European cornflower root extract as well as preparation method and application thereof in preparation of medicine for treating or preventing neurodegenerative diseases
By extracting and separating the roots of cornflower, a cornflower root extract with neuroprotective effects was prepared, which solved the problem of insufficient research on its material basis in the existing technology and realized the effective treatment and prevention of neurodegenerative diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG UYGUR AUTONOMOUS REGION DRUG RESEARCH INSTITUTE
- Filing Date
- 2026-03-16
- Publication Date
- 2026-04-14
AI Technical Summary
Current research on cornflower root mainly focuses on clinical applications and preliminary efficacy observations, lacking research on the material basis of its efficacy, which limits the exploration of its biological application value and the development of new drugs.
The root of *Centaurea rubra* was extracted by soaking in methanol, heating and refluxing, concentrated under reduced pressure, and then extracted with petroleum ether, dichloromethane and ethyl acetate. The extract was then separated by gradient elution using a silica gel column to obtain *Centaurea rubra* root extract, which was used to prepare neuroprotective drugs.
Cornflower root extract has a significant neuroprotective effect on a glutamate-induced HT22 cell injury model, improving cell survival and providing a cost-effective and safe neuroprotective drug option.
Smart Images
Figure CN121846162A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cornflower root technology, specifically to a cornflower root extract, its preparation method, and its application in the preparation of drugs for treating or preventing neurodegenerative diseases. Background Technology
[0002] European cornflower (Centaurea Behen Linn.) is an annual or perennial plant belonging to the genus Centaurea in the family Asteraceae. It typically grows on rocky slopes, in grasslands, and along roadsides. This medicinal herb has a mild, sticky taste and is believed to have various effects, including generating heat and dryness, tonifying the heart and strengthening yang, regulating the body and replenishing essence, refreshing the mind and spirit, drying dampness and consolidating essence, eliminating jaundice, and warming the uterus and promoting vitality. In traditional applications, it is primarily used to treat damp-cold or phlegmatic diseases, including palpitations, impotence, emaciation, low sperm count, irritability, premature ejaculation, spermatorrhea, jaundice, and cold uterus with a dark complexion.
[0003] Cornflower root is a commonly used medicinal herb in Uyghur medicine in Xinjiang, China, and has a long history of application in countries such as Iran, Turkey, India, and China. It is frequently used to treat jaundice, cystic fibrosis, and other diseases, and is also used in various compound preparations. Modern pharmacological studies have shown that cornflower root possesses various biological activities, including anti-inflammatory, cardiotonic, and sedative effects. As a medicinal resource, cornflower root has significant advantages such as easy availability, low cost, reliable clinical efficacy, and minimal side effects, making it highly valuable for development and application.
[0004] However, current research on cornflower root mainly focuses on clinical applications and preliminary efficacy observations, with relatively little research on the material basis of its efficacy. Therefore, it is of great significance to explore the potential biological application value of cornflower root, improve the quality of related products, and develop new drugs. 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 or preventing neurodegenerative diseases, overcoming the shortcomings of the prior art. It is the first to discover that a cornflower root extract has a significant neuroprotective effect on a glutamate-induced HT22 cell damage model.
[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: S1, Soak the roots of cornflower in methanol, then extract by heating and reflux to obtain an extract. Concentrate the extract under reduced pressure to obtain the total extract of cornflower roots. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by silica gel column gradient elution to obtain 4 fractions. The third fraction was concentrated to obtain cornflower root extract.
[0007] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: In step S1 above, 8 mL to 12 mL of methanol is added for every 1 g of cornflower root.
[0008] In step S3 above, the eluent used in gradient elution separation is a mixture of petroleum ether and ethyl acetate.
[0009] The volume ratios of petroleum ether and ethyl acetate mentioned above are 50 / 1, 25 / 1, 8 / 1, and 1 / 1, respectively.
[0010] In step S1 above, the temperature during reflux extraction is 50°C to 60°C, the reflux extraction is performed 3 times, and the extraction time for each extraction is 1 to 3 hours.
[0011] In step S1 above, the cornflower root must be dried and pulverized before use.
[0012] In step S1 above, the soaking temperature is room temperature and the soaking time is 2 to 5 hours.
[0013] The second technical solution of the present invention is achieved through the following measures: a cornflower root extract, prepared by the following method: S1, Soak the roots of cornflower in methanol, then extract by heating and reflux to obtain an extract. Concentrate the extract under reduced pressure to obtain the total extract of cornflower roots. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by silica gel column gradient elution to obtain 4 fractions. The third fraction was concentrated to obtain cornflower root extract.
[0014] The third technical solution 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 or preventing neurodegenerative diseases.
[0015] The root extract of *Centella asiatica* exhibits significant neuroprotective activity against glutamate-induced HT22 cell damage, effectively combating neuronal apoptosis induced by oxidative stress and improving HT22 cell survival. This significant neuroprotective activity makes *Centella asiatica* root extract a potential neuroprotective drug, offering a cost-effective and safe new natural drug option for treating neurodegenerative diseases. Attached Figure Description
[0016] Figure 1 This is a high-performance liquid chromatogram of the cornflower root extract in Example 10 of the present 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 chemicals mentioned in this invention are well-known and commonly used chemical reagents and chemicals 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: S1, Soak the roots of cornflower in methanol, then extract by heating and reflux to obtain an extract. Concentrate the extract under reduced pressure to obtain the total extract of cornflower roots. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by silica gel column gradient elution to obtain 4 fractions. The third fraction was concentrated to obtain cornflower root extract.
[0019] Example 2: As an optimization of the above example, in step S1, 8 mL to 12 mL of methanol is added for every 1 g of cornflower root.
[0020] Example 3: As an optimization of the above example, in step S3, the eluent used in gradient elution separation is a mixture of petroleum ether and ethyl acetate.
[0021] Example 4: As an optimization of the above examples, the volume ratios of petroleum ether and ethyl acetate are 50 / 1, 25 / 1, 8 / 1 and 1 / 1, respectively.
[0022] Example 5: As an optimization of the above example, in step S1, the temperature during heating and reflux extraction is 50°C to 60°C, the number of reflux extractions is 3, and the extraction time for each extraction is 1 hour to 3 hours.
[0023] Example 6: As an optimization of the above example, in step S1, the cornflower root is dried and pulverized before use.
[0024] Example 7: As an optimization of the above example, in step S1, the soaking temperature is room temperature and the soaking time is 2 to 5 hours.
[0025] Example 8: The cornflower root extract was obtained by the following method: S1, 1g of dried cornflower root was crushed and added to 8ml of methanol. It was soaked at room temperature for 2 hours, and then extracted by reflux at 50℃ to 60℃ 3 times, each time for 1 hour. The extracts were combined and concentrated under reduced pressure to obtain total cornflower root extract. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by gradient elution using a silica gel column, with a mixture of petroleum ether and ethyl acetate as the eluent. The volume ratios of the petroleum ether and ethyl acetate mixture were 50 / 1, 25 / 1, 8 / 1, and 1 / 1, respectively. The fraction with a volume ratio of 8 / 1 was collected and concentrated to obtain cornflower root extract.
[0026] Example 9: The cornflower root extract was obtained by the following method: S1, 1g of dried cornflower root was crushed and added to 12ml of methanol. It was soaked at room temperature for 5 hours, and then extracted by reflux at 50℃ to 60℃ 3 times, each time for 3 hours. The extracts were combined and concentrated under reduced pressure to obtain total cornflower root extract. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by gradient elution using a silica gel column, with a mixture of petroleum ether and ethyl acetate as the eluent. The volume ratios of the petroleum ether and ethyl acetate mixture were 50 / 1, 25 / 1, 8 / 1, and 1 / 1, respectively. The fraction with a volume ratio of 8 / 1 was collected and concentrated to obtain cornflower root extract.
[0027] Example 10: The cornflower root extract was obtained by the following method: S1, 1g of dried cornflower root was crushed and added to 10ml of methanol, soaked at room temperature for 3 hours, and then extracted by reflux at 50℃ to 60℃ 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain total cornflower root extract. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by gradient elution using a silica gel column, with a mixture of petroleum ether and ethyl acetate as the eluent. The volume ratios of the petroleum ether and ethyl acetate mixture were 50 / 1, 25 / 1, 8 / 1, and 1 / 1, respectively. The fraction with a volume ratio of 8 / 1 was collected and concentrated to obtain cornflower root extract.
[0028] The active ingredient of the cornflower root extract prepared in Example 10 of this invention was analyzed by ultra-high performance liquid chromatography / high resolution mass spectrometry / mass spectrometry (UHPLC / MS / MS). The instrument was a Thermo Fisher Scientific Ultimate 3000 / Q-Exactive UHPLC / MS / MS system. The HPLC conditions were as follows: the column was a WondaSil C column (250 mm × 4.6 mm, 5 μm, GL Sciences, Japan); gradient elution was performed using acetonitrile (A) - 0.1% formic acid water (B) as the mobile phase, the flow rate was 1 mL / min, the detection wavelength was 290 nm, the column temperature was 30 °C, and the injection volume was 10 μL. Mobile phase: Acetonitrile (A) - 0.1% formic acid water (B); Elution gradient: 5% to 30% A (0 to 6 min), 30% to 62% A (6 to 10 min), 62% to 62% A (10 to 18 min), 62% to 95% A (18 to 24 min).
[0029] Liquid chromatography, such as Figure 1 As shown, Figure 1 The peak at a retention time of 16.23 minutes is 9-oxo-10E,12Z-octadecadienoic acid, and its structural formula is: .
[0030] Therefore, the main active ingredient in the cornflower root extract of the present invention is phenolic acid, which has a good neuroprotective effect.
[0031] Experiment Example 1: Pharmacodynamic Experiment Experimental Methods: The cornflower root extract prepared in Example 10 of this invention was subjected to in vitro neuroprotective pharmacodynamic experiments using the CCK8 assay. The specific steps are as follows: HT22 cells were planted at 5 × 10⁶ cells per well. 3 Cells were seeded in 96-well cell culture plates and cultured for 24 h. The following groups were established: a blank group (complete culture medium), a control group (HT22 cells + complete culture medium), a model group (HT22 cells + complete culture medium + 4 μmol / L sodium glutamate), and an extract treatment group (HT22 cells + complete culture medium + cornflower root extract; the extract treatment group had three concentrations: 50 μg / mL, 100 μg / mL, and 200 μg / mL (low, medium, and high concentrations). After pre-incubation for 2 h, 4 μmol / L sodium glutamate was added to each concentration). Each group had three replicates. Cells were cultured for another 24 h, the culture medium was aspirated, and 100 μL of 10% CCK8 was added. The cells were incubated at 37°C for 0.5 h, and the absorbance of each well was measured at 450 nm. Cell viability was calculated using the cell viability formula.
[0032] Formula for calculating cell viability: Cell viability % = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.
[0033] Experimental results: The cell viability rates of the extract treatment group, model group, and control group are shown in Table 1.
[0034] As can be seen from the data in Table 1, compared with the model group, the cornflower root extract of the present invention has a significant neuroprotective effect on the glutamate-induced HT22 cell injury model at high concentrations, and can effectively counteract neuronal apoptosis caused by oxidative stress and improve the survival rate of HT22 cells.
[0035] In summary, the cornflower root extract of this invention exhibits significant neuroprotective effects against glutamate-induced HT22 cell damage, effectively combating neuronal apoptosis induced by oxidative stress and improving HT22 cell survival. This significant neuroprotective activity makes cornflower root extract a potential neuroprotective drug, and it is expected to provide a cost-effective and safe natural drug option for treating neurodegenerative diseases.
[0036] 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: S1, Soak the roots of cornflower in methanol, then extract by heating and reflux to obtain an extract. Concentrate the extract under reduced pressure to obtain the total extract of cornflower roots. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by silica gel column gradient elution to obtain 4 fractions. The third fraction was concentrated to obtain cornflower root extract.
2. The cornflower root extract according to claim 1, characterized in that... In step S1, 8 mL to 12 mL of methanol are added for every 1 g of cornflower root.
3. The cornflower root extract according to claim 1 or 2, characterized in that... In step S3, the eluent used in gradient elution is a mixture of petroleum ether and ethyl acetate.
4. The cornflower root extract according to claim 3, characterized in that... The volume ratios of petroleum ether and ethyl acetate are 50 / 1, 25 / 1, 8 / 1, and 1 / 1, respectively.
5. The cornflower root extract according to any one of claims 1 to 4, characterized in that... In step S1, during the reflux extraction, the temperature is 50°C to 60°C, the reflux extraction is performed 3 times, and the extraction time for each extraction is 1 to 3 hours.
6. The cornflower root extract according to any one of claims 1 to 5, characterized in that... In step S1, the cornflower root must be dried and pulverized before use.
7. The cornflower root extract according to any one of claims 1 to 6, characterized in that... In step S1, the soaking temperature is room temperature and the soaking time is 2 to 5 hours.
8. A method for preparing cornflower root extract according to any one of claims 2 to 7, characterized in that... Perform the following steps: S1, Soak the roots of cornflower in methanol, then extract by heating and reflux to obtain an extract. Concentrate the extract under reduced pressure to obtain the total extract of cornflower roots. S2, dilute the total extract of cornflower root with water to form a suspension, extract the suspension sequentially with petroleum ether, dichloromethane and ethyl acetate, concentrate the extracts respectively to obtain petroleum ether fraction extract, dichloromethane fraction extract and ethyl acetate fraction extract. S3, the dichloromethane fraction extract was separated by silica gel column gradient elution to obtain 4 fractions. The third fraction was concentrated to obtain cornflower root extract.
9. The use of a cornflower root extract according to any one of claims 1 to 7 in the preparation of a medicament for treating or preventing neurodegenerative diseases.