TWI615141B - Use of α-eleostearic acid for anti-aging - Google Patents

Abstract

The present invention provides a use of bitter gourd seed oil and a composition based thereon. The bitter gourd seed oil contains an effective dose of conjugated linoleic acid, and the bitter gourd seed oil has anti-aging effect. Thereby, the bitter gourd seed oil can be used to prepare an anti-aging composition.

Description

--Tung oil acid anti-aging use

The present invention relates to the use of a bitter gourd seed oil and compositions based thereon, in particular to the use of bitter gourd seed oil for anti-aging and anti-aging compositions.

In biology and medicine, aging refers to the phenomenon that the physiological state of an organism deteriorates with time. Aging causes the function or function of various parts of the body to decline or function, which makes the health function of the organism worse. Eventually it leads to the death of the organism. Modern people have a lot of stress in life, and the accumulation of long-term stress will accelerate the aging of the body and also cause many chronic diseases.

In recent years, the SIRT1 gene has been recognized as a longevity gene, and its encoded sirtuin-1 is an important NAD ⁺ -dependent deacetylase in mammals, which is scattered in the nucleus. And the cytoplasm, involved in many important physiological and pathological processes. Among them, the target proteins that can remove acetamidine-1 from p-amylase have p53, FOXO and Ku70, which are related to the threshold of apoptosis and the repair of stimulating cells. Cells that are lost by apoptosis during the lifetime of an organism may be an important factor in affecting aging, especially those that do not renew, such as the heart and brain. Studies have shown that in the fasting state (calorie restriction), the concentration of NAD ^{+ in} hepatocytes increases, which can promote the activity of deacetylase-1.

However, it is difficult for humans to adopt this intense diet. Therefore, in order to obtain the health effects of calorie restriction, it may be necessary to activate substances that can activate acetylase-1 to improve the global population due to aging and chronic diseases. The incidence is relatively increased, causing global health problems, and substances that can activate acetamylase-1 are used as normal body care to avoid medical expenses.

One aspect of the present invention is to provide a bitter melon seed oil for use in the preparation of an anti-aging pharmaceutical composition wherein the bitter gourd seed oil comprises an effective amount of conjugated linolenic acid (CLN). ).

According to the use of the aforementioned bitter gourd seed oil, the conjugated linoleic acid may be α-eleostearic acid (α-ESA).

According to the use of the aforementioned bitter gourd seed oil, the bitter gourd seed oil is extracted from bitter gourd seeds.

According to the use of the aforementioned bitter gourd seed oil, the anti-aging pharmaceutical composition is a sirtuin-1 activator.

According to the use of the aforementioned bitter gourd seed oil, wherein the anti-aging pharmaceutical composition is an nicotine phosphoribosyltransferase (nicotinamide phosphoribosyltransferase, NAMPT) active pharmaceutical composition.

According to the use of the aforementioned bitter gourd seed oil, wherein the anti-aging pharmaceutical composition is a nicotinamide adenine dinucleotide (NAD ⁺ )/reduced nicotine indoleamine adenine dinucleoside A pharmaceutical composition of the ratio of reduced form of nicotinamide adenine dinucleotide (NADH).

Another aspect of the present invention is to provide an anti-aging composition comprising a bitter gourd seed oil, and the bitter gourd seed oil contains an effective amount of conjugated linoleic acid.

According to the anti-aging composition described above, the conjugated linoleic acid is alpha-ternic acid.

The anti-aging composition according to the foregoing is in a form selected from the group consisting of a pharmaceutical composition, a feed composition, a beverage composition, a nutritional supplement composition, an edible composition, and a health care edible composition.

Thereby, the conjugated linoleic acid unique to the bitter gourd seed oil of the present invention increases the NAD ⁺ /NADH ratio by increasing the nicotine phosphoribosyltransferase activity, thereby activating the deacetylase-1 to achieve anti-aging. The efficacy of the conjugated linoleic acid-rich bitter melon seed oil of the present invention can be used for preparing an anti-aging pharmaceutical composition. The composition of the bitter gourd seed oil comprising an effective dose of conjugated linoleic acid can be used as an anti-aging pharmaceutical composition, feed composition, beverage composition, nutritional supplement composition, edible composition or health food composition. Has the potential to be used in the biomedical health market.

The above summary is intended to provide a simplified summary of the disclosure. In order for the reader to have a basic understanding of the disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. The description of the drawing is as follows: Figure 1 is an analysis of the fatty acid composition of bitter gourd seed oil; Figure 2A is a process of treating fatty acids. The results of deacetylation of intracellular FOXO1 protein; Figure 2B shows the quantitative results of intracellular acetylated FOXO1 protein/total FOXO1 protein after treatment of fatty acids; and Figure 3 shows the intracellular nicotine phosphoribosyl ribose group after treatment of fatty acids Figure of the results of transferase (NAMPT) activity; Figure 4 shows the intracellular nicotine indoleamine adenine dinucleotide (NAD ⁺ ) / reduced form of nicotine indoleamine adenine dinucleotide (NADH) after treatment of fatty acids Figure of the results of the ratio; and Figure 5 is a graph of the percentage of survival of nematodes after treatment of fatty acids.

The disclosure of the present specification proposes novel uses of bitter gourd seed oil, particularly the novel use of conjugated linoleic acid in bitter gourd seed oil. To evaluate the anti-aging effect of conjugated linoleic acid in bitter gourd seed oil by cell in vitro test, and further verify the anti-aging effect of conjugated linoleic acid in bitter gourd seed oil by increasing nicotine phosphoribosyltransferase (nicotinamide phosphoribosyltransferase; NAMPT) activity, increased nicotinamide adenine dinucleotide (NAD ⁺ ) / reduced form of nicotinamide adenine dinucleotide (NADH) The ratio, which in turn activates sirtuin-1, achieves anti-aging effects. The bitter gourd seed oil proposed in the present specification is a potential anti-aging substance, and particularly can be used as a substance for activating deacetylase-1, and can be used for preparing a pharmaceutical composition which is anti-aging.

The " Morordica charanita " described in this manual, also known as the melon, koi, squash or sorghum , is a vine herb of the Cucurbitaceae family of Momodisica and is the Asian people. Eat vegetables daily. Chinese pharmacology records the effects of clearing away heat, clearing eyesight, detoxifying and replenishing the kidney and moistening the spleen. Nowadays, the research has anti-tumor, anti-viral, anti-inflammatory, hypoglycemic or hypolipidemic therapeutic potentials. In recent years, studies on bitter gourd seeds have found that polypeptides extracted from bitter gourd seeds have physiological active ingredients that regulate blood sugar and anti-diabetes. In addition, bitter gourd seeds have physiological effects such as antibacterial, hypoglycemic, anti-cancer and anti-oxidation, and The physiological activity is due to the presence of special unsaturated fatty acids in the oil.

The "conjugated linolenic acid (CLN)" described in the present specification is an unsaturated fatty acid, and the natural polyunsaturated fatty acid has a double bond between the double bonds of the polyunsaturated fatty acid, and is conjugated. The appearance of a form (including cis cis or trans trans stereoisomers) is referred to as a conjugated fatty acid. Conjugated linoleic acid is a conjugated triene, more The number is present in the plant seeds.

The present invention will be further exemplified in the following specific examples to facilitate the general knowledge of the art to which the present invention pertains, and the present invention may be fully utilized and practiced without undue interpretation. The scope of the invention is limited, but is intended to illustrate how to practice the materials and methods of the invention.

<Test example> First, bitter gourd seed oil 1. Preparation of bitter gourd seed oil

In this experiment, bitter gourd seed oil was prepared from 10 different strains of bitter gourd. The dried bitter gourd seeds provided by the Hualien Agricultural Reform Institute are ground into powder by grinding coffee beans, and the bitter gourd seed powder and hexane (n-hexan, HPLC GRADE, ECHO) are soaked at room temperature for one day at a ratio of 1:10. It was filtered with a filter paper (whatman filter paper no. 1), and the filtrate was collected. The remaining bitter gourd residue was further soaked in the same ratio for two days, and then filtered, and the filtered filtrate was concentrated under reduced pressure to obtain the desired bitter gourd seed oil. The average yield of bitter melon seed oil of 10 samples was 28±5 g of bitter gourd seed oil (about 28%).

2. Analysis of fatty acid composition of bitter gourd seed oil

In this test example, the extracted bitter gourd seed oil was further analyzed for its fatty acid composition by gas chromatography (Gas Chromatograph). Since the structure of the conjugated linoleic acid in the bitter gourd seed oil is special, when the fatty acid is quantified by gas chromatography, the fatty acid methyl esterification needs to be carried out with caution. Avoid a reduction in the proportion of conjugated linoleic acid. The method of methyl esterification of fatty acids employs the NaOMe/methanol method, which is based on the procedure proposed by Chou YC et al. (Nutrition, 2012; 28: 803-811). The obtained fatty acid methyl ester (FAME) was dissolved in hexane, and fatty acid analysis was carried out using a gas chromatograph (Hewlett Packard 5890 GC). The conditions of gas chromatography were melting in DB-1. Flame ionization detection was carried out in a quartz capillary (60 m × 0.25 mm × 0.1 μm; Agilent), and the carrier gas was nitrogen (1.5 ml/min). The residence time of the fatty acid peak is then compared to the residence time of the standard to determine the fatty acid composition in the bitter gourd seed oil.

Please refer to Figure 1 and Table 1 below. Figure 1 is a graph showing the fatty acid composition of bitter gourd seed oil. Table 1 shows the ratio of each fatty acid in bitter gourd seed oil.

The results of Figure 1 and Table 1 show that five major fatty acids can be identified in bitter gourd seed oil, namely palmitic acid (3 ± 1%), stearic acid (36 ± 5%), oleic acid (5 ± 3%), linoleic acid (4 ± 1%) and alpha-ternic acid (46 ± 5%). Among them, α-tricoleic acid ( c 9, t 11, t 13-conjugated linoleic acid) is the most important fatty acid in bitter gourd seed oil, accounting for 46±5% of total fatty acids.

Second, the efficacy of bitter gourd seed oil against aging 1. Go to the effect of acetylated FOXO1

The transcription factor FOXO1 (Forkhead box protein O1) has physiological functions such as regulation of cell cycle and apoptosis, and FOXO1 protein is one of the target proteins for removing acetamyl group by deacetylase-1. In this test, the amount of acetylated FOXO1 in the cells after fatty acid treatment was analyzed by immunoprecipitation and western blotting to investigate whether bitter melon seed oil can activate deacetylase-1.

The cells used in this experiment were mouse hepatoma cell line H4IIEC3, which was purchased from the Bioresource Collection and Research Center (BCRC), and the cell culture medium was high in sugar (25 mmol/L D-). Glucose DMEM (Dulbecco's modified Eagle's medium) containing 20% horse serum, 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 unit/ml penicillin. The H4IIEC3 cells were cultured in the incubator with the above-mentioned cell culture medium at 5% CO ₂ and maintained at 37 ° C, and the cells were subcultured every 3 days.

Before the test, the cells were divided into three groups. H4IIEC3 cells were cultured in the above-mentioned cell culture medium at 37 ° C, 5% CO ₂ incubator until the cell coverage was about 70%, and then the cell culture medium was replaced with 1 The serum-free medium of % bovine serum albumin was cultured in a 5% CO ₂ incubator at 37 ° C until the next day, and the three groups of cells were respectively added with a carrier (absolute ethanol) and α-linolenic acid ( --linolenic [18:3(n-3)]) and α-linolenic acid + α-tricoleic acid. The group to which the carrier was added was a control group, and the group in which α-linolenic acid was added alone, wherein the final concentration of α-linolenic acid was 100 μM, and the group of α-linolenic acid and α-tallowic acid was added at the same time. The final concentration of α-linolenic acid was 75 μM, and the final concentration of α-tricoleic acid was 25 μM. The chemical structures of α-tricoleic acid and α-linolenic acid are as follows:

After H4IIEC3 cells were treated with the above fatty acid for 24 hours, H4IIEC3 cells were washed twice with PBS at 4 °C, and RIPA buffer solution containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Sigma) was added to dissolve the cells, and the cell lysate was taken. The supernatant was taken in a 1.5 ml microcentrifuge tube and centrifuged at 12,000 rpm for 10 minutes. The protein concentration was determined using a Bio-Rad protein assay kit, and the remaining protein extract was stored at -80 °C. The procedure of the immunoprecipitation method was to take 1 mg of the protein extract and mix it with Dynabeads Protein G (Invitrogen) conjugated with 8 μg of total FOXO1 protein antibody (Cell Signaling Technology) at 4 ° C for 12 hours, and then flush the total FOXO1 with 20 μl of the washing buffer solution. protein. The total FOXO1 protein expression and the acetylated FOXO1 protein expression were detected by Western blotting. The primary antibodies used were total FOXO1 protein antibody (Cell Signaling Technology) and acetylated FOXO1. Protein antibody (Lys259/262/271, Santa Cruz Biotechnology) with a dilution factor of 1:1000.

Please refer to Figures 2A to 2B. Figure 2A shows the results of deacetylation of FOXO1 protein in H4IIEC3 cells after fatty acid treatment, and Figure 2B shows the effect of acetylated FOXO1 protein/total FOXO1 protein in H4IIEC3 cells after fatty acid treatment. Quantitative results, in which IP represents the antibody used in the immunoprecipitation method, IB represents the antibody used in the Western blotting method, Ace-FOXO1 represents the acetylated FOXO1 protein antibody, T-FOXO1 represents the total FOXO1 protein antibody, and LN represents α-Linlinoleic acid, α-ESA represents α-tungolic acid, and there is a significant difference between the groups indicating different letters ( p < 0.05). The results showed that the amount of acetylated FOXO1 protein in H4IIEC3 cells was comparable to that of the control group in the group treated with α-linolenic acid alone, but the group of α-linolenic acid and α-tallowic acid was added at the same time, H4IIEC3 The amount of intracellular acetylated FOXO1 protein was significantly reduced, indicating that the cells treated with α-tricoleic acid had a higher degree of activation of deacetylase-1.

2. Increase the activity of nicotine phosphoribosyltransferase (NAMPT) activity

NAD ⁺ plays a very important role in many reactions in organisms, such as cofactors in redox reactions or as substrates in ADP-ribosylation reactions, providing ADP ribose. NAMPT is an enzyme that determines the rate of NAD ⁺ production on the NAD ⁺ biosynthetic recovery pathway and regulates the activity of deacetylase-1.

This test further analyzes the NAMPT activity in cells after fatty acid treatment to investigate whether bitter melon seed oil can activate deacetylase-1. H4IIEC3 cells were treated with the same fatty acid treatment steps as in Test Example 2 After 24 hours of treatment, the activity of NAMPT in H4IIEC3 cells was detected using a CycLex® NAMPT Colorimetric Assay Kit (CycLex Co.).

Please refer to Fig. 3 for the results of NAMPT activity in H4IIEC3 cells after treatment of fatty acids, wherein LN represents α-linolenic acid, α-ESA represents α-tallowonic acid, and * indicates significant difference ( p < 0.05). The results showed that the activity of NAMPT in H4IIEC3 cells was comparable to that of the control group in the group treated with α-linolenic acid alone, but the activity of NAMPT in H4IIEC3 cells was also added to the group of α-linolenic acid and α-teroleic acid. Significantly increased, especially between the group treated with α-linolenic acid alone and the group with α-linolenic acid and α-linoleic acid at the same time ( p <0.05), showing that via α-tricoleic acid The treated cells can increase the activity of NAMPT.

3. Increase NAD ⁺ /NADH ratio effect

All aging diseases and aging processes are associated with reduced citrate cycling activity, which is a biochemical reaction of mitochondria and is important for energy formation. Deacetylase-1 is one of the enzymes responsible for the removal of the acetamidine label on tissue proteins, and an increase in the ratio of NAD ⁺ or NAD ⁺ /NADH activates this enzyme.

This test further analyzes the ratio of NAD ⁺ /NADH in the cells after fatty acid treatment to investigate whether bitter melon seed oil can activate deacetylase-1. H4IIEC3 cells were treated for 24 hours in the same fatty acid treatment procedure as in Test Example 2, and the NAD ⁺ /NADH ratio in H4IIEC3 cells was detected by Biovision NAD ⁺ /NADH quantification kit (Milpitas).

Please refer to Figure 4 for the results of the ratio of NAD ⁺ /NADH in H4IIEC3 cells after treatment of fatty acids, where LN represents α-linolenic acid, α-ESA represents α-linoleic acid, and there are groups between different letters. Significant difference ( p < 0.05). The results showed that the ratio of NAD ⁺ /NADH in H4IIEC3 cells was comparable to that of the control group in the group treated with α-linolenic acid alone, but the group of α-linolenic acid and α-teroleic acid was added at the same time, and the NAD in H4IIEC3 cells. ^{The +} /NADH ratio is greatly increased, indicating that the NAD ⁺ /NADH ratio can be increased by cells treated with α-tricoleic acid.

It is known that deacetylase-1 is NAD ⁺ -dependent deacetylase, and the results from Test Example 2 to Test Example 2 show that α-tricoleic acid treatment can increase NAMPT activity and increase NAD ⁺ /NADH ratio, and then activate deacetylase-1 to deacetylate FOXO1, while α-tricoleic acid is the most important fatty acid in bitter gourd seed oil, so it can be inferred that bitter gourd seed oil can activate deacetylase-1 To achieve anti-aging effects.

4. Extend the effect of nematode life

It can be seen from the above test examples that the conjugated linoleic acid unique to the bitter gourd seed oil of the present invention has the effect of activating acetamylase-1, and the test example further investigates the bitter gourd seed oil of the present invention by the life test of the nematode. Whether the unique conjugated linoleic acid can prolong the lifespan of nematodes, once again verify its anti-aging effect.

Due to the ease of observation of the life cycle and morphology of nematodes, model organisms used to measure anti-aging activity have been used in the past. The nematode used in this experiment is Caenorhabditis elegans . Its adult length is about 1mm, mostly hermaphrodite, and its life cycle can be divided into eggs, four larval stages (L1-L4) and adults. The nematode medium used by Caenorhabditis elegans is NGM (Nematode Growth Medium). The test was divided into three groups, which were coated with NAG (100 μL of E. coli OP50 and pure ethanol) as control group, E.coli OP50+100 μM α-linolenic acid, and E.coli OP50. +75 μM α-linolenic acid + 25 μM α-tricoleic acid. Synchronized C. elegans (L4 larvae) were separately cultured in the above-mentioned NGM group, 20 in each group, cultured in a 25 ° C incubator, and the test medium was changed every 1 to 2 days, and dissected microscope Observe the growth situation, count the number of nematodes that survived every day, and determine the survival of the nematodes by touching the fine teeth of the toothbrush with a needle. If there is no reaction, it is considered dead.

Please refer to Fig. 5 for the results of the percentage of survival of nematodes after fatty acid treatment, wherein LN represents α-linolenic acid and α-ESA represents α-tallowic acid. The results showed that the group given 25 μM α-tricoleic acid had a higher survival rate than the other groups, and the fashion had a 45.5% survival rate on the 10th day of the test. The control group added to the vehicle had a survival rate of 10.6% on the 10th day of the test, indicating that α-tungonic acid prolonged the lifespan of the nematode, and once again proved to have anti-aging effect.

In summary, the conjugated linoleic acid unique to the bitter gourd seed oil of the present invention increases the NAD ⁺ /NADH ratio by increasing the NAMPT activity, thereby activating the deacetylase-1 to achieve anti-aging effect. And in the life test of nematodes, it was again proved that the conjugated linoleic acid unique to the bitter gourd seed oil of the present invention can prolong the life of the nematode. Therefore, the bitter gourd seed oil rich in conjugated linoleic acid of the present invention can be used for preparing an anti-aging pharmaceutical composition. The composition of the bitter melon seed oil comprising an effective dose of the conjugated linoleic acid can be used as an anti-aging pharmaceutical composition, a feed composition, a beverage composition, a nutritional supplement composition, an edible composition or a health food composition. Has the potential to be used in the biomedical health market.

The present invention has been disclosed in the above embodiments, but it is not intended to limit the invention, and the present invention can be modified and modified without departing from the spirit and scope of the invention. The scope is subject to the definition of the scope of the patent application.

Claims (5)

A use of alpha-eleostearic acid for the preparation of an anti-aging pharmaceutical composition. The use of α-tricoleic acid as described in claim 1, wherein the α-tungonic acid is extracted from bitter gourd seeds. The use of α-tricoleic acid as described in claim 1, wherein the anti-aging pharmaceutical composition is a sirtuin-1 activator. The use of α-tricoleic acid as described in claim 1, wherein the anti-aging pharmaceutical composition is a pharmaceutical composition for increasing the activity of nicotinamide phosphoribosyltransferase (NAMPT). The use of α-tricoleic acid as described in claim 1, wherein the anti-aging pharmaceutical composition is an nicotinamide adenine dinucleotide (NAD ⁺ )/reduced state A pharmaceutical composition of the ratio of reduced form of nicotinamide adenine dinucleotide (NADH).