-
槟榔(Areca catechu L.),别名傧郎、宾门等,是棕榈科多年生常绿乔木,原产马来西亚,广泛分布于南亚、东南亚[1]。我国主要栽种于海南岛、台湾、广东、广西和福建等地。槟榔是一种具有较高药用价值的中药,位居我国“四大南药”之首[2],主要用于治疗寄生虫病、消化不良、腹胀、腹痛、腹泻、水肿和黄疸等疾病[3]。同时,槟榔也是深受消费者喜爱的咀嚼食品[4]。据估计,在中国、南亚和东非等国家和地区,每天有超过4亿人以不同的食用方式咀嚼槟榔[5-6]。然而,咀嚼槟榔也具有一定的健康风险,例如嚼食槟榔与口腔癌关系密切[7],槟榔果的水提物和槟榔碱能显著地上调口腔黏膜成纤维细胞中c-jun原癌基因的表达[8],槟榔果的水提物能通过上调细胞间黏附分子−1(ICAM-1)的表达,而对体外培养的人类口腔黏膜成纤维细胞产生明显的增生作用,从而导致口腔黏膜下纤维化[9]。
槟榔的促消化、抗氧化、抗寄生虫、抗抑郁等药用活性与其包含的多种代谢产物密切相关[10]。槟榔中的化学成分主要包括糖类、脂质、蛋白质、粗纤维、多酚,同时包含0.2%~1.7%的生物碱[11]。PENG等[1]鉴定了槟榔中的59种化合物,包括生物碱、类黄酮、单宁、三萜和脂肪酸等,并发现吡啶类生物碱和缩合单宁是槟榔中的特征代谢物。槟榔中的吡啶类生物碱主要有槟榔碱、槟榔次碱、去甲基槟榔碱和去甲基槟榔次碱等4种,另外,还包含槟榔副碱、烟酸、烟酸甲酯、烟酸乙酯、N−甲基−1,2,5,6−四氢吡啶−3−羧酸乙酯、N−甲基哌啶−3−羧酸甲酯、N−甲基哌啶−3−羧酸乙酯、异去甲基槟榔次碱、高槟榔碱等9种生物碱[1]。近年来,随着分离手段的改进和检测技术的提高,TANG等[12]从槟榔中发现了3种新的生物碱,Arecatemine A、Arecatemine B和Arecatemine C,CAO等[13]报道了2种新的生物碱,分别为3−羧甲基丁酯吲哚1−N−β−D−吡喃葡萄糖苷和3−甲氧基−4−羟基苯基6−O−(2−氨基−1−氧代−3−苯基丙基)−β−D−吡喃葡萄糖苷。
目前,对槟榔碱的研究和报道主要集中在槟榔碱的药理、毒理活性及化学工艺合成上[14-16],其合成通路尚不清楚,这制约了对槟榔碱代谢调控与进化分子机理的深入研究,也阻碍了食用型低槟榔碱含量槟榔和药用型高槟榔碱含量槟榔的选育。代谢组学是研究物质合成通路的有效手段[17]。代谢组学本质上是指某一生物、组织或细胞中的所有低分子量代谢产物进行定性与定量分析的一门科学[18]。代谢组学研究中较常见的分析手段有色谱法、质谱法、核磁共振波谱法、紫外吸收光谱法和红外吸收光谱法等[19]。应用最广泛、最有效的是气相色谱−质谱(GC-MS)和液相色谱−质谱(LC-MS)[20]。色谱与质谱联用实现了从利用色谱进行物质分离到利用质谱进行物质鉴定的整个流程。其中,LC-MS能分析极性与相对分子质量较高及热稳定性差的化合物,具有前处理简单、检测范围广和检测效率高等优势,是现阶段代谢组学最常用的检测手段[21]。LC-MS检测的优势使其适合作为代谢通路解析的工具。因此,可利用基于LC-MS的代谢组学研究槟榔代谢物,并解析槟榔碱的合成通路。
本研究利用HPLC-OBITRAP-MS对槟榔各组织进行了非靶向代谢组检测,获得了大量的代谢信号,通过对代谢信号中二级碎片的结构注释,鉴定了超过150种代谢物,首次发现槟榔中存在糖基化的槟榔次碱。通过对代谢数据的定量分析,发现了氨基酸和生物碱空间分布的特异性。通过查阅槟榔碱结构类似物天然合成途径的文献,参考KEGG数据库中的信息,再结合实验获得的代谢物数据,推测了槟榔碱的可能合成途径,旨在为进一步探究槟榔碱的物质结构、来源、槟榔药理活性和毒理机制等提供参考。
Spatial Distribution of Arecoline Synthesis Precursors and Analysis of Arecoline Synthesis Pathway
-
摘要: 为探究槟榔中的生物活性成分,本研究以不同组织的槟榔为材料,利用高效液相色谱−质谱联用技术进行了非靶向代谢组学检测,经过定性分析,共获得了158个代谢物(包括氨基酸、脂质、核苷酸、生物碱和类黄酮等物质),首次在槟榔中发现槟榔次碱己糖苷的存在,并对氨基酸和生物碱进行了相对定量,发现芳香族氨基酸和支链氨基酸主要分布在种子中;烟酸衍生物在叶片中含量较高,槟榔次碱己糖苷和去甲基槟榔碱在外果皮中含量较高,其余生物碱均显示出在果实中尤其是种子中有较多的积累。基于烟酸、葫芦巴碱和槟榔碱的结构相似性,推测槟榔碱的合成通路是以天冬氨酸为起点,以烟酸、葫芦巴碱为基本骨架进行合成的。Abstract: To discover the bioactive components in Areca catechu L., metabolome of A. catechu L. was profiled by using high-performance liquid chromatography-mass spectrometry spectrometry-based on non-target metabolomics. A total of 158 metabolites were quantified, including amino acids, lipids, nucleotides, alkaloidsalkaloids, and flavonoids, and arecaidineacecainide hexosidehexoxide was discovered in A. catechu L. for the first time. The relative quantification of amino acids and alkaloids showed that aromatic amino acids and branched-chain amino acids were mainly distributed in the seeds of A. catechu L. Niacin derivatives were higher in the leaves; arecaidine hexoside and guvacoline were higher in the exocarp of the fruit; other alkaloids were highly accumulated in the fruits, especially in the seeds. According to the structural similarity of niacin, trigonelline, and arecoline, the synthesis pathway of arecoline is speculated as that L-aspartic acid is used as the starting point, and that niacin and trigonelline are used as the basic skeleton for the synthesis.
-
Key words:
- Areca catechu L. /
- arecoline /
- metabolome /
- spatial distribution /
- metabolic pathway
-
-
[1] PENG W, LIU Y J, WU N, et al. Areca catechu L. (Arecaceae): A review of its traditional uses, botany, phytochemistry, pharmacology and toxicology [J]. Journal of Ethnopharmacology, 2015, 164: 340 − 356. doi: 10.1016/j.jep.2015.02.010 [2] 曾琪. 槟榔化学成分的研究[D]. 长沙: 中南林业科技大学, 2007. [3] 国家药典委员会. 中华人民共和国药典[M]. 北京: 中国医药科技出版社, 2015. [4] HEATUBUN C D, DRANSFIELD J, FLYNN T, et al. A monograph of the betel nut palms (Areca: Arecaceae) of East Malaysia [J]. Botanical Journal of the Linnean Society, 2012, 168(2): 147 − 173. doi: 10.1111/j.1095-8339.2011.01199.x [5] GILANI A H, GHAYUR M N, SAIFY Z S, et al. Presence of cholinomimetic and acetylcholinesterase inhibitory constituents in betel nut [J]. Life Sciences, 2004, 75(20): 2377 − 2389. doi: 10.1016/j.lfs.2004.03.035 [6] 易攀, 汤嫣然, 周芳, 等. 槟榔的化学成分和药理活性研究进展[J]. 中草药, 2019, 50(10): 2498 − 2504. doi: 10.7501/j.issn.0253-2670.2019.10.034 [7] ZHANG X L, REICHART P A. A review of betel quid chewing, oral cancer and precancer in Mainland China [J]. Oral Oncology, 2007, 43(5): 424 − 430. doi: 10.1016/j.oraloncology.2006.08.010 [8] HO T J, CHIANG C P, HONG C Y, et al. Induction of the c-jun protooncogene expression by areca nut extract and arecoline on oral mucosal fibroblasts [J]. Oral Oncology, 2000, 36(5): 432 − 436. doi: 10.1016/S1368-8375(00)00031-2 [9] 冯云枝, 凌天牖. 槟榔提取物对口腔黏膜成纤维细胞表达细胞间粘附分子-1的影响[J]. 华西口腔医学杂志, 2002, 20(4): 241 − 243. doi: 10.3321/j.issn:1000-1182.2002.04.003 [10] 刘东林, 王小莹, 杨冰, 等. 槟榔药理毒理研究进展[J]. 中国中药杂志, 2013, 38(14): 2273 − 2275. [11] CHAVAN Y V, SINGHAL R S. Separation of polyphenols and arecoline from areca nut (Areca catechu L.) by solvent extraction, its antioxidant activity, and identification of polyphenols [J]. Journal of the Science of Food and Agriculture, 2013, 93(10): 2580 − 2589. doi: 10.1002/jsfa.6081 [12] TANG S N, ZHANG J, LIU D, et al. Three new areca alkaloids from the nuts of Areca catechu [J]. Journal of Asian Natural Products Research, 2017, 19(12): 1155 − 1159. doi: 10.1080/10286020.2017.1307187 [13] CAO M, YUAN H, DANIYAL M, et al. Two new alkaloids isolated from traditional Chinese medicine Binglang the fruit of Areca catechu [J]. Fitoterapia, 2019, 138(12): 104276. [14] GIRI S, IDLE J R, CHEN C, et al. A Metabolomic approach to the metabolism of the areca nut alkaloids arecoline and arecaidine in the mouse [J]. Chemical Research in Toxicology, 2006, 19(6): 818 − 827. doi: 10.1021/tx0600402 [15] BHANDARE A M, KSHIRSAGAR A D, VYAWAHARE N S, et al. Potential analgesic, anti-inflammatory and antioxidant activities of hydroalcoholic extract of Areca catechu L. nut [J]. Food and Chemical Toxicology, 2010, 48(12): 3412 − 3417. doi: 10.1016/j.fct.2010.09.013 [16] KIM H J, KO J W, CHA S B, et al. Evaluation of 13-week repeated oral dose toxicity of Areca catechu in F344/N rats [J]. Food and Chemical Toxicology, 2018, 114: 41 − 51. doi: 10.1016/j.fct.2018.02.015 [17] ROGACHEV I, AHARONI A. UPLC-MS-based metabolite analysis in tomato [J]. Methods in Molecular Biology, 2012, 860: 129 − 144. [18] SAITO K, MATSUDA F. Metabolomics for functional genomics, systems biology, and biotechnology [J]. Annual Review of Plant Biology, 2010, 61(1): 463 − 489. doi: 10.1146/annurev.arplant.043008.092035 [19] YANG Z, NAKABAYASHI R, OKAZAKI Y, et al. Toward better annotation in plant metabolomics: isolation and structure elucidation of 36 specialized metabolites from Oryza sativa (rice) by using MS/MS and NMR analyses [J]. Metabolomics, 2014, 10(4): 543 − 555. doi: 10.1007/s11306-013-0619-5 [20] FORCAT S, BENNETT M H, MANSFIELD J W, et al. A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress [J]. Plant Methods, 2008, 4(1): 16. doi: 10.1186/1746-4811-4-16 [21] LIN L Z, HARNLY J M. A screening method for the identification of glycosylated flavonoids and other phenolic compounds using a standard analytical approach for all plant materials [J]. Journal of Agriculture and Food Chemistry, 2007, 55(4): 1084 − 1096. doi: 10.1021/jf062431s [22] CUYCKENS F, CLAEYS M. Mass spectrometry in the structural analysis of flavonoids [J]. Journal of Mass Spectrometry: JMS, 2004, 39(1): 1 − 15. doi: 10.1002/jms.585 [23] STOBIECKI M. Application of mass spectrometry for identification and structural studies of flavonoid glycosides [J]. Phytochemistry, 2000, 54(3): 237 − 256. doi: 10.1016/S0031-9422(00)00091-1 [24] KATOH A, UENOHARA K, AKITA M, et al. Early steps in the biosynthesis of NAD in Arabidopsis start with aspartate and occur in the plastid [J]. Plant Physiology, 2006, 141(3): 851 − 857. doi: 10.1104/pp.106.081091 [25] ASHIHARA H, LUDWIG I A, KATAHIRA R, et al. Trigonelline and related nicotinic acid metabolites: occurrence, biosynthesis, taxonomic considerations, and their roles in planta and in human health [J]. Phytochemistry Reviews, 2014, 14(5): 765 − 798. [26] ZHENG X Q, HAYASHIBE E, ASHIHARA H. Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds [J]. Journal of Experimental Botany, 2005, 56(416): 1615 − 1623. doi: 10.1093/jxb/eri156 [27] MATSUI A, YIN Y, YAMANAKA K, et al. Metabolic fate of nicotinamide in higher plants [J]. Physiologia Plantarum, 2007, 131(2): 191 − 200. [28] ZHENG X Q, ASHIHARA H. Distribution, biosynthesis and function of purine and pyridine alkaloids in Coffea arabica seedlings [J]. Plant Science, 2004, 166(3): 807 − 813. doi: 10.1016/j.plantsci.2003.11.024 [29] CHEN W, GAO Y, XIE W, et al. Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism [J]. Nature Genetics, 2014, 46(7): 714 − 721. doi: 10.1038/ng.3007 [30] ASHIHARA H, DENG W W. Pyridine metabolism in tea plants: salvage, conjugate formation and catabolism [J]. Journal of Plant Research, 2012, 125(6): 781 − 791. doi: 10.1007/s10265-012-0490-x [31] KATAHIRA R, ASHIHARA H. Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.) [J]. Planta, 2009, 231(1): 35 − 45. doi: 10.1007/s00425-009-1023-2 [32] WAGNER R, FETH F, WAGNER K G. Regulation in tobacco callus of enzyme activities of the nicotine pathway: II. The pyridine-nucleotide cycle [J]. Planta, 1986, 168(3): 408 − 413. doi: 10.1007/BF00392369