CN112773825A

CN112773825A - Composition containing crabapple oil and application thereof in improving oxidation resistance of nerve cells and brain health

Info

Publication number: CN112773825A
Application number: CN202011190438.2A
Authority: CN
Inventors: 林咏翔; 李姿仪
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2019-11-01
Filing date: 2020-10-30
Publication date: 2021-05-11
Also published as: TWI767387B; CN112825990A; TW202118480A; TW202118503A

Abstract

The invention discloses an application of crabapple oil in preparation of an application for improving the oxidation resistance of nerve cells and brain health care. Wherein said Echinacea purpurea oil is prepared from seeds of an Echinacea purpurea by grinding, cold pressing at low temperature and filtering, and said Echinacea purpurea oil has an alpha-linolenic acid content of greater than 45%. The invention also provides a composition containing the Indian crabapple oil, which can be used for improving the oxidation resistance of nerve cells and promoting brain health.

Description

Composition containing crabapple oil and application thereof in improving oxidation resistance of nerve cells and brain health

Technical Field

The invention relates to an application of crabapple oil, in particular to an application of crabapple oil in preparation of medicines for improving the oxidation resistance of nerve cells and protecting brain.

Background

The acanthopanax papyrifera is known as Plukenetia volubilis, commonly known as "impressed Nut (Inca-Nut)," impressed Peanut (Inca-Peanout), "or" starfish fruit, "and is a perennial plant in the Euphorbiaceae family, which is characterized by trichomes (trichomes) on the leaves. The guava is native to the tropical regions of south america (surimi, venezuela, borlivia, columbia, ecuador, peru and northwest brazil) and to some of the archipelagic islands of the caribbean sea, whereas in south east asia it is mostly grown commercially.

The roasted seeds of the Plukenetia volubilis can be eaten as nuts, and the roasted leaves of the Plukenetia volubilis can be drunk as tea. The Indian fruit oil has light flavor of nut, and can be used for improving fragrance in cooking. At present, it is known that the crabapple oil has the effects of increasing high-density lipoprotein Cholesterol (HDL Cholesterol) in blood and preventing cardiovascular diseases.

Disclosure of Invention

In view of the above, in order to further actively promote the development and application of the crabapple oil in other aspects, a crabapple oil is provided, which can be applied to the preparation of a composition for improving the oxidation resistance of nerve cells and the brain health.

In some embodiments, an Indian crabapple oil is obtained by grinding, cold pressing, and filtering seeds of Indian crabapple, wherein the Indian crabapple oil contains alpha-Linolenic acid (alpha-Linolenic acid), and the content of the alpha-Linolenic acid in the Indian crabapple oil is more than 40% by weight.

In some embodiments, one of the crabapple oils may be a commercially available crabapple oil.

In some embodiments, the commercially available crabapple oil is that sold by Glint s.a.c. (peru).

In some embodiments, the use of an Indian crabapple seed oil for preparing a composition for improving the antioxidant capacity of nerve cells is disclosed, wherein the Indian crabapple seed oil is obtained by grinding Indian crabapple seed, cold pressing and filtering.

In some embodiments, the crabapple oil may delay neuronal cell oxidation.

In some embodiments, the guaiacum oil can slow the inflammatory response of nerve cells.

In some embodiments, the neuroprotection-associated gene comprises SOD2 gene or CAT.

In some embodiments, the concentration of the crabapple oil is 0.5% or more.

In some embodiments, the concentration of the crabapple oil is 1% or more.

In some embodiments, a use of an indian fruit oil for preparing a brain health composition, wherein the indian fruit oil is obtained from an indian fruit seed by crushing, cold pressing, and filtering.

In some embodiments, the use of the crabapple oil to prepare a discretionary composition.

In some embodiments, the use of the crabapple oil may be for the preparation of a composition for enhancing concentration or attention.

In some embodiments, the use of the crabapple oil to prepare a composition for enhancing cognitive ability is provided.

In some embodiments, the use of the crabapple oil to prepare a composition for improving clarity of an idea is provided.

In some embodiments, the use of the crabapple oil to prepare a memory enhancing composition.

In some embodiments, the memory may be long-term memory, short-term memory, image memory, and/or two-dimensional spatial memory.

In summary, the crabapple oil of any embodiment can be used to prepare a composition for improving the antioxidant capacity of nerve cells and protecting the brain. The crabapple oil of any of the examples can be used to prepare a composition for increasing the expression level of neuroprotective related genes (e.g., SOD2 gene and CAT gene) in cells. Use of the crabapple oil of any of the examples to prepare a discretionary composition. Use of the crabapple oil of any of the examples to prepare a focus or attention enhancing composition. Use of the crabapple oil of any of the examples to prepare a composition for enhancing cognitive ability. The use of the crabapple oil of any of the examples to prepare a composition for improving train of thought clarity. The use of the crabapple oil of any one of the examples to prepare a memory enhancing composition.

The invention is described in detail below with reference to the drawings and specific examples, but the invention is not limited thereto.

Drawings

FIG. 1 is a flow chart of an embodiment of a process for preparing an acanthopanax oil.

FIG. 2 is a diagram showing the neuroprotective related genes of example two and the expression ratios of the control group, the control group and the experimental group.

FIG. 3 is a diagram of the determination time scale of the color determination interference test in the third example.

FIG. 4 is a graph of the accuracy ratio of the reciprocal test of the digital span in example three.

FIG. 5 is a graph showing the response time ratio of the image memory test in the third example.

FIG. 6 is a diagram of the ratio of accuracy in the image memory test of the third example.

FIG. 7 is an exemplary diagram of an exemplary three-position interpretation memory test.

FIG. 8 is a histogram of the brain age derived from the location-based interpretation memory test results in the third example.

FIG. 9 shows the results of a questionnaire about the questions related to the definition of the concept in example three.

FIG. 10 is the results of a questionnaire survey on concentration-related questions in example three.

FIG. 11 shows the results of a questionnaire about memory-related questions in example three.

FIG. 12 is the results of a questionnaire survey on questions related to the degree of thought agility in example three.

Fig. 13 is the questionnaire survey results of the questions related to vitality and mental status in example three.

Figure 14 is an electron microscope image of liposomes of the example four.

Fig. 15 is an image of the results of a standing experiment for the four different formulations.

Detailed Description

Some embodiments of the present disclosure will be described below. The present disclosure may be embodied in many different forms without departing from the spirit thereof, and the scope of protection should not be limited to the details set forth in the specification.

Excel software was used for statistical analysis. Data are expressed as mean ± Standard Deviation (SD) and differences between groups are analyzed by student's t-test (student's t-test). In the drawings, the term "indicates a p value of less than 0.05, the term" indicates a p value of less than 0.01, and the term "indicates a p value of less than 0.001. As more "X", the more significant the statistical difference.

As used herein, the numerical values are approximate and all experimental data are shown to be within a range of plus or minus 10%, and more preferably within a range of plus or minus 5%.

As used herein, the term "extract" refers to the product produced by extraction. The extract may be presented as a solution dissolved in a solvent, or the extract may be presented as a concentrate or serum that is free or substantially free of solvent.

As used herein, "raw material for canola oil" refers generally to plant seeds, wherein the seeds may include virgin, dried or otherwise physically processed seeds to facilitate handling, which may further include intact, chopped, diced, milled, ground or otherwise processed seeds to affect the size and physical integrity of the raw material.

As used herein, the terms "cold pressing", "low temperature pressing" and "low temperature pressing" generally refer to the pressing of raw materials to extract fats and oils by means of a re-pressing process, wherein the re-pressing process is conducted at a temperature not exceeding 30 ℃. Wherein the pressing method is to use a pressure oil press to crush the fruits of the Plukenetia volubilis (Nippon) Sing with high pressure (15000 kg/unit) and discharge the oil.

Refer to fig. 1. In some embodiments, a crabapple oil is extracted by cold pressing of a crabapple oil raw material or a crabapple seed. In some embodiments, the adductor Oil (Sacha inchi (Plukenetia volubis) Oil) is obtained by subjecting an adductor Oil raw material, i.e., an adductor seed, to a pulverization procedure S01, a low-temperature extraction procedure S02, and a filtration procedure S03, in this order.

In some embodiments, the crabapple oil feedstock may be dehulled or shelled crabapple seeds. In some embodiments, the crabapple raw material may be fresh or dried crabapple seeds.

In some embodiments, the milling procedure S01 refers to whipping or rolling the crabapple oil raw material to break into powder or flakes. For example, the pulverization may be carried out by a juicer, a conditioner or a homogenizer. In some embodiments, the crushing process S01 may be performed simultaneously or before or after frying or steaming to improve oil extraction efficiency.

In some embodiments, the cold pressing procedure S02 refers to pressing the powdered or shredded crabapple oil material to extract its oil. In some embodiments, the temperature of the adducted fruit oil feedstock during the cold pressing process S02 is less than 30 degrees Celsius. In some embodiments, the cold pressing process S02 is performed by applying a vertical high pressure directly to the raw material of the addendum oil. In some embodiments, the pressure of the vertical high pressure extraction may be above 55 MPa. In some embodiments, the vertical high pressure extraction apparatus may apply a weight of over 600kg per unit area. In some embodiments, the powdered or shredded crabapple oil material is wrapped with cotton cloth into a circular cake shape to facilitate vertical high pressure extraction prior to cold pressing process S02. In other embodiments, the cold pressing procedure S02 includes applying high pressure (15000 kg/unit) to the raw material of the adductor oil through a pressure oil press to crush the fruit of the adductor and discharge the oil.

In some embodiments, the filtering process S03 is to pass the crude liquid of the processed crabapple oil obtained after cold pressing through a screen to filter out larger solids such as residue or precipitate to form a filtrate. For example, the screen may be a 400 mesh screen. In some embodiments, the crude de-fatliquor of the processed de-fatted nut oil obtained after cold pressing may be precipitated before the filtering step S03, so as to increase the efficiency of the filtering step S03. In some embodiments, the settling time may be 1 hour or until the liquid layer of the crude liquid of the added fruit oil becomes homogeneous.

In some embodiments, the guava oil comprises alpha-linolenic acid. In some embodiments, the alpha-linolenic acid content of the crabapple oil is greater than 40% by weight.

In some embodiments, the use of the Echinacea purpurea oil for preparing a composition for enhancing neuro-antioxidant ability is provided, wherein the Echinacea purpurea oil is obtained by enhancing expression level of a gene related to neuroprotection in cells, and the Echinacea purpurea oil is obtained by pulverizing, cold pressing and filtering. In some embodiments, the increased antioxidant capacity of a nerve refers to an increased ability of a nerve cell to resist free radicals. In some embodiments, the increase in antioxidant capacity of a nerve refers to an increase in the ability of a nerve cell to prevent damage.

In some embodiments, the neuroprotection-associated gene comprises at least one of the following genes: SOD2 gene (Superoxide Dismutase 2, GeneID:6648), CAT gene (Catalase, GeneID: 847).

In some embodiments, greater than 1% concentration of the Echinacea oil increases expression of neuroprotective related genes, thereby increasing neuronal antioxidant activity.

Free radicals such as Superoxide Anion (Superoxide) and Hydrogen Peroxide (Hydrogen Peroxide) cause axonal conduction inhibition and destruction of neuronal cells. Among them, the protein (superoxide dismutase) transcribed from the SOD2 gene decomposes superoxide into hydrogen peroxide, and the protein (catalase) transcribed from the CAT gene converts hydrogen peroxide into water and oxygen. The proteins transcribed from these two genes are important for scavenging oxides and free radicals. When the expression of these two genes in the cell is higher, the oxidation resistance of the cell is better.

In other words, the Echinacea purpurea oil can effectively improve the cellular oxidation resistance by increasing the expression of the two genes, thereby improving the resistance of the nerve to free radicals and protecting the nerve.

In some embodiments, the use of the crabapple oil may be for the preparation of a composition for brain health care. In some embodiments, the guava oil with brain health benefits comprises alpha-linolenic acid. In some embodiments, the alpha-linolenic acid content of the crabapple oil with brain health care capabilities is greater than 40% by weight.

In some embodiments, 1.5g of the crabapple oil taken daily can be used for brain health care to improve judgment, cognition, attention, memory, responsiveness, thinking agility, and/or thinking clarity.

In some embodiments, the composition can be a pharmaceutical. In other words, the medicine contains effective content of the Indian nutmeg oil.

In some embodiments, the aforementioned medicament may be formulated into a dosage form suitable for enteral or oral administration using techniques well known to those skilled in the art. Such dosage forms of administration include, but are not limited to: troches (tablets), tablets (troches), buccal tablets (lozenges), pills (pills), capsules (capsules), dispersible powders (dispersible granules), solutions, suspensions (suspensions), emulsions (emulsions), syrups (syrup), elixirs (elixir), syrups (syrup), and the like.

In some embodiments, the aforementioned medicament may be manufactured using techniques well known to those skilled in the art into a dosage form suitable for parenteral (parenteral) or topical (topologic) administration, including, but not limited to: injections (injections), sterile powders (sterile powders), external preparations (external preparation), and the like. In some embodiments, the medicament may be administered by a parenteral route (parenteral routes) selected from the group consisting of: subcutaneous injection (subecanal injection), intradermal injection (intraepithelial injection), and intralesional injection (intralesion).

In some embodiments, the pharmaceutical may further comprise a pharmaceutically acceptable carrier (pharmaceutical acceptable carrier) that is widely used in pharmaceutical manufacturing technology. For example, a pharmaceutically acceptable carrier can comprise one or more of the following agents: solvents (solvent), buffers (buffer), emulsifiers (emulsifying), suspending agents (suspending agent), disintegrating agents (disintegrant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients (excipient), stabilizers (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving), wetting agents (wetting agent), lubricants (lubricating), absorption delaying agents (absorption delaying agent), liposomes (liposome) and the like. The selection and amounts of such agents are within the skill and routine skill of those skilled in the art.

In some embodiments, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline (normal saline), Phosphate Buffered Saline (PBS), and aqueous alcohol-containing solutions (aqueous solution).

In some embodiments, the composition may be an edible composition. In some embodiments, the edible composition may be formulated into a food product or may be a food additive, i.e., added during the preparation of the food material by conventional methods to produce a food product, or added during the production of a food product. Herein, the food product may be a product formulated with edible material for ingestion by humans or animals.

In some embodiments, the food product may be, but is not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products), health foods (health foods) and dietary supplements (dietary supplements).

The first example is as follows: quantitation of alpha-linolenic acid in Indian fruit oil

Herein, the present invention is analyzed by Gas Chromatography (GC) after saponification and methyl esterification of the adducted fruit oil.

Materials and instruments

1. A gas chromatograph.

2. A detector: flame detector.

3. Chromatography tube: CP-Sil 88 capillary, inner membrane thickness 0.20 μm, inner diameter 0.25mm × 100m, or the same grade.

4. Reagent testing: pyrogallic acid (pyrogallic acid), ethanol (95%), hydrochloric acid, ammonia (28%), ether, petroleum ether, n-hexane, chloroform, sodium hydroxide, methanol, sodium chloride, anhydrous sodium sulfate, and 14% boron trifluoride methanol solution.

5. Fatty acid control standards: methyl 9,12, 15-cis-octadecatrienoate (9,12, 15-cis-octadecadienoic methyl ester).

6. Internal standard: glyceryl heneicosanoate (trihenicosanoin, 21: 0).

Preparation of 9,12,15-cis-octadecatrienoic acid methyl ester standard solution

Weighing about 50mg of each of the 9,12,15-cis-octadecatrienoic acid methyl ester reference standard substances, accurately weighing, dissolving with n-hexane, and metering to 10mL to serve as standard stock solution. was diluted with n-hexane for use as a standard solution.

Preparation of internal standard solution

About 100mg of an internal standard of glyceryl heneicosanoate was weighed, accurately weighed, dissolved in chloroform, and made to a volume of 10mL for use as an internal standard solution (hereinafter referred to as "standard solution").

Preparation of reagents

1.8.3M hydrochloric acid solution: 250mL of hydrochloric acid is slowly added into 110mL of detachable water, and the mixture is uniformly mixed.

2.1N sodium hydroxide in methanol: 4g of sodium hydroxide was taken and dissolved in methanol to make 100 mL.

3. Saturated sodium chloride solution: taking at least 40g of sodium chloride, adding 100mL of detachable water, and taking the upper layer liquid for standby after moderate stirring.

Preparation of a detection liquid for Echinacea purpurea oil

Weighing a proper amount of 10-20 mg of the crassula argentea fruit oil, adding 0.1mL of the internal standard solution, removing chloroform in a water bath at 40 ℃, and dissolving with 1mL of n-hexane.

Identification test and fatty acid contentMeasurement of

Each 1. mu.L of the test solution and the standard solution was measured accurately, and the solutions were injected into a gas chromatograph and subjected to gas chromatography under the following conditions. The conditions for the gas chromatography measurement were as follows:

1. temperature of the chromatography tube: initial temperature: at 170 ℃ for 40 min.

2. The heating rate is as follows: 3 ℃/min.

3. Final temperature: 200 ℃ for 50 min.

4. Temperature of the detector: at 300 ℃.

5. Injector temperature: at 250 ℃ to obtain a mixture.

6. Mobile phase gas helium flow rate: 0.75 mL/min.

7. The split ratio is as follows: 40: 1.

the retention time of the peak obtained by comparing the test solution of the adducted fruit oil with the standard solution is identified, and the content (%) of alpha-linolenic acid in the test solution is calculated according to the following calculation formula:

the content (%) of alpha-linolenic acid in the sample solution is (W _ FAMEx. times.F _ FAx. times.100)/W

Wherein W is the weight (g) of the sample; WFAMEx is the content (g) of 9,12,15-cis-octadecatrienoic acid methyl ester in the oil inspection liquid of the Indian fruit; FFax is for the conversion of methyl 9,12, 15-cis-octadecatrienoate to alpha-linolenic acid with the coefficients: 0.9520.

results of the experiment

According to the calculation result of the above formula, the alpha-linolenic acid content of the crabapple oil of the present invention is 48.1%.

Example two: cell experiment-Gene detection of inhibition of ROS production (Hydrogen peroxide treatment) by utilizing Mega-nut oilIn this case, the change in the content of active oxygen species in Mouse brain neuroblasts (Mouse brain neuroblastomas cells (Neuro2a)) after treatment with the added peanut oil was measured by using a fluorescent probe DCFH-DA in combination with a flow cytometer.

Materials and instruments

1. Cell lines: mouse brain neuroblasts (Mouse brain neuroblastomas cells (Neuro2a)) obtained from the american type culture collection ATCC; CCL-131.

2. Culture medium: dulbecco 'S Modified Eagle' S Medium (DMEM, available from Gibco, 11965-.

3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.

4. Hydrogen peroxide (H2O 2): purchased from Sigma-Aldrich, product number 95299-1L.

5. Trypsin (Trypsin-EDTA): 10 XTrypsin-EDTA (from Gibco) was diluted 10-fold with 1 XPBS solution.

6. Adding the fruit oil: the crabapple oil used in this experiment was purchased from Glint S.A.C. (Peru), product number 11002576, and it was prepared by crushing and rolling seeds of a crabapple, squeezing at a low temperature (lower than 30 ℃ C.) under a high pressure, and filtering.

Experimental procedure

The experiment will be divided into three groups of an experimental group, a blank control group (a group without the addition of the adducted fruit oil and without the treatment of the hydrogen peroxide), and a control group (a group without the addition of the adducted fruit oil and with the treatment of the hydrogen peroxide), and three repeated experiments are respectively carried out on each group:

1. neuro2a cells were plated at 1X 10 per well⁶In this manner, the culture medium was inoculated into 6-well plates each containing 2ml of the medium.

2. The culture plate was incubated at 37 ℃ with 5% CO2 for 24 hours.

3. The medium was removed.

4. To 2mL of the experimental medium of the experimental group, 20 μ L of the crabapple oil (peru Glint s.a.c., product No. 11002576) was added (i.e., the volume percentage of the crabapple oil in the cell culture medium was 1%), and H2O2 was added simultaneously, and reacted at 37 ℃ for 6 hours. Specifically, 35% wt of hydrogen peroxide was diluted to 100mM (10. mu.L of hydrogen peroxide was added to 990. mu.L of redistilled water), and then 20. mu.L of 100mM hydrogen peroxide was added to 2mL of cell culture plates.

5. The experimental medium in the control group was supplemented with 2mL of cell culture medium alone (i.e. without the crabapple oil) and reacted at 37 ℃ for 6 hours.

6. To the control experimental medium was added 2mL of cell culture medium alone (i.e., without the Echinacea oil) and H2O2 was added simultaneously and reacted at 37 ℃ for 6 hours. Specifically, 35% wt of hydrogen peroxide was diluted to 100mM (10. mu.L of hydrogen peroxide was added to 990. mu.L of redistilled water), and then 20. mu.L of 100mM hydrogen peroxide was added to 2mL of cell culture plates.

7. After the reaction, the supernatant was removed, and each well was rinsed 1 time with 1mL of 1 XPBS solution.

8. The cells were disrupted with a cell lysate (purchased from Geneaid corporation) to obtain a cell lysate.

9. RNA in the cell lysate was extracted using an RNA extraction reagent kit (available from Geneaid). Then, 2000 ng of the extracted RNA was used as a template

III reverse transcriptase (from Invitrogene) will perform reverse transcription to generate the corresponding cDNA.

10. Subsequently, cDNA was used as a template and a pair of primers (shown in Table 1 below) for amplifying a target gene was used to perform quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) using KAPA SYBR FAST kit (available from Sigma) in ABI StepOnePlus TM Real-Time PCR system (ABI StepOne Plus Real-Time PCR system, available from Thermo Fisher Scientific Co.) to quantify the mRNA expression amounts of SOD2 gene and CAT gene, thereby estimating the expression amounts of the proteins encoded by the respective genes. Wherein, the ABI StepOne Plus real-time PCR system is set with the conditions of reaction at 95 ℃ for 1 second and 60 ℃ for 20 seconds, and the total time is 40 cycles.

11. Thereafter, the relative expression amount of the target gene was measured by the 2-. DELTA.Ct method. The relative expression is defined as the fold change in RNA expression of a target gene in the experimental group relative to the same gene in the control group or control group. The method uses the cycle threshold of GAPDH gene as the cycle threshold (Ct) of the reference gene of the internal control, and the fold change is calculated according to the following formula:

target gene-Ct GAPDH of control group or target gene-Ct GAPDH of delta Ct ═ Ct experimental group

Target gene of delta-delta Ct experimental group-delta Ct control group

Multiple change 2^-ΔΔCtMean value of

Finally, the standard deviation was calculated using the STDEV formula of Excel software and analyzed in Excel software as a single-tailed Student t-test for statistically significant differences ([ p ] values < 0.05; < 0.01;. p values < 0.001). Wherein, the primer pair corresponding to the SOD2 gene is SOD2-F and SOD 2-R. The primer pair corresponding to CAT gene is CAT-F and CAT-R, and the primer pair corresponding to GADPH gene is GADPH-F and GADPH-R.

TABLE 1

Primer name	Sequence numbering	Sequence of
			SOD2-F	SEQ ID NO:1	GGAAGCCATC AAACGTGACTT
SOD2-R	SEQ ID NO:2	CCCGTTCCTT ATTGAAACCAAGC
			CAT-F	SEQ ID NO:3	TGGGATCTCG TTGGAAATAACAC
CAT-R	SEQ ID NO:4	TCAGGACGTA GGCTCCAGAAG
			GADPH-F	SEQ ID NO:5	CTGGGCTACA CTGAGCACC
GADPH-R	SEQ ID NO:6	AAGTGGTCGT TGAGGGCAATG

Here, the results of the corresponding gene quantification obtained in the three repeated experiments were averaged to obtain an average value, and then the average values of the control group and the experimental group were converted into relative expression (%) by regarding the average value of the control group as 1 (relative expression), as shown in fig. 2. Statistically significant differences between the measurements of the experimental group and the measurements of the control group were statistically analyzed by student t-test. In fig. 2, the p value is less than 0.001 in the experimental group compared with the control group, and the p value is less than 0.01 in the experimental group compared with the control group.

Results of the experiment

Referring to FIG. 2, compared to the control group (group treated with hydrogen oxide but without the addition of the Plukenetia volubilis oil), the experimental group treated with the Plukenetia volubilis oil showed significantly higher expression levels of the genes related to neuroprotection (i.e., SOD2 gene and CAT gene). Specifically, after the cranial nerve cells are firstly treated by hydrogen oxide and then treated by the seal fruit oil, the expression level of SOD2 gene is about 3.6 times higher than that of a control group only treated by hydrogen oxide, and the expression level of CAT gene is about 1.9 times higher than that of the control group. The result shows that the Indian fruit oil can effectively improve the expression quantity of SOD2 gene and CAT gene, can improve the oxidation resistance of cells and increase the capability of decomposing ROS, and has the effect of improving the oxidation resistance of nerve cells.

Example three: human body experiment-cognitive function detection

Using samples

The liquid state of the crabapple oil of the invention contains 15 mL/bag (containing 1.5g of crabapple oil (10% by weight), 7.5% of Arabic gum, 0.1% of soybean lecithin, 0.37% of corn starch gum, 0.1% of guar gum, 0.02% of DL-alpha-tocopheryl acetate, 13.0% of glycerol, 21.0% of D-xylitol, 0.045% of beta-carotene, 0.9% of citric acid, 0.087% of L-ascorbic acid (vitamin C), 0.35% of apple liquid flavor, 0.05% of potassium hexadienate, 0.52% of pineapple liquid flavor and 45.958% of water). In yet other embodiments, up to 5g of crabapple oil may be ingested by one person per day. Wherein the said oil is obtained from Glint S.A.C. (Peru), product number is 11002576, and it is prepared by crushing and rolling seeds of an Indian crabapple, squeezing at a temperature lower than 30 deg.C under a high pressure, and filtering.

Number of subjects and age

10 subjects between 25-55 years of age.

Experimental mode

Subjects consumed a liquid pack containing the inventive Echinacea oil (1.5 g of Echinacea oil) daily for 28 days (i.e., 4 weeks). And recording the test results of the subjects before (week 0) and 28 days after drinking according to different test items by using corresponding instruments and measuring methods. It should be noted that, except for the detection, the subject does not exercise or contact the items related to the detection in other ways, so as to reduce the influence on the detection result caused by the improvement of the familiarity with the detection content. In addition, the test time and state before and after the test of 28 days are the same (for example, if the test time before the test of 28 days is 9 am and breakfast is not eaten, the test time after the test of 28 days is 9 am and breakfast is not eaten) to reduce the interference factors caused by different physiological states and external environment.

Detecting items: the test subject is subjected to 1, color judgment interference test, 2, digital span inversion test, 3, image memory test, 4, position interpretation memory test and 5, cognitive function questionnaire feedback. The tests 1 to 3 are performed on the Cognitive Fun website (http:// Cognitive Fun. net /), and the tests 4 are performed on the Flash fabric website (http:// Flash fabric. com/f _ learning/bridge/tw _ bridge. html).

1. Color judgment interference test

After the test is started, the computer screen randomly displays an English word with a color (e.g., "" GREEN "") that is not necessarily the same as the color of the word (e.g., "" GREEN "") but the test subject needs to correctly input the color of the word instead of the color of the English word (YELLOW (YELLOW) as the correct answer of the previous example), wherein the test system only records the seconds of answering the question.

The total number of the tests is 20, the test results are the average of the second number of questions answered, and each subject is tested for 3 times. The test result presentation method is to set the average second of answer (judgment time) of 0 week of all subjects as 100%, calculate the percentage of the average second of answer (judgment time) of 28 days of all subjects, and present them as a histogram as shown in FIG. 3.

In addition, it is noted that all the testers can easily recognize all the English words with different colors in the test contents before and after drinking, and there is no obstacle to color judgment.

2. Digital span inversion test

When the test is initiated, the computer screen randomly presents a string of numbers (e.g., "514") that the subject must enter (in the example above, the subject should enter 415 "). Wherein the test system will record the correct rate of the subject.

The test is performed for one minute, the test result is the average value of the accuracy (number of answers/total number of questions), and each subject is tested for 3 times. The test result is presented by setting the average accuracy of all the subjects at week 0 as 100%, calculating the percentage of the average accuracy of all the subjects at day 28, and presenting the histogram as shown in FIG. 4.

3. Image memory test

When the test is started, the computer screen displays a series of pictures in time, and if the same pictures are seen in the display process, the test subject needs to click on the pictures or click on the blank key, and the test system records the number of right-to-right questions and the number of seconds for right-to-right questions.

The test was conducted for 1 minute, the test results were the accuracy (number of questions answered/total number of questions) and the response time (average of seconds of questions answered), and each subject was tested for 3 times. The test result is presented by setting the average response time of all subjects at week 0 as 100%, calculating the average accuracy percentage of all subjects at day 28, and presenting the result as a histogram shown in FIG. 5; and setting the average accuracy of all subjects at week 0 as 100%, calculating the average accuracy percentage of all subjects at day 28, and presenting the result as a histogram in FIG. 6.

4. Position interpretation memory test

After the test is started, several numbers appear on the screen at different positions, such as the content of the left frame in fig. 7. After about 0.7 seconds, the numbers on the screen will disappear, leaving only the positions of the numbers as the content of the right frame of fig. 7. At this time, the subject should click from small to large according to the number size. Based on the test results, including the response rate and the response time, the testing system will calculate the brain age of the subject. The test is not limited and each subject will be tested 10 times. The test results are presented by histogram showing the average brain age of all subjects at week 0 and the average brain age of all subjects at day 28 as shown in FIG. 8.

5. Cognitive function feedback questionnaire

Each subject was asked to perform a cognitive function questionnaire (as shown in table 2 below) before drinking the liquid pack (i.e., week 0) and after drinking the liquid pack for 4 weeks (i.e., week 4) to fill in feedback and presented and analyzed as pie charts in fig. 9-13 to assess the status of cognitive function improvement.

TABLE 2

Results of the experiment

Referring to fig. 3, 9 and 10, after drinking the liquid packet of the inventive crabapple oil for 4 weeks, the time for the subjects to judge in the color judgment interference test was reduced by 15.9% compared to week 0. According to the questionnaire feedback, after drinking the liquid packet of the inventive crabapple oil for 4 weeks, at least 50% of the subjects were improved from "poor" or "normal" to "good" in the definition of their own idea, and at least 70% of the subjects were improved from "poor" or "normal" to "good" in the attention-deficit of their own. Therefore, the inventive Indian buead seed oil has the effects of improving the definition, concentration, judgment and cognitive ability of the thought.

Referring to fig. 4 and 11, after drinking the liquid packet of the present crabapple oil for 4 weeks, the subject's accuracy in the digital span inversion test was improved by 5.5% compared to week 0. According to the questionnaire feedback, after drinking the liquid packet of the inventive crabapple oil for 4 weeks, 40% of the subjects had their own memory ratings from "poor", "poor" or "normal" to "good". Therefore, the inventive Indian buead oil has the effect of improving memory, especially short-term memory.

Referring to fig. 5, 6 and 12, after drinking the liquid packet of the inventive crabapple oil for 4 weeks, the response time of the subjects in the image memory test was reduced by 9.1% and the accuracy was improved by 13.5% compared to week 0. According to the questionnaire feedback, after drinking the liquid containing the present crataegus oil for 4 weeks, the evaluation of the self-thought agility of at least 41% of the subjects was improved from "poor" or "normal" to "good". Therefore, the crabapple oil of the present invention has the effects of improving image memory and thinking agility.

Referring to fig. 8 and 13, after drinking the liquid packet of the inventive crabapple oil for 4 weeks, the subjects had an average brain age reduction of 5 years as compared to week 0, as inferred by the results of the location interpretation memory test. According to the questionnaire feedback, at least 40% of the subjects rated a "poor" or "normal" to "good" for their own vitality and mental status after drinking the liquid packet of the inventive crabapple oil for 4 weeks. Therefore, the seal fruit oil of the invention has the effects of reducing brain age, improving vitality, mental state and two-dimensional space memory.

In conclusion, the crabapple oil of the present invention can effectively improve the attention, judgment, cognitive judgment and memory of the brain, wherein the memory includes short-term memory, image memory, digital memory and two spatial memories. Specifically, according to the above embodiments, the inventive crabapple oil can reduce the response time of cognitive interference judgment by 15.9%, improve the accuracy of short-term digital memory by 5.5%, reduce the response time of short-term image memory by 9.1%, improve the accuracy by 13.5%, and reduce the brain age by 5 years. In addition, the Indian buead seed oil can also improve mental state, mental capacity, sleep quality, anxiety resistance and stress resistance of users after drinking/eating.

Example four: composition standing stability test

Since the inventive Echinacea oil is mainly composed of lipids, a delamination phenomenon is easily caused when the inventive water-soluble composition is produced, and the stability of the composition in storage is concerned. Therefore, in order to improve the stability of the present invention, the liposome suspension with the capability of coating the present invention was tested for delamination after standing at room temperature (25 ℃) for one day. If the liposome suspension is a homogeneous non-stratified liquid, it represents that the liposome structure therein is relatively stable. On the other hand, if the liposome suspension is obviously layered and has more turbid and uneven color, the liposome contained in the liposome suspension is less stable in structure.

Wherein, the liposome with the capability of stably coating the Indian crabapple fruit oil is prepared by mixing lecithin, Arabic gum and water in a specific ratio and homogenizing by a specific pressure to form a liposome suspension. Wherein the liposome suspension comprises a plurality of liposomes having a stable structure. The prepared liposome has stable structure, can stably coat the effective components, and can be used for improving the biological absorptivity of the coated effective components.

Referring to FIG. 14, a cryo-electron microscope (cryo-TEM; JEOL, model JEM-1400) is used to observe liposomes in a liposome suspension, wherein the liposomes have a spherical shape and an outer diameter of about 200nm to 400 nm. And as can be seen from fig. 14, the same group of liposome suspensions contains a plurality of liposomes with different outer diameter lengths.

Here, A, B mixed liquids were prepared according to the formulation components and formulation ratios shown in table 3 below. The total weight of the mixed solution is 100 g. Firstly, mixing soybean lecithin, acetic acid DL-alpha-tocopherol ester, and the Indian fruit oil with part of water in a weight ratio (w/w) of 1: 4 and stirred at room temperature (25 deg.c) to confirm that the lecithin was dissolved in a portion of the water to form a first premix solution.

And, mixing gum arabic, guar gum, corn gum, sucralose, glycerin, potassium hexadienoate, D-xylitol, beta-carotene, citric acid, vitamin C, and the rest of water to form a second premix solution.

And mixing and stirring the first premix solution and the second premix solution uniformly to ensure that the components are completely dissolved in water, and quantifying the water to make the total weight 100 g to form a third premix solution. Next, the third premixed solution after the quantitative determination is sieved through a 80-mesh screen to form a mixed solution.

TABLE 3

Formulation components (weight%)	Group A	Group B
			Soybean lecithin	0.1	0.1
Arabic gum	7.5	2.5
			Guanhua bean glue (thickening agent)	0.1	0.1
Corn sugar glue (thickening agent)	0.37	0.37
			Acetic acid DL-alpha-tocopheryl alcohol ester	0.02	0.02
Glycerol	13.0	13.0
			D-xylitol	21.0	21.0
Beta-carotene	0.045	0.045
			Citric acid	0.9	0.9
L-ascorbic acid (vitamin C)	0.087	0.087
			Sucralose (sweetener)	0.87	0.87
Potassium hexadienate (preservative)	0.05	0.05
			Water (W)	45.958	50.958
Plukenetia volubilis oil	10	10

Then, the mixture was subjected to a sterilization program, and the set value of the sterilization program was sterilization at 95 ℃. + -. 5 ℃ for 30 minutes. And (3) when the temperature of the sterilized mixed solution is reduced to 50 ℃, homogenizing the cooled mixed solution by a homogenizing instrument (brand: GEA Niro Soavi, model: Panda Plus) to form liposome suspension, wherein the pressure value set in the homogenizing treatment is 350 bar.

Next, A, B the two liposome suspensions were allowed to stand at room temperature (25 ℃) for one day to observe whether or not the liposome suspensions were delaminated. Moreover, if the liposome suspension is a uniform non-stratified liquid, the liposome structure in the liposome suspension is relatively stable. On the other hand, if the liposome suspension is obviously layered and has more turbid and uneven color, the liposome contained in the liposome suspension is less stable in structure. In other words, the liposome suspension is significantly delaminated due to the rupture of the liposome coating and the lack of formation of liposomes, which means that the ratio is less suitable for the composition of the present invention.

Please refer to fig. 15. The liposome suspensions of group B produced significant stratification, representing insignificant emulsification, and the resulting liposome structures were unstable. While the liposome suspension of group A did not separate significantly, indicating significant emulsification. It can be seen that the stability of the composition containing the addendum oil prepared in this ratio is better when the mixture contains 7.5 wt% of gum arabic and the liposome suspension obtained by the pre-killing treatment of the mixture is not significantly layered.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims.

The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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Claims

1. A composition comprising an argania de, wherein the argania de is encapsulated by a liposome having stable encapsulation ability, and wherein the liposome having stable encapsulation ability comprises 0.1 wt% of lecithin and 3-8 wt% of acacia gum.

2. The composition of claim 1, wherein the composition comprises a brain-care composition and/or a neuroantioxidant-enhancing composition.

3. The application of the Indian buead seed oil in preparing the composition for improving the oxidation resistance of nerve cells is characterized in that the Indian buead seed oil is prepared by firstly performing a crushing procedure on seeds of Indian buead, then performing a cold pressing procedure on the seeds, and then performing a filtering procedure on the seeds.

4. The application of the Indian crabapple oil in preparing the brain health-care composition is characterized in that the Indian crabapple oil is prepared by firstly performing a crushing procedure on seeds of Indian crabapple, then performing a cold pressing procedure and then performing a filtering procedure.

5. The use of claim 3, wherein said composition for enhancing antioxidant capacity of nerves is for enhancing the antioxidant capacity of nerve cells by enhancing the ability of nerve cells to resist damage by free radicals.

6. The use according to claim 5, wherein the free radicals comprise superoxide or peroxide.

7. The use of claim 3, wherein said composition achieves an increase in the antioxidant capacity of said neural cells by increasing the expression level of a neuroprotection-associated gene in said cells.

8. The use of claim 5, wherein the neuroprotection-associated gene comprises the SOD2 gene or the CAT gene.

9. The use of claim 4, wherein said composition is for brain health by improving cognitive judgment, concentration, memory, responsiveness, mental agility, vitality, mental state and/or clarity of thinking.

10. The use of claim 9, wherein the memory comprises short term memory, digital memory, two dimensional spatial memory, or image memory.

11. The use according to claim 3 or 4, wherein said guava oil comprises at least 40% alpha-linolenic acid.

12. The use according to claim 3 or 4, wherein the composition is a medicament.

13. The use according to claim 3 or 4, wherein the composition is a food composition or a health food composition.