Benzofuran compound in valeriana and use thereof for treating diabetes

Abstract

The application discloses use of a benzofuran compound in a Chinese herb of Huaze Lan in treatment of diabetes. The benzofuran compound is separated from roots of the Chinese herb of Huaze Lan and is named as: 10 R -2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran. An improved preparation method of the benzofuran compound is used, roots of the Chinese herb of Huaze Lan are used as raw materials, and the improved preparation method comprises the following steps: green water solvent extraction, macroporous adsorption column chromatography and crystallization separation, and the improved separation method is green and efficient, has high purity and can be suitable for industrial production. The benzofuran compound is found to have a significant effect in treatment of diabetes through animal experiments, data analysis of blood glucose concentration, oral glucose tolerance and liver index of type 2 diabetes mice after administration. The benzofuran compound has a good effect in treatment of diabetes, and is expected to be developed into a new natural medicine for treatment of diabetes.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a traditional Chinese medicine extract 10 R Preparation method of 2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran and its use in the treatment of diabetes. Background Technology

[0002] The number of people with diabetes worldwide is growing rapidly, posing a global health challenge. According to the latest data from 2024, the number of people with diabetes globally has exceeded 800 million, with an age-standardized prevalence of 6.1%, of which 96% are type 2 diabetes. It is projected that the number of people with diabetes worldwide will increase to 1.31 billion by 2050.

[0003] Currently, both traditional Chinese medicine (TCM) and Western medicine have their advantages in treating diabetes. Western medicine primarily treats diabetes through oral hypoglycemic agents and insulin injections. Commonly used oral hypoglycemic agents include biguanides (such as metformin), sulfonylureas (such as glimepiride), thiazolidinediones (such as rosiglitazone), benzoic acid derivatives (such as repaglinide), and alpha-glucosidase inhibitors (such as acarbose). In recent years, novel targeted drugs such as GLP-1 receptor agonists and SGLT-2 inhibitors have also been gradually applied clinically. However, long-term use of some drugs may lead to symptoms such as diarrhea, weight changes, organ damage, and gastrointestinal reactions. TCM has unique advantages in the treatment of diabetes. Through holistic regulation and multi-target mechanisms, it can effectively improve blood glucose levels, alleviate symptoms, and prevent complications. In recent years, scholars at home and abroad have turned their attention to extracts of Chinese herbal medicines, discovering many new natural drugs with anti-diabetic effects. For example, natural extracts such as mulberry twig total alkaloid capsules have been developed into clinical anti-diabetic drugs. Therefore, the prospect of finding natural anti-diabetic drugs from Chinese herbal medicines is promising.

[0004] Huazelan Eupatorium chinense It belongs to the genus *Eupatorium* of the Asteraceae family, also known as Duoxugong, Tu Niuxi, and Liuyuexue. It is mainly distributed in Hubei, Hunan, and Guangdong provinces, and is a commonly used herb by the Tujia ethnic group in western Hubei and western Hunan, possessing the effects of clearing heat and detoxifying, soothing the liver and promoting blood circulation. Our research group has long been committed to the study of the chemical constituents and pharmacological activities of *Eupatorium fortunei*. Previously, through isolation and purification, we obtained a main component, which is compound 1 (10...) described in this invention. R-2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran (Chinese Invention Patent Publication No.: CN117186042A). This invention improves the separation and purification process of this compound. Literature review revealed no reported therapeutic effects on diabetic mice. The inventors previously reported that another main component of *Gnaphalium affine*, *Gnaphalium affine* (compound 3), simultaneously exhibits good inhibitory effects on α-glucosidase and PTP1B, and can serve as a lead compound targeting both diabetes (Chinese Invention Patent Publication No.: CN116103161A). Zhou Changxin et al. of Zhejiang University reported that *Gnaphalium affine* (compound 2) can promote glucose transport at C2C12 in human muscle cells (Chinese Invention Patent Publication No.: CN109232491A). This invention tested the therapeutic effects of three monomeric compounds on diabetic mice using a mouse model of diabetes. The results showed that the blood glucose levels in the mice treated with all three compounds generally decreased, and were all lower than those in the model group, indicating that all three compounds could improve blood glucose levels in T2DM (type 2 diabetes) mice. Compound 1 showed better hypoglycemic effects than compounds 2 and 3, and was superior to the positive control drug metformin, demonstrating good therapeutic efficacy for diabetes and showing promise as a new natural anti-diabetic drug. This invention patent discloses an improved preparation method for these compounds and their use in treating diabetes. Summary of the Invention

[0005] The first object of the present invention is to provide compound 1 (10 R An improved method for preparing 2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran, with a second objective being to provide its use in the preparation of medicaments for treating diabetes.

[0006] Benzofuran compounds, with the molecular formula C 13 H 14 O5, the structural formula is as follows:

[0007] Named: 10 R -2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran.

[0008] This invention provides a method for preparing the compound, comprising the following steps:

[0009] Step A: Crush the roots of Zephyranthes cusia, extract them by heating and reflux with pure water, filter the extract, and concentrate it under reduced pressure to obtain the extract.

[0010] Step B: Dissolve the extract obtained in Step A in an appropriate amount of water, then load it onto a macroporous adsorption resin chromatography column, elute with 30%-40% ethanol-water (volume ratio), combine the eluents, and concentrate under reduced pressure to obtain the extract.

[0011] Step C: Dissolve the extract obtained in step B completely in methanol, add pure water to make the methanol solution volume fraction 10%-40%, place it in a refrigerator at 4°C overnight, centrifuge the crystals, and dry them to obtain compound 1.

[0012] Furthermore, the aqueous solution described in step A is pure water.

[0013] Furthermore, the eluent mentioned in step B is 30%-40% ethanol-water.

[0014] Furthermore, the solvent mentioned in step C is 10%-40% methanol-water.

[0015] A method for preparing a drug for treating diabetes, wherein the drug is a benzofuran compound. Specifically, the active drug for treating diabetes refers to the benzofuran compound.

[0016] A composition for preparing a medicament for treating diabetes, said composition comprising the aforementioned benzofuran compounds.

[0017] The application of the benzofuran compounds in the preparation of drugs for treating diabetes that lower blood sugar and improve glucose tolerance.

[0018] The application of the benzofuran compounds in the preparation of drugs for treating diabetes that reduce liver indices and improve liver condition.

[0019] The drug is an oral or injectable preparation, and the dosage is 16~32 mg / kg / day.

[0020] The dosage form of the drug for treating diabetes is selected from one or more of the following dosage forms: tablets, capsules, granules, drop pills, suspensions, syrups, enteric-coated preparations, emulsion suspensions, and injections.

[0021] The present invention also discloses the use of compound 1 in the treatment of diabetes.

[0022] The advantages of this invention are as follows:

[0023] 1. This invention discloses that compound 1 has a significant therapeutic effect on diabetes, provides a new medical use for compound 1, and opens up new application areas for it.

[0024] 2. Compound 1 showed significant therapeutic effects on diabetes, mainly as follows: on day 28, the blood glucose level in the compound 1 administration group was 11.07±3.07 mmol / L, which was significantly lower than that in the model group (20.98±3.60 mmol / L) (P<0.01); it was also superior to the positive control drug metformin, and had a better hypoglycemic effect than compounds 2 and 3. Attached Figure Description

[0025] Figure 1 HPLC analysis chromatograms of compound 1 extracted with different solvents.

[0026] Figure 2 Effects of compounds 1-3 and the positive control drug metformin on blood glucose levels in type 2 diabetic mice. (* and ** indicate P < 0.05 and P < 0.01 compared to the normal control group, respectively; # and ## indicate P < 0.05 and P < 0.01 compared to the model group, respectively.)

[0027] Figure 3 Effects of compounds 1-3 and the positive control drug metformin on oral glucose tolerance in type II diabetic mice. (* and ** indicate P < 0.05 and P < 0.01 compared to the normal group, respectively; # and ## indicate P < 0.05 and P < 0.01 compared to the model group, respectively.) Detailed Implementation

[0028] The following embodiments are used to further explain and illustrate the present invention, but are not intended to limit the scope of protection of the present invention.

[0029] Example 1: Optimization of the preparation process of compound 1

[0030] Based on the polarity of compound 1, the extraction solvent in the first step of this invention is green pure water instead of 95% ethanol-water, as shown in the HPLC analysis chromatogram ( Figure 1 As can be seen, when pure water was used, compound 3 could not be extracted, compound 1 had a higher proportion in the extract, and activated carbon was not needed to remove impurities, which was more conducive to the precipitation of crystals.

[0031] The preparation method includes the following steps:

[0032] Step A: Crush the roots of Zephyranthes cusia to 20-40 mesh, extract with pure water by heating and reflux 5 times, 2 hours each time, combine the extracts, filter, and concentrate under reduced pressure to obtain extract a;

[0033] Step B: Dissolve extract a in water, load it onto a macroporous adsorption resin chromatography column, elute with 40% ethanol-water (volume ratio), combine the organic phases, and concentrate under reduced pressure to extract b.

[0034] Step C: After completely dissolving extract b in methanol, add 3 times the amount of pure water, place in a refrigerator at 4°C overnight, centrifuge the crystals, and dry to obtain compound 1.

[0035] Example 2: Animal experiments on the antidiabetic effects of compounds 1-3

[0036] 1. Experimental Materials

[0037] 1.1 Experimental animals: C57BL / 6J mice [provided by the Animal Experiment Center of China Three Gorges University, license number SCXK(E)2022-0061], weighing 18-20 g, 48 males.

[0038] 1.2 Experimental drugs: Compounds 1-3 (self-made, purity: HPLC > 95%), sodium carboxymethylcellulose (Maclean USP grade), metformin (Shanxi Qianyuan Pharmaceutical Group Co., Ltd., batch number: 202104, specification 20 mg)

[0039] 2. Experimental methods

[0040] 2.1 Animal experiment modeling and grouping: After 7 days of adaptive feeding, the mice were divided into a normal group and a model group. The normal group was fed with ordinary feed; the model group was fed with a high-fat diet (79% ordinary feed + 1% cholesterol + 10% egg yolk powder + 10% lard). After 8 weeks of induction feeding with the high-fat diet, the mice in the model group were intraperitoneally injected with STZ solution (50 mg / Kg, dissolved in 0.1 mol / L citrate buffer, pH 4.5) for 5 consecutive days. On the second day and one week after injection, the blood glucose of the mice in the model group was measured using a blood glucose meter respectively. When the fasting blood glucose was ≥ 11.1 mmoL / L for two consecutive times and the random blood glucose was ≥ 16.8 mmoL / L, the model was considered successful. The type II diabetic mice were randomly divided into four groups (10 mice in each group), including a model group (8 mice), a compound 1 administration group (10 mice), a compound 2 administration group (10 mice), a compound 3 administration group (10 mice), and a positive drug (metformin) administration group (10 mice). They were respectively given normal saline, compound 1 (16 mg / kg / d), compound 2 (16 mg / kg / d), compound 3 (16 mg / kg / d), and metformin solution (200 mg / kg / d), and the mice fed with normal diet were used as the normal group for 4 weeks. The type II diabetic mice were fed with a high-fat diet, and the NC mice were fed with ordinary feed. The water intake and food intake were monitored every two days, and the blood glucose was monitored every 7 days. After the experiment, all the mice were fasted for 12 hours, blood was collected from the eyes, the serum was collected by centrifugation at 12,000 rpm for 20 minutes in a 4℃ refrigerated centrifuge, the mice were euthanized with ether, and the liver was dissected. After labeling the serum and liver, they were stored in a -80℃ refrigerator for later use.

[0041] 2.2 Oral glucose tolerance test: After the last administration, oral glucose tolerance tests were performed on mice in the normal group, model group, and compound 1-3 administration groups. Mice were fasted for 12 hours and then orally administered glucose at 2 g / kg (BW). Blood glucose concentrations were measured by collecting serum from the tail vein at 0, 30, 60, 90, and 120 min after glucose administration. The area under the curve of glucose (AUCG) for each group was calculated using the following formula:

[0042]

[0043] In the formula: AUCG is the area under the glucose curve (mmol / L·h); A is the blood glucose concentration at 0 min; B is the blood glucose concentration at 30 min; C is the blood glucose concentration at 60 min; and D is the blood glucose concentration at 120 min.

[0044] 2.3 Liver Index Detection: The surface moisture of the dissected mouse livers was aspirated, and the livers were weighed using an electronic balance. The liver index (%) was calculated as: Liver weight / Body weight × 100%, yielding the mouse liver-to-body ratio.

[0045] 2.4 Statistical Processing: The data were statistically processed and inter-group difference tests were performed. ±S represents the mean. Differences between groups were analyzed using one-way ANOVA with SPSS statistical software. A p-value < 0.05 was considered statistically significant.

[0046] 3. Experimental Results

[0047] 3.1 Effects of compounds 1-3 and the positive control drug metformin on blood glucose levels in type II diabetic mice

[0048] The experimental results are shown in Figure 2 The results showed that blood glucose levels in the normal control group remained stable after 4 weeks of administration. Blood glucose levels in the model group remained consistently high. Blood glucose levels in all four administration groups (three administration groups and one positive control group) showed an overall decreasing trend and were all lower than those in the model group, indicating that all four administration groups could improve blood glucose levels in T2DM mice. On day 28, the blood glucose levels in the compound 1-3 administration groups were 11.07±3.07 mmol / L, 12.12±0.91 mmol / L, and 15.73±2.11 mmol / L, respectively. Compound 1 had a better hypoglycemic effect than compounds 2 and 3, and was significantly lower than the model group (20.98±3.60 mmol / L, P<0.01). Even at a lower dose, the compound 1 administration group showed a better hypoglycemic effect than the positive control group (18.64±4.48 mmol / L, P<0.01).

[0049] 3.2 Results of oral glucose tolerance test

[0050] The experimental results are shown in Figure 3 The results showed that blood glucose levels in the normal group mice initially increased and then decreased within 0–120 min. Blood glucose concentrations in both the model group and the drug-treated groups also initially increased and then decreased, reaching a maximum at 30 min. At 120 min, the blood glucose level in the model group mice was 27.81 ± 0.34 mmol / L. Compared with the model group, the fasting blood glucose levels in the compound 1–3 treated groups were 12.33 ± 0.67 mmol / L, 17.97 ± 1.52 mmol / L, and 24.33 ± 1.46 mmol / L, respectively, all significantly lower (P < 0.05). These results indicate that all three compounds significantly improved glucose tolerance in diabetic mice, repaired impaired glucose tolerance, and helped improve diabetic symptoms. Compound 1, in particular, showed better effects compared to compounds 2 and 3.

[0051] 3.3 Liver index results

[0052] The experimental results are shown in Table 1. The results indicate that the three compound administration groups significantly reduced the liver index compared with the model group (P < 0.05), suggesting that they help improve liver condition; in particular, compound 1 had a better effect than compounds 2 and 3.

[0053] Table 1. Effects of compounds 1-3 on liver index in type II diabetic mice

[0054]

[0055] * indicates P < 0.05 compared to the normal group; # indicates P < 0.05 compared to the model group.

[0056] In summary, this invention provides the application of compound 1 in the preparation of a drug for treating diabetes. Animal experiments conducted in this invention revealed that type 2 diabetic mice (model group) had a higher water intake than the normal group, consistent with the polydipsia symptom in diabetic patients. Compounds 1-3 reduced the daily water intake of mice, suggesting their role in improving diabetic symptoms; compound 1, in particular, showed a better effect than compounds 2 and 3. Type 2 diabetic mice (model group) also had a higher food intake than the normal group, consistent with the polyphagia symptom in diabetic patients. Compounds 1-3 reduced the daily food intake of mice, suggesting their role in improving diabetic symptoms; compound 1, in particular, showed a better effect than compounds 2 and 3. During the four weeks of administration, the blood glucose levels of the normal group mice remained stable. The blood glucose levels of the model group mice remained at a high level, while the blood glucose levels of the groups treated with compounds 1-3 showed an overall decreasing trend and were all lower than those of the model group mice, indicating that compounds 1-3 can improve blood glucose levels in type 2 diabetic mice. Compound 1 showed better hypoglycemic effects compared to compounds 2 and 3, with a blood glucose level of 11.07±3.07 mmol / L on day 28, significantly lower than the model group's 20.98±3.60 mmol / L (P<0.01). The compound 1 group, even at a lower dose, showed better hypoglycemic effects than the positive control group (18.64±4.48 mmol / L) (P<0.01). Oral glucose tolerance tests showed that blood glucose levels in normal mice initially increased and then decreased within 0–120 min. Blood glucose concentrations in both the model and drug-treated groups also initially increased and then decreased, reaching a maximum at 30 min. At 120 min, the fasting blood glucose level in the model group was 27.80±0.34 mmol / L. Compared to the model group, fasting blood glucose levels in all compound 1–3 groups were significantly lower. These results indicate that compounds 1–3 can significantly improve glucose tolerance in diabetic mice, repair impaired glucose tolerance, and help improve diabetic symptoms. Compound 1, in particular, showed better efficacy compared to compounds 2 and 3. Compared to the model group, all groups treated with compounds 1-3 significantly reduced liver indices, suggesting their role in improving liver function; compound 1, especially, demonstrated superior efficacy compared to compounds 2 and 3. These results indicate that compound 1 has a significant therapeutic effect on diabetic mice and holds promise for development into a novel natural anti-diabetic drug.

[0057] Although the present invention has been described in detail above through general description, specific embodiments, and activity experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The use of a benzofuran compound in the preparation of a medicament for treating diabetes, characterized in that, The molecular formula is C 13 H 14 O5, the structural formula is as follows: Named: 10 R -2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran.

2. The application according to claim 1, characterized in that, The preparation steps of benzofuran compounds include the following: A. Solvent extraction: The roots of Zephyranthes cusia were crushed and extracted by heating with water under reflux. The extract was then filtered and concentrated under reduced pressure to obtain extract a. B. Macroporous adsorption column chromatography separation: Dissolve extract a in water, load onto a macroporous adsorption resin column, wash with alcohol, combine the organic phases, and concentrate under reduced pressure to extract b. C. Separation by crystallization: After complete dissolution with methanol solution, the compound is cooled and crystallized to obtain the compound.

3. The application according to claim 2, characterized in that, The alcohol used in the alcohol washing of step B is a 30%-40% aqueous ethanol solution.

4. The application according to claim 2, characterized in that, The concentration of the methanol solution in step C is a 10-40% methanol aqueous solution.

5. The application according to claim 1, characterized in that, The application of benzofuran compounds in the preparation of drugs for treating diabetes that lower blood sugar and improve glucose tolerance.

6. The application according to claim 1, characterized in that, The application of the benzofuran compounds in the preparation of drugs for treating diabetes that reduce liver indices and improve liver condition.

7. The application according to claim 1, characterized in that, The drug is an oral or injectable preparation, and the dosage is 16~32 mg / kg / day.

8. The application according to claim 1, characterized in that, The dosage form of the drug for treating diabetes is selected from one or more of the following dosage forms: tablets, capsules, granules, drop pills, suspensions, syrups, enteric-coated preparations, emulsion suspensions, and injections.

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