An aporphine alkaloid compound isolated from the plant of ilicium religiosum and application thereof

Abstract

The application belongs to the field of biochemistry medicine, and discloses an aporphine alkaloid compound separated from a tree and application thereof. An aporphine alkaloid compound of the application has a structure as shown in formula I: the compound is extracted and separated from the tree, and through in-vitro cell experiments, it is shown that the compound has a significant effect of improving the decrease of MIN6 cell activity induced by palmitic acid and has a significant inhibitory effect on the pancreatic islet cell damage induced by palmitic acid. In addition, the in-vitro cell experiments also show that the compound has a significant effect of reducing the increase of triglyceride content of normal liver cells induced by palmitic acid, and it is confirmed that the compound has a significant lipid-lowering effect.

Description

Technical Field

[0001] This invention belongs to the field of biochemical medicine, specifically relating to an apophyte-like alkaloid compound isolated from the tamarisk tree and its applications. Background Technology

[0002] Metabolic associated fatty liver disease (MAFLD) is a chronic, non-communicable disease characterized by hepatic fat accumulation combined with at least one of the following three factors: overweight / obesity, type 2 diabetes, or metabolic dysfunction. Ectopic and excessive accumulation of fat in liver tissue, leading to hepatic steatosis, is the benign initiation of MAFLD. This stage does not involve significant liver damage and is reversible. However, 10-20% of simple fatty liver can progress to NASH, further developing into cirrhosis and hepatocellular carcinoma (HCC), potentially becoming the leading cause of liver transplantation in the future. Due to the extremely complex etiology of MAFLD, its pathogenesis remains poorly understood. The early "two-hit theory" has long been widely accepted, with lipid accumulation in hepatocytes representing the first hit, and insulin resistance (IR) being crucial in the development of hepatic lipid deposition. The steatosis caused by hepatocyte lipid accumulation increases the liver's susceptibility to other harmful factors, representing the second hit, which in turn leads to hepatocyte damage, inflammation, and fibrosis. With the deepening of MAFLD research in recent years, it has been found that the "two-hit theory" is insufficient to explain the complex molecular mechanisms and metabolic changes in MAFLD, and it has gradually been replaced by the "multiple-hit theory." The "multiple-hit theory" posits that multiple pathogenic factors, such as gut microbiota dysbiosis, nutritional factors, inflammatory responses, genetic and epigenetic factors, IR (inflammation resistance), and oxidative stress (OS), act in parallel or sequentially in a synergistic manner on genetically susceptible individuals to induce MAFLD. The "multiple-hit theory" has given us a more comprehensive understanding of MAFLD, but its pathogenesis still requires further in-depth research.

[0003] Litsea cubeba (Lauraceae family) is an evergreen tree belonging to the genus Litsea in the Lauraceae family. It is widely distributed in tropical and subtropical Asia, as well as subtropical North and South America. In China, it is mainly found in Guangdong, Hainan, and Yunnan provinces. Litsea cubeba is an important medicinal plant in my country; its leaves, roots, and bark are used in traditional medicines for their effects in clearing dampness and heat, reducing inflammation, disinfecting, stopping bleeding, and relieving pain. Summary of the Invention

[0004] The first aspect of this invention is to provide an apophene-like alkaloid compound with the structure shown in Formula I:

[0005]

[0006] A second aspect of this invention is to provide a method for extracting the above-mentioned apophene-like alkaloid compounds, comprising the following steps:

[0007] (1) The bark of the *Cynanchum paniculatum* tree was dried, cut into pieces, and extracted by heating and reflux with an organic solvent. The extract was filtered to obtain the total extract of *Cynanchum paniculatum*. The extract was concentrated under reduced pressure until there was no alcohol odor. The total extract was then taken and the pH was adjusted to 2-3 with sulfuric acid solution. Ethyl acetate was added for extraction, the upper organic phase was removed, and the lower layer was recovered. Sodium hydroxide solution was added to the lower layer to adjust the pH to 10-11. Ethyl acetate was added for extraction, the lower layer was removed, and the upper organic phase was recovered. The extract was concentrated under reduced pressure to obtain the total extract of ethyl acetate, i.e., the total alkaloid fraction.

[0008] (2) The total extract was subjected to silica gel column chromatography. Different ratios of dichloromethane-acetone were used as eluents for gradient elution. The eluent obtained by dichloromethane-acetone = 20:1 was collected, concentrated, and dried. The dried fractions were purified using Sephadex LH-20 with methanol as the eluent. Each 50 mL fraction was collected as a fraction, and a total of 35 fractions were obtained, which were numbered Fr.2b1-Fr.2b35 respectively.

[0009] (3) The obtained fractions Fr.2b25-Fr.2b29 were combined and further purified by semi-preparative high performance liquid chromatography to obtain the compound described in this invention.

[0010] Furthermore, the extractant used in step (1) is analytical grade methanol and / or 95% ethanol by volume; in step (2), the silica gel used for silica gel column chromatography has a particle size of 200-300 mesh; in step (2), the concentration is carried out by vacuum concentration and the drying is carried out by vacuum freeze drying.

[0011] Furthermore, the volume ratio of dichloromethane to acetone in step (2) is 100:0, 20:1, 10:1, 4:1, 2:1, and 1:1, respectively.

[0012] Furthermore, in step (3), the high performance liquid chromatography column is a YMC-Pack ODS-A column, the eluent volume ratio is 22:78 acetonitrile-water solution containing 0.5% formic acid and 0.1% triethylamine, the flow rate is 2 mL / min, the elution time is 40 min, and the retention time of the compound is 36.2 min.

[0013] A third aspect of this invention is to provide the use of the above-described apophene alkaloid compounds in the preparation of formulations that improve palmitic acid-induced decrease in MIN6 cell viability.

[0014] Furthermore, in the aforementioned application, the MIN6 cells are mouse pancreatic β-cell lines.

[0015] A fourth aspect of the present invention is to provide the use of the above-described apophene alkaloid compounds in the preparation of formulations that reduce palmitic acid-induced elevation of triglyceride levels in normal hepatocytes.

[0016] Furthermore, in the aforementioned application, normal hepatocytes are human liver normal cells HL-7702 cells.

[0017] Furthermore, the concentration of the apophene alkaloid compound is 0.39-50 μM.

[0018] The beneficial effects of this invention are:

[0019] The apophylloid alkaloid compound of this invention is a novel compound isolated from *Gnaphalium affine*. In vitro cell experiments showed that it significantly improved palmitic acid-induced decreased MIN6 cell viability and significantly inhibited palmitic acid-induced pancreatic islet cell damage. Furthermore, in vitro cell experiments also showed that it significantly reduced palmitic acid-induced elevation of triglyceride levels in normal hepatocytes, confirming its significant lipid-lowering effect. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 To investigate the effects of different treatments on palmitic acid-induced MIN6 cell viability.

[0022] Figure 2 The effects of different treatments on palmitic acid-induced triglyceride levels in HL-7702 cells. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] Take 20 kg of dried and cut bark of *Cynanchum paniculatum*, extract it by heating and reflux with 95% ethanol, filter to obtain total extract of *Cynanchum paniculatum*, concentrate under reduced pressure until no alcohol odor remains, and obtain total extract; take the obtained total extract, add sulfuric acid solution to adjust pH to 2-3 (1% sulfuric acid solution can be selected); add ethyl acetate for extraction, remove the upper organic phase, recover the lower layer, add sodium hydroxide solution to adjust pH to 10-11, add ethyl acetate for extraction, remove the lower layer, recover the upper organic phase, concentrate under reduced pressure, and obtain total extract of ethyl acetate (68 g), which is the total alkaloid fraction;

[0026] The obtained total extract was loaded using a silica gel dry method. First, the total extract extracted with ethyl acetate was dissolved in a small amount of methanol, then a small amount of silica gel was added and mixed thoroughly. After the organic solvent evaporated, the extract was finely ground and subjected to silica gel (200-300 mesh) column chromatography. Gradient elution was performed using different volume ratios of dichloromethane-acetone as eluents until colorless. The eluent with a dichloromethane-acetone ratio of 20:1 was collected, concentrated under reduced pressure, and freeze-dried under vacuum to obtain a dry powder of the effective fraction. The eluent ratios for each gradient and the naming of the resulting fractions are shown in Table 1.

[0027] Table 1. Nomenclature of fractions obtained from different gradient elution solutions

[0028]

[0029]

[0030] The obtained active fraction (Fr.2) was purified using a Sephadex LH-20 eluent, with methanol as the eluent. Each 50 mL fraction was collected as a separate fraction, resulting in 35 fractions, numbered Fr.2b1-Fr.2b35. Fractions Fr.2b25-Fr.2b29 were combined and further purified using semi-preparative high-performance liquid chromatography (HPLC) to yield one compound. The HPLC column was a YMC-PackODS-A column, and the eluent was acetonitrile-water solution containing 0.5% (v / v) formic acid and 0.1% (v / v) triethylamine (volume ratio 22:78). The flow rate was 2 mL / min, and the elution time was 40 min. The retention time of this compound was 36.2 min.

[0031] The compound was identified as an apophene alkaloid, with the molecular formula shown in Formula I:

[0032]

[0033] The high-resolution mass spectrometry data of the compounds described in this invention are shown in Table 2.

[0034] Table 2 High-resolution mass spectrometry data of the compounds of this invention

[0035]

[0036]

[0037] The spectral data of the compounds described in this invention are shown in Table 3.

[0038] Table 3. Spectral data of the compounds described in this invention.

[0039]

[0040] 1 The H NMR test conditions were 600MHz, DMSO-d6; 13 The C NMR test conditions were 150MHz, DMSO-d6.

[0041] Experimental method for improving palmitic acid-induced decrease in MIN6 cell viability using the compounds of this invention:

[0042] (1) MIN6 cell resuscitation and culture: After being taken out of the freezer at -80℃, the upper cryopreservation solution was discarded by centrifugation. The cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum, 50 μM mercaptoethanol and 1% penicillin and streptomycin. The cells were cultured in an incubator at 37℃ and 5% CO2 with a relative humidity of 95%. The medium was replaced with fresh medium every 24 to 36 hours depending on the cell growth. When the cells grew to 80% to 90%, they were passaged or cryopreserved.

[0043] (2) CCK-8 assay for cell viability: MIN-6 cells in good growth condition were evenly seeded into 96-well plates at a concentration of 1×10⁶ cells / well. 4 100 μL / well of RPMI-1640 medium (10% FBS, 50 μM mercaptoethanol, 10% P / S) was added to each cell / mL of the plate. The 96-well plates were incubated at 37°C and 5% CO2 for 24 h. The original medium was discarded, and the plates were divided into a blank group (CK), a model control group (with 300 μM PA), and an experimental group. The experimental group received 300 μM PA and was simultaneously treated with the compound to be screened. The solution was added at a rate of 1 × 10⁻⁶ cells / mL. 4 Add 100 μL of RPMI-1640 medium per well to each cell / mL and incubate for 24 h. Perform five replicates for each concentration gradient and use the mean of the blank group as a control.

[0044] (4) After the MIN-6 cells were treated with the above conditions, 100 μL of CCK-8 solution was added to each well of the 96-well incubator. The cells were then placed in an incubator at 37°C and 5% CO2 for 1 hour. Finally, the optical density (OD) was measured at a wavelength of 450 nm using a microplate reader, and cell viability was calculated.

[0045]

[0046] The experimental results are shown in Table 4 and Figure 1 .

[0047] Table 4. Effects of different treatments on MIN-6 cell viability

[0048]

[0049]

[0050] Note: Comparison between the blank control group and the model control group # P<0.05, ## P<0.01, ### P<0.001;

[0051] The experimental group was compared with the model control group. *P<0.05, **P<0.01, ***P<0.001.

[0052] The effects of apophene-like alkaloids isolated from the effective fraction obtained in this invention, diluted sequentially by 2-fold from 50 μM down to 0.39 μM, on the viability of palmitic acid-induced MIN6 cells were investigated at eight treatment concentrations. Figure 1 As shown: CK was the blank control group (without treatment with the compound and palmitic acid), and PA was the model control group (PA concentration of 300 μM / ml). Therefore, there was a significant difference between the blank control group and the model control group, indicating that the palmitic acid administration in the model group was successful in establishing the cell model. Compared with the model control group, the compound significantly improved the decrease in cell viability caused by palmitic acid in the experimental group at a concentration range of 0.39-6.25 μM, with the best effect observed at a concentration of 1.56 μM.

[0053] Experimental method for reducing palmitic acid-induced triglyceride levels in normal hepatocytes using the compounds of this invention:

[0054] (1) HL-7702 cell resuscitation and culture: After being removed from the freezer at -80℃, the cells were centrifuged and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics. The cells were then cultured in an incubator at 37℃ and 5% CO2 with a relative humidity of 95%. The medium was changed every 24–36 hours depending on the cell growth. When the cells reached 80%–90% cell growth, they were passaged or cryopreserved. After two stable passages, the cells were used for experimental research. The cell suspension was prepared at 2.0 × 10⁻⁶ cells / mL. 5 Cells were evenly seeded at a density of 1 ml per cell in a 12-well plate and cultured in a cell culture incubator.

[0055] (2) Each well was divided into the following groups: blank control group (10% BSA), model group (300 μM PA-BSA), and drug treatment group (300 μM PA-BSA + 0.3125 / 0.625 / 1.25 / 2.5 / 5 / 10 μM apophyte alkaloids), with 3 replicates per group. After inoculation, the 12-well plates were cultured for 24 h, then the original culture medium was removed and replaced with a mixture of BSA, PA-BSA and apophyte alkaloids at the target concentrations, and cultured for another 24 h. The cell culture medium was discarded, and the cells were washed twice with ice-cold PBS and collected.

[0056] (3) Measurement of intracellular TG levels: The intracellular TG levels of each treatment group were measured using a tissue cell triglyceride (TG) enzymatic assay kit (Beijing Pulilai Gene Technology Co., Ltd., E1013).

[0057] The experimental results are shown in Table 5 and Figure 2 .

[0058] Table 5. Effects of different treatments on palmitic acid-induced triglyceride levels in HL-7702 cells.

[0059] Compound processing methods Triglyceride content (μg / mg) Blank control group (CK) 17.95 Model group <![CDATA[84.76 ### ]]> Experimental group 1 (PA + compound - 0.3125 μM) <![CDATA[61.81 ** ]]> Experimental group 2 (PA + compound - 0.625 μM) <![CDATA[79.53 * ]]> Experimental group 3 (PA + compound - 1.25 μM) 91.79 Experimental group 4 (PA + compound - 2.5 μM) 92.44 Experimental group 5 (PA + compound - 5 μM) 95.15 Experimental group 6 (PA + compound - 10 μM) 106.34

[0060] Note: Comparison between the blank control group and the model group # P<0.05, ## P<0.01, ### P<0.001;

[0061] The experimental group and the model group showed *P<0.05, **P<0.01, ***P<0.001.

[0062] The effects of apophene-like alkaloids isolated from the effective fraction obtained in this invention, diluted sequentially by 2-fold from 10 μM down to 0.3125 μM (a total of 6 treatment concentrations), on palmitic acid-induced triglyceride levels in HL-7702 cells were investigated. Figure 2 As shown: the blank control group (without PA and compound treatment) showed a significant difference compared with the model group (PA concentration of 300 μM), indicating that the palmitic acid administration model was successfully established in the model group; compared with the model group, the compound significantly reduced the triglyceride content of normal hepatocytes induced by palmitic acid at concentrations of 0.3125 μM and 0.625 μM in the experimental group, indicating that the compound described in this invention has significant lipid-lowering activity.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An aporphine alkaloid compound characterized in that, Its structure is shown in Equation I: Equation I.

2. The method for extracting apophene-like alkaloids as described in claim 1, characterized in that, Includes the following steps: (1) The bark of the *Cynanchum paniculatum* tree was dried and cut into pieces. It was extracted by heating and reflux with analytical grade methanol and / or 95% ethanol. The extract was filtered to obtain the total extract of *Cynanchum paniculatum* tree. The extract was concentrated under reduced pressure until there was no alcohol odor. The total extract was obtained by adding sulfuric acid solution to adjust the pH to 2-3, adding ethyl acetate for extraction, removing the upper organic phase, and recovering the lower layer. The lower layer was adjusted to pH 10-11 with sodium hydroxide solution, and then extracted with ethyl acetate. The lower layer was removed, and the upper organic phase was recovered. The extract was concentrated under reduced pressure to obtain the total extract of ethyl acetate, i.e. the total alkaloids. (2) The total extract was subjected to silica gel column chromatography. Different ratios of dichloromethane-acetone were used as eluents for gradient elution. The volume ratios of dichloromethane-acetone were 100:0, 20:1, 10:1, 4:1, 2:1, and 1:1, respectively. The eluent obtained by dichloromethane-acetone at a ratio of 20:1 was collected, concentrated, and dried. The dried fractions were purified using Sephadex LH-20 with methanol as the eluent. Each 50 mL fraction was collected as a fraction, resulting in a total of 35 fractions, which were numbered Fr.2b1-Fr.2b35. (3) The obtained fractions Fr.2b25-Fr.2b29 were combined and further purified by semi-preparative high performance liquid chromatography to obtain the compound described in this invention. The high performance liquid chromatography column was a YMC-Pack ODS-A column, the eluent volume ratio was 22:78 acetonitrile-water solution containing 0.5% formic acid and 0.1% triethylamine, the flow rate was 2 mL / min, and the elution time was 40 min. The retention time of the compound was 36.2 min.

3. The extraction method as described in claim 2, characterized in that, In step (2), the silica gel used for silica gel column chromatography has a particle size of 200-300 mesh; in step (2), the concentration is carried out by vacuum concentration and the drying is carried out by vacuum freeze drying.

4. The use of the apophene alkaloid compound as described in claim 1 in the preparation of formulations that improve palmitic acid-induced decrease in MIN6 cell viability.

5. The application as described in claim 4, characterized in that, In the aforementioned application, the MIN6 cells are mouse pancreatic β-cell lines.

6. The application as described in claim 4, characterized in that, The concentration of the apophene alkaloid compound was 0.39-6.25 μM.

7. The use of the apophene alkaloid compound as described in claim 1 in the preparation of formulations that reduce palmitic acid-induced elevation of triglyceride levels in normal hepatocytes.

8. The application as described in claim 7, characterized in that, In the application described, normal hepatocytes are human liver normal cells HL-7702 cells.

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