DESC:FILED OF INVENTION
The present invention pertains to a skin care formulation comprising a plant extract derived from Emblica officinalis. This formulation is designed to enhance skin hydration, firmness, and elasticity, and to lighten skin spots while maintaining skin pH. Additionally, the formulation is effective in reducing wrinkles, expression lines, and facial skin spots, improving skin texture, and decreasing the size and appearance of skin pores.
BACKGROUND
The concept of beauty currently in force and sought by most people is that of young skin, without spots or wrinkles. However, with the advance of age, sun exposure, stress and pollution, the skin begins to undergo gradual alterations, characterizing skin aging.
Aging is the set of irreversible and inevitable physiological modifications accompanied by a change in the level of homeostasis. The physiological manifestation of aging is the gradual deterioration of function and responsiveness to environmental stresses. This manifestation is related both to a reduction in the total number of cells in the organism and to the disordered functioning of the many cells that remain. Collagen, a fundamental component of connective tissue, gradually becomes stiffer; elastin, another component of the same tissue, gradually loses its natural elasticity due to a reduction in the number of elastic fibers and other components in the connective tissue. The decrease in the functions of the connective tissue causes the fat layers under the skin to become uneven and the degeneration of the elastic fibers, combined with the slower rate of change and oxygenation of the tissues, causes the skin to dehydrate, resulting in wrinkles.
Even though there are clear differences between chrono-aging and photo-aging skin, mainly at molecular levels, some typical alterations of chrono-aging, such as decreased cellular life expectancy, decreased responses to growth factors, interruption in the synthesis of the extracellular matrix and elevation of proteolytic activity, are also observed in photo-aging. However, in photo-aging these alterations are more pronounced. As a consequence of these alterations, aging skin becomes thinner, more inflexible, less taut and more elastic.
Dry skin is characterized by a lack of shine and suppleness, with a rough appearance and a tendency to flake. This type of skin needs specific care, as lack of hydration can compromise the integrity of the skin.
The object of the present invention is to provide a skin care formulation comprising a plant extract composition, of Emblica Officinalis origin to provide an improvement in skin hydration, firmness and elasticity, spots lightening, maintenance of skin pH, reduction of wrinkles and expression lines.
OBJECTIVES OF THE INVENTION
The primary objectives of the present invention are to provide a skin care formulation that offers extensive benefits to improve skin health and appearance. Key objectives include:
Hydration Enhancement: Significantly increase skin hydration, thereby improving skin suppleness and reducing dryness.
Firmness and Elasticity: Enhance the firmness and elasticity of the skin, contributing to a more youthful and resilient skin texture.
Spot Reduction: Effectively lighten skin spots and improve overall skin tone uniformity, addressing issues of hyperpigmentation and discoloration.
Wrinkle and Line Reduction: Reduce the appearance of fine lines and wrinkles, providing an anti-aging effect and smoother skin texture.
Skin Texture Improvement: Improve the overall skin texture, making it smoother and more even.
Maintenance of Skin pH: Maintain the natural pH balance of the skin, essential for protecting the skin from irritants and pathogens.
Biochemical Enhancement: Increase the levels of critical skin proteins such as collagen and elastin at the mRNA level, indicating deep skin rejuvenation on a cellular basis.
These objectives aim to address the comprehensive needs of maintaining youthful, healthy, and radiant skin through a scientifically validated formulation derived from the extract of Emblica officinalis. The invention leverages traditional herbal knowledge alongside modern dermatological research to meet the growing consumer demand for effective, natural skin care solutions.
SUMMARY
This invention revolves around a novel skin care formulation derived from the extract of Emblica Officinalis (commonly known as Indian gooseberry). This formulation is engineered to enhance skin health. We disclose a skin care formulation comprising an extract of Emblica officinalis, wherein: the total polyphenol content of the extract is not less than 35%; the hydrolysable galloellagic tannins content of the extract is not less than 25%; the vitamin C content of the extract ranges from 1% to 1.5%; and the ellagic acid content of the extract is not less than 10%.
These components of skin care formulation are crucial for their antioxidant, anti-inflammatory, and skin-regenerative properties. The formulation can be processed into various product forms suitable for skin care, including creams, lotions, serums, gels, ointments, sprays, masks, cleansers, and patches. This versatility allows for broad application across different skin care routines and consumer needs.
Primary benefits and efficacy of said skin care formulation is as under:
Hydration and Elasticity: The formulation significantly increases skin hydration, firmness, and elasticity, essential for maintaining youthful and healthy skin.
Spot Reduction and Skin Tone Improvement: It effectively reduces the color intensity of skin spots and overall skin tone unevenness, enhancing the visual appearance of the skin.
Anti-Aging Properties: It contributes to the reduction of wrinkles and fine lines, notably improving skin texture and firmness.
Maintenance of Skin pH: The formulation helps in maintaining the natural pH of the skin, vital for skin health and protection against irritants and pathogens.
The efficacy of the formulation has been validated through various instrumental techniques and subjective analyses details of which are given here in brief:
Instrumental Assessments: Techniques such as cutometry and colorimetry have been used to objectively measure improvements in skin firmness, elasticity, and spot lightening.
Subjective Analysis: Perceived efficacy tests conducted with consumers have consistently reported improvements in skin texture, hydration, and overall health.
Gene Expression Impact: The formulation has shown promising results in vitro by increasing the mRNA levels of critical structural proteins such as collagen and elastin in human dermal fibroblast cells. This points to its effectiveness at the cellular level, contributing to its skin-rejuvenating properties.
Safety Profile: The skin care formulation has been tested for cytotoxicity and found to be safe for regular use, with a high threshold for cell viability.
Given its scientifically proven benefits and safety, this skin care formulation holds substantial potential for market success, catering to the growing consumer demand for natural and effective skin care solutions. This invention not only fulfils the market need for a natural, effective, and safe skin care product but also sets new standards in the cosmeceutical industry by combining traditional herbal knowledge with modern scientific research.
BRIEF DESCRIPTION OF THE DRAWINGS
Other features and advantages of the disclosure should become apparent from the detailed description of the invention read in conjunction with the figures of the following accompanying drawings.
FIG.1 represents assessment of skin hydration by corneometry after applying skin care formulation.
FIG.2 represents assessment of skin firmness by cutometry after applying skin care formulation.
FIG.3 represents assessment of skin elasticity by cutometry after applying skin care formulation.
FIG.4 represents assessment of spot lightening by colorimetry after applying skin care formulation.
FIG.5 represents assessment of skin pH after applying skin care formulation.
FIG.6-10 represents results obtained in the subjective analysis by perceived efficacy and the pleasantness after 28 days and 56 days in-home use of the skin care formulation.
FIG.11 represents mean values of wrinkles and expression lines after applying skin care formulation.
FIG.12 represents mean values for skin texture obtained at the baseline and after 28 and 56 days after applying skin care formulation.
FIG.13 represents mean values of pore intensity obtained at the baseline and after 28 and 56 days after applying skin care formulation.
FIG.14 represents mean values of surface spots obtained at the baseline and after 28 and 56 days after applying skin care formulation.
FIG.15 represents mean values of brown spots obtained at the baseline and after 28 and 56 days after applying skin care formulation.
FIG.16 represents mean values of UV spots obtained at the baseline and after 28 and 56 days after applying skin care formulation.
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed toward a skin care formulation derived from the extract of Emblica Officinalis.
This formulation enhances skin hydration, firmness, and elasticity, promotes the lightening of spots, and helps maintain the natural pH balance of the skin. Additionally, it effectively reduces wrinkles, expression lines, and improves the overall texture of the skin while diminishing pores and various types of facial spots.
The skin care formulation specifically incorporates an extract of Emblica officinalis. Extract of Emblica officinalis comprises total polyphenols, hydrolysable galloellagic tannins, vitamin C and ellagic acid.
Total polyphenol content in the extract composition is not less than 35%. Hydrolysable galloellagic tannins in the composition is not less than 25%. Vitamin C content in the composition is 1-1.5%. Ellagic acid content in the composition is not less than 10%.
In one embodiment, the preferred composition is 36.8% total polyphenol content, 25.8% hydrolysable galloellagictannins, 1.3% vitamin C and 12.5% Ellagic acid.
The skin care formulation comprising extract of Emblica officinalis is prepared according to the conventional method for producing a skin care formulation.
The skin care formulation can be processed into various product forms such as creams, lotions, serums, gels, ointments, sprays, masks, cleansers, and patches, utilizing conventional methods for skin care production.
In one embodiment invitro effect of the skin care formulation on gene expression levels of collagen, AQP-3 and elastin genes in Human Dermal Fibroblast cell line (HDF) cell line is studied. The results indicated an increased expression of collagen, elastin, and aquaporin-3 (AQP-3) genes. For instance, collagen mRNA levels increased by up to 1.29 folds, elastin by up to 1.31 folds, and AQP-3 by up to 1.20 folds at the highest tested concentration (31.25 µg/ml). The concentration required to inhibit cell growth by 50% (CTC50) is determined to be 110.14 µg/ml, indicating a moderate toxicity profile.
In another embodiment invitro effect of the skin care formulation on gene expression levels of collagen, AQP-3 and elastin genes in Human Keratinocyte cell line (HaCaT) is evaluated. Gene Expression: Similar to HDF, there is an increase in the expression of collagen, elastin, and AQP-3 with the skincare formulation. Collagen expression increased by up to 1.48 folds, elastin by up to 1.28 folds, and AQP-3 by up to 1.25 folds at the highest concentration.
Cytotoxicity: The CTC50 for HaCaT cells is slightly higher at 117 µg/ml, indicating a specific response different from HDF cells.
In one embodiment the skin care formulation is providing improvement in skin hydration, firmness and elasticity, spots lightening and maintenance of skin pH by instrumental techniques. The skin firmness and elasticity are assessed by cutometry technique (Cutometer® probe coupled to the Multi Probe MPA 580 Adapter, CKeletronics, Germany). The skin firmness is evaluated through the Uf (R0) parameter. It consists of maximum skin deformation after applying a force, including elastic and viscoelastic skin deformation. If the value of Uf is reduced, this averages that skin firmness increased. Skin elasticity is assessed using the Ur/Uf parameters that is the ratio between the immediate retraction (Ur) and the total deformation of the skin, including the viscous deformation of the skin, and corresponds to biological elasticity. The increase in skin elasticity means an increase in the Ur/Uf parameters that shows the increase of the properties of the elastic fibers. The spots lightening is assessed by the colorimetry technique (Colorimeter CL-400, Courage Khazaka). For this evaluation the values of L* and b* are recorded. Though the L* and b* values is possible to calculate the ITA° (Individual Typology Angle). The increase in ITA° values indicates spots lightening. Thus, an increase in ITA° values is expected after treatment with the skin care formulation. The maintenance of skin pH is assessed by pHmetry technique (Skin-pH-Meter®).
In another embodiment the efficacy of the skin care formulation is evaluated through subjective analysis by perceived efficacy. The subjective analysis by perceived efficacy is performed by applying a questionnaire answered by the research subjects after 28 days and 56 days of in-home use of the skin care formulation.
In another embodiment efficacy of the skin care formulation is analyzed by evaluating the wrinkles and expression lines, skin texture, skin pores and skin spots by image analysis using VISIA. The images of the face are obtained at the initial condition and after 28 and 56 days of in-home use of the skin care formulation. Wrinkles and expression lines are recognized by the software as furrows, folds or creases and quantified. The texture analysis is based on skin smoothness and considers the gradations in color compared to the skin tone, as well as the topographic aspect of the surface, indicating peaks and valleys. Pores are circular surface openings of sweat gland ducts. Due to shadowing, pores appear darker than the surrounding skin tone and are identified by their darker color and circular shape. The surface spots on the facial skin surface are typically red or brown lesions visible to naked eye that deviate from the uniform tone of the skin, like acne scars, hyperpigmentation and vascular lesions. The software can quantify the contrast between the tone of the skin and the colored spots. Brown spots are lesions that occur from an excess of melanin and are deeper within the skin such as hyperpigmentation, freckles, lentigines and melasma. Brown Spots are detected by VISIA’s RBX technology using cross polarized imaging and can be quantified by the software. The UV spots originates from the melanin that accumulates below the epidermis because of the sunlight damage. These spots are not always visible to the naked eye, and thus the UV beam, is used to detect them. The software can quantify the UV spots at the image generated by the UV flash.
The result showed that skin hydration is increased by 30.6% after 28 days and 33.1% after 56 days of in-home use. Skin firmness is increased by 20.8% after 28 days and 35.7% after 56 days and elasticity is increased by 10.0% after 28 days and 10.2% after 56 days. The skin care formulation provided a reduction in the skin spot color intensity by 7.6% after 28 days and 16.2% after 56 days. The formulation-maintained skin pH effectively without significant variations.
Based on subjective assessments, improvements are reported in terms of skin softness, healthiness, radiance, nourishment and texture by nearly all participants. Skin appears more youthful and feels firmer. Diminishes appearance of fine lines & wrinkles, crow’s feet lines near eyes and the visibly improves the appearance of skin discoloration.
These results collectively suggest that the skincare formulation effectively enhances skin properties at both the cellular and user-experience levels, confirming its potential benefits in skincare applications.
Examples
Example1
Invitro effect of the skin care formulation on gene expression levels of collagen, AQP-3 and elastin genes in Human Dermal Fibroblast cell line (HDF) cell line.
Collagen, AQP-3, and Elastin gene was estimated for the test formulation by gene expression method, where the levels of the expression of Collagen, AQP-3, and Elastin in Human Dermal Fibroblast cell line (HDF) were determined with respect to untreated HDF cells.
Cytotoxicity Studies:
The monolayer cell culture was trypsinized and the cell count was adjusted to 100,000 cells/ml using medium containing 10% FBS. To each well of the 96 well microtitre plate, 0.1ml of the diluted cell suspension was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µl of different test concentrations of skin care formulation (test drug) was added onto the partial monolayer in microtitre plates. The plates were then incubated at 37o C for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 ?l of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100 µl of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated and concentration of formulation needed to inhibit cell growth by 50% (CTC50) values is generated from the dose-response curves for each cell line.
RNA isolation and cDNAs synthesis
The HDF (Human Dermal Fibroblast cell line) cells treated with drug were subjected to cell lysis by treating with Tri-extract reagent. Chloroform was added, to isolate the total RNA from the sample and subjected for centrifugation. Out of the three distinct layers observed, upper layer was collected in fresh tube and equal volume of isopropanol was added and incubated at-200C for 10mins. After the incubation followed by centrifugation, appropriate volume of ethanol was added to resuspend the pellet. After incubation and centrifugation, the pellet was air dried and appropriate volume of TAE buffer was added. The isolated total RNA was further used for cDNAs synthesis. cDNAs was synthesized by priming with oligo dT primers followed by reverse transcriptase enzyme treatment. The cDNA thus synthesized was taken up for PCR for the amplification of Collagen, AQP-3 and Elastin and GAPDH (internal control).
RT-PCR procedure
The mRNA expression levels of Collagen, AQP-3 and Elastin were determined using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). 50µl of the reaction mixture was subjected to PCR for amplification of Collagen, AQP-3 and Elastin.
Results
Table 1: Cytotoxic properties of formulation against HDF cell line
Test Conc. (?g/ml) % Cytotoxicity CTC50 (?g/ml)
1000 74.34 ± 2.32 110.14 ± 2.85
500 68.36 ± 0.80
250 64.49 ± 1.89
125 52.65 ± 0.44
62.5 41.37 ± 1.53
31.25 30.31 ± 0.84
Table 2: The Quantitative gene expression level of Collagen normalized to GAPDH.
Test Sample Regulation in Terms of Folds
Collagen
Cell Control 1.00
Tiron (1 mg/mL) 1.14
Hyaluronic acid (0.1%) 1.16
Palmitoyl Oligopeptide (10 ppm) 1.18
Collagen (0.5%) 1.18
Test drug (7.8 µg/ml) 1.17
Test drug (15.6 µg/ml) 1.20
Test drug (31.25 µg/ml) 1.29
Table 3: The Quantitative gene expression level of Elastin normalized to GAPDH.
Test Sample Regulation in Terms of Folds
Elastin
Cell Control 1.00
Tiron (1 mg/mL) 1.12
Hyaluronic acid (0.1%) 1.17
Palmitoyl Oligopeptide (10 ppm) 1.18
Collagen (0.5%) 1.28
Test drug (7.8 µg/ml) 1.26
Test drug (15.6 µg/ml) 1.28
Test drug (31.25 µg/ml) 1.31
Table 4: The Quantitative gene expression level of AQP-3 normalized to GAPDH
Test Sample Regulation in Terms of Folds
AQP-3
Cell Control 1.00
Tiron (1 mg/mL) 1.15
Hyaluronic acid (0.1%) 1.15
Palmitoyl Oligopeptide (10 ppm) 1.14
Collagen (0.5%) 1.16
Test drug (7.8 µg/ml) 1.17
Test drug (15.6 µg/ml) 1.19
Test drug (31.25 µg/ml) 1.20
Study formulation, tested for in vitro cytotoxicity studies against HDF (Human Dermal Fibroblast cell line) by MTT assay exposing the cells to different combinations of study formulation. The CTC50 value of the study formulation on HDF cell line is 110.14 ± 2.85 µg/ml.
The cells treated with various test substances such as Tiron (1 mg/mL), Hyaluronic acid (0.1%), Palmitoyl Oligopeptide (10 ppm), Collagen (0.5%), lower concentration of test formulation 7.8 µg/ml, Medium concentration of test formulation - 15.6 µg/ml, and Higher concentration of test formulation -31.25 µg/ml. When the Semi Quantitative RT-PCR experiment was performed by using Collagen, AQP-3 and Elastin specific primers, the analysis revealed that upon treatment with the test substance the levels of Collagen, AQP-3 and Elastin mRNA was increased compared over the untreated cell control value.
At lower (7.8 µg/ml), medium (15.6 µg/ml) and higher concentrations (31.25 µg/ml), the levels of Collagen mRNA increased by 1.17 folds, 1.20 folds and 1.29 folds respectively relative to cell control. Similarly, the Elastin mRNA increased by 1.26 folds, 1.28 folds, and 1.31 folds relative to the cell control respectively when treated with lower, medium and higher concentrations of the test formulation. The mRNA of AQP-3 also showed an increase upon treatment with lower, medium and higher concentrations of test formulation. For AQP-3 the fold change with respect to the cell control is 1.17 folds, 1.19 folds, 1.20 folds at lower, medium and higher concentrations respectively.
Example 2
Invitro effect of the skin care formulation on gene expression levels of collagen, AQP-3 and elastin genes in Human Keratinocyte cell line (HaCaT).
Collagen, AQP-3 and Elastin gene was estimated for the skin care formulation by gene expression method, where the levels of the expression of Collagen, AQP-3 and Elastin in Human Keratinocyte cell line (HaCaT) were determined with respect to untreated HaCaT cells.
Cytotoxicity Studies:
The monolayer cell culture was trypsinized and the cell count was adjusted to 100,000cells/ml using medium containing 10% FBS. To each well of the 96 well microtitre plate, 0.1ml of the diluted cell suspension was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µl of different test concentrations of formulation (test drug) was added on to the partial monolayer in microtitre plates. The plates were then incubated at 37o C for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 µl of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37° C in 5% CO2 atmosphere. The supernatant was removed and 100 µl of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated and concentration of formulation needed to inhibit cell growth by 50% (CTC50) values is generated from the dose-response curves for each cell line.
RNA isolation and cDNAs synthesis
The HaCaT (Human Keratinocyte cell line) cells treated with test formulation were subjected to cell lysis by treating with Tri-extract reagent. Chloroform was added, to isolate the total RNA from the sample and subjected for centrifugation. Out of the three distinct layers observed, upper layer was collected in fresh tube and equal volume of isopropanol was added and incubated at-200C for 10mins. After the incubation followed by centrifugation, appropriate volume of ethanol was added to resuspend the pellet. After incubation and centrifugation, the pellet was air dried and appropriate volume of TAE buffer was added. The isolated total RNA was further used for cDNAs synthesis. cDNAs was synthesized by priming with oligo dT primers followed by reverse transcriptase enzyme treatment. The cDNA thus synthesized was taken up for PCR for the amplification of Collagen, AQP-3 and Elastin and GAPDH (internal control).
RT-PCR Procedure
The mRNA expression levels of Collagen, AQP-3 and Elastin were determined using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). 50µl of the reaction mixture was subjected to PCR for amplification of Collagen, AQP-3 and Elastin.
Table 5: Cytotoxic properties of test substance against HaCaT cell line
Test Conc. (?g/ml) % Cytotoxicity CTC50 (?g/ml)
1000 73.60 ± 2.69 117 ± 10.65
500 64.68 ± 1.33
250 63.64 ± 1.57
125 50.81 ± 1.05
62.5 38.42 ± 2.44
31.25 28.47 ± 1.44

Table 6: The Quantitative gene expression level of Collagen normalized to GAPDH
Test Sample Regulation in Terms of Folds
Collagen
Cell Control 1.00
Tiron (1 mg/mL) 1.05
Hyaluronic acid (0.1%) 1.20
Palmitoyl Oligopeptide (10 ppm) 1.32
Collagen (0.5%) 1.15
Test drug (7.8 µg/ml) 1.25
Test drug (15.6 µg/ml) 1.42
Test drug (31.25 µg/ml) 1.48
Table 7: The Quantitative gene expression level of Elastin normalized to GAPDH.
Test Sample Regulation in Terms of Folds
Elastin
Cell Control 1.00
Tiron (1 mg/mL) 1.11
Hyaluronic acid (0.1%) 1.18
Palmitoyl Oligopeptide (10 ppm) 1.20
Collagen (0.5%) 1.17
Test drug (7.8 µg/ml) 1.25
Test drug (15.6 µg/ml) 1.28
Test drug (31.25 µg/ml) 1.28
Table 8: The Quantitative gene expression level of AQP-3 normalized to GAPDH.
Test Sample Regulation in Terms of Folds
AQP-3
Cell Control 1.00
Tiron (1 mg/mL) 1.12
Hyaluronic acid (0.1%) 1.15
Palmitoyl Oligopeptide (10 ppm) 1.18
Collagen (0.5%) 1.13
Test drug (7.8 µg/ml) 1.14
Test drug (15.6 µg/ml) 1.22
Test drug (31.25 µg/ml) 1.25
Study formulation, tested for in vitro cytotoxicity studies against HaCaT (Human Keratinocyte cell line) by MTT assay exposing the cells to different combinations of study formulation. The CTC50 value of the test substance on the HaCaT cell line is 117±10.65 µg/ml.
The cells treated with various Study formulations such as Tiron (1 mg/mL), Hyaluronic acid (0.1%), Palmitoyl Oligopeptide (10 ppm), Collagen (0.5%), lower concentration of Study formulation - 7.8 µg/ml, Medium concentration of Study formulation - 15.6 µg/ml, and Higher concentration of Study formulation -31.25 µg/ml. When Semi-Quantitative RT-PCR experiment was performed by using Collagen, AQP-3 and Elastin specific primers, the analysis revealed that upon treatment with the Study formulation the levels of Collagen, AQP-3 and Elastin mRNA was increased compared over the untreated cell control value.
At lower (7.8 µg/ml), medium (15.6 µg/ml) and higher concentrations (31.25 µg/ml), the levels of Collagen mRNA was increased by 1.25 folds, 1,42 folds and 1.48 folds respectively relative to cell control. Similarly, the Elastin mRNA increased by 1.17 folds, 1.25 folds, and 1.28 folds relative to the cell control respectively when treated with lower, medium and higher concentrations.
The mRNA of AQP-3 also showed increase in upon treatment with lower, medium and higher concentrations of test formulation. For AQP-3 the fold change with respect to the cell control was 1.14 folds, 1.22 folds, 1.25 folds at lower, medium and higher concentrations respectively.
Example 3
Efficacy of skin care formulation via instrumental techniques, subjective analysis by perceived efficacy and images analysis by VISIA
28 subjects with dry skin were selected for the study and completed the study for up to 28 days. 25 subjects with dry skin completed the study for up to 56 days. The skin care formulation was applied twice a day to cover the face for 56 days. After 28 days and 56 days of facial application of the skin care formulation, skin hydration, skin firmness and elasticity, spots lightening and maintenance of skin pH were measured. Before the beginning of the measurements, the subjects remained for 30 minutes in air-conditioned environment at 22°C ± 2 and 55% ± 5 of relative humidity. The measurements were carried out at the beginning of the study and after 28 and 56 days of in-home use of the skin care formulation. The improvement in skin hydration was assessed by capacitance measurements through corneometry technique (Corneometer® 825). The skin firmness and elasticity were assessed by cutometry technique (Cutometer® probe coupled to the Multi Probe MPA 580 Adapter, CKeletronics, Germany). The skin firmness was evaluated through the Uf (R0) parameter and the skin elasticity was evaluated through the Ur/Uf (R7) parameter. The spots lightening was assessed by the colorimetry technique (Colorimeter CL-400, Courage Khazaka). Though the L* and b* values was possible to calculate de ITA° parameter and assess the reduction of spots color intensity. The maintenance of skin pH was assessed by pHmetry technique (Skin-pH-Meter®). The subjective analysis by perceived efficacy was performed by applying a questionnaire answered by the subjects after 28 days and 56 days of in-home use of the skin care composition. The methodology consisted of the evaluation of the wrinkles and expression lines, skin texture, pores and skin spots by image analysis using the VISIA. The images of the face were obtained at initial condition and after 28 and 56 days of in-home use of the skin care formulation.
Results
1. Instrumental techniques
• Assessment of skin hydration by corneometry
The mean values of capacitance obtained at the beginning of the study and after 28 and 56 days of in-home use of the skin care formulation were given in Fig 1. The significance of variation in skin hydration at each time of assessment for the skin care formulation was assessed by applying the bimodal paired Student’s t-test method, considering a 95% confidence interval, to the values of capacitance obtained at the beginning of the study, in relation to the values obtained after 28 and 56 days of in-home use.
The skin care composition provided a significant increase in skin hydration after 28 and 56 days of in-home use, indicating the improvement in skin hydration.
Skin hydration was increased by 30.6% after 28 days and 33.1% after 56 days of in-home use, when compared to the initial condition.
100% of the subjects presented improvement in skin hydration after 28 and 56 days of in-home use of the skin care formulation.
• Assessment of the skin firmness by cutometry
The mean values of Uf parameter obtained at the beginning of the study and after 28 and 56 days of in-home use of the product were given in Fig.2. The significance of improvement in skin firmness was evaluated by comparing the values of Uf parameter obtained at beginning of the study to the values obtained after 28 days, applying the paired Student’s t-test method, considering a 95% confidence interval. There was a significant decrease in the values of Uf parameter after 28 and 56 days of in-home use, indicating that the skin care formulation provided significant increase in skin firmness.
Skin firmness increased by 20.8% after 28 days and 35.7% after 56 days of in-home use. 100% of the subjects presented improvement in skin firmness after 28 days and 56 days of in-home use.
• Assessment of the skin elasticity by cutometry
The mean values of Ur/Uf parameter obtained at the beginning of the study and after 28 and 56 days of in-home use of the product are represented in Fig 3.
The skin care formulation provided improvement in the values of Ur/Uf parameter after 28 and 56 days of in-home use, indicating that the skin care formulation increased the skin elasticity.
Skin care formulation increased the skin elasticity by 10.0% after 28 days and 10.2% after 56 days of in-home use. 100% of the subjects presented improvement in skin elasticity after 28 and 56 days of in-home use.
• Assessment of spots lightening
Fig. 4 shows the mean values of ITA° (Individual Typology Angle) parameter obtained at the beginning of the study and after 28 and 56 days of in-home use of the product.
There was a significant increase in the values of ITA0 after 28 and 56 days of use of the skin care formulations, indicating that there was a significant reduction in the skin spots color intensity.
The skin care formulation provided a reduction in the skin spot color intensity after 28 and 56 days of in-home use by 7.6% after 28 days and 16.2% after 56 days.
64% of the subjects presented reduction in the skin spot color intensity after 28 days and 92% of them presented reduction in the skin spot color intensity after 56 days of in-home use.
• Assessment of maintenance of skin pH
The mean value of pH obtained throughout the study is represented in Fig. 5. The skin care formulation did not provide significant variation in the skin pH after 28 and 56 days, indicating the maintenance of the natural skin pH.
2. Subjective analysis by perceived efficacy
After 28 days of in-home use, 96% of the subjects noticed that the skin feels soft. 100% of the subjects noticed that the skin looks healthier. 100% of the subjects noticed that the skin feels hydrated. 100% of the subjects noticed that the skin feels healthier. 100% of the research subjects noticed that the improvement of skin texture. 100% of the subjects noticed that the skin is deeply moisturized. 96% of the subjects noticed that the skin appears radiant. 96% of the subjects noticed that the skin looks nourished. 100% of the subjects noticed that the skin feels nourished. 96% of the subjects noticed that the dull and lack luster skin appears revived with essential hydration. 100% of the subjects noticed that the skin feels smoother. 96% of the subjects noticed that the product leaves skin glowing. 96% of the subjects noticed that product revives appearance of fatigued skin. 100% of the subjects noticed that the plumps skin with hydration. 96% of the subjects noticed that the skin appears more youthful. 96% of the subjects noticed that the skin feels firmer. 93% of the subjects noticed the diminishes appearance of fine lines & wrinkles. 93% of the subjects noticed the diminishes appearance of crow’s feet lines near eyes. 93% of the subjects noticed the visibly improves the appearance of skin discoloration. 96% of the subjects noticed that product reduces the appearance of deep wrinkles. 100% of the subjects liked this product. 100% of the subjects recommend this product.
After 56 days of in-home use, 100% of the subjects noticed that the skin feels soft. 96% of the subjects noticed that the skin looks healthier. 100% of the subjects noticed that the skin feels hydrated. 100% of the subjects noticed that the skin feels healthier. 100% of the subjects noticed that the improvement of skin texture. 100% of the subjects noticed that the skin was deeply moisturized. 96% of the subjects noticed that the skin appears radiant. 100% of the subjects noticed that the skin looks nourished. 100% of the subjects noticed that the skin feels nourished. 100% of the subjects noticed that the dull and lackluster skin appears revived with essential hydration. 100% of the subjects noticed that the skin feels smoother. 96% of the subjects noticed that the product leaves skin glowing. 92% of the subjects noticed that product revives appearance of fatigued skin. 100% of the subjects noticed that the plumps skin with hydration. 96% of the subjects noticed that the skin appears more youthful. 96% of the subjects noticed that the skin feels firmer. 92% of the subjects noticed the diminishes appearance of fine lines & wrinkles. 92% of the subjects noticed the diminishes appearance of crow’s feet lines near eyes. 96% of the subjects noticed the visibly improves the appearance of skin discoloration. 92% of the subjects noticed that product reduces the appearance of deep wrinkles. 100% of the subjects liked this product. 100% of the subjects recommend this product.
The results obtained in the subjective analysis by perceived efficacy and the pleasantness after 28 days and 56 days in-home use of the product were represented in Fig-6-10.
3. Assessment via VISIA®
• Reduction of wrinkles and expression lines
The mean values of wrinkles and expression obtained during the study for the skin care formulation were shown in Fig 11.
After 28 days of in-home use of the skin care formulation, there was a significant reduction of the wrinkles and expression lines of 4.8%, reaching up to 11.1%. It was possible to observe that 64% of the subjects showed improvement in wrinkles and expression lines after 28 days of in-home use.
After 56 days of in-home use of the skin care formulation, there was a significant reduction of the wrinkles and expression lines of 5.0%, reaching up to 10.4%. It was possible to observe that 60% of the subjects showed improvement in wrinkles and expression lines after 56 days of in-home use.
• Skin texture
Fig.12 illustrates the mean values for skin texture obtained at the baseline and after 28 and 56 days of in-home use of the skin care formulation. After 28 and 56 days of in-home use of the skin care formulation, there was an improvement in the mean values for skin texture when compared with the initial condition.
• Skin pores reduction
Fig. 13 illustrates the mean values of pores intensity obtained during the study for the skin care formulation. After 28 days of in-home use of the skin care formulation, there was a significant reduction of pores of 6.5%, reaching up to 10.1%. It was possible to observe that 75% of the subjects showed a reduction of pores.
After 56 days of in-home use there was a significant reduction of pores of 6.7%, reaching up to 10.4%. It was possible to observe that 68% of the research subjects showed reduction of pores.
• Facial skin Spots
• Surface Spots
Fig 14 illustrates the mean values for surface spots obtained during the study for the skin care formulation. After 28 days of in-home use of the product there was a significant reduction in the values of Surface Spots of 2.2%, reaching up to 4.9%. It was possible to observe that 64% of the subjects showed a reduction of the facial skin surface spots.
After 56 days of in-home use of the skin care formulation there was a significant reduction in the values of surface spots of 2.8%, reaching up to 6.0%. It was possible to observe that 64% of the subjects showed reduction of the facial skin surface spots.
• Brown Spots
Fig. 15 illustrates the mean values of brown spots obtained during the study for the skin care formulation. After 28 days of in-home use of the skin care formulation there was a significant reduction in the values of Brown Spots of 3.9%, reaching up to 5.8%. It was possible to observe that 82% of the subjects showed reduction of the facial skin brown spots.
After 56 days of in-home use of the skin care formulation there was a significant reduction in the values of brown spots of 3.6%, reaching up to 6.4%. It was possible to observe that 72% of the subjects showed reduction of the facial skin brown spots.
• UV Spots
Fig. 16 illustrates the mean values for UV spots obtained during the study for the skin care formulation. After 28 days of in-home use of the skin care formulation there was a significant reduction in the values of UV Spots of 4.3%, reaching up to 8.6%. It was possible to observe that 75% of the subjects showed a reduction of the facial skin UV spots.
After 56 days of in-home use of the skin care formulation there was a significant reduction in the values of UV Spots of 7.7%, reaching up to 11.3%. It was possible to observe that 76% of the research subjects showed reduction of the facial skin UV spots.
Based on the results presented here, it is clear that the skin care formulation effectively enhances skin hydration, firmness, and elasticity, lightens spots, and maintains skin pH balance. Additionally, the formulation significantly improves the reduction of wrinkles and expression lines, refines skin texture, minimizes pores, and diminishes the appearance of facial skin spots. ,CLAIMS:1. A skin care formulation comprising an extract of Emblica officinalis, wherein: the total polyphenol content of the extract is not less than 35%; the hydrolysable galloellagic tannins content of the extract is not less than 25%; the vitamin C content of the extract ranges from 1% to 1.5%; and the ellagic acid content of the extract is not less than 10%.
2. The skin care formulation as claimed in claim 1, wherein the formulation is provided in one or more forms selected from the group consisting of: creams, lotions, serums, gels, ointments, sprays, masks, cleansers, and patches.
3. The skin care formulation as claimed in claim 1, wherein the formulation is capable of: a) increasing skin hydration; b) increasing skin firmness and elasticity; c) reducing the intensity of skin spot color; and d) maintaining skin pH.
4. The skin care formulation as claimed in claim 1, wherein the formulation is capable of : a) reducing wrinkles and expression lines; b) improving skin texture; c) reducing the size and appearance of skin pores and spots; d) reducing the visibility of surface spots, including facial skin surface spots; e) reducing facial skin brown spots; f) reducing facial skin UV spots; g) enhancing the skin's overall health, feel, and appearance, making it feel softer, smoother, and more hydrated; and h) diminishing the appearance of crow's feet lines near the eyes.
5. The skin care formulation as claimed in claim 1, wherein the formulation is capable of: a) increasing the levels of Collagen mRNA; b) increasing Elastin mRNA; and c) increasing the mRNA of AQP-3 (Aquaporin-3).
6. The skin care formulation as claimed in claim 1, wherein the formulation is capable of: a) making the skin feel firmer; b) diminishing the appearance of fine lines and wrinkles; c) visibly improving the appearance of skin discoloration; d) reducing the appearance of deep wrinkles; e) reviving the appearance of fatigued skin; and f) making the skin appear more youthful.
7. The skin care formulation as claimed in claim 1, wherein the formulation is capable of: a) deeply moisturizing the skin; b) making the skin appear radiant; and c) making the skin look nourished.