黑角珊瑚（海柳）挥发性成分提取及生物活性研究
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摘要
潮汕地区,位于我国东南部,濒临南海,地理位置独特,海洋资源丰富。很多海洋生物不仅可食用,因其本身的功效还可作为药用。我国自古就有食用海产、应用海洋药材的历史。潮汕民间亦有不少特色有效的海洋物种。
海柳(BC):别名角珊瑚,其外表虽与陆地上的柳树相似,但实则属于腔肠动物门、珊瑚纲(六放珊瑚亚纲)、角珊瑚目、黑角珊瑚科、黑角珊瑚属、日本黑角珊瑚Antipathes japonica Brook,多小枝黑角珊瑚Antipathes virgata Esper的群体分枝。在潮汕地区,海柳Antipathes japonica Brook作为特有的海洋生物材料,广泛用于烟嘴烟斗的加工制作中,且别有功效。历代本草都有关于海柳药用价值的记载,而目前对于其所含成分及生物活性的报道很少。本论文的目的是提取海柳挥发性成分(BCE),研究其生物活性,为BC进一步研发打下药理学基础。
方法:
1.采用二氧化碳超临界萃取(CO2-SFE)海柳烟嘴中挥发性成分。
2.运用气质联用(GC-MS)结合保留指数计算对提取的挥发性物质进行初步鉴定。
3.通过电感耦合等离子体发射光谱(ICPS)同时测定海柳中所含多种无机元素。
4. DPPH法、邻菲罗啉法体外测定BCE的抗氧化活性。
5. Kirby–Bauer (KB)纸片扩散法测试BCE抗菌敏感性;微量肉汤稀释法最小抑菌浓度(MIC)测定。
6.小鼠雾化吸入BCE(0.3mg/ml、1.5mg/ml、3mg/ml)后暴露于香烟烟雾中,复制急性肺损伤的动物模型。阳性药组雾化吸入Vc(3mg/ml),对照组和吸烟组(CS)雾化吸入生理盐水(NS)。实验结束后取肺组织,观察肺组织病理形态学变化,剩余组织匀浆后检测超氧化物歧化酶(SOD)和髓过氧化物酶(MPO)的活力及丙二醛(MDA)的含量。
结果:
1. BCE通过GC-MS分析,共鉴定出15种化学成分包括:磷酸三乙酯、丁羟甲苯、雪松醇、棕榈酸、角鲨烯、胆甾醇等。
2. ICPS测出海柳中所含P, Ca, Mg, S, B, Si, Fe, Cu, Zn, Ba等13种无机元素。
3.体外抗氧化实验中BCE有清除自由基DPPH和羟自由基作用。
4. BCE可以抑制革兰氏阳性菌:金黄色葡萄球菌S. aureus和表皮葡萄球菌S. epidermidis,对革兰氏阴性菌没有明显作用。测定BCE对该两种细菌的最小抑菌浓度MIC分别是:S. aureus 73μg/μl;S. epidermidis 37μg/μl。
5.肺组织病理学显示:急性肺损伤组小鼠肺组织呈现典型的炎症病理变化,包括肺泡充血,肺泡腔和血管壁中性粒细胞浸润,肺泡壁增厚等肺损伤性病变.BCE雾化吸入能够明显减轻肺损伤程度。给药组各剂量与模型组相比均能降低MPO和MDA水平,提高SOD的活性(P<0.05或P<0.01),且三个剂量呈量效关系。
结论:
首次提取了BCE,并研究了BCE的生物活性。通过体内、体外实验证明BCE所具有抗氧化活性、抗炎作用和抑菌能力。BCE对吸烟所致的急性呼吸道损伤具有一定保护作用。
Chaoshan area is located on the southeast coast of China in the subtropical zone. It has distinctive monsoon climates. The advantageous geographical location indicates the gestation of various species from sea to land. Some of them are used as traditional medicines in folk for treating some diseases.
Hailiu (Antipathes japonica Brook), one genera of black corals (BC), has some pharmaceutical functions used in Chinese folk, especially used as marine biotical materials for cigarette holders in Chaoshan area. At present, there was little known about the chemical constituents extracted of Hailiu. This study aimed to investigate the bioactivities of volatile constituents in BC and to explore the possible benefits of black corals cigarette holders (BCCHs).
Method:
1. The volatile constituents of BC were extracted by supercritical fluid extraction (SFE) with carbon dioxide (CO_2–SFE).
2. The BC extract (BCE) identified and analyzed the constituents by gas chromatography–mass spectrometry (GC–MS) combined with retention index.
3. Inorganic elements of BC were determined by inductively coupled plasma spectrometer (ICPS).
4. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) method and Phenanthroline-Fe2+ oxidative assay of hydroxyl radical were applied to evaluate in vitro antioxidation of BCE.
5. Antimicrobial susceptibility testing for BCE involved the Kirby–Bauer (KB) disk diffusion method. And a broth microdilution method was used to determine the minimal inhibitory concentration (MIC) against the susceptible microorganisms.
6. 60 mice were randomized into 6 groups. Before they were exposed to cigarette smoke (CS) in treatment groups mice were given BCE ultrasonic atomizing inhalation of 0.3mg/ml, 1.5mg/ml and 3mg/ml in normal saline (NS), respectively, for 10 minutes. While control group and CS group mice were treated NS,positive control group was administrated 6mg/ml of Vitamin C (Vc). All animals were euthanized via anesthetic overdose ip. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO) were analyzed. Histological and morphological studies of right upper lung were performed.
Result:
1. In total, 15 components were reliably identified in BC and found to be biologically active. These included triethyl phosphate, butylated hydroxytoluene, cedrol, n-hexadecanoic acid, squalene, and cholesterol.
2. 13 inorganic elements P, Ca, Mg, S, B, Si, Fe, Cu, Zn, Ba ect were determined by ICPS.
3. In the bioactivities tests, BCE showed a scavenging activity of DPPH and hydroxyl radicals.
4. The BCE exhibited moderate antimicrobial activity against the Gram-positive microorganisms. The results of the MIC determination indicated the BCE inhibited the microorganisms tested. S. aureus and S. epidermidis had a MIC of 73 and 37μg/μl, respectively.
5. Histopathological evaluation: Typical pathological inflammation lesions in the lung were observed in acute lung injury group,including alveolar congestion,hemorrhage,infiltration of neutrophils in the airspace or vessel wall,thickness of the alveolar wall. Pathological changes were relieved obviously in BCE treated groups. Compared with normal group,MPO and MDA in acute lung injury group were all higher(P<0.01),SOD was lower;MPO and MDA in BCE groups were all lower than acute lung injury model group (P<0.05 or P<0.01),SOD was higher.Meanwhile the BCE showed the dose dependent.
Conclution:
It is the first time to present some volatile constituents of BC. The functions of BC cigarette holders (BCCHs) are related to the active chemical compositions. The antioxidant and antimicrobial activities of BC from in vitro and in vivo may be protective for cigarette smokers who use BCCHs, especially for acute lung injury.
海柳(BC):别名角珊瑚,其外表虽与陆地上的柳树相似,但实则属于腔肠动物门、珊瑚纲(六放珊瑚亚纲)、角珊瑚目、黑角珊瑚科、黑角珊瑚属、日本黑角珊瑚Antipathes japonica Brook,多小枝黑角珊瑚Antipathes virgata Esper的群体分枝。在潮汕地区,海柳Antipathes japonica Brook作为特有的海洋生物材料,广泛用于烟嘴烟斗的加工制作中,且别有功效。历代本草都有关于海柳药用价值的记载,而目前对于其所含成分及生物活性的报道很少。本论文的目的是提取海柳挥发性成分(BCE),研究其生物活性,为BC进一步研发打下药理学基础。
方法:
1.采用二氧化碳超临界萃取(CO2-SFE)海柳烟嘴中挥发性成分。
2.运用气质联用(GC-MS)结合保留指数计算对提取的挥发性物质进行初步鉴定。
3.通过电感耦合等离子体发射光谱(ICPS)同时测定海柳中所含多种无机元素。
4. DPPH法、邻菲罗啉法体外测定BCE的抗氧化活性。
5. Kirby–Bauer (KB)纸片扩散法测试BCE抗菌敏感性;微量肉汤稀释法最小抑菌浓度(MIC)测定。
6.小鼠雾化吸入BCE(0.3mg/ml、1.5mg/ml、3mg/ml)后暴露于香烟烟雾中,复制急性肺损伤的动物模型。阳性药组雾化吸入Vc(3mg/ml),对照组和吸烟组(CS)雾化吸入生理盐水(NS)。实验结束后取肺组织,观察肺组织病理形态学变化,剩余组织匀浆后检测超氧化物歧化酶(SOD)和髓过氧化物酶(MPO)的活力及丙二醛(MDA)的含量。
结果:
1. BCE通过GC-MS分析,共鉴定出15种化学成分包括:磷酸三乙酯、丁羟甲苯、雪松醇、棕榈酸、角鲨烯、胆甾醇等。
2. ICPS测出海柳中所含P, Ca, Mg, S, B, Si, Fe, Cu, Zn, Ba等13种无机元素。
3.体外抗氧化实验中BCE有清除自由基DPPH和羟自由基作用。
4. BCE可以抑制革兰氏阳性菌:金黄色葡萄球菌S. aureus和表皮葡萄球菌S. epidermidis,对革兰氏阴性菌没有明显作用。测定BCE对该两种细菌的最小抑菌浓度MIC分别是:S. aureus 73μg/μl;S. epidermidis 37μg/μl。
5.肺组织病理学显示:急性肺损伤组小鼠肺组织呈现典型的炎症病理变化,包括肺泡充血,肺泡腔和血管壁中性粒细胞浸润,肺泡壁增厚等肺损伤性病变.BCE雾化吸入能够明显减轻肺损伤程度。给药组各剂量与模型组相比均能降低MPO和MDA水平,提高SOD的活性(P<0.05或P<0.01),且三个剂量呈量效关系。
结论:
首次提取了BCE,并研究了BCE的生物活性。通过体内、体外实验证明BCE所具有抗氧化活性、抗炎作用和抑菌能力。BCE对吸烟所致的急性呼吸道损伤具有一定保护作用。
Chaoshan area is located on the southeast coast of China in the subtropical zone. It has distinctive monsoon climates. The advantageous geographical location indicates the gestation of various species from sea to land. Some of them are used as traditional medicines in folk for treating some diseases.
Hailiu (Antipathes japonica Brook), one genera of black corals (BC), has some pharmaceutical functions used in Chinese folk, especially used as marine biotical materials for cigarette holders in Chaoshan area. At present, there was little known about the chemical constituents extracted of Hailiu. This study aimed to investigate the bioactivities of volatile constituents in BC and to explore the possible benefits of black corals cigarette holders (BCCHs).
Method:
1. The volatile constituents of BC were extracted by supercritical fluid extraction (SFE) with carbon dioxide (CO_2–SFE).
2. The BC extract (BCE) identified and analyzed the constituents by gas chromatography–mass spectrometry (GC–MS) combined with retention index.
3. Inorganic elements of BC were determined by inductively coupled plasma spectrometer (ICPS).
4. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) method and Phenanthroline-Fe2+ oxidative assay of hydroxyl radical were applied to evaluate in vitro antioxidation of BCE.
5. Antimicrobial susceptibility testing for BCE involved the Kirby–Bauer (KB) disk diffusion method. And a broth microdilution method was used to determine the minimal inhibitory concentration (MIC) against the susceptible microorganisms.
6. 60 mice were randomized into 6 groups. Before they were exposed to cigarette smoke (CS) in treatment groups mice were given BCE ultrasonic atomizing inhalation of 0.3mg/ml, 1.5mg/ml and 3mg/ml in normal saline (NS), respectively, for 10 minutes. While control group and CS group mice were treated NS,positive control group was administrated 6mg/ml of Vitamin C (Vc). All animals were euthanized via anesthetic overdose ip. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO) were analyzed. Histological and morphological studies of right upper lung were performed.
Result:
1. In total, 15 components were reliably identified in BC and found to be biologically active. These included triethyl phosphate, butylated hydroxytoluene, cedrol, n-hexadecanoic acid, squalene, and cholesterol.
2. 13 inorganic elements P, Ca, Mg, S, B, Si, Fe, Cu, Zn, Ba ect were determined by ICPS.
3. In the bioactivities tests, BCE showed a scavenging activity of DPPH and hydroxyl radicals.
4. The BCE exhibited moderate antimicrobial activity against the Gram-positive microorganisms. The results of the MIC determination indicated the BCE inhibited the microorganisms tested. S. aureus and S. epidermidis had a MIC of 73 and 37μg/μl, respectively.
5. Histopathological evaluation: Typical pathological inflammation lesions in the lung were observed in acute lung injury group,including alveolar congestion,hemorrhage,infiltration of neutrophils in the airspace or vessel wall,thickness of the alveolar wall. Pathological changes were relieved obviously in BCE treated groups. Compared with normal group,MPO and MDA in acute lung injury group were all higher(P<0.01),SOD was lower;MPO and MDA in BCE groups were all lower than acute lung injury model group (P<0.05 or P<0.01),SOD was higher.Meanwhile the BCE showed the dose dependent.
Conclution:
It is the first time to present some volatile constituents of BC. The functions of BC cigarette holders (BCCHs) are related to the active chemical compositions. The antioxidant and antimicrobial activities of BC from in vitro and in vivo may be protective for cigarette smokers who use BCCHs, especially for acute lung injury.
引文
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[7]张森林,毛天球.应用珊瑚/骨髓基质细胞/富血小板血浆组织工程骨修复兔颅骨缺损.中国口腔颌面外科杂志, 2010, 8 (1): 60-65.
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[11] Liang Li, Chang-Yun Wang, Chang-Lun Shao, Yue-Wei Guo, Guo-Qiang Li, Xue-Ping Sun, Lei Han, Hui Huang, Hua-Shi Guan. Sarcoglycosides A–C, new O-glycosylglycerol derivatives from the South China Sea soft coral Sarcophyton infundibuliform. Helv. Chim. Acta, 2009, 92: 1495-1502.
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[24]肖宗苗,王仁镇,李文旭.黑角珊瑚治疗骨伤疾病的临床观察.海南医学院学报, 2005, 2:135.
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[1]管华诗,耿美玉,王长云. 21世纪,中国的海洋药物.中国海洋药物, 2000, 4: 44-47.
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[4]艾小红;陈亿新;漆淑华;中国珊瑚化学成分与生物活性研究新进展广州大学学报(自然科学版), 5(1):49-56.
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[7]张森林,毛天球.应用珊瑚/骨髓基质细胞/富血小板血浆组织工程骨修复兔颅骨缺损.中国口腔颌面外科杂志, 2010, 8 (1): 60-65.
[8] Weinheimer A J, Spraggins R L. The occurrence of two new prostaglandin derivatives ( 15-epi-PGA2 and its acetate,methyl ester ) in the gorgonian Plexaura homomalla chemistry of coelenterates. XV [J]. Tetrahedron lett, 1969, 10: 5185-5188.
[9] Zhang W, Guo Y W, Gu Y C. Secondary metabolites from the South China Sea invertebrates: chemistry and biological activity [J]. Current Med.Chem, 2006, 13: 2041-2090.
[10] Lei Han, Chang-Yun Wang, Hui Huang, Chang-Lun Shao, Qing-Ai Liu, Jun Qi,Xue-Ping Sun, Peng Zhai, Yu-Cheng Gu. A new pregnane analogue from Hainan soft coral Scleronephthya gracillimum Kukenthal. Biochem. Syst. Ecol., 2010, 38: 243-246.
[11] Liang Li, Chang-Yun Wang, Chang-Lun Shao, Yue-Wei Guo, Guo-Qiang Li, Xue-Ping Sun, Lei Han, Hui Huang, Hua-Shi Guan. Sarcoglycosides A–C, new O-glycosylglycerol derivatives from the South China Sea soft coral Sarcophyton infundibuliform. Helv. Chim. Acta, 2009, 92: 1495-1502.
[12] Liang Li, Chang-Yun Wang, Chang-Lun Shao, Lei Han, Xue-Ping Sun, Jie Zhao, Yue-Wei Guo, Hui Huang, Hua-Shi Guan. Two new metabolites from the Hainan soft coral Sarcophyton crassocaule. J. Asian Nat. Prod. Res., 2009, 11(10): 851–855.
[13] Qiu Y, Qi S, Zhang S, Yang J, Xiao Z. New polyoxygenated steroids from the South China Sea gorgonian Echinogorgia aurantiaca. Pharmazie, 2006, 61(7):645-7.
[14] Qi S, Zhang S, Huang J, Xiao Z, Wu J, Li Q. Complete 1H and 13C NMR assignments of four new steroidal glycosides from a gorgonian coral Junceella juncea. Magn Reson Chem, 2005, 43(3):266-8.
[15] Yan P, Lv Y, van Ofwegen L, Proksch P, Lin W. Lobophytones A-G, new isobiscembranoids from the soft coral Lobophytum pauciflorum. Org Lett. 2010, 12(11):2484-7.
[16] Li Y, Carbone M, Vitale RM, Amodeo P, Castelluccio F, Sicilia G, Mollo E, Nappo M, Cimino G, Guo YW, Gavagnin M. Rare casbane diterpenoids from the Hainan soft coral Sinularia depressa. J Nat Prod, 2010, 26;73(2):133-8.
[17] Yan XH, Liu HL, Guo YW. Ximaosteroids A-D, new steroids from the Hainan soft coral Scleronephthya sp. Steroids, 2009, 74(13-14):1061-5.
[18] Yan XH, Liu HL, Huang H, Li XB, Guo YW. Steroids with Aromatic A-Rings from the Hainan Soft Coral Dendronephthya studeri Ridley. J Nat Prod, 2011,25;74(2):175-180.
[19] Aiello A, Fattorusso E, Menna M. Five new polar sterols from the black coral Antipathes subpinnata. Steroids, 1991, 56(10):513-517.
[20] Aiello A, Fattorusso E, Menna M. Four new bioactive polyhydroxylated sterols from the black coral Antipathes subpinnata. J Nat Prod, 1992, 55(3):321-325.
[21] Qi SH, Su GC, Wang YF, Liu QY, Gao CH. Alkaloids from the South China Sea Black Coral Antipathes dichotoma. Chem Pharm Bull (Tokyo), 2009, 57(1):87-88.
[22]苏国琛,张偲,漆淑华.黑角珊瑚的化学成分研究.中草药, 2008,39(11):1606-1609.
[23]葛洁虹,高程海,王萍,文良娟,漆淑华.南海二叉黑角珊瑚化学成分研究.中药材, 2010, 33(9):1403-1405
[24]肖宗苗,王仁镇,李文旭.黑角珊瑚治疗骨伤疾病的临床观察.海南医学院学报, 2005, 2:135.