宁夏枸杞Lycium barbarum L.为茄科Solanaceae枸杞属Lycium落叶灌木，具有很高的药用和食用价值。《本草纲目》记载:“春采叶，名天精草；夏采花，名长生草；秋采子，名枸杞子；冬采根，名地骨皮”。现代药理研究证实宁夏枸杞中的香豆素类化合物（东莨菪内酯和东莨菪苷）具有抗肿瘤、抗炎、降血糖、降血压等多种药理活性，然而宁夏枸杞中香豆素等物质的含量、分布规律及其生态意义尚不明确。本文以宁夏枸杞为研究对象，建立同时测定宁夏枸杞中香豆素等9种物质含量的UPLC-MS/MS方法，研究香豆素等9种物质在宁夏枸杞不同成熟度果实、组织部位、栽培品种及种植模式中的分布规律，研究盐碱胁迫对宁夏枸杞生长、香豆素等物质积累及抑制枸杞瘿螨为害的作用，旨在明确香豆素类物质在宁夏枸杞中的分布及其在适应盐碱环境中的作用，为宁夏枸杞综合开发利用及高品质药材的定向培育提供参考。主要研究结果如下：

1. 建立了同时测定宁夏枸杞中香豆素及苯丙酸等9种物质含量的UPLC-MS/MS方法。

16 min内实现对9种物质的有效分离，9种物质在各自的质量浓度范围内线性关系良好，线性相关系数均大于0.99，稳定性RSD<5.22%，重复性RSD<7.11%，在25和250 μg·ml^-1浓度下9种物质的精密度RSD<6.10%；平均加样回收率为96.67%~123.36%。

2. 初步明确了宁夏枸杞不同成熟度果实、组织部位、栽培品种及种植模式下香豆素等9种物质含量分布规律。

（1）宁夏枸杞中香豆素等9种物质在不同成熟度果实及组织部位含量各异。5种香豆素类物质（东莨菪内酯、东莨菪苷、秦皮素、秦皮苷、秦皮甲素）和2种苯丙酸（咖啡酸和绿原酸）均在青果中含量最高，并随果实成熟而下降，青果中各物质含量是红果的1.9~33.4倍，而另2种苯丙酸（阿魏酸和对香豆酸）则在成熟果实中含量更高。东莨菪内酯在叶片中含量高于根皮，而东莨菪苷在根皮中含量高于叶片，推测东莨菪苷由宁夏枸杞根部合成运输至叶片转化为东莨菪内酯发挥抗性。

（2）宁夏枸杞中东莨菪内酯及东莨菪苷在不同栽培品种中含量各异。常规种植模式下，东莨菪内酯在宁杞1号（叶片127.00 μg·g^-1）和杞鑫10号（青果316.33 μg·g^-1）中含量较高，东莨菪苷在宁杞7号（叶片1 888.33 μg·g^-1，青果96.97 μg·g^-1）中含量较高；有机种植模式下，东莨菪内酯在枸杞品系0909（叶片381.33 μg·g^-1，青果479.67 μg·g^-1）中含量较高，东莨菪苷在枸杞品系0909（叶片602.67μg·g^-1）和宁杞1号（青果183.33 μg·g^-1）中含量较高。

3）种植模式对宁夏枸杞中香豆素类物质含量的影响与环境胁迫强度有关。青海产区不同基地样品中，野生宁夏枸杞东莨菪内酯含量分别是栽培和荒弃宁夏枸杞的1.8和2.3倍。宁夏产区同一基地样品中，有机种植的宁夏枸杞东莨菪内酯含量是常规种植的1.4倍。

3. 证实了盐碱胁迫虽一定程度抑制宁夏枸杞生长，但促进香豆素类物质积累，并抑制枸杞瘿螨的为害。

（1）宁夏枸杞耐盐碱，但重度盐碱胁迫明显抑制植株生长和生物量积累，促进根系发育使根冠比增大。宁夏枸杞生长指标（株高和茎粗）和生物量参数（地上鲜重和地下鲜重）均与盐胁迫和碱胁迫呈负相关关系，而根冠比与二者均呈正相关关系，根冠比与地下鲜重相关性（r=0.352）大于与地上鲜重的相关性（r=-0.313）。

（2）盐碱胁迫会影响宁夏枸杞香豆素等物质积累，时间累积效应明显，且碱胁迫影响大于盐胁迫。碱胁迫可显著提高宁夏枸杞叶片中东莨菪内酯和东莨菪苷的积累，盐碱胁迫30 d的宁夏枸杞叶片中，二者均呈现出随碱浓度增大含量升高的趋势。碱胁迫条件下东莨菪苷积累效应更强，250 mmol·L^-1碳酸氢钠（NaHCO₃）处理30 d时，东莨菪内酯和东莨菪苷在宁夏枸杞叶片中积累量分别是对照组的5.3和20.6倍。

（3）碱胁迫会诱导宁夏枸杞根系分泌蓝色荧光物质，还会抑制枸杞瘿螨在叶片的为害。田间盐碱地虫瘿直径显著小于常规地，比常规地虫瘿直径小19.42%。室内碱胁迫后，虫瘿直径显著小于对照，pH值为9.50和10.00时虫瘿直径分别比对照组（pH=8.50）小27.47%和18.33%。

综上所述，本研究明确了宁夏枸杞中香豆素类物质主要分布于青果和叶片，且在胁迫较强的环境中积累量更高；碱胁迫会刺激宁夏枸杞积累香豆素等防御性次生代谢物质，且香豆素苷的积累效应强于香豆素；碱胁迫还会抑制枸杞瘿螨虫瘿生长。基于此，可初步推测香豆素类物质在宁夏枸杞环境适应中具有重要生态学意义，本发现可为宁夏枸杞高品质定向培育提供理论指导。研究发现宁夏枸杞青果和叶片中东莨菪内酯含量较高，根皮和叶片中东莨菪苷含量较高，对其资源开发利用具有指导意义。

Lycium barbarum L. is a deciduous shrub of Lycium from Solanaceae family, with high medicinal and edible value. "Compendium of Materia Medica" records: "Leaves picked in spring are called Tianjingcao, flowers picked in summer are called Changshengcao, fruits picked in autumn are called Goji, roots picked in winter are called Digupi". Modern pharmacological studies have confirmed that the coumarin substances (scopoletin and scopolin) in L. barbarum have various pharmacological activities such as anti-tumor, anti-inflammatory, hypoglycemic, and hypotensive. However, the content, distribution rules and ecological significance of coumarin and other substances in L. barbarum are still unclear. This paper used L. barbarum as the research material, a method for simultaneous determination of the content of 9 substances including coumarin in L. barbarum by Ultra High-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) was established. Based on this method, the distribution rules of 9 substances including coumarin in different ripeness fruits, tissue parts, strains and cultivation patterns of L. barbarum were studied. The effects of saline-alkali stress on the growth of L. barbarum, and the accumulation of substances such as coumarin and other substances and the inhibition of Aceria pallida Keifer were studied. In order to clarify the distribution rules of coumarins in L. barbarum and their role in adapting to saline-alkali environment, so as to provide reference for the comprehensive development and utilization of L. barbarum and the directional cultivation of high-quality medicinal materials. The main results were as follows:

1, A UPLC-MS/MS method was established for the simultaneous determination of 9 substances including coumarin and phenylpropionic acid in L. barbarum.

The method realized the effective separation of 9 kinds of substances within 16 minutes. The linear relationship of the 9 kinds of substances was good in their respective mass concentration range, the linear correlation coefficient was greater than 0.99, the stability RSDs were less than 5.22%, the repeatability RSDs were less than 7.11%, and the precision RSDs of the 9 kinds of components at 25 and 250 μg·ml^-1 were both less than 6.10%. The average recovery was 96.67%-123.36%.

2, The effects of different ripeness fruits, tissue parts, strains and cultivation modes of L. barbarum on the content of 9 substances including coumarin were preliminarily clarified.

(1) The contents of 9 substances including coumarin in L. barbarum were different in different ripeness fruits and tissue parts. The contents of 5 kinds of coumarins (scopoletin, scopolin, fraxetin, fraxin and esculin) and 2 kinds of phenylpropionic acids (caffeic acid and chlorogenic acid) were the highest in unripe fruits and decreased with fruit ripening. The contents of various substances in unripe fruits were 1.9-33.4 times higher than those in ripe fruits. While the contents of other 2 kinds of phenylpropionic acid (ferulic acid and p-coumaric acid) were higher in ripe fruits. The content of scopoletin in leaves was higher than that in root barks, while the content of scopolin in root barks was higher than that in leaves. It was speculated that L. barbarum synthesized scopolin from roots and transported to leaves and transformed into scopoletin to exert resistance.

(2) The contents of scopoletin and scopolin in L. barbarum were different in different strains. Under the conventional planting pattern, the higher content of scopoletin was found in Ningqi No.1 (127.00 μg·g^-1 in leaves) and Qixin No.10 (316.33 μg·g^-1 in unripe fruits), while the content of scopolin in Ningqi No.7 was higher (1 888.33 μg·g^-1 in leaves and 96.97 μg·g^-1 in unripe fruits). Under the organic planting pattern, the content of scopoletin in Goqipinxi No.0909 was higher (381.33 μg·g^-1 in leaves and 479.67 μg·g^-1 in unripe fruits), while the higher content of scopolin was found in Goqipinxi No.0909 (602.67 μg·g^-1 in leaves) and Ningqi No.1 (183.33 μg·g^-1 in unripe fruits).

(3) The effects of planting pattern on the content of coumarins in L. barbarum were related to the intensity of environmental stress. In the samples from different bases in Qinghai, the contents of scopoletin in wild samples L. barbarum were 1.8 and 2.3 times higher than those in cultivated and abandoned cultivated respectively. In samples from the same base in Ningxia, the content of scopoletin in organic cultivated samples L. barbarum was 1.38 times higher than that of conventional cultivated.

3. It was confirmed that saline-alkali stress inhibited the growth of L. barbarum, but promoted the accumulation of coumarins, and inhibited the damage of A. pallida.

(1) L. barbarum was tolerant to salt and alkali, but severe saline-alkali stress significantly inhibited plant growth and biomass accumulation, promoted root development and increased root-shoot ratio. The growth indexes (plant height and stem diameter) and biomass parameters (fresh weight above-ground and fresh-weight below ground) of L. barbarum were negatively correlated with salt stress and alkali stress, while the root-shoot ratio was positively correlated with both. The correlation between root shoot ratio and under-ground fresh weight (r=0.352) was greater than that with above-ground fresh weight (r=-0.313).

(2) Saline-alkali stress will affect the accumulation of coumarins and other substances in L. barbarum, and the time-accumulation effect is obvious, and the effect of alkali stress is greater than that of salt stress. Alkaline stress could significantly increase the accumulation of scopoletin and scopolin in L. barbarum leaves. In the L. barbarum leaves under saline-alkali stress for 30 days, the contents of both showed a trend of increasing with the increase of alkali concentration. The effect of scopolin accumulation was stronger than scopoletin under alkali stress. When treated with 250 mmol·L^-1 NaHCO₃ for 30 days, the accumulation of scopoletin and scopolin in L. barbarum leaves were 5.3 and 20.6 times higher than those in the control group, respectively.

(3) Alkaline stress could induce the roots of L. barbarum to secrete blue fluorescent substances, and also inhibited the damage of A. pallida on leaves. The diameter of galls in saline-alkali soils in the field was significantly smaller than that in conventional ones, which was 19.42% smaller than that in conventional ones. Under indoor alkali stress, the diameter of the galls was significantly smaller than that of the control, and the diameter of the galls at pH 9.50 and 10.00 were 27.47% and 18.33% smaller than those of the control group (pH=8.50), respectively.

To sum up, this study clarified that coumarins were mainly distributed in the unripe fruits and leaves of L. barbarum, and the accumulation was higher in the planting environment with stronger stress. Alkaline stress stimulated the accumulation of coumarin and other defensive secondary metabolites in L. barbarum, and the accumulation effect of coumarin glycosides was stronger than that of coumarin under alkali stress. Alkaline stress also inhibited the growth of A. pallida gall. Based on these results, it could be preliminarily speculated that coumarins have important ecological significance in the environmental adaptation of L. barbarum and provided a theoretical guidance for the high-quality directional cultivation of L. barbarum. The study also found that the content of scopoletin in the unripe fruits and leaves of L. barbarum were higher, and the content of scopolin in the root barks and leaves were higher, which has important guiding significance for the development and utilization of its medicinal resources.