Illumination method for improving quality of andrographis paniculata

Abstract

The application provides a light irradiation method for improving the quality of Andrographis paniculata, and belongs to the technical field of agricultural planting. The method for improving the quality of Andrographis paniculata provided by the application comprises the following steps: starting to irradiate Andrographis paniculata with blue light when the Andrographis paniculata sprouts for 15 days. The method for improving the quality of Andrographis paniculata provided by the application is simple and easy to implement, has low cost, can promote the growth and development of Andrographis paniculata plants, can effectively improve the accumulation of terpene lactone components of Andrographis paniculata, and further improves the quality of Andrographis paniculata, thereby providing strong technical support for the high-quality development of the Andrographis paniculata industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agricultural planting, and particularly relates to a light irradiation method for improving the quality of Andrographis paniculata. BACKGROUND

[0002] Andrographis paniculata, as a traditional Chinese medicinal material, has important application value in the fields of medicine, health products and feed additives due to its rich content of andrographolide, 14-deoxyandrographolide and other terpenoids with antiviral, anti-inflammatory and immunoregulatory activities. With the rapid development of the health industry, the market demand for high-quality Andrographis paniculata raw materials is continuously increasing. The content of effective components such as andrographolide and 14-deoxyandrographolide in Andrographis paniculata directly determines its quality and medicinal value. However, under the traditional planting mode, the content of effective components of Andrographis paniculata is easily affected by environmental factors, which becomes a key bottleneck restricting the stability of its medicinal effect and the improvement of its economic value. The existing planting methods of Andrographis paniculata mostly focus on traditional agronomic measures such as soil improvement, fertilizer application and irrigation management, and the systematic research and application of light, a key environmental factor, are relatively weak. There is currently a lack of systematic research on the precise regulation of specific light quality to improve the quality of Andrographis paniculata in this field, and a set of light regulation technical solutions that can be widely applied to practical production to precisely improve the quality of Andrographis paniculata has not yet been formed, which cannot meet the increasing market demand for high-quality Andrographis paniculata. In addition, since light color, duration and intensity will affect the growth and accumulation of effective components of Andrographis paniculata, but the optimal light color, duration and intensity required at different growth stages have not been clearly defined, which makes it difficult to optimize the quality of medicinal materials by reasonably arranging light color, time and intensity in the actual cultivation process, further restricting the development process of the standardized and high-quality planting of Andrographis paniculata. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a method for improving the quality of Andrographis paniculata, which can effectively improve the accumulation of terpenoid lactone components of Andrographis paniculata at low cost, thereby improving the quality of Andrographis paniculata and further improving the clinical application value of Andrographis paniculata.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0005] The present application provides a method for improving the quality of Andrographis paniculata, comprising the following steps: starting to irradiate Andrographis paniculata with blue light when Andrographis paniculata has been sprouted for 15 days.

[0006] Preferably, the light intensity of the blue light irradiation is 20-200 μmol / m 2 ·s.

[0007] Preferably, the photoperiod of the blue light irradiation is 12-16 hours of light per day and 8-12 hours of darkness per day.

[0008] Preferably, the number of days of the blue light irradiation is 42 days or more.

[0009] Preferably, the wavelength of the blue light is 380-500 nm.

[0010] Preferably, the quality comprises a growth index, a biomass index and / or an effective component content.

[0011] Preferably, the growth index comprises plant height, leaf width and / or leaf length.

[0012] Preferably, the biomass index comprises fresh weight of aboveground part.

[0013] Preferably, the effective component content comprises andrographolide content and / or 14-deoxyandrographolide content.

[0014] The application also provides application of the above method in up-regulating expression level of genes related to andrographolide synthesis pathway.

[0015] The application has the following beneficial effects:

[0016] The method for improving quality of Andrographis paniculata provided by the application is simple and easy to implement, has low cost, can promote growth and development of Andrographis paniculata plants, can effectively improve accumulation of andrographolide components, and thus improves the quality of Andrographis paniculata, thereby providing strong technical support for high-quality development of Andrographis paniculata industry. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is a photograph of Andrographis paniculata treated with different light qualities, wherein A is a representative photograph of Andrographis paniculata treated with different light qualities for 42 days (left) and single leaves of the plants (right), and the scale bar is 1 cm; B is a statistical result of plant height of different light quality treatment groups; C is a statistical result of leaf width of different light quality treatment groups; D is a statistical result of leaf length of different light quality treatment groups; E is a statistical result of fresh weight of aboveground part of Andrographis paniculata plants of different light quality treatment groups; wherein different lowercase letters on the column indicate significant difference at a confidence level of 95%;

[0018] Figure 2 Figure 2 is an HPLC chromatogram of Andrographis paniculata under different light quality treatment, wherein 1 is andrographolide, 2 is 14-deoxyandrographolide, and 3 is neoadrographolide;

[0019] Figure 3Figure 6 shows the effect of different light quality treatments on the content of active ingredients in Andrographis paniculata, wherein A is the statistical result of andrographolide content under different light quality treatments, B is the statistical result of 14-deoxyandrographolide content under different light quality treatments, and C is the statistical result of neoadrographolide content under different light quality treatments, wherein the different small letters on the column indicate significant differences at the 95% confidence level;

[0020] Figure 4 Figure 7 shows the gene expression level of Andrographis paniculata under blue light treatment, wherein A is a heatmap and clustering of differentially expressed genes, B is a volcano plot showing the up-regulation and down-regulation of gene expression in blue light and white light samples, and the unchanged gene expression, C is the GO enrichment of the top 50 differentially expressed genes (DEGs) before blue light and white light treatment, and D is the KEGG analysis of the enrichment of differentially expressed genes between Andrographis paniculata plants under blue light and white light treatment;

[0021] Figure 5Heatmap for biosynthetic pathway of andrographolides in A. paniculata, showing the expression levels of genes related to the biosynthesis of andrographolide and neoadrographolide. Heatmap analysis is shown as normalized gene expression (FPKM), where ACT is acetyl-CoA C-acetyltransferase, HMGS is 3-hydroxy-3-methylglutaryl-CoA synthase, HMGR is 3-hydroxy-3-methylglutaryl-CoA reductase, MK is MVA kinase, PMK is phospho-MVA kinase, MDC is diphospho-MVA decarboxylase, DXS is 1-deoxy-D-xylulose-5-phosphate synthase, DXR is 1-deoxy-D-xylulose-5-phosphate reductoisomerase, MCT is 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase, CMK is 4-(cytidine 5' diphospho)-2-C-methyl-D-erythritol kinase, MDS is 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HDS is 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, HDR is 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, GGPPS is (E,E,E)-geranylgeranyl diphosphate synthase, KSL is kaurene synthase-like, TPS is terpene synthase, and CPS is copalyl / labdadienyl diphosphate synthase.

[0022] Figure 6 qRT-PCR analysis of gene expression levels in A. paniculata under blue light treatment, where A-E represent the relative expression levels of representative genes in the diterpenoid biosynthetic pathway in A. paniculata plants under white light and blue light treatment, respectively, as determined by RT-qPCR method. * indicates significant difference, as determined by Student’s t-test: ** indicates p < 0.01; *** indicates p < 0.001.

[0023] In the above-mentioned figures, WL represents white light, BL represents blue light, RL represents red light, GL represents green light, and YL represents yellow light. DETAILED DESCRIPTION

[0024] The present application provides a method for improving the quality of Andrographis, comprising the following steps: starting to irradiate Andrographis with blue light when Andrographis has been sprouted for 15 days.

[0025] In the present application, the 15 days of sprouting refers to the time from the emergence of Andrographis seeds from the soil, the complete unfolding of cotyledons and the exposure of green color, which is recorded as the first day of sprouting, and the time is continuously counted thereafter. On the 15th day, monochromatic blue light is started to be irradiated. The device for emitting blue light in the present application is not particularly limited, and any device capable of emitting blue light in the art can be used. In some embodiments of the present application, the device for emitting blue light comprises an LED lamp.

[0026] In the present application, the light intensity of the blue light irradiation is preferably 20-200 μmol / m 2 ·s, more preferably 30-80 μmol / m 2 ·s, and further preferably 40-70 μmol / m 2 ·s. In the present application, the photoperiod of the blue light irradiation is preferably 12-16 hours of light per day and 8-12 hours of darkness per day. In some embodiments of the present application, the photoperiod of the blue light irradiation is 12 hours of light per day and 12 hours of darkness per day, 13 hours of light per day and 11 hours of darkness per day, 14 hours of light per day and 10 hours of darkness per day, 15 hours of light per day and 9 hours of darkness per day, or 16 hours of light per day and 8 hours of darkness per day. In the present application, the number of days of the blue light irradiation is preferably more than 42 days, and more preferably 42 days to the time of harvest. In the present application, the wavelength of the blue light is preferably 380-500 nm. In the present application, the temperature of the growth environment of Andrographis is preferably 25-30°C, more preferably 26-29°C, and further preferably 27-28°C. The present application does not have a particular limitation on the specific planting method of Andrographis other than the blue light irradiation, and any conventional planting and cultivation method for Andrographis in the art can be used, such as natural light irradiation or white light irradiation before the blue light irradiation.

[0027] In the present application, the quality preferably includes growth indicators, biomass indicators and / or effective component contents. The growth indicators preferably include plant height, leaf width and / or leaf length. The biomass indicators preferably include fresh weight of aboveground parts. The effective component contents preferably include andrographolide content and / or 14-deoxyandrographolide content.

[0028] The method provided by the application can improve the content of andrographolide, improve the quality of andrographis, reduce the production cost, improve the clinical application value of andrographis, and can be applied to the production of high-quality andrographis in a greenhouse.

[0029] The application also provides application of the method in up-regulating the expression level of genes related to the synthesis pathway of andrographis terpenoid lactones.

[0030] The technical solutions provided by the application will be described in detail in combination with examples below, but they should not be understood as limitations to the protection scope of the application.

[0031] In the following examples, all the conventional methods are used unless otherwise specified.

[0032] In the following examples, all the materials and reagents used are commercially available unless otherwise specified.

[0033] Example 1

[0034] A method for improving the quality of andrographis, comprising the following steps:

[0035] When the andrographis is 15 days old, the andrographis is irradiated with blue light with a light intensity of 20 μmol / m 2 ·s and a wavelength of 450-480 nm, 14 hours of light and 10 hours of darkness per day, for a total of 62 days.

[0036] Example 2

[0037] A method for improving the quality of andrographis, comprising the following steps:

[0038] When the andrographis is 15 days old, the andrographis is irradiated with blue light with a light intensity of 200 μmol / m 2 ·s and a wavelength of 380-500 nm, 16 hours of light and 8 hours of darkness per day, for a total of 42 days.

[0039] Example 3

[0040] A method for improving the quality of andrographis, comprising the following steps:

[0041] When the andrographis is 15 days old, the andrographis is irradiated with blue light with a light intensity of 54 μmol / m 2The lotus was irradiated with blue light of wavelength 450-470 nm for 12 hours of light and 12 hours of darkness per day for a total of 42 days.

[0042] Experimental Example 1

[0043] Take an appropriate amount of Andrographis paniculata seeds and sow them in a petri dish lined with filter paper. Place the dish in a culture room (temperature 28℃, humidity 65%) for 7 days. Then, transplant the Andrographis paniculata seedlings into flowerpots and place them in a plant growth chamber. Set the culture temperature to 28℃ and maintain the air humidity at around 65% (64%–66%). The light cycle should be 12 hours of light / 12 hours of darkness. Water regularly until the seedlings develop their first pair of true leaves (approximately 15 days after emergence). Then, proceed with the following treatments:

[0044] Five treatment methods were set up: white light (WL) at 56 μmol / m 2 ·s, Blue light (BL) 54μmol / m 2 The light was applied at 62 μmol / m²·s for green light (GL), 54 μmol / m²·s for red light (RL), and 62 μmol / m²·s for yellow light (YL), with a light cycle of 12 hours of light and 12 hours of darkness. The culture temperature remained at 28℃, and the air humidity was maintained at around 65% (64%–66%).

[0045] The plants were irradiated for a total of 42 days. On day 42, growth indicators (plant height, leaf width, leaf length), biomass indicators (fresh weight of aboveground parts), and the content of active ingredients (andrographolide, 14-deoxyandrographolide, neoandrographolide) were measured in different treatment groups. Details are as follows (each experiment below had 3 biological replicates, and each biological replicate had 30 method replicates; values ​​are expressed as mean ± standard error):

[0046] (1) Phenotypic determination of Andrographis paniculata in different light quality treatment groups

[0047] In each treatment group, more than 30 *Andrographis paniculata* plants with uniform growth were randomly selected to measure plant height, leaf length, and leaf width. Leaf length and width were measured using calipers, and plant height was measured using a ruler from the cotyledon to the growing point. The measurements were repeated three times, and the average value was taken. Results are as follows: Figure 1 As shown in A to D, compared with white light (WL), the quality of Andrographis paniculata was improved after treatment with blue light (BL), which was reflected in the increase of plant height, leaf width and leaf length.

[0048] (2) Fresh weight of Andrographis paniculata in different light quality treatment groups

[0049] After different light quality treatments, 30 uniformly growing *Andrographis paniculata* plants were selected. The above-ground and underground parts of the plants were separated, and the fresh weight of the above-ground parts was measured using an electronic analytical balance to determine the yield of *Andrographis paniculata*. The results are as follows:Figure 1 As shown in E, compared with white light (WL), the fresh weight of the aboveground parts of Andrographis paniculata was significantly increased after treatment with blue light (BL).

[0050] (3) Determination of the contents of andrographolide, 14-deoxyandrographolide, and neoandrographolide

[0051] After different light quality treatments, *Andrographis paniculata* was harvested. Following the detection methods for *Andrographis paniculata* in the 2020 edition of the Chinese Pharmacopoeia, high-performance liquid chromatography (HPLC) was used to determine the contents of andrographolide, 14-deoxyandrographolide, and neoandrographolide to ascertain the quality of *Andrographis paniculata*. The results are as follows: Figure 2 and Figure 3 As shown, by Figure 2 It can be seen that the chromatographic peak sizes of the active components in each group of Andrographis paniculata under different light quality treatments varied considerably. Figure 3 It can be seen that, compared with white light, the blue light irradiation group showed a significant increase in andrographolide and 14-deoxyandrographolide in Andrographis paniculata, with andrographolide increasing by 25% and 14-deoxyandrographolide increasing by 40%.

[0052] (4) Transcriptome analysis of changes in Andrographis paniculata gene expression levels in the blue light treatment group

[0053] Transcriptome analysis was performed on blue light treatment groups that significantly improved the quality of Andrographis paniculata. Three batches of each treatment group were analyzed to determine which signaling pathways blue light could promote the accumulation of terpene lactones in Andrographis paniculata. Results are as follows: Figure 4 and Figure 5 As shown. By Figure 4 It can be seen that after blue light treatment, the metabolic pathways in *Andrographis paniculata* were significantly enriched, and the biosynthetic pathways of secondary metabolites and terpenoids showed a certain degree of enrichment. Figure 5 It can be seen that most genes in the andrographolide biosynthesis pathway showed an upregulation trend in the blue light treatment group.

[0054] (5) Real-time quantitative PCR detection of the expression of genes involved in the synthesis pathway of andrographis paniculata terpenoid lactones in the blue light-treated group.

[0055] To further verify that blue light treatment can upregulate the expression of genes involved in the andrographis paniculata terpene lactone biosynthesis pathway, qRT-PCR was used to detect the expression levels of key enzyme genes in this pathway. The results are as follows: Figure 6 As shown, qRT-PCR detection revealed significant upregulation of the ApDXS3, ApDXR1, ApDXR3, ApMDS, ApHDR1, ApHDR2, ApTPS02, ApTPS03, ApTPS3-like, ApCPS2, ApGGPPS1, and ApGGPPS2 genes.

[0056] The above results show that during the planting of Andrographis paniculata, giving a certain blue light treatment can promote the growth and development of Andrographis paniculata and increase the content of andrographolide and 14-deoxyandrographolide, thereby improving the quality of Andrographis paniculata and having practical popularization and application value.

[0057] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for improving quality of Andrographis paniculata, characterized by, The method comprises the following steps: starting to irradiate Andrographis paniculata with blue light when Andrographis paniculata sprouts for 15 days; The blue light irradiation has a light intensity of 20 to 200 μmol / m 2 ·s; The photoperiod of the blue light irradiation is 12-16 hours of light per day and 8-12 hours of darkness per day; The number of days of the blue light irradiation is more than 42 days; The wavelength of the blue light is 380-500 nm; The quality comprises growth indexes, biomass indexes and / or effective component contents; The growth indexes comprise plant height, leaf width and / or leaf length; The biomass indexes comprise fresh weight of aboveground parts; The effective component contents comprise andrographolide content and / or 14-deoxyandrographolide content.

2. The method of claim 1 is applied to up-regulate the expression level of genes related to the synthesis pathway of Andrographis paniculata terpenoid lactones.

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