| Lycium barbarum L.is a deciduous shrub of 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·g1 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. |
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