Aconitic acid extracted from tremella fuciformis as well as preparation and application thereof

Abstract

The invention discloses a Homoaconitic acid extracted from tremella fuciformis as well as an extraction method and application of the Homoaconitic acid, and belongs to the field of research and development of natural medicines. The lappaconitic acid is obtained from tremella sporocarp through extraction, separation and purification for the first time, and the lappaconitic acid can serve as a novel phosphatase inhibitor to be used for developing drugs for treating bone diseases, cancers and inflammations or developing products for improving soil or water eutrophication.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine research and development, and specifically relates to homoaconitic acid extracted from Tremella fuciformis and an extraction method thereof, and application of the homoaconitic acid as a novel phosphatase inhibitor. Background Art

[0002] With the development of society, the incidence of metabolic diseases (such as obesity), cardiovascular diseases (such as arterial calcification), and bone and joint diseases (such as osteoarthritis) continues to rise, increasing the demand for novel therapeutic strategies. Alkaline phosphatase (ALP) is a metalloproteinase ubiquitous in diverse organisms (bacteria, plants, and animals). Under alkaline conditions, it nonspecifically catalyzes the hydrolysis of phosphate monoesters to produce inorganic phosphate and the corresponding alcohol. ALP isoenzymes include tissue-specific ALPs found in germ cells, intestine, and placenta, as well as nonspecific ALPs primarily found in various tissues, including the liver, bone, central nervous system, and kidney. ALP is used as an auxiliary diagnostic tool and an important biomarker for many diseases, such as inflammatory bowel disease, liver disease, biliary obstruction, obesity caused by disproportionate intracellular fat depots, prostate cancer bone metastasis, and vascular mineralization. Alkaline phosphatase inhibitors can be effective therapeutic agents for diseases such as arterial calcification, osteoarthritis, rickets, cholecystitis, renal osteodystrophy, and myocardial infarction. In addition, alkaline phosphatase is also widely present in water and soil environments, and plays an important role in the absorption and utilization of phosphorus by organisms such as algae, the formation of chitin in marine organisms, and the activity of soil microorganisms. Therefore, inhibiting the ALP activity of microorganisms in soil or water can affect the phosphorus cycle and reduce eutrophication. The currently approved alkaline phosphatase inhibitor levamisole has gradually withdrawn from the clinic due to its neurotoxicity, and although zinc / magnesium ion chelators can indirectly inhibit ALP activity by interfering with the metal cofactors of ALP, they cannot be used directly for treatment. Therefore, it is of great significance to develop new alkaline phosphatase inhibitors that are highly efficient, low-toxic and targeted. Summary of the Invention

[0003] The present invention aims to provide homoaconitic acid extracted from Tremella fuciformis (Tremella fungus), as well as its extraction method and application. Using Tremella fuciformis as a raw material, the present invention obtains homoaconitic acid through product extraction, separation, and purification. The preparation method is simple and easily scalable for industrial production. The resulting homoaconitic acid can be used as a novel phosphatase inhibitor.

[0004] To achieve the above object, the present invention adopts the following technical solutions: Homoaconitic acid, extracted from Tremella fuciformis, has a molecular formula of C7H8O6 and a structural formula as follows: . The basic carbon skeleton of homoaconitic acid is a chain structure composed of 4 carbon atoms, which includes a carbon-carbon double bond. The homoaconitic acid molecule contains 3 carboxyl groups, which are located on different carbon atoms in the chain structure.

[0005] The homoaconitic acid is obtained by using Tremella fuciformis fruiting bodies as raw materials through fruiting body treatment, product extraction, and product separation and purification. The specific extraction method includes the following steps: (1) Fruiting body processing: Dry and crush the fresh Tremella fuciformis fruiting bodies; (2) Product extraction: The Tremella powder obtained in step (1) is leached or extracted with a solvent, filtered, and concentrated to obtain a crude extract containing homoaconitic acid; (3) Product separation and purification: The crude extract containing homoaconitic acid obtained in step (2) is purified by chromatography to obtain homoaconitic acid.

[0006] Furthermore, the solvent used in step (2) is an aqueous solution of ethanol, methanol or acetone.

[0007] Furthermore, the volume concentration of ethanol, methanol or acetone in the solvent is 50-100%.

[0008] Furthermore, the chromatography technique in step (3) can adopt medium pressure or normal pressure preparative liquid chromatography.

[0009] Furthermore, when preparative liquid chromatography is used, reverse phase silica gel, gel LH-20 or normal phase silica gel is used as the chromatographic medium.

[0010] Furthermore, when preparative liquid chromatography is used, ethanol-water, methanol-water, acetone-water or acetonitrile-water is used as the mobile phase. Preferably, an ethanol-water solution containing 0-1 vol% formic acid is used as the mobile phase, wherein the volume ratio of ethanol to water is 50-95:5-50.

[0011] The obtained homoaconitic acid can be used as a phosphatase inhibitor for preparing therapeutic drugs for bone diseases, cancer, and inflammation, or for preparing products for improving soil or water eutrophication.

[0012] The beneficial effects of the present invention are: (1) The present invention is the first to prepare homoaconitic acid from Tremella fuciformis fruiting bodies. The raw material sources are wide and the preparation method is simple, which can be easily realized in industrial production.

[0013] (2) The homoaconitic acid obtained by the present invention has strong phosphatase inhibitory activity and can be used to develop therapeutic drugs for bone diseases, cancer and inflammation, or to prepare products for improving soil and water eutrophication. It has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound measured in Example 2.

[0015] Figure 2 is the carbon NMR spectrum of the compound measured in Example 2.

[0016] Figure 3 This is a high-resolution mass spectrometry analysis of the compound determined in Example 2 (positive ion mode).

[0017] Figure 4 This is a high-resolution mass spectrometry analysis of the compound determined in Example 2 (negative ion mode).

[0018] Figure 5 This is the standard curve drawn in Example 3.

[0019] Figure 6 This is the dose effect of the compound in Example 4 on inhibiting alkaline phosphatase activity. DETAILED DESCRIPTION

[0020] A method of extracting homoaconitic acid from Tremella fuciformis is as follows: (1) Fruiting body processing: Dry and crush the fresh Tremella fuciformis fruiting bodies; (2) Product extraction: The Tremella fuciformis powder obtained in step (1) is leached or extracted with an aqueous solution of ethanol, methanol or acetone (with a volume concentration of 50-100%), and then the solid matter is removed by filtration, centrifugation or membrane filtration, and then the solvent is removed by concentration to obtain a crude extract containing homoaconitic acid; (3) Product separation and purification: The crude extract containing homoaconitic acid obtained in step (2) is dissolved in methanol or ethanol, and subjected to medium-pressure or normal-pressure preparative liquid chromatography, using reversed-phase silica gel, gel LH-20 or normal-phase silica gel as the chromatographic medium, and ethanol-water, methanol-water, acetone-water or acetonitrile-water as the mobile phase for elution. The target peak with a retention time of about 7 minutes is collected at an absorption wavelength of 200-310 nm to obtain homoaconitic acid.

[0021] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0022] Example 1 Preparation of Homoaconitic Acid Fresh Tremella fuciformis (331.5 g) was oven-dried at 60°C and ground to obtain dry Tremella fuciformis powder (32.4 g). This powder was then added to 200 mL of 75 vol% ethanol solution, sonicated for 30 minutes, and then extracted overnight. This process was repeated three times to obtain a crude extract (2.6544 g). The crude extract was prepared into a 10 mg / mL sample solution using chromatographic-grade ethanol. The solution was then prepared using a preparative HPLC column (HYPRRGLD, 250 × 10 mm, 5 μm) using a 95 vol% aqueous ethanol solution (with 0.1% formic acid) as the mobile phase at a flow rate of 5 mL / min. The target peak, with a retention time of approximately 7 minutes, was collected at an absorption wavelength of 254 nm. The target compound (217.08 mg) was obtained.

[0023] Example 2 The compound obtained in Example 1 is a white powder, soluble in methanol and ethanol. 1 H-NMR, 13 C-NMR, DEPT, 1 H- 1 H COSY, HSQC and HMBC) were used for determination (Table 1). Data analysis showed that the compound contained 7 carbon signals, 3 carbonyl carbons: δ 168.6 (C1), δ 176.3 (C6) and δ 168.6 (C7), 2 olefin carbons: δ 129.3 (C2) and δ 147.1 (C3, possibly a quaternary carbon), and 2 methylenes: δ 24.4 (C4) and δ 34.1 (C5). In addition, the compound contained 1 low-field proton signal δ 6.79 ( d , J = 0.7 Hz, 1H) (H-2), two upfield alkyl type signal protons δ2.97 (m, 2H) (H-4) and δ2.44 (m, 2H) (H-5). From the HMBC spectrum, correlations between H-2 and C-1, C-3 and C-4, correlations between H-4 and C-1, C-2, C-3, C-5, C-6 and C-7, and long-range correlations between H-5 and C-3, C-4 and C-6 were observed. In the 1H-1H COSY spectrum, correlations between H-4 and H-5 were observed. Based on the above analysis and the lower field chemical shift value of H-2, the basic structure of the compound is speculated to be ( E )-but-1-ene-1,2,4-tricarboxylic acid. The compound was further subjected to high-resolution mass spectrometry (HRMS) detection, and the molecular formula of the compound was determined by Q-TOF MS to be C7H8O6. The hydrogenation mass spectrometry signal of the compound was detected in the positive ion mode. m / z [M+H] +189.0383 (theoretical value is C7H9O6, 189.0399) and sodium added mass spectrometry signal m / z [M+Na] + 211.0219 (theoretical value is C7H8NaO6, 211.0219); the deprotonated mass spectrometry signal of the compound can be detected in negative ion mode m / z [M−H] − 187.0243 (theoretical value is C7H7O6, 187.0243).

[0024] Combining NMR spectroscopy and mass spectrometry data, the obtained compound was identified as trans-homoaconitic acid with a molecular formula of C7H8O6 and a structural formula of: .

[0025] Table 1 NMR data of compounds (H spectrum 600 MHz, C spectrum 151 MHz, deuterated methanol)

[0026] Example 3 According to Table 2, 5 batches of fresh Tremella fuciformis (AE) from different sources were dried in an oven at 60°C, crushed, and then added with 200 mL of 75 vol% ethanol solution. After ultrasonication for 30 minutes, the mixture was extracted overnight and repeated three times to obtain a crude extract. The content of the target compound in Tremella fuciformis from different sources was then determined by HPLC. A small amount of the target compound obtained in Example 1 was first prepared into a 1 mg / mL solution with chromatographic grade ethanol. Then, 0, 5, 10, 15, 20, 25, and 30 μL were injected, respectively. Methanol-water (with 0.1% formic acid) was used as the mobile phase, the detection wavelength was 220 nm, the analysis time was 40 minutes, and each injection was repeated three times to obtain a standard curve of the target compound content and peak area (as shown in FIG1 ). Figure 1 ) and the regression equation: Y = 0.0218X - 0.0046 (Y is the content, X is the peak area). Next, each of the five crude extracts was prepared with chromatography-grade ethanol to a 10 mg / mL solution. 10 μL of each solution was injected and analyzed by HPLC using the same chromatographic method used for the standard curve. The content of the target compound in Tremella fuciformis from different sources was calculated based on the standard curve. The results showed that the content of the target compound in Tremella fuciformis from different sources ranged from 0.52% to 0.96%.

[0027] Table 2 Preparation of target compounds using Tremella fuciformis from different sources Content analysis of target compounds

[0028] Note: The compound content ratio refers to the proportion of its content in Tremella fuciformis dry powder.

[0029] Example 4 The inhibitory activity of the target compound against alkaline phosphatase was determined by UV spectrophotometry. ATP (adenosine 5'-triphosphate disodium salt) was used as a positive control. The compound was first prepared at a 12 mg / mL solution in chromatography-grade ethanol, and the positive control ATP was prepared at a 12 mg / mL solution in 10 mM Tris-HCl buffer (pH 8.52). The buffer, sample solution, and p-nitrophenylphosphate disodium hydrate (p-NPP) were then added sequentially to the experimental area of ​​a 96-well plate. After mixing, the plate was incubated at 37°C for 10 minutes in a biochemical incubator. Alkaline phosphatase was then added, and the reaction was continued at 37°C for another 10 minutes. Finally, the reaction was terminated by the addition of NaOH solution. The reaction systems of each treatment group were as follows: (1) blank group: 120 μL buffer + 20 μL solvent + 30 μL p-NPP + 10 μL NaOH; (2) negative control group: 110 μL buffer + 20 μL solvent + 30 μL p-NPP + 10 μL alkaline phosphatase + 10 μL NaOH; (3) positive blank group: 120 μL buffer + 20 μL ATP + 30 μL p-NPP + 10 μL NaOH; (4) positive control group: 110 μL buffer + 20 μL ATP + 30 μL p-NPP + 10 μL alkaline phosphatase + 10 μL NaOH; (5) sample blank group: 120 μL buffer + 20 μL sample + 30 μL p-NPP + 10 μL NaOH; (6) sample group: 110 μL buffer + 20 μL sample + 30 μL p-NPP + 10 μL Alkaline phosphatase + 10 μL NaOH. After the reaction is complete, set the microplate reader to a wavelength of 405 nm and measure the absorbance of the experimental area in the 96-well plate. Set up three parallel controls for each reaction system. Calculate the inhibition rate of each compound on alkaline phosphatase according to the following formula. The results are shown in Table 3: .

[0030] Table 3 Inhibition rate of alkaline phosphatase

[0031] As shown in Table 3, at the same concentration, the target compound has a strong inhibitory effect on alkaline phosphatase, and its inhibitory activity is significantly higher than that of the positive control ATP.

[0032] The dose effect of the compound's inhibitory activity on alkaline phosphatase was further determined. The specific procedure was to prepare the compound into sample solutions at five concentrations (2.5, 5, 10, 15, and 20 mg / mL) and test them using the same experimental conditions as above, with the final reaction concentrations being 0.2632, 0.5263, 1.0526, 1.5789, and 2.1053 mg / mL, respectively. The inhibition rates of the compound at different concentrations on alkaline phosphatase are shown in Tables 4 and Figure 6 .

[0033] Table 4 Inhibitory effects of compounds at different concentrations on alkaline phosphatase

[0034] The IC values ​​of the compounds for alkaline phosphatase inhibition were analyzed by SPSS software. 50 The concentration of the compound was 1.018 mg / mL, and within the experimental concentration range, the inhibitory activity was significantly positively correlated with its concentration (P < 0.01). The results showed that the compound had a significant inhibitory effect on alkaline phosphatase, with an inhibition rate higher than that of the positive control ATP, suggesting that it has the potential to be developed as an alkaline phosphatase inhibitor.

[0035] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A homoaconitic acid extracted from Tremella fuciformis, characterized in that: Its molecular formula is C7H8O6 and its structural formula is as follows: 。 2. A method for extracting homoaconitic acid as claimed in claim 1 from Tremella fuciformis, characterized in that: The following steps are involved: (1) Fruiting body processing: Dry and crush the fresh Tremella fuciformis fruiting bodies; (2) Product extraction: The Tremella powder obtained in step (1) is leached or extracted with a solvent, filtered, and concentrated to obtain a crude extract containing homoaconitic acid; (3) Product separation and purification: The crude extract containing homoaconitic acid obtained in step (2) is purified by chromatography to obtain the homoaconitic acid.

3. The method according to claim 2, characterized in that The solvent used in step (2) is an aqueous solution of ethanol, methanol or acetone.

4. Use of the homoaconitic acid as claimed in claim 1 as a phosphatase inhibitor.

5. The use according to claim 4, characterized in that The phosphatase inhibitor can be used to prepare therapeutic drugs for bone diseases, cancer, and inflammation, or to prepare products for improving soil or water eutrophication.

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