Process for the preparation of edible oils having neuroprotective effects and / or alleviating ad

Abstract

The application belongs to the technical field of natural product extraction and application, and discloses a preparation method of edible oil with nerve protection and / or AD alleviation, comprising: frying raw gardenia fruits at a temperature of 140-160 DEG C for 10-20 minutes, cooling, physically pressing the obtained scorched gardenia fruits below 50 DEG C, and obtaining edible oil after filtering and removing impurities. The prepared edible oil can significantly protect nerve cells damaged by H2O2, can significantly improve the spatial memory and learning ability in an APP / PS1 mouse model, can reduce the oxidative stress damage of brain tissue, can inhibit nerve inflammation, and can protect the structure and function of neurons. The preparation process is simple, green and safe, the raw material is a homologous plant of medicine and food, the source is wide, and the application provides a safe and effective daily dietary intervention way for the prevention and treatment of Alzheimer's disease and other neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and application technology, and in particular relates to a method for preparing edible oils with neuroprotective effects and / or alleviating AD. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common type of dementia in the elderly. Its pathological features mainly include β-amyloid protein deposition, tau protein hyperphosphorylation, and the resulting neuronal loss and synaptic dysfunction. Currently, the etiology of AD is not fully understood, and there is a lack of effective cures; existing drugs primarily focus on symptom relief. Therefore, finding safe, effective, and multi-targeted active ingredients or functional foods from natural products to intervene in AD has become a research hotspot.

[0003] Gardenia, an evergreen shrub belonging to the Rubiaceae family and the Gardenia genus, is used in traditional Chinese medicine for its heat-clearing, anti-inflammatory, calming, and sedative effects. Gardenia fruit oil is a natural oil extracted from gardenia fruit, rich in unsaturated fatty acids, phytosterols, iridoids, crocin, and other bioactive components. Current research on gardenia fruit oil focuses primarily on extraction process optimization, basic component analysis, and limited exploration of its antidepressant activity; however, systematic research on its neuroprotective effects, particularly its role in improving Alzheimer's disease (AD), is severely lacking. Furthermore, oil obtained by directly pressing raw gardenia fruit often has a slightly astringent odor, and the dissolution rate of some active ingredients is low. The refining process also results in a significant loss of active substances, hindering the effective development of the unique functional value of gardenia fruit oil. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for preparing edible oils with neuroprotective effects and / or to alleviate Alzheimer's disease (AD). This preparation method is simple to operate, green and safe, and suitable for industrialization. It fills the application gap of gardenia fruit oil in the field of intervention for neurodegenerative diseases and provides a new natural edible oil product solution for the prevention and treatment of Alzheimer's disease in daily diet.

[0005] To achieve the above objectives, the present invention provides a method for preparing edible oils with neuroprotective effects and / or alleviating AD, comprising the following steps: S1. Roasting and processing: Stir-fry raw gardenia fruit, let it cool, and you will get roasted gardenia fruit; S2. Low-temperature pressing: The roasted gardenia fruit is pressed at a low temperature, then filtered to remove impurities, to obtain edible oil with neuroprotective effects and / or to alleviate AD.

[0006] Preferably, in S1, the stir-frying temperature is 140-160℃ and the stir-frying time is 10-20 minutes; the surface of the charred gardenia fruit is charred brown, and the inner surface of the pericarp and the surface of the seeds are yellowish-brown; in S2, low-temperature pressing is physical pressing at a temperature below 50℃.

[0007] The present invention also provides an edible oil prepared by the above-described method for preparing an edible oil with neuroprotective effects and / or relieving AD.

[0008] This invention also provides the use of edible oils with neuroprotective and / or AD-relieving effects in the preparation of products for the prevention and / or treatment of neurodegenerative diseases.

[0009] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0010] Preferably, edible oils can prevent and / or treat neurodegenerative diseases by increasing the activity of superoxide dismutase and glutathione peroxidase in brain tissue, reducing the content of interleukin-6 and interleukin-1β in brain tissue, and improving the morphology and arrangement of neurons.

[0011] Preferably, the amount of edible oil added in the preparation of products for the prevention and / or treatment of neurodegenerative diseases is 300-500 μg / mL.

[0012] The present invention also provides a method for enhancing the neuroprotective effect of edible oil by adding edible oil with neuroprotective effect and / or alleviating AD prepared by the preparation method described above to the edible oil; or by using the edible oil infused with the roasted gardenia fruit described above.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects: 1) This invention is the first to use a process of roasting (stir-frying at 140-160℃ for 10-20 minutes) combined with low-temperature physical pressing (below 50℃) to prepare gardenia fruit oil. Compared with direct pressing of raw gardenia fruit or conventional refined oil, this process significantly improves the dissolution rate and bioavailability of active ingredients such as total iridoids, total crocin, total phenols, and total flavonoids in the oil. It also retains the unique roasted aroma and mild properties of roasted gardenia fruit, avoids organic solvent residues, and is green and safe.

[0014] 2) This invention elucidates the multi-target neuroprotective pathways of gardenia fruit oil: ① Antioxidant: enhances SOD and GSH-PX activity in brain tissue and reduces MDA content; ② Anti-inflammatory: significantly reduces the levels of pro-inflammatory factors IL-6 and IL-1β; ③ Neuroprotective: improves the morphology and arrangement of neurons in brain tissue, increases the number of Nissl bodies, and maintains the structural and functional integrity of neurons.

[0015] 3) The method for enhancing the neuroprotective effect of edible oil provided by the present invention can directly soak the charred gardenia fruit in ordinary edible oil, or directly mix the charred gardenia fruit oil prepared in the present invention with ordinary edible oil. The operation is simple, does not require complicated active ingredient separation and purification steps, and is easy to produce and promote.

[0016] 4) This invention uses gardenia as a medicinal and edible plant, which is widely available and inexpensive; the low-temperature spiral pressing preparation process is simple, green and environmentally friendly, with no organic solvent residue; and it has high safety. It can be made into oral or injectable preparations with pharmaceutically acceptable excipients, providing a brand-new natural drug candidate for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, which has both important theoretical significance and market promotion value.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 Line graphs showing the DPPH free radical scavenging rates of different gardenia fruit oils; Figure 2 The effect of different concentrations of H2O2 on the survival rate of PC12 cells is shown in Figure 1. ** represents P < 0.01. Figure 3 The figure shows the cytotoxicity of different gardenia fruit oils on PC12 cells. In the figure, A is raw gardenia fruit oil, B is charred gardenia fruit oil, C is raw water gardenia fruit oil, D is charred water gardenia fruit oil, and E is refined gardenia fruit oil. * represents P<0.05 and ** represents P<0.01. Figure 4 The effects of different gardenia fruit oils on H2O2-damaged PC12 cells are shown in the figure. A represents raw gardenia fruit oil, B represents charred gardenia fruit oil, C represents raw gardenia fruit oil, and D represents charred gardenia fruit oil. ## represents a significant difference from the control group (P<0.01), and * represents a significant difference from the H2O2-treated group (*P<0.05, **P<0.01). Figure 5 The bar chart shows the effects of various components of gardenia fruit oil on H2O2-damaged PC12 cells. In the figure, * indicates a significant difference from the control group, * represents P<0.05, and ** represents P<0.01; different letters indicate significant differences within the group, P<0.05. Figure 6The figure shows the effect of gardenia fruit oil on the cognitive function of APP / PS1 mice. In the figure, A is the result of the successful escape latency of mice after five days, B is the representative swimming trajectory of mice on the fifth day of training, C is the number of times mice crossed the platform during the exploration phase, D is the activity time of mice in the target quadrant during the exploration phase, E is the time of the first crossing of the platform during the exploration phase, and F is the representative swimming trajectory of mice during the exploration phase. ** represents P<0.01. Figure 7 The figure shows the effect of gardenia fruit oil on the success rate of Y-maze alternation in APP / PS1 mice. In the figure, A is the representative movement trajectory of each group of mice, and B is the percentage of correct alternations in each group of mice. ** represents P<0.01. Figure 8 The effect of GFO on HE staining results of brain tissue from APP / PS1 mice; Figure 9 The effect of GFO on Nissl staining results in brain tissue of APP / PS1 mice; Figure 10 The effect of gardenia fruit oil on oxidative stress levels in the cortex of APP / PS1 mice is shown in the figure. A represents SOD activity, B represents MDA content, C represents GSH-PX activity, * represents P<0.05, and ** represents P<0.01. Figure 11 The effect of gardenia fruit oil on the levels of pro-inflammatory factors in APP / PS1 mice is shown in the figure. A represents TNFα level, B represents IL-6 level, C represents IL-1β level, * represents P<0.05, and ** represents P<0.01. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0023] The PC12 cells used in the experiments of this invention were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. The specific methods for culture, thawing, passage, and cryopreservation are as follows.

[0024] PC12 cell culture: PC12 cells were cultured in high-glucose DMEM medium containing 1% penicillin-streptomycin mixture and 10% fetal bovine serum at 37°C with 5% CO2. Microscopic observation showed that the PC12 cells were in an adherent state.

[0025] PC12 cell resuscitation: Remove the cryovials containing PC12 cells from liquid nitrogen and immediately place them in a 37°C water bath for thawing. After thawing, wipe the surface of the cryovials with alcohol and transfer them to a clean bench. Transfer the PC12 cell suspension from the cryovials to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, add 1 mL of complete culture medium to resuspend the cells, and transfer them to a culture dish. Incubate in a 5% CO2 incubator at 37°C.

[0026] PC12 cell passage: When the cell density in the culture dish reaches approximately 85%, cell passage can be performed. Discard the waste liquid in the culture dish, wash the cells 2-3 times with sterile PBS, aspirate the PBS, add 1 mL of 0.25% trypsin, and incubate for 1 min to digest, allowing the cells to gradually detach from the culture dish wall. Immediately add at least 1.5 mL of high-glucose DMEM medium containing 1% penicillin-streptomycin mixture and 10% fetal bovine serum to terminate the digestion. Use a pipette to transfer the cells from the culture dish into the culture medium and then transfer them to sterile centrifuge tubes. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and passage at a 1:3 ratio.

[0027] PC12 cell cryopreservation: PC12 cells were digested during the logarithmic growth phase, following the same procedure as PC12 cell passage. After discarding the supernatant, 1 mL of cell cryopreservation solution was added to resuspend the cells, which were then transferred to cryovials. The cryovials were then subjected to programmed cooling and transferred to liquid nitrogen for preservation and future use.

[0028] Example 1 Preparation of processed gardenia fruit oil and raw gardenia fruit oil.

[0029] I. Processing of Gardenia Fruit.

[0030] (1) Preparation and processing of raw products.

[0031] Two varieties of Gardenia jasminoides, Gardenia jasminoides var. serrata and Gardenia jasminoides var. serrata, were selected. The raw product preparation and charring processing were carried out respectively, referring to the 2020 edition of the Chinese Pharmacopoeia. The specific procedures are as follows: Gardenia: Harvest the gardenia fruit when it is ripe and reddish-yellow in September-November. Remove the fruit stalks and impurities, steam until steam rises, take it out, and dry it.

[0032] Gardenia jasminoides: Harvest the gardenia jasminoides when the fruit is ripe and reddish-yellow from September to November. Remove the fruit stalks and impurities, steam until steam rises, take it out, and dry it.

[0033] Processed Gardenia: Set the temperature of the roasting machine to 150℃. After the temperature stabilizes, put in 1kg of raw Gardenia and stir-fry. After 15 minutes, the surface of the Gardenia fruit will turn dark brown, and the inner surface of the peel and the surface of the seeds will turn yellowish-brown. Remove and let cool.

[0034] Roasted Gardenia (processed): Set the temperature of the roasting machine to 150℃. After the temperature stabilizes, put in 1kg of raw Gardenia and stir-fry. After 15 minutes, the surface of the Gardenia fruit will turn dark brown, and the inner surface of the peel and the surface of the seeds will turn yellowish-brown. Remove and let cool.

[0035] (2) Preparation of gardenia fruit oil with different treatments.

[0036] The four types of gardenia fruit raw materials obtained—raw gardenia, charred gardenia, raw water gardenia, and charred water gardenia—were subjected to low-temperature physical pressing using a screw oil press, with the pressing temperature controlled below 50℃. After pressing, the raw gardenia fruit oil, charred gardenia fruit oil, raw water gardenia fruit oil, and charred water gardenia fruit oil were obtained respectively.

[0037] Meanwhile, the gardenia fruit oil from the tar water was refined through degumming, deacidification, decolorization, and deodorization to obtain refined gardenia fruit oil. A total of 5 gardenia fruit oil samples were obtained for subsequent quality and activity evaluation.

[0038] (3) Evaluation of the quality and activity of gardenia fruit oil under different treatments.

[0039] ① Physicochemical indicators and sensory evaluation.

[0040] Physicochemical indicators: Acid value was determined according to GB5009.229-2016, and peroxide value was determined according to GB5009.227-2016. The results showed that the acid value (all less than 3 mg / g) and peroxide value (all less than 0.25 g / 100 g) of the five gardenia fruit oils met the requirements of GB2716-2018 "National Food Safety Standard for Vegetable Oils", and possessed the basic quality of edible vegetable oils.

[0041] Sensory evaluation: Three core indicators were selected: color, caramel aroma, and bitterness. Twelve healthy sensory evaluators (six men and six women) scored the five gardenia fruit oils, and the scores were added together to obtain the total sensory evaluation score. The results showed that none of the five gardenia fruit oils had obvious unpleasant tastes. The caramelized gardenia fruit oil had a rich caramel aroma and scored significantly higher than the raw gardenia fruit oil of the same type. The refined gardenia fruit oil had the brightest color, but the caramel aroma was very faint.

[0042] ② Determination of active ingredient content: The contents of total iridoids, total crocin, total phenols, and total flavonoids in the five gardenia fruit oils were determined, and the results are shown in Table 1.

[0043] Table 1. Content of active ingredients in different gardenia fruit oils

[0044] Note: Different letters in the same row indicate significant differences in data (P<0.05), and "-" indicates that the component was not detected.

[0045] The results showed that processing significantly increased the dissolution of active ingredients in gardenia fruit oil. Specifically, the total crocin, total iridoids, total phenols, and total flavonoids in the processed charred gardenia fruit oil were all higher than those in the raw gardenia fruit oil of the same variety. Among them, the overall content of active ingredients in water gardenia fruit oil was significantly higher than that in mountain gardenia fruit oil, and the charred water gardenia fruit oil had the richest variety and highest content of active substances.

[0046] ③ In vitro antioxidant activity assay: Weigh an appropriate amount of DPPH reagent, dissolve it in anhydrous ethanol, and prepare a 0.101 mmol / mL DPPH solution (prepare fresh before use). Dilute various gardenia fruit oil samples to a series of concentrations with anhydrous ethanol, using anhydrous ethanol as a blank control. Mix 0.1 mL of DPPH solution with 0.1 mL of the test solution, shake well, and incubate in a dark room for 1 h. Measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging rate using the following formula, and calculate the IC50 of various gardenia fruit oils. 50 value.

[0047] DPPH free radical scavenging rate = [A0 - (A2 - A1)] / A0 × 100%.

[0048] In the formula: A0 is 0.1 mL DPPH solution + 0.1 mL anhydrous ethanol; A1 is 0.1 mL test solution + 0.1 mL anhydrous ethanol; A2 is 0.1 mL test solution + 0.1 mL DPPH solution.

[0049] The results are as follows Figure 1 As shown, the DPPH free radical scavenging rate gradually increased with the increase of oil concentration, among which the gardenia fruit oil with tar water showed the best in vitro antioxidant effect (IC50).50 2.19 mg / mL), followed by Gardenia jasminoides fruit oil (IC50). 50 2.35 mg / mL), raw gardenia fruit oil (IC50) 50 2.87 mg / mL), raw gardenia fruit oil (IC50) 50 4.25 mg / mL) and refined gardenia fruit oil (IC50) 50 (6.89 mg / mL), indicating that both the processed gardenia fruit oil and the processed gardenia fruit oil have outstanding antioxidant activity.

[0050] ④ Determination of neuroprotective activity of PC12 cells.

[0051] Establishment of H2O2-induced PC12 cell damage model and screening of gardenia fruit oil concentration: PC12 cells were cultured at 1×10⁶ cells per well. 5 Cells were seeded in 96-well plates and cultured adherently for 48 hours (the original culture medium was removed and replaced with fresh culture medium after the first 24 hours of culture). 100 μL of complete culture medium was added to each well in the control group, while 100 μL of different concentrations of H2O2 (40, 60, 80, 100, 120, 140, 160 μmol / L) diluted with complete culture medium was added to each well in the H2O2 damage group. The cells were then cultured in an incubator for another 2 hours. After culture, cell viability was detected using the MTT assay to determine the H2O2 concentration used for modeling.

[0052] The results are as follows Figure 2 As shown, compared with the control group, the cell survival rate decreased significantly with increasing H2O2 concentration. When the H2O2 concentration was 100 μmol / L, the cell survival rate was 57.53 ± 1.02%, close to the half-lethal rate. Therefore, this concentration was selected for subsequent experimental studies.

[0053] PC12 cells were spaced at 1 × 10⁶ cells per well. 5 After inoculating each well of a 96-well plate and incubating for 24 hours, the waste liquid was removed. In the control group, 100 μL of complete culture medium was added to each well, while in the sample group, 100 μL of gardenia fruit oil of different concentrations (raw gardenia fruit oil, charred gardenia fruit oil, raw water gardenia fruit oil, charred water gardenia fruit oil: 100-800 μg / mL; refined gardenia fruit oil: 200-2000 μg / mL) diluted with complete culture medium was added to each well. After treating PC12 cells for 24 hours, the cytotoxicity range of each gardenia fruit oil was determined by the MTT assay.

[0054] result Figure 3As shown, refined gardenia fruit oil (concentration range of 0-1200 μg / mL), raw gardenia fruit oil (concentration range of 0-600 μg / mL), charred gardenia fruit oil (concentration range of 0-700 μg / mL), raw water gardenia fruit oil (concentration range of 0-700 μg / mL), and charred water gardenia fruit oil (concentration range of 0-600 μg / mL) were selected for subsequent cell protection experiments.

[0055] Cell protection assay: PC12 cells were cultured at a concentration of 1 × 10⁶ cells per well. 5 Cells were seeded in 96-well plates and cultured adherently for 24 hours. After removing the waste liquid, samples were added according to the following groups: Control group: 100 μL of complete culture medium was added to each well and cultured for 24 hours, followed by another 100 μL of complete culture medium and culturing for 2 hours; H2O2 damage group: 100 μL of complete culture medium was added to each well and incubated for 24 hours, followed by another 100 μL of 100 μmol / L H2O2 and culturing for 2 hours; Sample group: 100 μL of complete culture medium containing gardenia fruit oil was added to each well and cultured for 24 hours, followed by another 100 μmol / L H2O2 and culturing for 2 hours. After culture, cell viability was assessed using the MTT assay.

[0056] The results are as follows Figure 4 As shown, raw gardenia fruit oil achieved the best cell protection effect at 400 μg / mL, increasing the survival rate of damaged cells by approximately 15.84%. Charred gardenia fruit oil increased the survival rate of damaged cells by approximately 17.50% at 300 μg / mL. Raw gardenia fruit oil increased the survival rate of damaged cells from 52.18±1.89% to 70.30±3.90% at 400 μg / mL. Charred gardenia fruit oil showed a significant cell protection effect at 100 μg / mL, increasing the survival rate of damaged cells from 51.27±0.51% to 88.93±1.04% at the optimal protective concentration of 400 μg / mL, an increase of approximately 26.76%. Refined gardenia fruit oil, however, showed a weaker cell protection effect, increasing the survival rate of damaged cells by only approximately 11.24%. In summary, the cell protection capabilities of various gardenia fruit oils are in the following order: charred gardenia fruit oil > raw gardenia fruit oil > charred mountain gardenia fruit oil > raw mountain gardenia fruit oil > refined gardenia fruit oil.

[0057] It is evident that the cell-protective effect of gardenia fruit oil is generally superior to that of gardenia fruit oil. Furthermore, the cell-protective effect of charred gardenia fruit oil, processed using traditional methods, is superior to that of unprocessed raw gardenia fruit oil. Refined gardenia fruit oil exhibits the worst cell-protective effect. Gardenia fruit oil is rich in unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid, as well as characteristic active ingredients completely lacking in edible oils (soybean oil, rapeseed oil, peanut oil, etc.). These components interact and influence each other, resulting in powerful antioxidant, anti-inflammatory, and neuroprotective activities, which is the material basis for the efficacy of gardenia fruit oil.

[0058] Gardenia jasminoides from Jiaoshui was selected as the core raw material for the preparation of edible oil (gardenia fruit oil), and all subsequent examples used gardenia jasminoides from Jiaoshui fruit oil as the research object.

[0059] Example 2 Determination of the protective effect of different components of gardenia fruit oil on H2O2-damaged PC12 cells.

[0060] Preparation of Gardenia Fruit Oil Components: ①Preparation of PGFO: Place the charred gardenia fruit into the feed inlet of a screw oil press and press it at a temperature of less than 50°C. After filtering to remove impurities, PGFO is obtained.

[0061] ② Preparation of deactivated PGFO: Take 0.50 g of PGFO sample, add 5 mL of n-hexane, and vortex. After the oil sample is fully dissolved, add 5 mL of 90% methanol solution, sonicate for 2 min, and then centrifuge at 8000×g for 10 min. After separation, recover the n-hexane phase and continue eluting the active substance. Repeat the above process 3 times. The collected n-hexane phase is then rotary evaporated at 35℃ to remove n-hexane, yielding deactivated PGFO.

[0062] ③Preparation of RGFO: Accurately weigh 100.00g of PGFO, heat to 60±5℃, slowly add 60mL of distilled water, stir at 60r / min for 20min, let stand and cool, then centrifuge (8000r / min, 20min) to separate the gel layer, and collect the upper degummed oil; heat 100.00g of degummed oil to 75℃, add 10mL of 15% NaOH solution, stir at 60r / min for 20min, let stand and cool, then centrifuge (8000r / min, 20min) to separate the soapstock, and collect the deacidified oil; heat 100.00g of deacidified oil to 60℃, add 5g of activated clay, kaolin, and activated carbon respectively, stir at 100r / min for 2h, let stand and cool, then centrifuge (8000r / min, 20min), discard the precipitate, and obtain RGFO.

[0063] PC12 cells were spaced at 1 × 10⁶ cells per well. 5Cells were seeded in 96-well plates and cultured adherently for 24 hours. After removing the waste liquid, samples were added according to the following groups: Control group: 100 μL of complete culture medium was added to each well and cultured for 24 hours, followed by another 100 μL of complete culture medium and culturing for 2 hours. H2O2 treatment group: 100 μL of complete culture medium was added to each well and incubated for 24 hours, followed by another 100 μL of 100 μmol / L H2O2 and culturing for 2 hours. Sample groups: 300 μg / mL, 400 μg / mL, and 500 μg / mL of complete culture medium containing gardenia fruit oil (PGFO, deactivated PGFO, and RGFO, respectively) were added to each well and cultured for 24 hours, followed by another 100 μmol / L H2O2 and culturing for 2 hours. Cell viability was assessed using the MTT assay after culture.

[0064] The results are as follows Figure 5 As shown, when PGFO, deactivated PGFO, and RGFO were applied to H2O2-damaged PC12 cells, PGFO significantly protected PC12 cells; and the cell-protective effect of PGFO was significantly higher than that of deactivated PGFO and RGFO. This indicates that the protective effect of gardenia fruit oil on PC12 cells is significantly reduced after the deactivation or refining process, due to the alteration of the content of active ingredients. This demonstrates that the active components in gardenia fruit oil can, to some extent, enhance the neuroprotective efficacy.

[0065] Meanwhile, deactivated PGFO showed significant cell protection at a concentration of 400 μg / mL, and increased cell viability by 10.43% at a concentration of 500 μg / mL. RGFO at the same concentration was less protective, increasing cell viability by only 5.53% at 500 μg / mL. Deactivated PGFO only altered the content of its active ingredients, while the refining process of gardenia fruit oil not only removed the active components but also altered the lipid components to some extent. This demonstrates that the lipid components in gardenia fruit oil are an important factor affecting the protection of nerve cells.

[0066] Example 3 Animal experiments have verified that gardenia fruit oil has a relieving effect on Alzheimer's disease (AD).

[0067] Four-month-old male APP / PS1 double transgenic mice (experimental group) and their littermates C57BL / 6 wild-type male mice (control group) were selected and acclimatized for one week under conditions of 25±2℃ and 50±5% relative humidity with a 12h / 12h light-dark cycle. They were then randomly divided into four groups: wild-type littermate control group (Control group), which was administered purified water by gavage; APP / PS1 model group (AD group), which was administered purified water by gavage; positive control group treated with donepezil hydrochloride (Positive group), which was administered donepezil hydrochloride dissolved in purified water (0.005g / kg BW / d) by gavage; and gardenia fruit oil treatment group (GFO group), which was administered gardenia fruit oil (0.5g / kg BW / d) by gavage.

[0068] The positive control drug and gardenia fruit oil required for gavage were prepared and used immediately. During the behavioral testing period, the intervention of gardenia fruit oil gavage continued for a total of 2 months.

[0069] I. Mouse behavioral experiments.

[0070] (1) Water maze experiment.

[0071] The Morris water maze was used to assess the learning and spatial memory abilities of mice. A gray water pool was used, with a fixed white circular platform hidden 1-2 cm underwater. Before the formal test, mice underwent five days of orientation navigation training, in which they were placed in different quadrants and then submerged in the water (24±1℃). If a mouse failed to find the platform within 60 seconds, it was guided to the platform to aid its learning and memory, and relevant data were recorded using an automated video tracking system. On the sixth day of the experiment, the platform was removed, and the mice were allowed to explore freely in the pool for 60 seconds. Real-time monitoring recorded the distance of their exploration path, the number of times they crossed the original platform location, and the time spent in the target quadrant.

[0072] Progressive cognitive decline is one of the core symptoms of Alzheimer's disease (AD). During orientation cruise training, the latency of successful escape in mice was recorded. Figure 6 As shown in Figure A, both the Positive and GFO groups showed beneficial effects as the number of training days increased. On the last day of training, mice in the Positive group found the platform in approximately 27.2 seconds, mice in the GFO group in approximately 23.51 seconds, mice in the Control group in 15.78 seconds, and mice in the AD group in 50.62 seconds. The movement trajectories of the mice on the fifth day are shown below. Figure 6 As shown in Figure B, compared with the AD group mice, the paths to the platform from the third quadrant were significantly shorter for the other groups of mice.

[0073] During the testing phase, after the platform was removed, the number of times mice in the Positive group and GFO group crossed the platform (e.g., Figure 6As shown in C) and the time spent in the target quadrant (e.g., Figure 6 The time to first cross the plateau (as shown in D) was significantly higher than that of the AD group mice, and the time to first cross the plateau (as shown in D) was also significantly higher. Figure 6 The values ​​shown in E were also significantly lower than those in the AD group.

[0074] Figure 6 As shown in Figure F, these are representative trajectories of mice in each group during the test. Mice in the Control group, Positive group, and GFO group all exhibited trajectories of continuously exploring the platform, while mice in the AD group exhibited trajectories of aimless movement.

[0075] (2) Y-maze experiment.

[0076] Before the experiment, mice were placed in a room to acclimatize for 30 minutes. The maze was cleaned with alcohol before each experiment to eliminate odor interference from the previous mouse. The mice were placed in the central area of ​​the Y-maze and allowed to explore freely for 8 minutes. A video tracking system recorded the animals' activities in the maze, including the number and order of entry into each arm. The number of correct alternations (the number of times the mouse consecutively entered three different arms of the Y-maze) was calculated, along with the spontaneous alternation rate. The formula was: Alternation rate = (Number of correct alternations / (Total number of arm entries - 2)) × 100% The Y-maze test assesses short-term spatial memory in mice by recording their alternating choices between the three arms. Under normal conditions, healthy mice tend to exhibit a preference for exploring new paths, while mice with impaired memory may exhibit a pattern of repeatedly choosing the same path. A successful spontaneous alternation is recorded when a mouse can consecutively enter all three arms of the Y-maze, indicating good spatial memory and an exploratory tendency. Results for each group of mice in the Y-maze experiment are as follows: Figure 7 As shown, the spontaneous alternation rate of mice in the Control group was 65.85±4.93%, while the spontaneous alternation rates of mice in the Positive group and GFO group were 49.28±2.23% and 47.42±7.57%, respectively. The spontaneous alternation rates of all three groups of mice were significantly higher than those of mice in the AD group (31.38±7.00%).

[0077] In conclusion, both behavioral experiments show that GFO can improve spatial memory and learning ability in AD mice to a certain extent, and GFO has a significant effect on improving behavioral disorders in AD mice.

[0078] After the behavioral experiments, blood was collected from the orbital sinus of mice into anticoagulant tubes. After standing, the blood was centrifuged at 5000 rpm for 10 min to separate the serum. The supernatant serum was collected into a 1.5 mL centrifuge tube and stored at -80°C for later use. After blood collection, the mice were euthanized by cervical dislocation. The skull was dissected layer by layer, and the entire brain tissue was harvested. The obtained brain specimens were immediately immersed in 4% paraformaldehyde fixative and fixed at 4°C for at least 24 hours. Subsequently, the tissues underwent pretreatment through gradient ethanol dehydration and xylene clearing. Finally, paraffin embedding technology was used to prepare tissue blocks for subsequent pathological testing. The remaining brain tissue was separated into cortex and hippocampus and placed in cryovials. The limbs of the mice were then fixed, and the chest was opened to remove the contents of the liver, kidneys, colon, and cecum. These tissues were immediately placed in cryovials for liquid nitrogen flash freezing and then transferred to a -80°C freezer for later use.

[0079] II. Staining of mouse brain tissue.

[0080] Preparation of paraffin sections of whole brain tissue: Mouse whole brain tissue was fixed with 4% paraformaldehyde at 4℃ for 24h, and then subjected to sequential treatments such as ethanol dehydration, xylene clearing, and paraffin infiltration. Finally, the tissue was embedded in paraffin using a paraffin embedding machine to prepare tissue paraffin blocks. The blocks were then continuously sliced ​​(4-6μm thick) using a microtome, mounted, and baked at 60℃ for 2h to obtain paraffin sections for subsequent staining analysis.

[0081] HE staining: First, dewax and rehydrate the paraffin sections by sequentially immersing them in xylene I, xylene II, and xylene III for 8 minutes each, followed by immersion in anhydrous ethanol I, anhydrous ethanol II, 85% ethanol, and 75% ethanol for 5 minutes each, and finally rinsing with tap water for 2 minutes. Next, hematoxylin staining is performed: after staining with hematoxylin for 6 minutes, rinse with water, differentiate with hydrochloric acid-ethanol for 1 second, rinse with water, and finally blue with ammonia solution for 15-30 seconds and rinse with water. Then, eosin staining is performed by immersing the sections in 85% ethanol for 1 minute for dehydration, followed by staining with eosin for 10-30 seconds. The stained sections are then sequentially immersed in anhydrous ethanol I for 30 seconds, anhydrous ethanol II for 3 minutes, anhydrous ethanol III for 3 minutes, xylene I for 3 minutes, and xylene II for 3 minutes until clear. The sections are then mounted with neutral resin, examined under a microscope, and the images are acquired and analyzed.

[0082] The results are as follows Figure 8As shown, neurons in the brain tissue of mice in the Control group exhibited regular and orderly sequences, with an intact overall tissue structure and no pathological abnormalities observed. Compared to the Control group, neurons in the cortical brain tissue of mice in the AD group showed reduced density and darker staining of cell nuclei. Neurons in other areas were loosely and irregularly arranged, indicating that the neurons in these areas may have suffered damage or undergone degenerative changes. After intervention with donepezil hydrochloride and gardenia fruit oil, the morphology and arrangement of neurons in the mouse brain tissue improved, becoming more normal and regular.

[0083] Nissl staining: The paraffin sections were dewaxed and hydrated, then stained in Nissl staining solution for 5 minutes, rinsed three times with pure water, rapidly dehydrated with anhydrous ethanol, cleared with xylene, mounted with neutral resin, examined under a microscope, and the images were acquired and analyzed.

[0084] The results are as follows Figure 9 As shown, the brain tissue of mice in the Control group had a large number of Nissl bodies, which were dark blue in color, and the neuronal cell bodies were intact with clear boundaries. In the brain tissue of mice in the AD group, the number of Nissl bodies was reduced and the color was lighter, and the distribution was sparser, especially in the cortical area and CA3 area of ​​the hippocampus. In the Positive and GFO groups, an increase in the number of Nissl bodies and a darker color could be observed in the brain tissue, which indicates that the internal structure and function of neurons were protected or restored to a certain extent.

[0085] III. Detection of oxidative stress indicators.

[0086] Accurately weigh mouse cerebral cortex tissue and add physiological saline at a weight (g):volume (mL) ratio of 1:9. Homogenize mechanically under ice-water bath conditions to prepare a 10% homogenate. Centrifuge at 3500 rpm for 10 min and collect the supernatant. Measure the corresponding indicators according to the instructions of the malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and nitric oxide (NO) assay kits.

[0087] The results are as follows Figure 10As shown, compared with the Control group mice, the SOD activity of the AD group mice was significantly decreased. After treatment with donepezil hydrochloride (Positive group) and gardenia fruit oil (GFO group), the SOD activity significantly increased, increasing by 46.93 and 56.07 U / mgprot, respectively. The MDA content in the cortex of the Control group mice was significantly lower than that of the AD group mice. However, gavage administration of donepezil hydrochloride and gardenia fruit oil to AD mice reduced the MDA content (26.14±6.12 nmol / mgprot) in AD mice to 19.64±0.89 and 21.44±2.17 nmol / mgprot, respectively. In addition, the GSH-PX activity of mice in the Control group (74.58±5.09U / mgprot) was significantly higher than that in the AD group (15.24±4.19U / mgprot), and the activity of mice in the Positive group (37.89±2.53U / mgprot) and the GFO group (30.42±1.30U / mgprot) was also significantly higher than that in the AD group.

[0088] IV. Detection of inflammatory factor indicators.

[0089] Mouse hippocampal tissue was lysed on ice using animal tissue lysis buffer, homogenized using a homogenizer, and centrifuged at 13000 rpm for 10 min after thorough lysis. The supernatant was collected. The corresponding indicators were measured according to the instructions of the ELISA kits for interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha).

[0090] The results are as follows Figure 11 As shown, donepezil hydrochloride significantly inhibited TNFα levels in the hippocampus of AD mice. While treatment with gardenia fruit oil in AD mice did not show a significant effect compared to the AD group, it did reduce TNFα levels to some extent. Compared to normal mice, AD mice had significantly elevated IL-6 (410.92±16.83 pg / mL) and IL-1β (231.64±39.65 pg / mL) levels. Treatment with donepezil hydrochloride and gardenia fruit oil reduced IL-6 levels to 245.42±39.93 and 278±67.64 pg / mL, respectively, and reduced IL-1β levels to 151.40±31.86 and 149.24±30.24 pg / mL, respectively.

[0091] In conclusion, gardenia fruit oil significantly alleviated cognitive dysfunction in APP / PS1 mice by reducing oxidative stress, inhibiting neuroinflammation, and improving neuronal structure and function.

[0092] This invention not only provides novel natural drug candidates for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, but also offers new ideas and references for the in-depth medicinal development of plant resources that are both food and medicine. It has significant theoretical value, clinical application value, and market promotion potential.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing edible oils with neuroprotective effects and / or those that alleviate AD, characterized in that, Includes the following steps: S1. Roasting and processing: Stir-fry raw gardenia fruit, let it cool, and you will get roasted gardenia fruit; S2. Low-temperature pressing: The roasted gardenia fruit is pressed at a low temperature, then filtered to remove impurities, to obtain edible oil with neuroprotective effects and / or to alleviate AD.

2. The preparation method according to claim 1, characterized in that, In S1, the stir-frying temperature is 140-160℃ and the stir-frying time is 10-20 minutes; the surface of the charred gardenia fruit is charred brown, and the inner surface of the pericarp and the surface of the seeds are yellowish-brown; in S2, low-temperature pressing is physical pressing at a temperature below 50℃.

3. An edible oil, characterized in that, It is prepared by the method for preparing edible oil with neuroprotective effect and / or AD relief as described in any one of claims 1-2.

4. The use of the edible oil according to claim 3 in the preparation of products for the prevention and / or treatment of neurodegenerative diseases.

5. The application according to claim 4, characterized in that, The neurodegenerative disease is called Alzheimer's disease.

6. The application according to claim 4, characterized in that, Edible oils can prevent and / or treat neurodegenerative diseases by increasing the activity of superoxide dismutase and glutathione peroxidase in brain tissue, reducing the content of interleukin-6 and interleukin-1β in brain tissue, and improving the morphology and arrangement of neurons.

7. The application according to claim 4, characterized in that, The amount of edible oil added in the preparation of products for the prevention and / or treatment of neurodegenerative diseases is 300-500 μg / mL.

8. A method for enhancing the neuroprotective effect of edible oils, characterized in that, Add the edible oil of claim 3 to the edible oil; or use the edible oil infused with the roasted gardenia fruit of claim 1.

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