European scabious root extract, process for its preparation and use thereof in the preparation of medicaments for the treatment of alzheimer's disease

Abstract

The present application relates to the technical field of Urtica dioica root extract, and is a kind of Urtica dioica root extract, its preparation method and application in preparing medicine for treating Alzheimer's disease, the Urtica dioica root extract of the present application is obtained after Urtica dioica root is extracted by organic solvent reflux, reduced pressure concentration and water washing.The Urtica dioica root extract of the present application can effectively improve the SH-SY5Y cell damage induced by A beta 25-35, reduce neuron apoptosis by inhibiting oxidative stress response, thereby significantly improving the survival rate of SH-SY5Y cells, the Urtica dioica root extract of the present application has certain potential in the research and development field of medicine for treating Alzheimer's disease, and provides a new strategy for developing safe and effective medicine for treating Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of cornflower root extract technology, specifically a cornflower root extract, its preparation method, and its application in the preparation of drugs for treating Alzheimer's disease. Background Technology

[0002] Cornflower (Centella asiatica) is an annual or perennial herb belonging to the genus Cornflower in the family Asteraceae. As a commonly used medicinal herb in the Uyghur medicine system, its roots have a long history of medicinal use, with extensive records of application in countries such as Iran, Turkey, India, and China. Traditional medical texts describe the herb as having a bland, sticky taste, and being warming in nature, possessing multiple effects including tonifying the heart and strengthening yang, regulating the body and replenishing essence, refreshing the mind and spirit, and drying dampness and consolidating essence. In clinical practice, cornflower root is often used to treat damp-cold or phlegmatic diseases, showing significant efficacy in treating palpitations, impotence, emaciation, low sperm count, irritability, premature ejaculation, jaundice, and cold uterus with a dark complexion. It is also an important component in commonly used compound preparations for treating jaundice, cystic fibrosis, and other diseases.

[0003] Modern pharmacological studies have preliminarily revealed the biological activities of cornflower root, indicating that it possesses various pharmacological effects such as anti-inflammatory, cardiotonic, and sedative properties. As a promising medicinal resource, cornflower root exhibits significant advantages such as wide availability, relatively low collection costs, definite clinical efficacy, and low toxicity. It aligns with the traditional drug development principles of "simplicity, convenience, effectiveness, and affordability," possessing high resource utilization value and potential for further in-depth research.

[0004] However, current research on cornflower root mainly focuses on case studies of clinical applications, with relatively weak research on the material basis of its medicinal effects. The lack of systematic isolation of its chemical components in existing technologies results in a lack of clear material basis for the development of related drug formulations, severely restricting the in-depth exploration of the medicinal value of cornflower root. This leaves a significant technological gap in the development of novel natural drugs using this herb. Summary of the Invention

[0005] This invention provides a cornflower root extract, its preparation method, and its application in the preparation of drugs for treating Alzheimer's disease. It overcomes the shortcomings of the prior art. The invention provides a cornflower root extract that can effectively improve Aβ25-35-induced SH-SY5Y cell damage and significantly improve the survival rate of SH-SY5Y cells, and can be applied in the field of preparing drugs for treating Alzheimer's disease.

[0006] One of the technical solutions of the present invention is achieved through the following measures: a cornflower root extract, obtained by the following method: The first step is to soak the roots of cornflower in an organic solvent, and then extract them by heating and reflux to obtain the extract. The second step is to concentrate the extract under reduced pressure and dry it to obtain the concentrate. The third step is to add water to the concentrate for washing, let it stand and separate into layers, discard the water layer, and obtain an oily substance, namely cornflower root extract.

[0007] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: In the first step above, 10 mL to 15 mL of organic solvent is added to each gram of cornflower root.

[0008] In the first step above, the soaking temperature is room temperature, and the soaking time is 2 to 3 hours.

[0009] In the first step above, during the reflux extraction, the reflux extraction temperature is 50℃ to 60℃, the reflux extraction time is 2.5 hours to 3.0 hours, and the number of reflux extractions is 3 to 4.

[0010] In the third step above, add water to the concentrate at a ratio of 20 to 30 times its mass.

[0011] In the third step above, the water is added and washed 2 to 3 times.

[0012] In the first step above, the cornflower root is dried and pulverized before use.

[0013] The second technical solution of the present invention is achieved through the following measures: a method for preparing cornflower root extract, carried out according to the following method: The first step is to soak the roots of cornflower in an organic solvent, and then extract them by heating and reflux to obtain the extract. The second step is to concentrate the extract under reduced pressure and dry it to obtain the concentrate. The third step is to add water to the concentrate for washing, let it stand and separate into layers, discard the water layer, and obtain an oily substance, namely cornflower root extract.

[0014] One of the technical solutions of the present invention is achieved through the following measures: the application of a cornflower root extract in the preparation of a drug for treating Alzheimer's disease.

[0015] The cornflower root extract of this invention is obtained by reflux extraction with an organic solvent, vacuum concentration, and washing with water. This cornflower root extract can effectively improve Aβ25-35 (Amyloid β-Protein 25-35)-induced SH-SY5Y cell damage by inhibiting oxidative stress and reducing neuronal apoptosis, thereby significantly improving the survival rate of SH-SY5Y cells. This cornflower root extract has potential in the development of Alzheimer's disease treatment drugs and provides a new strategy for developing safe and effective Alzheimer's disease treatments. Attached Figure Description

[0016] Figure 1 The high-performance liquid chromatogram of the cornflower root extract prepared in Example 8 of this invention. Detailed Implementation

[0017] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid; room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.

[0018] The present invention will be further described below with reference to embodiments: Example 1: The cornflower root extract was obtained by the following method: The first step is to soak the roots of cornflower in an organic solvent, and then extract them by heating and reflux to obtain the extract. The second step is to concentrate the extract under reduced pressure and dry it to obtain the concentrate. The third step is to add water to the concentrate for washing, let it stand and separate into layers, discard the water layer, and obtain an oily substance, namely cornflower root extract.

[0019] Example 2: As an optimization of the above example, in the first step, 10 mL to 15 mL of organic solvent is added to each gram of cornflower root.

[0020] Example 3: As an optimization of the above example, in the first step, the soaking temperature is room temperature and the soaking time is 2 to 3 hours.

[0021] Example 4: As an optimization of the above example, in the first step, during the heating and reflux extraction, the reflux extraction temperature is 50°C to 60°C, the reflux extraction time is 2.5 hours to 3.0 hours, and the number of reflux extractions is 3 to 4.

[0022] Example 5: As an optimization of the above example, in the third step, water with a mass of 20 to 30 times that of the concentrate is added to the concentrate.

[0023] Example 6: As an optimization of the above example, in the third step, the number of times water is added and washed is 2 to 3.

[0024] Example 7: As an optimization of the above example, in the first step, the cornflower root is dried and pulverized before use.

[0025] Example 8: The cornflower root extract is prepared as follows: The first step is to place each gram of cornflower root in 12 mL of acetone, soak it at room temperature for 2 hours, and then heat it at 50℃ to 60℃ and reflux for 2.5 hours. Repeat the reflux extraction 3 times and combine the extracts to obtain the extract. The second step is to concentrate the extract under reduced pressure, recover the solvent, and then dry it to obtain the concentrate. The third step is to add 20 mL of water to each gram of concentrate and stir until completely dispersed. Pour the mixture into a separatory funnel, let it stand and separate into layers, discard the lower water layer, and keep the upper oily insoluble layer. Repeat this washing process twice, and combine the upper oily insoluble layers, which is the cornflower root extract, labeled Y1.

[0026] Example 9: The cornflower root extract is prepared as follows: The first step is to place each gram of cornflower root in 10 mL of acetone, soak it at room temperature for 2 hours, and then heat it at 50℃ to 60℃ and reflux for 2.5 hours. Repeat the reflux extraction 3 times and combine the extracts to obtain the extract. The second step is to concentrate the extract under reduced pressure, recover the solvent, and then dry it to obtain the concentrate. The third step is to add 25 mL of water to each gram of concentrate and stir until completely dispersed. Pour the mixture into a separatory funnel, let it stand and separate into layers, discard the lower water layer, and keep the upper oily insoluble layer. Repeat this washing process twice, and combine the upper oily insoluble layers, which is the cornflower root extract, labeled as Y2.

[0027] Example 10: The cornflower root extract is prepared as follows: The first step is to place each gram of cornflower root in 15 mL of acetone, soak it at room temperature for 2 hours, and then heat it at 50℃ to 60℃ and reflux for 2.5 hours. Repeat the reflux extraction 3 times and combine the extracts to obtain the extract. The second step is to concentrate the extract under reduced pressure, recover the solvent, and then dry it to obtain the concentrate. The third step is to add 30 mL of water to each gram of concentrate and stir until completely dispersed. Pour the mixture into a separatory funnel, let it stand and separate into layers, discard the lower water layer, and keep the upper oily insoluble layer. Repeat this washing process twice, and combine the upper oily insoluble layers, which is the cornflower root extract, labeled Y3.

[0028] Comparative Example 1: The difference from Example 8 is that the third water washing step is not used, and the concentrate obtained in the second step is labeled as D1.

[0029] Comparative Example 2: The difference from Example 8 is that "each gram of cornflower root is placed in 12 mL of acetone" is replaced with "each gram of cornflower root is placed in 12 mL of petroleum ether". The resulting cornflower root extract is labeled as D2.

[0030] In Comparative Example 2, since the concentrate obtained by reflux extraction of cornflower root with petroleum ether and concentration under reduced pressure is incompatible with water and has a large difference in polarity, the water washing step has little effect and can be ignored.

[0031] Comparative Example 3: The difference from Example 8 is that "place each gram of cornflower root in 12 mL of acetone" is replaced with "place each gram of cornflower root in 12 mL of water"; and the water washing step is not used. The resulting cornflower root extract is labeled as D3.

[0032] In Comparative Example 3, the cornflower root was extracted with water reflux and concentrated under reduced pressure to obtain a concentrate. Since the concentrate itself is water-soluble, the water washing step was not used.

[0033] Experimental Example 1: The active ingredient analysis of the cornflower root extract of this invention was investigated.

[0034] Experimental methods and results: The active ingredients of the cornflower root extract prepared in Example 8 of this invention were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry-mass spectrometry (UHPLC-QE-MS). The liquid chromatogram of the cornflower root extract prepared in Example 8 of this invention is shown below. Figure 1 As shown, Figure 1 The peak at a retention time of 16.80 minutes is 9,12,13-trihydroxyoctadec-10-enoic acid, and its structural formula is: It is evident that the main active ingredient in the acetone extract of cornflower root of the present invention is unsaturated fatty acids.

[0035] Experimental Example 2: The present invention relates to the application of cornflower root extract in the preparation of a medicament for treating Alzheimer's disease.

[0036] Experimental Methods: The cornflower root extracts prepared in Examples 8 to 10 of this invention were subjected to in vitro anti-Alzheimer's disease pharmacodynamic experiments. The in vitro anti-Alzheimer's disease pharmacodynamic experiments were conducted using the CCK8 assay. Specific steps were as follows: SH-SY5Y cells (SH-SY5Y human neuroblastoma cell line) were cultured at 5 × 10⁶ cells per well. 3Cells were seeded in 96-well cell culture plates and cultured for 48 h. The following groups were established: a blank group (complete culture medium), a control group (SH-SY5Y cells + complete culture medium), a model group (SH-SY5Y cells + complete culture medium + 40 μM Aβ25-35 (β-amyloid protein 25-35 fragment, a widely used tool peptide in Alzheimer's disease (AD) research)), and a cornflower root extract treatment group (SH-SY5Y cells + complete culture medium + cornflower root extract prepared in Examples 8 to 10). The concentration of cornflower root extract in the treatment group was set at 200 μg / mL. After pre-incubation for 4 h, 40 μM Aβ25-35 was added to each well. Each group had three replicates. Cells were cultured for another 48 h. The culture medium was aspirated, and 100 μL of 10% CCK8 was added. The cells were incubated at 37°C for 1 h. The absorbance of each well was measured at 450 nm, and the cell viability was calculated using the cell viability formula. Cell survival rate calculation formula: Cell survival rate % = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0037] Experimental Results: The cell viability of the cornflower root extract treatment group of the present invention is shown in Table 1. As can be seen from Table 1, compared with the model group, the cornflower root extract prepared in Examples 8 to 10 of the present invention, when treated at a concentration of 200 μg / mL, effectively counteracted neuronal apoptosis induced by oxidative stress and improved the survival rate of SH-SY5Y cells in the Aβ25-35-induced SH-SY5Y cell injury model. Furthermore, compared with Comparative Example 1 (without the water washing step), the concentrate obtained in the second step of Examples 8 to 10 of the present invention, after water washing and purification, further improved the survival rate of SH-SY5Y cells. Compared with Comparative Example 2 (the organic solvent was petroleum ether), the cornflower root extract prepared in Examples 8 to 10 using acetone as the extraction solvent effectively improved the survival rate of SH-SY5Y cells.

[0038] In summary, the cornflower root extract of this invention is obtained by reflux extraction with an organic solvent, vacuum concentration, and washing with water. This cornflower root extract can effectively improve Aβ25-35-induced SH-SY5Y cell damage, reduce neuronal apoptosis by inhibiting oxidative stress, and thus significantly improve the survival rate of SH-SY5Y cells. This cornflower root extract has potential in the development of Alzheimer's disease treatment drugs and provides a new strategy for developing safe and effective Alzheimer's disease treatments.

[0039] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A cornflower root extract, characterized in that... Obtained using the following method: The first step is to soak the roots of cornflower in an organic solvent, and then extract them by heating and reflux to obtain the extract. The second step is to concentrate the extract under reduced pressure and dry it to obtain the concentrate. The third step is to add water to the concentrate for washing, let it stand and separate into layers, discard the water layer, and obtain an oily substance, namely cornflower root extract.

2. The cornflower root extract according to claim 1, characterized in that... The organic solvent is acetone.

3. The cornflower root extract according to claim 1 or 2, characterized in that... In the first step, add 10 mL to 15 mL of organic solvent to each gram of cornflower root.

4. The cornflower root extract according to claim 1, 2, or 3, characterized in that... In the first step, the soaking temperature is room temperature, and the soaking time is 2 to 3 hours.

5. The cornflower root extract according to any one of claims 1 to 4, characterized in that... In the first step, during the reflux extraction, the reflux extraction temperature is 50℃ to 60℃, the reflux extraction time is 2.5 hours to 3.0 hours, and the number of reflux extractions is 3 to 4.

6. The cornflower root extract according to any one of claims 1 to 5, characterized in that... In the third step, add water to the concentrate at a ratio of 20 to 30 times its mass.

7. The cornflower root extract according to any one of claims 1 to 6, characterized in that... In the third step, the water is added and washed 2 to 3 times.

8. The cornflower root extract according to any one of claims 1 to 7, characterized in that... In the first step, the cornflower root is dried and pulverized before use.

9. A method for preparing cornflower root extract according to any one of claims 2 to 8, characterized in that... Perform the following steps: The first step is to soak the roots of cornflower in an organic solvent, and then extract them by heating and reflux to obtain the extract. The second step is to concentrate the extract under reduced pressure and dry it to obtain the concentrate. The third step is to add water to the concentrate for washing, let it stand and separate into layers, discard the water layer, and obtain an oily substance, namely cornflower root extract.

10. The use of a cornflower root extract according to any one of claims 1 to 8 in the preparation of a medicament for treating Alzheimer's disease.

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