Iodocarbazole derivative and separation and extraction method and application thereof

Abstract

The invention discloses an iodo-carbazole derivative as well as a separation and extraction method and application thereof, and relates to the technical field of natural derivative compounds. According to the invention, bromine salt and iodine salt are respectively added into a culture medium, streptomyces sp. OUCMDZ-5511 is subjected to fermentation culture, according to the abundance difference of iodine and bromine, the chemical structure of bromo-carbazole obtained by separation is taken as a basis, the molecular weight after bromine substitution is converted into iodine substitution is taken as a target molecular weight, a target iodinated product is searched on LC-MS, and the target iodinated product is obtained through purification. The secondary metabolite is separated and purified by using the method as a tracking separation means, so that the problem that the bioavailability of the iodide by microorganisms is low is effectively solved; the iodo-carbazole derivative disclosed by the invention has a good application prospect in the aspects of preventing and treating neuroinflammation, cognitive impairment, Parkinson's disease, ischemic cerebral apoplexy and hemorrhagic cerebral apoplexy, is small in dosage and low in side effect, and can be used for research and development of related medicines and functional foods.

Description

Technical Field

[0001] This invention relates to the field of natural derived drugs and iodinated compounds, specifically to an iodocarbazole derivative, its separation and extraction method, and its application. Background Technology

[0002] Common neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, and Huntington's disease. Alzheimer's disease typically manifests as cognitive impairment in multiple areas, including memory, reaction time, language, and executive function. There is currently no cure; treatment primarily involves slowing disease progression using cholinesterase inhibitors and NMDA receptor antagonists, but these can cause various adverse reactions such as cerebral edema and hemorrhage. Parkinson's disease is characterized by degenerative damage to dopamine neurons in the substantia nigra pars compacta. It has an insidious onset and progressively worsens, with a course that can last for decades, severely impacting the quality of life for patients and their families. Clinically, treatment mainly involves slowing disease progression using levodopa, dopamine receptor agonists, and monoamine oxidase B inhibitors, but long-term use can lead to symptom fluctuations, drug resistance, and adverse reactions. Stroke is an acute cerebrovascular disease, which can be divided into ischemic stroke and hemorrhagic stroke. It is mainly caused by the blockage or sudden rupture of blood vessels in the brain, leading to brain tissue damage. Clinical manifestations include sudden limb paralysis, loss of consciousness, or sensory disturbances. Stroke, with its rapid onset and progression, has become a leading public health challenge posing a serious threat to public life and health. Clinically, while ischemic stroke can benefit from mechanical thrombectomy, the treatment window is narrow, and antiplatelet and thrombolytic drugs carry risks such as intracranial hemorrhage, gastrointestinal bleeding, and damage to nerves and the digestive tract mucosa. Hemorrhagic stroke currently still relies on surgical removal of the hematoma, which is highly invasive and has many postoperative complications. The commonly used drug mannitol can also cause adverse reactions such as electrolyte imbalance and kidney damage. To date, there are no safe and effective drugs for prevention and treatment. Therefore, finding natural active substances with fewer side effects and significant efficacy against neurodegenerative diseases and stroke is of great importance.

[0003] Marine Streptomyces are major contributors to new natural products in the marine actinomycetes phylum. New compounds discovered from them possess a variety of biological activities, such as anti-inflammatory, antibacterial, and antitumor effects. However, due to the extremely low bioavailability of iodides by microorganisms and the relatively low abundance of iodine in seawater, iodinated natural products are very rare, and research on their related activities is scarce, with few readily available isolation and extraction methods.

[0004] On the other hand, the dopaminergic neurons in the zebrafish brain are remarkably similar to those in humans. When exposed to neurotoxins such as 1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, zebrafish exhibit Parkinson's disease-like pathological phenotypes, including dopaminergic neuron loss and bradykinesia. Exposure to AlCl3 and scopolamine results in the detection of β-amyloid plaque deposition in the zebrafish brain, along with abnormal expression of genes associated with neurodegenerative diseases. Furthermore, the zebrafish's coagulation system and neurovascular unit structure are highly similar to those of mammals, accurately mimicking the pathological features of stroke. Most importantly, changes in dopaminergic neurons, plaques, and brain neurovasculature can be directly observed in juvenile zebrafish using transgenic fluorescent labeling or staining. This zebrafish disease model allows for the efficient discovery of compounds with anti-neurodegenerative disease and stroke activity.

[0005] Therefore, there is an urgent need to discover an active compound with few side effects and significant efficacy against neurodegenerative diseases and stroke, for the development of novel preventive or therapeutic drugs for neurodegenerative diseases and stroke. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide an iodocarbazole derivative, its separation and extraction method, and its application.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] An iodocarbazole derivative, wherein the iodocarbazole derivative is 5-iodo-6-methoxy-9-hydro-carbazole-1-ol, and its structural formula is shown in Formula I:

[0009]

[0010] This invention also includes a method for separating and extracting iodocarbazole derivatives, comprising the following steps:

[0011] ① After sterilization, sodium bromide was added to corn solid medium prepared with A1 liquid medium. Streptomyces sp. OUCMDZ-5511 was fermented at 28±0.5℃ for 30 days. Ethyl acetate was added, ultrasonically disrupted, and extracted. The ethyl acetate phase was concentrated to obtain ethyl acetate extract, which was then dried to obtain Streptomyces extract. 10 mg of the obtained streptomycin extract was added to 1-3 mL of methanol and centrifuged at 10000 rpm for 5 min. LC-MS analysis was performed to record the characteristic ion peaks of the brominated products in the fermentation products. The brominated carbazole derivative was then separated and purified.

[0012] The mass ratio of sodium bromide to corn solid culture medium was 3.3:100;

[0013] The inoculation volume for Streptomyces sp. OUCMDZ-5511 and corn solid medium is approximately 10 mL of bacterial culture per bottle of corn solid medium when the bacterial culture reaches the logarithmic growth phase.

[0014] The mass ratio of corn solid culture medium to ethyl acetate is 1:8–12;

[0015] ② Replace sodium bromide in step ① with potassium iodide, and keep the rest of the operation steps unchanged; when performing LC-MS analysis, combine the brominated carbazole derivative in step ① to infer the structure of the carbazole compound that the strain can use iodine atoms to form, and look for the molecular weight of the iodinated compound in the fermentation product.

[0016] The mass ratio of potassium iodide to corn solid culture medium was 7.5:100;

[0017] ③ Using the fermentation conditions of step ②, Streptomyces sp. OUCMDZ-5511 was fermented on a large scale, and the novel natural derivative of iodocarbazole, 5-iodo-6-methoxy-9-hydro-carbazole-1-ol, was obtained by column chromatography.

[0018] Preferably, in step ③, during column chromatography separation, the eluents selected are petroleum ether-dichloromethane and dichloromethane-methanol, respectively, wherein the volume ratios of petroleum ether and dichloromethane are 1:0, 10:1, 5:1, 1:1, and 0:1, respectively, and the volume ratios of dichloromethane and methanol are 80:1, 10:1, 1:1, and 0:1, respectively, resulting in nine fractions from Fr.1 to Fr.9. LC-MS shows that the target iodinated product is mainly enriched in the petroleum ether-dichloromethane fraction.

[0019] Secondly, fraction Fr.4, obtained by elution with petroleum ether-dichloromethane at a v / v ratio of 1:1, was separated by Sephadex LH-20 gel column chromatography in a MeOH solvent system to obtain nine subfractions. Fraction Fr.4.3 was purified by semi-preparative HPLC, C18 / AQUA, 50% MeCN-H2O, to obtain a new natural derivative of carbazole, 5-iodo-6-methoxy-9-hydro-carbazole-1-ol.

[0020] The present invention also includes the use of iodocarbazole derivatives in the preparation of drugs for the prevention and treatment of neuroinflammatory diseases.

[0021] The present invention also includes the use of iodocarbazole derivatives in the preparation of drugs for the prevention and treatment of cognitive impairment.

[0022] The present invention also includes the use of iodocarbazole derivatives in the preparation of drugs for the prevention and treatment of Parkinson's disease.

[0023] The present invention also includes the use of iodocarbazole derivatives in the preparation of drugs for the prevention and treatment of ischemic stroke.

[0024] The present invention also includes the use of iodocarbazole derivatives in the preparation of drugs for the prevention and treatment of hemorrhagic stroke.

[0025] The present invention has the following advantages over the prior art:

[0026] The iodocarbazole derivative of this invention is used to ferment Streptomyces sp. OUCMDZ-5511 by adding bromide and iodide salts to the culture medium. Based on the difference in iodine and bromine abundance, the chemical structure of the isolated bromocarbazole is used as the basis, and the molecular weight after bromine substitution to iodine substitution is used as the target molecular weight. The target iodinated product is searched on LC-MS and used as a tracking separation method to separate and purify the secondary metabolites of OUCMDZ-5511. The design is ingenious and effectively solves the problem of low microbial availability of iodides in the prior art. In addition, this invention designs a new method for successfully separating iodinated derivatives from fermentation products, providing a new approach for the subsequent separation and purification of natural iodinated derivatives.

[0027] The iodocarbazole derivative of this invention contains a 3-methylcarbazole matrix and iodine atoms, exhibiting significant anti-inflammatory activity. Neuroinflammation is a key factor in the occurrence and development of neurodegenerative diseases and stroke. The iodocarbazole derivative of this invention shows promising potential in the prevention and treatment of Parkinson's disease, cognitive impairment, ischemic stroke, and hemorrhagic stroke. It can be used in related drugs and functional foods, and requires low dosage with minimal side effects. Attached Figure Description

[0028] Figure 1 LC-MS analysis of solid-state fermentation products of Streptomyces sp. OUCMDZ-5511 after addition of 3.3% NaBr;

[0029] Figure 2 The structural formulas of a series of bromocarbazole compounds derived from the fermentation products of OUCMDZ-5511 are shown below.

[0030] Figure 3 To be Figure 2 Structural formulas of a series of iodocarbazole compounds after replacing bromine with iodine in the compound;

[0031] Figure 4 LC-MS analysis of solid-state fermentation products with different concentrations of KI added to OUC-MDZ 5511;

[0032] Figure 5The chemical structure diagram of 5-iodo-6-methoxy-9-hydro-carbazole-1-ol isolated from the deep-sea Streptomyces sp.OUCMDZ-5511;

[0033] Figure 6 To investigate the effects of different concentrations of samples on the migration of zebrafish immune cells to the lateral line thalamus;

[0034] (Indomethacin was the positive control group; 2.5 μmol / L and 5 μmol / L were the iodocarbazole-1-ol groups; the white rectangles represent the areas for counting immune cell counts at the lateral line thalamus.)

[0035] Figure 7 A statistical analysis of the number of immune cells migrating to the lateral line thalamus in zebrafish;

[0036] (Indomethacin was the positive control group, and 2.5 μmol / L and 5 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the neuroinflammation model group, ****P<0.0001)

[0037] Figure 8 This is a statistical analysis of the total swimming distance of zebrafish with cognitive impairment under alternating light and dark conditions (donepezil positive control group, 0.5 μmol / L and 1 μmol / L iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the cognitive impairment model group, ****P<0.0001).

[0038] Figure 9 A statistical analysis graph showing the changes in swimming speed of zebrafish with cognitive impairment under alternating light and dark conditions;

[0039] (The donepezil positive control group, and the 0.5 μmol / L and 1 μmol / L iodocarbazole-1-ol groups; the vertical axis represents the swimming speed of zebrafish per minute in different treatment groups, expressed as mean ± SEM, and the horizontal axis represents the duration of three light-dark alternation cycles.)

[0040] Figure 10 To investigate the effects of different concentrations of samples on monoaminergic neuron damage in Parkinson's disease-like zebrafish;

[0041] (Rosagiline was the positive control group; 0.5 μmol / L and 1 μmol / L were the iodocarbazole-1-ol groups; white brackets indicate the statistical region for the length of monoaminergic neurons.)

[0042] Figure 11Statistical analysis of the length of monoaminergic neurons in zebrafish with Parkinson's disease after different sample treatments (rasagiline was the positive control group, 0.5 μmol / L and 1 μmol / L were iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the Parkinson's disease model group, **P<0.01, ****P<0.0001)

[0043] Figure 12 The effect of different concentrations of samples on Parkinson's disease-like behavior in zebrafish;

[0044] (Rosagiline was the positive control group, and 0.5 μmol / L and 1 μmol / L were the iodocarbazole-1-ol groups; the black line represents the slow swimming trajectory, the green line represents the medium swimming trajectory, and the red line represents the fast swimming trajectory.)

[0045] Figure 13 Statistical analysis of the total swimming distance of Parkinson's disease-like zebrafish after different sample treatments;

[0046] (Rasagiline was the positive control group, and 0.5 μmol / L and 1 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the Parkinson's disease model group, ***P<0.001, ****P<0.0001)

[0047] Figure 14 Effects of different concentrations of samples on the area of ​​thrombus in the brain of zebrafish (aspirin was the positive control group, and 1 μmol / L and 2 μmol / L were the iodocarbazole-1-ol groups; the white dashed box represents the statistical area of ​​the thrombus).

[0048] Figure 15 Statistical analysis of cerebral thrombosis area in zebrafish (aspirin was the positive control group, 1 μmol / L and 2 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the ischemic stroke model group, ****P<0.0001)

[0049] Figure 16 Effects of different concentrations of samples on the area of ​​cerebral hemorrhage in zebrafish (valsartan was the positive control group, and 1 μmol / L and 2 μmol / L were iodocarbazole-1-ol groups).

[0050] Figure 17 Statistical analysis of the area of ​​brain hemorrhage in zebrafish (valsartan was the positive control group, and 1 μmol / L and 2 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the hemorrhagic stroke model group, ***P<0.001, ****P<0.0001). Detailed Implementation

[0051] The purpose of this invention is to provide an iodocarbazole derivative, its separation and extraction method, and its application. The invention will be further described below with reference to specific embodiments.

[0052] The Streptomyces sp. OUCMDZ-5511 of this invention was provided by Professor Zhu Weiming's research group at the College of Pharmaceutical Sciences, Ocean University of China.

[0053] In this invention, C18 / AQUA is the model of the chromatographic column.

[0054] In this invention, the solvent ratio of the mobile phase is 50% MeCN-H2O, and the volume ratio of MeCN to H2O is 1:1.

[0055] Example 1

[0056] Isolation and extraction of iodocarbazole derivatives

[0057] Streptomyces sp. OUCMDZ-5511 was fermented using 3.3% sodium bromide as the bromide salt, added to corn solid medium prepared with A1 liquid medium after sterilization. After static incubation at 28℃ in a fermentation chamber for 30 days, 500 mL of ethyl acetate was added for disruption and extraction. The ethyl acetate phase was concentrated and dried to obtain the Streptomyces extract. 10 mg of the extract was dissolved in 1–3 mL of analytical grade methanol (if in suspension, the sample needs to be centrifuged at 10000 rpm for 5 min), and analyzed by LC-MS. The results are shown below. Figure 1 Characteristic ion peaks (M:M+2 = 1:1) and (M:M+2:M+4 = 1:2:1) of the brominated product were found in the fermentation product. Bromocarbazole was isolated and purified, with the structural formula shown below. Figure 2 As shown.

[0058] The inoculation volume for Streptomyces sp. OUCMDZ-5511 and corn solid medium is 10 mL of bacterial culture per bottle of corn solid medium when the bacterial culture reaches the logarithmic growth phase (approximately 10 mL per bottle).

[0059] The mass ratio of corn solid culture medium to ethyl acetate is 1:8–12;

[0060] To avoid the influence of other halide ions in seawater and to allow microorganisms to metabolize and utilize bromide ions to the maximum extent, the above-mentioned corn solid culture medium is fermented with distilled water.

[0061] Because microorganisms have extremely low natural availability of iodides, and the abundance of iodine in seawater is relatively low (almost a thousand times lower than that of bromine), iodinated natural products are very rare. To obtain more rare iodinated natural products with excellent activity, sodium bromide in the corn culture medium was replaced with potassium iodide, while other conditions remained unchanged, causing strain OUCMDZ-5511 to produce iodocarbazole. Based on the isolated chemical structure of bromocarbazole, the molecular weight after bromine substitution to iodine substitution was used as the target molecular weight. The structural formula after bromine substitution to iodine substitution is shown below. Figure 3 As shown. Target iodinated products were identified using LC-MS and used as a tracking separation method to separate and purify the secondary metabolites of OUCMDZ-5511.

[0062] Using corn solid medium prepared with A1 liquid medium as the basal medium, five different concentrations of KI were added: 3.3%, 5.0%, 7.5%, 10%, and 15%. After fermentation, the products were analyzed by LC-MS. Based on the work on the isolation of bromine products using OUCMDZ-5511, it was speculated that if the strain could utilize iodine atoms, the structure would likely be a carbazole compound, such as 4-iodo-3-methoxy-9H-carbazole, which has a similar structure to compounds 1-3. Based on this, the molecular weights of iodinated compounds could be found in the fermentation products, such as... Figure 4 As shown in the figure, the red part has a molecular weight of 323, which is the predicted iodinated compound 4-iodo-3-methoxy-9H-carbazole. Furthermore, the yield of the target iodinated product is the highest at a KI addition concentration of 7.5%, so 7.5% is determined to be the optimal KI addition concentration.

[0063] Following large-scale fermentation under these conditions, preliminary column chromatography separation of the crude extract from *Streptomyces* sp. OUCMDZ-5511 was performed using a reduced-pressure normal-phase silica gel column. Petroleum ether-dichloromethane (1:0–0:1) and dichloromethane-methanol (1:0–0:1) were used sequentially as mobile phases for gradient elution, yielding nine fractions, Fr.1–Fr.9. The selected volume ratios of petroleum ether to dichloromethane were 1:0, 10:1, 5:1, 1:1, and 0:1, respectively, and the volume ratios of dichloromethane to methanol were 80:1, 10:1, 1:1, and 0:1, respectively.

[0064] LC-MS showed that the target iodinated product was mainly enriched in the low-polarity fraction (Fr.4). Fraction Fr.4 (petroleum ether-dichloromethane, v / v = 1:1, V = 1.5 L, 0.8 g) was separated by Sephadex LH-20 gel column chromatography in pure methanol to obtain nine subfractions. Fraction Fr.4.3 (43 mg) was purified by semi-preparative HPLC (C18 / AQUA, 50% acetonitrile-water) to obtain a novel natural derivative of iodocarbazole, 5-iodo-6-methoxy-9-hydro-carbazole-1-ol (2.0 mg, tR 9.6 min, hereinafter referred to as iodocarbazole-1-ol), with the following structural formula: Figure 5 As shown.

[0065] Example 2

[0066] Prepare zebrafish

[0067] Zebrafish strains suitable for the experiment were selected, and healthy, sexually mature individuals were placed in a mating tank at a 1:1 female-to-male ratio, separated by a partition. The tanks were placed in darkness, and the partition was removed the following day before the lights were turned on. Light stimulation was applied to induce ovulation, with ovulation occurring within half an hour to minimize developmental differences between embryos. Fertilized eggs were collected, disinfected, and cleaned, then placed in zebrafish culture water with 0.2 ppm methylene blue added. The tanks were cultured at 28±0.5℃ under controlled light conditions with a 14h light / 10h dark cycle. One-third of the embryo culture water was replaced every 24 hours, and dead embryos were removed. During the experiment, zebrafish of appropriate developmental stages were selected for testing under a stereomicroscope.

[0068] The zebrafish strains can be selected from Tg(zlyz:EGFP) zebrafish, AB strain zebrafish, and Tg(slc18a2:GFP) zebrafish.

[0069] Example 3

[0070] Comparative analysis of the anti-neuroinflammatory activity of iodocarbazole-1-ol with other drugs

[0071] Tg(zlyz:EGFP) zebrafish embryos were obtained using the method described in Example 2. At 3 days post-fertilization (dpf), normally developing zebrafish were placed in 24-well plates and randomly divided into a blank control group, a neuroinflammation model group, an indomethacin positive control group, and two iodocarbazole-1-ol groups, with three replicates per group and ten fish per replicate. The blank control group and the neuroinflammation model group were given zebrafish culture water, the indomethacin positive control group was given 20 μmol / L indomethacin solution, and the two iodocarbazole-1-ol groups were given 2.5 μmol / L and 5 μmol / L iodocarbazole-1-ol solutions, respectively. All zebrafish were placed in a constant temperature incubator at 28 ± 0.5 °C. After 2 hours of culture, except for the blank control group, all other groups were replaced with 40 μmol / L copper sulfate solution and cultured for another 45 minutes. Ten zebrafish from each group were selected, and side views of the zebrafish were acquired under an Olympus fluorescence microscope, as shown below. Figure 6 As shown. Statistical analysis was performed on the number of immune cells migrating to the lateral line thalamus of zebrafish, such as... Figure 7 As shown, indomethacin was the positive control group, and 2.5 μmol / L and 5 μmol / L were the iodocarbazole-1-ol groups; the white rectangles represent the statistical regions of immune cell counts at the lateral line nerve thalamus of zebrafish. The results are expressed as mean ± SEM. Compared with the blank control group, ####P<0.0001; compared with the neuroinflammation model group, ****P<0.0001. Figure 6 and Figure 7 It can be seen that copper sulfate can induce neuroinflammatory response in zebrafish, manifested as the migration and aggregation of zebrafish immune cells towards the lateral line thalamus. The number of zebrafish immune cells migrating to the lateral line thalamus was significantly reduced in the iodocarbazole-1-ol pretreatment group, indicating that it has significant anti-neuroinflammatory activity. Moreover, the effective concentrations (2.5 μmol / L and 5 μmol / L) are both lower than those of the clinical drug indomethacin (20 μmol / L), and the effect of 5 μmol / L is better.

[0072] Example 4

[0073] Analysis of the effect of iodocarbazole-1-ol on improving cognitive impairment

[0074] AB strain zebrafish embryos were obtained using the method described in Example 2. At 3 days post-fertilization (dpf), normally developing zebrafish were placed in 24-well plates and randomly divided into a blank control group, a cognitive impairment model group, a donepezil positive control group, and two iodocarbazole-1-ol treatment groups, with three replicates per group and ten fish per replicate. The blank control group was treated with zebrafish culture water, the cognitive impairment model group with 80 μmol / L aluminum chloride solution, the donepezil positive control group with a combination of 80 μmol / L aluminum chloride and 4 μmol / L donepezil solution, and the two iodocarbazole-1-ol groups with a combination of 80 μmol / L aluminum chloride and 0.5 μmol / L or 1 μmol / L iodocarbazole-1-ol solution, respectively. All zebrafish were cultured in a constant temperature incubator at 28 ± 0.5 °C, with the culture medium changed every 24 hours, for a continuous treatment until 6 days post-fertilization (dpf). Six to eight juvenile zebrafish were randomly selected from each group, washed twice with zebrafish culture water, and transferred to 48-well plates (one fish per well). They were placed in a Zebrabox for 10 minutes to acclimatize, and their behavioral patterns were collected using a zebrafish juvenile behavior analyzer. The behavioral test lasted a total of 60 minutes, including three light-dark cycles (10 minutes of light, 10 minutes of darkness), with behavioral data output every minute. Zebralab software was used to analyze the total distance the zebrafish moved during the light, darkness, and light-dark cycles. Figure 8 As shown, donepezil served as the positive control group, and 0.5 μmol / L and 1 μmol / L were iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the cognitive impairment model group, ****P<0.0001. The speed changes of zebrafish during the light-dark cycle were analyzed, and the results are as follows... Figure 9 As shown, donepezil was the positive control group, and 0.5 μmol / L and 1 μmol / L were the iodocarbazole-1-ol groups; the vertical axis represents the swimming speed of zebrafish in different treatment groups per minute, expressed as mean ± SEM, and the horizontal axis represents the duration of three light-dark alternation cycles.

[0075] Figure 8 and Figure 9The results indicate that aluminum chloride solution can induce a cognitive impairment model in zebrafish, manifested by a significant reduction in both total swimming distance and speed during alternating light and dark cycles, particularly pronounced in the dark environment. The change in speed upon transitioning from darkness to light also showed a significant decrease, indicating a marked decline in responsiveness and thus cognitive impairment. Iodocarbazole-1-ol treatment significantly increased the total swimming distance of zebrafish, along with both total swimming distance and speed in the dark environment and the change in speed upon transitioning from darkness to light. This demonstrates that iodocarbazole-1-ol can significantly improve cognitive impairment in zebrafish. Its effective concentrations (0.5 μmol / L and 1 μmol / L) are lower than those of the clinical drug donepezil (4 μmol / L), with 1 μmol / L showing better efficacy.

[0076] Example 5

[0077] Analysis of the anti-Parkinson's disease effect of iodocarbazole-1-ol

[0078] Tg(slc18a2:GFP) zebrafish embryos were obtained using the method in Example 2. At 1 day post-fertilization (dpf), normally developing zebrafish were demembranes and randomly divided into a blank control group, a Parkinson's disease model group, a rasagiline positive control group, and two iodocarbazole-1-ol groups, with three replicates per group and ten fish per replicate. The blank control group was treated with zebrafish culture water; the Parkinson's disease model group was treated with 60 μmol / L 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution; the rasagiline positive control group was treated with a mixture of 60 μmol / L 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1 μmol / L rasagiline solution; and the two iodocarbazole-1-ol groups were treated with a mixture of 60 μmol / L 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 0.5 μmol / L or 1 μmol / L iodocarbazole-1-ol solution, respectively. The zebrafish from each group were placed in a constant temperature incubator at 28±0.5℃, with the culture medium changed every 24 hours. At 4 days post-flop (dpf), 10 juvenile fish from each group were randomly selected, and brain images were acquired using an Olympus inverted fluorescence microscope. Figure 10 As shown, rasagiline served as the positive control group, while 0.5 μmol / L and 1 μmol / L were iodocarbazole-1-ol groups; the white brackets indicate the statistical regions for the length of zebrafish monoaminergic neurons. Changes in monoaminergic neuron length were measured and calculated using Image-Pro Plus software, and statistical analysis was performed using GraphPad Prism. Figure 11 As shown, rasagiline was the positive control group, and 0.5 μmol / L and 1 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the Parkinson's disease model group, **P<0.01, ****P<0.0001.

[0079] Figure 10 and Figure 11 Note: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine can induce a zebrafish Parkinson's disease model, characterized by a significant decrease in the length of fluorescently labeled monoaminergic neurons in Tg(slc18a2:GFP) zebrafish, i.e., neuronal loss. The length of monoaminergic neurons in zebrafish treated with iodocarbazole-1-ol significantly increased, indicating that iodocarbazole-1-ol can inhibit monoaminergic neuronal loss and has anti-Parkinson's disease activity. Its effective concentration (0.5 μmol / L and 1 μmol / L) is less than or equal to that of the clinical drug rasagiline (1 μmol / L).

[0080] When zebrafish reached 5 days post-flop (dpf) after being grouped and treated according to the above methods, 10-15 juvenile fish were randomly selected from each group, washed twice with zebrafish culture water, and placed in 48-well plates, one fish per well, to acclimatize in a Zebrabox for 10 minutes. During formal testing, the behavior recording time was set to 20 minutes, and the data acquisition frequency was set to automatic recording once per minute. Figure 12 As shown, the swimming trajectories of zebrafish changed over 20 minutes. Rasagilan served as the positive control group, while 0.5 μmol / L and 1 μmol / L were iodocarbazole-1-ol groups. The black line represents slow swimming trajectories, the green line represents medium-speed swimming trajectories, and the red line represents fast swimming trajectories. Zebralab software was used to process and analyze the total swimming distance of zebrafish in each group over 20 minutes. The results are as follows: Figure 13 As shown, rasagiline was the positive control group, and 0.5 μmol / L and 1 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the Parkinson's disease model group, ***P<0.001, ****P<0.0001.

[0081] Figure 12 and Figure 13 Note: Zebrafish exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exhibited significantly reduced swimming trajectory and total swimming distance, displaying Parkinson's disease-like behavior. Compared to the Parkinson's disease model group, zebrafish treated with iodocarbazole-1-ol showed significantly increased swimming trajectory and total distance, indicating that iodocarbazole-1-ol possesses anti-Parkinson's disease activity.

[0082] Example 6

[0083] Analysis of the anti-ischemic stroke effect of iodocarbazole-1-ol

[0084] AB line zebrafish embryos were obtained using the method in Example 2. At 2 days postpartum (dpf), the normally developing zebrafish were demembranous and randomly divided into a blank control group, an ischemic stroke model group, an aspirin positive control group, and two iodocarbazole-1-ol groups, with 3 replicates in each group and 10 fish in each replicate. In this study, the blank control group was treated with zebrafish culture water, the ischemic stroke model group was treated with 1.9 μmol / L ponatinib solution, the aspirin positive control group was treated with a combination of 1.9 μmol / L ponatinib and 124.9 μmol / L aspirin solution, and the two iodocarbazole-1-ol groups were treated with 1.9 μmol / L ponatinib and 1 μmol / L or 2 μmol / L iodocarbazole-1-ol solution, respectively. All zebrafish were placed in a constant temperature incubator at 28±0.5℃. After 24 h of culture, the culture medium was aspirated, and the zebrafish were stained with o-anisidine for 10 min under dark conditions. The staining was performed three times with dimethyl sulfoxide, followed by fixation with 4% paraformaldehyde. Images of the zebrafish brain were then acquired under a Zeiss microscope. Figure 14 As shown, aspirin served as the positive control group, while 1 μmol / L and 2 μmol / L were the iodocarbazole-1-ol groups; the white dashed boxes represent the statistical areas of cerebral thrombus area. The cerebral thrombus area of ​​zebrafish was calculated using Image-Pro Plus software, and statistical analysis was performed using GraphPad Prism. Figure 15 As shown, aspirin was the positive control group, and 1 μmol / L and 2 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the ischemic stroke model group, ****P<0.0001.

[0085] Figure 14 and Figure 15 Note: Ponatinib can induce an ischemic stroke model in zebrafish, resulting in a significant increase in the thrombus area in the zebrafish brain. Treatment with iodocarbazole-1-ol significantly reduced the thrombus area in the zebrafish brain, indicating that iodocarbazole-1-ol has significant anti-ischemic stroke activity, and the effective concentrations (1 μmol / L and 2 μmol / L) are much lower than those of the clinical drug aspirin (124.9 μmol / L).

[0086] Example 7

[0087] Analysis of the anti-hemorrhagic stroke effect of iodocarbazole-1-ol

[0088] AB strain zebrafish embryos were obtained using the method described in Example 2. At 1 day post-flop (dpf), normally developing zebrafish were demembranes and randomly divided into a blank control group, a hemorrhagic stroke model group, a valsartan positive control group, and two iodocarbazole-1-ol treatment groups, with three replicates per group and ten fish per replicate. The blank control group received zebrafish culture water, the hemorrhagic stroke model group received 1.5 μmol / L atorvastatin solution, the valsartan positive control group received a co-treatment of 1.5 μmol / L atorvastatin and 10 μmol / L valsartan solution, and the two iodocarbazole-1-ol groups received a co-treatment of 1.5 μmol / L atorvastatin and 1 μmol / L or 2 μmol / L iodocarbazole-1-ol solution, respectively. All zebrafish were placed in a constant temperature incubator at 28 ± 0.5 °C. After 24 hours of culture, ten zebrafish from each group were selected, and zebrafish brain images were acquired using a Zeiss fluorescence microscope, as shown below. Figure 16 As shown, valsartan served as the positive control group, while 1 μmol / L and 2 μmol / L were iodocarbazole-1-ol groups. The white dashed boxes represent the statistical areas of cerebral hemorrhage. The area of ​​cerebral hemorrhage was measured using Image-Pro Plus software, and statistical analysis was performed using GraphPad Prism. Figure 17 As shown, valsartan was the positive control group, and 1 μmol / L and 2 μmol / L were the iodocarbazole-1-ol groups; compared with the blank control group, ####P<0.0001; compared with the hemorrhagic stroke model group, ***P<0.001, ****P<0.0001.

[0089] Figure 16 and Figure 17 Note: Atorvastatin can induce a hemorrhagic stroke model in zebrafish, manifested as significant hemorrhage in the telencephalon, diencephalon, and midbrain regions. Treatment with iodocarbazole-1-ol significantly reduced the area of ​​cerebral hemorrhage in zebrafish, indicating its significant anti-hemorrhagic stroke activity, and the effective concentrations (1 μmol / L and 2 μmol / L) are lower than those of the clinical drug valsartan (10 μmol / L).

[0090] It can be seen that the novel natural derivative of this invention, 5-iodo-6-methoxy-9-hydro-carbazole-1-ol, possesses anti-neuroinflammatory, neurodegenerative disease, and stroke-related activities. It has broad application prospects in related drugs and functional foods for anti-neuroinflammatory, neurodegenerative disease, and stroke purposes.

Claims

1. An iodocarbazole derivative, characterized in that: The iodocarbazole derivative is 5-iodo-6-methoxy-9-hydro-carbazole-1-ol, and its structural formula is shown in Formula I:

2. The method for separating and extracting the iodocarbazole derivative according to claim 1, characterized in that: Includes the following steps: ① After sterilization, sodium bromide was added to corn solid medium prepared with A1 liquid medium. Streptomyces sp. OUCMDZ-5511 was fermented at 28±0.5℃ for 30 days. Ethyl acetate was added, ultrasonically disrupted, and extracted. The ethyl acetate phase was concentrated to obtain ethyl acetate extract, which was then dried to obtain Streptomyces extract. 10 mg of the obtained streptomycin extract was added to 1-3 mL of methanol and centrifuged at 10000 rpm for 5 min. LC-MS analysis was performed to record the characteristic ion peaks of the brominated products in the fermentation products. The brominated carbazole derivative was then separated and purified. The mass ratio of sodium bromide to corn solid culture medium was 3.3:100; The mass ratio of corn solid culture medium to ethyl acetate is 1:8–12; ② Replace sodium bromide in step ① with potassium iodide, and keep the rest of the operation steps unchanged; when performing LC-MS analysis, combine the brominated carbazole derivative in step ① to infer the structure of the carbazole compound that the strain can use iodine atoms to form, and look for the molecular weight of the iodinated compound in the fermentation product. The mass ratio of potassium iodide to corn solid culture medium was 7.5:100; ③ Using the fermentation conditions of step ②, Streptomyces sp. OUCMDZ-5511 was fermented on a large scale, and the novel natural derivative of iodocarbazole, 5-iodo-6-methoxy-9-hydro-carbazole-1-ol, was obtained by column chromatography.

3. The method for separating and extracting iodocarbazole derivatives according to claim 2, characterized in that: In step ③, during column chromatography separation, the eluents selected were petroleum ether-dichloromethane and dichloromethane-methanol, respectively. The volume ratios of petroleum ether to dichloromethane were 1:0, 10:1, 5:1, 1:1, and 0:1, respectively, and the volume ratios of dichloromethane to methanol were 80:1, 10:1, 1:1, and 0:1, respectively. Nine fractions, Fr.1 to Fr.9, were obtained. LC-MS showed that the target iodinated product was mainly enriched in the petroleum ether-dichloromethane fraction. Secondly, fraction Fr.4, obtained by elution with petroleum ether-dichloromethane at a v / v ratio of 1:1, was separated by Sephadex LH-20 gel column chromatography in a MeOH solvent system to obtain nine subfractions. Fraction Fr.4.3 was purified by semi-preparative HPLC, C18 / AQUA, 50% MeCN-H2O, to obtain a new natural derivative of carbazole, 5-iodo-6-methoxy-9-hydro-carbazole-1-ol.

4. The application of the iodocarbazole derivative according to claim 1, characterized in that: Application in the preparation of drugs for the prevention and treatment of neuroinflammatory diseases.

5. The application of the iodocarbazole derivative according to claim 1, characterized in that: Application in the preparation of drugs for the prevention and treatment of cognitive impairment.

6. The application of the iodocarbazole derivative according to claim 1, characterized in that: Application in the preparation of drugs for the prevention and treatment of Parkinson's disease.

7. The application of the iodocarbazole derivative according to claim 1, characterized in that: Application in the preparation of drugs for the prevention and treatment of ischemic stroke.

8. The application of the iodocarbazole derivative according to claim 1, characterized in that: Application in the preparation of drugs for the prevention and treatment of hemorrhagic stroke.

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