BR  >> Vol. 8 No. 3 (May 2019)

    阳春砂叶片受病害诱导产生更多的挥发性萜类
    Leaves of Amomum villosum Lour. Produce More Volatile Terpenoids Induced by Disease

  • 全文下载: PDF(1359KB) HTML   XML   PP.196-203   DOI: 10.12677/BR.2019.83026  
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作者:  

魏洁书,付 裕:中山大学新华学院药学院,广东 广州;
李 萌,赵海莹,杨锦芬:广州中医药大学中药资源科学与工程研究中心/岭南中药资源教育部重点实验室(广州中医药大学),广东广州

关键词:
阳春砂病害挥发性萜类单萜Amomum villosum Lour. Disease Volatile Terpenoid Monoterpenoid

摘要:

目的:分析阳春砂病变叶片中的挥发性萜类,探究阳春砂叶片受病害刺激后挥发性萜类代谢的响应情况,为阳春砂抗病相关的萜类代谢及其抗病机理的研究奠定基础。方法:分别提取阳春砂病变叶片中健康部位和病变部位的挥发性萜类,采用GC-MS方法进行分析。结果:阳春砂叶片病变部位能检测到更多的挥发性萜类,有10种单萜和1种倍半萜仅在病变部位检测到,如葑烯、香芹醇、龙脑烯醛等,大多为含氧单萜。与健康部位相比,侧柏烯、莰烯、柠檬烯、松香芹醇的含量有显著性提高(p < 0.05),其中柠檬烯的含量有极显著性提高(p < 0.01),是健康部位的5.27倍。结论:阳春砂叶片受病害诱导产生更多的挥发性萜类,其种类增加,部分含量显著提高,这些挥发性萜类可能参与了阳春砂对病害的防御。

Objective: To analyze the volatile terpenoids composition in the pathological leaves of Amomum villosum Lour. and to explore the response of metabolism of volatile terpenoids while the leaves are stimulated by pathogenic bacteria. Method: Volatile terpenoids in the healthy and diseased parts of pathological leaves of A. villosum were extracted respectively and detected by GC-MS. Result: More species of volatile terpenoids were detected in the diseased part, including ten monoterpenoids and one sesquiterpenoid, as fenchene, carveol, campholenal etc., most of which were oxygenated monoterpenoids. The contents of 3-thujene, camphene, D-limonene and pinocarveol in the diseased part were significantly higher than those in the healthy part (p < 0.05), especially the content of D-limonene in the diseased part was 5.27 times that in the health part (p < 0.01). Conclusion: Different quality and quantity volatiles terpenoids were released when the leaves of A. villosum were infected. More species of volatile terpenoids were induced and the contents of some volatile terpenoids had been significantly increased. These volatile terpenoids may be involved in the resistance to disease in A. villosum.

1. 引言

阳春砂(Amomum villosum Lour.)是著名的南药,具有化湿开胃、温脾止泻、理气安胎的功效,用于湿浊中阻、脘痞不饥、脾胃虚寒、呕吐泄泻、妊娠恶阻、胎动不安。主要生长在云南、广西、广东、海南、福建等省区的热带和亚热带地区的林中荫湿处。阳春砂在生长过程中常常会发生叶斑病或叶枯病,多由病原菌引起,在其生理、组织结构和形态上发生一定的病变特征 [1] [2] 。

植物利用次生代谢对外界胁迫做出响应。萜类化合物是植物次生代谢产物,其分布广泛,种类繁多,也是植物抵御胁迫的重要物质 [3] 。阳春砂芳香化湿、行气止痛的主要药效物质为挥发油,其主要成分为单萜、倍半萜等挥发性萜类 [4] 。课题组曾利用茉莉酸甲酯喷施阳春砂叶片后发现MeJA对阳春砂萜类合成上游途径的关键酶基因AvHMGR (3-hydroxy-3-methylglutaryl Coenzyme A reductase)、AvDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase)和AvDXS (1-deoxy-D-xylulose-5-phosphate synthase)的表达均有促进作用 [5] ,且挥发性萜类如α-蒎烯、柠檬烯含量都有显著性提高 [6] ,说明阳春砂的萜类代谢受胁迫信号分子茉莉酸的诱导。阳春砂病害胁迫下的萜类次生代谢变化未见报道,本研究就阳春砂病变叶片中挥发性萜类变化情况进行分析,探究阳春砂叶片受病害刺激后挥发性萜类的响应情况,为阳春砂中防御性萜类挥发信号分子及其抗病机理的研究奠定基础。

前人曾采用水蒸气蒸馏法对阳春砂叶片挥发油成分进行研究 [7] [8] ,本研究采用溶剂法直接提取阳春砂叶片中的挥发性萜类物质,该方法高效,样品用量少,条件稳定可控。基于溶剂法样品用量少的特点,本研究取同一叶片的健康部位和病变部位,分别提取挥发性萜类成分进行比较,着重考察病变部位相对于健康部位挥发性萜类成分和含量的变化。

2. 实验材料

阳春砂病变叶片采自广州中医药大学大学城校区阳春砂种植园。病变叶片如图1

Figure 1. The pathological leaves of Amomum villosum Lour.

图1. 阳春砂病变叶片。注:图中标尺为1 cm

3. 实验方法

3.1. 挥发性萜类提取

分别将阳春砂病变叶片的健康部位与病变部位在液氮下研磨成细粉,每个部位设置4个重复,每个样品称取0.2 g粉末,加入1 mL正己烷,密封涡旋振荡,超声处理30 min,40℃水浴1 h。10,000 r/min离心5 min,取上层有机相待测。

3.2. 气相色谱–质谱条件及定量方法

采用安捷伦7890B/5977A气相色谱–质谱检测器和安捷伦HP5-MS石英毛细管柱(30 m × 250 μm × 0.25 μm film thickness),进样量1 μL,以速度为1 mL/min的氦气为载气,起始温度为35℃并保持5 min,以12℃/min速度升至300℃,保持5 min。采用EI源的质谱,化合物结构和名称利用NIST14/Wiley275质谱库和标准化合物验证;单萜使用β-蒎烯作为外标,倍半萜和二萜使用石竹烯作为外标,分别制作标准曲线进行定量。

3.3. 统计分析方法

采用SPSS19.0统计软件进行数据的统计分析。所得数据用均数 ± 标准差表示,多个样本均数的比较采用单因素方差分析(One-Way ANOVA)。P < 0.05为有统计学差异,P < 0.01为极显著差异。

4. 结果与分析

4.1. 病变部位与健康部位所含挥发性萜类总体情况分析

在阳春砂叶片病变部位中,共检测到20种挥发性萜类,其中单萜类化合物18种,倍半萜类化合物2种;健康部位中共检测到12种挥发性萜类,其中单萜类化合物9种,倍半萜类化合物2种,二萜类化合物1种。结果见表1图2。从结果可以看出,当阳春砂叶片病变后,所产生的挥发性萜类在质与量上均不同于健康部位。

(a) (b)

Figure 2. The percentages of volatile terpenoid. (a) The healthy part; (b) The diseased part

图2. 挥发性萜类的百分比。(a) 健康部位;(b) 病变部位

Table 1. Volatile terpenoid of the healthy and diseased part from the pathological leaves in A. villosum

表1. 阳春砂病变叶片中健康部位与病变部位的挥发性萜类

*为病变部位和健康部位在P < 0.05水平差异有统计学意义;**为P < 0.01;n.d.为未检测到;FW为鲜重。

4.2. 病变部位与健康部位挥发性萜类种类比较

病变部位比健康部位检测到更多的挥发性萜类(图2),有10种单萜和1种倍半萜仅在病变部位检测到,单萜包括葑烯(Fenchene)、对伞花烃(Cymene)、侧柏二烯(Thujadiene)、香芹醇(Carveol)、龙脑烯醛(Campholenal)、马鞭草烯醇(Verbenol)、松香芹酮(Pinocarvone)、侧柏醇(4-Thujanol)以及2,8-p-Menthadien-1-ol和p-Mentha-1(7),8-dien-2-ol,其中包括7种含氧单萜(表1),分别有醇类,如香芹醇、马鞭草烯醇、侧柏醇、2,8-p-Menthadien-1-ol、p-Mentha-1(7),8-dien-2-ol和醛类(龙脑烯醛)、酮类(松香芹酮)。病变部位中新增的含氧萜类化合物较不含氧萜类化合物种类多。新增加的倍半萜为香橙烯(Alloaromadendrene),属于双环倍半萜。以上萜类除了龙脑烯醛、对伞花烃、香橙烯曾在砂仁叶挥发油中检测到以外 [7] [8] ,其他新增加的成分均为首次在阳春砂叶片中检测到。病变部位与健康部位相比,没有检测到叶绿醇(phytol),与病变部位已变黄有关(图1)。

4.3. 病变部位与健康部位挥发性萜类含量比较

阳春砂病变叶片不同部位均检测到β-蒎烯(β-Pinene),其含量最高(图2),病变部位为1037.73 ng/mg,占所有检测到的萜类成分53.40%,健康部位为854.71 ng/mg,占48.17%,其次为α-蒎烯(α-Pinene),病变部位和健康部位分别为644.07 ng/mg和452.57 ng/mg,这2种萜类在病变部位的含量相对健康部位有所提高,但不具有显著性差异。与健康部位对比,病变部位的侧柏烯(3-Thujene)、莰烯(Camphene)和松香芹醇(Pinocarveol)的含量有显著性提高,分别是健康部位的1.52倍、1.78倍和5.56倍,柠檬烯(D-Limonene)的含量与健康部位相比有极显著性差异,是健康部位的5.27倍(表1)。从总量上看,健康部位检测到的挥发性萜类总量为1600.55 ng/mg,病变部位为2154.52 ng/mg,挥发性萜类总量比健康部位更高,这与阳春砂叶片病变后萜类次生代谢变得更活跃有关。病变部位检测到的单萜总量高于健康部位,而倍半萜总量则低于健康部位。

5. 讨论

阳春砂是富含萜类的姜科植物,其叶片本身就含有种类较丰富的挥发性萜类。本研究发现阳春砂叶片病变部位相对健康部位能产生更多的挥发性萜类,推测阳春砂叶片受到病原菌攻击后,启动防御机制,萜类次生代谢变得更活跃,产生更多的挥发性萜类,这些挥发性萜类可能参与了阳春砂对病害的防御。植物在生物或者非生物的逆境下,其次生代谢物会增加,特别是挥发性萜类,这些萜类成分可能对病原菌或昆虫有一定的毒性,同时也可能具有引诱害虫天敌的作用,以防御微生物或昆虫的侵袭 [9] [10] [11] 。阳春砂病变叶片中检测到新增加的萜类化合物,大部分属于含氧单萜。含氧萜类包括酚类、醛类、酮类、醇类、酯类,萜类碳骨架的亲脂性和官能团的亲水性是判断抗菌活性的重要指标,它们的次序是:酚类 >醛类 > 酮类 > 醇类 > 酯类 > 碳氢类 [12] 。病变部位检测到11种新增加的萜类,其中7种为含氧萜类化合物,较不含氧萜类化合物种类多,这也与Kalemba D等的研究相符,推测含氧萜类的增加可提高阳春砂叶片对病害的抗性。

植物为了有效地保护自身免受病原菌的侵害而长期进化产生的一系列化学防御物质,如萜类化合物,其具有不同的生理功能,多数萜类都有抑菌、杀菌的作用 [13] ,其中单萜、倍半萜化合物具有广谱抗菌活性,如薄荷精油中的单萜香芹醇(Carveol) [14] 、香唐松草(Dracocephalum foetidum)中的单萜柠檬烯 [15] 、广藿香(Pogostemon cablin Benth.)中的倍半萜广藿香醇(Patchouli Alcohol) [16] 等。本研究发现,阳春砂叶片病变部位与健康部位相比,侧柏烯、莰烯、柠檬烯、松香芹醇的含量都有显著性提高,α-蒎烯、β-蒎烯含量也有提高。据文献报道,侧柏烯具有抗菌作用 [17] ,α-蒎烯、β-蒎烯、莰烯、柠檬烯具有抑菌杀菌作用以及驱虫作用 [18] [19] [20] [21] [22] 。病变叶片中检测到新增加的倍半萜香橙烯(Alloaromadendrene),关于其生理活性也有相关报道,如从4种龙脑香属植物的原油树脂中分离得到香橙烯,其在昆虫防御中起着重要作用 [23] ,杜鹃花中的香橙烯具有一定的抗菌活性 [24] [25] 。其他植物中这些挥发性萜类对逆境的响应和抗逆作用也有报道,如对鱼腥草(Hottuynia cordata)进行UV-B诱导,发现柠檬烯及其他单萜类化合物在短时间内得到大量地积累 [26] ,受伤的青蒿(Artemisia annua)幼嫩叶片中β-蒎烯含量显著提高 [27] 。前人研究也表明不同单萜物质的抗菌谱和抗菌能力都有可能不同 [28] ,故而阳春砂病变叶片中多种单萜类物质含量的提高,可能起到综合的抗菌效果。

萜类合酶(terpene synthase, TPS)是萜类合成的关键酶,病原菌或者昆虫等特异诱导萜类物质的产生,与萜类合酶的催化密切相关。如杨树(Populus)叶片受昆虫侵害后,其叶片诱导了TPS基因的表达和挥发性萜类的释放 [29] 。研究表明,多数萜类合酶能催化产生多个产物 [30] 。课题组已从阳春砂中克隆和鉴定了两个萜类合酶——AvPS (pinene synthase,蒎烯合酶)和AvBPPS (bornyl diphosphate synthase,龙脑基二磷酸合酶)基因,它们的产物都不止一个,其中AvBPPS有多个副产物 [31] 。阳春砂病变后的叶片检测到种类更多的萜类,可能是病害胁迫刺激了萜类次生代谢,使某些萜类合酶比正常情况下合成更多样的产物。

植物能够在特定时间合成与释放不同的挥发性萜类,如巨冷杉(Abies grandis)受到虫害侵扰后,单萜、倍半萜和二萜的生物合成在时间上存在先后差异 [32] ;利马豆在黑暗中受到侵害时只能释放少量β-罗勒烯(β-ocimene),而在次日拂晓才开始大量释放 [33] ,表明植物在受到刺激后,会在不同时间点释放不同挥发性萜类。本研究病变叶片中α-蒎烯、β-蒎烯含量虽有增加,但并未发现显著性差异,这可能与挥发性萜类的释放时间和积累有关。

6. 结论

本研究比较了阳春砂叶片病变部位与健康部位中的挥发性萜类,发现阳春砂的挥发性萜类的合成受病害刺激变得活跃,产生较多的可能具有抗菌活性的含氧萜类,为阳春砂抗病相关的萜类代谢及其抗病机理的研究奠定基础。

基金项目

广东省普通高校青年创新人才项目(2017KQNCX258);国家自然科学基金面上项目(81872954);中山大学新华学院校级科研一般项目(2018YB015)。

NOTES

*通讯作者。

文章引用:
魏洁书, 付裕, 李萌, 赵海莹, 杨锦芬. 阳春砂叶片受病害诱导产生更多的挥发性萜类[J]. 植物学研究, 2019, 8(3): 196-203. https://doi.org/10.12677/BR.2019.83026

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