• 百种中国杰出学术期刊
  • 中国精品科技期刊
  • 中国高校百佳科技期刊
  • 中国高校精品科技期刊
  • 中国国际影响力优秀学术期刊
  • 中国科技核心期刊

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

硬毛棘豆根的抑菌活性成分研究

叶生伟 胡嘉隽 胡子龙 赵龙 郝楠 田向荣

叶生伟, 胡嘉隽, 胡子龙, 赵龙, 郝楠, 田向荣. 硬毛棘豆根的抑菌活性成分研究[J]. 农药学学报, 2022, 24(2): 289-297. doi: 10.16801/j.issn.1008-7303.2021.0158
引用本文: 叶生伟, 胡嘉隽, 胡子龙, 赵龙, 郝楠, 田向荣. 硬毛棘豆根的抑菌活性成分研究[J]. 农药学学报, 2022, 24(2): 289-297. doi: 10.16801/j.issn.1008-7303.2021.0158
YE Shengwei, HU Jiajun, HU Zilong, ZHAO Long, HAO Nan, TIAN Xiangrong. Studies on the antimicrobial constituents from the roots of Oxytropis hirta Bunge[J]. Chinese Journal of Pesticide Science, 2022, 24(2): 289-297. doi: 10.16801/j.issn.1008-7303.2021.0158
Citation: YE Shengwei, HU Jiajun, HU Zilong, ZHAO Long, HAO Nan, TIAN Xiangrong. Studies on the antimicrobial constituents from the roots of Oxytropis hirta Bunge[J]. Chinese Journal of Pesticide Science, 2022, 24(2): 289-297. doi: 10.16801/j.issn.1008-7303.2021.0158

硬毛棘豆根的抑菌活性成分研究

doi: 10.16801/j.issn.1008-7303.2021.0158
基金项目: 国家重点研发计划 (2017YFD0201105).
详细信息

Studies on the antimicrobial constituents from the roots of Oxytropis hirta Bunge

Funds: National Key R&D Program of China(2017YFD0201105).
  • 摘要: 为明确硬毛棘豆Oxytropis hirta Bunge根的农用抑菌活性成分,用75%乙醇浸提硬毛棘豆根,得到粗提物;再分别以石油醚、乙酸乙酯与正丁醇为溶剂对粗提物进行萃取,得到不同溶剂萃取相;通过活性追踪法对抑菌活性好的乙酸乙酯相运用现代柱层析与波谱学技术进行了分离与结构鉴定,并对所分离的化合物进行了抑菌活性评价。结果表明:硬毛棘豆根乙酸乙酯相抑菌活性最好,在质量浓度为1 × 103 mg/L时对水稻纹枯病菌Thanatephorus cucumeris和番茄灰霉病菌Botrytis cinerea的抑制率分别为57.2%和55.4%,对烟草青枯病菌Pseudomonas solanacearum与猕猴桃溃疡病菌Pseudomonas syringae pv. actinidae的抑菌圈分别为21.8 mm和19.0 mm。从乙酸乙酯相分离鉴定出6个化合物,分别为β-香树脂醇 ( 1 )、3-oxo-azukisapogenol ( 2 )、5α-豆甾-9(11)-烯-3β-醇 ( 3 )、豆甾-4-烯-3,6-二酮 ( 4 )、3β,22β,24-三羟基齐墩果-12-烯 ( 5 )和azukisapogenol ( 6 )。抑菌活性测试结果表明:化合物 1 对金黄色葡萄球菌Staphylococcus aureus的最低抑菌浓度 (MIC)为50 mg/L,对烟草青枯病菌、猕猴桃溃疡病菌和枯草芽孢杆菌Bacilus subtilis的MIC为100 mg/L;化合物 6 对水稻纹枯病菌和番茄灰霉病菌的EC50值分别为117.4mg/L和86.2 mg/L。硬毛棘豆根乙酸乙酯相对供试植物病原真菌和细菌有较好的抑制活性,其中三萜类是其主要活性成分之一,具备进一步开发为杀菌剂的潜力。
  • 1  硬毛棘豆根中分离鉴定的化合物结构1 ~ 6

    1.  Chemical structures of compounds 1-6 from Oxytropis hirta

    表  1  硬毛棘豆根75%乙醇粗提物及其不同萃取相对植物病原真菌的抑制作用

    Table  1.   Inhibition of 75% ethanol extract and its different phases from O. hirta against plant pathogenic fungi

    病原真菌
    Pathogenic fungi
    抑制率
    Inhibition rate/%
    粗提物
    Crude extract
    石油醚相
    Petroleum ether
    phase
    乙酸乙酯相
    Ethyl acetate phase
    正丁醇相
    n-Butanol phase
    水相
    Water phase
    辣椒疫霉 P. capsici 17.2 ± 2.3 b 10.1 ± 2.7b 25.2 ± 1.2 a 20.6 ± 3.7 a 12.1 ± 2.6 b
    苹果炭疽病菌 C. gloeosporioides 24.0 ± 1.5 b 15.6 ± 0.3 c 33.4 ± 0.7 a 30.2 ± 2.1 a 15.4 ± 1.1 c
    小麦赤霉病菌 F. graminearum 27.5 ± 0.7 b 12.7 ± 1.7c 37.3 ± 1.4 a 32.3 ± 0.2 a 17.3 ± 1.0 c
    小麦全蚀病菌 G. graminis 33.3 ± 1.1 a 9.3 ± 1.2 c 35.3 ± 0.6 a 29.6 ± 1.1 b 20.2 ± 0.3 b
    水稻纹枯病菌 T. cucumeris 45.7 ± 1.0 b 16.3 ± 0.5 d 57.2 ± 2.1 a 50.2 ± 1.7 ab 23.4 ± 0.5 c
    番茄灰霉病菌 B. cinerea 52.9 ± 2.1 a 14.3 ± 1.4 d 55.4 ± 2.2 a 46.7 ± 1.1 b 32.4 ± 2.2 c
    注:表中数据为3次重复结果的平均值 ± SD,同行数据后不同小写字母表示经Duncan氏新复极差法检验不同处理在P < 0.05水平上差异显著。Note: Data were expressed as mean ± SD, and repeated for three times. Different lower-case letters in each line indicate significant differences between treatments by Duncan’s new multiple range test (P < 0.05).
    下载: 导出CSV

    表  2  硬毛棘豆根75%乙醇粗提物及其不同萃取相对植物病原细菌的抑制作用

    Table  2.   The inhibition of 75% ethanol extract and its different phases from O. hirta against plant pathogenic bacteria

    病原细菌
    Pathogenic bacteria
    抑菌圈直径
    Antibacterial circle diameter/mm
    粗提物
    Crude extract
    石油醚相
    Petroleum ether phase
    乙酸乙酯相
    Ethyl acetate phase
    正丁醇相
    n-Butanol phase
    水相
    Water phase
    链霉素
    streptomycin
    白菜软腐病菌 E. carotovora 10.2 ± 0.1 b 8.2 ± 0.1 c 10.3 ± 0.2 b 9.1 ± 0.1 bc 6.5 ± 0.1 c 22.1 ± 0.1 a
    烟草青枯病菌 P. solanacearum 13.7 ± 0.2 b 10.2 ± 0.1 b 21.8 ± 0.2 a 15.4 ± 0.1 b 7.7 ± 0.1 c 23.2 ± 0.1 a
    猕猴桃溃疡病菌 P. syringae 17.5 ± 0.1 ab 7.1 ± 0.1 c 19.0 ± 0.2 ab 13.8 ± 0.2 b 10.6 ± 0.2 b 21.1 ± 0.2 a
    注:粗提物和链霉素的质量浓度分别为1 × 103 mg/L和100 mg/L。数据为3次重复结果的平均值 ± SD,同行数据后不同小写字母表示经Duncan氏新复极差法检验不同处理在P < 0.05水平上差异显著。Note: The concentrations of the crude extracts and streptomycin are 1 × 103 mg/L and 100 mg/L, respectively. Data were expressed as mean ± SD, and repeated for three times. Different lower-case letters in each line indicate significant differences between treatments by Duncan’s new multiple range test (P < 0.05).
    下载: 导出CSV

    表  3  化合物1 ~ 6的碳谱数据 (CDCl3, 125 MHz)

    Table  3.   13C NMR data of compounds 1-6 (CDCl3, 125 MHz)

    No.123456
    138.838.937.535.838.638.3
    227.134.531.934.227.926.0
    379.2215.372.0199.781.180.3
    438.951.232.2125.743.042.4
    555.355.840.0161.356.155.8
    618.519.421.3202.518.718.4
    732.832.429.947.133.332.9
    840.039.846.134.539.939.8
    947.846.6141.051.247.947.7
    1037.138.936.840.036.936.6
    1123.724.0121.921.124.023.8
    12121.9122.732.239.4122.5122.6
    13145.3144.142.642.8144.1144.1
    1441.942.757.056.842.341.6
    1528.626.124.524.226.126.9
    1626.227.028.528.229.927.2
    1732.732.656.356.137.632.4
    1847.446.212.212.145.046.5
    1947.040.319.617.746.440.5
    2031.242.036.436.330.742.3
    2134.929.019.318.941.729.0
    2237.335.934.234.176.835.9
    2328.322.229.426.322.622.4
    2415.766.050.446.164.764.4
    2515.816.326.329.416.416.0
    2617.016.720.119.317.116.7
    2726.325.919.020.025.625.8
    2827.428.323.323.328.428.1
    2933.5184.612.112.233.0181.9
    3023.919.320.219.2
    下载: 导出CSV

    表  4  化合物对6种植物病原真菌的抑制作用

    Table  4.   Antifungal effects of compounds against six plant phytopathogenic fungi

    病原真菌
    Phytopathogenic fungi
    抑制率
    Inhibition rate/%
    135630%丁香菌酯 SC
    coumoxystrobin 300 SC
    辣椒疫霉 P. capsici 46.2 ± 1.4
    苹果炭疽病菌 C. gloeosporioides 39.7 ± 1.3
    小麦赤霉病菌 F. graminearum 17.6 ± 1.4 b 43.2 ± 0.6 a
    小麦全蚀病菌 G. graminis 10.3 ± 0.5 b 15.7 ± 1.0 b 9.7 ± 0.3 c 13.7 ± 1.1 b 47.6 ± 0.7 a
    水稻纹枯病菌 T. cucumeris 15.6 ± 0.2 c 43.6 ± 2.3 b 52.1 ± 1.1 a
    番茄灰霉病菌 B. cinerea 12.3 ± 0.3 b 9.6 ± 0.1 c 8.7 ± 1.1 c 62.4 ± 1.7 a 60.1 ± 2.4 a
    注:“-” 表明未起到抑制效果;化合物与30%丁香菌酯SC的有效成分质量浓度均为100 mg/L。同行数据后不同小写字母表示经Duncan氏新复极差法检验不同处理在P < 0.05水平上差异显著 (n = 3)。Note: “-” indicates that there is no inhibitory effect. The concentrations of the test compounds and effective constituent of coumoxystrobin 300 SC are 100 mg/L. Different lower-case letters in each line indicate significant differences between treatments by Duncan’s new multiple range test (P < 0.05, n = 3).
    下载: 导出CSV

    表  5  化合物6对水稻纹枯病菌与番茄灰霉病菌的EC50

    Table  5.   EC50 value of compound 6 against T. cucumeris and B. cinerea

    病原真菌
    Phytopathogenic fungi
    毒力回归方程
    Virulence equation
    χ2EC50/(mg/L)95%CI/(mg/L)
    水稻纹枯病菌 T. cucumeris y = –2.5 + 1.2x 1.8 117.4 99.0~148.8
    番茄灰霉病菌 B. cinerea y = –2.7 + 1.4x 1.1 86.2 70.3~104.7
    下载: 导出CSV

    表  6  化合物对3种植物病原细菌的抑制作用

    Table  6.   Antibacterial activities of compounds against three plant pathogenic bacteria

    病原细菌
    Pathogenic bacteria
    1356链霉素
    streptomycin
    白菜软腐病菌 E. carotovora + + +
    烟草青枯病菌 P. solanacearum + + +
    猕猴桃溃疡病菌 P. syringae + + +
    注: 96孔板单孔中菌液浑浊且显粉红色为 “ + ”,清澈透明为 “−”;化合物和链霉素的质量浓度分别为200 mg/L和100 mg/L。Note: Single-well inoculum in 96-well plate is turbid and pink as “ + ”, clear and transparent as “−”. The concentrations of the test compounds and streptomycin are 200 mg/L and 100 mg/L, respectively.
    下载: 导出CSV

    表  7  化合物1对5种细菌的最低抑制浓度

    Table  7.   Minimum inhibitory concentration of compound 1 against five bacteria

    病原细菌
    Pathogenic bacteria
    质量浓度
    Concentration/(mg/L)
    200100502512.5
    白菜软腐病菌 E. carotovora + + + +
    烟草青枯病菌 P. solanacearum + + +
    猕猴桃溃疡病菌 P. syringae + + +
    枯草芽孢杆菌 B. subtilis + + +
    金黄色葡萄球菌 S. aureus + +
    注:96孔板单孔中菌液浑浊且显粉红色为 “+”,清澈透明为 “−”。Note: Single-well inoculum in 96-well plate is turbid and pink as “+”, clear and transparent as “−”.
    下载: 导出CSV
  • [1] CANTRELL C L, DAYAN F E, DUKE S O. Natural products as sources for new pesticides[J]. J Nat Prod, 2012, 75: 1231-1242. doi: 10.1021/np300024u
    [2] 车传亮, 杨冬燕, 万川, 等. 分子插件法及其在农药分子设计中的应用[J]. 农药学学报, 2017, 19(5): 533-542.

    CHE C L, YANG D Y, WAN C, et al. The plug-in molecules method and its application in designing of novel agrochemicals[J]. Chin J Pestic Sci, 2017, 19(5): 533-542.
    [3] 白明纲, 包晓华, 吴香杰. HPLC法测定硬毛棘豆中山柰素的含量[J]. 中国民族医药杂志, 2008, 14(11): 56-57. doi: 10.3969/j.issn.1006-6810.2008.11.037

    BAI M G, BAO X H, WU X J. Determination of kaempferolin herba Oxytropis hirtae by HPLC[J]. J Med Pharm Chin Minor, 2008, 14(11): 56-57. doi: 10.3969/j.issn.1006-6810.2008.11.037
    [4] 李玉林, 廖志新, 杜玉枝, 等. 棘豆属植物化学成分研究概况[J]. 天然产物研究与开发, 2002, 14(5): 75-79. doi: 10.3969/j.issn.1001-6880.2002.05.021

    LI Y L, LIAO Z X, DU Y Z, et al. A survey of the studies on chemical constituents of Oxytropis species[J]. Nat Prod Res Dev, 2002, 14(5): 75-79. doi: 10.3969/j.issn.1001-6880.2002.05.021
    [5] AMIRKHANOVA A S, USTENOVA G O. Review of the current status of study Oxytropis[J]. Asian J Pharm Clin Res, 2018, 11(4): 50-55. doi: 10.22159/ajpcr.2018.v11i4.23656
    [6] ZHANG M R, JIANG K, YANG J L, et al. Flavonoids as key bioactive components of Oxytropis falcata Bunge, a traditional anti-inflammatory and analgesic Tibetan medicine[J]. Nat Prod Res, 2020, 34(23): 3335-3352. doi: 10.1080/14786419.2019.1574786
    [7] LI M X, LAN Z H, WEI L L, et al. Phytochemical and biological studies of plants from the genus Oxytropis[J]. Rec Nat Prod, 2012, 6(1): 1-20.
    [8] 陈醒, 杨光明, 蔡宝昌. 棘豆属植物生物碱类成分结构特征和生理活性研究进展[J]. 南京中医药大学学报, 2011, 27(1): 95-97. doi: 10.3969/j.issn.1000-5005.2011.01.028

    CHEN X, YANG G M, CAI B C. Research advances in structures and biological activities of alkaloids in Oxytropis[J]. J Nanjing Univ Tradit Chin Med, 2011, 27(1): 95-97. doi: 10.3969/j.issn.1000-5005.2011.01.028
    [9] 曹光荣, 李绍君, 段得贤, 等. 黄花棘豆有毒成分的分离与鉴定[J]. 西北农林科技大学学报(自然科学版), 1989, 17(3): 1-8.

    CAO G R, LI S J, DUAN D X, et al. The isolation and identification of toxic componets from Oxytropis ochrocephala[J]. J Northwest Agric For Univ (Nat Sci Ed), 1989, 17(3): 1-8.
    [10] 童军卫, 刘补兴, 胡小铭, 等. 苦马豆素对胶质瘤细胞U87凋亡及HSP90/AKT通路的影响[J]. 实用药物与临床, 2018, 21(3): 241-245.

    TONG J W, LIU B X, HU X M, et al. Influence of swainsonine on glioma cell U87 apoptosis and HSP90/AKT pathway[J]. Pract Pharm Clin Remedies, 2018, 21(3): 241-245.
    [11] 张兴, 王兴林, 王胜宝, 等. 西北地区杀虫植物资源初步调查[J]. 甘肃农业大学学报, 1993, 28(1): 93-98.

    ZHANG X, WANG X L, WANG S B, et al. Preliminary investigation on the resources of botanical insecticides in north-western China[J]. J Gansu Agric Univ, 1993, 28(1): 93-98.
    [12] 郭蓉, 郭亚洲, 王帅, 等. 中国天然草地有毒植物及其放牧家畜中毒病研究进展[J]. 畜牧兽医学报, 2021, 52(5): 1171-1185.

    GUO R, GUO Y Z, WANG S, et al. Advances in research on poisonous plants and grazing livestock poisoning diseases of natural grassland in China[J]. Acta Vet et Zootechnica Sin, 2021, 52(5): 1171-1185.
    [13] 孟根小, 奥乌力吉, 霍万学, 等. 多叶棘豆体外抗菌作用部位的筛选研究[J]. 中国中医药信息杂志, 2016, 23(12): 51-54. doi: 10.3969/j.issn.1005-5304.2016.12.013

    MENG G X, AO W L J, HUO W X, et al. Study on screening for antibacterial active site of Oxytropis myriophylla in vitro[J]. Chin J Inform Tradit Chin Med, 2016, 23(12): 51-54. doi: 10.3969/j.issn.1005-5304.2016.12.013
    [14] 杨震发, 袁呈山, 杨顺义, 等. 黄花棘豆抑制植物病原菌的活性成分研究[J]. 植物保护, 2014, 40(6): 59-64. doi: 10.3969/j.issn.0529-1542.2014.06.011

    YANG Z F, YUAN C S, YANG S Y, et al. Antifungal activity and isolation of active ingredients in Oxytropis ochrocephala[J]. Plant Prot, 2014, 40(6): 59-64. doi: 10.3969/j.issn.0529-1542.2014.06.011
    [15] LI Y, YE S, HU Z, et al. Identification of anti-TMV active flavonoid glycosides and their mode of action on virus particles from Clematis lasiandra Maxim[J]. Pest Manag Sci, 2021, 77(11): 5268-5277. doi: 10.1002/ps.6569
    [16] HAO N, HAN L R, LI Y T, et al. New 8-O-4' neolignans and their antibacterial activity from the whole plants of Clematis lasiandra[J]. ACS Omega, 2020, 5(31): 19661-19666. doi: 10.1021/acsomega.0c02339
    [17] 王静, 叶敏, 范黎明, 等. 菌丝生长速率法筛选纤维素降解菌的研究[J]. 中国农学通报, 2013, 29(33): 323-326. doi: 10.3969/j.issn.1000-6850.2013.33.057

    WANG J, YE M, FAN L M, et al. Screening of cellulose degrading fungi by mycelium growth rate method[J]. Chin Agric Sci Bull, 2013, 29(33): 323-326. doi: 10.3969/j.issn.1000-6850.2013.33.057
    [18] VINCENT J G, VINCENT H W, MORTON J. Filter paper disc modification of the Oxford cup penicillin determination[J]. Exp Biol Med, 1944, 55(3): 162-164. doi: 10.3181/00379727-55-14502
    [19] WIEGAND I, HILPERT K, HANCOCK R E. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances[J]. Nat Protoc, 2008, 3(2): 163-175. doi: 10.1038/nprot.2007.521
    [20] 方琴, 黄初升, 陈希慧, 等. 几种猕猴桃属植物中乌苏烷型三萜化合物的谱学研究[J]. 广西师范学院学报(自然科学版), 2007, 24(4): 53-60.

    FANG Q, HUANG C S, CHEN X H, et al. Survey on the spectral features of ursane triterpense in Actinidia planties[J]. J Guangxi Teachers Education Univ (Nat Sci Ed), 2007, 24(4): 53-60.
    [21] 邓可众, 熊英, 高文远. 半边莲的化学成分研究[J]. 中草药, 2009, 40(8): 1198-1201. doi: 10.3321/j.issn:0253-2670.2009.08.007

    DENG K Z, XIONG Y, GAO W Y. Chemical constituents of Lobelia chinensis[J]. Chin Tradit Herb Drugs, 2009, 40(8): 1198-1201. doi: 10.3321/j.issn:0253-2670.2009.08.007
    [22] CHEN W H, QI H Y, SHI Y P. 24-Hydroxyoleanane-type triterpenes from the aerial parts and roots of Oxytropis falcata[J]. J Nat Prod, 2009, 72(8): 1410-1413. doi: 10.1021/np900199x
    [23] 徐秀芝, 张承忠, 李冲. 锁阳化学成分的研究[J]. 中国中药杂志, 1996, 21(11): 676-677,704. doi: 10.3321/j.issn:1001-5302.1996.11.018

    XU X Z, ZHANG C Z, LI C. Studies on chemical components of Cynomorium songaricum Rupr[J]. China J Chin Mater Med, 1996, 21(11): 676-677,704. doi: 10.3321/j.issn:1001-5302.1996.11.018
    [24] SHEN C C, SYU W J, LI S Y, et al. Antimicrobial activities of naphthazarins from Arnebia euchroma[J]. J Nat Prod, 2002, 65(12): 1857-1862. doi: 10.1021/np010599w
    [25] 李娆娆, 王彩芳, 雷沛霖, 等. 槐花炭脂溶性及水溶性部位化学成分研究[J]. 中国中药杂志, 2010, 35(5): 607-609.

    LI R R, WANG C F, LEI P L, et al. Chemical constituents in Flos Sophorae Carbonisatus[J]. China J Chin Mater Med, 2010, 35(5): 607-609.
    [26] OKAWA M, YAMAGUCHI R, DELGER H, et al. Five triterpene glycosides from Oxytropis myriophylla[J]. Chem Pharm Bull (Tokyo), 2002, 50(8): 1097-1099. doi: 10.1248/cpb.50.1097
    [27] 巩红飞, 杨爱梅, 柳军玺, 等. 甘肃棘豆的化学成分研究[J]. 中草药, 2010, 41(2): 187-190.

    GONG H F, YANG A M, LIU J X, et al. Studies on chemical constituents of Oxytropis kansuensis[J]. Chin Tradit Herb Drugs, 2010, 41(2): 187-190.
    [28] SUN R Q, JIA Z J, CHENG D L, et al. Four saponins from Oxytropis ochrocephala[J]. Planta Med, 1992, 58(2): 211-213. doi: 10.1055/s-2006-961432
  • 加载中
图(1) / 表(7)
计量
  • 文章访问数:  95
  • HTML全文浏览量:  43
  • PDF下载量:  18
  • 被引次数: 0
出版历程
  • 收稿日期:  2021-08-10
  • 录用日期:  2021-10-11
  • 网络出版日期:  2021-10-26
  • 刊出日期:  2022-04-10

目录

    /

    返回文章
    返回