Illumination method for improving quality of andrographis paniculata

Abstract

The invention provides an illumination method for improving the quality of andrographis paniculata, and belongs to the technical field of agricultural planting. The method for improving the quality of the andrographis paniculata comprises the following step of irradiating the andrographis paniculata with blue light 15 days after seedling emergence of the andrographis paniculata. The method for improving the quality of the andrographis paniculata is simple, easy to implement and low in required cost, growth and development of andrographis paniculata plants can be promoted, accumulation of andrographis paniculata terpene lactone components can be effectively improved, the quality of the andrographis paniculata is improved, and powerful technical support is provided for high-quality development of the andrographis paniculata industry.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural planting, and particularly relates to an illumination method for improving the quality of Andrographis paniculata. Background Art

[0002] Andrographis paniculata is a traditional Chinese medicinal material. It has important application value in the fields of medicine, health products and feed additives because it is rich in terpenoid compounds such as andrographolide and 14-deoxyandrographolide with antiviral, anti-inflammatory and immunomodulatory activities. With the rapid development of the health industry, the market demand for high-quality andrographis paniculata raw materials continues to grow. The content of active ingredients such as andrographolide and 14-deoxyandrographolide in andrographis paniculata directly determines its quality and medicinal value. However, under the traditional planting model, the content of active ingredients in andrographis paniculata is easily affected by fluctuations in environmental factors, which has become a key bottleneck restricting the stability of its efficacy and the improvement of its economic value. Existing andrographis paniculata planting methods 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. Currently, there is a lack of systematic research on methods for precisely controlling specific light quality to improve the quality of Andrographis paniculata. A comprehensive light control technology solution that can be widely applied in actual production to precisely improve the quality of Andrographis paniculata has not yet been established, making it difficult to meet the market's growing demand for high-quality Andrographis paniculata. Furthermore, since light color, duration, and intensity can affect Andrographis paniculata's growth and the accumulation of its active ingredients, the optimal light color, duration, and intensity required for different growth stages have not yet been clearly defined. This makes it difficult to optimize the quality of the medicinal material by rationally arranging light color, duration, and intensity during actual cultivation, further hindering the development of standardized and high-quality Andrographis paniculata cultivation. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for improving the quality of Andrographis paniculata, which can effectively increase the accumulation of Andrographis paniculata terpenoid lactone components 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 object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for improving the quality of Andrographis paniculata, comprising the following steps: starting to irradiate Andrographis paniculata with blue light 15 days after Andrographis paniculata emerges.

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

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

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

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

[0010] Preferably, the quality includes growth index, biomass index and / or active ingredient content.

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

[0012] Preferably, the biomass indicator includes the fresh weight of the aboveground part.

[0013] Preferably, the active ingredient content includes andrographolide content and / or 14-deoxyandrographolide content.

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

[0015] Beneficial effects of the present invention:

[0016] The method for improving the quality of Andrographis paniculata provided by the present invention is simple and easy to implement, has low cost, can promote the growth and development of Andrographis paniculata plants, and can also effectively increase the accumulation of Andrographis paniculata terpenoid lactone components, thereby improving the quality of Andrographis paniculata, and providing strong technical support for the high-quality development of the Andrographis paniculata industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 3. Effects of different light quality treatments on the phenotypes of Andrographis paniculata. A is a representative photograph of Andrographis paniculata treated with different light qualities for 42 days (left) and a single leaf of these plants (right). The scale bar is 1 cm. B is the statistical results of plant height under different light quality treatments. C is the statistical results of leaf width under different light quality treatments. D is the statistical results of leaf length under different light quality treatments. E is the statistical results of the fresh weight of the aboveground part of Andrographis paniculata plants under different light quality treatments. Different lowercase letters above the columns indicate significant differences at the 95% confidence level.

[0018] Figure 2 HPLC chromatograms of Andrographis paniculata under different light quality treatments, where 1 is andrographolide, 2 is 14-deoxyandrographolide, and 3 is neoandrographolide;

[0019] Figure 3The effect of different light quality treatments on the content of active ingredients in Andrographis paniculata, where 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 neoandrographolide content under different light quality treatments. Different lowercase letters on the columns indicate significant differences at the 95% confidence level.

[0020] Figure 4 Transcriptome analysis of gene expression levels in Andrographis paniculata under blue light treatment, where A is the heat map and clustering of differentially expressed genes, B is the volcano plot showing up- and down-regulation of gene expression in blue and white light samples, as well as unchanged gene expression, C is the GO enrichment of the top 50 differentially expressed genes (DEGs) under blue and white light treatments, and D is the KEGG analysis of the enrichment of differentially expressed genes between Andrographis paniculata plants under blue and white light treatments;

[0021] Figure 5Figure 3 Heat map of the andrographolide biosynthesis pathway in Andrographis paniculata, representing the expression levels of genes related to the biosynthesis of andrographolide and neoandrographolide. The heat map analysis is shown as normalized gene expression (FPKM). ACT is acetyl-CoA C-acetyltransferase, HMGS is 3-hydroxy-3-methylglutaryl-CoAsynthase, 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, and MCT is 2-C-methyl-D-erythritol-4-phosphate. cytidylyltransferase, CMK is 4-(cytidine5′diphospho)-2-C-methyl-D-erythritol kinase, MDS is 2-C-methyl-D-erythritol-2,4-cyclodiphosphatesynthase, HDS is 4-hydroxy-3-methylbut-2-enyldiphosphatesynthase, HDR is 4-hydroxy-3-methylbut-2-enyldiphosphatereductase, GGPPS is (E,E,E)-geranylgeranyl diphosphate synthase, KSL is kaurene synthase-like, TPS is terpene synthase, CPS is copalyl / labdadienyldiphosphate synthase;

[0022] Figure 6 This is qRT-PCR analysis of gene expression levels in Andrographis paniculata under blue light treatment. A to E are the relative expression levels of representative genes in the diterpenoid lactone biosynthesis pathway in Andrographis paniculata plants under white light and blue light treatment, respectively, determined by RT-qPCR. * indicates significant difference. Student's t-test: ** indicates p < 0.01; *** indicates p < 0.001.

[0023] In the above drawings, 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 invention provides a method for improving the quality of Andrographis paniculata, comprising the following steps: starting to irradiate Andrographis paniculata with blue light 15 days after Andrographis paniculata emerges.

[0025] In the present invention, the 15th day of emergence refers to the time from when the Andrographis paniculata seeds germinate and emerge from the soil, when the cotyledons are fully expanded and reveal green, which is recorded as the first day of emergence. The time is continuously counted, and on the 15th day, monochromatic blue light irradiation is started. The present invention does not specifically limit the device for emitting blue light, and any device that can emit blue light conventionally in the art can be used. In some embodiments of the present invention, the device for emitting blue light includes an LED lamp.

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

[0027] In the present invention, the quality preferably includes growth indicators, biomass indicators and / or active ingredient content; the growth indicators preferably include plant height, leaf width and / or leaf length; the biomass indicators preferably include fresh weight of the aboveground part; the active ingredient content preferably includes andrographolide content and / or 14-deoxyandrographolide content.

[0028] The method provided by the present invention can, while ensuring the healthy growth of Andrographis paniculata plants, directionally increase the content of andrographolide substances, significantly improve the quality of Andrographis paniculata, reduce production costs, and increase the clinical application value of Andrographis paniculata. It can be applied to the production of high-quality Andrographis paniculata in a greenhouse.

[0029] The present invention also provides the use of the above method for upregulating the expression levels of genes involved in the andrographolide lactone synthesis pathway. In the present invention, the genes preferably include ApDXS3, ApDXR1, ApDXR3, ApMDS, ApHDR1, ApHDR2, ApTPS02, ApTPS03, ApTPS3-like, ApCPS2, ApGGPPS1, and / or ApGGPPS2.

[0030] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1

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

[0035] When Andrographis paniculata seedlings emerged 15 days ago, the light intensity was 20 μmol / m 2 ·s, and irradiated Andrographis paniculata with blue light of wavelength of 450-480nm, with light period of 14 hours and dark period of 10 hours per day, for a total of 62 days.

[0036] Example 2

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

[0038] When Andrographis paniculata seedlings emerged 15 days ago, the light intensity was 200 μmol / m 2 ·s, and irradiate Andrographis paniculata with blue light of wavelength of 380-500nm, with light period of 16 hours and dark period of 8 hours per day, for a total of 42 days.

[0039] Example 3

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

[0041] When Andrographis paniculata seedlings emerged 15 days ago, the light intensity was 54 μmol / m 2·s, and irradiate Andrographis paniculata with blue light of wavelength of 450-470nm, with 12 hours of light and 12 hours of darkness per day, for a total of 42 days.

[0042] Test Example 1

[0043] An appropriate amount of Andrographis paniculata seeds were sown in a culture dish lined with filter paper and cultured in a culture room (temperature 28°C, humidity 65%) for 7 days. The Andrographis paniculata seedlings were then transplanted into pots and placed in a plant growth room with a culture temperature set at 28°C and an air humidity maintained at approximately 65% (64%-66%). The photoperiod was 12 hours light / 12 hours dark. Watering was performed regularly until the seedlings grew their first pair of true leaves (calculated from the time of emergence, 15 days after emergence). The following treatments were then carried out:

[0044] Set up 5 treatment methods: using white light (WL) 56μmol / m 2 s, blue light (BL) 54 μmol / m 2 ·s, green light (GL) 62μmol / m2·s, red light (RL) 54μmol / m2·s, and yellow light (YL) 62μmol / m2·s were irradiated, with a light cycle of 12 hours light / 12 hours dark, the culture temperature remained at 28°C, and the air humidity was maintained at around 65% (64%~66%).

[0045] Irradiation was performed for a total of 42 days. On the 42nd day, the growth indicators (plant height, leaf width, leaf length), biomass (fresh weight of aboveground parts), and active ingredient content (andrographolide, 14-deoxyandrographolide, and neoandrographolide) of Andrographis paniculata plants in the different treatment groups were measured. The details are as follows (each experiment below had three biological replicates, each biological replicate had 30 method replicates, and 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, randomly selected andrographis paniculata plants (>30 plants) with the same growth were measured for plant height, leaf length and leaf width. The leaf length and leaf width were measured with a vernier caliper, and the plant height was measured from the cotyledon to the growth point with a ruler. The results were repeated 3 times and the average value was taken. Figure 1 As shown in Figures A to D, compared with white light (WL), the quality of Andrographis paniculata was improved after treatment with blue light (BL), as reflected by the increase in plant height, leaf width, and leaf length.

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

[0049] After the different light quality treatments, 30 Andrographis paniculata plants with the same growth were selected, the aboveground and underground parts of the plants were separated, and the fresh weight of the aboveground parts of Andrographis paniculata was weighed using an electronic analytical balance to determine the Andrographis paniculata yield. Figure 1 As shown in Figure E, compared with white light (WL), the fresh weight of the aerial part of Andrographis paniculata was significantly increased after blue light (BL) treatment.

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

[0051] After the different light quality treatments, Andrographis paniculata was harvested and the content of andrographolide, 14-deoxyandrographolide and neoandrographolide in Andrographis paniculata was detected by high performance liquid chromatography (HPLC) according to the Andrographis paniculata detection method under the 2020 edition of the Chinese Pharmacopoeia to determine the quality of Andrographis paniculata. Figure 2 and Figure 3 As shown by Figure 2 It can be seen that the chromatographic peak sizes of the active ingredients in each group of Andrographis paniculata under different light quality treatments are quite different. Figure 3 It can be seen that compared with white light, the andrographolide and 14-deoxyandrographolide in the Andrographis paniculata group treated with blue light increased significantly, 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] The blue light treatment group with significant improvement in the quality of Andrographis paniculata was selected for transcriptome analysis. Three batches were made for each treatment group to analyze the signal pathways through which blue light can promote the accumulation of Andrographis paniculata terpenoid lactones. Figure 4 and Figure 5 As shown. Figure 4 It can be seen that after blue light treatment, the metabolic pathways in Andrographis paniculata were significantly enriched, and the secondary metabolite biosynthesis pathway and terpenoid synthesis pathway showed a certain degree of enrichment. Figure 5 It can be seen that most of the genes in the andrographolide lactone biosynthesis pathway showed an up-regulation trend in the blue light treatment group.

[0054] (5) Real-time fluorescence quantitative (qRT-PCR) detection of the expression of genes in the Andrographis paniculata lactone synthesis pathway in the blue light treatment group

[0055] To further verify that blue light treatment can upregulate the expression of genes in the biosynthesis pathway of andrographolide lactones, qRT-PCR was used to detect the expression levels of key enzyme genes in the biosynthesis pathway of andrographolide lactones. Figure 6 As shown, qRT-PCR detection showed that ApDXS3, ApDXR1, ApDXR3, ApMDS, ApHDR1, ApHDR2, ApTPS02, ApTPS03, ApTPS3-like, ApCPS2, ApGGPPS1, and ApGGPPS2 genes were significantly upregulated.

[0056] The above results show that during the cultivation process of Andrographis paniculata, giving a certain amount of 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 promotion and application value.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for improving the quality of Andrographis paniculata, characterized in that: The method comprises the following steps: starting to irradiate the Andrographis paniculata with blue light 15 days after the Andrographis paniculata seedlings emerge.

2. The method according to claim 1, characterized in that The light intensity of the blue light irradiation is 20 to 200 μmol / m 2 ·s.

3. The method according to claim 1, characterized in that The photoperiod of the blue light irradiation is 12 to 16 hours of light per day and 8 to 12 hours of darkness per day.

4. The method according to claim 1, wherein The number of days for the blue light irradiation is more than 42 days.

5. The method according to claim 1, wherein The wavelength of the blue light is 380-500 nm.

6. The method according to claim 1, characterized in that The quality includes growth index, biomass index and / or active ingredient content.

7. The method according to claim 6, characterized in that The growth indicators include plant height, leaf width and / or leaf length.

8. The method according to claim 6, characterized in that The biomass index includes the fresh weight of the aboveground part.

9. The method according to claim 6, characterized in that The active ingredient content includes andrographolide content and / or 14-deoxyandrographolide content.

10. Use of the method according to any one of claims 1 to 9 in up-regulating the expression level of genes related to the synthesis pathway of andrographolide lactones.

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