A siderophore compound and its preparation and application

Abstract

The present invention relates to the field of microbial drug technology, specifically to a siderophore compound (hydroxamate siderophore compound), its preparation method, and application. The compound is the following compound 1-3; the hydroxamate siderophore provided by the present invention has significant iron chelation ability and promotes seed germination and plant growth activity. They are ideal compounds for use as therapeutic agents for iron overload diseases and growth regulators for promoting crop growth. The compound can be produced by fermentation with special actinomycetes, its preparation method is simple and efficient, and the resulting product is high in purity. The invention has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial medicines, and in particular to an iron carrier compound (a hydroxamate iron carrier compound) and a preparation method and application thereof. Background Art

[0002] Iron is an essential element for many metabolic systems in living organisms. It is a component of organic molecules such as cytochromes and ferric redox proteins, and is also a cofactor and activator for many enzymes. Therefore, iron deficiency in organisms will seriously affect their physiological metabolic processes, and iron overload will also lead to biological dysfunction and organ damage. Generally speaking, the iron required by organisms is Fe 2+ , and most of the iron in nature is in the form of Fe 3+ Therefore, the absorption and transport of iron play an important role in the growth and development of organisms.

[0003] Siderophores are low-molecular-weight compounds produced by bacteria or fungi with metal chelating abilities. Their transport function and ability to regulate iron levels have broad applications in enhancing beneficial microbial colonization and pathogen inhibition, promoting plant iron uptake, biosignaling, and developing siderophore-antibiotic conjugates. For example, siderophores produced by Aspergillus niger, Penicillium citrinum, and Trichoderma harzianum can increase the stem and root length of chickpeas. Siderophores produced by Bacillus subtilis effectively inhibit Fusarium oxysporum, the pathogen of pepper wilt. The siderophore deferrioxamine B inhibits the growth of Trypanosoma brucei. Furthermore, numerous studies have demonstrated that regulating ferroptosis plays a key role in a variety of conditions, including tumor suppression and immunity, neurodegenerative diseases, tissue and organ damage, inflammatory and infectious diseases. Therefore, siderophores are potentially useful in research on diabetes, neurodegenerative diseases, anticancer agents, and the novel coronavirus. In particular, the current "Trojan Horse" program formed by the coupling of iron carriers and antibiotics has led to the discovery of new trihydroxamate iron carrier-ciprofloxacin and tricatechin iron carrier-amoxicillin coupled drugs, making the coupling of drugs and iron carriers a research hotspot with huge development prospects.

[0004] Siderophores are classified into four types based on their chemical structure and iron chelating groups: catecholate, hydroxamate, carboxylate, and mixed siderophores. Hydroxamate siderophores are primarily produced by Streptomyces and fungi. It is worth mentioning that the hydroxamate siderophore, deferoxamine, has been used for half a century as an iron chelator approved by the Food and Drug Administration. Its agent, deferoxamine B mesylate, can be used to treat chronic iron overload, such as transfusion-induced hemosiderosis, severe thalassemia, and chronic anemia, as well as acute iron poisoning or aluminum overload. This patent provides novel hydroxamate siderophores with higher activity prepared from a newly discovered rare actinomycete species (Streptomonas nanhaiense 12A09), and invents the application of these novel siderophores. Summary of the Invention

[0005] The present invention aims to provide an iron carrier compound (hydroxamate iron carrier compound) and a preparation method and application thereof.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A siderophore compound, wherein the compound is the following compound 1-3:

[0008]

[0009] A method for preparing siderophore compounds, using Streptomonospora nanhaiensis 12A09 T ) through fermentation, culture and purification to obtain the compound 1-3.

[0010] The Streptomonospora nanhaiensis 12A09 T ) See the literature Int. J. Syst. Evol. Microbiol. 2013. 64: 4447-4455 (https: / / doi.org / 10.1099 / ijs.0.052704-0).

[0011] Specifically:

[0012] (1) Streptomonospora nanhaiensis 12A09 T ) were inoculated onto Gao's solid medium No. 1 and cultured for 3-6 days for activation; the activated strain was inoculated into 50 mL of NO2 medium using the digging method and cultured at 25°C-35°C, 180-220 rpm, and shaken for 2-5 days to serve as the fermentation seed liquid;

[0013] (2) The seed solution obtained above was aseptically inoculated into NO2 medium and cultured at 25°C-35°C, 180-220 rpm, and shaking for 5-10 days;

[0014] (3) The fermentation broth obtained above was separated into solid and liquid, 20-50 g of macroporous adsorption resin HP20 was added to the fermentation supernatant, and adsorption was carried out under shaking at room temperature for 2-4 h. The adsorption resin was then collected and eluted with methanol to obtain a siderophore extract;

[0015] (4) The siderophore extract was separated by silica gel column chromatography, and gradient elution was performed with a dichloromethane:methanol (v / v) ratio of 100:0 to 0:100, and the fractions with a ratio of 95:5 to 60:40 were collected to obtain compound 1-3 according to claim 1.

[0016] In step (4), the collected components are separated by silica gel column chromatography, and the fraction FA1 is eluted with a volume ratio of dichloromethane to methanol of 95:5-84:16, and the fraction FA2 is eluted with a volume ratio of dichloromethane to methanol of 83:17-60:40;

[0017] Fraction FA1 was separated on Sephadex LH-20 and eluted with a solvent having a volume ratio of 1:1 of dichloromethane to methanol. 60-90 mL of the eluate was collected as subfraction Fr2, and 120-150 mL of the eluate was collected as subfraction Fr7.

[0018] The subfraction Fr2 was purified by semi-preparative HPLC, eluting with 40-50% aqueous methanol at a flow rate of 1.5-3.0 mL / min to obtain compound 1 (t R 13-17min)、4(t R 18-22min) and 5(t R 25-30min); the sub-fraction Fr7 was eluted with 40-50% aqueous methanol at a flow rate of 1.5-3.0mL / min to obtain compound 6 (t R 15-20min);

[0019] Fraction FA2 was separated by Sephadex LH-20 and eluted with a solvent of dichloromethane:methanol in a volume ratio of 1:1, and 50-180 mL of the eluate was collected. The eluate was purified by semi-preparative HPLC with 20-40% methanol in water at a flow rate of 1.5-3.0 mL / min to obtain compound 2 (t R 7-10.5min) and 3(t R 11-16min).

[0020] The NO2 liquid culture medium is composed of 10g starch, 20g glucose, 10g yeast extract, 10g tryptone, 3g beef extract, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 2g calcium carbonate, and 1L of aged seawater containing 3% sea salt per liter of water, with a pH of 7.0;

[0021] An application of the siderophore compound, wherein the compounds 1-3 are used for preparing potential iron-removing agents.

[0022] An application of the siderophore compound, wherein the compounds 1-3 are used in the preparation of a crop seed germination promoting agent, a crop growth promoting agent, or a siderophore-antibiotic coupled drug.

[0023] An application of the compounds prepared by the method, wherein the compounds 4, 5 and 6 are used in the preparation of a crop seed germination promoting agent, a crop growth promoting agent or an iron carrier-antibiotic coupled drug.

[0024] The advantages of the present invention are:

[0025] The hydroxamate siderophores of the present invention are produced by special strains through specific fermentation conditions and processes, and are different from the strains that previously produced hydroxamate siderophores at the family and genus classification level. Because different fermentation media and culture conditions have a significant impact on the production of metabolites, the culture media and culture conditions of the present invention can significantly increase the structural diversity and yield of hydroxamate siderophores, and can produce siderophores with novel structures. For example, compounds 1-3 differ from previously reported siderophore compounds in terms of cyclization and substituents, and their structural changes have a significant impact on biological activity. The hydroxamate siderophores prepared by the present invention are significantly more active than deferoxamine mesylate, a drug for treating iron overload, and have good application prospects.

[0026] The hydroxamate siderophores of the present invention are found for the first time to be able to promote plant seed germination and plant growth activity, and are ideal compounds for promoting crop growth as growth regulators.

[0027] The compound of the present invention can be produced by fermentation with special actinomycetes, and the preparation method is simple and efficient, and the obtained product has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention provides the detection results of siderophores produced by strain 12A09 under different fermentation conditions (A: different fermentation medium; B: different liquid volumes in fermentation shake flasks).

[0029] Figure 2 The structures of compounds 1-6 provided in the examples of the present invention.

[0030] Figure 3Compound 1-3 provided in the embodiments of the present invention 1 H- 1 Significant correlation between H COSY and HMBC.

[0031] Figure 4 This is the iron chelating activity of compounds 1-6 provided in the examples of the present invention. DETAILED DESCRIPTION

[0032] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the protection content of this patent is not limited to this.

[0033] Example 1 Preparation method of siderophore produced by fermentation of Streptomyces nanhaiense 12A09

[0034] A new species of Streptomonospora nanhaiensis 12A09 isolated from deep-sea sediments of the South China Sea was used. T ) was prepared, the strain is described in Int. J. Syst. Evol. Microbiol. 2013. 64: 4447-4455, and its deposit number is CCTCC AB 2013140. The strain 12A09 was inoculated into different culture media NO2, NM2, ISP3 and PSA (see Figure 1 ), the ability to produce siderophores was determined by the chromium azosulfonate (CAS) plate method, and the Figure 1 A shows that the activity of producing iron carriers in NO2 medium is the strongest; then we further screened the effect of different loading volumes of NO2 medium on its ability to produce iron carriers. The study showed that the strongest ability to produce iron carriers was achieved when the loading volume was 600 mL ( Figure 1 B).

[0035] In summary, the strain has the strongest activity in producing siderophores on NO2 medium. Figure 1 ), molecular network analysis based on HPLC-MS / MS showed that the iron carrier diversity was the richest and most novel, so it was fermented and prepared according to the optimized conditions.

[0036] First, strain 12A09 was inoculated onto Gao's solid medium No. 1 and cultured for 3-5 days for activation. The activated strain was then inoculated into 50 mL of NO2 medium using the slug method. The culture was shaken at 28°C, 180 rpm, for 3 days to serve as the fermentation seed liquid. The cultured seed liquid was then aseptically inoculated into a 3-L shake flask containing 600 mL of NO2 medium and cultured at 28°C, 180 rpm, for 7 days, resulting in a total fermentation volume of approximately 49 L. After fermentation, the fermentation broth was centrifuged to separate the solid and liquid phases. The supernatant was then added to 40 g of macroporous adsorption resin HP20 and adsorbed at room temperature for 2 hours with shaking. The adsorption resin was then collected and eluted with methanol to yield 43.07 g of siderophore extract.

[0037] The siderophore extract was further separated by silica gel column chromatography, and gradient elution was performed with a dichloromethane:methanol (v / v) ratio of 100:0 to 0:100, and fractions FA1 and FA2 were collected with a dichloromethane:methanol volume ratio of 90:10 to 85:15.

[0038] Fraction FA1 was separated on Sephadex LH-20 and eluted with a solvent having a volume ratio of 1:1 of dichloromethane and methanol. 73-84 mL of the eluate was collected as subfraction Fr2, and 133-144 mL of the eluate was collected as subfraction Fr7. Subfraction Fr2 was purified by semi-preparative HPLC using 45% aqueous methanol at a flow rate of 2.0 mL / min to obtain compound 1 (t R 14.2-15.8min)、4(t R 19.1-21.1min) and 5(t R 25.5-27.5min); eluting the subfraction Fr7 with 45% aqueous methanol at a flow rate of 2.0 mL / min, compound 6 (t R 17.5-19.0min).

[0039] Fraction FA2 was separated by Sephadex LH-20 and eluted with a solvent of dichloromethane:methanol in a volume ratio of 1:1, collecting 61-168 mL of the eluate. The eluate was purified by semi-preparative HPLC with 30% methanol in water at a flow rate of 2.0 mL / min to obtain compound 2 (t R 9.5-10.8min) and 3(t R 11.8-12.5min).

[0040] Example 2: Structural analysis of the hydroxamate siderophores 1-6 obtained above

[0041] Compound 1 is a white powder. The molecular ion peak given by HR-ESI-MS is [M+Na] + m / z 591.3458, the molecular formula of the compound was determined to be C 27 H 48 O7N6, unsaturation degree is 7, UV(MeOH)λ max :230nm. Infrared spectrum shows OH peak at 3291cm -1 and amide peak at 3705 cm -1 and 3671cm -1 . Combined 1 H-NMR (DMSO-d6, 600 MHz) gave six active hydrogen proton signals (δ H 9.59, 7.76, 7.75, 7.74, 7.73, 7.72), and many methylene proton signals (δ H 1.22-3.47). 13 C-NMR (DMSO-d6, 150 MHz) data showed the presence of 27 carbon signals, of which 6 were ester or amide carbonyl carbon signals (δ C 171.4, 171.4, 171.3, 171.2, 171.2, 171.2), and 21 methylene sp 3 Hybridized carbon signal. Compound 1 H- 1 The main correlations between H COSY and HMBC are as follows Figure 3 .from 1 H- 1 The H COSY spectrum results showed that H-3 / H-4, H-7 / H-8 / H-9 / H-10 / H-11, H-14 / H-15, H-18 / H-19 / H-20 / H-21 / H-22, H-25 / H-26, H-29 / H-30 / H-31 / H-32 / H-33 were related. HMBC showed that H-7 was remotely related to C-9, H-8 to C-10, H-11 to C-9, H-11 to C-13, and H-18 to C-20. In summary, the chemical structure of compound 1 is as follows: Figure 2 , a new compound not reported in the literature. It is a derivative of the compound terragine E, with an additional amide bond and a missing -CO-NOH group. Compound 1 is named desferrioxamine G1. 1 H-NMR (DMSO-d6, 600 MHz) and 13 C-NMR (DMSO-d6, 150 MHz) data are shown in Table 1.

[0042] Compound 2 is a white powder. The molecular ion peak given by HR-ESI-MS is [M+Na]+ m / z 575.3507, the molecular formula of the compound was determined to be C 27 H 48 O6N6, unsaturation degree is 7, UV(MeOH)λ max :210nm. Infrared spectrum shows amide peak at 3705cm -1 and 3671cm -1 and 3290cm -1 . Combined 1 H-NMR (CD3OD-d4, 600 MHz) gave 21 methylene proton signals (δ H 1.34-3.18). 13 C-NMR (CD3OD-d4, 150 MHz) data showed the presence of 21 carbon signals, including 6 ester or amide carbonyl carbon signals (δ C 171.0-174.4), and 21 methylene sp 3 Hybridized carbon signal. Compound 1 H- 1 The main correlations between HCOSY and HMBC are as follows Figure 3 .from 1 H- 1 The H COSY spectrum results showed that H-3 / H-4, H-7 / H-8 / H-9 / H-10 / H-11, H-14 / H-15, H-18 / H-19 / H-20 / H-21 / H-22, H-25 / H-26, H-29 / H-30 / H-31 / H-32 / H-33 were correlated. HMBC showed that H-7 was remotely correlated with C-5, H-7 with C-9, H-8 with C-10, and H-9 with C-11. In summary, the chemical structure of compound 2 is as follows: Figure 2 , a new compound not reported in the literature. It is a derivative of the aforementioned compound desferrioxamine G1, with an additional amide bond and a missing -CO-NOH group. Compound 2 is named desferrioxamine G2. 1 H-NMR (CD3OD-d4, 600 MHz) and 13 C-NMR (CD3OD-d4, 150 MHz) data are shown in Table 1.

[0043] Compound 3 is a white powder. The molecular ion peak given by HR-ESI-MS is [M+Na] + m / z 393.2459, the molecular formula of the compound was determined to be C 18 H 34 O4N4, unsaturation is 4, UV(MeOH)λ max:220nm, indicating that the molecule contains a, b unsaturated carbonyl groups. The infrared spectrum shows an amide peak at 3705cm -1 and 3671cm -1 and 3290cm -1 . Combined 1 H-NMR (CD3OD-d4, 600 MHz) gave 12 methylene proton signals (δ H 1.36-3.16), two methyl double proton signals (δ H 1.94)(6H, s). 13 C-NMR (CD3OD-d4, 150 MHz) data showed the presence of 18 carbon signals, including 4 ester or amide carbonyl carbon signals (δ C 174.3, 174.3, 173.0, 173.0), and 12 methylene sp 3 hybrid carbon signal, and two overlapping methyl carbon signals (δ C 22.3). Compound 1 H- 1 The main correlations between H COSY and HMBC are as follows Figure 3 .from 1 H- 1 The HCOSY spectrum results showed that H-4 / H-5 / H-6 / H-7 / H-8, H-11 / H-12, H-15 / H-16, / H-17 / H-18 / H-19 were correlated. HMBC showed that H-4 and C-6, H-5 and C-7, H-6 and C-8, H-8 and C-10 were remotely correlated. In summary, the chemical structure of compound 3 is as follows Figure 2 , which is a new compound not reported in the literature. Compound 3 was named desferrioxamine G3. 1 H-NMR (CD3OD-d4, 600 MHz) and 13 C-NMR (CD3OD-d4, 150 MHz) data are shown in Table 1.

[0044] Table 1 Compounds 1-3 1 H and 13 C NMR data (600 / 150 MHz, δ in ppm)

[0045]

[0046]

[0047] a Measured in DMSO-d6.

[0048] b Measured in CD3OD-d4.

[0049] Compound 4 is a white powder. 1 H-NMR (DMSO-d6, 600 MHz) gave four active hydrogen proton signals (δ H 7.74, 7.73, 7.72, 7.71) and two active hydrogen proton signals (δ H 9.58), and 21 methylene proton signals (δ H 1.20-3.45). 13 C-NMR (DMSO-d6, 150 MHz) data showed the presence of 27 carbon signals, including 6 ester or amide carbonyl carbon signals and 21 methylene sp 3 The hybridized carbon signal indicates that it is also a desferrioxamine-type substance. Further comparison shows that the above data are basically consistent with the data of terragine E in the literature, so compound 4 is identified as terragine E ( Figure 2 ). 1 H-NMR (DMSO-d6, 600 MHz) and 13 C-NMR (DMSO-d6, 150 MHz) data are shown in Table 2.

[0050] Compound 5 is a white powder. 1 H-NMR (DMSO-d6, 600 MHz) gave three active hydrogen proton signals (δ H 7.71) and three active hydrogen proton signals (δ H 9.58), and 21 methylene proton signals (δ H 1.20-3.45). 13 C-NMR (DMSO-d6, 150 MHz) data showed the presence of 27 carbon signals, including 6 ester or amide carbonyl carbon signals and 21 methylene sp 3 The hybridized carbon signal indicates that it is also a desferrioxamine-type substance. Further comparison shows that the above data are basically consistent with the data of desferrioxamine E in the literature, so compound 5 is identified as desferrioxamine E ( Figure 2 ). 1 H-NMR (DMSO-d6, 600 MHz) and 13 C-NMR (DMSO-d6, 150 MHz) data are shown in Table 2.

[0051] Compound 6 is a white powder. 1 H-NMR (DMSO-d6, 600 MHz) gave three active hydrogen proton signals (δ H7.71) and three active hydrogen proton signals (δ H 9.61, 9.57, 9.57), and 20 methylene proton signals (δ H 1.20-3.45). 13 C-NMR (DMSO-d6, 150 MHz) data showed the presence of 26 carbon signals, including 6 ester or amide carbonyl carbon signals and 20 methylene sp 3 The hybridized carbon signal indicates that it is also a desferrioxamine-type substance. Further comparison shows that the above data are basically consistent with the desferrioxamine D2 data in the literature, so compound 6 is identified as desferrioxamine D2 ( Figure 2 ). 1 H-NMR (DMSO-d6, 600 MHz) and 13 C-NMR (DMSO-d6, 150 MHz) data are shown in Table 2.

[0052] Table 2 Compounds 4-6 1 H and 13 C NMR data (600 / 150 MHz, δ in ppm, DMSO-d6)

[0053]

[0054] Example 3 Determination of iron chelating ability of siderophores

[0055] Preparation of CAS detection solution (100 mL): Add 6 mL of 10 mM HDTMA aqueous solution to a 100 mL volumetric flask. While stirring with a glass rod, slowly add 1.5 mL of an iron-containing solution (1 mM ferric chloride hexahydrate prepared in 10 mM HCl) and 7.5 mL of a 2 mM CAS (chrome azurol S) aqueous solution. Slowly and carefully add 10 mL of 0.5 M anhydrous piperazine solution to 6.25 mL of 12 M hydrochloric acid. Rinse the buffer solution (pH 5.6) into the volumetric flask and dilute to 100 mL with water to provide 100 mL of CAS detection solution.

[0056] The monomeric compounds 1-6 obtained in the above examples were used as test samples, and deferoxamine mesylate was used as a control. Serial half-diluted samples were prepared in a 96-well round-bottom microtiter plate. 100 μL of the test compound sample and 100 μL of CAS detection solution were added to each well. The reaction was allowed to proceed at 25°C for 4 h. The color change was the end point of the reaction. The absorbance at 630 nm was measured using a microplate reader to quantify the residual CAS-Fe in each well. 3+The experiment was repeated three times for each sample, and the solution was zeroed with double-distilled water. When iron exchange is slow, a CAS shuttle solution was used. 4 mM 5-sulfosalicylic acid was added to the above solution to rapidly chelate iron. In this case, absorbance at 630 nm was measured within 6 hours. The decrease in the concentration of the CAS-iron-containing complex was used to assess the chelator's ability to compete for iron.

[0057] The results are as follows Figure 4 As shown, the horizontal axis is the concentration of the sample to be tested, and the vertical axis is the relative absorbance. When the absorbance of the sample was measured at pH 5.6 and 630 nm, the Fe-CAS-HDTMA complex had almost no absorption because there was no iron carrier binding to Fe. 3 + . As the concentration of the test sample increases, the relative absorbance continues to decrease. Due to the low stability of the complex, Fe will be immediately transferred to the iron carrier, and the concentration of the CAS-iron-containing ion complex in the reaction system is decreasing. When the concentration of the test sample reaches 1200μM, the concentration of the CAS-iron-containing ion complex in the reaction system reaches the lowest value, thereby evaluating the ability of the test sample to compete for iron. The results show that the iron chelating activity of compounds 1-6 is higher than that of the standard substance deferoxamine mesylate. Among them, the new compound desferrioxamine G3(3) has the highest iron scavenging activity, ED 50 =53.17μM, followed by compounds terragine E(4) and desferrioxamine D2(6), ED 50 The iron scavenging activity of the new compounds desferrioxamine G1 (1) and desferrioxamine G2 (2) was also significantly higher than that of the control agent, which were 72.37 μM and 75.39 μM, respectively. The iron scavenging activity of the compound desferrioxamine E (5) was ED 50 The concentration of 109.74 μM was also higher than the positive control drug deferoxamine mesylate at 117.08 μM.

[0058] Example 4 Siderophore activity in promoting seed germination

[0059] First, the monomer compounds 1-6 prepared in the above examples and the positive control 3-indoleacetic acid were dissolved in methanol solution, and sterile water was used to prepare a test solution with a concentration of 0.01 μM (methanol ratio 1%). Then, the pakchoy seeds and rice seeds were sterilized with 75% alcohol for 5 minutes and washed 5 times with distilled water. The treated seeds (N=60) were then placed in a glass culture dish containing 9 cm cellulose filter paper, and 5 mL of the test solution was added to each culture dish. After incubation in the dark at 24°C for 48 hours, the seed germination rate was calculated. When the radicle protruded more than 2 mm, the seeds were considered to have germinated.

[0060] The results of the seed germination experiment are shown in Table 3. Compounds 1-6 exhibited varying degrees of activity in promoting pakchoi seed germination at a concentration of 0.01 μM (germination growth rate 1.9% to 7.4%). Compounds 3 and 4 showed significantly stronger activity than the positive control, 3-indoleacetic acid. Compounds 1, 5, and 6 exhibited significant activity in promoting rice seed germination at a concentration of 0.01 μM (germination growth rate 9.5% to 14.3%), comparable to the positive control, 3-indoleacetic acid.

[0061] Table 3 Seed germination promoting activity of compounds 1-6 and 3-indoleacetic acid (0.01 μM)

[0062]

[0063] Note: “ / ” represents no activity in promoting seed germination.

[0064] Example 5 Siderophore activity in promoting plant growth

[0065] First, the monomer compounds 1-6 and the control deferoxamine mesylate prepared in the above examples were dissolved in methanol solution, and sterile water was used to prepare test solutions with concentrations of 0.1 μM and 0.01 μM, respectively (methanol ratio 1%). Then, the pakchoy seeds and lettuce seeds were sterilized with 75% alcohol for 5 minutes and washed 5 times with distilled water. The treated seeds (N=60) were then placed in a glass culture dish containing 9 cm cellulose filter paper, and 5 mL of the test solution was added to the culture dish. Incubate in the dark at 24 ° C for 48 hours to germinate. After 2 days of germination, the seeds were carefully removed and the taproot length was measured.

[0066] The results of the plant growth-promoting activity test are shown in Table 4. Compounds 2-5 showed significant growth-promoting activity for pakchoi seeds (root length 5.30-7.01 cm) at different concentrations, but no growth-promoting activity was observed for compounds 1 and 6. Compounds 1-3 and 5-6 showed significant growth-promoting activity for lettuce seeds (root length 4.77-6.29 cm) at different concentrations, but no growth-promoting activity was observed for compound 4.

[0067] Table 4 Plant growth promoting activity of compounds 1-6 (root length / cm)

[0068]

Claims

1. A siderophore compound, characterized in that: The compounds are the following compounds 1-3: 。 2. A method for preparing a siderophore compound, characterized in that: (1) Transfect the South China Sea Streptomyces 12A09 ( Streptomonospora nanhaiensis 12A09 T ) were inoculated onto Gao's No. 1 solid medium and cultured for 3-6 days for activation; the activated strain was inoculated into 50 mL of NO2 medium using the digging method and cultured at 25°C-35°C and 180-220 rpm for 2-5 days to serve as the fermentation seed liquid; (2) The seed solution obtained above was aseptically inoculated into NO2 culture medium and cultured at 25℃-35℃ and 180-220 rpm for 5-10 days; (3) The fermentation liquid obtained above was separated into solid and liquid, and 20-50 g of macroporous adsorption resin HP20 was added to the fermentation supernatant. The mixture was adsorbed at room temperature for 2-4 h under shaking, and then the adsorption resin was collected and eluted with methanol to obtain the siderophore extract. (4) The siderophore extract was separated by silica gel column chromatography according to the dichloromethane:methanol ( v / v ) was gradient eluted from a volume ratio of 100:0 to 0:100, and fractions FA1 and FA2 were collected from a volume ratio of dichloromethane to methanol of 95:5 to 84:

16. (5) Fraction FA1 was separated by Sephadex LH-20 and eluted with a solvent having a volume ratio of 1:1 of dichloromethane to methanol. 60-90 mL of the eluate was collected as subfraction Fr2, and 120-150 mL of the eluate was collected as subfraction Fr7. The subfraction Fr2 was purified by semi-preparative HPLC, eluting with 40-50% methanol in water at a flow rate of 1.5-3.0 mL / min to obtain compound 1 (t R 13-17 min), 4 (t R 18-22 min) and 5 (t R 25-30 min); the sub-fraction Fr7 was eluted with 40-50% methanol aqueous solution at a flow rate of 1.5-3.0 mL / min to obtain compound 6 (t R 15-20 min); Fraction FA2 was separated on Sephadex LH-20 and eluted with a solvent of dichloromethane:methanol in a volume ratio of 1:1, and 50-180 mL of the eluate was collected; the eluate was purified by semi-preparative HPLC with 20-40% methanol in water at a flow rate of 1.5-3.0 mL / min to obtain compound 2 (t R 7-10.5min) and 3 (t R 11-16 min).

3. The method for preparing the siderophore compound according to claim 2, wherein: The NO2 liquid culture medium is composed of 10 g starch, 20 g glucose, 10 g yeast extract, 10 g tryptone, 3 g beef extract, 0.5 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 2 g calcium carbonate, and 1 L of 3% sea salt aged sea water per liter of water, with a pH of 7.

0.

4. A use of the siderophore compound according to claim 1, characterized in that: The compounds 1-3 are used in preparing agents for promoting crop seed germination and for preparing agents for promoting crop growth.

5. The use of the following compound, characterized in that: The use of compound 4, compound 5 and compound 6 as a preparation of a crop seed germination promoting agent or a crop growth promoting agent respectively; The structural formula of compound 4-6 is as follows: 。

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