Composition with nerve protection function, lavender extract as well as preparation method and application of lavender extract

Abstract

The invention relates to the technical field of natural medicine, in particular to a composition with a nerve protection function, a lavender extract and a preparation method and application of the lavender extract, and the composition with the nerve protection function comprises luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A and rosmarinic acid. The lavender extract contains luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A and rosmarinic acid, and is prepared according to the following method: adding a required amount of an extraction solvent into crushed lavender, heating, refluxing and extracting, concentrating and drying the extract, and concentrating to obtain the lavender extract. The lavender extract containing the composition with the nerve protection function is obtained. The composition with the neuroprotection function and the lavender extract have good effects in BV-2 cell viability influence experiments and zebra fish Alzheimer disease model experiments, and a new solution is provided for research of drugs for preventing and treating Alzheimer disease.

Description

Technical Field

[0001] This invention relates to the field of natural medicine technology, specifically to a composition and its application with neuroprotective function, as well as a lavender extract containing such a composition, its preparation method, and its application. Background Technology

[0002] Research on Alzheimer's disease treatments currently faces multiple severe challenges. Traditional cognitive enhancers such as donepezil and memantine can only treat symptoms, improving cognitive symptoms but not stopping disease progression. Emerging disease-modifying therapies such as aducanatumab and lencanemab, while targeting the pathological mechanisms, still have limited and controversial clinical efficacy. Novel monoclonal antibody drugs can cause severe amyloid-associated imaging abnormalities (ARIA), including potentially fatal side effects such as cerebral edema and cerebral hemorrhage. Traditional drugs also commonly cause adverse reactions such as gastrointestinal reactions and neuropsychiatric symptoms. Drug development in this field faces almost hopeless challenges; the success rate was only 0.4% between 1995 and 2021. The reasons include the biological barrier of the blood-brain barrier limiting drug entry into the central nervous system, the slow disease progression leading to lengthy clinical trial cycles, extremely high research and development costs, and insufficient understanding of the complex pathological mechanisms of Alzheimer's disease, resulting in repeated failures of drug development strategies based on single hypotheses (such as the β-amyloid hypothesis). These systemic problems collectively constitute a major bottleneck in the field of Alzheimer's disease drug treatment.

[0003] Lavender( Lavandula ) is a member of the Lamiaceae family ( Lamicaceae Lavender is an important aromatic and medicinal plant with a long history of use and extensive medicinal value. The genus *Lavender* contains 39 known species, widely distributed throughout the world, including North Africa, France, Spain, and Italy. Some species have been introduced and cultivated in China and other parts of the world. As a traditional medicinal plant, lavender is renowned for its unique aromatic properties.

[0004] Recent studies have shown that the bioactive substances in lavender are not limited to its essential oil components. Aqueous or ethanolic extracts of lavender exhibit significant bioactivity in areas such as antioxidation, anti-inflammation, and neuroprotection. However, compared to research on essential oils, exploration of the bioactive components of these extracts has been significantly insufficient. This research bias may stem from traditional research focusing primarily on the aromatic properties of lavender while neglecting the important value of other active ingredients.

[0005] The medicinal value of lavender is widely recognized. The European Medicines Agency's Herbal Medicinal Products Committee has approved the use of narrow-leaved lavender flowers in the preparation of herbal teas and oral formulations, primarily for relieving stress and improving sleep quality. In traditional Chinese medicine, the use of lavender dates back to ancient times. The *Chinese Materia Medica* records its application in ancient texts such as the *Thirty-Six Volumes of Hui Prescriptions*, mainly for improving symptoms such as pain, neurasthenia, and forgetfulness. Currently, lavender is used in modern pharmaceutical preparations such as *Compound Gao Zi Ban Tablets* and *Aiweixin Oral Liquid*.

[0006] To clarify the current research status of non-essential oil components of lavender, a dual-track research strategy of "component isolation and activity evaluation" was adopted. Through a systematic analysis of relevant literature published between 2007 and 2024, the focus was on the isolation and identification of non-volatile components from lavender plants and the bioactivity of their extracts. Existing research indicates that lavender plants contain 151 non-volatile small molecule compounds, mainly including flavonoids, phenolic acids, lignans, and triterpenoids. Bioactivity studies have shown that lavender extracted with different solvents exhibits significant antioxidant, anti-inflammatory, neuroprotective, anti-diabetic, and anti-tumor effects.

[0007] Inflammation is a complex defense and repair response of the body to external stimuli such as pathogens, irritants, or infections. Modern medical research shows that this protective response plays an important role in the occurrence and progression of many age-related chronic diseases, including cardiovascular disease, diabetes, Alzheimer's disease, and some cancers. Of particular note is that chronic inflammation is a key factor in neurodegenerative diseases; peripheral inflammatory imbalance leads to neuroinflammation, affecting glial cells and subsequently damaging neurons. Oxidative stress also plays a crucial role in this process.

[0008] Lavender species exhibit significant antioxidant and neuroprotective effects, with different extracts demonstrating strong anti-inflammatory and neuroprotective activity. Narrow-leaved lavender has a long history of anti-inflammatory medicinal use, is a raw material in the European Pharmacopoeia, and is the only lavender species included in the Chinese Materia Medica. Although current research has preliminarily revealed the anti-inflammatory activity of narrow-leaved lavender, most efficacy evaluations are based on crude extracts, and a deeper understanding of the underlying substances responsible for its anti-inflammatory activity is still lacking. Summary of the Invention

[0009] This invention provides a composition with neuroprotective function, a lavender extract, a method for preparing the same, and its application, overcoming the shortcomings of the prior art and providing a composition and lavender extract with neuroprotective function that can be used to treat and prevent Alzheimer's disease.

[0010] One of the technical solutions of the present invention is achieved by the following measures: a composition with neuroprotective function, comprising: luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A and rosmarinic acid.

[0011] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: The mass ratio of the above-mentioned luteolin-7-O-glucoside, apigenin-7-O-glucoside, rosmarinic acid and salvianolic acid A is (0.5 to 2): (4 to 8): (50 to 80): (2 to 6).

[0012] The second technical solution of the present invention is achieved through the following measures: the application of a composition with neuroprotective function in the treatment and prevention of Alzheimer's disease.

[0013] The third technical solution of the present invention is achieved by the following measures: a lavender extract containing a composition with neuroprotective function, wherein the total amount of luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A and rosmarinic acid in the lavender extract is 6 mg / g to 9 mg / g.

[0014] The following are further optimizations and / or improvements to the third technical solution of the above invention: The lavender extract containing the composition with neuroprotective function described above was prepared by the following method: The required amount of extraction solvent was added to the pulverized lavender, and the mixture was heated under reflux for extraction. After the extract was concentrated and dried, a lavender extract containing a composition with neuroprotective function was obtained.

[0015] The extraction solvent used above is ethanol, which is 55% to 65% by volume.

[0016] The above-mentioned heating and reflux extraction is carried out at 80℃ to 90℃, with reflux extraction 1 to 3 times, and each reflux is 40 min to 90 min.

[0017] The ratio of lavender to extraction solvent in the above single reflux extraction is 100g:1000mL to 2000mL.

[0018] The fourth technical solution of the present invention is achieved through the following measures: a method for preparing a lavender extract containing a composition with neuroprotective function, comprising the following steps: The required amount of extraction solvent was added to the pulverized lavender, and the mixture was heated under reflux for extraction. After the extract was concentrated and dried, a lavender extract containing a composition with neuroprotective function was obtained.

[0019] The following are further optimizations and / or improvements to the fourth technical solution of the invention described above: The extraction solvent used above is ethanol, which is 55% to 65% by volume.

[0020] The above-mentioned heating and reflux extraction is carried out at 80℃ to 90℃, with reflux extraction 1 to 3 times, and each reflux is 40 min to 90 min.

[0021] The ratio of lavender to extraction solvent in the above single reflux extraction is 100g:1000mL to 2000mL.

[0022] The fifth technical solution of the present invention is achieved through the following measures: the application of a lavender extract containing a composition with neuroprotective function in the treatment and prevention of Alzheimer's disease.

[0023] This invention provides a composition with neuroprotective function, identifying the neuroprotective function of a combination of four compounds: luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid, and applying it to drugs for the prevention and treatment of Alzheimer's disease. Additionally, a method for effectively extracting this combination of four compounds from narrow-leaved lavender is provided. The obtained extract showed good results in experiments affecting BV-2 cell activity and in zebrafish Alzheimer's disease model experiments, providing a new solution for the research of drugs for the prevention and treatment of Alzheimer's disease. Detailed Implementation

[0024] 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.

[0025] The present invention will be further described below with reference to embodiments: Example 1: The neuroprotective composition comprises: luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid.

[0026] Example 2: As an optimization of the above example, the mass ratio of luteolin-7-O-glucoside, apigenin-7-O-glucoside, rosmarinic acid and salvianolic acid A is (0.05 to 0.15): (0.4 to 0.8): (5.0 to 8.0): (0.2 to 0.6).

[0027] Example 3: Application of this neuroprotective composition in the treatment and prevention of Alzheimer's disease.

[0028] Example 4: The lavender extract containing a composition with neuroprotective function, wherein the total amount of luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A and rosmarinic acid in the lavender extract is 6 mg / g to 9 mg / g.

[0029] Example 5: As an optimization of the above examples, the lavender extract containing the composition with neuroprotective function was prepared by the following method: The required amount of extraction solvent was added to the pulverized lavender, and the mixture was heated under reflux for extraction. After the extract was concentrated and dried, a lavender extract containing a composition with neuroprotective function was obtained.

[0030] Example 6: As an optimization of the above examples, the extraction solvent is ethanol with a volume percentage of 55% to 65%.

[0031] Example 7: As an optimization of the above example, the heating and reflux extraction is carried out at 80°C to 90°C, and the reflux extraction is carried out 1 to 3 times, with each reflux lasting 40 to 90 minutes.

[0032] Example 8: As an optimization of the above examples, the ratio of lavender and extraction solvent in a single reflux extraction is 100g:1000mL to 2000mL.

[0033] Example 9: A method for preparing the lavender extract containing a composition with neuroprotective function, comprising the following steps: The required amount of extraction solvent was added to the pulverized lavender, and the mixture was heated under reflux for extraction. After the extract was concentrated and dried, a lavender extract containing a composition with neuroprotective function was obtained.

[0034] The extraction solvent used above is ethanol, which is 55% to 65% by volume.

[0035] The above-mentioned heating and reflux extraction is carried out at 80℃ to 90℃, refluxed 1 to 3 times, with each reflux lasting 40 to 90 minutes. The ratio of lavender to extraction solvent in each reflux extraction is 100g:1000mL to 2000mL.

[0036] Example 10: Application of the lavender extract containing the composition with neuroprotective function in the treatment and prevention of Alzheimer's disease.

[0037] Example 11: The neuroprotective composition comprises, by weight, 0.11 parts of luteolin-7-O-glucoside, 0.66 parts of apigenin-7-O-glucoside, 7.7 parts of rosmarinic acid, and 0.44 parts of salvianolic acid A.

[0038] Example 12: The neuroprotective composition comprises, by weight, 0.11 parts of luteolin-7-O-glucoside, 0.55 parts of apigenin-7-O-glucoside, 6.6 parts of rosmarinic acid, and 0.55 parts of salvianolic acid A.

[0039] Example 13: A lavender extract containing a composition with neuroprotective function was prepared according to the following steps: Weigh 50.0 g of narrow-leaved lavender sample and extract twice, sequentially, with 750 mL and 500 mL of 60% (v / v) ethanol at 85 °C for 60 min each time. Combine the two extracts, concentrate under vacuum by rotary evaporation, and then freeze-dry to obtain a lavender extract containing a composition with neuroprotective function.

[0040] Example 14: A lavender extract containing a composition with neuroprotective function was prepared according to the following steps: Weigh 50.0 g of narrow-leaved lavender sample and extract twice, sequentially, with 750 mL and 500 mL of 65% (v / v) ethanol at 80 °C for 60 min each time. Combine the two extracts, concentrate under vacuum by rotary evaporation, and then freeze-dry to obtain a lavender extract containing a composition with neuroprotective function.

[0041] Comparative Example 1: The difference from Example 13 is that 30% ethanol by volume was used for extraction.

[0042] Comparative Example 2: The difference from Example 13 is that 80% ethanol by volume was used for extraction.

[0043] Experimental Example 1: The extracts from Examples 13 and 14 were analyzed by liquid chromatography-triple quadrupole mass spectrometry to obtain the contents of luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A and rosmarinic acid in the extracts. The results are shown in Table 1.

[0044] Experiment Example 2: Effect of BV-2 Cell Viability Experiment (1) BV-2 cell culture: DMEM high-glucose complete medium was prepared by mixing basal medium, 10% (v / v) premium fetal bovine serum, and 1% (v / v) penicillin-streptomycin. BV-2 cells were seeded in T25 culture flasks and cultured at 37°C, 5% CO2, and saturated humidity, with the medium changed every 24 hours. Cells were passaged when the cell density reached 70% to 80%, and cells in the logarithmic growth phase were selected for subsequent experiments.

[0045] (2) Aβ 1-42 Aging Prepare pre-cooled hexafluoroisopropanol (HFIP), and add Aβ. 1-42 The powder was completely dissolved to prepare a colorless, transparent solution with a concentration of 1 mg / mL. After standing on ice for 10 minutes, Aβ was... 1-42 Aliquot the HFIP solution into clean centrifuge tubes and evaporate the HFIP at room temperature in a fume hood until a clear, transparent film forms at the bottom of the tubes. Store at -80°C. Before use, first dilute Aβ with DMSO. 1-42 Dissolve to 5 mg / mL, then dilute to 5 μg / mL with DMEM high-glucose complete medium. Pre-incubate at 4 °C for 24 h, centrifuge at 14,000 × g for 10 min, and use the supernatant for experiments.

[0046] (3) Cell grouping and anti-inflammatory activity detection Cells were divided into a blank control group and an Aβ group. 1-42 The control group, positive control (donepezil) group, and Examples 11 to 14 and Comparative Examples 1 to 2. The blank control group was a complete culture medium containing cells, Aβ 1-42 The group was added with Aβ containing 5 μmol / L 1-42 The complete culture medium was used, and the positive control group was given a complete culture medium solution containing 5 μmol / L donepezil.

[0047] BV-2 cells in the logarithmic growth phase were seeded into 96-well plates (seeding density 5 × 10⁶). 4 (4 replicates per group) After 18 hours of culture, cell viability in each group was detected using the CCK-8 assay.

[0048] Subsequently, except for the blank control group, all other groups were treated with Aβ containing 5 μmol / L. 1-42 A cell damage model was established by incubation in complete culture medium for 6 hours. Subsequently, cells containing Aβ were incubated... 1-42 The complete culture medium was replaced with the complete culture medium of Examples 11 to 14 and Comparative Examples 1 to 3, and cultured for another 24 hours. Cell viability in each group was detected using the CCK-8 assay, and the Aβ levels of BV-2 cells in Examples 11 to 14 and Comparative Examples 1 to 3 were evaluated based on the absorbance (OD) values ​​at 450 nm. 1-42 The improvement effect of the damage model is shown in Table 2.

[0049] In this embodiment, the dosage concentrations of the compositions of Examples 11 and 12 were 0.11 mg / L of luteolin-7-O-glucoside, 0.66 mg / L of apigenin-7-O-glucoside, 7.7 mg / L of rosmarinic acid, 0.44 mg / L of salvianolic acid A, and 0.11 mg / L of luteolin-7-O-glucoside, 0.55 mg / L of apigenin-7-O-glucoside, 6.6 mg / L of rosmarinic acid, and 0.55 mg / L of salvianolic acid A. 0.1 g of each of the extracts of Examples 13 and 14, as well as Comparative Examples 1 and 2, were prepared into solutions with a final concentration of 1 g / L before administration.

[0050] β-amyloid (Aβ1-42)-induced neuroinflammation is a key pathological process in the development of neurodegenerative diseases. This invention evaluated the effects of Examples 11-14 and Comparative Examples 1-2, and donepezil on Aβ1-42 using cell models. 1-42 The effects of stimulating the secretion of inflammatory factors in BV-2 microglia were investigated. The results showed that the extracts from Example 13, at concentrations of luteolin-7-O-glucoside 0.11 mg / L, apigenin-7-O-glucoside 0.66 mg / L, rosmarinic acid 6.9 mg / L, and salvianolic acid A 0.4 mg / L, had a good protective effect on neuroinflammatory model cells.

[0051] Experiment Example 3: Anti-Alzheimer's Disease Activity Experiment Zebrafish were placed at a density of 30 zebrafish per well in 6-well culture plates and incubated at a constant temperature of (28±1)°C. Each experiment was repeated three times.

[0052] The experimental groups were as follows: zebrafish treated with aluminum chloride formed the model group; zebrafish treated with aluminum chloride and donepezil formed the positive control group; zebrafish treated with 0.1% DMSO formed the negative control group; and zebrafish treated with different concentrations of extract and aluminum chloride formed the experimental group.

[0053] After treatment, the zebrafish's movement trajectories were recorded over 60 minutes using a behavior analyzer. All experiments included three 60-minute light / dark phase cycles (10 minutes of light phase and 10 minutes of dark phase). The zebrafish's movement speed was analyzed, and the average speed over 60 minutes was calculated (to assess motor dysfunction). Speed ​​differences during light-dark transitions were compared (to assess reaction time), and statistical analysis was performed on the movement speed and speed differences for each experimental group during light-dark transitions. The results are shown in Table 3 (SD in Table 3 represents standard deviation, and p-value represents significance level). In this experimental example, the dosage concentrations of the compositions in Examples 11 and 12 were 0.11 mg / L of luteolin-7-O-glucoside, 0.66 mg / L of apigenin-7-O-glucoside, 7.7 mg / L of rosmarinic acid, and 0.44 mg / L of salvianolic acid A, respectively. The extracts from Examples 13 and 14, as well as Comparative Examples 1 and 2, were each prepared into solutions with a concentration of 1 g / L before administration.

[0054] The present invention evaluated the bioactivity of Examples 11 to 14 and Comparative Examples 1 to 2 in a zebrafish AD model. The results showed that the extract of Example 13 had the best effect on restoring the motor function of the AD zebrafish model at the following administration concentrations: luteolin-7-O-glucoside 0.11 mg / L, apigenin-7-O-glucoside 0.66 mg / L, rosmarinic acid 6.9 mg / L, and salvianolic acid A 0.4 mg / L.

[0055] Luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid are compounds with complex biological regulatory characteristics, involving multiple biological pathways and closely related to inflammation, cancer, neurodegeneration, and diabetic complications. Lavender components play an important role in the pathogenesis research of Alzheimer's disease. This invention has determined the neuroprotective functions of luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid in lavender components and provided the optimal extraction method for the effective components.

[0056] In summary, based on research into the pathogenesis of Alzheimer's disease involving lavender components, this invention has identified the neuroprotective function of a combination of four compounds: luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid, and applied them to the development of drugs for the prevention and treatment of Alzheimer's disease. Furthermore, this invention provides an effective method for extracting these four compounds from narrow-leaved lavender. The obtained extract showed good results in experiments affecting BV-2 cell activity and in zebrafish Alzheimer's disease model experiments, providing a new solution for the research of drugs for the prevention and treatment of Alzheimer's disease.

[0057] The above technical features constitute various 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 composition with neuroprotective function, characterized in that... include: Luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid.

2. The composition with neuroprotective function according to claim 1, characterized in that... The mass ratio of luteolin-7-O-glucoside, apigenin-7-O-glucoside, rosmarinic acid, and salvianolic acid A is 0.5 to 2:4 to 8:50 to 80:2 to 6.

3. The use of a composition with neuroprotective function according to claim 1 or 2 in the treatment and prevention of Alzheimer's disease.

4. A lavender extract containing the composition with neuroprotective function as described in claim 1 or 2, characterized in that... The total amount of luteolin-7-O-glucoside, apigenin-7-O-glucoside, salvianolic acid A, and rosmarinic acid in lavender extract ranges from 6 mg / g to 9 mg / g.

5. The lavender extract containing a composition with neuroprotective function according to claim 4, characterized in that... It is prepared by the following method: The required amount of extraction solvent was added to the pulverized lavender, and the mixture was heated under reflux for extraction. After the extract was concentrated and dried, a lavender extract containing a composition with neuroprotective function was obtained.

6. The lavender extract containing a composition with neuroprotective function according to claim 5, characterized in that... The extraction solvent is ethanol, which is 55% to 65% by volume.

7. The lavender extract containing a composition with neuroprotective function according to claim 5 or 6, characterized in that... The heating and reflux extraction is carried out at 80℃ to 90℃, and the reflux extraction is carried out 1 to 3 times, with each reflux lasting 40 to 90 minutes.

8. The lavender extract containing a composition with neuroprotective function according to claim 7, characterized in that... The ratio of lavender to extraction solvent in a single reflux extraction is 100g:1000mL to 2000mL.

9. A method for preparing a lavender extract containing a composition having neuroprotective function according to claim 4, 6, 7, or 8, characterized in that... Includes the following steps: The required amount of extraction solvent was added to the pulverized lavender, and the mixture was heated under reflux for extraction. After the extract was concentrated and dried, a lavender extract containing a composition with neuroprotective function was obtained.

10. The use of a lavender extract containing a composition with neuroprotective function according to claims 4 to 8 in the treatment and prevention of Alzheimer's disease.

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