Enzymatic preparation of dihydroisoquercitrin and its application in prevention and treatment of cardiovascular and cerebrovascular diseases and quality control of ginkgo leaves

Abstract

The application discloses an enzyme preparation method of dihydroisoquercitrin and application of the dihydroisoquercitrin in prevention and treatment of cardiovascular and cerebrovascular diseases and quality control of ginkgo leaves, and belongs to the technical field of biological medicines. The enzyme preparation method takes taxifolin as a substrate, takes S-adenosyl methionine as a methyl donor, and carries out a methylation reaction under catalysis of catechol-O-methyltransferase to obtain dihydroisoquercitrin through purification. A pharmaceutical composition containing dihydroisoquercitrin, taxifolin and / or artemetin is used for preventing or treating cardiovascular and cerebrovascular diseases such as atherosclerosis, myocardial ischemia, hypertension, vascular cognitive impairment and the like. Pharmacokinetic experiments show that oral bioavailability of dihydroisoquercitrin, taxifolin and artemetin is significantly higher than that of traditional flavonols. The dihydroisoquercitrin, taxifolin and artemetin are used as index components to establish an HPLC or LC-MS / MS quality control method for ginkgo leaf extract or preparation thereof, and a new technical scheme is provided for development of high oral bioavailability cardiovascular and cerebrovascular drugs and quality control of ginkgo products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an enzymatic preparation method of dihydroisorhamnoside and its application in the prevention and treatment of cardiovascular and cerebrovascular diseases and in the quality control of Ginkgo biloba leaves. Background Technology

[0002] Ginkgo leaves ( Ginkgo biloba Ginkgo biloba extract is a widely used herbal medicine globally, approved in European countries such as Germany and France for the treatment of cognitive impairment and peripheral vascular disease. In China, more than 180 ginkgo products have been approved for clinical use. The pharmacological activity of ginkgo leaf extract is mainly attributed to its flavonoids and terpene lactones, with total flavonoids accounting for more than 24% of the extract. For a long time, domestic and international research on ginkgo flavonoids has focused primarily on flavonols, including quercetin, kaempferol, and isorhamnetin and their glycosides. However, the oral bioavailability of these flavonols is low, typically below 10%, severely limiting their in vivo efficacy. For example, the oral bioavailability of quercetin in humans is usually less than 5%, and kaempferol and isorhamnetin also have similar issues. The main reason for the low bioavailability is that these flavonol glycosides are difficult to be directly absorbed in the intestine. They need to be hydrolyzed into aglycones by intestinal flora before they can be absorbed, and after absorption, they are rapidly metabolized by glucuronidation or sulfation.

[0003] Dihydroflavonols are the reduced forms of flavonols with their C2-C3 double bonds. Representative compounds include taxifolin (also known as dihydroquercetin), aromadendrin (also known as dihydrokaempferol), and dihydroisorhamnetin. Existing literature reports that taxifolin and aromadendrin are widely found in pine trees, fruits, and vegetables, possessing various biological activities such as antioxidant, anti-inflammatory, anticancer, and cardiovascular protection. However, there are currently no reports on the presence of these dihydroflavonol compounds in Ginkgo biloba extract or their systematic pharmacokinetic characteristics. In particular, the presence, preparation methods, and pharmacokinetic studies of dihydroisorhamnetin (the dihydroreduced form of isorhamnetin) in Ginkgo biloba extract are lacking, leading to insufficient understanding and utilization of its potential value.

[0004] While commercially available standards for dihydroisorhamnoside are available in the current technology, they are primarily obtained through plant extraction, with no reports of chemical synthesis or biocatalytic preparation methods. Traditional plant extraction methods are cumbersome, time-consuming, and yield-low, severely hindering pharmacological research and standardized application of this compound. Regarding quality control of Ginkgo biloba extract, current pharmacopoeias (such as the Chinese Pharmacopoeia, the United States Pharmacopeia, and the European Pharmacopoeia) all use the content of total flavonol glycosides and terpene lactones as core indicators. Specifically, the Chinese Pharmacopoeia typically stipulates a total flavonol glycoside content of no less than 24.0% and a terpene lactone content of no less than 6.0%; while the United States Pharmacopeia and the European Pharmacopoeia stipulate a total flavonol glycoside content between 22.0% and 27.0%, and a terpene lactone content between 5.4% and 6.6% (EP) or 5.4% and 12.0% (USP), respectively. This system primarily focuses on flavonols, failing to adequately cover other types of flavonoids such as dihydroflavonols. Existing characteristic spectral technologies (such as CN113995776B) are still limited to flavonol glycosides, excluding dihydroflavonols, and are not correlated with pharmacokinetic parameters. The aforementioned quality control standards based on chemical component content fail to correlate with pharmacokinetic exposure characteristics or bioavailability, making it difficult to comprehensively and scientifically reflect the product's true in vivo exposure level and potential efficacy.

[0005] Therefore, developing an enzymatic preparation method for dihydroisorhamnoside, revealing the oral bioavailability characteristics of piperidin, hesperidin, and dihydroisorhamnoside, and using it as a supplementary indicator for the quality control of Ginkgo biloba extract, has significant scientific and practical value. Summary of the Invention

[0006] In view of the technical problems in the existing technology, such as the lack of efficient and green preparation methods for dihydroisorhamnone, low oral bioavailability of flavonoid components, and imperfect quality control system of Ginkgo biloba extract, this invention aims to provide an enzymatic preparation method for dihydroisorhamnone and its application in the prevention and treatment of cardiovascular and cerebrovascular diseases and the quality control of Ginkgo biloba extract.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses an enzymatic method for preparing dihydroisorhamnoin, using piperidine as a substrate and S-adenosylmethionine as a methyl donor, and carrying out a methylation reaction under the catalysis of catechol-O-methyltransferase, and then purifying to obtain dihydroisorhamnoin.

[0008] The catechol-O-methyltransferase is derived from recombinant human COMT, recombinant rat COMT, or animal liver fluid.

[0009] Preferably, the animal liver cytosolic fluid is rat liver cytosolic fluid. This source of COMT enzyme has high activity, low preparation cost, and is easy to obtain. It can be prepared in batches by differential centrifugation, which can ensure the catalytic efficiency of the enzymatic synthesis reaction and batch-to-batch consistency, thereby ensuring the stable production of dihydroisorhamnoside.

[0010] Preferably, the concentration of taurine in the reaction system is 50–100 μM, the concentration of S-adenosylmethionine is 0.5–2 mM, the reaction temperature is 35–40℃, and the pH of the reaction system is 7.0–8.0. This achieves the optimal ratio of substrate to methyl donor, ensuring high conversion rate while avoiding raw material waste, and possesses technical advantages for industrial application.

[0011] Preferably, the methylation reaction is carried out in Tris-HCl buffer, HEPES buffer, or phosphate buffer.

[0012] Preferably, the purification includes two steps: organic solvent extraction and preparative high-performance liquid chromatography (HPLC). The organic solvent extraction uses ethyl acetate, methyl tert-butyl ether, butyl acetate, or dichloromethane; the preparative HPLC uses a C18 preparative column, with an acetonitrile-water system or a methanol-water system as the mobile phase, and a detection wavelength of 280–360 nm.

[0013] Secondly, the present invention provides dihydroisorhamnoside obtained by the above-mentioned enzymatic preparation method.

[0014] The purity of the dihydroisorhamnoside is ≥98%, preferably ≥99%.

[0015] Thirdly, the present invention provides a pharmaceutical composition comprising the aforementioned dihydroisorhamnoside, as well as piperidine and / or hesperidin, and a pharmaceutically acceptable carrier.

[0016] The mass ratio of dihydroisorhamnoside to taurine is 1:10 to 10:1.

[0017] Preferably, the mass ratio of dihydroisorhamnoside to taurine is 1:5 to 5:1.

[0018] Preferably, the pharmaceutical composition further comprises ginkgo leaf extract or an extract of it rich in dihydroflavonol glycosides.

[0019] Fourthly, this invention provides the use of the dihydroisorhamnoside or the pharmaceutical composition thereof in the preparation of drugs for the prevention and / or treatment of cardiovascular and cerebrovascular diseases. Based on pharmacokinetic experiments in mice, it has been demonstrated that dihydroisorhamnoside in Ginkgo biloba extract has high oral bioavailability, and this application has a clear pharmacodynamic basis and the technical advantage of high in vivo exposure.

[0020] The cardiovascular and cerebrovascular diseases mentioned include atherosclerosis, myocardial ischemia, myocardial infarction, hypertension, myocardial hypertrophy, heart failure, coronary heart disease, diabetic cardiomyopathy, ischemic stroke, sequelae of cerebral hemorrhage, transient ischemic attack, vascular dementia, cerebral arteriosclerosis, chronic cerebral ischemia, peripheral vascular disease, venous thrombosis, and other cardiovascular and cerebrovascular diseases related to oxidative stress.

[0021] The dosage form of the drug is any one of tablets, capsules, granules, oral liquids, sustained-release formulations, nano-formulations, or targeted colon-release formulations.

[0022] Fifthly, the present invention provides a quality control method for Ginkgo biloba extract or its preparations, wherein one or more of the following are used as indicator components: piperidine, hesperidin, and dihydroisorhamnoside, and their content in the Ginkgo biloba extract or its preparations is determined by high performance liquid chromatography or liquid chromatography-mass spectrometry.

[0023] Preferably, the total taxane content is 0.5‰ to 1.0‰, the total hesperidin content is 2‰ to 5‰, and the total dihydroisorhamnoside content is 0.1‰ to 0.4‰.

[0024] In the high-performance liquid chromatography method, the detection wavelength of the ultraviolet detector is 360 nm; the mobile phase is a mixed solvent of aqueous and organic phases; the liquid chromatography-mass spectrometry technique adopts negative ion mode to perform multiple reaction monitoring on taurine, hesperidin and dihydroisorhamnoside.

[0025] Preferably, the quality control method further includes a step of acid hydrolysis pretreatment of the ginkgo leaf extract or its preparation.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a novel enzymatic method for the preparation of dihydroisorhamnine, which for the first time proposes using piperidine as a substrate, S-adenosylmethionine as a methyl donor, and catechol-O-methyltransferase as a catalyst to prepare dihydroisorhamnine via an enzymatic methylation reaction. This method offers a completely new preparation route for dihydroisorhamnine, featuring mild reaction conditions, simple operation, and high product specificity, overcoming the shortcomings of traditional plant extraction methods such as cumbersome steps, low yield, and poor purity.

[0027] The dihydroisorhamnoside product directly obtained by the enzymatic preparation method provided by this invention can achieve a purity of over 99% after preparative HPLC purification. 1 The structure was confirmed by H NMR and MS spectral data, providing a well-defined and reliable standard and active pharmaceutical ingredient for subsequent pharmacological activity studies, pharmacokinetic experiments and drug development.

[0028] For the first time, the oral bioavailability characteristics of dihydroflavonols in Ginkgo biloba extract have been systematically revealed: total piperidin 26.4%, total hesperidin 22.2%, and total dihydroisorhamnoside 386%. In particular, the total exposure of dihydroisorhamnoside reached 386% of that of intravenous administration, far exceeding 100%, overturning the traditional understanding in this field that "flavonoids have low oral bioavailability" and confirming a dual high-exposure mechanism of "direct absorption + prodrug conversion".

[0029] The pharmaceutical composition provided by this invention combines three dihydroflavonols with high oral bioavailability to exert a synergistic effect, providing a formulation basis for the prevention and treatment of cardiovascular and cerebrovascular diseases with a combination of multiple active ingredients.

[0030] The quality control method for Ginkgo biloba extract or its preparations provided by this invention uses one or more of the following as indicator components: piperidin, hesperidin, and dihydroisorhamnoside, and determines their content by HPLC or LC-MS / MS. The technical advantage of this quality control method is that it overcomes the shortcomings of existing Ginkgo biloba quality control systems that only use flavonol glycosides and terpene lactones as indicators, incorporating highly bioavailable active ingredients into the quality control process, thus enabling quality evaluation to better reflect the product's in vivo exposure characteristics and true efficacy. Attached Figure Description

[0031] Figure 1 The chemical structural formulas of piperidine, hesperidin and dihydroisorhamnoside are shown. Figure 2 A schematic diagram illustrating the reaction principle of COMT enzyme-catalyzed methylation of taurine to dihydroisorhamnetin; Figure 3 The time-matter curves for the conversion of taxifolin (TXF) to dihydroisorhamnetin (DHISR) by COMT enzyme are shown. In the figure, A is the peak area of ​​the substrate taxifolin as a function of time (substrate consumption curve), and B is the peak area of ​​the product dihydroisorhamnetin as a function of time (product formation curve). Figure 4 The figures show the progress curves of the COMT enzyme-catalyzed reaction at different SAM concentrations. In the figure, A is the consumption curve of the substrate xatuspin (TXF) over time, and B is the formation curve of the product dihydroisorhamnoside (DHISR) over time.

[0032] Figure 5 HPLC chromatogram of dihydroisorhamnoside preparative purification; Figure 6 dihydroisorhamnoside 1 H NMR spectrum; Figure 7 The image shows the LC-MS / MS mass spectrum of dihydroisorhamnoside. Figure 8 The following are plasma concentration-time curves of three dihydroflavonols after oral administration of GBE50 to mice. In the figure, A is the concentration-time curve of total taurine in plasma after oral administration of GBE50 to mice, B is the concentration-time curve of total hesperidin in plasma after oral administration of GBE50 to mice, and C is the concentration-time curve of total dihydroisorhamnoside in plasma after oral administration of GBE50 to mice. Figure 9 The bar chart shows the oral bioavailability of three dihydroflavonols and flavonols. t-QCT, t-KMF, and t-ISR refer to total quercetin, total kaempferol, and total isorhamnetin, respectively, while t-TXF, t-AMD, and t-DHISR refer to total piperidin, total hesperidin, and total dihydroisorhamnetin, respectively.

[0033] Figure 10 The LC-MS / MS chromatograms for identifying piperidin, hesperidin, and dihydroisorhamnoside in Ginkgo biloba extract (GBE50) are shown. (A)–(D) are the total ion chromatograms of GBE50 before acid hydrolysis and the extracted ion chromatograms of the three dihydroflavonols; (E)–(H) are the total ion chromatograms of GBE50 after acid hydrolysis and the extracted ion chromatograms of the three dihydroflavonols. The responses of each component were significantly enhanced after acid hydrolysis. (I) 50% methanol blank solution; (J) piperidin reference standard (m / z 303.0 / 285.0); (K) hesperidin reference standard (m / z 287.0 / 259.0); (L) enzymatically synthesized product dihydroisorhamnoside (m / z 317.0 / 125.0). Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. 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 should fall within the scope of protection of the present invention.

[0035] Taxifolin (CAS No.: 480-18-2) reference standard was purchased from the National Institutes for Food and Drug Control (purity ≥98%), and can also be purchased from suppliers such as Sigma-Aldrich (USA) and Shanghai Aladdin Biochemical Technology Co., Ltd. through commercial channels. The taxifolin substrate used in the enzymatic synthesis reaction also comes from the above source and does not require further purification.

[0036] Aromadendrin (CAS No.: 480-20-6) reference standard was purchased from Shanghai Tongtian Biotechnology Co., Ltd. (purity ≥98%).

[0037] S-Adenosylmethionine (SAM, CAS No.: 29908-03-0) was purchased from Sigma-Aldrich (purity ≥98%), dissolved in ice-cold tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4), and then aliquoted. Store at 80℃ for later use, and avoid repeated freeze-thaw cycles.

[0038] Catechol-O-methyltransferase (COMT) was derived from rat hepatic cytoplasm (RLC) and prepared in-house according to the method described in Example 1. Male Sprague-Dawley rats used for RLC preparation were purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd., weighing 200–250 g and SPF grade. The animal experimental protocol was approved by the institution's animal ethics committee before implementation.

[0039] Ginkgo biloba extract (GBE50) was purchased from Shanghai Xingling Pharmaceutical Co., Ltd., with a total flavonol glycoside content ≥24% and a terpene lactone content ≥6%. Shuxuening injection was purchased from Langzhi Group Wanrong Pharmaceutical Co., Ltd., with a specification of 10 mL / vial (containing 8.4 mg of flavonol glycosides and 1.4 mg of terpene lactones).

[0040] Laboratory animals (male ICR mice, weighing 18–22 g) were purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd., and housed in a barrier-controlled animal facility at a temperature of 22±2℃ and a humidity of 50%±10%, with a 12-hour light / 12-hour dark cycle and free access to food and water. All animal experiments complied with the relevant regulations of the laboratory animal management and use committee of the institution.

[0041] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1: Enzymatic preparation of dihydroisorhamnoside (1) Preparation of rat hepatic cytoplasm (RLC) Male Sprague-Dawley rats (200-250 g) were sacrificed, and their livers were quickly removed and rinsed in ice-cold physiological saline until no blood was visible. The liver tissue was minced and 3 times its volume (w / v) of pre-chilled homogenization buffer (50 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM DTT, pre-chilled at 4°C) was added, and homogenized in an ice bath. The homogenate was centrifuged at 9,000 × g for 20 min at 4°C, and the supernatant was collected. This supernatant was then centrifuged at 105,000 × g for 60 min at 4°C. The resulting supernatant was the rat hepatic cytosol (RLC). The RLC was aliquoted into centrifuge tubes. Store at 80℃ for later use. Protein concentration in RLC was determined using the Bradford method, with bovine serum albumin as a standard.

[0042] (2) Enzymatic synthesis reaction The total volume of the reaction system was 10 mL, and its composition was as follows: piperidine (substrate): 80 μM, RLC protein prepared in step (1): 1 mg / mL, S-adenosylmethionine (SAM, methyl donor): 1 mM, Tris-HCl buffer (pH 7.4): 50 mM, DTT: 1 mM, MgCl₂ 2: 2 mM. Add the above components sequentially to the reaction vessel and mix thoroughly. Incubate the reaction mixture in a 37°C air bath with shaking for 4 h.

[0043] The chemical structural formulas of piperidine, hesperidin, and dihydroisorhamnoside are attached. Figure 1 As shown. Among them, taxifolin B ring has a 3′,4′-dihydroxy structure (ortho-dihydroxy structure), which is a natural substrate of catechol-O-methyltransferase (COMT); dihydroisorhamnetin is a product of methylation of the 3′-hydroxyl group of taxifolin B ring; aromadendrin is an analog of taxifolin B ring lacking an ortho-hydroxyl group.

[0044] The reaction principle of COMT enzyme-catalyzed methylation of taurine to dihydroisorhamnoside is as follows: Figure 2 As shown, in the presence of COMT enzyme and the methyl donor S-adenosylmethionine (SAM), piperidin can be selectively 3′-O-methylated to generate dihydroisorhamnoin. This reaction is carried out under mild conditions (37°C, pH 7.4), with high regioselectivity (preferring 3′-O-methylation over 4′-O-methylation) and few byproducts.

[0045] (3) Monitoring of reaction process and verification of enzyme catalytic activity Samples were taken at different time points (5, 15, 30, 60, 90, 120, 180 min), the reaction was terminated with ethyl acetate, and the samples were extracted by shaking (1500 rpm) for 5 min, centrifuged at 13000 rpm for 7 min, and the supernatant was collected and dried under nitrogen at room temperature. The nitrogen-dried sample tubes containing the analyte were reconstituted with acetonitrile, and the resulting samples were extracted by shaking (1500 rpm) for 5 min, centrifuged at 13000 rpm for 7 min, and the supernatant was analyzed by LC-MS / MS to determine the peak areas of the substrate xacutanein (TXF) and the product dihydroisorhamnoside (DHISR). Figure 3 The results demonstrate that COMT can efficiently catalyze this methylation reaction, with a substrate conversion rate of approximately 99.3% after 120 min.

[0046] To further investigate the effect of the methyl donor SAM concentration on catalytic efficiency, this invention also set up reaction systems with different SAM concentrations (100, 200, 500, and 1000 μM), with other conditions remaining the same (80 μM taurine, 1 mg / mL rat liver cytosol, pH 7.4, 37℃). Samples were taken at 0, 60, 120, and 180 min to determine the peak areas of the substrate and product. The results are attached. Figure 4 As shown, the peak area of ​​the substrate TXF decreases with reaction time, and the rate of decrease accelerates significantly with increasing SAM concentration. The highest substrate conversion rate was observed within 120 min when the SAM concentration was 1000 μM, indicating that a higher SAM concentration is beneficial for the methylation reaction. This result further validates the SAM-dependent catalytic characteristics of COMT. Considering both catalytic efficiency and cost, a SAM concentration of 1 mM is preferred.

[0047] (4) Reaction termination and product enrichment After the enzymatic synthesis reaction was completed, 3 times the volume of ethyl acetate was added to terminate the reaction. The reaction solution was extracted three times with ethyl acetate, shaken, and centrifuged at 12,000×g for 10 min at 4℃. The supernatant was collected, concentrated under reduced pressure (40℃ water bath, rotary evaporation), and evaporated to dryness. The residue was redissolved in acetonitrile.

[0048] Preparative high-performance liquid chromatography (prep-HPLC) purification: The chromatographic conditions were as follows: Column: Shiseido Capcell Pak C18 AQ S3 column (5 μm particle size, 250 mm × 10 mm inner diameter); Mobile phase: Phase A was acetonitrile-water (volume ratio 1:99, containing 0.4 mM formic acid); Phase B was pure acetonitrile; Gradient elution program: 0–5 min, the proportion of Phase B linearly increased from 40% to 50%; 5–9 min, the proportion of Phase B linearly decreased from 50% to 40%; Flow rate: 3 mL / min; Detection wavelength: 360 nm; Injection volume: 500 μL.

[0049] The HPLC chromatogram of dihydroisorhamnoside preparative purification is shown below. Figure 5 As shown, the chromatographic peak with a retention time of approximately 6.76 min is the substrate piperidine (TXF), and the chromatographic peak with a retention time of approximately 8.05 min is the target product dihydroisorhamnoside (DHISR). The product peak was well separated from the substrate peak and adjacent impurity peaks, and the fraction with a retention time of approximately 8.05 min was collected. The collected solution was concentrated under reduced pressure and freeze-dried. The product was heated at 50℃ and 0.1 mbar to obtain a white powdery solid. The purity of the product was determined to be 99.2% and the yield (based on piperidine) was 65.3% by HPLC area normalization.

[0050] Example 2: Structural identification of the enzymatically synthesized product The off-white powdery solid obtained in Example 1 was subjected to 1H NMR spectroscopy (NMR spectroscopy). 1 ¹H NMR and mass spectrometry (MS) analysis.

[0051] 1 H NMR measurement conditions: A 300 MHz NMR spectrometer was used, with CD3OD as the solvent. The measurement results are shown in [reference needed]. Figure 6 And Table 1: δ 4.57 (1H, d, J=11.6 Hz, H-2), 5.89 (1H, d, J=2.1 Hz, H-3), 5.89 (1H, d, J=2.1 Hz, H-6), 5.92 (1H, d, J=2.1 Hz, H-8), 7.11 (1H, d, J=1.8 Hz,H-2′), 6.83 (1H, d, J=8.1 Hz, H-5′), 6.97 (1H, dd, J=8.1, 1.8 Hz, H-6′), 3.88(3H, s, OCH3).

[0052] LC-MS / MS determination conditions: electrospray ionization (ESI) source, negative ion mode. See [link to results]. Figure 7 And Table 1: m / z 317 [MH] - Major fragment ion: m / z 289 [MH-CO] m / z 125 [1,3,5-trihydroxybenzene] The synthesized product was confirmed to be dihydroisorhamnetin.

[0053] Table 1: Liquid chromatography-tandem mass spectrometry (LC / MS / MS) and proton nuclear magnetic resonance (NMR) of the synthesized product dihydroisorhamnoside 1 HNMR data

[0054] From the above 1 1H NMR and MS data confirmed that the synthesized product was dihydroisorhamnetin.

[0055] Example 3: Pharmacokinetic Study of Three Dihydroflavonols in Mice (1) Laboratory animals Male ICR mice, weighing 18–22 g, were housed in an environment with a temperature of 22±2℃, humidity of 50%±10%, and a 12-hour light / 12-hour dark cycle, with free access to food and water. They were fasted for 12 hours before the experiment, but water was allowed.

[0056] (2) Dosing regimen Mice were randomly divided into an oral administration group and an intravenous administration group.

[0057] Oral administration group (n=6 / time point): Ginkgo biloba extract GBE50 (180 mg / kg) was administered by gavage. GBE50 was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) solution.

[0058] Intravenous group (n=3 / time point): Shuxuening injection (8 mL / kg) was administered via tail vein, diluted with 5% glucose injection.

[0059] (3) Blood sample collection Blood samples were collected from the oral administration group at 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours before and after administration.

[0060] Blood samples were collected from the intravenous group at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 hours before and after drug administration.

[0061] Blood was collected from the posterior orbital venous plexus and deposited into heparinized centrifuge tubes. The blood samples were anticoagulated with heparin and centrifuged at 4°C, 3000×g for 10 min to separate the plasma. Store at 70℃ for later use.

[0062] (4) Sample processing Add 50 µL of an aqueous solution containing 20 mM ascorbic acid and 4 M hydrochloric acid, shake at 1500 rpm for 3 min, and then hydrolyze by shaking in an 80 °C water bath for 30 min. After cooling the hydrolysate, add 1 mL of ethyl acetate, vortex for 10 min, and centrifuge at 13,000 rpm for 7 min at 4 °C. Collect the upper organic phase. Dry the organic phase under a nitrogen stream, redissolve the residue in 40 μL of pure acetonitrile, and inject 20 μL for analysis.

[0063] (5) LC-MS / MS analysis conditions Chromatographic column: Phenomenex Gemini C18 column (50 mm × 2.0 mm, 5 μm). Mobile phase: Phase A was acetonitrile-water (v / v 1:99, containing 0.2 mM formic acid), and Phase B was acetonitrile-water (v / v 90:10, containing 0.2 mM formic acid). Gradient elution program: 0–3 min, 5% B → 98% B; 3–6 min, 98% B hold; 6–6.1 min, 98% B → 5% B; 6.1–10 min, 5% B hold. Flow rate: 0.3 mL / min.

[0064] Mass spectrometry conditions: electrospray ionization source, negative ion mode, multiple reaction monitoring (SRM) mode. The monitoring ion pairs for each compound are as follows: Taxodiacetic acid (total taxodiacetic acid, t-TXF): m / z 303 → 125; Hesperidin (total hesperidin, t-AMD): m / z 287 → 125; Dihydroisorhamnoside (total dihydroisorhamnoside, t-DHISR): m / z 317 → 125.

[0065] (6) Calculation of pharmacokinetic parameters Pharmacokinetic parameters were calculated using Kinetica 2000 (version 3.0, Philadelphia, PA) with a non-compartmental model selected. C max The measured maximum plasma drug concentration is represented by the area under the plasma drug concentration-time curve (AUC). 0-t Calculated using the trapezoidal method, T max This refers to the time to peak plasma concentration after drug administration. Oral bioavailability F is calculated using the following formula: F = (AUC p.o. × Dose i.v. ) / (AUC i.v. × Dose p.o. ) × 100%, where, F Oral bioavailability represents the percentage of a drug that is absorbed into the systemic circulation after oral administration. AUC (Area Under the Curve) reflects the total exposure of the drug into the systemic circulation. p.o. Area under the plasma concentration-time curve (per os, oral administration) for the oral administration group; AUC i.v. The area under the plasma concentration-time curve for the intravenously administered group (intravenous injection). Intravenously administered drugs are considered to have entered 100% systemic circulation and are used as a reference for calculating absolute bioavailability. p.o. This is the oral dosage; Dose i.v. This is the dosage for intravenous administration.

[0066] (7) Experimental results The main pharmacokinetic parameters of the three dihydroflavonols in mice are shown in Table 2, and the plasma concentration-time curves are attached. Figure 8 (A: Total taurine, B: Total hesperidin, C: Total dihydroisorhamnoside), see attached bar chart for bioavailability comparison. Figure 9 .

[0067] Table 2: Major pharmacokinetic parameters of three dihydroflavonols in mice

[0068] Depend on Figure 8 and Figure 9 As shown in Table 2, the three dihydroflavonols described in this invention have high bioavailability, especially the total dihydroisorhamnoside. F The value reached as high as 386%, far exceeding 100%. This confirms the synergistic amplification effect of its dual sources (dihydroisorhamnoside and dihydroisorhamnoside derived from the COMT-catalyzed conversion of taurine in vivo).

[0069] Example 4: Optimization of Enzymatic Synthesis Reaction Conditions (1) Optimization of substrate concentration and SAM concentration Enzymatic synthesis reactions were performed using different substrate concentrations of taxonomycin (50, 80, 100 μM) and SAM concentrations (0.5, 1.0, 2.0 mM), with other conditions the same as in Example 1. After 2 hours of reaction, the relative yield of dihydroisorhamnoside was calculated based on the chromatographic peak area (with the 80 μM taxonomycin + 1.0 mM SAM group as 100%). The results are shown in Table 3.

[0070] Table 3: Relative yields at different substrate and SAM concentrations

[0071] The results showed that the highest yield of dihydroisorhamnoside was achieved when the concentration of taxine was 80–100 μM and the concentration of SAM was 1–2 mM. Considering both cost and efficiency, the optimal concentrations were 80 μM taxine and 1 mM SAM.

[0072] (2) pH optimization With fixed concentrations of 80 μM for taurine and 1 mM for SAM, reactions were carried out for 4 hours at different pH values ​​(6.0, 6.5, 7.0, 7.4, and 8.0). The relative yield was calculated with the pH 7.4 group as 100%. The results are shown in Table 4.

[0073] Table 4: Effect of different pH values ​​on yield (37℃)

[0074] (3) Temperature optimization With fixed concentrations of 80 μM for taurine and 1 mM for SAM, the reactions were carried out for 4 hours at different temperatures (25, 30, 37, 42, and 50 °C). The relative yield was calculated with the yield of the 37 °C group as 100%. The results are shown in Table 5.

[0075] Table 5: Effect of different temperatures on yield (pH 7.4)

[0076] The results showed that the optimal reaction conditions were pH 7.0–7.4 and temperature 37 °C.

[0077] Example 5: Pharmaceutical composition (tablets) containing dihydroflavonols Take 10 mg of dihydroisorhamnoside prepared in Example 1, and mix it with 20 mg of taurine (purchased from the National Institutes for Food and Drug Control) and 15 mg of hesperidin (purchased from Sigma-Aldrich). Add 80 mg of microcrystalline cellulose, 10 mg of crospovidone, and 2 mg of magnesium stearate, and mix well. Compress the mixture into tablets using a single-punch tablet press via direct compression. Each tablet weighs approximately 137 mg and contains 10 mg of dihydroisorhamnoside, 20 mg of taurine, and 15 mg of hesperidin.

[0078] The pharmaceutical composition provided in this embodiment is based on a synergistic high-exposure mechanism of "direct absorption + prodrug conversion": in vivo, piperidin can be partially converted into dihydroisorhamnoside by COMT enzyme catalysis, which further significantly increases the systemic exposure level of the latter, with a mass ratio of 1:2.

[0079] Example 6: Determination of the content of three dihydroflavonols in Ginkgo biloba leaf extract (1) Preparation of test solution Accurately weigh approximately 40 mg of Ginkgo biloba extract GBE50 and place it in a round-bottom flask. Add 25 mL of a methanol-25% hydrochloric acid solution (volume ratio 4:1) and mix well. Heat under reflux in an 80°C water bath for 30 minutes, then rapidly cool to room temperature. Transfer the hydrolysate to a 50 mL volumetric flask, dilute to the mark with 50% methanol, and mix well. Filter the above solution through a 0.45 μm filter membrane, and use the filtrate as the test solution.

[0080] (2) Preparation of reference solution Accurately weigh appropriate amounts of taxonomyl, hesperidin, and dihydroisorhamnoside reference standards, dissolve and dilute them in methanol to prepare mixed reference solutions of various concentrations. The concentration ranges of each reference standard are: taxonomyl 0.05~5 μg / mL, hesperidin 0.1~10 μg / mL, and dihydroisorhamnoside 0.02~2 μg / mL.

[0081] (3) HPLC-UV analysis conditions Chromatographic column: Waters Symmetry C18 column (150 mm × 4.6 mm, 5 μm). Column temperature: 20℃. Mobile phase: water-methanol (50:50 v / v, containing 2 mM phosphoric acid), isocratic elution. Flow rate: 1.2 mL / min. Detection wavelength: 360 nm. Injection volume: 10 μL.

[0082] (4) Mass spectrometry conditions (LC-MS / MS method, used for higher sensitivity or structural confirmation) Ion source: Electrospray ionization (ESI) source, negative ion mode; Monitored ion pairs: Taxodiacein m / z 303→125; Hesperidin m / z 287→125; Dihydroisorhamnoside m / z 317→125; Collision energy: 27 30 eV.

[0083] (5) Content determination results The LC-MS / MS chromatograms for identifying piperidin, hesperidin, and dihydroisorhamnoside in Ginkgo biloba leaf extract are attached. Figure 10 As shown, under the above conditions, the retention times of the chromatographic peaks of taxine, hesperidin, and dihydroisorhamnoside were 4.6 min, 5.2 min, and 5.3 min, respectively. The contents of each component were calculated using the external standard method, and the results (based on the dried extract) were: total taxine 0.76‰ (w / w), total hesperidin 3.2‰ (w / w), and total dihydroisorhamnoside 0.24‰ (w / w). The contents of the three components in this batch of samples were all within the recommended range of this invention (total taxine 0.5‰~1.0‰, total hesperidin 2‰~5‰, and total dihydroisorhamnoside 0.1‰~0.4‰), indicating a high exposure potential after oral administration.

[0084] Example 7: Comparison of in vitro metabolic stability of three dihydroflavonols Taxostatin, hesperidin, and dihydroisorhamnoside were co-incubated with mouse liver microsomes (0.5 mg protein / mL) at 37°C. Samples were taken at 0, 5, 15, 30, and 60 minutes to determine the remaining content. With 0 minutes as 100%, the remaining percentage at each time point was calculated. The results are shown in Table 6. Table 6: Content determination at different incubation times

[0085] The above results indicate that dihydroisorhamnoside has significantly better in vitro metabolic stability than piperidin and hesperidin, with a residual content still above 50% after 60 minutes. Higher metabolic stability corresponds to higher systemic exposure levels.

[0086] Example 8: Protective effect of three dihydroflavonols against H2O2-induced H9c2 cardiomyocyte injury Rat H9c2 cardiomyocytes were seeded in 96-well plates and cultured to 80% confluence. Cells were pretreated for 24 hours with different concentrations of piperidin, hesperidin, or dihydroisorhamnoside (0.1, 1, 10, 50 μM), followed by oxidative damage induced by 200 μM H2O2, and cultured for another 24 hours. Cell viability was determined using the MTT assay, and the results are shown in Table 7.

[0087] Table 7: Cell viability of each treatment group

[0088] The above results indicate that piperidin, hesperidin, and dihydroisorhamnoside can all protect H9c2 cardiomyocytes from H2O2-induced oxidative damage in a concentration-dependent manner, with dihydroisorhamnoside showing the most significant protective effect. This suggests that dihydroisorhamnoside possesses superior cardiomyocyte protective activity. These results, along with dihydroisorhamnoside's highest oral bioavailability (386%) and optimal metabolic stability, support its potential as a therapeutic agent for cardiovascular and cerebrovascular diseases.

[0089] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for the enzymatic production of dihydroisoquercetin, characterized in that, Using piperidine as a substrate and S-adenosylmethionine as a methyl donor, a methylation reaction was carried out under the catalysis of catechol-O-methyltransferase, and the resulting product was purified to obtain dihydroisorhamnoside.

2. The enzymatic method of preparing dihydroisoquercetin according to claim 1, characterized by, The catechol-O-methyltransferase is derived from recombinant human COMT, recombinant rat COMT, or animal liver fluid.

3. The enzymatic method of preparing dihydroisoquercetin according to claim 1, characterized by, The concentration of taurine in the reaction system was 50–100 μM, the concentration of S-adenosylmethionine was 0.5–2 mM, the reaction temperature was 35–40 ℃, and the pH of the reaction system was 7.0–8.

0.

4. Dihydroisorhamnoside obtained by the enzymatic preparation method according to any one of claims 1 to 3.

5. A pharmaceutical composition, characterized by, It comprises dihydroisorhamnoside as described in claim 4, as well as taurine and / or hesperidin, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein, The mass ratio of dihydroisorhamnoside to taurine is 1:10 to 10:

1.

7. The use of the dihydroisorhamnetin of claim 4 or the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament for the prevention and / or treatment of cardiovascular and cerebrovascular diseases.

8. Use according to claim 7, characterized in that, The cardiovascular and cerebrovascular diseases mentioned are atherosclerosis, myocardial ischemia, myocardial infarction, hypertension, myocardial hypertrophy, heart failure or coronary heart disease, ischemic stroke, sequelae of cerebral hemorrhage, transient ischemic attack, vascular dementia, cerebral arteriosclerosis or chronic cerebral ischemia.

9. A method for quality control of a ginkgo biloba leaf extract or a preparation thereof, characterized by, One or more of the following ingredients—taperitonein, hesperidin, and dihydroisorhamnoside—were used as indicator components, and their content in Ginkgo biloba extract or its preparations was determined by high performance liquid chromatography or liquid chromatography-mass spectrometry.

10. The quality control method according to claim 9, characterized in that, The total fructose content is 0.5‰ to 1.0‰, the total hesperidin content is 2‰ to 5‰, and the total dihydroisorhamnoside content is 0.1‰ to 0.4‰.

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