.5 (C-1), 115.9 (C-2), 114.1 (C-5), 56.6 (8-OCH3)。以上數(shù)據(jù)與文獻(xiàn)(李藥蘭等,2006)報道的對羥基間甲氧基苯甲酸(4-hydroxy-3-methoxy-benzoic acid)的數(shù)據(jù)基本一致,故鑒定化合物7為對羥基間甲氧基苯甲酸(4-hydroxy-3-methoxy-benzoic acid)。
化合物8 白色粉末,分子式為C5H5NO。ESI-MS m/z: 96.04? [M + H]+。 1H-NMR (600 MHz, DMSO-d6) δH: 9.76 (1H, s, 4-OH), 7.74 (2H, d, J = 8.6 Hz, H-2/6), 6.91 (2H, d, J = 8.2 Hz, H-3/5)。 13C-NMR (150 MHz, DMSO-d6) δC: 160.7 (C-4), 149.8 (C-2/6), 115.9 (C-3/5)。以上數(shù)據(jù)與文獻(xiàn) (王偉忠,2008)報道的4-羥基吡啶(4-pyridinol)的數(shù)據(jù)基本一致,故鑒定化合物8為4-羥基吡啶(4-pyridinol)。
化合物9 白色粉末,分子式為C7H6O3。ESI-MS m/z: 139.03? [M + H]+。 1H-NMR (600 MHz, CD3OD) δH: 7.87 (2H, d, J = 8.7 Hz, H-2/6), 6.80 (2H, d, J = 8.7 Hz, H-3/5)。 13C-NMR (150 MHz, CD3OD) δC: 171.6 (C-7), 163.1 (C-4), 133.0 (C-2/6), 124.1 (C-1), 116.0 (C-3/5)。以上數(shù)據(jù)與文獻(xiàn)(李艷茸等,2014)報道的對羥基苯甲酸(p-hydroxybenzoic acid)的數(shù)據(jù)基本一致,故鑒定化合物9為對羥基苯甲酸(p-hydroxybenzoic acid)。
化合物10 紅棕色固體,分子式為C34H44O19。ESI-MS m/z: 779.25? [M+Na]+。 1H-NMR (600 MHz, CD3OD) δH: 7.62 (1H, d, J = 15.8 Hz, Caf H-7″), 7.10 (1H, d, J = 2.0 Hz, Caf H-2″), 6.99 (1H, dd, J = 8.2, 2.1 Hz, Caf H-6″), 6.82 (1H, d, J = 8.2 Hz, Caf H-5″), 6.75~6.71 (2H, m, Agl H-2/5), 6.60 (1H, dd, J = 8.1, 2.1 Hz, Agl H-6), 6.31 (1H, d, J = 15.9 Hz, Caf H-8″), 5.21 (1H, d, J = 1.8 Hz, Rha H-1), 4.98 (1H, m, Glu H-4′), 4.94 (1H, d, J = 2.3 Hz, Api H-1′), 4.40 (1H, d, J = 7.9 Hz, Glu H-1′), 4.02 (1H, m, Agl H-8), 3.96~3.90 (2H, m, Rha H-2, Api H-4′a), 3.83 (1H, m, Glu H-3′), 3.80~3.69 (4H, m, Glu H-5′/6′, Api H-2′/4′b), 3.64~3.60 (1H, m, Rha H-3), 3.58 (2H, s, Api H-5′), 3.51 (1H, dd, J = 11.2, 5.7 Hz, Glu H-2′), 3.35~3.30 (2H, m, Rha H-4/5), 2.82 (2H, m, Agl H-7), 1.11 (3H, d, J = 6.2 Hz, Rha H-6)。 13C-NMR (150 MHz, CD3OD) δC: 168.3 (Caf C-9″), 149.8 (Caf C-4″), 148.2 (Caf C-7″), 146.8 (Caf C-3″), 146.1 (Agl C-3), 144.7 (Agl C-4), 131.6 (Agl C-1), 127.7 (Caf C-1″), 123.4 (Caf C-6″), 121.5 (Agl C-6), 117.3 (Agl C-2), 116.7 (Caf C-5″), 116.5 (Agl C-5), 115.4 (Caf C-2″), 114.8 (Caf C-8″), 111.1 (Api C-1′), 104.2 (Glu C-1′), 103.1 (Rha C-1), 81.8 (Glu C-3′), 80.7 (Api C-3′), 78.3 (Api C-2′), 76.2 (Glu C-2′), 75.2 (Api C-4′), 74.6 (Glu C-5′), 73.8 (Rha C-4), 72.4 (Rha C-2), 72.4 (Agl C-8), 72.1 (Rha C-3), 70.9 (Glu C-4′), 70.5 (Rha C-5), 68.5 (Glu C-6′), 65.7 (Api C-5′), 36.7 (Agl C-7), 18.5 (Rha C-6)。以上數(shù)據(jù)與文獻(xiàn)(Yamasaki et al., 2007)報道的連翹酯苷B (forsythoside B) 的數(shù)據(jù)基本一致,故鑒定化合物10為連翹酯苷B (forsythoside B)。
化合物11 黃綠色固體,分子式為C36H48O20。ESI-MS m/z: 823.27? [M +Na]+。 1H-NMR (600 MHz, DMSO-d6) δH: 7.54 (1H, d, J = 15.8 Hz, Acyl H-7″), 7.29 (1H, d, J = 2.0 Hz, Acyl H-2″), 7.09 (1H, dd, J = 8.2, 2.0 Hz, Acyl H-6″), 6.79 (1H, d, J = 8.1 Hz, Acyl H-5″), 6.70 (2H, d, J = 8.1 Hz, Agl H-2/5), 6.58 (1H, dd, J = 8.1, 2.1 Hz, Agl H-6), 6.42 (1H, d, J = 15.9 Hz, Acyl H-8″), 5.03 (1H, d, J = 1.7 Hz, Rha H-1), 4.76 (1H, d, J = 2.9 Hz, Api H-1′), 4.68 (1H, t, J = 9.7 Hz, Glu H-4′), 4.42 (1H, d, J = 7.9 Hz, Glu H-1′), 4.25 (1H, dd, J = 7.9, 3.8 Hz, Agl H-7), 3.80 (3H, s, 3″-OCH3), 3.78 (1H, s, Agl H-8a), 3.69 (3H, m, Glu H-5′, Rha H-2, Api H-2′), 3.62 (3H, m, Api H-3′/4′), 3.57~3.48 (3H, m, Agl H-8b, Glu H-2′/3′), 3.40~3.30 (2H, m, Glu H-6′, Rha H-5), 3.26 (3H, m, Rha H-3, Api H-5′), 3.13 (3H, s,7-OCH3), 3.09 (1H, d, J = 9.4 Hz, Rha H-4), 0.98 (3H, d, J = 6.2 Hz, Rha H-6)。 13C-NMR (150 MHz, DMSO-d6) δC: 165.8 (Acyl C-9″), 149.5 (Acyl C-3″), 147.9 (Acyl C-4″), 145.8 (Agl C-4), 145.2 (Acyl C-7″), 145.0 (Agl C-3), 129.7 (Agl C-1), 125.5 (Acyl C-1″), 123.3 (Acyl C-6″), 118.1 (Agl C-6), 115.5 (Agl C-5, Acyl C-5″), 114.1 (Agl C-2), 113.9 (Acyl C-8″), 111.1 (Acyl C-2″), 109.1 (Api C-1′), 102.8 (Glu C-1′), 101.3 (Rha C-1), 82.3 (Agl C-7), 78.9 (Api C-3′), 78.8 (Glu C-3′), 75.9 (Api C-2′), 74.3 (Glu C-2′), 73.7 (Agl C-8), 73.4 (Api C-4′), 72.8 (Glu C-5′), 71.7 (Rha C-4), 70.5 (Rha C-2), 70.4 (Rha C-3), 69.3 (Glu C-4′), 68.8 (Rha C-5), 67.1 (Glu C-6′), 63.2 (Api C-5′), 56.0 (7-OCH3), 55.6 (3″-OCH3), 18.1 (Rha C-6)。以上數(shù)據(jù)與文獻(xiàn)(Xu et al., 2019)報道的 nepetifosides D的數(shù)據(jù)基本一致,故鑒定化合物11為 nepetifosides D。
化合物12 橙紅色膏體,分子式為C29H36O15。ESI-MS m/z: 647.21? [M+Na]+。 1H-NMR (600 MHz, CD3OD) δH: 7.57 (1H, d, J = 15.8 Hz, Caf H-7″), 7.05 (1H, d, J = 2.1 Hz, Caf H-2″), 6.89 (1H, dd, J = 8.2, 2.1 Hz, Caf H-6″), 6.78 (1H, d, J = 8.2 Hz, Caf H-5″), 6.69 (1H, d, J = 2.0 Hz, Agl H-2), 6.65 (1H, d, J = 8.0 Hz, Agl H-5), 6.54 (1H, dd, J = 8.0, 2.0 Hz, Agl H-6), 6.30 (1H, d, J = 15.9 Hz, Caf H-8″), 5.20 (1H, d, J = 1.7 Hz, Rha H-1), 4.51 (1H, dd, J = 11.9, 2.2 Hz, Glu H-6a′), 4.39~4.36 (1H, m, Glu H-6b′), 4.34 (1H, d, J = 7.9 Hz, Glu H-1′), 4.04~3.93 (3H, m, Agl H-8b, Rha H-2/ 4), 3.75~3.69 (2H, m, Agl H-8a, Rha H-3), 3.58~3.55 (1H, m, Glu H-5′), 3.54 (1H, d, J = 8.9 Hz, Glu H-3′), 3.42 (1H, m, Glu H-4′), 3.35~3.30 (1H, m, Glu H-2′), 2.83~2.73 (2H, m, Agl H-7), 1.26 (3H, d, J = 6.2 Hz, Rha H-6)。13C-NMR (150 MHz, DMSO-d6) δC: 169.2 (Caf C-9″), 149.5 (Caf C-4″), 147.2 (Caf C-7″), 146.7 (Caf C-3″), 146.0 (Agl C-3), 144.6 (Agl C-4), 131.4 (Agl C-1), 127.6 (Caf C-1″), 123.2 (Caf C-6″), 121.3 (Agl C-6), 117.1 (Agl C-2), 116.5 (Agl C-5), 116.4 (Caf C-5″), 115.1 (Caf C-2″), 114.8 (Caf C-8″), 104.3 (Glu C-1′), 102.6 (Rha C-1), 83.9 (Glu C-3′), 75.6 (Glu C-2′), 75.3 (Glu C-5′), 73.9 (Rha C-4), 72.4 (Agl C-8), 72.3 (Rha C-3), 72.2 (Rha C-2), 70.3 (Glu C-4′), 70.0 (Rha C-5), 64.6 (Glu C-6′), 36.6 (Agl C-7), 17.9 (Rha C-6)。以上數(shù)據(jù)與文獻(xiàn)(Saimaru & Orihara, 2010)報道的異毛蕊花苷 (isoacteoside)的數(shù)據(jù)基本一致,故鑒定化合物12為異毛蕊花苷(isoacteoside)。
化合物13 橙紅色膏體,分子式為C29H36O15。ESI-MS m/z: 647.21? [M+Na]+。 1H-NMR (600 MHz, DMSO-d6) δH: 7.46 (1H, d, J = 15.8 Hz, Caf H-7″), 7.03 (1H, d, J = 2.0 Hz, Caf H-2″), 6.97 (1H, dd, J = 8.2, 2.1 Hz, Caf H-6″), 6.76 (1H, d, J = 8.1 Hz, Caf H-5″), 6.65~6.61 (2H, m, Agl H-2/5), 6.49 (1H, dd, J = 8.1, 2.1 Hz, Agl H-6), 6.20 (1H, d, J = 15.9 Hz, Caf H-8″), 4.72 (1H, m, Glu H-4′), 4.35 (1H, d, J = 7.9 Hz, Glu H-1′), 3.88 (1H, dd, J = 9.2, 6.4 Hz, Rha H-2), 3.72~3.07 (10H, m, Agl H-8, Rha/Glu-H), 2.76~2.63 (2H, m, Agl H-7), 0.96 (3H, d, J = 6.1 Hz, Rha H-6)。 13C-NMR (150 MHz, DMSO-d6) δC: 166.0 (Caf C-9″), 148.8 (Caf C-3″), 145.8 (Caf C-7″), 145.2 (Agl C-3, Caf C-4″), 143.8 (Agl C-4), 129.4 (Agl C-1), 125.7 (Caf C-1″), 121.7 (Caf C-6″), 119.8 (Agl C-6), 116.5 (Caf C-5″), 116.0 (Agl C-2), 115.7 (Agl C-5), 114.9 (Caf C-8″), 113.8 (Caf C-2″), 102.5 (Glu C-1′), 101.5 (Rha C-1), 79.3 (Glu C-3′), 74.7 (Glu C-5′), 74.7 (Glu C-2′), 71.9 (Rha C-4), 70.8 (Rha C-2), 70.6 (Rha C-3), 70.5 (Agl C-8), 69.4 (Rha C-5), 69.0 (Glu C-4′), 61.0 (Glu C-6′), 35.2 (Agl C-7), 18.4 (Rha C-6)。以上數(shù)據(jù)與文獻(xiàn)(Lan et al., 2018)報道的毛蕊花苷(acteoside)的數(shù)據(jù)基本一致, 故鑒定化合物13為毛蕊花苷(acteoside)。
化合物14 黃色固體,分子式為C35H46O19。ESI-MS m/z: 793.26? [M+Na]+。 1H-NMR (600 MHz, DMSO-d6) δH: 7.54 (1H, d, J = 15.8 Hz, Acyl H-7″), 7.29 (1H, d, J = 2.0 Hz, Acyl H-2″), 7.09 (1H, dd, J = 8.3, 2.0 Hz, Acyl H-6″), 6.79 (1H, d, J = 8.1 Hz, Acyl H-5″), 6.59 (2H, m, Agl H-2/5), 6.50 (1H, dd, J = 8.1, 2.1 Hz, Agl H-6), 6.41 (1H, d, J = 15.9 Hz, Acyl H-8″), 5.03 (1H, d, J = 1.7 Hz, Rha H-1), 4.78 (1H, d, J = 2.9 Hz, Api H-1′), 4.64 (1H, t, J = 9.7 Hz, Glu H-4′), 4.38 (1H, d, J = 7.9 Hz, Glu H-1′), 3.80 (3H, s, 3″-OCH3), 3.76~3.63 (7H, m, Glu H-3′/5′, Api H-2′/3′/4′, Rha H-2), 3.57~3.48 (2H, m, Agl H-8), 3.32~3.18 (6H, m, Api H-5′, Glu H-2′/6′, Rha H-3″/5) , 3.10 (1H, t, J = 9.4 Hz, Rha H-4), 2.80~2.65 (2H, m, Agl H-7), 0.98 (3H, d, J = 6.2 Hz, Rha H-6)。 13C-NMR (150 MHz, DMSO-d6) δC: 166.3 (Acyl C-9″), 150.0 (Acyl C-3″), 148.4 (Acyl C-4″), 146.2 (Acyl C-7″), 145.5 (Agl C-3), 144.0 (Agl C-4), 129.6 (Agl C-1), 126.0 (Acyl C-1″), 123.7 (Acyl C-6″), 120.0 (Agl C-6), 116.8 (Agl C-2), 116.0 (Agl C-5), 115.7 (Acyl C-8″), 114.3 (Acyl C-5″), 111.5 (Acyl C-2″), 109.6 (Api C-1′), 102.7 (Glu C-1′), 101.7 (Rha C-1), 79.3 (Glu C-3′, Api C-3′), 76.4 (Api C-2′), 74.8 (Glu C-2′), 73.9 (Api C-4′), 73.3 (Glu C-5′), 72.1 (Rha C-4), 71.0 (Rha C-2), 70.8 (Rha C-3), 70.8 (Agl C-8), 69.9 (Glu C-4′), 69.2 (Rha C-5), 67.6 (Glu C-6′), 63.6 (Api C-5′), 56.1 (3″-OCH3), 35.5 (Agl C-7), 18.6 (Rha C-6)。以上數(shù)據(jù)與文獻(xiàn)(Jia & Gao, 1993)報道的 pedicularioside M的數(shù)據(jù)基本一致,故鑒定化合物14為 pedicularioside M。
化合物15 黃綠色固體,分子式為C35H46O20。ESI-MS m/z: 809.26? [M+Na]+。 1H-NMR (500 MHz, DMSO-d6) δH: 7.47 (1H, d, J = 15.8 Hz, Caf H-7″), 7.03 (1H, d, J = 2.1 Hz, Caf H-2″), 6.98 (1H, dd, J = 8.3, 2.1 Hz, Caf H-6″), 6.76 (1H, d, J = 8.1 Hz, Caf H-5″), 6.71~6.68 (2H, m, Agl H-2/5), 6.58 (1H, dd, J = 8.0, 2.0 Hz, Agl H-6), 6.20 (1H, d, J = 15.9 Hz, Caf H-8″), 5.02 (1H, d, J = 1.5 Hz, Rha H-1), 4.76 (1H, d, J = 2.8 Hz, Api H-1′), 4.68 (1H, t, J = 9.7 Hz, Glu H-4′), 4.42 (1H, d, J = 7.8 Hz, Glu H-1′), 4.25 (1H, dd, J = 7.8, 3.9 Hz, Agl H-7), 3.79 (1H, d, J = 9.3 Hz, Agl H-8a), 3.72~3.57 (7H, m, Glu H-3′/5′, Api H-2′/3′/4′, Rha H-2), 3.57~3.46 (2H, m, Agl H-8b, Glu H-6′a), 3.36~3.17 (6H, m, Api H-5′, Glu H-2′/6′, Rha H-3/5), 3.13 (3H, d, J = 6.1 Hz,7-OCH3), 3.09 (1H, d, J = 9.4 Hz, Rha H-4), 0.96 (3H, d, J = 6.2 Hz, Rha H-6)。 13C-NMR (125 MHz, DMSO-d6) δC: 165.7 (Caf C-9″), 148.5 (Caf C-4″), 145.8 (Caf C-7″), 145.6 (Caf C-3″), 145.2 (Agl C-4), 145.0 (Agl C-3), 129.7 (Agl C-1), 125.5 (Caf C-1″), 121.4 (Caf C-6″), 118.1 (Agl C-6), 115.8 (Caf C-5″), 115.4 (Agl C-5), 114.8 (Caf C-2″), 114.0 (Agl C-2), 113.4 (Caf C-8″), 109.1 (Api C-1′), 102.8 (Glc C-1′), 101.2 (Rha C-1), 82.2 (Agl C-7), 78.9 (Glc C-3′), 78.8 (Api C-3′), 75.9 (Api C-2′), 74.3 (Glc C-2′), 73.7 (Agl C-8), 73.4 (Api C-4′), 72.7 (Glc C-5′), 71.6 (Rha C-4), 70.5 (Rha C-5), 70.4 (Rha C-3), 69.3 (Glc C-4′), 68.7 (Rha C-5), 67.0 (Glc C-6′), 63.2 (Api C-5′), 56.0 (7-OCH3), 18.1 (Rha C-6)。分析質(zhì)譜和NMR數(shù)據(jù),發(fā)現(xiàn)化合物15與連翹酯苷B的結(jié)構(gòu)相近(Wang et al., 2005),主要區(qū)別在于化合物15多了1個甲氧基結(jié)構(gòu)片段信號 [δH 3.13 (3H, d, J = 6.1 Hz,7-OCH3),δC 56.0 (7-OCH3)],以及由甲氧基引起的C-7位次甲基CH的化學(xué)位移向低場位移的信號δC 73.4 (Agl C-8),82.2 (Agl C-7),129.7 (Agl C-1), HMBC譜上可觀測δH 3.13 ( 3H, d, J = 6.1 Hz,7-OCH3 )與δC 82.2( Agl C-7 )遠(yuǎn)程相關(guān)的信號,進(jìn)一步確定甲氧基連在苯乙醇結(jié)構(gòu)片段Agl C-7位,故鑒定化合物15為 β-甲氧基連翹酯苷B (β-methoxy forsythoside B)。
4 討論與結(jié)論
本研究采用多種現(xiàn)代色譜分離技術(shù)和波譜鑒定手段從我國特有植物全緣葉紫珠的根、莖提取物中分離鑒定了15個化合物,包括3個甾體類(1、2、5),2個三萜類(4、6),6個苯乙醇苷類(10-15),1個黃酮類(3),2個苯甲酸類(7、9)和1個生物堿(8)。本研究結(jié)果表明,苯乙醇苷類化合物是全緣葉紫珠的特征性物質(zhì)成分,該類化合物是由苯乙醇苷元、咖啡?;⑵咸烟?鼠李糖/芹菜糖等糖基取代衍生而成的天然水溶性糖苷(Tian et al., 2021)。現(xiàn)代藥理研究表明,苯乙醇苷類物質(zhì)具有抗氧化、改善記憶、保肝、神經(jīng)保護、抗腫瘤等多種藥理作用,在治療阿爾茨海默病、改善記憶力等方面的發(fā)展?jié)摿τ葹橥怀觯↙ee et al., 2006)。紫珠屬植物作為常見的民間藥,其最早便是被用于止血。余婧等(2015)研究表明廣東紫珠發(fā)揮止血作用的主要成分為苯乙醇苷類,本研究發(fā)現(xiàn)的苯乙醇苷類化合物大多為首次從全緣葉紫珠中得到,其中化合物11和化合物14為首次從紫珠屬中報道。因此,本研究不僅豐富了全緣葉紫珠植物的次生代謝化合物庫,也為進(jìn)一步挖掘全緣葉紫珠中結(jié)構(gòu)新穎的止血活性成分及其藥理機制研究奠定了物質(zhì)基礎(chǔ)。
本研究結(jié)果發(fā)現(xiàn),全緣葉紫珠的石油醚萃取部位的化學(xué)成分主要為甾體、萜類等小極性物質(zhì),結(jié)合文獻(xiàn)研究發(fā)現(xiàn):α-香樹脂醇(化合物4)對白背飛虱雌蟲具有較好的殺滅效果;β-谷甾醇(化合物5)不僅對Aedes aegypti和 Culex quinquefasciatus 2種蚊蟲具有較好的殺滅效果(Chenniappan & Kadarkari, 2012; Angajala & Subashini, 2018),還對煙曲霉菌、黑曲霉菌、根霉菌的生長有一定的抑制作用(Prince et al., 2019);熊果酸(化合物6)可通過減少浮游細(xì)菌在表面的附著力和破壞其生物膜的形成達(dá)到抑菌作用(Sycz et al., 2022);對羥基苯甲酸(化合物9)通過促進(jìn)氧自由基的產(chǎn)生,抑制銅綠微囊藻的生長(張庭廷等,2008)。因此,我們推測全緣葉紫珠脂溶性物質(zhì)在植物病害防控方面具有一定的應(yīng)用前景,為進(jìn)一步開發(fā)和利用該植物提供了一定的科學(xué)依據(jù)。
2020版《中國藥典》收錄的紫珠屬物種包括廣東紫珠、裸花紫珠、大葉紫珠、杜虹花,它們均為常見的喬木型或灌木型類群,這些類群往往喜歡生長在路旁、林緣等易受外界干擾的向陽環(huán)境,而全緣葉紫珠作為紫珠屬僅有的幾個藤本型類群之一,往往生活在林下環(huán)境,通??颗示壠渌参锏靡垣@得陽光和生長空間。這些特定的林下環(huán)境脅迫是否影響全緣葉紫珠次生代謝活動?本研究表明全緣葉紫珠中的15個化合物中就有6個化合物為紫珠屬首次報道,這些意外的發(fā)現(xiàn)將激勵著我們進(jìn)一步加深對全緣葉紫珠天然產(chǎn)物成分的挖掘和藥理活性研究。
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(責(zé)任編輯 鄧斯麗 周翠鳴)
收稿日期:? 2022-05-03
基金項目:? 國家自然科學(xué)基金(31760045,31970220); 廣西自然科學(xué)基金(2018GXNSFAA281132,2018GXNSFAA281146); 廣西甘蔗生物學(xué)重點實驗室開放課題項目(GXKLSCB-202004)。
第一作者: 馬文杰(1996-),碩士研究生,主要從事藥用植物天然產(chǎn)物化學(xué)研究,(E-mail)mawenjie1018@163.com。
*通信作者:? 蘇志維,博士,研究員,碩士研究生導(dǎo)師,主要從事天然產(chǎn)物化學(xué)等研究,(E-mail)suzw@gxtcmu.edu.cn。