Application of butyrolactone metabolic ketone I in preparation of antioxidant drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of butyrolactone metabolic ketone I separated from a rat excrement metabolite of an aspergillus terreus secondary metabolite butyrolactone I in preparation of an antioxidant drug. The compound has an obvious anti-oxidation effect; the raw materials for preparing the compound are rich in resources, the extraction and separation technical difficulty is small, the solvent can be recycled, and the production cost is low.

Description

[0001] This application is a divisional application with application number 201910558609.3, filed on June 26, 2019. The name of the invention at the time of application was “A dibenzophenone compound, its preparation method and application”. Technical Field

[0002] The invention belongs to the field of medical technology, and particularly relates to the application of butyrolactone metabolite ketone I in the preparation of antioxidant drugs. Background Art

[0003] Butyrolactone I is the main active secondary metabolite of Aspergillus terreus. It was first isolated by Kiriyama from Aspergillus terreus var. africanus IFO 8835 in 1977. Because of its five-membered unsaturated lactone ring in its structure, it was named butyrolactone I.

[0004] Previous studies have shown that butyrolactone I has clear and selective cyclin-dependent kinase (CDK) inhibitory activity. Its in vitro inhibitory mechanism for CDC2 and CDK2 has also been elucidated, making it a potential anti-tumor drug candidate. Butyrolactone I and its derivatives have also been shown to be a class of multifunctional natural active substances with a wide range of pharmacological activities.

[0005] We administered butyrolactone I orally to rats, collected their urine and feces, and used various chemical and chromatographic methods to separate and identify the metabolites of butyrolactone I in vivo. We speculated on the transformation process of butyrolactone I in vivo and measured the pharmacological activities of these metabolites to understand the structural changes after drug metabolism and the relationship between these changes and efficacy and toxicity. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of butyrolactone metabolite ketone I in the preparation of antioxidant drugs.

[0007] The present invention is to separate new benzophenone compounds from rat fecal metabolites of butyrolactone I, a secondary metabolite of Aspergillus terreus, and its application. The chemical structural formula (I) of the benzophenone compounds is:

[0008]

[0009] Wherein, R1 is selected from hydrogen, (C1~C6) alkyl, (C1~C6) alkoxy, halogenated (C1~C4) alkyl or (C1~C4) alkoxy, (C1~C6) alkylthio, amino substituted with mono- or di-(C1~C6 alkyl), (C1~C6) alkylamido, (C1~C6) alkylsulfinyl, (C1~C6) alkylacyl, halogen.

[0010] Furthermore, the difference between the number of carbon atoms of R1 and R2 is within 3.

[0011] Furthermore, the difference between the number of carbon atoms of R3 and R4 is within 3.

[0012] Furthermore, R1 is hydrogen and / or R2 is hydrogen.

[0013] Furthermore, R3 is a methyl group and / or R4 is a methyl group.

[0014] Furthermore, its chemical structural formula (II) is:

[0015]

[0016] Named butyrolactone metabolite ketone I.

[0017] A pharmaceutical composition comprises the compound and a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.

[0018] Application of the pharmaceutical composition or the compound in the preparation of antioxidant health foods and / or antioxidant drugs.

[0019] A preparation method for the compound comprises: (1) orally administering butyrolactone I to an animal, collecting its urine and / or feces, and extracting the mixture with an organic solvent; and (2) eluting and separating the butyrolactone metabolite ketone I using chromatography.

[0020] In the preparation method, the animal is a rodent; and / or the organic solvent is ethyl acetate.

[0021] The preparation method comprises the following steps: (1) chromatography on a silica gel column followed by elution, and / or (2) chromatography on a C18 reverse phase column followed by elution.

[0022] The raw material resources for preparing the compound of the present invention are abundant, the extraction and separation technology is easy to use, the solvent can be recycled, and the production cost is low.

[0023] The preparation method of the above compound is as follows:

[0024] (1) Extraction: 40 Wistar rats, half male and half female, weighing 200 ± 20 g, were fed with 40 mg kg-1 of the drug by gavage after 1 week of acclimatization. The rats were placed in metabolic cages, one rat per cage, and feces were collected over a 24-hour period. During the experiment, the rats were fed with starch buns and drinking water containing 4‰ normal saline and 1‰ glucose solution. Feces were collected every morning for 24 hours and stored in a -80°C refrigerator. Cumulative gavage administration was continued for 4 weeks, with the drug administered once a day. A total of 8 g of extract was obtained from the ethyl acetate extract of the feces.

[0025] (2) Separation: The extract was subjected to silica gel column chromatography, with a gradient elution of dichloromethane-methanol system in a volume ratio of 100:0-100:50, and thin layer chromatography detection. The fractions containing the new compound were collected, and then subjected to gradient elution of petroleum ether-ethyl acetate system in a volume ratio of 100:0-0:100, and finally subjected to preparative liquid chromatography on a C18 reverse phase column, with elution of methanol-water in a volume ratio of 68:32, to obtain the new compound butyrolactone metabolite ketone I.

[0026] The new compound of the present invention, butyrolactone metabolite ketone I, colorless oil (methanol), UV (MeOH) λ max at 209 nm. HR-ESI-MS gave [M+Na] at m / z 333.1447 + Peak, the molecular formula is estimated to be C 20 H 22 O3. 1 H-NMR and 13 C-NMR data are shown in Table 1.

[0027] Experimental studies have shown that the compound of the present invention has significant antioxidant effects and can therefore be used to prepare antioxidant drugs.

[0028] Table 1 Butyrolactone metabolite ketone I 1 H and 13 C NMR data

[0029]

[0030] Structural elucidation of the new compound butyrolactone metabolite ketone I.

[0031] like Figure 1 As shown, the new compound of the present invention 1 H NMR, 13 The structure of the compound was determined by C NMR, 2D-NMR (HSQC, HMBC) spectra, and HR-ESI-MS spectra. Specifically:

[0032] Colorless oil (methanol), UV (MeOH) λ max :209nm. Infrared spectroscopy (IR) suggests the presence of hydroxyl groups (3431cm-1 ), double bond (1638cm -1 ) and other functional groups. HR-ESI-MS gave [M+Na] at m / z 333.1447. + Peak (calcd.333.1467), its molecular formula was determined to be C 20 H 22 O3.

[0033] 1 The aromatic region of the H-NMR (400 MHz, DMSO-d6) spectrum gives a group of proton signals of the AA′BB′ coupling system δ H 6.91 (2H, d, J = 8.4 Hz, H-2′, 6′), 6.68 (2H, d, J = 8.4 Hz, H-3′, 5′), it is speculated that there is a para-substituted benzene structure; δ H 6.76 (1H, dd, J = 8.0, 2.0 Hz, H-6″), 6.75 (1H, d, J = 2.0 Hz, H-2″), 6.70 (1H, d, J = 8.0 Hz, H-5″), suggesting the presence of a 1,3,4-trisubstituted benzene ring structure. H 1.65 (3H, s, H-10″) and 1.67 (3H, s, H-11″), a double bond hydrogen signal δ H 5.23 (1H, t, J = 7.2 Hz, H-8") and the coupled hydrogen signal δ H 3.16 (2H, d, J = 7.2 Hz, H-7″), suggesting the presence of an isopentenyl fragment.

[0034] 13 The C-NMR (100 MHz, DMSO-d6) spectrum gave 14 aromatic or double bond carbon signals, which were consistent with the carbon numbers of the two benzene ring fragments and the isopentenyl fragment speculated above. Compared with butyrolactone I, compound butyrolactone metabolite ketone I reduced the carbon signals of -COOCH3 and lactone structure fragments, and the keto carbonyl carbon signal δ C 206.9 (C-2) and two methylene carbon signals δC 47.8 (C-1) and 48.0 (C-3). The above information suggests that the lactone ring is opened and decarboxylation rearrangement occurs.

[0035] The HSQC spectrum provides information on all hydrogen-carbon bonds in the structure, as shown in Table 1.

[0036] In the HMBC spectrum, the proton signal δ H 3.61 (2H, s, H-1) and carbon signal δ C 206.9(C-2), 125.2(C-1'), 130.9(C-2') correlation, δH 3.59 (2H, s, H-3) and carbon signal δ C 206.9 (C-2), 128.2 (C-6") and C-2' (δc 130.9) related to H-7" (δ H 3.16) is associated with carbon signals C-4″ (δc 154.0), C-3″ (δc 127.8) and C-8″ (δc 123.3), H-10″ (δ H 1.67) is related to C-9″ (δc 131.5) and C-8″ (δc 123.3), H-11″ (δ H 1.65) is associated with C-9″ (δc 131.5) and C-8″ (δc 123.3), and the active hydrogen signal 4′-OH (δ H 9.21) is related to C-4′ (δc 156.5) and C-5′ (δc 115.6), 4″-OH (δ H 9.19) related to C-4″ (δc 154.0) and C-5″ (δc 115.1). The structure of the compound was determined to be 1-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-3-(4-hydroxyphenyl)propan-2-one.

[0037] This compound is a new diphenylacetone compound that has not been reported in the literature. The relevant NMR data are shown in Table 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 New compounds 1 H NMR spectrum;

[0039] Figure 2 New compounds 13 C NMR spectrum;

[0040] Figure 3 HSQC spectra of new compounds;

[0041] Figure 4 HMBC spectra of new compounds;

[0042] Figure 5 HR-ESI-MS spectra of the new compounds. DETAILED DESCRIPTION

[0043] The present invention is further described below by way of examples so that those skilled in the art can more fully understand the present invention, but the present invention is not limited in any way.

[0044] Example 1: 40 Wistar rats, half male and half female, weighing 200±20 g, were fed for one week and then orally administered with 40 mg·kg -1 The rats were placed in metabolic cages, one rat per cage, and urine and feces were collected for 24 hours. During the experiment, the rats were fed starch-based steamed bread and their drinking water consisted of 4% saline and 1% glucose solution. Feces were collected every morning for 24 hours and stored in a -80°C refrigerator. Cumulative oral administration was continued for 4 weeks, with administration once daily, for a total of approximately 14 g. A total of 8 g of extract was obtained from the fecal ethyl acetate extract. The extract was eluted using silica gel column chromatography with a gradient of dichloromethane-methanol in a volume ratio of 100:0-100:50, followed by thin-layer chromatography detection. Fractions containing the new compound were collected and then eluted using a gradient of petroleum ether-ethyl acetate in a volume ratio of 100:0-0:100, followed by preparative liquid chromatography on a C18 reverse-phase column with a volume ratio of methanol-water of 68:32, at a flow rate of 3 ml / min and detection at 210 nm, yielding 80 mg of the new compound, butyrolactone metabolite 1.

[0045] Example 2: Antioxidant activity test of the new compound butyrolactone metabolite ketone I

[0046] 1. Experimental materials and instruments

[0047] Instruments: Microplate reader: Thermo scientific, USA; 96-well plate: Nest Biotech, USA; Precision electronic balance: Shimadzu, Japan; Micropipette: 100-1000 μL, Thermo, USA;

[0048] Reagents: DPPH (Sigma), ABTS (J&K Chemicals), PBS (Solarbio), L-ascorbic acid (Aldrich Chemicals).

[0049] 2. Test methods

[0050] DPPH method:

[0051] Accurately weigh 3.94 mg of DPPH, dissolve it in anhydrous ethanol, and dilute it to a 10 ml brown volumetric flask. Then dilute it 5 times to make a solution with a concentration of 0.2 mM. Measure its absorbance at 517 nm so that its absorbance value is around 0.7. L-ascorbic acid and the test compound are respectively made into concentrations of 100, 50, 25, 10, 5, and 1 μg / ml with anhydrous ethanol. Use a pipette to draw 100 μL of each into a 96-well plate, and then add 100 μL of different concentrations of the test sample solution dissolved in ethanol (100, 50, 25, 10, 5, 1 μg / ml). After each is thoroughly mixed, stand at room temperature in the dark for 30 minutes, and measure the absorbance value of each well at 517 nm using a microplate reader; the activity of each compound in scavenging DPPH free radicals is calculated using the following formula:

[0052] DPPH scavenging activity (%) = [1-(S 样品 -SB 空 ) / (C 对照 -CB 空白 )]×100%.

[0053] Among them, sample S: sample solution 100 μL + DPPH 100 μL;

[0054] Negative control C: 100 μL of anhydrous ethanol + 100 μL of DPPH;

[0055] Blank control CB: 200 μL of anhydrous ethanol.

[0056] Positive control: L-ascorbic acid

[0057] ABTS method:

[0058] Preparation of 0.01mol / L PBS: Dissolve 0.395g NaCl, 0.1g KCl, 0.12g KH2PO4, and 0.9g K2HPO4 in 400ml distilled water, adjust the pH to 7.4 with HCl, and finally make up to 500ml. Place in the refrigerator and keep refrigerated until use.

[0059] Preparation of potassium persulfate (2.45 mmol / L): Weigh 0.0066 g of potassium persulfate and dilute to 10 ml with distilled water.

[0060] Preparation of ABTS solution: Weigh 0.0384 g of ABTS and dilute to 10 ml with prepared PBS.

[0061] Generation of ABTS+· free radicals: Mix the prepared potassium persulfate solution and ABTS solution in a 1:1 volume ratio and incubate at room temperature in the dark for 12–16 hours to generate ABTS+·. Dilute this solution with 0.01 M PBS (pH 7.4). When the absorbance at 734 nm reaches 0.70 (±0.02), equilibrate at 30°C for 30 minutes and measure the absorbance.

[0062] L-ascorbic acid and the test compound were prepared with anhydrous ethanol to concentrations of 100, 50, 25, 10, 5, 2, and 1 μg / ml, respectively.

[0063] ABTS+· (150 μL) solution was placed in a 96-well plate, and 100 μL of ethanol solution of the test sample of different concentrations (100, 50, 25, 10, 5, 2, and 1 μg / ml) was added. After thorough mixing and standing in the dark for 20 minutes, the absorbance of each well was measured at 734 nm using a microplate reader. The ABTS+· free radical scavenging activity of the compound was calculated using the following formula:

[0064] ABTS+·Scavenging activity (%) = [1-(S 样品 -SB 空 ) / (C 对照 -CB 空白 )]×100%.

[0065] Sample S: 100 μL sample solution + ABTS + 150 μL;

[0066] Negative control C: 100 μL of anhydrous ethanol + 150 μL of PBS;

[0067] Blank control CB: 100 μL of anhydrous ethanol + ABTS + 150μL.

[0068] Positive control: L-ascorbic acid

[0069] 3. Experimental results

[0070] The antioxidant capacity of compound butyrolactone metabolite ketone I was evaluated using DPPH and ABTS free radical scavenging methods. The results showed (Table 2) that it exhibited significant free radical scavenging capacity.

[0071] Table 2 Antioxidant activity of butyrolactone metabolite ketone I (IC 50 ,μM)

[0072]

[0073] Vc a(ascorbic acid)was used as positive control in test of antioxidantactivities.

[0074] b ND:Not detect。

Claims

1. The application of butyrolactone metabolite ketone I in the preparation of antioxidant drugs, characterized in that: The preparation method of butyrolactone metabolite ketone I comprises: (1) administering butyrolactone I orally to an animal, collecting the urine and / or feces thereof, and extracting the urine and / or feces with an organic solvent; and (2) eluting and separating the butyrolactone metabolite ketone I with a chromatography method.

2. The use of the butyrolactone metabolite ketone I according to claim 1 in the preparation of antioxidant drugs, characterized in that: The structural formula of the butyrolactone metabolite ketone I is shown in formula (I):

3. The preparation method according to claim 1, wherein: The animal is a rodent; and / or the organic solvent is ethyl acetate.

4. The preparation method according to claim 1, wherein: The chromatography is (1) silica gel column chromatography followed by elution, and / or (2) C18 reverse phase column chromatography followed by elution.

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