A shikimic acid compound, its preparation method and application

Abstract

This invention relates to a shikimic acid compound, its preparation method, and its applications. This shikimic acid compound is stable and easy to store. In vitro pharmacological experiments have confirmed its stability (IC50). 50 The value was 0.0014 mol / L, indicating that it has good α-glucosidase inhibitory activity. This shikimic acid compound or its derivatives or its pharmaceutical salts can be used in hypoglycemic drugs and are expected to be developed as lead compounds for new treatments of diabetes.

Description

Technical Field

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

[0002] Diabetes mellitus is a metabolic disease caused by a deficiency in insulin secretion or impaired biological function, or both, due to genetic factors or environmental factors. Its main characteristic is hyperglycemia. Long-term hyperglycemia can lead to a series of complications, such as cardiovascular disease and kidney disease. It is the third leading cause of serious threats to human health after malignant tumors and cardiovascular and cerebrovascular diseases.

[0003] Currently, there are many medications for treating diabetes, such as insulin, biguanides, and sulfonylureas. While these drugs are very effective at controlling blood sugar, they usually have varying degrees of toxic side effects, and long-term use can cause damage to the patient's body and lead to drug resistance. Alpha-glucosidase inhibitors slow down glucose formation by inhibiting the activity of alpha-glucosidase, thereby reducing the incidence of diabetes and having a good preventive effect on diabetic complications. They are widely used as first-line drugs in the clinical treatment of type 2 diabetes. However, it has been reported that alpha-glucosidase inhibitors such as acarbose and their analogues can also produce some adverse side effects, such as gastrointestinal damage and allergic reactions. Therefore, finding new alpha-glucosidase inhibitors with mild or no adverse effects is very important for the treatment of diabetes. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a drug for treating diabetes. The shikimic acid compounds of this invention have good α-glucosidase inhibitory activity and can be used to treat diabetes.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: a shikimic acid compound, wherein the structural formula of the shikimic acid compound is:

[0006]

[0007] The beneficial effects of this invention are: the shikimic acid compound of this invention (named Orthacanthain A) is stable and easy to store, and in vitro pharmacological experiments have confirmed that it has an IC50 concentration of 1,000 mg / L. 50 The value was 0.0014 mol / L (IC50 value of the positive control acarbose). 50 The value was 0.0019 mol / L, indicating that it has good α-glucosidase inhibitory activity. This shikimic acid compound, Orthacanthain A, or its derivatives, or its pharmaceutical salts, can be used in hypoglycemic drugs and are expected to be developed as lead compounds for the treatment of diabetes.

[0008] To achieve the second objective mentioned above, this invention provides a method for preparing shikimic acid compounds, comprising:

[0009] After crushing and extracting the leaves of *Cephalotaxus fortunei*, the filtrate was defatted. The defatted filtrate was then separated sequentially by Sephadex LH-20 gel column chromatography, MCI gel CHP 20P column chromatography, Sephadex LH-20 gel column chromatography, Diaion HP20SS column chromatography, Toyopearl HW-40F column chromatography, and Chromatorex C18 column chromatography. The eluent for all separations was a mixture of methanol and water.

[0010] The beneficial effects are as follows: The potent α-glucosidase inhibitor obtained by extracting and separating the leaves of *Cinnamomum yuanjiangense* from the leaves of this invention is simple to prepare and easy to operate. Moreover, the extraction is carried out using leaves as raw materials, which are abundant and have good potential economic benefits. Furthermore, the obtained α-glucosidase inhibitor Othacanthain A is stable and easy to store, and has good α-glucosidase inhibitory activity. It is expected to be used as a lead compound to develop new drugs for the treatment of diabetes.

[0011] Furthermore, the eluent for the first Sephadex LH-20 gel column chromatography separation is a mixture of methanol and water with methanol volume contents of 0%, 20%, 40%, 60%, 80%, and 100% respectively.

[0012] The eluent for the MCI gel CHP 20P column chromatography separation is a mixture of methanol and water with methanol volume contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

[0013] The eluent for the second Sephadex LH-20 gel column chromatography separation was a mixture of methanol and water with methanol volume contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

[0014] The eluent for the Diaion HP20SS column chromatography separation is a mixture of methanol and water with methanol volume contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

[0015] The eluent for the Toyopearl HW-40F column chromatography separation is a mixture of methanol and water with methanol volume contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

[0016] The eluent for the Chromatorex C18 column chromatography separation is a mixture of methanol and water with a methanol volume content of 30%.

[0017] Furthermore, the extraction includes: extracting with methanol at a volume concentration of 65-75% 2-3 times, each extraction lasting 6-8 days, and then concentrating the filtrates under reduced pressure.

[0018] The reagent used for degreasing is petroleum ether.

[0019] To achieve the third objective mentioned above, this invention provides a medicinal salt of shikimic acid compounds, wherein the medicinal salt is a salt synthesized from the aforementioned shikimic acid compounds with organic acids and / or inorganic acids.

[0020] To achieve the fourth objective mentioned above, this invention provides a pharmaceutically acceptable esterified derivative of shikimic acid compounds, wherein the pharmaceutically acceptable esterified derivative is prepared by esterification reaction of the aforementioned shikimic acid compounds.

[0021] To achieve the fifth objective mentioned above, this invention provides the application of shikimic acid compounds in the preparation of hypoglycemic drugs.

[0022] It should be noted that the shikimic acid compound Orthacanthain A, or its derivatives, or its 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.

[0023] Furthermore, the hypoglycemic drug includes a pharmaceutically acceptable carrier and the shikimic acid compound and / or a derivative of the shikimic acid compound.

[0024] It should be noted that derivatives of shikimic acid compounds include pharmaceutically acceptable salts of shikimic acid compounds. Attached Figure Description

[0025] Figure 1 The structural formula of the shikimic acid compound (Orthacanthain A) of this invention is shown below;

[0026] Figure 2 The shikimic acid compound (Orthacanthain A) of the present invention 1 H-NMR spectrum;

[0027] Figure 3 The shikimic acid compound (Orthacanthain A) of the present invention 13 C-NMR spectrum;

[0028] Figure 4 The HSQC spectrum of the shikimic acid compound (Orthacanthain A) of the present invention is shown below.

[0029] Figure 5 The HMBC spectrum of the shikimic acid compound (Orthacanthain A) of the present invention is shown below.

[0030] Figure 6 The image shows the HREIMS spectrum of the shikimic acid compound (Orthacanthain A) of this invention. Detailed Implementation

[0031] 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.

[0032] Example

[0033] A shikimic acid compound (Orthacanthain A) and its preparation method, comprising:

[0034] (1) After crushing 5.5 kg of dried Yuanjiang Conifer leaves, it was extracted three times by soaking in 40 L of 70% methanol for 7 days each time. After filtration, the filtrates were combined and then concentrated under reduced pressure to obtain 2.4 L of aqueous solution. The aqueous solution was defatted four times with 2.5 L of petroleum ether. The defatted aqueous solution was then concentrated under reduced pressure to remove the petroleum ether.

[0035] (2) The aqueous solution after removing petroleum ether was separated by Sephadex LH-20 gel (10cm×50cm) column chromatography. It was eluted sequentially with 2L of a mixture of methanol and water at different concentrations (the volume fractions of methanol were 0%, 20%, 40%, 60%, 80%, and 100%). The same components were combined by TLC monitoring to obtain 7 fractions (Fr.1~7).

[0036] (3) Fr.4 (227.0 g) was separated by MCI gel CHP 20P (7.5 cm × 29.5 cm) column chromatography. The fractions were eluted sequentially with 1.5 L of a mixed solution of methanol and water of varying concentrations (the volume fractions of methanol were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). The same components were combined by TLC monitoring to obtain 11 fractions (Fr.4.1 to 4.11).

[0037] (4) Fr.4.1 (57.1 g) was separated by Sephadex LH-20 gel chromatography (5.0 cm × 25.0 cm). The fractions were eluted sequentially with 0.8 L of a mixture of methanol and water at different concentrations (the volume fractions of methanol were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). The same components were combined by TLC monitoring to obtain 10 fractions (Fr.4.1.1 to 4.1.10).

[0038] (5) Fr.4.1.8 (28.8 g) was separated by Diaion HP20SS (4.0 cm × 36.5 cm) column chromatography. The fractions were eluted sequentially with 0.8 L of a mixed solution of methanol and water of varying concentrations (the volume fractions of methanol were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). The same components were combined by TLC monitoring to obtain four fractions (Fr.4.1.8.1 to 4.1.8.4).

[0039] (6) Fr.4.1.8.1 (12.8 g) was separated by Toyopearl HW-40F (3.0 cm × 16.5 cm) column chromatography. The fractions were eluted sequentially with 0.2 L of a mixed solution of methanol and water of varying concentrations (where the volume fractions of methanol were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). The same components were combined by TLC monitoring to obtain 5 fractions (Fr.4.1.8.1.1 to 4.1.8.1.5).

[0040] (7) Fr.4.1.8.1.2 (2.8 g) was separated by Chromatorex C18 (2.0 cm × 29.5 cm) column chromatography and eluted isocratically with a mixture of 1.0 L of methanol and water (in which the volume fraction of methanol was 30%) to obtain compound Othacanthain A (249.1 mg).

[0041] Identification of shikimic acid compound (Orthacanthain A):

[0042] Figure 2 It is the compound Orthacanthain A. 1 H-NMR spectrum; Figure 3 It is the compound Orthacanthain A 13 C-NMR spectrum; Figure 4 This is the HSQC spectrum of compound Orthacanthain A; Figure 5 This is the HMBC spectrum of compound Orthacanthain A; Figure 6 This is the HREIMS spectrum of the compound Orthacanthain A. Shikimic acid (Orthacanthain A) is a white amorphous powder with a bright spot under 254 nm UV light. It reacts with 1% FeCl3-EtOH colorimetric reagent to form brown spots. The HREIMS spectrum shows its quasi-molecular ion peak at m / z: 1399.2083 [M+H]. + (Calculated value 1399.2109, C) 63 H 51 O 37 The molecular formula is C. 63 H 50 O 37 The degree of unsaturation is 39. 1 H-NMR (500MHz, acetone-d6), 13 C-NMR (125MHz, acetone-d6) data are shown in Table 1 below.

[0043] Table 1. NMR data of shikimic acid compound Orthacanthain A (in acetone-d6)

[0044]

[0045]

[0046] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional and two-dimensional NMR data, the structural formula of the compound was deduced as follows: Figure 1 As shown, it is named OthacanthainA.

[0047] Activity assay of Orthacanthain A:

[0048] Prepare a 50 mM, pH 6.8 solution of 20×PBS phosphate buffer. Use 1 mmol / L p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate. Prepare an enzyme solution with an activity of 0.5 U / mL using 10 U / mg α-glucosidase. Use acarbose as a positive control. Add 50 μL of PBS phosphate buffer to a 96-well plate. Add 40 μL of sample (Orthacanthain A) and PBS to the enzyme reaction group and blank group, respectively. Add 10 μL of α-glucosidase, mix well, and incubate at 37°C for 5 min. Then add 20 μL of 1 mmol / L PNPG solution and incubate at 37°C for 30 min. Measure the absorbance at 405 nm using a microplate reader. The α-glucosidase inhibition rate is calculated using the formula: α-glucosidase inhibition rate = [1-(A...] 反应组 -A 反应对照组 ) / (A空白组 -A 空白对照组 Calculate the enzyme activity inhibition rate by multiplying the result by 100%. Repeat the above experiment three times in parallel, take the average value, and calculate the IC50. 50 value.

[0049] Activity results:

[0050] IC50 of the positive drug acarbose 50 The IC50 of Orthacanthain A is 0.0019 mol / L. 50 The concentration was 0.0014 mol / L, showing strong α-glucosidase inhibitory activity, which can be effectively used to prepare hypoglycemic drugs, opening up new pathways for hypoglycemic drugs and new uses for *Castanopsis fargesii*.

[0051] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0052] 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 shikimic acid compound, characterized by, The structural formula of the shikimic acid compound is: 。 2. The method for preparing shikimic acid derivatives according to claim 1, wherein, The method comprises the following steps: After Yuanjiang acer leaf is crushed and extracted, the filtrate is defatted, and the defatted filtrate is sequentially separated by a first Sephadex LH-20 gel column chromatography, a MCI gel CHP 20P column chromatography, a second Sephadex LH-20 gel column chromatography, a Diaion HP20SS column chromatography, a Toyopearl HW-40F column chromatography, and a Chromatorex C18 column chromatography, and the eluent is a mixture of methanol and water.

3. The method according to claim 2, wherein the eluent of the first Sephadex LH-20 gel column chromatography is a mixture of methanol and water with the volume content of methanol being 0%, 20%, 40%, 60%, 80% and 100% in sequence. The eluent of the MCI gel CHP 20P column chromatography is a mixture of methanol and water with the volume content of methanol being 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence. The eluent of the second Sephadex LH-20 gel column chromatography is a mixture of methanol and water with the volume content of methanol being 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence. The eluent of the Diaion HP20SS column chromatography is a mixture of methanol and water with the volume content of methanol being 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence. The eluent of the Toyopearl HW-40F column chromatography is a mixture of methanol and water with the volume content of methanol being 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence. The eluent of the Chromatorex C18 column chromatography is a mixture of methanol and water with the volume content of methanol being 30%.

4. The method of claim 2, wherein the macerating comprises: The Yuanjiang acer leaf is extracted with methanol with a volume concentration of 65-75% for 2-3 times, each time for 6-8 days, and the filtrate is combined and concentrated under reduced pressure. The reagent used for defatting is petroleum ether.

5. A pharmaceutically acceptable salt of a shikimic acid compound, characterized in that, The medicinal salt is a salt synthesized from the shikimic acid compound and an organic acid and / or an inorganic acid.

6. The shikimic acid compound according to claim 1 in the preparation of a blood sugar-lowering drug.

7. The use of the shikimic acid derivative according to claim 6 for the preparation of a hypoglycemic agent, characterized in that, The blood sugar-lowering drug comprises a pharmaceutically acceptable carrier and the shikimic acid compound.

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