CN114149985A - Preparation method and application of molecular motor - Google Patents
Preparation method and application of molecular motor Download PDFInfo
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- CN114149985A CN114149985A CN202111477618.3A CN202111477618A CN114149985A CN 114149985 A CN114149985 A CN 114149985A CN 202111477618 A CN202111477618 A CN 202111477618A CN 114149985 A CN114149985 A CN 114149985A
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a preparation method and application of a molecular motor, wherein the molecular motor is prepared by using thermophilic bacteria, and a differential centrifugation method and a density gradient centrifugation method are combined, so that macromolecular impurities such as cell fragments, organelles, globulin and the like are removed, and the molecular motor with higher purification and function is obtained. The molecular motor prepared by the invention has various action paths to realize the effects of tightening and resisting aging, has no adverse effect related to oxidation, can inhibit the activities of MMP-1 and MMP-9, can promote the expression of growth factors GDF11 and TGF-beta 1, can inhibit the expression of Bax gene and promote the expression of Bcl-2, reduces the loss of rDNA copy number, and has certain effects of resisting ultraviolet injury, promoting proliferation, resisting apoptosis, resisting aging and tightening the skin on epidermal and dermal tissues.
Description
Technical Field
The invention belongs to the field of biological and high polymer materials, and relates to a preparation method and application of a molecular motor, which have high energy conversion efficiency and high purification. And to the use of the prepared molecular motors in the cosmetic field.
Background
Cellular aging is a major aging process characterized by irreversible growth arrest, resistance to apoptosis, production of aging-related secretory phenotypes (SASP), mitochondrial dysfunction, and alterations in DNA and chromatin. Cellular senescence can be classified as acute senescence, such as preventing excessive fibrosis at the wound site; chronic aging, such as persistent macromolecular damage (protein misfolding, DNA damage, epigenetic changes, telomere shortening, etc.) caused by stress responses in the body; and (3) treating aging, such as body stress reaction caused by normal cell injury caused by radiotherapy and chemotherapy.
Multiple modes of induction of cellular senescence include DNA damage, active metabolites, inflammation, oncogenes, mitogens, protein toxic stress and damage-related molecular patterns. Wherein:
1) the DNA Damage Response (DDR) is central to the induction of cellular senescence. DDR can be caused by telomere shortening or dysfunction, mutations, irradiation, and alkylating agents.
2) The active metabolites are elevated, including active oxygen, fatty acids, glucose, and ceramides.
3) Protein aggregation, unfolded protein response and mTOR activation are associated with protein toxic stress and loss of protein homeostasis.
These inducers ultimately mediate the cellular senescence phenotype through mechanisms that converge on pathways that activate p 16. sup. INK4a/Rb and/or p53/p21, depending on the inducer and cell type.
Growth factors are generally considered to be a subset of cytokines, and are signaling proteins that stimulate cell growth, differentiation, survival, inflammation, and tissue repair. They can be secreted by adjacent cells, tissues, glands, and even cancer cells. All cells require a series of growth factors to maintain proliferation and viability, stimulate proliferation of epidermal, epithelial and embryonic cells, promote wound healing, bone remodeling, reduce skin laxity, improve skin elasticity and tone, and exhibit moisturizing and antioxidant properties when applied to the facial skin of elderly participants.
The molecular motor, namely ATP synthetase, is a family of membrane protein complexes for catalyzing and synthesizing ATP by using transmembrane electrochemical gradient, catalyzes the synthesis of energy substance ATP in cells, is a key structure for supporting photosynthesis and respiration, converts energy released by an electron transfer chain into transmembrane proton (H +) gradient in the respiration or photosynthesis process, and then enables ADP + Pi to synthesize ATP by virtue of ATP synthetase along the proton gradient.
F1F0ATPase has very high energy conversion efficiency, and as a naturally occurring nano-scale energy conversion device, the catalytic process of ATP synthase is reversible, and can hydrolyze ATP and synthesize ATP. The primary function of ATP synthase molecules under physiological conditions is to synthesize ATP. Because the working efficiency of the motor is far higher than that of the molecular motor which is manually designed at present, the motor also gets attention in the research fields of nano science, mechanics, micro electro mechanical system and the like in recent years. In the field of biomedicine, molecular motors are currently used mainly as nanosensors for the research and detection of harmful chemicals in pathological diagnosis and biomedical detection, in view of their high biological potential.
Disclosure of Invention
The molecular motor is a natural source and is non-toxic, is prepared by fermenting thermophilic bacteria and is used for evaluating the influence on cell aging of non-damaged skin for the first time, and the molecular motor has interference and reversal effects on natural aging (endogenous aging) and has an anti-aging effect.
The first object of the present invention is to provide a method for manufacturing a molecular motor, which is efficient, and which can manufacture a molecular motor more resistant to heat, having more efficient energy conversion capability, and which can obtain a molecular motor with higher degree of purification and function by improving the method for manufacturing a molecular motor.
The second purpose of the invention is to provide the application of the molecular motor prepared by the preparation method, and the application of the molecular motor in the aspects of compact, anti-aging, repairing and anti-inflammation of cosmetics.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of making a molecular motor, the method comprising the steps of:
1) inoculating the cultured bacteria into a culture medium for secondary culture;
2) centrifugally collecting thalli from the bacteria obtained by re-culturing in the step 1), re-suspending the thalli by using a buffer solution A, and crushing for multiple times to obtain a cell crushing solution;
3) extracting and purifying the cell disruption solution obtained in the step 2) by a differential centrifugation method and a density gradient centrifugation method to obtain a molecular motor;
alternatively, the preparation method comprises the following steps:
a) taking the cultured bacteria, and centrifugally collecting somatic cells;
b) suspending the bacterial cells obtained in the step a) by using a buffer solution B;
c) adding PMSF, carrying out ice bath ultrasonic crushing, carrying out first centrifugation to obtain supernatant, carrying out second centrifugation on the supernatant, and removing the centrifuged supernatant to obtain the F-containing product1F0-a complex of atpases;
wherein, the bacteria in the steps 1) and a) are thermophilic bacteria.
In the invention, the differential centrifugation method and the density gradient centrifugation method are combined, so that macromolecular impurities such as cell debris, organelles, globulin and the like are removed, and a molecular motor with higher purification and function is obtained.
F1F0ATPase is the model with excellent stress resistance in the current research model of ATPase, but maintains the activityThe solution system is required to be a suspension containing water, various ions, biological membrane lipids, membrane proteins and other substances, and the properties of the suspension are completely different from those of the protein solution used for manufacturing the water-soluble protein chip.
The energy conversion efficiency of a general molecular motor is about 60%, and the molecular motor prepared by the method can reach 100%.
In a preferred embodiment of the present invention, the thermophilic bacteria includes one of thermophilic thermus thermophilus, thermophilic roseococcus, thermophilic bacillus, thermophilic streptococcus, sulfolobus solfataricus, marigold thermatopae and pyrococcus, and the inoculation ratio of the thermophilic bacteria is 1: 98-105.
As a preferable embodiment of the present invention, in step 2), the buffer a comprises the following raw materials: 0.1-0.8mol/L PBS pH8.0, 0.1-20mmol/L MgCl2，0.1-5mmol/L DTT。
As a preferable embodiment of the present invention, in step B), the buffer B comprises the following raw materials: 45-55mM Tricine-NaOH, pH7.8-8.2, 0.2-0.3M sucrose solution, 3-5mM MgCl2And (3) solution.
As a preferable scheme of the invention, in the step 3), the differential centrifugation method is centrifugation for 40 minutes at 1-6 ℃ and 3000 Xg at 600-; the density gradient centrifugation method comprises the following steps: adding the precipitate obtained by the differential centrifugation method into sucrose solutions with the mass percent concentration of 20 percent, 30 percent, 40 percent, 50 percent and 60 percent respectively, performing ultracentrifugation at the temperature of 1-6 ℃ for 60-90min by using 300,000 Xg at the temperature of 150,000, and collecting liquid in the region with the mass percent concentration of 40-50 percent of the sucrose solution.
As a preferred embodiment of the present invention, in step c), the final concentration of PMSF is 1mmol/L, and the ultrasonication conditions are as follows: the amplitude is 40-60%, the ultrasonic treatment is carried out for 5-10s, the ultrasonic treatment is stopped for 6-10s, and the time is 25-35 min.
In a preferred embodiment of the invention, in step c), the first centrifugation is performed at 20,000 Xg for 25-35min and the second centrifugation is performed at 180,000 Xg for 85-95 min.
The second aspect of the present invention provides the use of the molecular motor prepared by the above-described preparation method in the cosmetic field.
As a preferred embodiment of the present invention, the use of the molecular motor in the preparation of a cosmetic for tightening anti-aging.
In a preferred embodiment of the present invention, the molecular motor inhibits MMP-1 and MMP-9 activities.
As a preferred embodiment of the invention, the molecular motor can promote the expression of the growth factors GDF11 and TGF-beta 1.
As a preferred scheme of the invention, the molecular motor can reduce rDNA copy number loss, and has certain effects of resisting ultraviolet injury, promoting proliferation, resisting apoptosis, resisting aging and tightening skin on epidermal and dermal tissues.
In the invention, the molecular motor prepared by the invention has multiple action paths to realize the effects of tightening and resisting aging.
Compared with the prior art, the invention has the following beneficial effects:
1) the preparation method is efficient, the prepared molecular motor is more heat-resistant and has high-efficiency energy conversion capability, and the molecular motor with higher purification and function can be obtained by improving the preparation method of the molecular motor;
2) the molecular motor prepared by the invention has various action paths to realize the effects of tightening and resisting aging, has no adverse effect related to oxidation, can inhibit the activities of MMP-1 and MMP-9, can promote the expression of growth factors GDF11 and TGF-beta 1, can inhibit the expression of Bax gene and promote the expression of Bcl-2, reduces the loss of rDNA copy number, and has certain effects of resisting ultraviolet injury, promoting proliferation, resisting apoptosis, resisting aging and tightening the skin on epidermal and dermal tissues.
Drawings
FIG. 1 is the relative expression level of mRNA in epidermal tissue of the present invention.
FIG. 2 is the relative expression level of the mRNA in dermal tissue according to the present invention.
FIG. 3 is the normalized copy number of ribosomal DNA and mitochondrial DNA.
FIG. 4 is the oxidative stress-related indicator assay.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a preparation method of a molecular motor, which comprises the following steps:
1) taking cultured bacteria (mainly thermophilic bacteria, mainly thermophilic thermus) and culturing the strains: thermus thermophilus is inoculated into the culture medium according to the proportion of 1:100, and the culture is continued for 48 h.
2) Wall breaking: centrifuging at 4,000 rpm for 30min at 4 deg.C after fermentation to collect thallus; homogenizing with homogenization buffer (0.1-0.8mol/L PBS pH8.0, 0.1-20mmol/L MgCl20.1-5mmol/L DTT), and crushing the suspended bacteria for 3 times at 0-6 ℃ under the ultrahigh pressure of 500-1000M to obtain cell crushing liquid.
3) And extracting and purifying by using a differential centrifugation method and a density gradient centrifugation method.
Specifically, the density gradient centrifugation method comprises the steps of: centrifuging the obtained cell disruption solution for 40 minutes at 1-6 ℃ and 3000 Xg for 600-. And collecting the liquid C, and removing the sucrose to obtain the molecular motor.
Molecular motor activity test method (coupling method): nucleoside Diphosphate (NDP) is one of the high-energy phosphate hydrolysates of nucleoside triphosphate, pyruvate dehydrogenase (PK) and Lactate Dehydrogenase (LDH) are common coupling enzymes used to measure the production of NDP, the reaction substrate is Nicotinamide Adenine Dinucleotide (NADH) and needs to be accomplished by Adenosine Triphosphate (ATP) hydrolysis, and the amount of ATP hydrolysis is determined by measuring the decrease in optical density of NADH at 340nm (enzyme activity: ATPase hydrolysis activity is defined as the amount of ATP hydrolyzed at 37 ℃ per minute by 1. mu.L enzyme).
Particle size comparison at different sugar densities, molecular motor activity comparison is shown in table 1.
Table 1 comparison of the activity (ATP hydrolysis) of the resulting molecular motors centrifuged at different density gradients (twice):
example 2
This embodiment provides another method for preparing a molecular motor, including:
taking cultured bacteria (mainly thermophilic bacteria, mainly thermophilic thermus). The cells were collected by low speed centrifugation using 50mM Tricine-NaOH, pH8.0, 0.25M sucrose, 4mM MgCl2Suspending the solution, adding PMSF with final concentration of 1mmol/L, carrying out ice-bath ultrasonic disruption for 30min (amplitude is 55%, ultrasonic disruption is carried out for 5s, and stopping for 8s), carrying out ultrasonic disruption on cells, firstly centrifuging at high speed (20,000 g) for 30min to obtain supernatant, then centrifuging the supernatant at ultra high speed (180,000 g) for 90min, and removing the supernatant to obtain the final product containing F1F0-a complex of ATPases.
Example 3
In this example, experiments were performed on the in vitro anti-aging efficacy of the sample molecular motor from several perspectives, such as matrix metalloproteinase in the expression level of the relevant mRNA, growth factors related to cellular senescence, and gene influences related to autophagy and apoptosis regulation, respectively:
in the experiment, a T-Skin in vitro artificial full-thickness Skin model (3D Skin model) is selected, and half of the control and molecular motor sample groups are used for evaluating and researching the in vitro anti-aging effect of the detected sample, namely the molecular motor stock solution prepared in the embodiment 1. Experiments show that a control group and a molecular motor group are administered by adopting an epidermal smearing mode, smearing is repeated for 1 time every 24 hours, after 5 days are continued, the relative expression quantity of mRNA (messenger ribonucleic acid) of MMP-1, MMP-9, GDF-11, TGF-beta 1, Bax, Bcl-2, Beclin-1 and Caspase-3 of each group of skin models is detected by real-time fluorescence quantitative PCR, and the detection items of the control group and the molecular motor group are shown in a table 2.
TABLE 2 mRNA expression test items for control group and molecular Motor group
Referring to fig. 1 and 2, fig. 1 is the relative expression level of epidermal tissue mRNA, indicating p <0.05 compared to control; denotes p <0.01 compared to control.
Figure 2 is the relative expression level of dermal tissue mRNA, indicating p <0.05 compared to control; denotes p <0.01 compared to control.
Wherein:
1) mRNA expression of MMP-1 and MMP-9:
matrix metalloproteinases are the most prominent enzymes responsible for the appearance of signs of aging such as wrinkles and fine lines in the skin. Wherein MMP-1 is collagenase closely related to human skin aging, and can degrade collagen and elastin in dermis. The primary function of MMP-9 is to degrade and remodel the extracellular matrix with MMP-1. Based on the important role of matrix metalloprotease in skin aging process, it is a development direction of many cosmetic raw materials to inhibit the activities of MMP-1 and MMP-9.
Compared with the control group, the relative expression amounts of two matrix metalloproteinase mRNAs in the epidermal tissue of the molecular motor group and the relative expression amount of MMP-9mRNA of the dermal group are respectively reduced, and the difference has statistical significance, and referring to fig. 1 and fig. 2, the experimental result fully proves that the molecular motor can effectively reduce the expression of MMP-1 and MMP-9 in the epidermal layer and the dermal layer. The two matrix metalloproteinases are the most important enzymes causing skin aging symptoms such as shrinkage, fine wrinkles and the like in the field of photoaging, are closely related to the degradation of extracellular matrixes such as collagen, elastin and the like in a dermis, and inhibiting the activity of the enzymes is a way for delaying aging. Experiments show that the molecular motor can effectively reduce the expression of MMP-1 and MMP-9, and has better effect of delaying senility on cosmetics.
2) Growth factor TGF-beta modulation:
both GDF11 and TGF-. beta.1 belong to members of the transforming growth factor-beta (TGF-. beta.) family, which belongs to a group of factors that regulate cell growth and differentiation, and are generally present in higher amounts in tissues in which cell differentiation is active. Among them, GDF11 is a key regeneration factor of senescent cells. GDF11 can improve neurodegenerative and neurovascular diseases, increase skeletal muscle volume and enhance muscle strength, and its efficacy in reversing aging in clinical applications is also one of its broad biological effects. TGF-. beta.1 is a potent stimulator of tissue regeneration, and TGF-. beta.1 activates fibroblasts to produce collagen in studies related to wound healing. GDF11 expression has a tendency to decrease in mice and humans with increasing age. In vivo animal experiments, TGF-beta 1 is expressed positively in skin tissues of rats in the young group and negatively in the old group, so GDF11 and TGF-beta 1 are considered to be important endogenous regulators of skin repair and regeneration.
In the experiment of the present invention, the relative expression levels of GDF11 mRNA in epidermal tissue, GDF11 and TGF-. beta.1 mRNA in dermal tissue were increased and the difference was statistically significant, respectively, as compared with the control group, see FIGS. 1 and 2.
The molecular motor has the function of promoting the expression of growth factors GDF11 and TGF-beta 1, and the high expression of GDF11 and TGF-beta 1 predicts the potential of the molecular motor in the functions of promoting proliferation and repair.
3) Apoptosis and autophagy element regulation
Since apoptosis and autophagy are interactively regulated in signal regulation, there are many common regulatory elements. The invention selects the most representative elements of Bax, Bcl-2, Beclin-1 and Caspase-3 for preliminary exploration. Beclin-1 plays an important role in regulating and controlling apoptosis and autophagy, and Bcl-2 influences the activity of the cell by forming a complex with the Beclin-1. Apoptosis and autophagy are interactively regulated in signal regulation, having many common regulatory elements. Among these regulatory elements, Bax, Bcl-2, Beclin-1 and Caspase-3 are independent and closely related to each other, and play an important role in apoptosis and autophagy.
3.1) inhibition of apoptosis:
bax and Bcl-2 are members of Bcl protein family, Bcl-2 is a protein for inhibiting apoptosis, and the biological effect of Bax is to antagonize Bcl-2, thereby promoting apoptosis and shortening the survival cycle of cells. The ratio of the two thus determines whether the cell goes towards apoptosis.
In the present invention, the mRNA expression level of Bax was significantly reduced in the molecular motor group, and Bcl-2 was significantly increased and statistically significant in both the dermal and epidermal layers, compared to the control group. The results show that the Bax/Bcl-2 ratio is remarkably reduced, and Caspase-3 has no difference compared with a control group, so that the molecular motor product has the tendency of inhibiting the apoptosis of epidermal and dermal layers.
3.2) modulation of autophagy:
bcl-2 is also actively involved in the regulation of autophagy, and the relative amounts of Bcl-2 and Beclin-1 bound to each other in the cell determine the level of autophagy of the cell to some extent. Due to phosphorylated Beclin-1, lipase VPS34 was activated, inducing autophagy. Thus, when the Bcl-2 and Beclin-1 binding is increased, the cell autophagy is inhibited. Autophagy is a key mechanism for prolonging life, and is resistant to premature aging in skin tissues.
In the experiments of the invention, the expression level of mRNA in the epidermis was significantly reduced in the expression level of Beclin-1, which was statistically significant compared to the control group, and the expression level of Beclin-1 in dermal tissue was below the detection limit. Increased Bcl-2 and decreased Beclin-1 expression levels are predictive of inhibition of autophagy in cells.
The experiment results show that when the epidermis of the sample molecule motor stock solution is smeared, compared with a control group, the relative expression quantity of two matrix metalloproteinase mRNAs in the epidermal tissue of the molecule motor group is respectively and obviously reduced, and the difference has statistical significance; decrease in expression level of MMP-1 and MMP-9 in dermal tissue. The mRNA expression level of Bax gene in the dermis or epidermis is obviously reduced compared with the control group; the expression level of Bcl-2 is obviously increased compared with that of the control group, and the statistical significance is achieved. Compared with a control group, the expression level of Beclin-1 in epidermis is obviously reduced, and the statistical significance is achieved;
therefore, the molecular motor prepared by the method has the effect of inhibiting the expression of MMP-1 and MMP-9 genes, has the effect of promoting the expression of growth factors GDF11 and TGF-beta 1, and has the tendency of inhibiting apoptosis from the viewpoint of anti-aging effect. Presents certain trends related to ultraviolet injury resistance, proliferation promotion, apoptosis resistance and aging resistance on epidermis and dermis tissues.
Example 4
In the embodiment, the in vitro anti-aging effect of the sample is tested according to the detection indexes of the relative length of the sample molecular motor to the telomere of the epidermis and the dermal cell, rDNA and mtDNA copy number.
An in vitro artificial full-thickness Skin model (3D Skin model) of T-Skin is selected, and half of the control and molecular motor sample groups are used for evaluating and researching the in vitro anti-aging effect of a detected sample, namely the molecular motor stock solution (POLARKI) prepared in example 1. Setting a control group and a molecular motor group in an experiment, applying the drug in an epidermal application mode, repeatedly applying the drug for 1 time every 24 hours, and detecting rDNA copy number and mtDNA standardized copy number of each group of skin models by real-time fluorescent quantitative PCR after 5 continuous days; detecting the relative length of telomeres by a double-color fluorescence quantitative PCR method; the test items of the control group and the molecular motor group are shown in Table 3.
TABLE 3 control and molecular Motor groups
Ribosomal dna (rdna) copy number: rDNA is the most abundant housekeeping gene in cells, is involved in ribosome assembly and protein synthesis, is one of the weakest regions of eukaryotic cell genome due to the characteristics of a repetitive sequence, and the reduction of the copy number of ribosomal DNA can influence the functions of cells and has a certain correlation with human aging.
Mitochondria are the factories of cellular energy production, the centers of free radical metabolism, and the gradual decline of the rate of ATP production by mitochondria is also a marker of cellular senescence. Mitochondrial function is closely related to the quantity and quality of mitochondrial dna (mtdna), and therefore mitochondrial dna (mtdna) copy number can be an important indicator of mitochondrial function. mtDNA copy number variation can cause mitochondrial dysfunction, mitochondrial productivity capacity is reduced, and finally cell functional decline is caused.
Referring to fig. 3, in the epidermal and dermal tissues, compared with the control group, the molecular motor group ribosomal DNA and mitochondrial DNA sample groups are all higher than the control group, and especially, the ribosome 28s, 18s and 5.8s are more than twice as high in the control group, which has certain biological significance.
The experimental results show that when the epidermis of the molecular motor stock solution subjected to the detection sample is smeared, compared with a control group, the molecular motor group rDNA28s, 18s and 5.8s have more than twice of the control group, and have biological significance.
Therefore, the molecular motor prepared by the method reduces rDNA copy number loss from the perspective of anti-aging effect. Presents certain trends related to ultraviolet injury resistance, proliferation promotion, apoptosis resistance and aging resistance of skin tissues.
Example 5
In the embodiment, the anti-aging effect of the sample molecular motor on the in vitro whole skin model is respectively detected from the aspects of several oxidative stress indexes of GSH, GSH-Px, SOD and MDA, and experiments are carried out;
a T-Skin in vitro artificial full-thickness Skin model (3D Skin model) is selected, and half of the control and molecular motor sample groups are used for evaluating and researching the in vitro anti-aging effect of a detected sample, namely the molecular motor stock solution prepared in the embodiment 1. The test is to set a control group and a molecular motor group, to administer the drug by adopting a mode of smearing epidermis, to repeatedly smear for 1 time every 24 hours, and to detect the relevant indexes of oxidative stress GSH, GSH-Px, SOD and MDA of each group of skin models by using a biochemical kit after 5 continuous days, wherein the detection items of the control group and the molecular motor group are shown in a table 4.
TABLE 4 mRNA expression test items for control group and molecular Motor group
The relative indexes of oxidative stress are detected through biochemical level, and the defense and elimination capacity of the sample to free radicals is evaluated.
Referring to fig. 4, in epidermal and dermal tissues, there was no significant difference in the four indices of molecular motor group GSH, GSH-PX, SOD, MDA compared to the control group. The results suggest that the sample did not cause oxidative damage to skin tissue.
The experimental result shows that when the molecular motor group is subjected to epidermal smearing of the molecular motor stock solution of the test sample, compared with the control group, the molecular motor group is detected by the oxidative stress related index, and no oxidative damage is observed.
Therefore, the molecular motor prepared by the method has multiple action paths to realize the effects of tightening and resisting aging, has no adverse effect related to oxidation, can inhibit the activities of MMP-1 and MMP-9, can promote the expression of growth factors GDF11 and TGF-beta 1, can inhibit the expression of Bax genes and promote the expression of Bcl-2, reduces the loss of rDNA copy number, and has certain effects of resisting ultraviolet injury, promoting proliferation, resisting apoptosis, resisting aging and making skin compact.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (13)
1. A method of manufacturing a molecular motor, comprising the steps of:
1) inoculating the cultured bacteria into a culture medium for secondary culture;
2) centrifugally collecting thalli from the bacteria obtained by re-culturing in the step 1), re-suspending the thalli by using a buffer solution A, and crushing for multiple times to obtain a cell crushing solution;
3) extracting and purifying the cell disruption solution obtained in the step 2) by a differential centrifugation method and a density gradient centrifugation method to obtain a molecular motor;
alternatively, the preparation method comprises the following steps:
a) taking the cultured bacteria, and centrifugally collecting somatic cells;
b) suspending the bacterial cells obtained in the step a) by using a buffer solution B;
c) adding PMSF, carrying out ice bath ultrasonic crushing, carrying out first centrifugation to obtain supernatant, carrying out second centrifugation on the supernatant, and removing the centrifuged supernatant to obtain the F-containing product1F0-a complex of atpases;
wherein, the bacteria in the steps 1) and a) are thermophilic bacteria.
2. The method of claim 1, wherein the thermophilic bacteria comprises one of thermophilic thermus bacteria, thermophilic roseococcus, thermophilic bacillus, thermophilic streptococcus, sulfolobus, marigold thermatopae and pyrococcus, and the inoculation ratio of the thermophilic bacteria is 1: 98-105.
3. The method according to claim 1 or 2, wherein in step 2), the buffer solution a comprises the following raw materials: 0.1-0.8mol/L PBS pH8.0, 0.1-20mmol/L MgCl2，0.1-5mmol/LDTT。
4. The method according to claim 1 or 2, wherein in step B), the buffer solution B comprises the following raw materials: 45-55mM Tricine-NaOH, pH7.8-8.2, 0.2-0.3M sucrose solution, 3-5mM MgCl2And (3) solution.
5. The method as claimed in claim 1 or 2, wherein the differential centrifugation method in step 3) is centrifugation at 600-; the density gradient centrifugation method comprises the following steps: adding the precipitate obtained by the differential centrifugation method into sucrose solutions with the mass percent concentration of 20 percent, 30 percent, 40 percent, 50 percent and 60 percent respectively, performing ultracentrifugation at the temperature of 1-6 ℃ for 60-90min by using 300,000 Xg at the temperature of 150,000, and collecting liquid in the region with the mass percent concentration of 40-50 percent of the sucrose solution.
6. The method for preparing a molecular motor according to claim 1 or 2, wherein in step c), the final concentration of PMSF is 1mmol/L, and the ultrasonication conditions are as follows: the amplitude is 40-60%, the ultrasonic treatment is carried out for 5-10s, the ultrasonic treatment is stopped for 6-10s, and the time is 25-35 min.
7. The method of claim 1 or 2, wherein in step c), the first centrifugation is performed at 20,000 Xg for 25-35min, and the second centrifugation is performed at 180,000 Xg for 85-95 min.
8. Use of a molecular motor prepared by the method according to any one of claims 1 to 7 in the cosmetic field.
9. Use of a molecular motor according to claim 8, characterized in that it is used in the preparation of a cosmetic for tightening anti-aging.
10. Use of a molecular motor according to claim 9, wherein the molecular motor inhibits MMP-1, MMP-9 activity.
11. The use of a molecular motor according to claim 9, wherein the molecular motor promotes the expression of the growth factors GDF11 and TGF- β 1.
12. The use of a molecular motor according to claim 9, wherein the molecular motor inhibits the expression of Bax gene and promotes the expression of Bcl-2.
13. The use of the molecular motor according to claim 9, wherein the molecular motor reduces rDNA copy number loss, exhibits certain anti-UV damage, pro-proliferative, anti-apoptotic, anti-aging and skin-tightening related effects on epidermal and dermal tissues.
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