| Andrographis paniculata is a globally well-known Chinese herbal medicinal plant,the main chemical components of which are diterpene lactones and flavonoids.Diterpene lactones have been widely explored,but flavonoids,especially the accumulation and distribution,are rarely reported.Previous evidences have supported the anti-inflammatory,anti-cancer,anti-atherosclerosis and other pharmacological functions of flavonoids,which can be applied in the treatment of fungal infections and Alzheimer’s disease.This study aims to explore the distribution and accumulation of flavonoids in Andrographis paniculata,which provides references for further research on Andrographis paniculata.Through the traditional chromatographic separation methods,12 compounds were separated from the ethanol extract of Andrographis paniculata,which were further identified by modern spectroscopy methods.They were skullcapflavone I,7-O-methyldihydrowogonin,7-O-methylwogonin,5-hydroxy-7,8,2’-trimethoxyflavone,5-hydroxy-7,8,2’,5’-tetramethoxyflavone,skullcapflavone I 2’-O-β-D-glucopyranoside,5,4’-dihydroxy-7-methoxyflavone-8-O-β-D-pyranoglucoside,andrographolide,neoandrographolide,dehydroandrographolide,deoxyandrographolide and hexacosanic acid.All these compounds were already known compounds.In this paper,the flavonoids in Andrographis paniculata were analyzed for the first time by thin-layer chromatography.Using skullcapflavone I,7-O-methyldihydrowogonin,7-O-methylwogoninand5-hydroxy-7,8,2’,5’-tetramethoxyflavone as the references,and petroleumed ether-dichloromethane-acetone-methanol（6:2:1:0.1）as the developing agent,the plate（Merck G）was developed under the conditions of 4℃and 47%humidity.It was then taken out and dried,sprayed with1%aluminum trichloride ethanol solution,and inspected at 254 nm.The results showed7-O-methyldihydrowogonin and 7-O-methylwogonin could be clearly observed,while skullcapflavone I and 5-hydroxy-7,8,2’,5’-tetramethoxyflavone were more difficult to be observed.TLC could be preliminarily employed to judge the contents of the four flavonoids in medicinal materials and decoction pieces of Andrographis paniculata.In this paper,the Gemini C18 column（4.6×250 mm,5μm）was used as the chromatographic column at room temperature,with acetonitrile-methanol-1%phosphoric acid water（40:4:56）as the mobile phase at the flow rate of 0.8 m L/min and detected at 285 nm.The contents of the four flavonoids,ie skullcapflavone I,7-O-methyldihydrowogonin,7-O-methylwogoninand5-hydroxy-7,8,2’,5’-tetramethoxyflavone were simultaneously determined for the first time by HPLC,which were precisely,stably and repeatedly measured and could be used for the determination of the above-mentioned flavonoids in Andrographis paniculata.The above-mentioned HPLC method was used to determine the contents of the above four flavonoids in medicinal materials and decoction pieces of Andrographis paniculata.The content of 7-O-methyldihydrowogonin in them ranged from 0.0161 to0.0906 mg·g-1and 0.0215 to 0.0534 mg·g-1,respectively.The content of 7-O-methylwogonin in them ranged from 0.0046 to 0.0272 mg·g-1 and 0.0110 to 0.0240 mg·g-1,respectively.The content of skullcapflavone I in them ranged from 0.0023 to 0.0233mg·g-1 and 0.0042 to 0.0121 mg·g-1,respectively.The content of 5-hydroxy-7,8,2’,5’-tetramethoxyflavone in them ranged from 0.0009 to 0.0156 mg·g-1 and 0.0026 to 0.0129mg·g-1.The total contents of four flavonoids in them ranged from 0.0282 to 0.1308 mg·g-1 and 0.0492 to 0.0918 mg·g-1,respectively.In order to obtained the best harvest time of Andrographis paniculata,the accumulation dynamics of flavonoids and diterpene lactones in Andrographis paniculata were analyzed for the first time as well.The accumulation of the four flavonoids in the root,stem and leaf of Andrographis paniculata was measured,respectively.Throughout the growth period,the contents of skullcapflavone I,7-O-methyldihydrowogonin and 5-hydroxy-7,8,2’,5’-tetramethoxyflavone and the total contents of four flavonoids in the root gradually increased with the growth of the plant,and their contents ranged from0.0217 to 0.2765 mg·g-1,0.0416 to 0.2576 mg·g-1,0.0086 to 0.1357 mg·g-1 and 0.3054to 0.8042 mg·g-1,respectively.However,the content of 7-O-methylwogonin in the root gradually decreased from 0.2335 mg·g-1 to 0.1344 mg·g-1 as the plant growth.The contents of skullcapflavone I,7-O-methyldihydrowogonin,7-O-methylwogonin and 5-hydroxy-7,8,2’,5’-tetramethoxyflavone and the total contents of four flavonoids in the stem and leaf gradually decreased as the plant growth.The contents of the four flavonoids and the total contents of four flavonoids in the stem ranged from 0.0004 to 0.0058 mg·g-1,0.0028 to 0.3811 mg·g-1,0.0008 to 0.4004 mg·g-1,0.0112 to 0.0394 mg·g-1 and 0.0040to 0.8267 mg·g-1,respectively,which were 0.0022 to 0.0137 mg·g-1,0.0004 to 0.0127mg·g-1,0.0008 to 0.0359 mg·g-1,0 mg·g-1 and 0.0025 to 0.0623 mg·g-1 in the leaf,respectively.In the root,the contents of andrographolide and deoxyandrographolide reached the peak before budding,and that of neoandrographolide reached the highest value after fruit maturation,while the content of dehydroandrographolide increased to the highest in the seedling stage,and the total contents of four diterpene lactones increased to the highest before budding stage and in the fruit-set period.In the stem,the contents of the four diterpene lactones and the total contents of four diterpene lactones gradually increased to the highest during the rapid growth period,and then gradually decreased.In the leaf,the contents of andrographolide,deoxyandrographolide,dehydroandrographolide and the total contents of four diterpene lactones gradually increased,which yielded the maximum at the fruit ripening stage,seedling stage,before budding stage and before budding stage,respectively.Then,they gradually decreased.The content of neoandrographolide in the whole growth period was relatively low and stable.The flavonoids in Andrographis paniculata mainly accumulate in the root,and the diterpene lactones in Andrographis paniculata mainly accumulate in the leaf.When the flavonoids in Andrographis paniculata are used as the inspection index,the above-ground part of Andrographis paniculata in the seedling stage and the underground part of Andrographis paniculata in the fruit ripening stage are better.In order to assess the relationship between the accumulation of flavonoids in Andrographis paniculata and other traits,flavonoids,diterpene lactones,plant height,number of stem nodes,number of primary tillers,number of secondary tillers and leaf-to-stem ratio were used as variables,after data standardized,their correlations were assessed using SPSS.Significant correlations among the four flavonoids in the root,stem and leaf were examined（P<0.01）.In the root,stem and leaf,the absolute value of the correlation coefficient among the four flavonoids ranged from 0.835 to 0.997,0.864 to0.977,and 0.947 to 0.954,respectively.Negative correlations among skullcapflavone I,7-O-methyldihydrowogonin and 5-hydroxy-7,8,2’,5’-tetramethoxyflavone in the root and among the four flavonoids in the stem and leaf were identified,while the 7-O-methylwogonin in the root had a positive correlation with the four flavonoids in the stem and leaf.All the four flavonoids in the stem and leaf all presented significant positive correlations with each other（P<0.01）.Increased accumulations of skullcapflavone I,7-O-methyldihydrowogonin and 5-hydroxy-7,8,2’,5’-tetramethoxyflavone in the root indicated reduced accumulations of the 7-O-methylwogonin in the root and the four flavonoids in the stem and leaf.Through the correlation analysis between the flavonoids in Andrographis paniculata and other traits,significant correlations among the flavonoids in Andrographis paniculata,the plant height and the number of first-level tillers were identified（P<0.01）.The correlation coefficients between the four flavonoids in root and the number of stem nodes were higher than those of other traits,which were 0.874,0.986,-0.991 and 0.903,respectively.The correlation coefficients between the four flavonoids in the stem and the leaf-to-stem ratio were higher than those of other traits,with 0.948,0.991,0.858 and 0.973,respectively.The correlation coefficients between skullcapflavone I in the leaf,7-O-methylwogonin in the leaf and leaf-to-stem ratio were higher than those of other traits,which were 0.940 and 0.987,respectively.The correlation coefficient between 7-O-methyldihydrowogonin in the leaf and the number of primary tillers was higher than that of other traits,which was-0.965.The correlations between flavonoids in Andrographis paniculata,dehydroandrographolide in the root,and deoxyandrographolide in the stem and leaf were more significant than those of other diterpene lactones.It’s suggested that there were close connections between the accumulations of flavonoids in Andrographis paniculata,dehydroandrographolide in the root,and deoxyandrographolide in the stem and leaf. |
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