A phenolic acid compound, its preparation and application

Abstract

The present application relates to a kind of phenolic acid compound and its preparation and application, the structural formula of the phenolic acid compound is: it is confirmed by in vitro pharmacological experiment, the phenolic acid compound has good alpha-glucosidase inhibiting activity, wherein IC 50 Value is 4.022±0.199mmol / L, can be used for preparing alpha-glucosidase inhibitor drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a phenolic acid compound and its preparation and application. Background Technology

[0002] Diabetes mellitus is a group of multifactorial metabolic diseases characterized by chronic hyperglycemia, primarily including type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Type 2 diabetes mellitus (T2DM) is a disease typically associated with a range of metabolic disorders and cardiovascular risk factors, such as obesity, insulin resistance, dyslipidemia, hypertension, atherosclerosis, prethrombotic states, and endothelial dysfunction, collectively known as metabolic syndrome (MS). To date, T2DM has been treated with specific monomodal compounds that control hyperglycemia by increasing insulin secretion (secreting agents) or insulin action (insulin sensitizers) or slowing glucose uptake. Long-term hyperglycemia in diabetic patients can cause chronic damage and dysfunction to various tissues in the body, particularly the eyes, kidneys, heart, blood vessels, and nerves, seriously endangering human health.

[0003] Currently, acarbose and voglibose, commonly used in clinical practice, have shown good efficacy in delaying the progression of type 2 diabetes and increasing the likelihood of impaired glucose tolerance returning to normal. However, side effects still exist, including digestive problems such as bloating, diarrhea, and vomiting. A small number of patients may experience adverse reactions such as hypoglycemia, hypertension, retinopathy, and neuropathy. Therefore, finding new alpha-glucosidase inhibitors is an important task in the field of drug development, requiring continuous scientific research and innovation to provide better drug options and personalized treatment plans.

[0004] *Castanopsis mekongensis* is a tree that can reach up to 25 meters in height and 1 meter in diameter at breast height. Its branches are grayish-brown, and the one-year-old branches, petioles, leaf undersides, and inflorescence axes are densely covered with short hairs. Two-year-old branches have fewer hairs. It is abundant and mainly found in southern and southwestern Yunnan province. It grows in evergreen broad-leaved forests in mountainous areas below approximately 2000 meters in altitude. The wood of this species also belongs to the *Castanopsis* group, but its detailed structure is unknown. Currently, there are no reports on the chemical composition of *Castanopsis mekongensis*, and even fewer reports on components with α-glucosidase inhibitory activity. Therefore, it is necessary to provide a method for preparing and applying α-glucosidase inhibitory active ingredients from *Castanopsis mekongensis*, offering a new option for hypoglycemic drugs and addressing the shortcomings of existing technologies. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a phenolic acid compound, its preparation, and its application. The objective is to provide a phenolic acid compound with good α-glucosidase inhibitory activity and its preparation method.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] Firstly, a phenolic acid compound, wherein the structural formula of the phenolic acid compound is as follows:

[0008]

[0009] The novel phenolic acid component with α-glucosidase inhibitory activity provided by the present invention was first isolated by the inventors from the leaves of the plant Castanopsis mekongensis. This novel phenolic acid component can be named mekongensis A.

[0010] The beneficial effects of this invention are: in vitro pharmacological experiments have confirmed that the phenolic acid compounds have good α-glucosidase inhibitory activity, wherein IC50... 50 The value was 4.022±0.199mmol / L, which can be used to prepare α-glucosidase inhibitor drugs.

[0011] Secondly, a method for preparing a phenolic acid compound includes the following steps:

[0012] (1) After crushing the Mekong River cone, extract it to obtain the total extract;

[0013] (2) The total extract is defatted to obtain a defatted aqueous extract;

[0014] (3) The defatted water extract was sequentially separated by first gel column chromatography, resin column chromatography, second gel column chromatography and chromatographic column chromatography. Each chromatography was performed by gradient elution with a mixture of methanol and water to obtain phenolic acid compounds.

[0015] The beneficial effects of adopting the above scheme are: the above extraction method is simple, easy to operate, and environmentally friendly; and the Mekong River cone is used as raw material for extraction, which is rich in resources and has good potential economic benefits. In addition, the new hypoglycemic component mekongensis A is stable and easy to store; its α-glucosidase inhibitory activity is good, and it is expected to be used as a lead compound to develop new hypoglycemic drugs.

[0016] Further, the specific extraction method in step (1) is as follows: extraction is performed 2-3 times with an aqueous solution of alcohol compounds, each extraction lasting 6-8 days. The filtrates are combined and then distilled under reduced pressure at a constant temperature to obtain the total extract. The volume ratio of alcohol compounds to water in the aqueous solution is (70-90):(30-10). The extraction method includes reflux extraction, continuous reflux extraction, ultrasonic extraction, percolation extraction, or maceration extraction, etc. The particle size of the pulverized material is 20-60 mesh.

[0017] The beneficial effects of adopting the above-mentioned further scheme are: the above extraction method is more conducive to the extraction of compound mekongensis A and maintains its original biological activity; the above-mentioned particle size is more conducive to the extraction of phenolic acid compounds.

[0018] Furthermore, the alcohol compound includes any one or a mixture of at least two of ethanol, methanol, and propanol; the isothermal vacuum distillation temperature is 45±5℃, and the pressure is 50KPa~200KPa. For example, a rotary evaporator is used for vacuum distillation.

[0019] Furthermore, the reagent used for degreasing in step (2) is petroleum ether, the volume ratio of the total extract to the petroleum ether is 1:0.3-0.4, and the degreasing extraction is performed 2-3 times.

[0020] The beneficial effects of adopting the above-mentioned further scheme are: petroleum ether extraction can effectively remove impurities such as lipids and pigments, which is beneficial to the next step of separation.

[0021] Further, in step (3), the first gel column and the second gel column are Sephadex LH-20; the resin column is Diaion HP20SS small-pore resin; and the chromatographic column is Chromatorex C 18 Chromatographic column; the volume ratio of the methanol and water mixture is 0% to 100%.

[0022] The beneficial effect of adopting the above-mentioned further scheme is that the above-mentioned chromatography column material can better separate phenolic acid compounds from other compounds, which is beneficial to the purification of subsequent steps.

[0023] Furthermore, the Mekong Cone includes at least one of the following: dried or fresh Mekong Cone leaves, Mekong Cone branches, Mekong Cone stems, and Mekong Cone bark.

[0024] Thirdly, a pharmaceutical salt of a phenolic acid compound, wherein the pharmaceutical salt is a salt synthesized from the phenolic acid compound.

[0025] Fourthly, a pharmaceutically acceptable esterified derivative of a phenolic acid compound, wherein the pharmaceutically acceptable esterified derivative is prepared by esterification of the phenolic acid compound.

[0026] Fifthly, the application of the phenolic acid compounds in the preparation of hypoglycemic drugs.

[0027] It should be noted that the phenolic acid compounds, their derivatives, or their pharmaceutical salts provided by this invention can be combined with excipients or carriers permitted in formulations or pharmaceuticals to prepare drugs or pharmaceutical compositions with α-glucosidase inhibitory activity that can be used to treat diabetes. These drugs or pharmaceutical compositions can be in dosage forms such as tablets, granules, or capsules; they can also be formulated using methods known in the modern pharmaceutical industry, such as nano-, controlled-release, and sustained-release formulations.

[0028] Furthermore, the hypoglycemic drug comprises a pharmaceutically acceptable carrier and the phenolic acid compound and / or a derivative of the phenolic acid compound and / or a pharmaceutically acceptable salt of the phenolic acid compound. Attached Figure Description

[0029] Figure 1 The compound mekongensis A of this invention 1 H-NMR (500MHz, Methanol-d4) spectrum;

[0030] Figure 2 The compound mekongensis A of this invention 13 C-NMR (125MHz, Methanol-d4) spectrum;

[0031] Figure 3 The HSQC spectrum of the compound mekongensis A of this invention;

[0032] Figure 4 The HMBC spectrum of the compound mekongensis A of this invention;

[0033] Figure 5 The compound mekongensis A of this invention 1 H- 1 H COSY spectrum;

[0034] Figure 6 This is the HREIMS spectrum of the compound mekongensis A of this invention. Detailed Implementation

[0035] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0036] Example

[0037] 1. Extraction of the phenolic compound mekongensis A

[0038] 1.1 Plant source

[0039] The leaf samples used for extraction were collected in Gasa Town, Jinghong City, Yunnan Province in April 2022. They were identified by Kunming Caizhi Biotechnology Co., Ltd. as leaves of Castanopsis mekongensis. The specimens are preserved in the Guangxi Key Laboratory of Plant Functional Substances and Sustainable Utilization of Resources.

[0040] 1.2 Extraction and Separation

[0041] 5.5 kg of dried Mekong River cone leaves were pulverized into suitable sizes using a pulverizer. The leaves were extracted three times with 40 L of 70% methanol solution at room temperature, each extraction lasting 7 days. The extracts were combined and distilled under reduced pressure at 45 °C to obtain a total extract (909 g). This was extracted three times with petroleum ether to obtain 358 g of the petroleum ether fraction and 510 g of the aqueous fraction. The aqueous fraction was subjected to gradient elution using a Sephadex LH-20 gel column (9.5 cm × 32 cm) with methanol-water solution (0%–100%, V / V), specifically with eluents of 0%, 20%, 40%, 60%, 80%, and 100% methanol-water solution. The eluents were combined under the guidance of thin-layer chromatography to obtain seven fractions, Fr.1–Fr.7.

[0042] Fr.2 was eluted using a Diaion HP20SS column (4 cm × 32 cm) with a gradient elution of methanol-water solution (0%–100%, V / V), successively using 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v) methanol-water solution as eluent, yielding five fractions: Fr.21–Fr.25. Fr.2.2 was then eluted using a Sephadex LH-20 column (2.2 cm × 25 cm) with a gradient elution of 0%, 10%, 20%, 30%, 40%, 50%, 60%, and 70% (v / v) methanol-water solution as eluent, yielding Fr.222. Fr.222 was finally purified by Chromatorex C... 18 A column (2.2 cm × 25 cm) was eluted sequentially with methanol aqueous solutions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, and 70% by volume to obtain pure phenolic compound mekongensis A (15 mg).

[0043] 1.3 Identification of mekongensis A

[0044] Figure 1 It is compound mekongensis A. 1H-NMR (500MHz, Methanol-d4) spectrum; Figure 2 It is compound mekongensis A. 13 C-NMR (125MHz, Methanol-d4) spectrum; Figure 3 This is the HSQC spectrum of compound mekongensis A; Figure 4 This is the HMBC spectrum of compound mekongensis A; Figure 5 It is compound mekongensis A. 1 H- 1 H COSY spectrum; Figure 6 This is the HREIMS spectrum of compound mekongensis A. It is a white amorphous powder with a bright spot under 254 nm UV light, and reacts with 1% FeCl3-EtOH colorimetric reagent to form a blue-black spot. The HREIMS spectrum shows its quasi-molecular ion peak at m / z: 351.0358 [MH]. - (Calculated value 351.0292, C) 15 H 12 O 10 The molecular formula is C. 15 H 12 O 10 The degree of unsaturation is 10. 1 H-NMR (500MHz, Methanol-d4), 13 The C-NMR (125MHz, Methanol-d4) data are shown in Table 1 below.

[0045] Table 1. NMR data of compound mekongensis A (in methanol-d4)

[0046]

[0047]

[0048] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional and two-dimensional NMR data, the structural formula of the compound is derived as shown in formula (Ⅰ), and it is named mekongensis A;

[0049]

[0050] 2. Detection of α-glucosidase inhibitory activity

[0051] 2.1 Experimental Materials

[0052] α-Glucosidase (Sigma-Aldrich, Inc., USA); Acarbose (Shanghai Yuanye Biotechnology Co., Ltd.); p-nitrobenzene-α-D-glucopyranoside (pNPG, Shanghai Yuanye Biotechnology Co., Ltd.); Phosphate buffer (PBS, Beijing Solarbio Technology Co., Ltd.); SP-MAX3500FL multi-functional microplate reader (Shanghai Flash Spectrum Biotechnology Co., Ltd.); XS205 DualRange analytical balance (Mettler-Toledo Group, Zurich, Switzerland).

[0053] 2.2 Experimental Methods

[0054] The experiment was divided into a blank control group containing only the substrate, a blank group with added enzyme, a reaction control group with added sample, and a reaction group with added sample and enzyme. 20×PBS phosphate buffer was diluted 20-fold to prepare a solution with a concentration of 50 mmol / L. -1 Using this PBS solution as the solvent system, at 1 mmol·L -1 Using p-nitrophenyl-α-D-glucopyranoside PNPG as a substrate, 10 U·mg of the purchased product was used. -1 α-glucosidase was prepared to have an enzyme activity of 0.5 U·mL. -1 The enzyme solution was prepared and acarbose was used as a positive control. 50 μL of PBS phosphate buffer was added to each well of a 96-well plate. 40 μL of different concentrations of sample (8 mg / mL) were added to both the reaction group and the control group. -1 4mg mL -1 2mg mL -1 1 mg mL -1 0.5 mg mL -1 0.25 mg mL -1 0.125 mg / mL -1 and 0.0625 mg mL -1 Add equal volumes of PBS solution to the blank group and blank control group, add 10 μL of α-glucosidase to the blank group and reaction group, and add equal volumes of PBS solution to the blank control group and reaction control group respectively. Shake the 96-well plate thoroughly, mix well, incubate at 37°C for 5 min, and then add 20 μL of 1 mmol·L⁻¹ PBS solution. -1 The PNPG solution was reacted in a constant temperature incubator at 37℃ for 30 min, and the absorbance was measured at a wavelength of 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0055] The α-glucosidase inhibition rate was calculated using the formula: α-glucosidase inhibition rate = [1 - (Reaction group A - Reaction control group A) / (Blank group A - Blank control group A)] × 100%. The experiment was repeated three times in parallel, and the IC50 was calculated using GraphPad Prism software. 50 value.

[0056] 2.3 Activity Results

[0057] mekongensis A IC 50 It was 4.022 ± 0.199 mmol·L. -1 It exhibits strong α-glucosidase inhibitory activity, which can be effectively used in the development of hypoglycemic drugs, opening up new avenues for hypoglycemic drugs and new uses for Mekong River plants.

[0058] In summary, in vitro pharmacological experiments have confirmed that the phenolic acid compounds possess good α-glucosidase inhibitory activity, with IC50 showing a significant effect. 50 The value was 4.022±0.199mmol / L, which can be used to prepare α-glucosidase inhibitor drugs.

[0059] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A phenolic acid compound, characterized in that, The phenolic acid compound has a structural formula as shown in the following formula: 。 2. The method according to claim 1, wherein the phenolic acid compound is prepared by the method comprising the steps of: The method comprises the following steps: ​ (1) crushing the Meigong cone and then performing extraction to obtain total extract; (2) performing degreasing on the total extract to obtain a water extract after degreasing; (3) sequentially performing first gel column chromatography separation, resin column chromatography separation, second gel column chromatography separation and chromatography column chromatography separation on the water extract after degreasing, wherein each chromatography separation is performed by gradient elution using a mixture of methanol and water to obtain the phenolic acid compound.

3. The method for preparing a phenolic acid compound according to claim 2, characterized in that, In step (1), the extraction is performed by using an aqueous solution of an alcohol compound for 2-3 times, each time for 6-8 days, and then performing constant-temperature reduced-pressure distillation on the combined filtrate to obtain the total extract; the volume ratio of the alcohol compound to water in the aqueous solution of the alcohol compound is (70-90):(30-10).

4. The method for preparing a phenolic acid compound according to claim 3, characterized in that, The alcohol compound includes any one or a mixture of at least two of ethanol, methanol and propanol; the constant-temperature reduced-pressure distillation is performed at a temperature of 45±5℃ and a pressure of 50KPa-200KPa.

5. The method for preparing a phenolic acid compound according to claim 2, characterized in that, In step (2), the reagent used for degreasing is petroleum ether or n-hexane; when the reagent used for degreasing is petroleum ether, the volume ratio of the total extract to the petroleum ether is 1:0.3-0.4, and the degreasing extraction is performed for 2-3 times.

6. The method for preparing a phenolic acid compound according to claim 2, characterized in that, The first gel column and the second gel column in step (3) are Sephadex LH-20; the resin column is small-pore resin Diaion HP20SS; and the chromatographic column is Chromatorex C 18 a chromatographic column; and the volume ratio of the mixture of methanol and water is 0% to 100%.

7. The method for preparing a phenolic acid compound according to claim 2, characterized in that, The Meigong cone includes at least one of dry or fresh Meigong cone leaves, Meigong cone branches, Meigong cone stems and Meigong cone peels.

8. A pharmaceutically acceptable salt of a phenolic acid compound, characterized in that, The medicinal salt is a salt synthesized from the phenolic acid compound of claim 1.

9. Use of the phenolic acid compound of claim 1 in the preparation of a blood sugar-lowering drug.

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