CN109758444A - Application of the 2- methyl substituted fatty acid at anti-oxidant aspect - Google Patents
Application of the 2- methyl substituted fatty acid at anti-oxidant aspect Download PDFInfo
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- CN109758444A CN109758444A CN201910236884.3A CN201910236884A CN109758444A CN 109758444 A CN109758444 A CN 109758444A CN 201910236884 A CN201910236884 A CN 201910236884A CN 109758444 A CN109758444 A CN 109758444A
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Abstract
The present invention relates to anti-oxidation tech field, more particularly, to a kind of 2- methyl substituted fatty acid anti-oxidant aspect application.The 2- methyl substituted fatty acid prepares the application in oxidation resistant food and/or health care product, and is preparing the application in anti-oxidation medicine.The present invention passes through using Caenorhabditis elegans as model animal, juglone is as oxidant, it was found that 2- methyl substituted fatty acid has significant antioxidation biology activity, time-to-live of Caenorhabditis elegans under the conditions of Acute oxidative injury can significantly be extended by showing, 2- methyl substituted fatty acid can realize antioxidation by adjusting internal ROS, it can be used in preparing oxidation resistant food and/or health care product, and be used to prepare in the drug etc. for preventing or treating the disease as caused by oxidative stress.
Description
Technical field
The present invention relates to anti-oxidation tech fields, more particularly, to a kind of 2- methyl substituted fatty acid at anti-oxidant aspect
Using.
Background technique
Aging is a kind of natural law, and the physical function for being mainly manifested in people with advancing age different degrees of moves back
Change.This process of aging is regulated and controled by many factors, and in many factors for causing aging, oxidativestress damage, that is, free radical declines
Old theory is widely approved.Excessive generate of free radical is the principal element for generating oxidative stress, and wherein active oxygen (ROS) is right
The damage of body is maximum.In the causes of senescence reported, " free radical theory " is thought during aging, can produce in body
Raw excessive active oxygen radical (ROS) leads to the damage of DNA and tissue, and then accelerates aging rate.The aging of organism
Journey is the free radical accumulation that the histocyte of body constantly generates as a result, free radical can cause DNA damage so as to cause mutation,
Induced tumor is formed.Free radical is the intermediate product of eubolism, and respond is very strong, can make many kinds of substance in cell
It aoxidizes, damages biomembrane, additionally it is possible to make the macromolecules cross-linkings such as protein, nucleic acid, influence its normal function.
Thus, a kind of natural materials with antioxidation are provided to be of great significance.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide 2- methyl substituted fatty acids in the application of anti-oxidant aspect, and 2- methyl replaces fatty
Acid has significant antioxidation biology activity, can significantly extend survival of the Caenorhabditis elegans under polar oxygenated damaging condition
Time, 2- methyl substituted fatty acid can realize antioxidation by adjusting internal ROS.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention provides a kind of application of 2- methyl substituted fatty acid in oxidation resistant food and/or health care product.
As in a specific embodiment, the present invention provides a kind of 2- methyl substituted fatty acids to prepare oxidation resistant food
And/or the application in health care product.
2- methyl substituted fatty acid belongs to natural products, is rich in 2- methyl substituted fatty acid in common fruit.This hair
It is bright by using Caenorhabditis elegans as model animal, it is significant to find that 2- methyl substituted fatty acid has as oxidant for juglone
Antioxidation biology activity, show when can significantly extend survival of Caenorhabditis elegans under the conditions of Acute oxidative injury
Between, 2- methyl substituted fatty acid can realize antioxidation by adjusting internal ROS.
Preferably, 2- methyl substituted fatty acid is selected fromWherein, n be selected from 1,2 or 3, i.e., 2-Methyl Butyric Acid,
2 methyl valeric acid, 2 methyl caproic acid.As n=1,2- methyl substituted fatty acid isAs n=2,2- methyl
Substituted fatty acid isAs n=3,2- methyl substituted fatty acid is2- methyl takes
Fat subsitutes acid includes It is any one or more of.
As in different embodiments, 2- methyl substituted fatty acid can be independently operated, using above-mentioned several fatty acid
Can be used it is several be used in mixed way, such asMixing,Mixing,Mixing and three kinds of changes
Object is closed to be used in mixed way.
The present invention is turned into antioxygen in fatty acid entities by lot of experiment validation, methyl the position of substitution and substitution alkyl length
With most important.Carbon atom number is that the straight chain fatty acid (formic acid, acetic acid, propionic acid, butyric acid, valeric acid and caproic acid) of 1-6 cannot increase
The anti-oxidation stress ability of strong Caenorhabditis elegans, 2- methyl replace propionic acid, 2- ethyl replace butyric acid, 2- ethyl replace caproic acid with
And 3- methyl replaces butyric acid not have interior antioxidation action.
Above-mentioned 2- methyl substituted fatty acid has significant antioxidation biology activity, can significantly extend Caenorhabditis elegans
Time-to-live under polar oxygenated damaging condition realizes antioxidation by adjusting internal ROS.
Above-mentioned 2- methyl substituted fatty acid can prepare the production for preventing or repairing the cellular damage as caused by oxidative stress
Product.
It preferably, further include edible auxiliary materials in oxidation resistant food and/or health care product.2- methyl can such as be replaced to fat
It is sour to be mixed with common edible auxiliary materials, antioxidant health-care product is prepared.
Preferably, the food and/or health care product are liquid condition, and the concentration of the 2- methyl substituted fatty acid is 60-
100μM.It is furthermore preferred that the concentration of the 2- methyl substituted fatty acid is 80 μM.
The present invention also provides a kind of 2- methyl substituted fatty acids to prepare the application in anti-oxidation medicine.
Preferably, above-mentioned anti-oxidation medicine is for preventing or treating the disease as caused by oxidative stress.
Oxidative stress refers to that under the effect of destructive stimulus factor, ROS is generated excessive or dysbolism is occurred and is more than endogenous
Property Antioxidative Defense System when eliminating ability to it, ROS increases in vivo and participates in the formation of oxidative biological macromolecular, induces base
Because of mutation, protein denaturation and lipid peroxidation, and then damage lysosome, mitochondria etc., eventually lead to the oxidative damage of cell.
The disease as caused by oxidative stress includes diabetes, hypertension, tumour, lung inflammation etc..
Level of lipid in Diabetes Mellitus increases, and is positively correlated with intercellular adhesive moleculer-1, surface
Oxidative stress plays an important role in the generation, development of diabetic vascular complications.
The height that essential hypertension, renovascular hypertension and accelerated hypertension, salt-sensitive hypertension, cyclosporine induce
There is oxidative stress enhancing, oxidative stress can cause and maintain hypertension for blood pressure and pre-eclampsia etc..
ROS can the related enzyme systems repaired of damage dna and DNA, activate proto-oncogene, cause intracellular multi-signal molecule and
The exception of its regulatory factor, eventually leads to cell carcinogenesis.Experiment finds that active o content and reaction are anti-oxidant in breast cancer tissue
Horizontal index is significantly raised, illustrates that breast cancer tissue is in oxidative stress status etc..
Products of oxidative stress can cause neutrophil leucocyte to be detained in lung microcirculation, raise and activate, and activated transcription because
Sub such as nuclear factor kappaB, transforming growth factor-β1, and then the release of inflammatory mediator is adjusted, therefore, oxidative stress has bright with lung inflammation
True correlation.
2- methyl substituted fatty acid of the invention can adjust internal ROS, significantly increase anti-oxidation stress ability, thus energy
Preparation is enough in the drug for preventing or treating the disease as caused by oxidative stress.
Preferably, the dosage form of drug includes capsule, tablet, powder, granule, injection, oral solution, appointing in pill
It is a kind of.
In practical applications, added according to the actual demand of pharmaceutical dosage form using 2- methyl substituted fatty acid as active constituent
Add corresponding auxiliary material, the pharmaceutical dosage form of demand is prepared.
Preferably, when preparing drug, 2- methyl substituted fatty acid is mixed with phosphate buffer solution, compound concentration is
The mother liquor of 60-100mM, mother liquor dilute to obtain the solution for the 2- methyl substituted fatty acid that concentration is 60-100 μM with water.Wherein, phosphorus
The pH of hydrochlorate buffer solution is preferably 6.0, and concentration is preferably 1-2M, more preferably 1.5M.If drug is liquid preparation, drug
PH is 6.0.
Preferably, 2- methyl substituted fatty acid includes any one of 2-Methyl Butyric Acid, 2 methyl valeric acid, 2 methyl caproic acid
Or it is a variety of.
Compared with prior art, the invention has the benefit that
(1) 2- methyl substituted fatty acid has significant antioxidation biology activity, realizes that antioxygen is turned by adjusting internal ROS
With；
(2) present invention is by lot of experiment validation, and methyl the position of substitution and substitution alkyl length are to antioxygen in fatty acid entities
Change effect is most important, and the present invention has significant antioxidation biology activity using specific 2- methyl substituted fatty acid, can
It is the significant time-to-live for extending Caenorhabditis elegans under polar oxygenated damaging condition, anti-oxidant by adjusting internal ROS realization
Effect；
(3) 2- methyl substituted fatty acid of the invention can adjust internal ROS, significantly increase anti-oxidation stress ability, energy
It is enough in and prepares oxidation resistant food and/or health care product, and be used to prepare and preventing or treating the disease as caused by oxidative stress
The drug of disease.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is provided in an embodiment of the present invention using Caenorhabditis elegans as the schematic diagram of animal pattern；
Fig. 2 is that each strain N2, daf-2 and clk-1 mutant nematode is exposed to 200 μM of juglones in the embodiment of the present invention 4
In survivorship curve；
Fig. 3 be in the embodiment of the present invention formic acid as administration group (N2+1) and blank control group (N2+Vehicle) by N2 line
Worm is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Fig. 4 be in the embodiment of the present invention acetic acid as administration group (N2+2) and blank control group (N2+Vehicle) by N2 line
Worm is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Fig. 5 be in the embodiment of the present invention propionic acid as administration group (N2+3) and blank control group (N2+Vehicle) by N2 line
Worm is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Fig. 6 be in the embodiment of the present invention butyric acid as administration group (N2+4) and blank control group (N2+Vehicle) by N2 line
Worm is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Fig. 7 be in the embodiment of the present invention valeric acid as administration group (N2+5) and blank control group (N2+Vehicle) by N2 line
Worm is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Fig. 8 be in the embodiment of the present invention caproic acid as administration group (N2+6) and blank control group (N2+Vehicle) by N2 line
Worm is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Fig. 9 be in the embodiment of the present invention isobutyric acid as administration group (N2+7) and blank control group (N2+Vehicle) by N2
Nematode is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 10 is 2-Methyl Butyric Acid in the embodiment of the present invention as administration group (N2+8) and blank control group (N2+
Vehicle N2 nematode) is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 11 is 2 methyl valeric acid in the embodiment of the present invention as administration group (N2+9) and blank control group (N2+
Vehicle N2 nematode) is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 12 is 2 methyl caproic acid in the embodiment of the present invention as administration group (N2+10) and blank control group (N2+
Vehicle N2 nematode) is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 13 is 2 Ethylbutanoic acid in the embodiment of the present invention as administration group (N2+11) and blank control group (N2+
Vehicle N2 nematode) is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 14 is 2 ethyl hexanoic acid in the embodiment of the present invention as administration group (N2+12) and blank control group (N2+
Vehicle N2 nematode) is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 15 is 3 Methylbutanoic acid in the embodiment of the present invention as administration group (N2+13) and blank control group (N2+
Vehicle N2 nematode) is exposed to oxidative stress survivorship curve in 200 μM of juglones；
Figure 16 is DPPH in vitro anti-oxidation experimental result in the embodiment of the present invention, and wherein A is 2-Methyl Butyric Acid group, and B is
2 methyl valeric acid group, C are 2 methyl caproic acid group；
Figure 17 is ABTS in vitro anti-oxidation experimental result in the embodiment of the present invention, and wherein A is 2-Methyl Butyric Acid group, and B is
2 methyl valeric acid group, C are 2 methyl caproic acid group；
Figure 18 is antioxidation in vitro cooperative experiment test result in the embodiment of the present invention, and wherein A is 2-Methyl Butyric Acid group, B
For 2 methyl valeric acid group, C is 2 methyl caproic acid group；
Figure 19 is the internal ROS real-time monitoring of 2-Methyl Butyric Acid and valeric acid in the embodiment of the present invention as a result, wherein A is to be based on
H2ROS real-time monitoring fluorescence intensity figure in-DCFDA line probe polypide, B are based on H2ROS fluorogram in-DCFDA line probe polypide
Top figure is 2-Methyl Butyric Acid group respectively in picture, A and B, and lower section figure is valeric acid group respectively in A and B；
Figure 20 is that N2 nematode in the embodiment of the present invention (antibiotic inactivates OP50) is exposed to oxidative stress in 200 μM of juglones
Survivorship curve；Wherein, A is 2-Methyl Butyric Acid group, and B is 2 methyl valeric acid group, and C is 2 methyl caproic acid group；
Figure 21 is that N2 nematode (high-temperature inactivation OP50) is exposed to oxidative stress life in 200 μM of juglones in the embodiment of the present invention
Deposit curve；Wherein, A is 2-Methyl Butyric Acid group, and B is 2 methyl valeric acid group, and C is 2 methyl caproic acid group.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with the drawings and specific embodiments, but
Be it will be understood to those of skill in the art that it is following described embodiments are some of the embodiments of the present invention, rather than it is whole
Embodiment is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Based on the embodiments of the present invention, ability
Domain those of ordinary skill every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same
Or production firm person is not specified in instrument, is the conventional products that can be obtained by commercially available purchase.
The portion of reagent information used in the embodiment of the present invention can choose as follows:
Butyric acid (Butyric acid), isobutyric acid (Isobutyric acid), 2-Methyl Butyric Acid (2-Methylbutyric
Acid), 2 Ethylbutanoic acid (2-Ethylbutyric acid), valeric acid (Pentanoic acid), 2 methyl valeric acid (2-
Methylpentylic acid), 3 methylvaleric acid (3-Methylbutanoic acid), caproic acid (Hexanoic acid), 2-
Methylhexanoic acid (2-Methylhexanoic acid), 2 ethyl hexanoic acid (2-Ethylhexanoic acid), 1,1- diphenyl-
2- trinitrophenyl-hydrazine (DPPH) and 2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS), juglone purchase
It buys in Shanghai Aladdin reagent Co., Ltd；
Caenorhabditis elegans is purchased from Caenorhabditis Genetics Center (CGC)；
It can choose in the embodiment of the present invention for part device information used in vitro and in vivo experiments as follows:
Microplate reader: model SYNERGY-H1；
Stereo microscope: model Motic SMZ-161；
Inverted fluorescence microscope: model Nikon-Ts2；
Nematode culture case: model LICHEN -250E.
Embodiment 1
A certain amount of 2-Methyl Butyric Acid is taken, the phosphate buffer (pH=6.0) of 1.5M is added, is uniformly mixed, is formed dense
Degree is the mother liquor of 80mM；It then is 80 μM with the concentration that deionized water dilutes mother liquor to 2-Methyl Butyric Acid, as working solution.
Embodiment 2
A certain amount of 2 methyl valeric acid is taken, the phosphate buffer (pH=6.0) of 1.5M is added, is uniformly mixed, is formed dense
Degree is the mother liquor of 80mM；It then is 80 μM with the concentration that deionized water dilutes mother liquor to 2 methyl valeric acid, as working solution.
Embodiment 3
A certain amount of 2 methyl caproic acid is taken, the phosphate buffer (pH=6.0) of 1.5M is added, is uniformly mixed, is formed dense
Degree is the mother liquor of 80mM；It then is 80 μM with the concentration that deionized water dilutes mother liquor to 2 methyl caproic acid, as working solution.
Embodiment 4
The internal antioxygenic property of 2- methyl substituted fatty acid
1, Caenorhabditis elegans strain and synchronization:
Nematode strain includes N2 (Bristol), clk-1 (mq130), daf-2 (e1368), hidden bar beautiful to different lines
Nematode carries out synchronization processing；20 or so adults are taken, 4h of laying eggs is transferred in nematode solid medium (NGM) culture medium, obtains
The worm's ovum of synchronization.
2, the foundation of the vivo oxidation Stress model based on Caenorhabditis elegans
NGM culture medium is prepared: weigh 0.5g sodium chloride, the agar of 1g peptone and 4g are added in the conical flask of 500mL,
It is then respectively adding the MgSO of the 1M of 200 μ L4With the CaCl of 1M2.Phosphate buffer (the PH of the 1.5M of 3.25mL is then added
=7.0), finally with deionized water constant volume to 200mL, 121 DEG C of high-temperature sterilization 30min.Then culture medium 60 DEG C are cooled to add
Enter prepared juglone (6.5mg juglone being dissolved in 3mL dehydrated alcohol, ultrasonic dissolution obtains prepared juglone)
1mL is stirred continuously, and is subsequently poured into culture dish, whole sterile working and to be protected from light processing, ultimately forms 200 μM of final concentration
Juglone NGM culture medium.The adult of day1 (is administered the 80 of embodiment 1-3 in administration group by synchronization Caenorhabditis elegans respectively
μM 2- methyl substituted fatty acid as administration group) and the culture medium of blank control group in 20 DEG C culture 3 days after, be transferred to containing recklessly
In the culture medium of peach quinone, each plate is transferred to 50 Caenorhabditis elegans, and whole process is protected from light.It is observed continuously under the microscope per small
When death condition, until nematode is all dead；Survivorship curve is drawn with Graphpad Prism 6.0.
3, internal ROS detection
(1) ROS end point determination: compound concentration is the H of 50mM2- DCFDA solution (is kept in dark place).Administration group (is divided respectively
Not Gei Yao embodiment 1-3 80 μM of 2- methyl substituted fatty acid as administration group) and control group nematode with buffer, (nematode is slow
Fliud flushing M9) it is flushed in the centrifuge tube of 1mL, it is centrifuged 1 minute in 3000r/min, removes supernatant, in triplicate.The 4mM of 50 μ L is added dropwise
Sodium azide solution to glass slide on agar membrane on.Then, 30 nematodes are chosen to be put into wherein, are anaesthetized.After 1min, lead to
Fluorescence microscope is crossed to take pictures.
(2) ROS real-time monitoring: using administration group (respectively be administered embodiment 1-3 80 μM of 2- methyl substituted fatty acid as
Administration group) and the Caenorhabditis elegans of control group clean according to the method described above, in triplicate.96 orifice plate of black is taken, each pre- use
The nematode buffer M9 of 50mL is added in hole, then chooses respectively into 50 nematodes.Then, the H of 50 μ L is added in each hole2-
DCFDA (4mM) probe after mixing gently, carries out fluorescence detection using microplate reader (exciting light 485nm emits 530nm).Often
10min detection is primary, persistently detects 140min.
Embodiment 5
In vitro anti-oxidation detection
1, DPPH (1,1- diphenyl -2- trinitrophenyl-hydrazine) in vitro anti-oxidation is tested
The preparation of DPPH test fluid: taking DPPH1mg to be dissolved in 20mL dehydrated alcohol, ultrasonic 5min.
A0Value detection: absorption value is surveyed after taking the above-mentioned DPPH solution of 2mL and 1mL dehydrated alcohol to be sufficiently mixed at 519nm, is
A0。
The detection of A value: take the above-mentioned DPPH test fluid of 2mL and the working solution in 1/ embodiment of 1mL embodiment, 2/ embodiment 3 abundant
Absorption value at 519nm is surveyed after mixing, is A.Experiment is independent in triplicate.
ROS clearance rate=(A0-A)/A0* 100%
2, ABTS (2,2- joins nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts) in vitro anti-oxidation experiment
ABTS test fluid is prepared: taking the ABTS (7.4M) and K of 2mL respectively2S2O8(2.6M) is mixed overnight under the conditions of being protected from light.
ABTS mixed liquor after overnight is diluted with 95% ethyl alcohol according to 1 ﹕ 40, ABTS test fluid is obtained.
A0Value detection: it after taking 0.8mL ABTS test fluid addition 0.2mL dehydrated alcohol to be sufficiently mixed, is detected at 734nm
Absorption value is A0。
The detection of A value: take 0.8mL ABTS test fluid that the working solution in 1/ embodiment of 0.2mL embodiment, 2/ embodiment 3 is added
After being sufficiently mixed, absorption value is detected at 734nm.Experiment is independent in triplicate.
ROS clearance rate=(A0-A)/A0* 100%
Experimental example 1
Data processing is carried out using Graphpad Prism 6.0, P value < 0.05 is with statistical difference, P value <
0.01 is with significant statistical difference.Experiment is independent more than three times to be repeated.
1, the building of oxidative stress experimental model
(Redox Biology, 11 (2017) 708-714 and Ageing Research in existing research
Reviews, 12 (2013) 918-930), juglone, which can make to be exposed in the model animal short time therein, generates a large amount of ROS,
Acute oxidative injury is caused, its death is caused.In this research, as shown in Figure 1, being used using Caenorhabditis elegans as animal pattern
Final concentration of 200 μM of juglone is that oxidant constructs anti-oxidant appraisement system.As shown in Fig. 2, compared with N2, daf-2
(e1368) nematode of mutant strain survival ability under stressed condition is stronger, and the nematode viability of clk-1, mq130 mutant strain
Ability is weak, P < 0.0001.The validity of the appraisement system is demonstrated by both the above mutant strain nematode.
2, the internal anti-oxidant research based on Caenorhabditis elegans
In order to which comparative illustration difference fatty acid and its substituted fatty acid under the conditions of Caenorhabditis elegans oxidative stress to surviving
13 kinds of fatty acid in table 1 are tested in the influence of ability referring to the detection method of the internal antioxygenic property of embodiment 4,
Oxidative stress survivorship curve is drawn respectively, as shown in Fig. 3-Figure 15.
The fatty acid and substituted fatty acid and internal antioxidant activity that 1 anti-oxidation stress of table uses
Note: wherein internal antioxidant activity, " ﹢ ", which is represented, has internal antioxidant activity, and "-", which represents, does not have internal antioxygen
Change activity
Fig. 3-8 is formic acid, acetic acid, propionic acid, butyric acid, valeric acid and caproic acid respectively as administration group, and N2 nematode is exposed to 200
Oxidative stress survivorship curve in μM juglone, it can be seen that formic acid, acetic acid, propionic acid, butyric acid, valeric acid and caproic acid cannot enhance
The anti-oxidation stress ability of Caenorhabditis elegans.Wherein, formic acid, acetic acid, propionic acid, butyric acid, valeric acid and caproic acid are respectively as administration
For group compared with blank control group, P value is respectively P1=0.1325, P2=0.1427, P3=0.2018, P4=0.0002, P5=
0.0002, P6=0.0452.
Fig. 9 is isobutyric acid as administration group, and N2 nematode is exposed to oxidative stress survivorship curve in 200 μM of juglones, from
It is found that isobutyric acid cannot enhance the anti-oxidation stress ability of Caenorhabditis elegans in figure.Wherein, isobutyric acid is as administration group and sky
White control group is compared, and P value is P7=0.3422.
Figure 10-12 is 2-Methyl Butyric Acid in embodiment 1-3,2 methyl valeric acid, 2 methyl caproic acid respectively as administration group,
N2 nematode is exposed to oxidative stress survivorship curve in 200 μM of juglones, it can be seen that 2-Methyl Butyric Acid, 2 methyl valeric acid,
2 methyl caproic acid can significantly improve the anti-oxidation stress ability of Caenorhabditis elegans.Wherein, 2-Methyl Butyric Acid, 2- methylpent
Respectively as administration group compared with blank control group, P value is respectively P for acid, 2 methyl caproic acid8< 0.0001, P9< 0.0001, P10
< 0.0001.
Figure 13-15 is 2 Ethylbutanoic acid, 2 ethyl hexanoic acid, 3 Methylbutanoic acid respectively as administration group, by the exposure of N2 nematode
The oxidative stress survivorship curve in 200 μM of juglones, it can be seen that 2 Ethylbutanoic acid, 2 ethyl hexanoic acid, 3 Methylbutanoic acid are equal
Without interior antioxidation action.Wherein, 2 Ethylbutanoic acid, 2 ethyl hexanoic acid, 3 Methylbutanoic acid are respectively as administration group and sky
White control group is compared, and P value is respectively P11=0.1118, P12=0.9822, P13=0.0002.
From the above it is found that methyl the position of substitution and replace alkyl length to the interior antioxidation action of fatty acid extremely
Close it is important, while illustrate 2-Methyl Butyric Acid, 2 methyl valeric acid, 2 methyl caproic acid antioxidation specificity.
3, antioxidation in vitro detects
In order to further probe into the interior antioxidation action mechanism of 2- methylfatty acid, by external anti-in embodiment 5
Oxidisability detection method, DPPH and two kinds of ABTS experiments have detected its external ability and ascorbic collaboration for directly removing ROS
Antioxidation.
Figure 16 is DPPH in vitro anti-oxidation experimental result, and Figure 17 is ABTS antioxidation in vitro experimental result, can from figure
Know, the 2-Methyl Butyric Acid, 2 methyl valeric acid and 2 methyl caproic acid in embodiment 1-3 all do not have the external energy for directly removing ROS
Power.Wherein, abscissa respectively represents the 2-Methyl Butyric Acid (8) of various concentration (8,80,800 and 8000 μM), 2 methyl valeric acid (9)
With 2 methyl caproic acid (10), 80 μM of cysteines (cys) are used as positive control.
Figure 18 is antioxidation in vitro cooperative experiment test result, it can be seen that 2-Methyl Butyric Acid in embodiment 1-3,
2 methyl valeric acid and 2 methyl caproic acid cannot enhance the oxidation resistance of endogenous classics anti-oxidant Vitamins C.Wherein, horizontal
Coordinate respectively represents the 2-Methyl Butyric Acid (8), 2 methyl valeric acid (9) and 2 methyl caproic acid of various concentration (8,80 and 800 μM)
(10) it is used in mixed way with 0.1mg/mL vitamin C, 0.1mg/mL vitamin C (Vc) is as control.
Through the above results it is found that methyl replaces the antioxidant activity of short chain fatty acids not stem from its own removing having
ROS free radical ability, living source act in vivo in it.
By taking the valeric acid in the 2-Methyl Butyric Acid and table 1 in embodiment 1 as an example, through the ROS real-time monitoring in embodiment 4, prison
The result is shown in Figure 19 is surveyed, wherein A is based on H2ROS real-time monitoring fluorescence intensity figure in-DCFDA line probe polypide, B are based on H2-
ROS fluorescent image in DCFDA line probe polypide.It can be seen that 2-Methyl Butyric Acid can significantly reduce ROS water in nematode body
It is flat, antioxidation can be realized by adjusting internal ROS.
Experimental example 2
Escherichia coli are inactivated by two methods of antibiotic (ampicillin) and high temperature, further probe into 2- first
The interior antioxidation action mechanism of base fatty acid.
Wherein, antibiotic colibacillus deactivating experimental method includes: to connect OP50 strain in 50mL fluid nutrient medium
(Luria-Bertani, LB) shakes bacterium under the conditions of 37 DEG C overnight.Then 1mL ampicillin (5mg/mL) will be added in bacterium solution,
37 DEG C, under conditions of 180r/min overnight.It takes above-mentioned inactivated bacterial liquid is a little in super-clean bench, is added drop-wise to blank NGM culture surface,
Whether all 37 DEG C overnight, check Escherichia coli OP50 thoroughly inactivation.It has sterilized centrifugation finally, the bacterium solution of inactivation is packed into 50mL
Guan Zhong, 3 000r/min are centrifuged 10min.After abandoning supernatant, the sterile LB of 5mL is added, carries out paving bacterium after vortex 1min.
High-temperature inactivation Escherichia coli experimental method includes: to connect OP50 strain in 50mL LB culture medium, mistake under the conditions of 37 DEG C
Night shakes bacterium.Then bacterium solution is put into autoclave, 120 DEG C of sterilizing 20min.Above-mentioned inactivated bacterial liquid is a little, drop is taken in super-clean bench
It is added to blank NGM culture surface, whether all 37 DEG C overnight, check Escherichia coli OP50 thoroughly inactivation.Finally, by the bacterium of inactivation
Liquid is fitted into 50mL in sterile centrifugation tube, and 3000r/min is centrifuged 10min.After abandoning supernatant, after the addition sterile LB of 5mL, vortex 1min
Carry out paving bacterium.
The experimental results showed that as shown in figures 20-21, respectively antibiotic inactivation and high-temperature inactivation group, in embodiment 1-3
2- methylfatty acid is exposed to oxidative stress in 200 μM of juglones respectively as administration group, N2 nematode (antibiotic inactivates OP50)
Survivorship curve.It can be seen from the figure that either antibiotic inactivation group or high-temperature inactivation group neither influences in embodiment 1-3
The antioxidation of 2- methylfatty acid all significantly improves the ability of the anti-oxidation stress of Caenorhabditis elegans.Wherein, antibiosis
Plain inactivation group, respectively as administration group compared with blank control group, P value is divided for 2-Methyl Butyric Acid, 2 methyl valeric acid, 2 methyl caproic acid
It Wei not < 0.0001, < 0.0001, < 0.0001；High-temperature inactivation group, 2-Methyl Butyric Acid, 2 methyl valeric acid, 2 methyl caproic acid point
Not Zuo Wei administration group compared with blank control group, P value is respectively < 0.0001, < 0.0001, < 0.0001.
The present invention is turned into antioxygen in fatty acid entities by lot of experiment validation, methyl the position of substitution and substitution alkyl length
With most important, the present invention has significant antioxidation biology activity using specific 2- methyl substituted fatty acid, can be significant
Extend time-to-live of the Caenorhabditis elegans under polar oxygenated damaging condition, realize antioxidation by adjusting internal ROS,
Can be used for preparing the product for preventing or repairing the cellular damage as caused by oxidative stress, or preparation for prevent or treat by
Drug, health care product of disease caused by oxidative stress etc..
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
- Application of the 1.2- methyl substituted fatty acid in oxidation resistant food and/or health care product.
- 2. application according to claim 1, which is characterized in that the 2- methyl substituted fatty acid include 2-Methyl Butyric Acid, 2 methyl valeric acid, 2 methyl caproic acid are any one or more of.
- 3. application according to claim 1 or 2, which is characterized in that also wrapped in the oxidation resistant food and/or health care product Include edible auxiliary materials.
- 4. application according to claim 1 or 2, which is characterized in that the food and/or health care product are liquid condition, institute The concentration for stating 2- methyl substituted fatty acid is 60-100 μM.
- 5. application according to claim 4, which is characterized in that the concentration of the 2- methyl substituted fatty acid is 80 μM.
- 6.2- methyl substituted fatty acid is preparing the application in anti-oxidation medicine.
- 7. application according to claim 6, which is characterized in that the anti-oxidation medicine is for preventing or treating to be answered by oxidation Disease caused by swashing.
- 8. application according to claim 7, which is characterized in that the disease as caused by oxidative stress include diabetes, Any one of hypertension, tumour, lung inflammation.
- 9. application according to claim 6, which is characterized in that the dosage form of the anti-oxidation medicine include capsule, tablet, Any one of powder, granule, injection, oral solution, pill.
- 10. according to the described in any item applications of claim 6-9, which is characterized in that the 2- methyl substituted fatty acid includes 2- Methylbutanoic acid, 2 methyl valeric acid, 2 methyl caproic acid are any one or more of.
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| CN105232506A (en) * | 2015-11-04 | 2016-01-13 | 温州医科大学 | Application of butyric acid and salts thereof in preparation of medicine for treating or preventing gastric ulcer |
| CN108125940A (en) * | 2017-12-28 | 2018-06-08 | 崔明 | Valeric acid is being prepared for preventing and/or treat the purposes in the drug of neoplasm radiotheraphy secondary reaction |
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| EP2030616A1 (en) * | 2007-08-31 | 2009-03-04 | Asan Laboratories Company (Cayman) Limited | Short-chain fatty acid derivatives for treating pruritus |
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| CN104940181A (en) * | 2015-06-29 | 2015-09-30 | 齐锦生 | Application of beta-hydroxybutyric acid or pharmaceutically acceptable salt thereof |
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