Application of jasmonic acid in improving indigo content, indigotin content and quinazoline content in isatis indigotica

Abstract

The application belongs to the technical field of cultivating Isatis indigotica Fort, and particularly relates to application of jasmonic acid to increasing content of indigo and / or indigotin in Isatis indigotica Fort. The application can effectively increase the content of indigo and indigotin in Isatis indigotica Fort by spraying jasmonic acid on Isatis indigotica Fort seedlings, and the content is rapidly increased. The test results show that, compared with a control group without applying jasmonic acid, the indigotin content in leaf blades of 1.5-2 month old Isatis indigotica Fort seedlings can be increased from 0 mu g / g to 356.94-744.13 mu g / g after 24-48 hours of applying jasmonic acid on Isatis indigotica Fort; the indigo content can be increased from 126.17 mu g / g to 173.27 mu g / g after 48 hours of applying jasmonic acid; and the quinazoline content can be increased from 75.67 mu g / g to 218.36-279.65 mu g / g. It can be seen that the application provides a technical basis for cultivating Isatis indigotica Fort with health care functions.

Description

Application of Jasmonic Acid in Increasing the Contents of Indigo, Indican and Quinazoline in Isatis indigotica Technical Field

[0001] The present invention belongs to the technical field of Isatis indigotica cultivation, and particularly relates to the application of jasmonic acid in increasing the contents of indigo, indican and quinazoline in Isatis indigotica. Background Art

[0002] Isatis indigotica Fortune belongs to the genus Isatis of the family Brassicaceae, and is a famous medicinal plant and natural dye plant. It is native to China and is cultivated throughout China, mainly distributed in Hebei, Jiangsu, Xinjiang and other places.

[0003] The dry powder, mass or granule prepared from the leaves or stems and leaves of Isatis indigotica is called Qingdai, which is both a famous traditional Chinese medicine and a natural dye. The main components of Qingdai are indigo and indirubin. Among them, indigo is formed by the hydrolysis of indican into 3-hydroxyindole and then air oxidation. It has the effects of clearing heat and detoxifying, cooling blood and removing macules, purging fire and relieving convulsion, enhancing immunity, etc.

[0004] In many regions of China, Isatis indigotica is used as a functional vegetable due to its rich medicinal components, and has the effects of supplementing vitamins, clearing heat and anti-inflammatory, and preventing colds. In addition, indigo and indican in the leaves of Isatis indigotica are also used by ethnic minorities in Yunnan and Guizhou regions for food dyeing. Therefore, finding an effective way to effectively increase the effective components of indigo and indican in the leaves of Isatis indigotica provides an important basis for the production and utilization of Isatis indigotica, and also has important significance for its scientific cultivation management. At present, there is no report on the related methods for increasing indigo and indican in the leaves of Isatis indigotica. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of jasmonic acid in increasing the contents of indigo, indican and quinazoline in Isatis indigotica. By applying jasmonic acid to Isatis indigotica, the contents of indigo, indican and quinazoline in Isatis indigotica can be effectively increased.

[0006] The present invention provides the application of jasmonic acid in one or more of increasing the contents of indigo, indican and quinazoline in Isatis indigotica.

[0007] Preferably, the Isatis indigotica includes Isatis indigotica leaves.

[0008] Preferably, the jasmonic acid is used in the form of a jasmonic acid solution, and the concentration of the jasmonic acid solution is 10 - 20 mM.

[0009] The present invention also provides a method for increasing one or more of the contents of indigo, indican and quinazoline in Isatis indigotica, including:

[0010] Spraying jasmonic acid on Isatis indigotica seedlings growing for 1.5 - 2 months.

[0011] Preferably, the jasmonic acid is used in the form of a jasmonic acid solution with a concentration of 10-20 mM.

[0012] Preferably, the spraying volume of jasmonic acid is 0.5-2 mL / plant.

[0013] Preferably, the jasmonic acid is sprayed between 9:00 AM and 12:00 PM.

[0014] Preferably, the jasmonic acid is sprayed at a temperature of 25-30°C.

[0015] Preferably, the woad includes woad leaves.

[0016] Preferably, the jasmonic acid is sprayed 1-2 days before harvesting the Isatis indigotica leaves.

[0017] Beneficial effects:

[0018] This invention provides the application of jasmonic acid in increasing the content of one or more of indigo, indoside, and quinazoline in Isatis tinctoria. Specifically, this invention also provides a method for increasing the content of one or more of indigo, indoside, and quinazoline in Isatis tinctoria, comprising: spraying jasmonic acid onto Isatis tinctoria seedlings that have grown for 1.5-2 months. This invention, by spraying jasmonic acid onto Isatis tinctoria seedlings, can effectively increase the content of indigo, indoside, and quinazoline in Isatis tinctoria, and the increase is rapid. The experimental results showed that, compared with the control group without jasmonic acid, applying jasmonic acid to Isatis indigotica for 24-48 hours increased the indigo glycoside content in Isatis indigotica leaves from 0 μg / g to 356.94-744.13 μg / g; the indigo content also increased from 126.17 μg / g to 173.27 μg / g after 48 hours of jasmonic acid application; and the quinazoline content in Isatis indigotica leaves increased from 75.67 μg / g to 218.36 μg / g (10 mM jasmonic acid treatment for 48 hours) - 241.49 μg / g (20 mM jasmonic acid treatment for 24 hours) after 24-48 hours of jasmonic acid application. Therefore, this invention provides a technical basis for the cultivation of Isatis indigotica for both edible and medicinal purposes with health benefits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 shows the standard curve for the determination of indoside in Test Example 1;

[0021] Figure 2 is the standard curve for determining indigo in Test Example 1;

[0022] Figure 3 shows the standard curve for the determination of quinazoline in Test Example 1;

[0023] Figure 4 shows the results of the determination of indigo glycoside content in Isatis indigotica leaves in Examples 1-3 and Comparative Example 1;

[0024] Figure 5 shows the results of the determination of indigo content in Isatis indigotica leaves in Examples 1-3 and Comparative Example 1;

[0025] Figure 6 shows the results of the determination of quinazoline content in Isatis indigotica leaves in Examples 1-3 and Comparative Example 1;

[0026] Figure 7 shows the HPLC analysis of the Isatis indigotica sample in Comparative Example 1;

[0027] Figure 8 shows the HPLC analysis chromatogram of the Isatis indigotica sample in Example 1;

[0028] Figure 9 shows the HPLC analysis chromatogram of the Isatis indigotica sample in Example 2. Detailed Implementation

[0029] This invention provides the application of jasmonic acid in increasing the content of indigo, indoside, and quinazoline in Isatis tinctoria, preferably in increasing the content of indigo, indoside, and quinazoline in Isatis tinctoria. The Isatis tinctoria described in this invention preferably includes Isatis tinctoria leaves; the jasmonic acid is preferably used in the form of a jasmonic acid solution, the concentration of which is preferably 10-20 mM. The Isatis tinctoria described in this invention preferably includes tetraploid Isatis tinctoria or diploid Isatis tinctoria, more preferably diploid Isatis tinctoria.

[0030] Specifically, the present invention also provides a method for increasing the content of one or more of indigo, indoside and quinazoline in woad, comprising: spraying jasmonic acid onto woad seedlings that have grown for 1.5-2 months.

[0031] In this invention, the *Isatis indigotica* preferably includes *Isatis indigotica* leaves; the jasmonic acid is preferably used in the form of a jasmonic acid solution, the concentration of which is preferably 10-20 mM; the preparation method of the jasmonic acid solution preferably includes: dissolving jasmonic acid in a trace amount of anhydrous ethanol, and then diluting it with distilled water to 10-20 mM; the spraying volume of the jasmonic acid is preferably 0.5-2 mL / plant, more preferably 1 mL / plant. This invention preferably uses a spray bottle for spraying. This invention preferably sprays the jasmonic acid 1-2 days before harvesting the *Isatis indigotica* leaves, and preferably between 9:00 AM and 12:00 PM; the spraying preferably includes foliar spraying. This invention involves spraying jasmonic acid 1-2 days before harvesting, which helps to rapidly increase the content of indigo, indoside, and quinazoline in Isatis tinctoria, thereby improving its quality. This avoids the weakened effect of spraying too early, which leads to a reduced increase in the content of indigo, indoside, and quinazoline, and the insufficient increase in the content of indigo, indoside, and quinazoline, which leads to a insufficient increase in the content of indigo, indoside, and quinazoline. The reason for spraying between 9:00 and 12:00 is that Isatis tinctoria grows more vigorously during this period and is more sensitive to external stimuli. Spraying with jasmonic acid can effectively stimulate Isatis tinctoria to accumulate a higher content of indigo, indoside, and quinazoline.

[0032] In this invention, when increasing the indigo content in Isatis tinctoria, it is preferable to spray a 10-20 mM jasmonic acid solution 2 days before harvesting, and more preferably a 10 mM jasmonic acid solution; when increasing the quinazoline content in Isatis tinctoria, it is preferable to spray a 10-20 mM jasmonic acid solution 1 day before harvesting, and more preferably a 20 mM jasmonic acid solution.

[0033] In this invention, the preferred temperature for jasmonic acid spraying is 25-30℃ to ensure the normal growth and metabolism of Isatis indigotica.

[0034] In this invention, the isatis seedlings preferably include isatis seedlings obtained from seed cultivation; the cultivation steps of the isatis seedlings obtained from seed cultivation preferably include: soaking and germinating isatis seeds to obtain isatis seedlings;

[0035] The woad seedlings were transplanted and cultivated to obtain the woad seedlings obtained from seed cultivation.

[0036] In this invention, isatis seeds are preferably soaked and germinated in a petri dish lined with filter paper. The filter paper is preferably in two layers, and each petri dish preferably contains 25 isatis seeds, which are then soaked in 7 mL of distilled water. The soaking and germination process is preferably carried out in an incubator, and the preferred conditions include: a photoperiod of 12L:12D; a light intensity of 1000-6000 lux, more preferably 3000 lux; a temperature of 10-35℃, more preferably 25℃; and a time of 3-7 days, more preferably 5 days. The isatis seeds used in this invention are preferably black, mature, and plump seeds. The variety of isatis is not specifically limited; for example, the variety used in this embodiment is Isatis indigotica Fortune.

[0037] After obtaining the Isatis indigotica seedlings, the present invention preferably transplants and cultivates the Isatis indigotica seedlings to obtain Isatis indigotica seedlings. The present invention preferably transplants the Isatis indigotica seedlings into humus soil for planting and cultivation; the specific cultivation conditions are not particularly limited and can be carried out according to conventional cultivation methods in the art.

[0038] This invention, by spraying jasmonic acid onto Isatis indigotica seedlings, can effectively increase the content of indigo, indoside, and quinazoline in Isatis indigotica, and the increase is rapid. The inventors hypothesize that the mechanism by which jasmonic acid increases the content of indigo and indoside in Isatis indigotica lies in the fact that jasmonic acid upregulates the expression of the indigo synthesis gene in Isatis indigotica seedlings, prompting the plant to actively produce indigo and / or indoside, as well as secondary metabolites such as quinazoline, in response to external stress or stimuli.

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] A method for increasing the levels of indigo, indigo glycosides, and quinazoline in Isatis indigotica includes the following steps:

[0042] Step 1: Select black, mature, and plump diploid seeds of cultivated woad (Isatis indigotica Fortune);

[0043] Step 2: Place the whole seed in a petri dish lined with two layers of filter paper, add 7 mL of distilled water, and culture under alternating light and dark conditions. The light culture conditions are: continuous light for 12 hours, light intensity of 3000 lux, and light culture temperature of 25℃. The dark culture conditions are: continuous darkness for 12 hours, dark culture temperature of 25℃, and germinate for 5 days.

[0044] Step 3: Transplant the woad seedlings into humus soil and water and fertilize them normally according to the conventional management methods for woad. Cultivate for 1.5 months. On the morning of the day before harvest, from 9:00 to 12:00, spray the leaves of the woad seedlings with 10mM jasmonic acid, with a spraying amount of 10mL / 10 plants.

[0045] Step 4: After 24 hours, the woad seedlings were harvested, and the contents of indigo, indigo glycoside, and quinazoline in the leaves were determined by HPLC.

[0046] Example 2

[0047] A method for increasing the levels of indigo, indigo glycosides, and quinazoline in Isatis indigotica includes the following steps:

[0048] Step 1: Select mature, plump, black cultivated woad (Isatis indigotica Fortune) seeds;

[0049] Step 2: Place the whole seed in a petri dish lined with two layers of filter paper, add 7 mL of distilled water, and culture under alternating light and dark conditions. The light culture conditions are: continuous light for 12 hours, light intensity of 3000 lux, and light culture temperature of 10-35℃. The dark culture conditions are: continuous darkness for 12 hours, dark culture temperature of 10-35℃, and germinate for 5 days.

[0050] Step 3: Transplant the woad seedlings into humus soil and water and fertilize them normally according to the conventional management methods for woad. Cultivate for 1.5 months. On the morning of the day before harvest, from 9:00 to 12:00, spray the leaves of the woad seedlings with 10mM jasmonic acid, with a spraying amount of 10mL / 10 plants.

[0051] Step 4: After 48 hours, the woad seedlings were harvested, and the contents of indigo, indigo glycoside, and quinazoline in the leaves were determined by HPLC.

[0052] Example 3

[0053] A method for increasing the levels of indigo, indigo glycosides, and quinazoline in Isatis indigotica includes the following steps:

[0054] Step 1: Select black, mature, and plump diploid seeds of cultivated woad (Isatis indigotica Fortune);

[0055] Step 2: Place the whole seed in a petri dish lined with two layers of filter paper, add 7 mL of distilled water, and culture under alternating light and dark conditions. The light culture conditions are: continuous light for 12 hours, light intensity of 3000 lux, and light culture temperature of 10-35℃. The dark culture conditions are: continuous darkness for 12 hours, dark culture temperature of 10-35℃, and germinate for 5 days.

[0056] Step 3: Transplant the woad seedlings into humus soil, water and fertilize normally, and cultivate for 2 months. On the morning of the day before harvest, from 9:00 to 12:00, spray the woad seedlings with 20mM jasmonic acid at a rate of 10mL per 10 plants.

[0057] Step 4: After 24 hours, the woad seedlings were harvested, and the contents of indigo, indigo glycoside, and quinazoline in the leaves were determined by HPLC.

[0058] Comparative Example 1

[0059] A method for increasing the levels of indigo, indigo glycosides, and quinazoline in Isatis indigotica includes the following steps:

[0060] Step 1: Select black, mature, and plump diploid seeds of cultivated woad (Isatis indigotica Fortune);

[0061] Step 2: Place the whole seed in a petri dish lined with two layers of filter paper, add 7 mL of distilled water, and culture under alternating light and dark conditions. The light culture conditions are: continuous light for 12 hours, light intensity of 3000 lux, and light culture temperature of 10-35℃. The dark culture conditions are: continuous darkness for 12 hours, dark culture temperature of 10-35℃, and germinate for 5 days.

[0062] Step 3: Transplant the woad seedlings into humus soil, water and fertilize normally, and cultivate for 1.5 months. On the morning of the day before harvest, spray the woad seedlings with tap water from 9:00 to 12:00, at a rate of 2 mL per seedling.

[0063] Step 4: After 24 hours and 48 hours, the woad seedlings were harvested, and the contents of indigo, indigo glycoside, and quinazoline in the leaves were determined by HPLC.

[0064] Test Example 1

[0065] The contents of indigo, indigo glycoside, and quinazoline in the leaves of Isatis indigotica in Examples 1-3 and Comparative Example 1 were determined using HPLC. The steps are as follows:

[0066] 1) Preparation of standard solutions and construction of standard curves: Accurately weigh approximately 1 mg each of indigo glycoside, indigo, and quinazoline standards, and place them in a 10 mL Erlenmeyer flask. Add 8 mL of N,N-dimethylformamide (DMF), sonicate for 30 min to dissolve, and then bring the volume to 10 mL. Prepare five concentration gradient standard solutions using this method. When constructing the standard curve, use the standard concentration (mg·mL⁻¹) as the reference value. -1Using the x-axis as the horizontal axis and peak area (A) as the y-axis, regression analysis was performed to calculate the regression equation and correlation coefficient for each standard. Standard curve: Indican standard curve formula: Y = 18764.79832X + 18.94950(R² + π / 2)² 2 =0.99744); Indigo standard curve formula Y = 31498.85124X + 71.13721(R) 2 =0.99960); H-quinazoline standard curve formula Y = 17631.64459X + 14.37054(R) 2 =0.9990 (as shown in Figure 1-3).

[0067] 2) Preparation of the test solution: Take the jasmonic acid-treated Isatis indigotica sample, wash it with water, gently wipe it dry with absorbent paper, wrap it in aluminum foil, and immediately immerse it in liquid nitrogen for 15 min. Then, quickly weigh about 200-500 mg and transfer it to a 2 mL centrifuge tube. Add 1 mL of DMF and two small steel balls (d = 4 mm), and grind them in a fully automated rapid sample grinder (Tissuelyser-24, Shanghai Jingxin Industrial Development Co., Ltd.). After grinding, add 7 mL of DMF, mix with a shaker, and extract ultrasonically for 30 min. After cooling to room temperature, make up to 10 mL, centrifuge for 10 min at 12000 rpm, and filter the supernatant through a 0.45 μm microporous membrane for later use.

[0068] 3) Chromatographic conditions: The HPLC instrument was an Agilent 1260 high-performance liquid chromatograph, and the column was an Agilent Technologies C18 column, model: Agilent ZORBAX Rx-C18, 4.6×250mm, 5μm. Mobile phase A was water, and mobile phase B was methanol, using a gradient elution method: 0 min: 10% methanol / 90% water; 30 min-50 min: 80% methanol / 20% water. Flow rate: 1.0 ml / min, column temperature: 30℃, detection wavelength: 280 nm, injection volume: 10 μL.

[0069] The results of indigo glycoside, indigo, and H-quinazoline determined in Examples 1-3 and Comparative Example 1 are shown in Figures 4-6; the HPLC chromatograms of Isatis indigotica leaves detected in Comparative Example 1 and Examples 1-2 are shown in Figures 7-9, where Figures 7-9 are the HPLC chromatograms of Isatis indigotica leaves after treatment with 10 mM jasmonic acid for 24 h and 20 mM jasmonic acid for 24 h, respectively.

[0070] As shown in Figure 4, compared with the Isatis indigotica sprayed with distilled water in Comparative Example 1 (CK), the indoside content in the Isatis indigotica leaves of Example 1 increased from 0 μg / g to 356.94 μg / g after spraying with 10 mM jasmonic acid for 24 h; the indoside content in the Isatis indigotica leaves of Example 2 increased from 0 μg / g to 744.13 μg / g after spraying with 10 mM jasmonic acid for 48 h; and the indoside content in the Isatis indigotica leaves of Example 3 increased from 0 μg / g to 600.01 μg / g after spraying with 20 mM jasmonic acid for 24 h.

[0071] As shown in Figure 5, compared with the isatis indigotica sprayed with distilled water in Comparative Example 1 (CK), the indigo content in the isatis indigotica leaves of the isatis in Example 2 increased from 126.17 μg / g to 173.27 μg / g after being sprayed with 10 mM jasmonic acid for 48 h.

[0072] As shown in Figure 6, compared with the Isatis indigotica sprayed with distilled water in Comparative Example 1 (CK), the quinazoline content in the Isatis indigotica leaves of Example 1 increased from 75.67 μg / g to 241.49 μg / g after spraying with 10 mM jasmonic acid for 24 h; the quinazoline content in the Isatis indigotica leaves of Example 2 increased from 75.67 μg / g to 218.36 μg / g after spraying with 10 mM jasmonic acid for 48 h; and the quinazoline content in the Isatis indigotica leaves of Example 3 increased from 75.67 μg / g to 279.65 μg / g after spraying with 20 mM jasmonic acid for 24 h.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for increasing the content of one or both of indoside and quinazoline in Isatis tinctoria, characterized in that, The process includes the following steps: spraying jasmonic acid onto the leaves of Isatis indigotica that have grown for 1.5-2 months; spraying jasmonic acid 1-2 days before harvesting the leaves; the jasmonic acid is used in the form of a jasmonic acid solution with a concentration of 10-20 mM.

2. A method for increasing the indigo content, indoside content, and quinazoline content in Isatis tinctoria, characterized in that, The process includes the following steps: spraying jasmonic acid onto the leaves of Isatis indigotica that have grown for 1.5-2 months; spraying jasmonic acid two days before harvesting the leaves; the jasmonic acid is used in the form of a jasmonic acid solution with a concentration of 10 mM.

3. The method according to claim 1 or 2, characterized in that, The spraying volume of jasmonic acid is 0.5-2 mL per plant.

4. The method according to claim 1 or 2, characterized in that, The jasmonic acid was sprayed between 9:00 AM and 12:00 PM.

5. The method according to claim 1 or 2, characterized in that, The jasmonic acid is sprayed at a temperature of 25-30℃.

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