Recombinant human elastin and its preparation method and application

Abstract

The present invention relates to a recombinant human elastin protein, and its preparation method and application. The recombinant human elastin protein comprises one or more elastin domains in the amino acid sequence of the full-length human elastin protein as shown in SEQ ID NO.1, as well as one or more of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. The amino acid sequence of the recombinant human elastin protein is shown in SEQ ID NO.2. The nucleotide sequence is a recombinant plasmid, a recombinant vector, or a recombinant bacterium. The preparation method comprises: synthesizing the nucleotide sequence as shown in SEQ ID NO.1, constructing a recombinant protein, inducing expression, centrifuging, purifying, and drying to obtain the recombinant human elastin protein as shown in SEQ ID NO.2. The application is to detect and evaluate the anti-aging, anti-wrinkle, and firming effects of the recombinant human elastin protein, as well as in cosmetics. The preparation method of the present invention is simple, the protein expression level is high, and the cosmetic product has anti-aging, anti-wrinkle, and firming effects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a preparation method of recombinant human elastin and its application. Background Art

[0002] Elastin is a crucial extracellular matrix protein in the human body, second only to collagen in abundance. Elastin not only imparts elasticity to tissues but also plays a crucial role in regulating various cellular functions, including cell adhesion, proliferation, differentiation, chemotaxis, and migration. Elastin has garnered significant attention in the fields of medical aesthetics and skincare.

[0003] Currently, elastin is primarily produced through animal tissue extraction and genetic recombination. However, animal tissue-derived elastin is prone to excessive degradation, disrupting the structure and function of natural elastin. It also poses challenges such as immunogenicity and the risk of disease transmission. Genetic recombination-based elastin production offers advantages such as low cost and ease of scalable production, and holds great promise for future development.

[0004] To this end, the present invention provides a method for preparing recombinant human elastin and an example of its application in cosmetics. The amino acid sequence of the recombinant human elastin is entirely derived from human elastin and has a secondary structure and properties similar to those of natural elastin. Furthermore, by optimizing and standardizing the methods and means for testing the anti-aging, anti-wrinkle and firming efficacy of cosmetic raw materials, the recombinant human elastin prepared by the present invention is stabilized in quality and determined in efficacy. Adding an appropriate amount of recombinant human elastin to cosmetics can address problems such as skin sagging, decreased skin elasticity and wrinkles, achieving anti-aging, anti-wrinkle and firming efficacy, repairing aging skin and keeping the skin youthful and healthy. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art by disclosing a method for preparing recombinant human elastin and its application. Specifically, the present invention discloses a method for preparing recombinant human elastin with anti-aging, anti-wrinkle, and firming properties, and its application in the cosmetics field. The method comprises: a method for preparing recombinant human elastin with anti-aging, anti-wrinkle, and firming properties; and testing methods using cell-based assays such as human fibroblast proliferation assays, human fibroblast migration assays, and human fibroblast extracellular matrix (ECM) gene expression assays. The recombinant human elastin produced by the present invention is screened for efficacy and optimized for efficacy testing to ensure the quality and activity of the resulting recombinant human elastin. The recombinant human elastin with anti-aging, anti-wrinkle and firming effects prepared by the present invention is highly active, non-toxic and non-irritating, has significant anti-aging, anti-wrinkle and firming effects, and can significantly promote cell proliferation; the present invention also proposes a method for using the recombinant human elastin in cosmetics. Adding an appropriate amount of the recombinant human elastin with anti-aging, anti-wrinkle and firming effects to cosmetics can increase skin elasticity, effectively solve problems such as skin sagging, decreased skin elasticity and wrinkles, and achieve anti-aging, anti-wrinkle and firming effects.

[0006] The purpose of the present invention and the technical problems solved are achieved by the following technical solutions: According to the present invention, a recombinant human elastin protein comprises one or more elastin domains in the amino acid sequence of the full-length human elastin protein as shown in SEQ ID NO.1.

[0007] The purpose of the present invention and the solution to its technical problems can be further achieved by adopting the following technical measures.

[0008] Preferably, the recombinant human elastin consists of one or more elastin domains in the amino acid sequence of human full-length elastin as shown in SEQ ID NO.1, and one or more of a secretion tag, a histidine tag, a fluorescent protein and a protease cleavage site.

[0009] Preferably, the amino acid sequence of the recombinant human elastin is as shown in SEQ ID NO.2.

[0010] Preferably, the nucleic acid is a recombinant plasmid, a recombinant vector or a recombinant bacterium.

[0011] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0012]

[0013]

[0014] The amino acid sequence of the recombinant human elastin as shown in SEQ ID NO.2 is as follows:

[0015] SPEAQAAAAAKAAKYGAAGAGVLGGLVPGAPGAVPGVPGTGGVPGVGTPAAAAAKAAAKAAQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGVAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAG ADEGVRRSLSPELREGDPSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK.

[0016] The objectives of the present invention and the technical problems solved therein are also achieved by the following technical solutions. According to the present invention, a method for preparing the recombinant human elastin as described above is provided, the method comprising the following steps: S1: synthesizing a nucleotide sequence encoding the recombinant human elastin as shown in SEQ ID NO.1; S2: inserting the nucleotide sequence as shown in SEQ ID NO.1 into a pET28a plasmid to construct a recombinant vector, and introducing the nucleotide sequence into Escherichia coli BL21 to screen and obtain recombinant engineered bacteria; S3: transferring the engineered bacteria into LB medium, expanding the culture, and inducing expression by adding IPTG; S4: collecting the bacterial precipitate by centrifugation, dispersing it with a nickel ion affinity chromatography column eluent, homogenizing it multiple times with a high-pressure homogenizer to fully disrupt the bacterial cells, centrifuging the disrupted liquid, and collecting the supernatant; S5: transferring the obtained supernatant to a nickel ion affinity chromatography column to purify the elastin solution; S6: removing imidazole from the solution by dialysis, and freeze-drying the solution to obtain the recombinant human elastin as shown in SEQ ID NO.2.

[0017] Preferably, the conditions for the expanded culture in step S3 are 34-45°C for 10-12h; the conditions for adding IPTG are when OD 600When the concentration reaches 0.8-1.2, IPTG is added at a final concentration of 1 mM; the expression is induced at 25°C for 12 hours; the conditions for centrifuging the bacterial precipitate in step S4 are 1500-5000 rps, 0-8°C, and 15-45 minutes to collect the bacterial precipitate; the conditions for multiple homogenization with a high-pressure homogenizer are 800 bar, 4°C, and 40 Hz for 2-3 times; the conditions for centrifuging the broken liquid are 8000 rps, 4°C, and 20 minutes; the dialysis method in step S6 is dialyzed using 0.02M PBS.

[0018] Preferably, the optimal temperature for the expanded culture in step S3 is 37°C.

[0019] Preferably, the optimal conditions for centrifugation to collect bacterial precipitate in step S4 are 3000 rps, 4°C, and 30 min.

[0020] The purpose of the present invention and the technical problems solved therein are achieved by adopting the following technical solutions: According to the present invention, the above-mentioned recombinant human elastin is used to detect and evaluate the anti-aging, anti-wrinkle and firming effects of the recombinant human elastin.

[0021] Preferably, the application includes but is not limited to one or more combinations of cell experiments such as human fibroblast proliferation assay, human fibroblast migration assay, and expression of human fibroblast extracellular matrix (ECM)-related genes.

[0022] The purpose of the present invention and the technical problems solved therein are further achieved by adopting the following technical solutions: The present invention proposes an application of the above-mentioned recombinant human elastin in cosmetics.

[0023] Preferably, the application in cosmetics is the application of the recombinant human elastin as shown in SEQ ID NO. 2 as any one of the additives for anti-wrinkle and firming essence, anti-wrinkle and firming lotion, anti-wrinkle and firming toner, anti-wrinkle and firming cream, and anti-wrinkle and firming mask.

[0024] Furthermore, cosmetics can be prepared by the preparation method of the present invention, wherein the additive of the cosmetics includes the above-mentioned SEQ ID NO. 2.

[0025] Compared with the prior art, the present invention has obvious advantages and beneficial effects. By means of the above technical solution, compared with the prior art, the present invention has at least the following advantages and effects:

[0026] First, the amino acid sequence of this recombinant human elastin with anti-aging, anti-wrinkle and firming effects is completely derived from human elastin, and has a secondary structure and properties similar to natural elastin.

[0027] Second, the recombinant human elastin with anti-aging, anti-wrinkle and firming effects is efficiently expressed in E. coli, making it easy to achieve large-scale production and obtaining a high-purity protein. The invention has low technical difficulty, a simple preparation method, energy-saving and labor-saving properties, and high production efficiency.

[0028] Third, the recombinant human elastin with anti-aging, anti-wrinkle and firming effects is highly active, has significant anti-aging, anti-wrinkle and firming effects, and can significantly promote cell proliferation and migration. Adding this compound to cosmetics can increase skin elasticity, effectively solve problems such as skin sagging, decreased skin elasticity and wrinkles, and achieve anti-aging, anti-wrinkle and firming effects.

[0029] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following preferred embodiments are specifically cited and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the effect of the recombinant human elastin of Examples 1-3 of the present invention and Comparative Examples 1-3 on the proliferation of HSF cells.

[0031] Figure 2 This is the effect of the recombinant human elastin of Examples 1-3 of the present invention and Comparative Examples 1-3 on the migration ability of HSF cells.

[0032] Figure 3 This is the effect of the recombinant human elastin of Examples 1-3 of the present invention and Comparative Examples 1-3 on the expression level of Elastin gene in the extracellular matrix.

[0033] Figure 4 This is the effect of the recombinant human elastin of Examples 1-3 of the present invention and Comparative Examples 1-3 on the expression of extracellular matrix MMP 3 genes.

[0034] Figure 5 This is the effect of the recombinant human elastin of Examples 1-3 of the present invention and Comparative Examples 1-3 on the expression level of the extracellular matrix MMP 9 gene. DETAILED DESCRIPTION

[0035] To further illustrate the technical means and effects of the present invention to achieve the intended purpose, the following detailed description of the preparation method of recombinant human elastin and its application, features, and effects is provided below in conjunction with the accompanying drawings and preferred embodiments.

[0036] The following examples illustrate the present invention, but the present invention is not limited to these examples. Modifications to the specific embodiments of the present invention or equivalent replacements of some technical features without departing from the spirit of the present invention should be included in the scope of the technical solution claimed in the present invention.

[0037] In this example, no specific techniques or conditions are specified, and the operations were performed according to conventional techniques and instrument specifications in the art. All reagents or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0038] The present invention provides a recombinant human elastin protein, which comprises one or more elastin domains in the amino acid sequence of the full-length human elastin protein as shown in SEQ ID NO.1.

[0039] The recombinant human elastin is composed of one or more elastin domains in the amino acid sequence of the full-length human elastin as shown in SEQ ID NO. 1, and one or more of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site.

[0040] The amino acid sequence of the recombinant human elastin is shown in SEQ ID NO.2.

[0041] The nucleic acid is a recombinant plasmid, a recombinant vector or a recombinant bacterium.

[0042] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0043]

[0044] The amino acid sequence of the recombinant human elastin as shown in SEQ ID NO.2 is as follows:

[0045] SPEAQAAAAAKAAKYGAAGAGVLGGLVPGAPGAVPGVPGTGGVPGVGTPAAAAAKAAAKAAQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGVAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAG ADEGVRRSLSPELREGDPSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK.

[0046] Example 1

[0047] This embodiment provides a method for preparing recombinant human elastin with anti-aging, anti-wrinkle and firming effects, comprising the following steps:

[0048] 1. A method for preparing recombinant human elastin with anti-aging and anti-wrinkle effects, comprising the following key steps:

[0049] S1. synthesizing a nucleotide sequence encoding the recombinant human elastin;

[0050] S2. The above nucleotide sequence was inserted into the pET28a plasmid to construct a recombinant vector, which was then introduced into Escherichia coli BL21 to screen for recombinant engineered bacteria;

[0051] S3. Transfer the engineered bacteria into LB medium and culture at 37℃ for 10-12h. 600 When the concentration reached 0.8-1.2, IPTG was added to a final concentration of 1 mM and the expression was induced at 25°C for 12 h;

[0052] The bacterial precipitate was collected by centrifugation at S4.3000 rps, 4°C, 30 min, and dispersed with the eluate from the nickel ion affinity chromatography column. The cells were homogenized 2-3 times with a high-pressure homogenizer at 800 bar, 4°C, 40 Hz to fully disrupt the cells. The supernatant was collected by centrifugation at 8000 rps, 4°C, 20 min.

[0053] S5. The obtained supernatant is transferred to a nickel ion affinity chromatography column to purify the elastin solution;

[0054] S6. Remove imidazole from the solution by dialysis with 0.02 M PBS, and freeze-dry to obtain recombinant human elastin.

[0055] 2. Conduct efficacy tests on the recombinant human elastin for anti-aging, anti-wrinkle and firming effects;

[0056] Specifically, appropriate amounts of recombinant human elastin were used to conduct cell experiments such as human fibroblast proliferation assay, human fibroblast migration assay, and human fibroblast extracellular matrix (ECM)-related gene expression assay to detect and evaluate the anti-aging, anti-wrinkle and firming effects of the recombinant human elastin.

[0057] 3. Analyze the experimental data and select compositions that have no precipitation, no flocs, no odor, and outstanding anti-aging, anti-wrinkle and firming effects. Add them to basic formulas such as cosmetic essences, essences, and essence creams to produce recombinant human elastin with anti-wrinkle and firming effects. This can be used as an additive in any of the cosmetic anti-wrinkle and firming essences, anti-wrinkle and firming lotions, anti-wrinkle and firming toners, anti-wrinkle and firming creams, and anti-wrinkle and firming masks.

[0058] Example 2

[0059] The difference between this embodiment and embodiment 1 is that in S3, the engineered bacteria are transferred into TB culture medium for expansion and culture. The other parts are the same as those in embodiment 1.

[0060] Example 3

[0061] The difference between this embodiment and embodiment 1 is that, in S3, the engineered bacteria are transferred to SB culture medium for expansion and culture. The other parts are the same as those in embodiment 1.

[0062] Comparative Example 1

[0063] The difference between this comparative example and Example 1 is that in S3, the final concentration of IPTG is 1.2 mM, and the other parts are the same as in Example 1.

[0064] Comparative Example 2

[0065] The difference between this example and Example 1 is that in S3, the final concentration of IPTG is 0.8 mM, and the other parts are the same as Example 1.

[0066] Comparative Example 3

[0067] The difference between this example and Example 1 is that in S3, the final concentration of IPTG is 0.6 mM, and the other parts are the same as in Example 1.

[0068] The following efficacy tests were performed on the compositions in Examples 1-3 and Comparative Examples 1-3:

[0069] 1. Recombinant human elastin HSF cell proliferation experiment

[0070] Experimental method: The recombinant human elastin prepared in Examples 1-3 and Comparative Examples 1-3 was diluted to 10% (v / v) with DMEM basal medium as the test solution. HSF cells in the logarithmic growth phase were cultured at 1×10 5 cells / mL, 100 μL / well were inoculated into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 24 h; the old culture medium was discarded, and the cells were washed once with PBS solution. Then, 100 μL of the test solution was added to the cell solution in each well. Fetal bovine serum (10% FBS) was added to the positive control group, and an equal volume of DMEM basal medium was added to the blank group. The cells were cultured at 37°C for 48 h; 10 μL of CCK8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 h; the absorbance was measured at 450 nm with 630 nm as the reference wavelength, and the measurement results were recorded. The specific test results are shown in the table. Figure 3 .

[0071] Wherein: the absorbance of the sample group is As, 450nm - As, 630nm; the absorbance of the blank group is A0, 450nm - A0, 630nm.

[0072] Depend on Figure 1 It can be seen that the recombinant human elastin prepared in Examples 1-3 and Comparative Examples 1-3 at a concentration of 10% both significantly promoted HSF cell proliferation, and the recombinant human elastin prepared in Examples 1-3 had the most significant proliferation-promoting effect, while the effect of Comparative Examples 1-3 on HSF cell proliferation was weaker.

[0073] 2. Recombinant human elastin HSF cell migration assay

[0074] Experimental method: The recombinant human elastin prepared in Examples 1-3 and Comparative Examples 1-3 was diluted to 10% (v / v) with DMEM basal medium as the test solution; then the following operation was performed: HSF cells in the logarithmic growth phase were plated in a 12-well plate. After 24 hours, the cells covered the bottom and were gently scratched at the bottom with a 200 μL pipette tip to simulate human skin damage; the culture medium was discarded and the detached cells were gently rinsed with PBS; then culture medium containing different concentrations of the test sample was added, and a blank control group and a positive control group were set up at the same time; microscope photos were taken at 0h and 48h, and then the scar area and healing rate were calculated using ImageJ software, and the measurement results were recorded. The specific test results are shown in Figure 2 .

[0075] Mobility = (scratch area at 0 h - scratch area at 48 h) / scratch area at 0 h

[0076] Depend on Figure 2It can be seen that the recombinant human elastin prepared in Examples 1-3 and Comparative Examples 1-3 all have a certain promoting effect on HSF cell migration; Examples 1-3 have the most significant promoting effect on HSF cell migration, which is comparable to that of the positive control group of 10% fetal bovine serum, while Comparative Examples 1-3 have a weaker effect on HSF cell proliferation.

[0077] 3. Detection of recombinant human elastin extracellular matrix gene expression

[0078] Experimental method: The recombinant human elastin prepared in Examples 1-3 and Comparative Examples 1-3 was diluted to 10% (v / v) with DMEM basal culture medium as the test solution; then the following operation was performed: HSF cells were plated on a 6-well plate and cultured for 24 hours, and then replaced with complete culture medium containing different concentrations of the test sample. After 24 hours, total RNA was extracted with TRIzon, and RNA purity and concentration were detected using NanoDrop. Subsequently, cDNA was synthesized and amplified by PCR, and the target gene band was confirmed by agarose gel electrophoresis analysis. Finally, the cDNA was used for Real-Time PCR quantitative detection to obtain the effect of the test sample on the expression of extracellular matrix genes in HSF cells. The 2^-ΔΔCT method was used for data processing. For specific experimental results, see Figure 3-5 .

[0079] Depend on Figure 3-5 As can be seen, the recombinant human elastin produced in Examples 1-3 and Comparative Examples 1-3 promoted the expression of the extracellular matrix gene (elastin) to varying degrees, with the recombinant human elastin produced in Examples 1-3 having the most significant promoting effect. The recombinant human elastin produced in Examples 1-3 and Comparative Examples 1-3 inhibited the expression of extracellular matrix genes (MMP 3, MMP 9) to varying degrees, with the recombinant human elastin produced in Examples 1-3 having the most significant inhibitory effect. This indicates that the recombinant human elastin produced in the present invention can influence the expression of extracellular matrix genes, thereby exerting its firming and anti-wrinkle effects.

[0080] In summary, the present invention relates to a recombinant human elastin protein, its preparation method, and its application. The recombinant human elastin protein of the present invention is highly active, non-toxic, and non-irritating, exhibits significant anti-aging, anti-wrinkle, and firming effects, and can significantly promote cell proliferation and migration. The preparation method of the present invention is simple, and the protein expression level is high. When applied to cosmetics, adding an appropriate amount of recombinant human elastin protein with anti-aging, anti-wrinkle, and firming effects to the cosmetics can increase skin elasticity, effectively addressing issues such as skin sagging, decreased skin elasticity, and wrinkles, achieving anti-aging, anti-wrinkle, and firming effects.

[0081] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A recombinant human elastin, characterized by: The amino acid sequence of the recombinant human elastin is shown in SEQ ID NO.2; the nucleotide sequence encoding the recombinant human elastin is shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that: The recombinant vector is a recombinant plasmid; the recombinant plasmid comprises the nucleotide sequence encoding the recombinant human elastin as shown in SEQ ID NO.

1.

3. A recombinant bacterium, characterized in that The recombinant bacterium comprises the nucleotide sequence encoding the recombinant human elastin as shown in SEQ ID NO. 1; or the recombinant vector according to claim 2.

4. A method for preparing recombinant human elastin according to claim 1, characterized in that: The preparation method comprises the following steps: S1: Synthesizing the nucleotide sequence encoding the recombinant human elastin as shown in SEQ ID NO.1; S2: Insert the nucleotide sequence shown in SEQ ID NO.1 into the pET28a plasmid to construct a recombinant vector, and introduce it into Escherichia coli BL21 to screen and obtain recombinant engineered bacteria; S3: The engineered bacteria were transferred into LB medium, cultured, and IPTG was added to induce expression; S4: Collect bacterial precipitates by centrifugation, disperse them with nickel ion affinity chromatography column eluent, homogenize them multiple times with a high-pressure homogenizer to fully disrupt the bacteria, centrifuge the disrupted liquid, and collect the supernatant; S5: transferring the obtained supernatant to a nickel ion affinity chromatography column to purify the elastin solution; S6: removing imidazole from the solution by dialysis, and freeze-drying to obtain the recombinant human elastin as shown in SEQ ID NO.

2.

5. The preparation method according to claim 4, characterized in that: The conditions for the expanded culture in step S3 are 34-45° C. for 10-12 hours; The condition for adding IPTG is when OD 600 When it reaches 0.8-1.2, add IPTG to a final concentration of 1 mM; The conditions for inducing expression were 25°C for 12 h; The conditions for collecting the bacterial precipitate by centrifugation in step S4 are 1500-5000 rps, 0-8°C, and 15-45 minutes to collect the bacterial precipitate; The conditions for multiple homogenization with a high-pressure homogenizer are 800 bar, 4°C, and 40 Hz for 2-3 times; The conditions for centrifuging the disrupted liquid were 8000 rps, 4°C, 20 min; The dialysis method in step S6 is performed using 0.02M PBS.

6. Use of the recombinant human elastin as claimed in claim 1 in cosmetics.

7. The use according to claim 6, characterized in that: The recombinant human elastin described in the claims is used as an additive in any one of anti-wrinkle and firming essence, anti-wrinkle and firming lotion, anti-wrinkle and firming toner, anti-wrinkle and firming cream, and anti-wrinkle and firming mask.

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