CN112656696B - Composition of molecular motor lipid vesicles and preparation method and application thereof - Google Patents
Composition of molecular motor lipid vesicles and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a composition of molecular motor lipid vesicle for improving the solubility and the long-acting property of a molecular motor and promoting the absorption, a preparation method and application thereof, wherein the assembly structure of the molecular motor lipid vesicle is a closed structure or a cell compartment containing molecular motor ATP synthase and a lipid bilayer thereof, and the closed structure or the cell compartment is provided with biomembrane property and is also called vesicle, and the vesicle (niosolnes) is also called liposome in the past.
Description
Technical Field
The invention belongs to the field of biological and high molecular materials, and particularly relates to a composition of molecular motor lipid vesicles, a preparation method and application thereof, wherein the composition has the effects of improving the solubility and the long-acting property of a molecular motor and promoting the absorption of drugs or functional components.
Background
The molecular motor is a nano system which is composed of biological macromolecules and utilizes chemical energy to perform mechanical work. Generally, one class of ATP synthase proteins is the family of membrane protein complexes that catalyze the synthesis of ATP using an electrochemical gradient across the membrane, which drives ATP synthase to synthesize ATP. In the organism, small molecules of the breakdown products of sugars and fats are oxidized and broken down in mitochondria to release energy, which is stored for the most part in a molecule called Adenosine Triphosphate (ATP) to be used by the organism.
The molecular motor is also a group of transmembrane proteins which are usually embedded in mitochondria, chloroplasts and bacterial plasma membranes, and the biological membrane is the most basic structural and functional unit cell of organisms, takes a lipid bilayer closed vesicle as a basic framework structure, and has hydrophilic and hydrosulfuric properties, wherein all layers are separated by water. ATP synthase (molecular motor) is a transmembrane protein, and its content is generally low and it is unstable alone, easy to change structure and lose activity, and its structure has hydrophobic subunit, so water solubility is not high, thus affecting its use. Therefore, the preparation of lipid vesicles with lipid vesicles, i.e. with closed structures or cellular compartments with biofilm-like properties, usually at the molecular motor of ATP synthase, is advantageous for maintaining its activity and its use.
The molecular motor carried lipid bilayer membrane has been applied to the research of a carrier and a sensor used as a medicine, and document 1 ZL 200410098929.9 mentions an in vitro reconstructed molecular motor liposome as an adjustable molecular motor micropower biosensor.
The naturally-existing lipid molecular layer membrane has incomparable advantages of other drug carriers, for example, the erythrocyte membrane is applied to the drug carrier, and has good biocompatibility and biodegradability, so that the stability and the biological targeting property of the drug can be improved, the half-life period of the drug is prolonged, and the lipid molecular layer membrane has slow release and reduces the toxic and side effects of the drug. In addition, artificially constructed vesicular liposomes mainly comprising phospholipids and cholesterol are widely used.
However, the conventional bilayer membrane has poor mechanical properties, is easy to break, and has a very limited lifetime, which can be maintained for only a few hours (rarely exceeding 8 hours) at normal temperature. And the existing conditions are very harsh, so that the traditional bilayer is not developed in practical application. The artificial simple phospholipid bilayer liposome, also called an artificial membrane, is also unstable and has the defects of easy oxidation, leakage, poor storage property, easy aggregation and precipitation and the like in vitro; the application is limited because the medicine is easily degraded by some enzymes in the body and phagocytized by macrophages so as not to reach target tissues to exert effective action and the like.
Disclosure of Invention
The invention aims to provide a molecular motor lipid vesicle composition which can improve the stability and the antioxidant property of a lipid membrane so as to improve the practical application performance in order to overcome the defects of poor mechanical property, easy breakage and limited service life of the traditional bilayer membrane.
The second aspect of the invention is to provide a preparation method of the composition of the molecular motor lipid vesicle.
The third aspect of the invention also provides the application of the molecular motor lipid vesicle prepared by the method in the field of cosmetics.
In order to achieve the purpose, the invention adopts the following technical scheme:
a molecular motor lipid vesicle composition comprising molecular motor lipid bodies having bilayers, a charged polymer, a biological growth factor and an antioxidant enzyme;
wherein, the polymer with charge, the biological growth factor and the antioxidant enzyme are self-assembled and embedded into a bilayer liposome by utilizing the reconstruction of a biological membrane, and the liposome is embedded with a molecular motor.
In the invention, the assembly structure of the recombinant molecular motor lipid vesicle is a closed structure or a cell compartment containing molecular motor ATP synthase and a lipid bilayer thereof, has biomembrane properties, and is also called as a vesicle, wherein the vesicle (niosolnes) is also called as a lipoid, refers to a drug-carrying system which is composed of nonionic surfactants (with or without cholesterol) and has extremely similar in vivo and in vitro properties with the liposome, and avoids the defects of purification, easy oxidative degradation and the like of the liposome adopting phospholipid.
The biological membrane obtained by the invention is complete or fragment, has a lipid bilayer structure in form, mainly comprises lipid, proteins such as F0F 1-ATPase, SOD (thermophilic SOD), charged polymers, biological growth factors and the like, and a small amount of saccharides are combined on the lipid or the proteins through covalent bonds.
In the invention, the charged polymer is distributed on the surface of the bilayer structure, and the molecular motor lipid vesicle composition can be far away from each other by the principle that like charges repel each other, so that the problem of forming precipitates due to agglomeration is avoided;
a thermophilic SOD is embedded in the surface of the bilayer structure, and the SOD is used as a superoxide dismutase for preventing the molecular motor lipid vesicle composition from being oxidized;
the biological growth factor can provide a targeting effect on skin cells, promote skin wounds and improve the activity of epidermal cells.
According to one embodiment of the invention, the charged polyamino acid is a negatively charged amino acid polymer; the amino acid polymer comprises one of polyglutamic acid or polyaspartic acid.
According to one embodiment of the invention, the charged polyamino acid is a positively charged amino acid polymer comprising one of polyhistidine, polylysine or polyarginine.
According to an embodiment of the present invention, the polymer with a charge is one of polysialic acid, hyaluronic acid, chondroitin sulfate, heparan sulfate, chitosan, which have a negative charge.
According to one embodiment of the invention, the biological growth factor is NGF or KGF.
According to one embodiment of the present invention, the antioxidant enzyme is SOD produced by thermophilic bacteria.
According to an embodiment of the present invention, the thermophilic bacteria includes one of thermophilic thermus, streptococcus thermophilus, bacillus stearothermophilus, streptococcus thermophilus, thermophilic sporotrichum and streptomyces thermophilus.
More preferably, the present invention utilizes biofilm reconstitution self-assembly to intercalate amino acids, biological growth factors and SOD.
Most preferably, the present invention embeds polyglutamic acid, biological growth factors and SOD using biofilm reconstitution self-assembly.
According to one embodiment of the invention, the biological growth factor is NGF or KGF.
More preferably, the biological growth factor of the present invention is selected for KGF-2.
A preparation method of the molecular motor lipid vesicle composition comprises the following steps:
1) Collecting thalli;
2) And (3) crushing thalli: crushing the thalli obtained in the step 1) to obtain cell crushing liquid;
3) Preparing heat-resistant SOD: centrifuging the cell disruption solution obtained in the step 2) to obtain a precipitate A and a supernatant B; forming a suspension solution of the precipitate A by using a buffer solution B, performing enzymolysis reaction, centrifuging to obtain a supernatant solution, and freeze-drying to obtain SOD powder;
4) Preparation of F0F 1-ATPase molecule motor liposome corpuscle biomembrane: centrifuging the supernatant B obtained in the step 3) for multiple times to obtain a precipitate B, and preparing to obtain an F0F1-ATP enzyme molecular motor liposome corpuscle biomembrane;
5) Preparation of self-assembled film insertion: dissolving a polymer with charges, biological growth factors and SOD powder obtained in the step 3) in anhydrous carbamyl, adding imine, magnetically stirring in an ice bath, dissolving octadecylamine in DMF, slowly dropping into the solution, charging nitrogen, and reacting; after the reaction is finished, excess water-alcohol mixed liquor and deionized water are used for dialysis in sequence, and a water system membrane is used for filtration and drying to obtain a compound;
6) Preparing a recombined molecular motor film: and (3) matching the F0F 1-ATPase molecular motor liposome biological membrane obtained in the step 4) with the compound obtained in the step 5), dissolving the compound in chloroform, adding PBS solution with the same volume, incubating in a water bath, and removing chloroform under reduced pressure to obtain the recombinant molecular motor membrane.
According to the inventionIn a preferred embodiment, the buffer solution a comprises the following raw materials: 1-80mM tris. HCl,0.1-5.0mM beta-mercaptoethanol, 0.l-2mM EDTA, 0.1-0.5M sucrose and 1-10mM MgCl 2 (ii) a The buffer solution B comprises the following raw materials: 1-20mM tris. HCl,0.1-5.0mM beta-mercaptoethanol and 0.1-2mM EDTA.
An application of the molecular motor lipid vesicle composition in the cosmetic field.
After the molecular motor is assembled, the recombinant molecular motor membrane is improved as follows:
1) The molecular motor improves the stability of the molecular motor under the action of the liposome, and the structure can synthesize ATP in an extracellular environment to provide energy for cells and improve the cell activity;
2) SOD (thermophilic SOD), polyglutamic acid: the antioxidant capacity of the lipid vesicle is improved, and lipid peroxidation is prevented;
3) Polyglutamic acid has large negative rational electricity, and like charges repel each other, so that lipid coagulation and precipitation are prevented;
4) KGF is a keratinocyte growth factor, can provide a targeting effect on skin cells, can promote skin wounds and improve the activity of epidermal cells.
The method comprises the following steps of (1) reconstructing a natural molecular motor liposome double-layered membrane by using SOD, polyglutamic acid and KGF to obtain a more stable and skin-targeted molecular motor biological membrane for skin application, wherein the specific method comprises the following steps: the molecular motor liposome with the bilayer is prepared by breaking the wall of biological bacteria, and KGF, SOD (thermophilic SOD) and polyglutamic acid are embedded by utilizing the characteristic of biofilm reconstruction self-assembly to obtain the stable, efficient and targeted recombinant molecular motor liposome vesicle.
Compared with the prior art, the invention has the following beneficial effects:
the reconstructed self-assembled molecular motor vesicle comprises ATP synthetase, antioxidant SOD, targeted epidermal growth factor KGF and anti-agglomeration efficient humectant polyglutamic acid (the moisture retention capacity is 4 times of that of hyaluronic acid) with negative charge on a lipid membrane, and has the characteristics of providing energy for cells to improve fine and smooth activity, promoting cell growth and wound healing in a targeting manner, efficiently preserving moisture, stabilizing products and the like.
Drawings
Fig. 1 is a schematic diagram of a molecular motor lipid vesicle composition of the invention.
In the figure, 1. Molecular motor liposome; 2. a biological growth factor; 3. an antioxidant enzyme; 4. a polymer having an electrical charge.
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.
The reagents used in the present invention are commercially available, and the equipment used is also commercially available.
Referring to fig. 1, the present invention provides a molecular motor lipid vesicle composition comprising a molecular motor liposome 1 having a bilayer, a charged polymer 4, a biological growth factor 2 and an antioxidant enzyme 3;
wherein, the polymer with charge, the biological growth factor and the antioxidant enzyme are self-assembled and embedded into a bilayer liposome by utilizing the reconstruction of a biological membrane, and the liposome is embedded with a molecular motor.
The invention also provides a preparation method of the molecular motor lipid vesicle, which comprises the following steps:
1) Collecting thalli;
the thallus used in the step 1) comprises one of thermophilic thermus bacteria, streptococcus thermophilus, bacillus stearothermophilus, streptococcus lactis, thermophilic sporotrichum and streptomyces thermophilus.
Preferably, the thermophilic bacterium is a thermophilic thermus.
2) And (3) crushing thalli: crushing the thalli obtained in the step 1) to obtain cell crushing liquid;
the cell disruption used in step 2) may be performed by a mechanical method, for example: high-pressure homogenizing crushing, oscillating bead crushing, high-speed stirring bead grinding crushing, ultrasonic crushing and impact crushing; or by non-mechanical methods such as osmotic shock fragmentation, freeze-thaw fragmentation, enzymatic fragmentation, and chemical fragmentation.
In the invention, in order to improve the antioxidant capacity of the lipid vesicle and prevent lipid peroxidation, superoxide dismutase (SOD) is adopted.
3) Preparing heat-resistant SOD: centrifuging the cell disruption solution obtained in the step 2) to obtain a precipitate A and a supernatant B; forming a suspension solution of the precipitate A by using a buffer solution B, reacting, centrifuging to obtain a supernatant solution, and freeze-drying to obtain SOD powder;
4) Preparation of F0F 1-atpase molecular motor liposome corpuscle biofilm: centrifuging the supernatant B obtained in the step 3) for multiple times to obtain a precipitate B, and preparing to obtain an F0F1-ATP enzyme molecular motor liposome corpuscle biomembrane;
5) Preparation of self-assembled film insertion: dissolving the charged polymer, biological growth factors and the SOD powder obtained in the step 3) in anhydrous carbamyl, adding imine, magnetically stirring in ice bath, dissolving octadecylamine in DMF, slowly dropping into the solution, charging nitrogen, and reacting; after the reaction is finished, excessive water-alcohol mixed solution and deionized water are used for dialysis in sequence, and water system membrane filtration and drying are carried out to obtain a compound;
according to one embodiment of the invention, the charged polyamino acid is a negatively charged amino acid polymer; the amino acid polymer comprises one of polyglutamic acid or polyaspartic acid.
According to one embodiment of the invention, the charged polyamino acid is a positively charged amino acid polymer comprising one of polyhistidine, polylysine or polyarginine.
According to an embodiment of the present invention, the polymer with charge is one of polysialic acid, hyaluronic acid, chondroitin sulfate, heparan sulfate, chitosan with negative charge.
More preferably, the charged polymer is polyglutamic acid.
According to one embodiment of the invention, the biological growth factor is NGF or KGF.
More preferably, the biological growth factor of the present invention is selected for KGF-2.
According to one embodiment of the present invention, the antioxidant enzyme is SOD produced by thermophilic bacteria.
According to an embodiment of the present invention, the thermophilic bacteria includes one of thermophilic thermus, streptococcus thermophilus, bacillus stearothermophilus, streptococcus thermophilus, thermophilic sporotrichum and streptomyces thermophilus.
6) Preparing a recombined molecular motor film: and (3) matching the F0F1-ATP enzyme molecular motor liposome corpuscle biomembrane obtained in the step (4) with the compound obtained in the step (5), dissolving in chloroform, adding PBS solution with the same volume, incubating in a rotary evaporator water bath, and decompressing to remove chloroform to obtain the recombined molecular motor membrane.
The molecular motor biomembrane has a lipid bilayer structure, so that the molecular motor biomembrane has the effects of moisturizing, whitening and resisting aging when being applied to the field of cosmetics, and achieves the effects of improving stability, solubility and durability and promoting absorption.
Example 1
The embodiment provides a preparation method of a molecular motor lipid vesicle, which comprises the following steps:
1) And (3) collecting thalli: inoculating streptococcus thermophilus into a culture medium, culturing at 75 ℃ to the late logarithmic phase, and collecting thalli;
2) And (3) crushing thallus: freezing and storing the thallus obtained in step 1) at-80 deg.C, adding 500g thallus into 1L buffer solution (80mM tris. HCl, PH8.0, containing 1.0mM beta-mercaptoethanol, 0.lmM EDTA 0.25M sucrose, 4mM MgCl 2 ) By ultrasonic waveTreating the bacterial liquid to achieve crushing;
3) Centrifuging, preparing heat-resistant SOD: the cell disruption solution was centrifuged at high speed (20000 g) for 30min to obtain a precipitate A and a supernatant B. Precipitate A was treated with 500mL of a buffer (15mM tris. HCl, PH7.6, containing 1.0 mM. Beta. -mercaptoethanol, 0.lmM EDTA) to which was added 20M MgCl 2 8mL of the solution and 10mg of DNaseI are placed at room temperature for reaction for 30 minutes, DNase is used for decomposing DNA, and then centrifugation is carried out at 8500rpm to obtain supernatant (namely SOD solution), and the supernatant is freeze-dried to obtain SOD;
4) Molecular motor liposome biofilm preparation: preparing by differential centrifugation, collecting supernatant B, centrifuging at 8500rpm at 4 deg.C, collecting supernatant C, and centrifuging at 180000 Xg and 4 deg.C for 90min at ultra-high speed; taking the lower precipitate, diluting with 10 times of 0.1M PBS buffer solution, centrifuging at 50000 Xg and 4 deg.C with Percoll supernatant, and sucking the lower layer, which is F0F1-ATP enzyme molecule motor liposome corpuscle biomembrane;
5) Preparing a self-assembly film material insertion material: dissolving the SOD powder (20mg), KGF-2 mu g and polyglutamic acid (100 mg) obtained in the step 3) in 10mL of anhydrous methionyl, adding 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 80mg of N-hydroxysuccinimide, magnetically stirring for 2 hours in an ice bath, dissolving 72mg of octadecylamine in 5mLDMF, slowly dropping the solution into the polyglutamic acid solution, filling nitrogen, reacting at 30 ℃ for 28H, after the reaction is finished, filling into a dialysis bag (molecular weight cut-off is 3500), dialyzing excessive water-alcohol mixed solution for 1 day in sequence, dialyzing with deionized water for 1 day, filtering with a water system membrane with the diameter of 0.45 mu m, and drying to obtain a polyglutamic acid compound;
6) Preparing a recombined molecular motor film: reassembling the molecular motor biomembrane by adopting a reverse phase evaporation method, and mixing the molecular motor biomembrane obtained in the step 4) with the polyglutamic acid compound 2 obtained in the step 5): 1, dissolving in chloroform with 4 times of volume, adding PBS solution with the same volume, incubating in a water bath for 20min at 50 ℃ and 80rpm on a rotary evaporator, and removing chloroform under reduced pressure to obtain the recombinant molecular motor membrane.
Example 2
This example is identical to example 1, with the only difference that polylysine is used instead of polyglutamic acid.
Comparative example 1
The liposome is prepared by using a conventional method, wherein the mass ratio of phospholipid to cholesterol is 5.
The molecular motor liposomes of examples 1-3 were tested against the liposomes of comparative example 1 and the results are shown in table 1.
TABLE 1 test results
As can be seen from table 1, the molecular motor liposomes prepared in examples 1 to 3 of the present invention have better stability and antioxidant property than the liposomes of comparative example 1, because the molecular motor improves the stability of the molecular motor under the action of the liposomes, and the structure can synthesize ATP in the extracellular environment to provide energy for cells and improve cell viability, SOD (thermophilic SOD), polyglutamic acid: the biological membrane containing the molecular motor has a lipid bilayer structure, so that the biological membrane has the effects of moisturizing, whitening and resisting aging when being applied to the field of cosmetics, and achieves the effects of improving stability, improving solubility and long-acting property of the molecular motor and promoting absorption.
Example 4
Cosmetic additive benefit (moisture)
(1) Randomly selecting ten people, selecting a position of 10cm multiplied by 10cm from the center of the back of the left hand and the right hand of each person, smearing the recombined molecular motor membrane obtained in the embodiment 1 and re-dissolved in PBS with the volume of 500 mu L by the hand of any side, and smearing PBS with the same volume as the reference on the hand of the person;
(2) After the hands are continuously smeared at the same positions for two weeks, the water content of the skin at two sides is tested by a skin moisture tester;
(3) The results show that the average skin moisture content of the back of the hand coated with the recombinant molecular motor film is 51%, the average skin moisture content of the back of the hand coated with PBS is 43%, and the recombinant molecular motor film can effectively moisturize the skin as a cosmetic additive.
Example 5
Cosmetic additive beneficial effects (whitening)
(1) Taking 0.5 mmol/L L-DOPA as a substrate, in a activity detection system of 1mL 0.05mol/L phosphate buffer solution, firstly adding 20 mu L of the recombined molecular motor membrane (dissolved in DMSO solution) obtained in the embodiment 1 into a 1.5mL pear-shaped tube, then adding 400 mu L of the substrate solution which is preserved in a constant-temperature water bath at 30 ℃ in advance, supplementing the volume to 970 mu L by using a buffer solution, then adding 30 mu L of tyrosinase aqueous solution, immediately mixing uniformly, taking PBS as a control, measuring a linear increase of an absorbance value with time when the wavelength is 475nm within 1min at the constant temperature at 30 ℃, and obtaining the enzyme activity from the linear slope;
(2) The result shows that the inhibition activity of the recombined molecular motor film on the tyrosinase is 39%, the inhibition activity of the tyrosinase of the control group is almost not obvious, and the recombined molecular motor film as a cosmetic additive can effectively promote the whitening.
Example 6
Cosmetic additive benefit (anti-aging)
(1) Randomly selecting ten people in the age range of forty-five to fifty-five, selecting a position of 10cm multiplied by 10cm from the middle of the back of the left hand and the right hand of each person, smearing the recombinant molecular motor membrane obtained in the embodiment 1 and re-dissolved in PBS with the volume of 500 mu L by the hand of any one side, and smearing PBS with the same volume as the control;
(2) After the skin is continuously smeared at the same positions of two hands for four weeks, the anti-aging conditions of the skins at two sides are tested by a skin elasticity tester;
(3) The results show that the average skin elasticity of the back of the hand coated with the recombinant molecular motor film is 35%, the average skin elasticity of the back of the hand coated with PBS is 27%, and the recombinant molecular motor film can effectively resist aging as a cosmetic additive.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (3)
1. A molecular motor lipid vesicle composition, which comprises a molecular motor lipid body with a bilayer, polyglutamic acid, a biological growth factor KGF and an antioxidant enzyme, wherein the antioxidant enzyme is SOD prepared from thermophilic bacteria;
wherein, polyglutamic acid with negative charge, biological growth factor KGF and antioxidase are self-assembled and embedded into a bilayer liposome by utilizing biomembrane reconstruction, and a molecular motor is embedded into the liposome;
the preparation method of the molecular motor lipid vesicle composition comprises the following steps:
1) Collecting thalli, wherein the thalli is streptococcus thermophilus;
2) And (3) crushing thalli: putting the thalli obtained in the step 1) into a buffer solution A, and crushing to obtain a cell crushing solution;
3) Preparing SOD: centrifuging the cell disruption solution obtained in the step 2) to obtain a precipitate A and a supernatant B; the precipitate A is suspended in buffer B and MgCl is added 2 Reacting the solution with DNaseI, centrifuging to obtain a supernatant solution, and freeze-drying to obtain SOD powder;
4) Preparation of F0F 1-atpase molecular motor liposome corpuscle biofilm: centrifuging the supernatant B obtained in the step 3) for multiple times to obtain a precipitate B, and preparing to obtain an F0F1-ATP enzyme molecular motor liposome corpuscle biomembrane;
5) Preparation of self-assembled film insertion: dissolving polyglutamic acid with negative charges, biological growth factor KGF and SOD powder obtained in the step 3) in anhydrous methyl aminoacyl, adding imine, magnetically stirring in an ice bath, dissolving octadecylamine in DMF, slowly dropping into the solution, filling nitrogen, and reacting; after the reaction is finished, excess water-alcohol mixed liquor and deionized water are used for dialysis in sequence, and a water system membrane is used for filtration and drying to obtain a compound;
6) Preparing a recombined molecular motor film: and (3) matching the F0F 1-ATPase molecular motor liposome biological membrane obtained in the step 4) with the compound obtained in the step 5), dissolving the compound in chloroform, adding PBS solution with the same volume, incubating in a water bath, and removing chloroform under reduced pressure to obtain the recombinant molecular motor membrane.
2. The molecular motor lipid vesicle composition of claim 1, wherein the buffer solution a comprises the following raw materials: 1-80mM tris. HCl,0.1-5.0mM beta-mercaptoethanol, 0.l-2mM EDTA, 0.1-0.5M sucrose and 1-10mM MgCl 2 (ii) a The buffer solution B comprises the following raw materials: 1-20mM tris. HCl,0.1-5.0mM beta-mercaptoethanol and 0.1-2mM EDTA.
3. Use of a molecular motor lipid vesicle composition according to any of claims 1-2 for the preparation of a cosmetic product.
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