Field of Invention
The present invention relates to cosmetic products, more specifically to antiaging products.

Background and Prior Art of the Invention

Dermal aging results in skin losing its ability to regenerate, while simultaneously suffering from damage caused by free radicals and ultraviolet rays. Aged skin becomes wrinkled, saggy, dry, rough and chapped. Stress is one of the major causes of degradation of human cells. This degradation manifests itself in speedy aging and premature cell death. There is always a need for developing new compositions which provides efficient antiaging properties.

β-Carotene is an organic compound and classified as a terpenoid. It is a strongly-coloured red-orange pigment abundant in plants and fruits. β-Carotene is also the substance in carrots that colours them orange. As a carotene with beta-rings at both ends, it is the most common form of carotene. Beta-carotene can be found in fruits, vegetables, and whole grains. Beta-carotene is used to decrease asthma symptoms caused by exercise; to prevent certain cancers, heart disease, cataracts, and age related macular degeneration (AMD); and to treat AIDS, alcoholism, Alzheimer’s disease, depression, epilepsy, headache, heartburn, high blood pressure, infertility, Parkinson’s disease, rheumatoid arthritis, schizophrenia, and skin disorders including psoriasis and vitiligo.

Lycopene (from the Greek word lykopersikon, meaning tomato) is a bright red carotene and carotenoid pigment and phytochemical found in tomatoes and other red fruits and vegetables, such as red carrots, watermelons, grapefruits, & papayas (but not strawberries or cherries). Although lycopene is chemically a carotene, it has no vitamin A activity.

Lycopene is not an essential nutrient for humans, but is commonly found in the diet, mainly from dishes prepared with tomato sauce. When absorbed from the stomach, lycopene is transported in the blood by various lipoproteins and accumulates in the liver, adrenal glands, and testes.

Because preliminary research has shown an inverse correlation between consumption of tomatoes and cancer risk, lycopene has been considered a potential agent for prevention of some types of cancers, particularly prostate cancer.

A recent application US 20100004330 teaches a food or drink which provides anti-ageing, anti-cancer, anti-heart disease, vision-enhancing effects of antioxidant substances. US’330 dislcoses that certain anti-oxidative supplements when added in certain ratios in food or drink in certain ratio result in mutual interference of absorption and/or biological effect but are more effective in reducing free-radicals. The food or drink contains an appropriate quantity of the following effective content: vitamin C (water-soluble), β-carotene (lipid-soluble), lycopene (lipid-soluble), lutein (lipid-soluble) and surfactants.

The scavenging capacity of combinations of lycopene, ß-carotene, vitamin E, and vitamin C on the free radical 2,2-diphenyl-1-picrylhydrazyl (dpph), by J. Chen, J. Shi . Macnaughton, Y. Kakuda, S.J. Xue, Y. Ma, M. Zhang, Y. Jiang, Journal of Food Biochemistry, Volume 33, Issue 2, pages 232–245, April 2009 mentions that Lycopene, a very powerful antioxidant present in fruits and vegetables, is the most efficient quencher of singlet oxygen among the carotenoids. There are other natural antioxidants such as carotenoids, vitamin C, tocopherols and polyphenolics in fruits and vegetables that are consumed together and that therefore have potential for synergistic interactions. Synergistic effects were determined by combining lycopene with other antioxidants (vitamin E, vitamin C, β-carotene) in various ratios. A synergistic effect was observed when all four antioxidants were present in combination at high lycopene activity. A synergistic effect was also observed when vitamin E and vitamin C were used in combination at high vitamin E concentrations. It appeared that certain combination ratios and concentration levels of these antioxidants have effects to enhance the antioxidant activity, compared to the individual compound, which suggests that similar combinations in the diet or in functional food products might provide greater health beneficial effects.

The present applicant in their previous application 1079/KOL/2009 had found that the combination of beta carotene and lycopene provides enhanced antioxidant effect where the synergistic combination is operable even at lower concentrations whereby the colour impact from the mixture is minimized such that the antioxidant activity is maintained and yet the colour is not compromised.

Known anti aging agents show activity at low levels and such activity is not increased on increasing the concentration. On the contrary the activity decreases beyond such small threshold level. Surprisingly it has now been found that the actives (Beta Carotene and Lycopene) are active at higher concentration than the known compounds and the activity is also higher, mainly in terms of increased cells proliferation. Accordingly the present inventors have found a novel composition to prevent dermal aging through cell proliferative route wherein the actives have increased activity with increased concentration unlike the prior art compounds.

Object of the invention

It is an object of the invention to provide new compositions with cell proliferative activity.
It is yet another object of the invention to provide compositions which have antiaging activity.

Summary of the invention

The present invention provides an antiaging composition comprising β-carotene and lycopene in a ratio of 80:20, said β-carotene and lycopene being present at a concentration from 1µM to 50 µM

Brief Description of Accompanying Figures

Fig 1 illustrates the cell proliferation at 1 hour by combination of β-Carotene and lycopene (80:20 ratio).
Fig 2 illustrates the cell proliferation at 2 hour by combination of β-Carotene and lycopene (80:20 ratio).
Fig 3 illustrates the cell proliferation at 3 hour by combination of β-Carotene and lycopene (80:20 ratio).
Fig 4 illustrates the cell proliferation at 4 hour by combination of β-Carotene and lycopene (80:20 ratio).
Fig 5 illustrates cell proliferating effects of Carotenoid, Lycopene and mixture of Carotenoid and Lycopene at 80:20 ratio at different concentrations
Fig 6 illustrates the comparative study of cell proliferation activity of combination of Beta carotene and Lycopene (80:20 ratio) with Catechin at 1 hr time point
Fig 7 illustrates the comparative study of cell proliferation activity of combination of Beta carotene and Lycopene (80:20 ratio) with Catechin at 2 hr time point
Fig 8 illustrates the comparative study of cell proliferation activity of combination of Beta carotene and Lycopene (80:20 ratio) with Catechin at 3 hr time point

Fig 9 illustrates the comparative study of cell proliferation activity of combination of Beta carotene and Lycopene (80:20 ratio) with Catechin at 4 hr time point

Fig 10 illustrates the cytotoxic activity of catechin up to 100 µM.

Detailed Description of the Present Invention

The present invention provides a composition comprising β-Carotene and Lycopene that shows enhanced cell proliferation at higher concentrations.

Generally the biologically active ingredients do not show positive activity at higher concentrations. Further it had been observed that after a certain concentration no improvement in the activity of the actives is observed, inspite of increasing their concentrations. However the present inventors have surprisingly found that the actives of the present invention did not show decreasing activity at higher concentration but on contrary stimulated cell proliferation. The actives in the present invention provide an increase in their activity along with increase in concentration.

It suggested that generally proliferation declines as cells age. Many popular anti-aging concepts are based on accelerating cell division. They stimulate skin cells to divide; thereby increasing the cells’ “turnover rate” which help the cells to get rejuvenated themselves. Accordingly the actives of the present invention in a composition provide antiaging properties.

The essential features of the present invention are
1. Cell proliferation by carotenoids
2. Replenish the dying cells
3. Positively influencing the skin antiaging process

The present inventors have found that the composition comprising β-Carotene and Lycopene in 80:20 ratio at different molar concentrations (most preferably at 50 µM) provides unexpected enhancement of cell proliferation. Accordingly the composition of the present invention provides superior antiaging properties. According to the present invention the concentration of the β-carotene and lycopene in 80:20 ratio in the composition is from 1 µM to 50 µM, preferably 10 µM to 50 µM and most preferably 50µM.

The actives, β-Carotene and Lycopene may be present in the composition in amounts 0.428 µg (1µM) to 21.44 µg (50 µM) of Beta carotene/ml and 0.107 µg (1µM) to 5.36 µg (50 µM) of Lycopene/ml. wherein the ratio of β-Carotene and Lycopene is 80:20.

The present invention is adapted for use in as personal care compositions such as antiaging/ anti wrinkle products. The various personal care compositions includes creams, lotion, gel, roll-ons, serums, balms, paste, mousse, sticks, wipes, suspensions, solutions, etc which promote cell proliferation activity and anti aging applications. Accordingly the personal care compositions can comprise actives such as combination of β-Carotene and Lycopene at a concentration of 50µM or higher along with art known cosmetic excipients.

The formulation of the present invention can be prepared by known conventional processes. For example a skin care cream formulation in accordance with the present invention can be prepared by a process comprising steps of :
i) mixing the water phase ingredients and heating to 75-80 º C,
ii) mixing the oil phase ingredients and heating to 75-80 º C,
iii) when both phases reach 75-80ºC, the oil phase is added to the water phase and homogenized,
iv) the resultant mixture is cooled under normal stirring,
v) further preservatives, viscosity modifiers are added and mixed
vi) when the temperature is below 40 º C β-carotene and lycopene (80:20 ratio) are added and mixed.
vii) finally temperature is brought below 37 º C and fragrances are added and mixed well to provide the final formulation.

The present invention is now illustrated by way of non-limiting examples.

Example 1
The activity of the combination of β-Carotene and Lycopene (80:20) was studied. Viability of cultured cells was determined by reduction of MTT (Sigma) to formazan (Mosmann, 1983). Cells were seeded in 96-well plates and cultured for 24 h. After treatment with the combination of β-Carotene and lycopene (80:20 ratio) at different concentration such as 1µM, 10µM, 50µM, MTT (5 mg/ml in PBS, 100 μl) was added to each well. Cells were incubated at 37oC for 30 min and DMSO (100 μl) was added to dissolve the formazan crystals. The absorbance was measured at 560nm using an ELISA reader (Thermo Fisher, Germany).
Figures 1, 2, 3, 4 and Table 1 demonstrates enhancement of cell proliferation when treated with combination of β-Carotene and Lycopene (80:20).

Table 1: Assessment of cell proliferation at different time periods by combination of β-Carotene and lycopene (80:20 ratio) in fibroblast cells by Total Cell count

Time
Control 80/20
β- Carotene and
Lycopene (1µM) 80/20
β- Carotene and Lycopene (10 µM) 80/20
β- Carotene and Lycopene (50 µM)
1 Hr (50+52+40+50)/4
= 48 (55+38+35+33)/4
= 54.25 (33+48+55+46)/4
= 45.50 (44+50+40+42)/4
= 44.00
2 Hr (40+55+56+50)/4
=51.5 (55+46+51+55)/4
= 51.25 (54+49+61+43)/4
= 51.75 (65+58+66+81)/4
= 67.50
3 Hr (55+46+61+56)/4
=54.5 (48+44+45+60)/4
= 54.50 (52+50+60+45)4
= 51.75 (56+65+72+64)/4
= 64.25
4 Hr
(48+56+61+56)/4
=54.5 (46+52+56+58)/4
= 54.50 (46+52+56+58)/4
= 52.25 (53+49+62+45)/4
= 62.50

Example 2

The activity of the combination of β-Carotene and Lycopene (80:20) comparing it with individual components were studied. Viability of cultured cells was determined by reduction of MTT (Sigma) to formazan (Mosmann, 1983). Cells were seeded in 96-well plates and cultured for 24 h. After treatment with β-Carotene, Lycopene and the combination of β-Carotene and lycopene (80:20 ratio) at different concentration such as 1µM, 10µM, 50µM, MTT (5 mg/ml in PBS, 100 μl) was added to each well. Cells were incubated at 37oC for 30 min and DMSO (100 μl) was added to dissolve the formazan crystals. The absorbance was measured at 440 nm using an ELISA reader (Thermo Fisher, Germany).

Fig 5 demonstrates enhancement of cell proliferation when treated with combination of β-Carotene and Lycopene (80:20) at 50µM.

Example 3

The individual cell proliferative activity of the ß-carotene and lycopene was studied using MTT assay. Viability of cultured cells was determined by reduction of MTT (Sigma) to formazan (Mosmann, 1983). Cells were seeded in 96-well plates and cultured for 24 h. After treatment with the combination of β-Carotene and lycopene (80:20 ratio) and catechin at different concentration such as 1µM, 10µM, 50µM, MTT (5 mg/ml in PBS, 100 μl) was added to each well. Cells were incubated at 37oC for 30 min and DMSO (100 μl) was added to dissolve the formazan crystals. The absorbance was measured at 560nm using an ELISA reader (Thermo Fisher, Germany).

The activity of the combination of ß-carotene and lycopene was then compared to that of catechin a known antioxident, result of which are shown in Figures 6 to 9. The results demonstrate that the combination of ß-carotene and lycopene is comparable to the activity of known antioxidant catechin and the combination of β-Carotene and lycopene (80:20 ratio) shows further increase in cell proliferating activity at concentration of 50µM.

Example 4

The anti-proliferative activity of catachin at varying concentration up to 100 µM was studied using MTT assay. The result obtained is shown in Figure 10. It is evident from Figure 10 that there is no further improvement in the antiproliferative activity of catechin beyond 10 µM.

Accordingly from the above it is evident that whereas increase in concentration of catechin beyond 10µM lowers its activity (fig 10) but that of combination of ß-carotene and lycopene there is increase in activity beyond 10 µM and rather the best activity is at 50µM (fig 6-9)

Example 5
Acream comprising the β-carotene and lycopene in a ratio of 80:20 at a concentration in accordance with the present invention i.e 50 µM is prepared. The constituents of the said cream formulation are provided in Table 2.

Table 2 Ingredients that are present in the skin care formulation (cream) are as follows:

S.No. INCI Name % w/w Function
1 B carotene 0.8 Active
2 Lycopene 0.2 Active
3 Pasteurized water* 84.18 Water phase
4 Ammonium Acryloyl - dimethyltaurate / VP Copolymer 0.05 Water Phase
5 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 0.17 Water phase
6 Xanthan gum 0.10 Water phase
7 Disodium EDTA 0.10 Water phase
8 Magnesium sulfate 0.70 Water phase
9 C12-15 Alkyl Benzoate 3.00 Oil phase
10 Potassium Cetyl Phosphate (and) Hydrogenated Palm Glycerides 2.00 Oil phase
11 Cetostearyl alcohol 1.50 Oil phase
12 Glyceryl Stearate (and) PEG-100 Stearate 2.00 Oil phase
13 Triethanolamine 0.20 Viscosity modifiers
14 Hydroxyethyl Acrylate / Sodium Acryloyldimethyl Taurate Copolymer (and) Isohexadecane (and) Polysorbate 60 3.00 Viscosity modifiers
15 Phenoxyethanol 0.70 Preservatives
16 Ethylhexyl Glycerin 0.50 Preservatives
17 Fragrance 0.80 Fragrance

The above formulation is prepared according to the method as set below, the amounts of the components being provided in the above table:
Phase A: Water phase ingredients are weighed and heated to 75-80 º C,
Phase B: Oil phase ingredients are weighed and heated it to 75-80 º C,
Phase C: When both phases reach 75-80 º C, phase B is added to phase A and homogenized for 15 to 20 min,
Phase D: The mixture is cooled under normal stirring,
Phase E: When the temperature reaches below 50 º C preservatives, viscosity modifiers are added and mixed,
Phase F: When the temperature reaches below 40 º C the β carotene and lycopene of 50 micro molar concentration (80:20 ratio) are added and mixed,
Phase G: When temperature is below 37 º C fragrance are added and mixed for 10 min.
The resultant formulation is a skin care cream where the concentration of β- carotene and lycopene in the final product is 50 µM.

WE CLAIM:

1. An antiaging composition comprising β-carotene and lycopene in a ratio of 80:20, said β-carotene and lycopene being present at a concentration from 1 µM to 50 µM.
2. The antiaging composition as claimed in claim 1 wherein said concentration of said β-carotene and lycopene is preferably 10 to 50 µM.
3. The antiaging composition as claimed in claim 1 wherein said concentration of said β-carotene and lycopene is more preferably 50 µM.
4. The antiaging composition as claimed in claim 1 wherein said composition has enhanced antiproliferative activity in fibroblast cells.
5. The antiaging composition as claimed in claim 1, wherein said composition is in the form selected from cream, lotion, gel, roll-ons, serums, balms, paste, mousse, sticks, wipes, suspensions and solutions.

ABSTRACT

An antiaging composition comprising β-carotene and lycopene in a ratio of 80:20, said β-carotene and lycopene being present at a concentration from 1 µM to 50 µM.