Preparation method and application of alkaloid aplysingoniopora A

Abstract

The application discloses a preparation method and application of a novel alkaloid compound aplysingoniopora A. The alkaloid compound aplysingoniopora A is a novel compound, has good inhibiting effect on DLD-1 and HT-29 of colorectal cancer, and through in-depth research, application scenarios of the compound aplysingoniopora A in the field of improving immunity and bacteriostasis are improved. The application provides an alternative compound for development of a new marine medicine and provides medicinal value in the field of disease treatment.

Description

Technical Field

[0001] This invention relates to the field of marine biomedicine technology, and to a method for preparing a novel alkaloid, alysingoniopora A, and its applications in multiple fields. Background Technology

[0002] Due to the diverse characteristics of marine habitats, corals have developed chemical defense systems constructed from various secondary metabolites in extreme environments. The potential medicinal value of these small molecule compounds has increasingly attracted the attention of chemists and pharmacologists, and has been a hot topic in marine natural product research for many years. Malignant tumors are a class of diseases that seriously threaten human life and health. Because current clinically used cancer treatments, such as alkylating agents, antibiotics, herbal medicines, antimetabolites, and hormones, exhibit drawbacks such as drug resistance, multidrug resistance, time-dependent effects, and significant toxic side effects, scientists are prompted to search for structurally novel antitumor drugs.

[0003] The research group isolated a novel alkaloid, aplysingoniopora A, from the polyps of the columnar hornhole coral in the Beibu Gulf. This alkaloid has anti-tumor effects and can be used as a new anti-tumor drug. The group also studied the immunomodulatory effects and other functions of the alkaloid, providing new possibilities for the application of the novel alkaloid aplysingoniopora A. Summary of the Invention

[0004] The purpose of this invention is to provide a new class of alkaloid compounds, their preparation methods, and applications in preparing pharmaceutical compositions. The invention also aims to study the applications of these compounds, expand their application areas, accelerate their application, and provide new directions for the pharmaceutical field.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A novel alkaloid, alysingoniopora A, has the following chemical structural formula:

[0007]

[0008] The present invention also includes the application of the novel alkaloid alysingoniopora A in the preparation of anticancer related drugs.

[0009] Furthermore, the colorectal cancer cells are DLD-1 and / or HT-29.

[0010] The present invention also includes the use of the novel alkaloid Aplysingoniopora A in the preparation of immune-enhancing drugs.

[0011] The present invention also includes the application of the novel alkaloid alysingoniopora A in the preparation of antibacterial agents.

[0012] Furthermore, the antibacterial agent can inhibit Escherichia coli (Escherichia coli). Escherichia coli Staphylococcus aureus Staphylococcus aureus Bacillus subtilis ( Bacillus subtilis ), brewer's yeast ( Saccharomyces cerevisiae ) and / or enterospermia enterica subspecies cholerae serotype ( Salmonella enterica subsp. enterica serovar choleraesuis ).

[0013] This invention also includes a method for preparing the novel alkaloid alysingoniopora A, comprising the following steps:

[0014] (1) After crushing the columnar horn-hole coral polyps, the crude extract of the sample was obtained by ethanol extraction.

[0015] (2) The crude extract obtained was separated by normal phase silica gel column chromatography under reduced pressure, and eluted with chloroform-methanol solution. The eluent was collected, concentrated and dried under reduced pressure, and the components were combined to obtain 7 component extracts Fr.A1~Fr.A7.

[0016] (3) The obtained fractional extract Fr.A5 was separated by medium-pressure normal phase silica gel column chromatography, and eluted sequentially with chloroform-methanol solution. The eluent was collected, concentrated and dried under reduced pressure, and the fractions were combined to obtain 5 fractional extracts Fr.A5-1~Fr.A5-5.

[0017] (4) The obtained fraction extract Fr.A5-4 was separated by ODS column chromatography after pretreatment. It was eluted sequentially with methanol-water solution. The eluent was collected, examined by TLC, and the color was developed. The colored eluent was concentrated to dryness under reduced pressure to obtain the concentrated fraction Fr.A5-4-4 for later use.

[0018] (5) The component extract Fr.A5-4-4 obtained in step (4) was separated by semi-preparative high performance liquid chromatography to obtain the novel alkaloid aplysingoniopora A.

[0019] The present invention has the following beneficial effects:

[0020] 1. The alkaloid compound 1 (aplysingoniopora A) of this invention is a novel compound that exhibits inhibitory activity against tumor cells and can serve as a lead compound for the preparation of antitumor drugs to treat malignant tumors. In-depth research on this compound revealed that it also shows good inhibitory effects against DLD-1 and HT-29 in colorectal cancer. Furthermore, the novel compound (aplysingoniopora A) was found to have even better antibacterial and immune-enhancing effects. This in-depth study expands the application scenarios of compound (aplysingoniopora A), provides a candidate compound for the development of new antitumor drugs, and is of great significance for the development of marine medicinal resources in China. Attached Figure Description

[0021] Figure 1 The structural formula of compound alysingoniopora A; Detailed Implementation

[0022] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0023] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features. Example 1

[0024] (1) Preparation and verification of the alkaloid compound alysingoniopora A of the present invention.

[0025] Take columnar horn-hole coral Goniopora columna One hundred g of hydroid sample was pulverized and extracted three times with 95% ethanol, each time soaking for three days. The amount of 95% ethanol used was 3 to 5 times that of the sample. The extract was filtered, and the filtrates were combined and concentrated under reduced pressure to obtain 20 g of crude ethanol extract.

[0026] The crude extract of the sample was separated by normal-phase silica gel column chromatography under reduced pressure. The effective column volume Veffective = 120 cm20 3 The extracts were eluted sequentially with chloroform-methanol solutions at volume ratios of 9:1, 8:2, 7:3, 1:1, 3:7, 2:8, and 0:10. The eluents were collected, concentrated and dried under reduced pressure, analyzed by TLC, and the fractions were combined to obtain seven fractional extracts, Fr.A1-Fr.A7.

[0027] The fractional extract Fr.A5 (1.2 g) was separated by medium-pressure normal-phase silica gel column chromatography, with a column volume Veffective = 40 cm2. 3The sample was eluted sequentially with chloroform-methanol at volume ratios of 8:2, 7:3, and 1:1 at a flow rate of 15 min / mL. The eluent was collected, concentrated and dried under reduced pressure, analyzed by TLC, and the fractions were combined to obtain five fractional extracts Fr.A5-1 to Fr.A5-5.

[0028] The fractional extract Fr.A5-4 (128 mg) was separated by ODS column chromatography after pretreatment, with a column volume Veffective = 10 cm2. 3 The sample was eluted sequentially with methanol-water gradients of 1:9, 3:7, 1:1, 7:3, 9:1 and 10:0 to obtain several elution fractions. The fractions were examined by TLC and developed. The elution fractions were concentrated to dryness under reduced pressure to obtain the concentrate fraction Fr.A5-4-4 (35 mg) for later use.

[0029] The component extract Fr.A5-4-4 was prepared by semi-preparative high-performance liquid chromatography using a COSMOSIL C18 semi-preparative column (250 mm × 10 mm, 5...). μ m, Shanghai Qinzhen Biotechnology Co., Ltd.), the preparation conditions were: isocratic elution separation with a methanol / pure water volume ratio of 21:79, a flow rate of 3 min / mL, and a retention time of t. R =16~17 min. Compound (4 mg) was obtained.

[0030] (2) Structural analysis of compound alysingoniopora A

[0031] Nuclear magnetic resonance (NMR) analysis confirmed the chemical structure, physicochemical properties, and spectral data of the compound alysingoniopora A of this invention as follows: alysingoniopora A: yellow powder; 1 H NMR (500 MHz, DMSO- d 6, J inHz) δ H : 7.55 (H-4, s), 7.49 (H-1, d, J =8.4 Hz), 7.27 (H-6, s), 7.16 (H-2, d, J =8.5 Hz), 4.25 (H-11, t, J =6.2 Hz), 3.72 (H2-9, m), 3.94 (H-13, m), 3.69 (H2-14, d), 2.03 (H-12a, m), 1.91 (H-12b, m). 13 C NMR (125 MHz, DMSO-d 6) δ C : 177.0(C-16), 173.9 (C-10), 161.4 (C-15), 139.0 (C-5), 127.4 (C-8), 126.2 (C-6), 123.2 (C-2), 120.8 (C-1), 115.3 (C-4), 116.1 (C-3), 109.7 (C-7), 54.6 (C-13), 53.3 (C-11), 49.3 (C-14), 39.7 (C-12), 33.9 (C-9); HR-ESI-MS: m / z 406.0515 [M+H] - .

[0032] (3) Test of the inhibitory activity of compound aplysingoniopora A on tumor cell growth

[0033] The isolated compound: aplysingoniopora A, structural formula see [link to structural formula]. Figure 1 Therefore, the inhibitory effect of this compound on colorectal cancer cells was studied. Specifically, three types of colorectal cancer cells, DLD-1, HT-29, and SW480PDC, were seeded in 96-well plates at a density of 3000 cells per well. The inhibitory effect of different concentrations of the compound on the proliferation of DLD-1, HT-29, and SW480PDC cells was determined using the sulfonylrhodamine B (SRB) method; cisplatin was used as a positive control. All colorectal cancer cells, DLD-1 and HT-29, were cultured in RPMI-1640 medium; SW480PDC cells were cultured in L15 medium. All cells were cultured in an incubator at 37°C and CO2 saturated humidity. Cells after three passages were used for the experimental research of this invention. Tumor cells were seeded in 96-well plates, and after 24 hours of adhesion, different concentrations of alysingoniopora A were added. After 72 hours of drug treatment, SRB staining was performed, and the absorbance at 515 nm was measured using an ELISA reader to detect the survival rate of tumor cells and calculate the IC50 of lysingoniopora A in inhibiting tumor cell proliferation. 50 value.

[0034] The results are shown in Table 1:

[0035] Table 1. Inhibitory effects of alkaloid Aplysingoniopora A on different colorectal cancer cells (IC50, 100 mg / kg / dL). 50 (μM)

[0036]

[0037] As can be seen from the experimental results in Table 1, when IC 50 A value less than 5 indicates that the corresponding cell inhibition rate is effective. Aplysingoniopora A has an inhibitory effect on colorectal cancer cells DLD-1 and HT-29, but no inhibitory effect on colorectal cancer cells SW480PDC. Compared with the positive control cisplatin, aplysingoniopora A has a more significant inhibitory effect on colorectal cancer cells DLD-1 and HT-29, but no significant inhibitory effect on colorectal cancer cells SW480PDC. Example 2

[0038] The immune-enhancing effects of compound Aplysingoniopora A are as follows:

[0039] Mice of similar weight were randomly divided into 9 groups: normal control group, high-dose and low-dose compound groups, model group and levamisole group; 10 mice in each group.

[0040] Preparation methods for high and low dose groups: The compounds were dissolved in DMSO (cell grade) to prepare the low dose group: 1 mg / kg; and the high dose group: 10 mg / kg; cyclophosphamide solution 0.01 ml / g; levamisole solution 30 mg / kg.

[0041] The normal control group was injected intraperitoneally with 10 ml / (kg·d) of physiological saline for 3 consecutive days. The high- and low-dose compound groups, the model group, and the levamisole group were injected intraperitoneally with an equal volume of cyclophosphamide for 3 consecutive days. Starting on day 4, the normal control group and the model group were administered an equal volume of physiological saline by gavage for 4 consecutive days; the high- and low-dose compound groups were administered the same for 4 consecutive days. On day 8, mice were injected with 0.1 ml / 10 g of Indian ink via the tail vein. At 1 (t1) and 5 (t2) min later, 20 μl of blood was collected via the orbital vein and added to 2 ml of 0.1% Na2CO3 solution. The absorbance was measured at 680 nm using a 722 spectrophotometer. After blood collection, the mice were euthanized by cervical dislocation, and the liver and spleen were weighed. The clearance index (K) and phagocytic index (α) were calculated using the following formulas.

[0042] K = (logA1 - logA5) / (t2 - t1) = log (A1 / A5) / 4

[0043] α = Body weight / (Liver weight + Spleen weight) × K 1 / 3

[0044] Effects on cell proliferation:

[0045] Mice were euthanized by cervical dislocation, soaked in 75% ethanol solution for 5 min, and their spleens were aseptically removed. The spleens were washed with Hanks' solution, cut into small pieces on a 100-mesh wire mesh, and lightly ground with a sterile glass syringe core. After rinsing, filtering, and centrifuging, red blood cells were destroyed with Tris-NH4Cl, incubated on ice, centrifuged, and washed twice with RPMI 1640 medium. Cells were then prepared into 1 × 10⁻⁶ cells / mL solution. 6 Spleen lymphocytes were suspended at 180 μl / well in 96-well plates. 20 μl of the corresponding solution was added to each well, followed by 20 μl of Con A (5 μg / ml). Wells containing only 20 μl of RPMI 1640 medium (without cells) served as blank controls, wells containing only 20 μl of RPMI 1640 medium as negative controls, and wells containing only 20 μl of Con A (5 μg / ml) as positive controls. Cells were cultured in a CO2 incubator for 3 days. Then, 20 μl of MTT (5 mg / ml) was added, and the cells were cultured further. After centrifugation and discarding the supernatant, 180 μl of DMSO was added to each well, and the mixture was vortexed. The A value was measured at 570 nm using a microplate reader. The results are shown in Table 2-3.

[0046] Table 2. Clearance index and phagocytic index of mice in each group

[0047]

[0048] Note: aa: compared with the normal control group, P<0.01; b: compared with the model group, P<0.05.

[0049] Compared with the normal control group, the carbon clearance index and phagocytic index of the model group were significantly decreased; compared with the model group, both the levamisole group and the high-dose compound group significantly increased the clearance index and phagocytic index of immunosuppressed mice, but the difference in phagocytic index between the two groups was not significant.

[0050] Table 3. Proliferation levels of splenic lymphocytes in each group of mice

[0051]

[0052] Note: Compared with the negative control group, aa: P < 0.01; compared with Con A group, b: P < 0.05; bb: P < 0.01.

[0053] Table 3 shows that, compared with the negative control group, 5 μg / ml Con A significantly induced the proliferation of mouse T lymphocytes. The compound significantly enhanced the ability of Con A to induce the proliferation of mouse T lymphocytes, with the most significant enhancement effect observed at a final concentration of 10 mg / kg. The compound also has a good effect on enhancing the immunity of mice. Example 3

[0054] Compound: alysingoniopora A, details are as follows:

[0055] Four wells were made on the culture medium of the tested bacteria. 1 ml of each compound at different concentrations (1 mg / kg, 3 mg / kg, 5 mg / kg) was injected into the wells. For the blank control group, distilled water was injected. The plates were incubated for 3 minutes and then placed in a 37°C incubator overnight. The diameter of the inhibition zone was measured and recorded the next day. The tested bacteria were: *Escherichia coli* (…). Escherichia coli Staphylococcus aureus Staphylococcus aureus Bacillus subtilis ( Bacillus subtilis ), brewer's yeast ( Saccharomyces cerevisiae ) and entero Salmonella enterica subspecies cholerae serotype ( Salmonella enterica subsp. enterica serovar choleraesuis The results obtained by averaging are shown in Table 4:

[0056] Table 4. Antibacterial effects of different alkaloids on different tested bacteria

[0057]

[0058] As shown in Table 4, the compound has inhibitory effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, yeast, and Salmonella, with the inhibitory effect being Staphylococcus aureus > Escherichia coli > yeast > Bacillus subtilis > Salmonella.

[0059] In summary, compound aplysingoniopora A has a significant inhibitory effect on colorectal cancer cells DLD-1 and HT-29, a good effect on enhancing the immunity of mice, and a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, yeast and Salmonella.

[0060] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. An alkaloid, alysingoniopora A, characterized in that, Its chemical structural formula is as follows: 。 2. The application of the alkaloid alysingoniopora A as described in claim 1 in the preparation of anticancer related drugs, characterized in that, The cancer targeted by the anticancer-related drug is colorectal cancer, and the cells targeted by the colorectal cancer are DLD-1 and / or HT-29.

3. The use of the alkaloid Aplysingoniopora A as described in claim 1 in the preparation of immune-enhancing drugs.

4. The application of the alkaloid alysingoniopora A as described in claim 1 in the preparation of an antibacterial agent, characterized in that, The antibacterial agent can inhibit Escherichia coli. Escherichia coli Staphylococcus aureus Staphylococcus aureus Bacillus subtilis Bacillus subtilis brewer's yeast Saccharomyces cerevisiae and / or Salmonella enterica subspecies enterica serotype 1 (swine cholera serotype) Salmonella enterica subsp. enterica serovar Choleraesuis .

5. The method for preparing the alkaloid alysingoniopora A as described in claim 1, characterized in that, The preparation method includes the following steps: (1) After crushing the columnar horn-hole coral polyps, the crude extract of the sample was obtained by ethanol extraction. (2) The crude extract obtained was separated by normal phase silica gel column chromatography under reduced pressure, and eluted with chloroform-methanol solution. The eluent was collected, concentrated and dried under reduced pressure, and the components were combined to obtain 7 component extracts Fr.A1~Fr.A7. (3) The obtained fractional extract Fr.A5 was separated by medium-pressure normal phase silica gel column chromatography, and eluted sequentially with chloroform-methanol solution. The eluent was collected, concentrated and dried under reduced pressure, and the fractions were combined to obtain 5 fractional extracts Fr.A5-1~Fr.A5-5. (4) The obtained fraction extract Fr.A5-4 was separated by ODS column chromatography after pretreatment. It was eluted sequentially with methanol-water solution. The eluent was collected, examined by TLC, and the color was developed. The colored eluted fraction was concentrated to dryness under reduced pressure to obtain the concentrated fraction Fr.A5-4-4 for later use. (5) The alkaloid aplysingoniopora A was obtained by semi-preparative high performance liquid chromatography separation of the component extract Fr.A5-4-4 obtained in step (4).

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