Alkaloid compound and application thereof

Abstract

The present invention belongs to the technical field of drug or functional food application. The present invention discloses a compound of the following structural formula and the use of the compound in preparing a drug for treating fatty liver disease or a functional food for alleviating fatty liver disease. Also disclosed is Eurotium cristatum ( Eurotium cristatum )SXHBTBU1934 and a method for preparing the compound.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and food technology, and particularly relates to a compound extracted and separated from Eurotium cristatum and its application in preparing a drug for treating fatty liver disease or a functional food for alleviating fatty liver disease. Background Art

[0002] Microorganisms have played a crucial role in drug development, with examples including penicillin, streptomycin, avermectin, and lovastatin. Since Fleming's discovery of penicillin in 1928, it has become a life-saving drug. World War II ushered in the industrialization of penicillin. Genetic methods have been used to continuously improve culture media and fermentation conditions, enabling microorganisms to mass-produce penicillin along desired synthetic routes. Microorganisms have made significant contributions to human health and drug development.

[0003] Nonalcoholic fatty liver disease (NAFLD) is a clinical and pathological syndrome characterized by fatty degeneration and fat accumulation in hepatocytes without a history of excessive alcohol consumption. It is closely associated with hyperlipidemia and insulin resistance and can progress to steatohepatitis, cirrhosis, and hepatocellular carcinoma. While there are currently no approved treatments for NAFLD, drug development in this area is highly active and holds broad promise.

[0004] Fuzhuan tea is a type of black tea among the six major types of tea. Under the action of microorganisms, this fermented tea undergoes profound physiological and biochemical changes, thus forming a unique flavor. Summary of the Invention

[0005] The invention extracts and separates an alkaloid compound from Eurotium cristatum, which can be used in preparing a drug for treating fatty liver disease or a functional food for alleviating fatty liver disease.

[0006] The present invention first provides a compound, the structural formula of which is as follows:

[0007] .

[0008] Furthermore, the present invention also provides the compounds and their analogs, derivatives, prodrugs, metabolites and active salts thereof, wherein representative acid addition salts include but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Likewise, basic nitrogen-containing groups can be quaternized with lower alkyl halides such as the chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; long chain halides such as the chlorides, bromides, and iodides of decyl, dodecyl, tetradecyl, and octadecyl; aryl alkyl halides such as benzyl bromide and phenethyl bromide, and the like.

[0009] Examples of acids that can form pharmaceutically acceptable acid addition salts with the compounds of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid.

[0010] The compounds provided by the present invention and their analogs, derivatives, prodrugs, metabolites and active salts are used in preparing drugs for treating fatty liver disease, and as food additives for developing functional foods for alleviating fatty liver disease.

[0011] The present invention also provides Eurotium cristatum ( Eurotium cristatum )SXHBTBU1934, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC NO.21026.

[0012] Further provided is a method of using the Eurotium cristatum ( Eurotium cristatum ) SXHBTBU1934 is a method for preparing a compound of the following formula, comprising fermenting and culturing the aforementioned Eurotium cristatum, and extracting the compound from the fermentation product:

[0013] .

[0014] Preferably, the Eurotium cristatum ( Eurotium cristatum) SXHBTBU1934 is inoculated into a sterilized culture medium containing tea leaves, rice, corn, brown rice, wheat, orange peel, and grapefruit peel, and the culture is statically fermented at 25-32°C for 7-30 days; then, the compound is extracted from the fermentation product.

[0015] More specifically, the fermented product is soaked in ethanol overnight, filtered, and then evaporated to dryness using a rotary evaporator. The product is then extracted with ethyl acetate and deionized water three times to obtain an organic layer, which is then evaporated to dryness. The organic layer is weighed and dissolved in a mixed solvent of dichloromethane and methanol. The sample is mixed with a crude fermentation extract and column chromatography silica gel (100-200 mesh) at a weight ratio of 1:1.3-1.7, and vacuum column chromatography is performed for separation.

[0016] Column chromatography was performed using an organic solvent system with different ratios of n-hexane and dichloromethane, with a volume ratio of n-hexane:dichloromethane of 2:98, to obtain a sample; separation was performed using gel Sephadex LH-20 column chromatography, with the eluent being n-hexane:dichlorohexane (volume ratio of 2:1), to obtain fraction 3; preferably, purification was performed using reversed-phase high-performance liquid chromatography, with the chromatographic column being an Agilent reversed-phase C18 column and the mobile phase being 40% acetonitrile in water-100% acetonitrile (time for 20 minutes) to obtain the pure compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Phylogenetic tree constructed based on calmodulin gene sequences.

[0018] Figure 2 It is the reverse phase high performance liquid chromatogram of the compound.

[0019] Figure 3 is the mass spectrum of the compound.

[0020] Figure 4 This is the H NMR spectrum of the compound.

[0021] Figure 5 It is the carbon NMR spectrum of the compound.

[0022] Figure 6 The half-inhibitory concentration of the compound on lipid accumulation in the HepG2 cell model.

[0023] Biomaterial deposit information:

[0024] The present invention is the cristatum ( Eurotium cristatum )SXHBTBU1934, which is deposited in the General Microbiology Center of China Culture Collection of Microorganisms, abbreviated as CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 21, 2020, and the deposit number is: CGMCC NO.21026. DETAILED DESCRIPTION

[0025] The present invention is further described below with reference to specific examples in order to provide a better understanding of the present invention, but the present invention is not limited thereto.

[0026] Example 1

[0027] 1. Isolation of Fungi

[0028] According to the method of the literature (Yang Ruijuan, Wang Qiaomei, Peng Wenshu, Ji Aibing, Zhang Wenjie, Yan Liang, Isolation and identification of microorganisms in 8 commercially available "golden flower" teas, Tropical Agricultural Sciences, 2019, 39 (10), 81-88), Eurotium cristatum was isolated from the commercially available Fu tea in Xinxianxi District, Xi'an City, Shaanxi Province. Eurotium cristatum )SXHBTBU1934.

[0029] The specific process is as follows:

[0030] Strain isolation:

[0031] Source of isolated strains: The Fuzhuan tea used was purchased from the Xinxian West District Market in Xi'an, Shaanxi Province.

[0032] Culture medium used:

[0033] Malt extract agar medium (MEA, per liter of medium): malt extract 30.0 g, peptone 5.0 g, agar 15.0 g, pH 6.0;

[0034] Sabouraud dextrose agar (SDA) medium (per liter): peptone 5.0 g, tryptone, glucose 40.0 g, agar 15.0 g, pH 4.0 - 6.0 (adjust to 6.0, not too low);

[0035] When the sterilized culture medium is not hot enough to burn the back of your hand (about 40°C), add chloramphenicol and streptomycin to a final concentration of 0.2 mg / mL (prepared in advance, filtered through a 0.22µm microporous membrane, and stored at -20°C), mix well, and pour into a plate to inhibit bacterial growth.

[0036] Separation method:

[0037] Weigh 10g of Fuzhuan tea, add 100ml of sterile water, soak for 10 minutes, and shake to disperse the tea leaves. Take 200 μL of the suspension (3 dilutions of 10 -3 , 10 -4 , 10 -5), spread onto isolation medium MEA and SDA, and invert and incubate at 28°C. After the second day of incubation, observe the plates for fungal colonies and pick them out promptly. Observe them daily thereafter and pick out new fungal colonies as soon as they appear.

[0038] Strain identification:

[0039] After 7 days of culture on MEA plate culture medium, the colonies were approximately circular with a bulge in the center and were brown in color. The color became lighter from the center to the outside, from brown to brown, golden yellow to light yellow. A small amount of liquid droplets oozed out of the colonies, and the hyphae extended in all directions. The hyphae morphology was obvious and the edges were relatively neat. The back of the colony was smooth and had no ridges. As the culture time increased, the pigment produced made the culture medium appear dark brown.

[0040] Use an inoculation needle to cut about 1 cm from a 7-day-old culture dish. 2 Place the bacterial cells in a centrifuge tube, add 800 μL of DNA extraction reagent, and add a steel ball (to facilitate cell disruption). Place the centrifuge tube symmetrically in a SCIENTZ-48 high-throughput tissue grinder to disrupt the bacterial cells. Genomic DNA of the test strain is obtained and amplified by PCR. The calmodulin gene sequence is amplified using the universal fungal primers mdAD1 (5'-GCCGACTCTTTGACTGAAGAGC-3') and cmdQ1 (5'-GCATCATGAGCTGGACGAATTC-3').

[0041] PCR amplification system (50 μL): 5 μL of 10× Buffer, 5 μL of 2.5 mM dNTPs, 5 μL of 25 mM MgCl, 5 μL of template DNA, 0.5 μL each of primers ITS4 and ITS5, 0.5 μL of Taq DNA polymerase, and 4.5 μL of ddH2O. The reaction procedure was as follows: 37 cycles of initial denaturation at 94°C for 4 min, denaturation at 94°C for 1 min, annealing at 55°C for 4 min, and extension at 72°C for 1.5 min, followed by a 10-min extension at 72°C. The reaction was then stored at 25°C.

[0042] The calmodulin gene sequence of the strain was 100% similar to that of NRRL4222, so the strain was identified as Eurotium cristatum ( Eurotium cristatum ), a phylogenetic tree was constructed based on the calmodulin gene sequence. Figure 1 This strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms with the accession number CGMCC NO.21026 on December 21, 2020.

[0043] Fermentation culture of fungi

[0044] The activated Eurotium cristoides is prepared into a spore suspension, which is then inoculated onto any culture medium suitable for the growth of Eurotium cristoides, such as sterilized tea leaves (30% distilled water by weight of tea leaves), rice (100% distilled water by weight of rice), corn (100% distilled water by weight of corn), brown rice (100% distilled water by weight of brown rice), wheat (100% distilled water by weight of wheat), orange peel (40% distilled water by weight of orange peel), or grapefruit peel (40% distilled water by weight of grapefruit peel), and is cultured for static fermentation at 25-32°C (28°C in this embodiment) for 7-30 days (19 days in this embodiment).

[0045] Preparation of active compounds

[0046] The fermented product is extracted using methods such as methanol, ethanol, ethyl acetate, acetone, dichloromethane, ether, supercritical extraction, etc. (but not limited to the above methods) to obtain an extract.

[0047] The specific method is as follows: Fermentation is performed on culture media such as tea, rice, corn, brown rice, wheat, orange peel, or grapefruit peel for 7-30 days (19 days in this example). Each culture medium has been analyzed by liquid chromatography. The fermentation product (including the bacteria and culture medium) that produces this compound is then soaked in ethanol overnight. After filtration, it is evaporated to dryness on a rotary evaporator. Extraction is then performed three times with a 1:1 ratio of ethyl acetate to deionized water. The resulting organic layer is then evaporated to dryness, weighed, and dissolved in a mixed solvent of dichloromethane and methanol. The sample is then mixed with a crude fermentation extract and column chromatography silica gel (100-200 mesh) in a weight ratio of 1:1.5. After the organic solvent is evaporated, the sample is separated by vacuum column chromatography.

[0048] The column chromatography was eluted with an organic solvent system of n-hexane and dichloromethane in different ratios, with the volume ratios being 100% n-hexane (fraction 1), 1:99 (n-hexane: dichloromethane, sample A was obtained, fraction 2), 2:98 (n-hexane: dichloromethane, sample was obtained, fraction 3), 3:97 (n-hexane: dichloromethane, sample was obtained, fraction 4), 4:96 (n-hexane: dichloromethane, sample was obtained, fraction 5), 5:95 (n-hexane: dichloromethane, sample was obtained, fraction 6), 6:94 (n-hexane: dichloromethane, sample was obtained, fraction 7), 8:92 (n-hexane: dichloromethane, sample was obtained, fraction 8), and 10:90 (n-hexane: dichloromethane, sample was obtained, fraction 9), to obtain 8 fractions with increasing polarity.

[0049] Sample B was separated by gel Sephadex LH-20 column chromatography with n-hexane:dichlorohexane (volume ratio of 2:1) as eluent. One fraction was collected per 10 ml and five fractions were obtained after merging according to the HPLC analysis results.

[0050] Fraction 3 was purified by reverse-phase high performance liquid chromatography using an Agilent reverse-phase C18 column and a mobile phase of 40% acetonitrile in water-100% acetonitrile (for 20 minutes) to obtain a compound.

[0051] Structural identification of compounds

[0052] The purity of the obtained compound was determined by HPLC. Figure 2 The structures of samples with a purity greater than 98% were identified by mass spectrometry and nuclear magnetic resonance (NMR). The NMR was determined using a Bruker AVANCE DRX-500 NMR instrument, and the mass spectrometry was determined using a Waters G2-XS mass spectrometer.

[0053] The mass spectrum of the target compound is shown in Figure 3 The high-resolution quasi-molecular ion peak in the positive ion mode is [M+H] + m / z 408.2289.

[0054] The H NMR spectrum of the target compound is shown in Figure 4 , specific data δ H (CDCl3): 9.65 (s), 7.47 (s), 7.22 (s), 7.19 (d, J = 8.0 Hz), 7.08 (dd, J = 8.0, 8.0 Hz), 6.99 (d, J = 8.0Hz), 6.48 (d, J = 12.5 Hz), 6.07, (dd, J = 17.5, 10.5 Hz), 5.17 (d, J = 10.5Hz), 5.15 (d, J = 17.5 Hz), 4.1129 (m), 3.24 (d, J= 14.5 Hz), 3.01 (d, J= 4.5Hz), 2.92 (m), 1.60 (d, J= 7.0 Hz), 1.55 (s, ), 1.51 (s), 1.40 (s).

[0055] The carbon NMR spectrum of the target compound is as follows Figure 5 , specific data δ C (CDCl3) :19.2, 21, 25, 27.5, 27.5, 29.8, 33.7, 39.3, 51.8, 60.4, 64.4, 103.2, 112.7, 112 .9, 117.8, 121.1, 122.2, 122.7, 124, 126.3, 134.2, 144.3, 144.6, 160.1, 165.8;

[0056] The molecular structure of the target compound was finally determined as follows:

[0057] .

[0058] 4. Effects of Compounds on Fat Accumulation in the HepG2 Model

[0059] In vitro steatosis cell models are widely used to investigate lipid accumulation in hepatocytes during non-alcoholic fatty liver disease (NAFLD) due to their limited influencing factors and controllable experimental conditions. These models are often developed using the HepG2 hepatoma cell line by inducing the accumulation of oleic acid and palmitic acid mixtures at varying ratios and concentrations. Hepatic lipid accumulation and steatosis refer to the excessive accumulation of triglycerides within hepatocytes, making lipid accumulation in hepatocytes a sensitive indicator for evaluating anti-NAFLD activity.

[0060] To simulate the development of non-alcoholic fatty liver disease (NAFLD) in vitro, this study used oleic acid (OA)-induced human hepatocellular carcinoma HepG2 cells to establish a NAFLD model. The effects of compounds on lipid accumulation in HepG2 cells were assessed using Bodipy fluorescence assays, while their effects on cell viability were measured using colorimetric methods to evaluate their anti-NAFLD activity.

[0061] HepG2 cells were cultured conventionally in DMEM supplemented with 10% fetal bovine serum and double-streptomycin (100 U / ml penicillin and 100 U / ml streptomycin) at 37°C in a 5% CO2 incubator. When the cell density reached 80%, the cells were subcultured at a 1:3 ratio. When the cells reached logarithmic growth, they were plated in 96-well plates (100 µg per well) and cultured overnight at 37°C.

[0062] After the cells were cultured overnight, the supernatant was discarded and culture medium containing 0.25 mM oleic acid (90 μg) was added and incubated for 24 h. After the model was successfully established, the model group was replaced with conventional cell culture medium.

[0063] Drug-treated group: The model group successfully induced by oleic acid was added with 10 μl / well of the test compound at different concentrations (concentrations were: 0.0001 μg / ml, 0.001 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 1000 μg / ml) and incubated for 24 h.

[0064] Normal saline (10 μl / well) was added to the normal control group (cells not induced with oleic acid) and the model control group (the model group successfully induced with oleic acid was not added with drugs as the control group).

[0065] Oil Red O staining was performed as follows: After 24 hours of cell culture, the supernatant was discarded, the cells were washed two to three times with PBS buffer, and fixed with 4% paraformaldehyde for 15 to 20 minutes. The fixative was discarded, and 50 μl of Oil Red O working solution was added per well. The cells were incubated at room temperature for 1 hour. The staining solution was discarded, and the cells were washed three to five times with PBS to remove excess impurities and precipitates. HepG2 cell morphology and lipid droplet distribution were observed under a microscope, and photographs were taken to record the experimental results.

[0066] Calculation method: Inhibition rate (%) =

[0067] The experimental results are as follows Figure 6 The half-inhibitory concentration IC of the compound on lipid accumulation in the HepG2 cell model was calculated. 50 It is 0.052µg / ml.

[0068] Therefore, the present invention extracts and separates a new structural alkaloid compound from Eurotium cristatum, which reduces lipid deposition in HepG2 cells in an in vitro cell model and can be used to develop drugs for treating non-alcoholic fatty liver disease.

Claims

1. A compound of the following structural formula and its active salt: 。 2. The compound according to claim 1 and its active salt, characterized in that The active salt is selected from the group consisting of acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate.

3. Use of the compound according to claim 1 or 2 and its active salt in the preparation of a drug for treating fatty liver.

4. Eurotium cristatum ( Eurotium cristatum )SXHBTBU1934, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC NO.21026.

5. A method of using the Eurotium cristatum as claimed in claim 4 ( Eurotium cristatum ) SXHBTBU1934 is a method for preparing a compound of the following formula: ； The described Eurotium cristatum ( Eurotium cristatu m) inoculating SXHBTBU1934 onto a sterilized culture medium containing tea leaves, rice, corn, brown rice, wheat, orange peel, or grapefruit peel, and culturing the culture at 25-32° C. for 7-30 days; and then extracting the compound from the fermentation product; Then, the fermented product was soaked in ethanol overnight, filtered, and evaporated to dryness using a rotary evaporator. The organic layer was extracted three times with ethyl acetate and deionized water, and then evaporated to dryness. The organic layer was weighed and dissolved in a mixed solvent of dichloromethane and methanol. The sample was mixed at a weight ratio of fermentation crude extract to column chromatography silica gel = 1:1.3-1.7, and vacuum column chromatography was performed for separation. Column chromatography was performed using an organic solvent system of n-hexane and dichloromethane in different ratios, and the sample was obtained by elution with n-hexane:dichloromethane in a volume ratio of 2:98; The sample was separated by gel Sephadex LH-20 column chromatography, with n-hexane:dichlorohexane as the eluent, where the volume ratio of n-hexane:dichlorohexane was 2:

1. One fraction was collected per 10 ml and five fractions were obtained after merging according to the results of HPLC analysis. Fraction 3 was purified by reverse-phase high performance liquid chromatography using an Agilent reverse-phase C18 column and a mobile phase of 40% acetonitrile in water-100% acetonitrile with a retention time of 20 minutes to obtain the pure compound.

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