CN113332456A - Detection method for anti-wrinkle and wrinkle-reducing activity of skin care raw material and application thereof - Google Patents
Detection method for anti-wrinkle and wrinkle-reducing activity of skin care raw material and application thereof Download PDFInfo
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Abstract
The invention provides a detection method for anti-wrinkle and wrinkle-reducing activity of a skin care raw material and application thereof, wherein the detection method comprises the following steps: (1) culturing Escherichia coli in a liquid culture medium, mixing the bacterial liquid with the skin care raw material to be tested, and coating the mixture on an NGM flat plate for storage; (2) carrying out synchronization on nematodes, and then cracking to obtain larvae; (3) inoculating the larvae obtained in the step (2) to the NGM flat plate obtained in the step (1) for culture, then picking out, recording the swing times of the head of nematode parameters, and judging the anti-wrinkle and wrinkle-lightening activity of the skin care raw material to be detected; the step (1) and the step (2) do not distinguish the sequence. The method for detecting the anti-wrinkle and wrinkle-reducing activity of the skin care raw material provided by the invention has the advantages of short experimental period, high detection speed, capability of screening the raw material at high flux and low cost.
Description
Technical Field
The invention belongs to the field of cosmetic raw material detection, particularly relates to a method for detecting anti-wrinkle and wrinkle-reducing activity of a skin care raw material and application thereof, and particularly relates to a method for detecting anti-wrinkle and wrinkle-reducing activity of a skin care raw material with low cost and high speed and application thereof.
Background
With the increasing demand of consumers for anti-wrinkle cosmetics, the research and development of anti-wrinkle products in enterprises are increasing, and the anti-wrinkle cosmetics occupy more and more important positions in the whole cosmetic industry. The detection of the wrinkle resistance of the cosmetic raw material can improve the development efficiency of the anti-wrinkle cosmetic. At present, the anti-wrinkle and wrinkle-reduction efficacy of cosmetics is mainly tested by a 3D skin model and a human body efficacy test, an animal model experiment method is not established, and the anti-wrinkle performance of cosmetic raw materials cannot be rapidly and efficiently tested.
Liuyang and the like summarize the existing evaluation methods for the anti-wrinkle effect of cosmetics, and comprise a biochemical method, a cell experimental method, a human body trial experimental method and the like (Liuyang, Deng Yimei, Zhao Hua. cosmetic anti-wrinkle effect evaluation method [ J ]. daily chemical science 2015,38(004):18-21), but the methods have the problems of overlarge difference with the physiological state of a human body, high cost and low efficiency. Therefore, how to provide a method for detecting the anti-wrinkle and wrinkle-weakening activity of skin care raw materials, which has low cost and high speed and is close to the physiological state of a human body, becomes a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting the anti-wrinkle and wrinkle-reducing activity of a skin care raw material and application thereof, and particularly provides a method for detecting the anti-wrinkle and wrinkle-reducing activity of the skin care raw material with low cost and high speed and application thereof. The method for detecting the anti-wrinkle and wrinkle-reducing activity of the skin care raw material provided by the invention has the advantages of short experimental period, high detection speed, capability of screening the raw material at high flux and low cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a method for detecting anti-wrinkle and wrinkle-reducing activity of a skin care raw material, which comprises the following steps:
(1) culturing Escherichia coli in liquid culture medium, mixing the bacterial liquid with skin care raw material to be tested, and coating on NGM (nematode growth solid culture medium) plate for preservation;
(2) carrying out synchronization on nematodes, and then cracking to obtain larvae;
(3) inoculating the larvae obtained in the step (2) to the NGM flat plate obtained in the step (1) for culture, then picking out, recording the swing times of the head of nematode parameters, and judging the anti-wrinkle and wrinkle-lightening activity of the skin care raw material to be detected;
the step (1) and the step (2) do not distinguish the sequence.
The detection method adopts the nematodes as model organisms for detection, reduces the production cost, shortens the experimental period, improves the detection speed and can screen raw materials at high flux.
Preferably, the liquid medium in step (1) is LB liquid medium.
Preferably, the volume ratio of the bacterial liquid to the skin care raw material to be tested in the step (1) is (92-98): (2-8), wherein the part of the bacterial liquid can be 92, 93, 94, 95, 96, 97 or 98, etc., and the part of the skin care raw material to be tested can be 2, 3, 4, 5, 6, 7 or 8, etc., but the invention is not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.
Preferably, the skin care raw material to be tested in step (1) comprises a neuroleptic peptide compound.
The detection method utilizes the nematode model to detect the ability of neuropeptides to block nerve and muscle nerve impulses, so as to evaluate the effects of the raw materials on resisting wrinkles and removing wrinkles, and the effect is closer to the physiological state of a human body.
Preferably, the nematode in step (2) is caenorhabditis elegans.
The left side and the right side of the caenorhabditis elegans body are basically symmetrical, four muscles distributed in four quadrants penetrate through the whole body and are tightly connected with a nervous system to control the body of the caenorhabditis elegans to bend towards the dorsal side or the ventral side, and the muscles are connected independently except the head; the neuroinhibitory peptides are potent inhibitors of neurotransmission, and can block impulse conduction at neuromuscular junction by inhibiting release of acetylcholine vesicles at the neuromuscular junction, so that muscle paralysis is caused to achieve the effect of smoothing skin wrinkles; by adopting the mode of co-culturing the caenorhabditis elegans and the skin care raw material to be detected and then detecting the movement capacity of the caenorhabditis elegans, the anti-wrinkle and wrinkle-lightening activity of the skin care raw material to be detected can be clearly and visually detected and compared.
Preferably, the culturing period in step (3) is 2 to 5 days, such as 2 days, 3 days, 4 days or 5 days, but not limited to the above-mentioned values, and other values not listed in the above-mentioned range of values are also applicable.
Preferably, said sorting out in step (3) further comprises treating the nematodes with a stimulant.
The stimulant is adopted to treat the nematodes, so that the movement capability of the nematodes can be improved, and the inhibition effect of the skin care raw materials to be detected on the muscle movement of the nematodes can be more clearly and definitely reflected.
Preferably, the stimulant comprises neostigmine mesylate.
Preferably, the step (3) of recording the number of swings of the head of the nematode parameter further comprises recording the movement track of the nematode.
Preferably, the step (3) of recording the number of swings of the head of the nematode parameter further comprises measuring the enzyme activity of the nematode nerve-associated enzyme.
The detection items are verified from multiple angles and mutually verified, so that the accuracy of the detection method is improved.
Preferably, the nematode nerve-associated enzyme comprises calcineurin (CaN) and/or acetylcholinesterase (AchE).
Acetylcholinesterase is a key enzyme in biological nerve conduction. It can degrade acetylcholine between cholinergic synapses and terminate the excitatory action of neurotransmitter on postsynaptic membranes, thus ensuring the normal transmission of nerve signals in organisms. When the activity of the nematode AchE enzyme is inhibited, the Ach accumulated in the nematode body can not be decomposed in time, so that the Ach at the nerve-muscle joint is reduced, the excitation impulse between motor nerves and muscles is blocked, and the movement capacity of the nematode is reduced.
The CaN has the functions of promoting the release of neurotransmitters by neurons and the development of muscle tissues, and enhances the capacity of releasing Ach into the muscle tissues. Ca in vivo when nematodes are stimulated by neuroleptic peptides+Ion channels are opened, resulting in Ca+Large amount of external influx of cytosolic, Ca+The increase in the concentration in the cytoplasm activates the enzymatic activity of CaN, and thus the activity of CaN increases.
In another aspect, the invention also provides the application of the method for detecting the anti-wrinkle and wrinkle-reducing activity of the skin care raw material in the detection of the cosmetic raw material.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for detecting the anti-wrinkle and wrinkle-reducing activity of a skin care raw material, which adopts nematodes as model organisms for detection, reduces the production cost, shortens the experimental period, improves the detection speed and can screen the raw material at high flux; by adopting the mode of co-culturing the caenorhabditis elegans and the skin care raw material to be detected and then detecting the movement capacity of the caenorhabditis elegans, the anti-wrinkle and wrinkle-lightening activities of the skin care raw material to be detected can be clearly and visually detected and compared; the stimulant is adopted to treat the nematodes, so that the movement capacity of the nematodes can be improved, and the inhibition effect of the skin care raw materials to be detected on the muscle movement of the nematodes can be more clearly and definitely reflected; the method is verified from multiple angles and mutually verified, so that the accuracy of the detection method is improved.
Drawings
FIG. 1 is a graph showing the results of determination of head oscillation frequency of nematodes under natural conditions, wherein A, B, C and D represent the results of the tests of the groups A, B, C and D, respectively;
FIG. 2 is a graph showing the results of measurement of head wobble frequency of nematodes under excitation, wherein A, B, C and D represent the results of the tests in groups A, B, C and D, respectively;
FIG. 3 is a graph of the movement trace of nematodes in a control group under natural conditions;
FIG. 4 is a graph of the locus of movement of group D nematodes under natural conditions;
FIG. 5 is a graph of the movement trace of nematodes in a control group under excitatory stimulation;
FIG. 6 is a graph of the movement traces of group D nematodes under excitatory stimulation;
FIG. 7 is a graph showing the results of determination of acetylcholinesterase activity in nematodes, wherein A, B, C and D represent the results of the tests in groups A, B, C and D, respectively;
FIG. 8 is a graph showing the results of activity assays for calcineurin in nematodes, wherein A, B, C and D represent the results of the tests in groups A, B, C and D, respectively.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
In the following examples, the red scorpion toxin, conotoxin, and snake venom-like peptide were purchased from Zhejiang volume tidal volume and volume, respectively;
coli OP50 is a uracil synthesis-deficient strain purchased from CGC;
caenorhabditis elegans was purchased from the Caenorhabditis elegans Genetics Center (CGC), USA;
the following examples used the following starting materials:
(1M) Potassium phosphate buffer
KH2PO4 108.39g
K2HPO4 35.69g
Adding water to 1L, and adjusting pH to 6.0.
② M9 buffer solution
Water was added to 1L and sterilized at 121 ℃ for 15 minutes.
③ LB liquid culture medium
Tryptone 10g/L
Sodium chloride 10g/L
Yeast powder 5g/L
Prepared with distilled water, adjusted to pH 7.0 with 1M sodium hydroxide solution and sterilized at 121 ℃ for 15 minutes.
Fourthly, the solid culture medium (NGM) for the growth of the nematodes is counted by 1L
After shaking, sterilization was carried out at 121 ℃ for 30 minutes and at 80 ℃ for 15 minutes, and then the following solutions (cholesterol filter sterilized, and the rest sterilized at high temperature) were added.
1M CaCl2 l mL
1M MgSO4 l mL
5mg/mL Cholesterol dissolved in ethanol l mL
The mixture was left for 2 days under 1M potassium phosphate.
Lysate
0.1g NaOH and 1.3mL NaClO were dissolved in 4mL distilled water and mixed well.
Examples
The embodiment provides a method for detecting anti-wrinkle and wrinkle-reducing activities of skin care raw materials, and the peptides to be detected are red scorpion toxin (group A), conotoxin (group B), snake venom-like peptide (group C) and red scorpion toxin and conotoxin compound polypeptide (the volume ratio is 9:1, group D).
The method comprises the following specific steps:
(1) cultivation of E.coli OP50
Taking OP50 strain to streak on LB plate, picking single colony in 10mL LB liquid culture medium, 37 deg.C, 200rpm, shaking culture for 12h, to OD600Equal to 0.4, for inoculation of NGM to feed normal group nematodes.
(2) Coating of coli OP50
The inoculum (60 mm diameter plate plus 100. mu.L) was added to each NGM plate and spread evenly on the NGM plate using a sterile spreader or glass tube bottom, taking care that the inoculum edge should be about 0.5cm from the plate edge. The bacterial coated NGM plates were ready for use overnight at 25 ℃.
(3) Nematode phasic lysis
The caenorhabditis elegans pregnant worm growth plates were rinsed with 1mL M9 buffer and the blow beating repeated until most of the adults and eggs on the plates were eluted from the lawn. The aqueous solution containing the pregnant insect was aspirated into a 2mL centrifuge tube and 1mL nematode lysis solution was added. The centrifuge tube was shaken for several seconds, then left to stand, and left to stand for 2 minutes and shake for 1 time, and then the operation was continued for 3 times. The mixture was centrifuged at 3000rpm for 60s in a low speed centrifuge. The waste supernatant was aspirated and retained to 0.2mL of lysis solution. The buffer M9 was added to the scale of 2mL in the centrifugal tube containing the lysate, and the tube was shaken for a few seconds. The operation was carried out twice in succession. After centrifugation and discarding of the supernatant, nematode eggs were pipetted from the bottom of the centrifuge tubes onto E.coli OP 50-coated NGM plates. In a sterile area, fertilized eggs in the lysed nematodes basically develop into L4 larvae after about 48 hours, and synchronization is completed for the experiment.
(4) Test grouping and drug treatment:
in the control group, 95. mu.L of E.coli OP50 bacterial liquid and 5. mu. L M9 were mixed and applied. And (4) drying the bacterial-coated NGM plate in the dark, sealing the membrane, and storing at 4 ℃ for later use.
Preparation of experimental group medicine NGM: 95 μ L of E.coli OP50 bacterial liquid was mixed with 5 μ L of the test substance (A, B, C, D group) uniformly (neuropeptide loading volume fraction is 5%). A pipette gun pipetted 100. mu.L of the inoculum gently centered on each NGM plate.
(5) Measuring the head swing frequency of the nematode under natural conditions:
synchronized nematodes were picked into each group of media in control and experimental groups, 30 per plate, 3 per group in parallel. After 3 days of drug intervention, the nematodes were picked onto food-free NGM plates, and after 1min of nematode locomotion, the number of oscillations of the head of each nematode in 30s time was observed and recorded under a stereoscopic microscope. Selecting 15 insects for recording on each plate, and removing one maximum value and one minimum value from each plate
(6) Measuring the head swing frequency of the nematode under excitation stimulation:
and (3) picking synchronized nematodes into culture media of a control group and an experimental group, and collecting the nematodes in a 2mL centrifuge tube after 3 days of drug intervention on the nematodes to obtain 1mL nematode aqueous suspension of 3000 nematodes/mL. 1mL of neostigmine mesylate at a concentration of 0.5mg/mL was added to the aqueous nematode suspension. And (3) after 4 hours of neostigmine methosulfate treatment, taking nematode liquid into an NGM plate without food, and observing and recording the swinging times of the head of each nematode in 30s time under a stereoscopic microscope after the nematodes recover to move for 1 min. 15 worms were picked for each plate and recorded, one maximum and one minimum being removed from each plate.
(7) And (3) determination of nematode motion tracks:
and (3) picking synchronized nematodes into culture media of a control group and a group D, treating the nematodes according to the natural conditions and the excitation stimulation method after the nematodes are subjected to drug intervention for 3 days, observing the movement track of each group of nematodes on an NGM plate, taking a picture, and comprehensively analyzing the movement performance of each group of nematodes.
(8) And (3) determining the nematode nerve-related enzyme activity:
synchronized L4 nematodes were picked into the control and experimental groups of medium, 30 per plate, 3 per group in parallel. Collecting nematodes after 3 days of drug intervention, collecting the nematodes on the plate with sterile PBS to a 2mL centrifuge tube for washing, centrifuging at low speed to remove supernatant, repeatedly metering to 1mL after three times, and performing ultrasonication in ice water bath (ultrasound for 3s, interval for 3s, and working time for 3 min). Centrifuging at low temperature (4 deg.C, 12000r/min, 15min), collecting supernatant, and standing at 4 deg.C. The determination of enzyme activity is carried out according to the instruction of a calcineurin (CaN) kit and a Solebao acetylcholinesterase (AchE) kit of Nanjing institute of bioengineering.
The results are summarized as follows:
the measurement results of the head swing frequency of the nematodes under natural conditions and excitation stimulation are shown in fig. 1-2, and it can be found from the figure that peptides to be measured under natural conditions can block the nerve and muscle nerve impulse capability to realize the muscle relaxation effect, the red scorpion toxin can inhibit the head swing frequency of the nematodes by 24.06%, the conotoxin can inhibit the head swing frequency of the nematodes by 17.30%, the snake venom like peptide can inhibit the head swing frequency of the nematodes by 9.02%, and the red scorpion toxin and conotoxin compound polypeptide with the mixing volume ratio of 9:1 can inhibit the head swing frequency of the nematodes by 33.08%; under the excitation condition, the red scorpion toxin can inhibit the head swing frequency of the nematode to 9.72 percent, the conopeptides can inhibit the head swing frequency of the nematode to 5.80 percent, the snake-like venom peptides can inhibit the head swing frequency of the nematode to 8.02 percent, and the red scorpion toxin and conopeptides compound polypeptide with the mixing volume ratio of 9:1 can inhibit the head swing frequency of the nematode to 17.59 percent.
The results of the nematode motion trail measurement are shown in fig. 3-6, wherein fig. 3 and 4 are the motion trail graphs of the nematodes in the control group and the group D under natural conditions, respectively, and fig. 5 and 6 are the motion trail graphs of the nematodes in the control group and the group D under excitation, respectively. The locomotory behavior of nematodes on agar media is typical of sinusoidal movements, and is achieved by the coordinated contractions of the muscle of the dorsal and abdominal body walls of the nematodes (FIG. 3). In the graph of fig. 4, the nematode is affected by the red scorpion and the conopeptide, the movement ability of the nematode is inhibited, the nematode cannot normally swing the body, the movement curve tends to be smooth, and no obvious wave peak exists. The compound peptide of the red scorpion peptide and the conopeptide can block the normal movement of the nematodes to a certain extent, so that the nematodes can crawl after trailing. Neostigmine methosulfate treated nematodes still exhibited sigmoidal overall locomotor morphology, but the amplitude of oscillation was greater than that of untreated nematodes, with intermittent transient irregular wriggling (fig. 5). After the compound polypeptide of the red scorpion and the conopeptide is acted by neostigmine methosulfate, the body bending amplitude of the nematode is recovered to a certain extent, but the tail swing is still inflexible and the motion track is relatively gentle (figure 6).
The determination results of the nematode nerve-related enzyme activity are shown in figures 7-8, wherein figure 7 is a diagram of the determination results of the nematode acetylcholinesterase activity, and figure 8 is a diagram of the nematode calcineurin activity. Acetylcholinesterase (AchE) is a key enzyme that hydrolyzes acetylcholine (Ach), maintaining normal muscle physiology. As shown in FIG. 7, compared with the control group, the activity of the nematode AchE enzyme in each experimental group is reduced, and particularly, the AchE enzyme activity of the red scorpion and conopeptide compound polypeptide group is remarkably reduced by 50.11 percent (p)<0.05). Calcineurin (CaN) is directly affected by Ca in cell signaling+The regulation of (1) is involved in nerve signal conduction and regulates the adaptation of the body to stimulation during exercise. As shown in FIG. 8, compared with the control group, the CaN enzyme activity of the nematodes in each group of the experiment group is increased, and particularly, the CaN enzyme activity of the red scorpion and conopeptide compound polypeptide group is significantly increased by 2.14 times (p)<0.05). The change of the activity of the nematode nerve-related enzyme is consistent with the prior apparent data of the movement capacity of the nematode.
The data show that the detection method provided by the invention can effectively detect the anti-wrinkle and wrinkle-reducing activities of the skin care raw materials. By adopting the nematodes as model organisms for detection, the production cost is reduced, the experimental period is shortened, the detection speed is improved, and the raw materials can be screened at high flux; by adopting the mode of co-culturing the caenorhabditis elegans and the skin care raw material to be detected and then detecting the movement capacity of the caenorhabditis elegans, the anti-wrinkle and wrinkle-lightening activities of the skin care raw material to be detected can be clearly and visually detected and compared; the stimulant is adopted to treat the nematodes, so that the movement capacity of the nematodes can be improved, and the inhibition effect of the skin care raw materials to be detected on the muscle movement of the nematodes can be more clearly and definitely reflected; from a plurality of angles, mutual verification is achieved, and the accuracy of the detection method is improved.
The applicant states that the present invention is illustrated by the above examples to describe the method for detecting anti-wrinkle activity of a skin care material and the application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention is implemented only by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A detection method for anti-wrinkle and wrinkle-reducing activities of skin care raw materials is characterized by comprising the following steps:
(1) culturing Escherichia coli in a liquid culture medium, mixing the bacterial liquid with the skin care raw material to be tested, and coating the mixture on an NGM flat plate for storage;
(2) carrying out synchronization on nematodes, and then cracking to obtain larvae;
(3) inoculating the larvae obtained in the step (2) to the NGM flat plate obtained in the step (1) for culture, then picking out, recording the swing times of the head of nematode parameters, and judging the anti-wrinkle and wrinkle-lightening activity of the skin care raw material to be detected;
the step (1) and the step (2) do not distinguish the sequence.
2. The method for detecting anti-wrinkle and wrinkle-reducing activities of skin care raw materials according to claim 1, wherein the liquid culture medium in step (1) is LB liquid culture medium.
3. The method for detecting the anti-wrinkle and wrinkle-reduction activities of skin care raw materials according to claim 1 or 2, wherein the volume ratio of the bacterial liquid in the step (1) to the skin care raw material to be detected is (92-98): (2-8).
4. The method for detecting anti-wrinkle and wrinkle reduction activities of skin care raw material according to any one of claims 1-3, wherein the skin care raw material to be detected in step (1) comprises a neuroleptic peptide compound.
5. The method for detecting anti-wrinkle activity of a skin care raw material according to any one of claims 1 to 4, wherein the nematode in step (2) is caenorhabditis elegans.
6. The method for detecting anti-wrinkle and wrinkle reduction activities of skin care raw materials according to any one of claims 1 to 5, wherein the incubation period in step (3) is 2 to 5 days;
preferably, said sorting out in step (3) further comprises treating the nematodes with a stimulant;
preferably, the stimulant comprises neostigmine mesylate.
7. The method for detecting anti-wrinkle activity of a skin care raw material according to any one of claims 1 to 6, wherein the step (3) of recording the number of swings of the head with the nematode parameter further comprises recording the movement trace of the nematode.
8. The method for detecting anti-wrinkle and wrinkle reduction activities of skin care raw material according to any one of claims 1 to 7, wherein the step (3) of recording the number of swings of head of nematode parameters further comprises measuring the enzyme activity of nematode nerve-related enzyme.
9. The method of claim 8, wherein the nematode-related enzymes comprise calcineurin and/or acetylcholinesterase.
10. Use of a method of testing a skin care material for anti-wrinkle and wrinkle reduction activity according to any one of claims 1-9 in a cosmetic material test.
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| 田雨等: ""久效磷对秀丽隐杆线虫运动、学习和觅食行为的影响"", 《环境科学研究》 * |
| 马娜娜等: ""硫柳汞对秀丽隐杆线虫神经行为及生长发育的影响"", 《毒理学杂志》 * |
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