TW202118503A - Composition containingplukenetiavolubilisoil and its uses of enhancing the antioxidant capacity of nerve cells and brain health - Google Patents

Abstract

Use ofPlukenetia volubilis Oil for the Enhancement of the Antioxidant Capacity of Nerve cells and Brain health wherein thePlukenetia volubilis Oil is obtained from cold-compressing and filtering crushedPlukenetia volubilis seeds. The present invention also discloses a composition containingPlukenetia volubilis Oil that has the uses of enhancing the antioxidant capacity of nerve cells and brain health.

Description

Composition containing inca fruit oil and its use for improving nerve cell antioxidant capacity and brain health care

The present invention relates to a use of Sacha indica oil, in particular to the use of preparation to enhance the antioxidant capacity of nerve cells and brain health care.

The scientific name of Inca fruit is Plukenetia volubilis , commonly known as "Inca-Nut", "Inca-Peanut" or "Star fruit". It is a perennial plant in the Euphorbiaceae family. One of its characteristics is the leaf Plant hairs (trichomes). Inca fruit is native to tropical regions of South America (Suriname, Venezuela, Bolivia, Colombia, Ecuador, Peru, and northwestern Brazil) and parts of the Caribbean Sea, while it is mostly grown commercially in Southeast Asia.

The roasted inca seeds can be eaten as nuts, and the roasted inca leaves can be brewed as tea. Inca oil has a slightly nutty flavor and can be used to enhance the aroma in cooking. It is currently known that Sacha Inchi Oil has the effects of raising HDL Cholesterol in the blood and preventing cardiovascular diseases.

In view of this, in order to more actively promote the development and application of other aspects of inca oil, an inca oil is proposed, which can be used to prepare a composition for improving the antioxidant capacity of nerve cells and brain health.

In some embodiments, an inca fruit oil is obtained by crushing, cold pressing, and filtering an inca fruit seed, wherein the inca fruit oil contains alpha-linolenic acid, and wherein the inca fruit oil The content of oil alpha-linolenic acid exceeds 40% by weight.

In some embodiments, an inca oil may be a commercially available inca oil.

In some embodiments, the commercially available inca oil is an inca oil sold by Glint S.A.C. (Peru).

In some embodiments, an inca oil is used for the preparation of a composition for enhancing the antioxidant capacity of nerve cells, wherein the inca oil is one of the compositions that achieves the antioxidant capacity of ascending nerve cells by increasing the expression of neuroprotection-related genes in cells Purpose, and the inca fruit oil is obtained by crushing, cold pressing and filtering an inca fruit seed.

In some embodiments, Sacha indica oil can delay the oxidation of nerve cells.

In some embodiments, Sacha indica oil can slow down the inflammatory response of nerve cells.

In some embodiments, neuroprotection-related genes include SOD2 gene or CAT.

In some embodiments, the concentration of Sacha indica oil is above 0.5%.

In some embodiments, the concentration of inca oil is more than 1%.

In some embodiments, an inca fruit oil is used to prepare a brain health care composition, wherein the inca fruit oil is obtained by crushing, cold pressing, and filtering an inca fruit seed.

In some embodiments, Sacha indica oil can be used to prepare a composition for improving judgment.

In some embodiments, Sacha indica oil can be used to prepare a composition that enhances concentration or attention.

In some embodiments, Sacha indica oil can be used to prepare a composition for enhancing cognitive ability.

In some embodiments, Sacha indica oil can be used to prepare a composition for improving the clarity of thinking.

In some embodiments, Sacha indica oil can be used to prepare memory-enhancing compositions.

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

In summary, the inca oil of any embodiment can be used to prepare a composition for enhancing the antioxidant capacity of nerve cells and brain health. The Sacha indica oil of any embodiment can be used to prepare a composition that increases the expression of neuroprotection-related genes (such as SOD2 gene and CAT gene) in cells. The inca oil of any embodiment can be used to prepare a composition for improving judgment. The inca oil of any embodiment can be used to prepare a composition for improving concentration or attention. The inca oil of any embodiment can be used to prepare a composition for enhancing cognitive ability. The inca oil of any embodiment can be used to prepare a composition for improving the clarity of thinking. The inca oil of any embodiment can be used to prepare a memory-enhancing composition.

The following will describe some specific implementation aspects of this case. Without departing from the spirit of this case, this case can still be practiced in many different forms, and the scope of protection should not be limited to the conditions specified in the specification.

In this case, Excel software was used for statistical analysis. The data are expressed as mean±standard deviation (SD), and the differences between groups are analyzed by student’s t-test. In the diagram, "*" represents the p-value is less than 0.05, "**" represents the p-value is less than 0.01, and "***" represents the p-value is less than 0.001. The more "*", the more significant the statistical difference.

The values used in this article are approximate values, all experimental data are expressed in the range of plus or minus 10%, the best is in the range of plus or minus 5%.

As used herein, the term "extract" refers to a product prepared by extraction. The extract can be presented in the form of a solution dissolved in a solvent, or the extract can be presented as a concentrate or essence containing no or substantially no solvent.

As used herein, "Inca fruit oil material" generally refers to plant seeds, where the seeds may include raw, dried or processed by other physical methods to facilitate processing, and may further include whole, chopped, diced, and milled , Ground or otherwise processed to affect the size and physical integrity of the raw material seeds.

As the terms used herein, "cold pressing", "low-temperature extraction", and "low-temperature extraction" generally refer to the extraction of oil from raw materials by heavy pressing, and the temperature during the heavy pressing does not exceed 30°C. Among them, the method of heavy pressing is a pressure oil press, which uses high pressure (15000 kg/unit) to crush the fruit of the Inca fruit and discharge its oil.

Refer to Figure 1. In some embodiments, an inca fruit oil is obtained by squeezing the inca fruit oil raw materials or the inca fruit seeds by cold pressing. In some embodiments, Sacha inchi (Plukenetia volubilis ) Oil is obtained by sequentially performing the crushing procedure S01, the low-temperature extraction procedure S02, and the filtering procedure S03 as the raw material of Sacha inchi oil, that is, the Sacha inchi seed.

In some embodiments, the raw material of inca fruit oil may be inca fruit seeds that are peeled or contain shells. In some embodiments, the raw material of inca fruit may be fresh or dried seeds of inca fruit.

In some embodiments, the pulverizing process S01 refers to whipping or rolling the raw material of inca oil until it is split into powder or fragments. For example, a juicer, a conditioner, or a homogenizer can be used for crushing. In some embodiments, frying or steaming may be performed at the same time or before or after the crushing process S01 is performed to improve the efficiency of oil extraction.

In some embodiments, the cold pressing procedure S02 refers to pressing the powdered or fragmented inca oil raw material to squeeze its oil. In some embodiments, the temperature of the Sacha inchi oil raw material during the cold pressing process S02 is lower than 30 degrees Celsius. In some embodiments, the cold pressing procedure S02 is to directly apply vertical high pressure to the raw material of inca oil for extraction. In some embodiments, the pressure of the vertical high-pressure extraction can be above 55Mpa. In some embodiments, the vertical high-pressure extraction device can apply a gravity of more than 600 kg per unit area. In some embodiments, before the cold pressing process S02, the powdered or fragmented Inca oil raw material is covered with cotton cloth into a circular cake shape to facilitate vertical high-pressure extraction. In other embodiments, the cold pressing procedure S02 is to apply high pressure (15000 kg/unit) to the raw material of inca oil through a pressure oil press, crush the inca fruit and drain the oil.

In some embodiments, the filtering procedure S03 refers to passing the crude inca oil obtained after cold pressing through a screen to filter out larger solids such as residues or sediments to form a filtrate. For example, the screen may be a 400 mesh screen. In some embodiments, the crude inca oil obtained after cold pressing may be precipitated before performing the filtering process S03 to increase the efficiency of the filtering process S03. In some embodiments, the precipitation time can be 1 hour or until the liquid layer of the crude inca oil becomes a uniform state.

In some embodiments, Sacha indica oil contains alpha-linolenic acid. In some embodiments, the weight percentage of the alpha-linolenic acid content of the inca oil exceeds 40%.

In some embodiments, Sacha butter is used to prepare a composition for enhancing nerve antioxidant capacity, wherein Sacha butter is used to increase the expression of neuroprotection-related genes in cells to enhance nerve antioxidant capacity, and Sacha butter is processed through It is made by crushing, cold pressing and filtering. In some embodiments, the enhancement of nerve antioxidant capacity refers to the enhancement of nerve cells' ability to resist free radicals. In some embodiments, the improvement of nerve antioxidant capacity refers to the improvement of the ability of nerve cells to prevent damage.

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

In some embodiments, inca oil at a concentration of 1% or more can increase the expression of neuroprotection-related genes, thereby enhancing the antioxidant capacity of nerve cells.

Superoxide (Superoxide Anion) and Hydrogen Peroxide (Hydrogen Peroxide) and other free radicals can cause axon conduction to be blocked and damage neuronal cells. Among them, the protein transcribed by the SOD2 gene (superoxide dismutase) will decompose superoxide into hydrogen peroxide, and the protein transcribed by the CAT gene (catalase) will convert hydrogen peroxide into water And oxygen. The proteins transcribed from these two genes are very important for scavenging oxides and free radicals. The higher the expression level of these two genes in the cell, the better the antioxidant capacity of the cell.

In other words, by increasing the expression of the above two genes, Sacha Inchi Oil can effectively enhance cellular anti-oxidation, so as to enhance the nerve's resistance to free radicals, and then achieve the function of protecting nerves.

In some embodiments, Sacha indica oil can be used to prepare brain health care compositions. In some embodiments, the inca oil with brain health capabilities contains alpha-linolenic acid. In some embodiments, the weight percentage of the alpha-linolenic acid content of the sachacha oil with brain health care ability exceeds 40%.

In some embodiments, daily consumption of 1.5g of Sacha inchi oil can be used for brain health care, thereby improving judgment, cognitive ability, concentration, memory, reaction ability, thinking agility, and/or clarity of thinking.

In some embodiments, the aforementioned composition may be a pharmaceutical product. In other words, this medicine contains an effective content of Sacha inchi oil.

In some embodiments, the aforementioned medicines can be manufactured into a dosage form suitable for intestinal or oral administration by using techniques well known to those skilled in the art. These dosage forms include, but are not limited to: tablet, troche, lozenge, pill, capsule, dispersible powder or fine granule ( granule, solution, suspension, emulsion, syrup, elixir, slurry and the like.

In some embodiments, the aforementioned medicines can be manufactured into a dosage form suitable for parenterally or topically by using techniques well known to those skilled in the art. These dosage forms include , But not limited to: injection, sterile powder, external preparation and the like. In some embodiments, the drug may be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, Intradermal injection and intralesional injection.

In some embodiments, the pharmaceutical product may further include a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more of the following reagents: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrant Disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

In some embodiments, the pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline, and phosphate buffered saline (PBS) , Aqueous solution containing alcohol (aqueous solution containing alcohol).

In some embodiments, the aforementioned composition may be an edible composition. In some embodiments, the edible composition can be made into a food product or can be a food additive, which means that a food product is made by adding it during food preparation by a conventional method, or it can be used in a food product. Added during the production process. Here, the food product may be a product formulated with edible materials for human or animal ingestion.

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

Example 1: Inca Fruit Oil alpha- Linolenic acid quantification

Here, after saponification and methyl esterification of the inca oil of the present invention, it is analyzed by gas chromatograph (GC).

Materials and instruments 1. Gas chromatograph 2. Detector: flame ion detector. 3. Chromatography tube: CP-Sil 88 capillary, inner membrane thickness 0.20 μm, inner diameter 0.25mm × 100 m, or equivalent. 4. Reagents: 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 complex solution. 5. Standard for fatty acid control: 9,12,15-cis-octadecatrienoic methyl ester (9,12,15-cis-octadecatrienoic methyl ester). 6. Internal standard: Triheneicosanoin (triheneicosanoin, 21:0).

9,12,15 cis - standard solution prepared octadecatrienoic acid methyl ester: cis-9,12,15-weighed - octadecatrienoic acid methyl ester using standard controls each about 50 mg, accurately weighed, dissolved in n-hexane, and dilute to 10 mL, as the standard stock solution. Before use, dilute it with n-hexane and use it as a standard solution.

Preparation of internal standard solution: Weigh about 100 mg of the internal standard of glyceryl behenate, accurately weigh it, dissolve it in chloroform, and dilute to 10 mL for use as an internal standard solution (hereinafter referred to as standard solution).

Reagent preparation: 1. 8.3M hydrochloric acid solution: Take 250 mL of hydrochloric acid, slowly add 110 mL of deionized water, and mix well. 2. 1N sodium hydroxide methanol solution: Take 4 g of sodium hydroxide and dissolve it in methanol to make 100 mL. 3. Saturated sodium chloride solution: Take at least 40 g of sodium chloride, add 100 mL of deionized water, stir moderately, and take the upper layer for use.

Preparation of inca oil test solution: Weigh an appropriate amount of 10-20 mg of inca oil in this case, add 0.1 mL of the internal standard solution, place it in a 40℃ water bath to remove the chloroform, and dissolve it with 1 mL of n-hexane.

Identification test and determination of fatty acid content: Accurately measure 1 μL each of the test solution and the standard solution, and inject them into the gas chromatograph, and perform gas chromatography under the following conditions. The conditions of gas chromatography determination are as follows: 1. Chromatography tube temperature: initial temperature: 170℃, 40 min. 2. Heating rate: 3℃/min. 3. Final temperature: 200℃, 50 min. 4. Detector temperature: 300℃. 5. Injector temperature: 250℃. 6. The mobile phase gas helium flow rate: 0.75 mL/min. 7. Split ratio: 40:1.

其中，W為取樣分析檢體之重量(g)；W_FAMEx 為印加果油檢液中9,12,15-順式-十八碳三烯酸甲基酯之含量(g)；F_Fax 為9,12,15-順式-十八碳三烯酸甲基酯轉換為alpha-亞麻酸之係數，其係數為：0.9520。Comparing the retention time of the peaks obtained from the Inca fruit oil test solution and the standard solution, determine the content of alpha-linolenic acid in the test solution (%) according to the following calculation formula: The content of alpha-linolenic acid in the test solution (%) =

Among them, W is the weight of the specimen for sampling and analysis (g); W _FAMEx is the content of 9,12,15-cis-octadecatrienoic acid methyl ester (g) in the inca oil test solution; F _Fax is The coefficient for converting 9,12,15-cis-octadecatrienoic acid methyl ester to alpha-linolenic acid is 0.9520.

Experimental result

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

Example 2: Cell experiment - Inca Oil Inhibition ROS Generation (hydrogen peroxide treatment) genetic testing

Here, the fluorescent probe DCFH-DA was used in conjunction with flow cytometry to measure the changes in the content of reactive oxygen species in mouse brain neuroblastoma cells (Neuro2a) after being treated with Saguaro oil.

Materials and equipment 1. Cell line: Mouse brain neuroblastoma cells (Neuro2a), taken from the American Type Culture Collection ATCC; Cat. CCL-131. 2. Medium: Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, DMEM, purchased from Gibco, 11965-092) is added with additional ingredients to make it contain 10 vol% FBS (fetal bovine Serum, purchased from Gibco, 10437-028), 3.7g /L Sodium bicarbonate (purchased from Sigma, S5761-500G), 1% antibiotic Antibiotic-Antimycotic penicillin-streptomycin (Gibco, #15240062). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. Hydrogen peroxide (H ₂ O ₂ ): purchased from Sigma-Aldrich, product model 95299-1L. 5. Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) is diluted 10 times with 1X PBS solution. 6. Inca fruit oil: The inca fruit oil used in this experiment is purchased from Glint SAC (Peru), the product number is 11002576, and its preparation method is that the seeds of an inca fruit are crushed and rolled and then subjected to vertical high pressure. It is extracted at low temperature (below 30 degrees Celsius) and filtered.

Experimental steps

The experiment will be divided into three groups: the experimental group, the blank control group (the group that has not added inca oil, nor the group treated with hydrogen peroxide), and the control group (the group that has not added inca oil, but has been treated with hydrogen peroxide). Three replicate experiments were performed in each group: 1. Neuro2a cells were seeded in a 6-well culture dish containing 2ml medium ^{per well at 1×10 6 cells per well.} 2. Place the culture plate under 5% CO ₂ and 37°C for 24 hours. 3. Remove the medium. 4. Add 20 μL of Inchi oil (Peruvian Glint SAC, product number 11002576) to the 2mL experimental medium of the experimental group (that is, the volume percentage of Inchi oil in the cell culture medium is 1%), and simultaneously add H ₂ O ₂ , And react at 37°C for 6 hours. Specifically, 35% wt of hydrogen peroxide is first diluted to 100 mM (10 μL of hydrogen peroxide is added to 990 μL of double distilled water), and then 20 μL of 100 mM hydrogen peroxide is added to a 2 mL cell culture dish. 5. Add 2 mL of cell culture medium (that is, without Sacha oil) to the experimental medium of the control group and react at 37°C for 6 hours. 6. Add a simple 2 mL cell culture medium (that is, without Sacha inchi oil) to the experimental medium of the control group, and simultaneously add H ₂ O ₂ , and react at 37°C for 6 hours. Specifically, 35% wt of hydrogen peroxide is first diluted to 100 mM (10 μL of hydrogen peroxide is added to 990 μL of double distilled water), and then 20 μL of 100 mM hydrogen peroxide is added to a 2 mL cell culture dish. 7. After the reaction, after removing the supernatant, rinse each well with 1 mL of 1X PBS solution once. 8. Break the cells with cell lysate (purchased from Geneaid) to obtain cell lysate. 9. Use RNA extraction reagent kit (purchased from Geneaid) to extract RNA from cell lysate. Next, take the extracted RNA of 2000 nanograms (ng) as a template, and use SuperScript®III reverse transcriptase (purchased from Invitrogene) to perform reverse transcription to generate the corresponding cDNA. 10. Later, use cDNA as a template and use the primer pair used to amplify the target gene (as shown in Table 1 below) in the ABI StepOnePlusTM Real-Time PCR system (ABI StepOne Plus real-time PCR system, purchased from Thermo Fisher Scientific) KAPA SYBR FAST kit (purchased from Sigma) was used to perform quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) to quantify the mRNA expression of SOD2 gene and CAT gene, and then infer each The expression level of the protein encoded by the gene. Among them, the setting conditions of the ABI StepOne Plus real-time PCR system are 95°C for 1 second, 60°C for 20 seconds, a total of 40 cycles. 11. Thereafter, use the 2- ^ΔΔCt method to determine the relative expression level of the target gene. The so-called relative expression level is defined as the fold change of the RNA expression level of a target gene in the experimental group relative to the same gene in the control group or the control group. This method uses the cycle threshold of the GAPDH gene as the reference gene cycle threshold (Ct) of the internal control, and calculates the fold change according to the following formula: △Ct = Ct _{experimental group target gene / control group or control group target gene} -Ct _GAPDH △ △Ct= Target gene of △Ct experimental group-fold change _{of target gene of} △Ct _{control group} ^{= 2 -ΔΔCt average value.} Finally, use the STDEV formula of Excel software to calculate the standard deviation, and use the one-tailed Student t-test in Excel software Analyze whether there is a statistically significant difference (*p value <0.05; **p value <0.01; ***p value <0.001). Among them, the primer pairs corresponding to the SOD2 gene are SOD2-F and SOD2-R. The primer pairs corresponding to the CAT gene are CAT-F and CAT-R, and the primer pairs corresponding to the GADPH gene are GADPH-F and GADPH-R.

Table 1 Primer name Serial number sequence 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 quantitative results of the corresponding genes obtained from three repeated experiments are averaged to obtain the average value, and then the average value of the control group and the experimental group is converted into relative performance by taking the average value of the control group as 1 (relative performance amount) Quantity (%), as shown in Figure 2. The statistically significant difference between the measurement results of the experimental group and the measurement results of the control group is statistically analyzed by the student t-test. In Figure 2, "***" represents that the p-value of the experimental group is less than 0.001 when compared with the control group, and "**" represents that the p-value of the experimental group is less than 0.01 when compared with the control group.

Experimental result

Referring to Figure 2, the expression levels of neuroprotection-related genes (i.e. SOD2 gene and CAT gene) in the experimental group after treatment with inca oil compared to the control group (the group treated with hydrogen oxide but without the addition of inca oil) Both are significantly improved. Specifically, after the cranial nerve cells were treated with hydrogen oxide and then treated with the inca oil of this case, the SOD2 gene expression was about 3.6 times higher than that of the control group treated with only hydrogen oxide, while the CAT gene expression was higher. The control group was about 1.9 times higher. This result shows that Sacha butter can effectively enhance the expression of SOD2 gene and CAT gene, can enhance the antioxidant capacity of cells and increase the ability to decompose ROS, and has the effect of enhancing the antioxidant capacity of nerve cells.

Example 3: Human experiment - Cognitive function test

Use samples

Liquid pack containing the inca oil of the present invention 15mL/pack (contains 1.5g inca oil (10% by weight), 7.5% gum arabic, 0.1% soy lecithin, 0.37% corn sugar gum, and 0.1 Guanhua bean gum %, DL-α-tocopherol acetate 0.02%, glycerol 13.0%, D-xylitol 21.0%, β-carotene 0.045%, citric acid 0.9%, L-ascorbic acid (vitamin C) 0.087%, apple liquid flavor 0.35%, potassium hexadienoate 0.05%, pineapple liquid flavor 0.52%, water 45.958%). In some other embodiments, each person can take up to 5g of Sacha inchi oil per day. Among them, this inca fruit oil is purchased from Glint SAC (Peru), the product number is 11002576, and its preparation method is that an inca fruit seed is crushed and pressed, and then squeezed at a vertical high pressure and low temperature (below 30 degrees Celsius) , And then filtered.

Number and age of subjects

10 subjects aged 25-55 years old.

Experimental method

The subjects drank one liquid pack containing the inca oil of the present invention (each containing 1.5 g of inca oil) daily for 28 days (ie 4 weeks). And before the start of drinking (week 0) and 28 days after drinking, according to different test items, use the corresponding equipment and measurement methods to record the test results of the subjects. It should be noted that, except for the test, the subjects did not practice or touch items related to the test in other ways to reduce the impact on the test results due to the increase in the familiarity of the test content. In addition, the time and status of the test before drinking and 28 days after drinking were the same (for example, if the test time before drinking was 9 am and no breakfast was eaten, the test time after drinking 28 days was also the same. It is 9 o'clock in the morning and the time and state of not eating breakfast) to reduce the interference factors due to different physiological states and the external environment.

Test items: 1. Color judgment interference test, 2. Digital span inversion test, 3. Image memory test, 4. Position interpretation memory test, 5. Cognitive function questionnaire feedback. The test from 1. to 3. above is conducted on the Cognitive Fun website (http://cognitivefun.net/), and the test for 4. is on the Flash Fabrica website (http://flashfabrica.com/f_learning/brain/tw_brain .html).

1. Color Judgment Interference Test

When the test starts, the computer screen will randomly display a colored English word (for example: "GREEN"), but the color of the English word may not be the same as the color stated in the word (for example: the word color of the word "GREEN") It is yellow), and the subject must correctly enter the color of the text instead of the color described in the English word (the correct answer in the above example is YELLOW). The test system will only record the number of seconds to answer the correct question.

There are a total of 20 questions in this test. The test result is the average number of seconds to answer the correct questions, and each subject takes 3 tests. The test result presentation method is to set the average answer seconds (judgment time) of all subjects in week 0 as 100%, and calculate the percentage of the average answer seconds (judgment time) of all subjects on the 28th day, as a long bar The graph is shown in Figure 3.

In addition, it is particularly noted that all testers can easily recognize all color English words in the test content before and after drinking, and there is no obstacle to judging the color.

2. Numerical Span Inversion Test

When the test starts, a random string of numbers (for example: "514") will appear on the computer screen, and the testee needs to input this string of numbers (in the previous example, the subject should input 415"). Among them, the test system will record the correct rate of subjects.

This test is conducted for one minute, and the test result is the average of the correct rate (number of correct answers/total number of questions), and each subject will take 3 tests. The test result presentation method is to set the average correct rate of all subjects at week 0 as 100%, and calculate the percentage of the average correct rate of all subjects on the 28th day, which is presented as a bar graph as shown in Figure 4.

3. Image memory test

When the test starts, a series of pictures will be displayed on the computer screen within a period of time. If they see exactly the same picture during the display, the subject must click on the picture or click the blank button, and the test system will record the correct answer. Count and the number of seconds to answer the question correctly.

This test is conducted for 1 minute. The test results are correct rate (number of correct answers/total number of questions) and response time (average number of seconds for correct answers), and each subject takes 3 tests. The test result presentation method is to set the average response time of all subjects at week 0 as 100%, calculate the percentage of the average correct rate of all subjects on the 28th day, and present it as a bar graph as shown in Figure 5; and The average correct rate of the test subjects at week 0 was set as 100%, and the percentage of the average correct rate of all subjects on the 28th day was calculated and presented as a bar graph as shown in Figure 6.

4. Location Interpretation Memory Test

After the test begins, several numbers will appear in different positions on the screen at the same time, as shown in the left frame of Figure 7. After about 0.7 seconds, the number on the screen will disappear, leaving only the position of the number as shown in the right frame of Figure 7. At this time, the subjects need to click on the numbers in order from small to large. Based on the test results, including the correct answer rate and reaction time, the test system will automatically calculate the brain age of the subject. There is no time limit for this test, and each subject will take 10 tests. The test results are presented in a bar graph as shown in Figure 8. The average brain age of all subjects at week 0 and the average brain age of all subjects at day 28 are presented as a bar graph.

5. Cognitive function feedback questionnaire

All subjects were asked to take a cognitive function questionnaire (shown in Table 2 below) before drinking the liquid pack (ie week 0) and 4 weeks after drinking the liquid pack (ie week 4) Go to Figure 13 to present and analyze it to evaluate the improvement of cognitive function.

Table 2 Mental state Very bad difference ordinary it is good well A. Recent vitality and mental state 1 2 3 4 5 B. Recent state of palpitation and anxiety 1 2 3 4 5 C. Thinking about the state of agility in the near future 1 2 3 4 5 D. The state of clear thinking in the near future 1 2 3 4 5 E. Recent memory status 1 2 3 4 5 F. Recent work concentration status 1 2 3 4 5 G. Recent stress resistance status 1 2 3 4 5 H. Recent sleep state 1 2 3 4 5

Experimental result

Please refer to Fig. 3, Fig. 9 and Fig. 10, compared to the 0th week, after drinking the liquid pack containing the inca oil of the present invention for 4 weeks, the judgment time of the subjects in the color judgment interference test was reduced by 15.9%. According to the feedback from the questionnaire, at least 50% of the subjects rated their own mental clarity from “poor” or “normal” to “good” after drinking the liquid pack containing the inca oil of the present invention for 4 weeks, and at least 70% of the subjects rated their own concentration from "poor" or "normal" to "good". It can be seen that the inca oil of the present invention has the effect of improving the clarity of thinking, concentration, judgment and cognitive ability.

Please refer to Fig. 4 and Fig. 11, compared to week 0, after drinking the liquid pack containing the inca oil of the present invention for 4 weeks, the accuracy of the subjects in the digital span inversion test increased by 5.5%. According to the feedback from the questionnaire, 40% of the subjects rated their own memory from "very poor", "poor" or "normal" to "good" after drinking the liquid pack containing the inca oil of the present invention for 4 weeks. It can be seen that the inca oil of the present invention has the effect of improving memory, especially short-term memory.

Please refer to Fig. 5, Fig. 6 and Fig. 12, compared to week 0, after drinking the liquid pack containing the inca oil of the present invention for 4 weeks, the reaction time of the subjects in the image memory test was reduced by 9.1%, and The accuracy rate is increased by 13.5%. According to the feedback from the questionnaire, at least 41% of the subjects rated their thinking agility from “poor” or “normal” to “good” after drinking the liquid pack containing the inca oil of the present invention for 4 weeks. It can be seen that the inca oil of the present invention has the effect of improving image memory and thinking agility.

Please refer to Figure 8 and Figure 13, compared to the 0th week, after drinking the liquid pack containing the inca oil of the present invention for 4 weeks, the brain age estimated by the subjects through the results of the position interpretation memory test decreased on average 5 years old. According to the feedback from the questionnaire, at least 40% of the subjects rated their vitality and mental state from "very poor" or "normal" to "good" after drinking the liquid pack containing the inca oil of the present invention for 4 weeks. It can be seen that the inca oil of the present invention has the effects of reducing brain age, improving vitality, mental state and memory in two-dimensional space.

In summary, the inca oil of the present invention can effectively improve brain concentration, judgment, cognitive judgment, and memory. The memory includes short-term memory, image memory, digital memory, and spatial memory. Specifically, according to the foregoing embodiments, the Sacha inchi oil of the present invention can reduce the reaction time of cognitive interference with judgment by 15.9%, increase the accuracy of short-term digital memory by 5.5%, and reduce the reaction time of short-term image memory by 9.1% And the correct rate is increased by 13.5%, and the brain age is reduced by 5 years. In addition, Inca fruit oil can also improve the mental state, brain power, sleep quality and anti-anxiety and anti-stress ability of users after drinking/eating.

Example 4: Test of the static stability of the composition

Since the inca oil of the present invention is mainly composed of lipids, delamination is likely to occur when the water-soluble composition containing the inca oil of the present invention is produced, which in turn causes doubts about the stability of the composition in storage. Therefore, in order to improve the stability of the inca fruit of the present invention, it is tested whether the liposome suspension with the ability to coat the inca fruit oil is allowed to stand for one day at room temperature (25°C). If the liposome suspension is a homogeneous liquid without stratification, it means that the liposome structure in it is relatively stable. Conversely, if the liposome suspension has obvious stratification and the color is turbid and uneven, it means that the structure of the liposomes contained in it is relatively unstable.

Among them, the preparation method of liposomes with the ability to stably coat inca oil is to mix specific proportions of lecithin, acacia and water, and homogenize under specific pressure to form a liposome suspension. Among them, the liposome suspension contains a plurality of liposomes with stable structures. Here, the prepared liposome has a stable structure, can stably coat the active ingredients, and can be used to improve the bioabsorption rate of the coated active ingredients.

Please refer to Figure 14, which is the use of cryo-electron transmission microscopy (cryo-TEM; brand: JEOL, model: JEM-1400) to observe the liposomes in the liposome suspension, the shape of which is spherical , And the outer diameter length is about 200nm~400nm. And it can be seen from Figure 14 that the same set of liposome suspension contains multiple liposomes with different outer diameter lengths.

Here, the mixtures of groups A and B are configured according to the formula ingredients and formula ratios in Table 3 below. The total weight of the mixture is 100 grams. First, mix soybean lecithin, DL-α-tocopherol acetate, inca oil and part of water at a weight ratio (w/w) of 1:4, and stir at room temperature (25°C) to confirm that the lecithin is dissolved In part of the water to form the first premixed solution.

In addition, acacia gum, Guanhua bean gum, corn sugar gum, sucralose, glycerin, potassium adipate, D-xylitol, β-carotene, citric acid, vitamin C and other water are mixed to form a second premixed solution .

In addition, the first pre-mixed solution and the second pre-mixed solution are mixed and evenly stirred to ensure that each component is completely dissolved in water, and the total weight is 100 grams with water to form a third pre-mixed solution. Next, the quantified third premixed solution was sieved with 80 meshes to form a mixed solution.

table 3 Formula composition (wt%) Group A Group B Soy Lecithin 0.1 0.1 Gum arabic 7.5 2.5 Guanhua Bean Gum (Thickener) 0.1 0.1 Corn gum (thickener) 0.37 0.37 DL-α-tocopherol acetate 0.02 0.02 glycerin 13.0 13.0 D-xylitol 21.0 21.0 β-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 hexadienoate (preservative) 0.05 0.05 water 45.958 50.958 Inca oil 10 10

Then, the mixture is subjected to a sterilization program, and the setting value of the sterilization program is 95°C±5°C for 30 minutes. When the sterilized mixture is cooled to 50°C, homogenize the cooled mixture with a homogenizer (brand: GEA Niro Soavi, model: Panda Plus) to form a liposome suspension. The pressure value is 350 bar.

Next, the liposome suspensions of the two groups A and B were allowed to stand at room temperature (25°C) for one day to observe whether the liposome suspensions were stratified. Moreover, if the liposome suspension is a homogeneous liquid without stratification, it means that the liposome structure in it is relatively stable. Conversely, if the liposome suspension has obvious stratification and the color is turbid and uneven, it means that the structure of the liposomes contained in it is relatively unstable. In other words, due to the rupture of the coating layer of the liposomes, no liposomes were formed, and the liposome suspension was significantly stratified, indicating that this ratio is less suitable for the composition of the Sacha indica oil of the invention.

Refer to Figure 15. The liposome suspension of group B has obvious stratification, which means that the emulsification effect is not significant, and the resulting liposome structure is unstable. However, the liposome suspension of group A has no obvious stratification, which means that the emulsification effect is significant. It can be seen that when the mixed liquid contains 7.5 weight percent gum arabic, and the liposome suspension obtained by the pre-killing treatment has no obvious stratification, the stability of the inca oil-containing composition made at this ratio Better.

Although the technical content of the present invention has been disclosed in the preferred embodiments as above, it is not intended to limit the present invention. Anyone who is familiar with this technique and makes some changes and modifications without departing from the spirit of the present invention should be covered by the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

no.

Fig. 1 is a flow chart of an embodiment of the method for preparing Inca oil. Figure 2 is a graph showing the ratio of neuroprotection-related genes and performance of control group, control group, and experimental group in Example 2. Fig. 3 is a graph of the judgment time ratio of the color judgment interference test in the third example. Figure 4 is a graph of the percentage of accuracy of the reciprocal test of the number span in the third example. Figure 5 is a graph of the reaction time ratio of the image memory test in Example 3. Figure 6 is a graph showing the percentage of correctness of the image memory test in the third example. Fig. 7 is a schematic diagram of an example of the positional interpretation memory test in example three. Fig. 8 is a bar graph of the brain age estimated by the results of the positional interpretation memory test in Example 3. Figure 9 shows the results of a questionnaire survey on issues related to clarity of thinking in Example 3. Figure 10 shows the results of the questionnaire survey on issues related to concentration in the third example. Figure 11 shows the results of the questionnaire survey on memory-related issues in the third example. Figure 12 shows the results of the questionnaire survey on questions related to thinking agility in the third example. Figure 13 shows the results of a questionnaire survey on vitality and mental state in the third example. Figure 14 is an electron microscope image of liposomes in Example 4. Figure 15 is an image of the static experiment results of different formulations in Example 4.

Claims (13)

A composition containing inca oil, wherein the inca oil is coated by a liposome with stable coating ability, and wherein the liposome with stable coating ability comprises 0.1 weight percent of lecithin and 3 weight percent Percent~8% by weight of gum arabic. The composition according to claim 1, wherein the composition comprises a brain health care composition and/or a nerve-raising antioxidant composition. A kind of inca oil is used to prepare a composition for enhancing the antioxidant capacity of nerve cells, wherein the inca oil is prepared by a crushing process, a cold pressing process, and then a filtering process Become. An inca oil is used for preparing a brain health-care composition, wherein the inca oil is prepared by a crushing process, a cold pressing process, and a filtering process. The use according to claim 3, wherein the nerve cell anti-oxidant composition enhances the nerve cell's anti-oxidant ability by increasing the nerve cell's ability to resist free radical damage. The use according to claim 5, wherein the free radical comprises superoxide or peroxide. The use according to claim 3, wherein the composition improves the antioxidant capacity of the nerve cell by increasing the expression of neuroprotection-related genes in the cell. The use according to claim 5, wherein the neuroprotection-related gene comprises SOD2 gene or CAT gene. The use according to claim 4, wherein the composition achieves the brain health care by improving cognitive ability, concentration, memory, reaction ability, thinking agility, vitality, mental state and/or clarity of thinking. The use according to claim 9, wherein the memory includes short-term memory, digital memory, two-dimensional spatial memory, or image memory. Such as the use of claim 3 or 4, wherein the inca fruit oil contains at least 40% or more of alpha-linolenic acid. Such as the use of claim 3 or 4, wherein the composition is a medicine. Such as the use of claim 3 or 4, wherein the composition is a food composition or a health food composition.

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