MANGIFERIN AS A PROTECTIVE AGENT AGAINST MITOCHONDRIAL DNA DAMAGE AND SKIN-CARE COMPOSITIONS COMPRISING SAME

Abstract

The invention relates to compounds that provide protective effects against mitochondrial DNA damage. In particular, it relates to mangiferin, ideally in the form of a naturally-occurring mangiferin extract, and to mangiferin-containing compositions and their use as protective agents against mitochondrial DNA damage. Aspects of the invention relate to an additive for a skin care composition, wherein the additive comprises a complex of mangiferin with β-cyclodextrin or a β-cyclodextrin derivative. The invention also relates to skin care compositions comprising the mangiferin complexes, as well as to methods of their preparation. In embodiments, the additive can be used in a sunscreen formulation to protect against UV-induced mtDNA damage.

Claims

1. A topical composition comprising a compound having the following general formula I: ##STR00011## wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 is selected from the group consisting of —H, —OH and a glucosyl radical, or an ester or pharmaceutically acceptable salt thereof, for the prevention of mitochondrial DNA damage.

2. A topical composition as in claim 1 wherein the compound is mangiferin, having the formula: ##STR00012## or isomers or derivatives or an ester thereof or one of its pharmaceutically acceptable salts.

3. (canceled)

4. (canceled)

5. (canceled)

6. A topical composition as in claim 1 wherein the composition is a pharmaceutical composition.

7. A topical composition as in claim 1 wherein the composition is a cosmetic composition.

8. A topical composition as in claim 1 wherein the composition is in the form of a cream, oil, ointment or lotion.

9. An additive for a skin care composition, wherein the additive comprises a complex of mangiferin with β-cyclodextrin or a β-cyclodextrin derivative.

10. An additive as claimed in claim 9, wherein the β-cyclodextrin derivative is selected from hydroxypropyl-β-cyclodextrin, sulfobutylether β-cyclodextrin and mono (6-ethylene-diamino-6-deoxy)-β-cyclodextrin.

11. An additive as claimed in claim 9, wherein the mangiferin-βcyclodextrin complex has a stoichiometric ratio of 1:1.

12. An additive as claimed in claim 9, for the prevention of mitochondrial DNA damage.

13. A skin care composition comprising the additive of claim 9.

14. A skin care composition as claimed in claim 13, wherein the additive is included in the composition such that the concentration of the mangiferin in the composition is from 1 to 10% (w/w).

15. A skin care composition as claimed in claim 13, wherein the composition comprises the mangiferin-βcyclodextrin complex in an aqueous phase.

16. A skin care composition as claimed in claim 13, wherein the composition is in the form of an emulsion.

17. A skin care composition as claimed in claim 13, wherein the composition further comprises at least one additional ingredient selected from an organic UV filter, an inorganic UV filter, an emulsifier, a suspending agent, a film former and a skin conditioner.

18. A skin care composition as claimed in claim 13, wherein the skin care composition is in the form of a cream, lotion, oil, ointment or spray.

19. A method of preparing an additive as claimed in claim 9, the method comprising: preparing a solution of β-cyclodextrin or a β-cyclodextrin derivative in water; adding mangiferin to the solution; allowing the complex to form at a temperature between 30° C. and 50° C. for between 3 to 6 hours; and removing solids by filtration.

20. A method as claimed in claim 19, wherein the β-cyclodextrin or β-cyclodextrin derivative is added at a concentration of from 1 to 3% (w/w).

21. A method as claimed in claim 19, wherein the mangiferin is added at a concentration of from 0.1 to 0.3%.

22. A method as claimed in claim 19, wherein the β-cyclodextrin or β-cyclodextrin derivative is β-cyclodextrin.

23. A method as claimed in claim 19, wherein the β-cyclodextrin or β-cyclodextrin derivative is a β-cyclodextrin derivative selected from hydroxypropyl-β-cyclodextrin, sulfobutylether β-cyclodextrin and mono (6-ethylene-diamino-6-deoxy)-β-cyclodextrin.

25. A method of preparing a skin care composition comprising: preparing a water phase comprising the additive of claim 9; preparing an oil phase; adding the water phase to the oil phase to form an initial water-in-oil emulsion and subsequently an oil-in-water emulsion.

25. The method of claim 24, comprising subjecting the emulsion to at least one microfluidisation step.

26. A method for preventing mitochondrial DNA damage in mammals, comprising the steps of: administering a topical composition comprising a compound having the following general formula I: ##STR00013## wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 is selected from the group consisting of —H, —OH and a glucosyl radical, or of one of the esters or its pharmaceutically acceptable salts.

27. A method for preventing mitochondrial DNA damage as in claim 26 wherein the compound is mangiferin, having the formula: ##STR00014## or isomers or derivatives or an ester thereof or one of its pharmaceutically acceptable salts.

28. A topical composition as in claim 2, wherein the compound is a naturally-occurring mangiferin extract.

29. A topical composition as in claim 28 wherein the mangiferin extract is present at an effective concentration for protecting against DNA damage.

30. A topical composition as in claim 29 wherein the mangiferin extract is present at an effective concentration of 1000 μg/ml or greater.

31. A topical composition as in claim 30 wherein the mangiferin extract is present at an effective concentration of between 1000 μg/ml and 5000 μg/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings where like parts are provided with corresponding reference numerals and in which:

[0024] FIG. 1 shows the structure of mangiferin;

[0025] FIG. 2 shows viability assays for a range of mangiferin extract concentrations;

[0026] FIG. 3 shows MtDNA damage in primary human skin fibroblasts and human skin cell line (HDFn) cells following UV irradiation;

[0027] FIG. 4 shows mtDNA damage in human skin cell line (HDFn) cells following UV irradiation in the presence of mangiferin;

[0028] FIG. 5 shows mtDNA damage in human skin cell line (HDFn) cells following UV irradiation in the presence of commercial sun screen products (SPF0, SPF15 and SPF30);

[0029] FIG. 6 is a UV absorbance scan for mangiferin at 1000 μg/ml;

[0030] FIG. 7 shows UV absorbance scans for mangiferin-βcyclodextrin complexes;

[0031] FIG. 8 shows basal respiration of a mangiferin-containing sunscreen formulation and control formulations (water-based formulation, leading commercial product formulation) as measured on the Seahorse analyser;

[0032] FIG. 9 shows mitochondrial respiration of a mangiferin-containing sunscreen formulation and control formulations (water-based formulation, leading commercial product formulation) as measured on the Seahorse analyser;

[0033] FIG. 10 shows glycolytic index of a mangiferin-containing sunscreen formulation and control formulations (water-based formulation, leading commercial product formulation) as measured on the Seahorse analyser.

DETAILED DESCRIPTION

[0034] The present invention relates to a topical composition comprising a compound of formula I:

##STR00005##

wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 is selected from the group consisting of —H, —OH and a glucosyl radical, or of one of the esters or its pharmaceutically acceptable salts, for the prevention of mitochondrial DNA damage.

[0035] In an embodiment, the compound of formula I is mangiferin, having the formula:

##STR00006##

or isomers or derivatives or an ester thereof, or one of its pharmaceutically acceptable salts.

[0036] The compound may be a naturally-occurring plant extract.

[0037] The compound may be a naturally-occurring mangiferin extract.

[0038] Optionally, the mangiferin extract is present at an effective concentration for protecting against DNA damage.

[0039] Optionally, the mangiferin extract is present at an effective concentration of 1,000 μg/ml or greater.

[0040] Optionally, the mangiferin extract is present at an effective concentration of between 1,000 μg/ml and 5,000 μg/ml.

[0041] Optionally, the composition is a pharmaceutical composition.

[0042] Alternatively, the composition is a cosmetic composition.

[0043] The composition may be in the form of a cream, oil, ointment or lotion.

[0044] The invention also relates to the use of the compounds described above to protect the mitochondrial DNA of living cells from damage.

[0045] The damage may be from reactive oxygen species (ROS), oxidative stress, exposure to UVA and/or UVB etc.

[0046] Preferably the living cells are mammalian cells. More preferably the living cells are human skin cells.

[0047] According to an aspect of the present invention there is provided a method for preventing mitochondrial DNA damage in mammals, comprising the steps of: administering a topical composition comprising a compound having the following general formula I:

##STR00007##

wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 is selected from the group consisting of —H, —OH and a glucosyl radical, or of one of the esters or its pharmaceutically acceptable salts.

[0048] Preferably the compound is mangiferin, having the formula:

##STR00008##

or isomers or derivatives or an ester thereof or one of its pharmaceutically acceptable salts.

[0049] Preferably the compound is a naturally occurring plant extract.

[0050] Most preferably the compound is naturally occurring mangiferin extract.

[0051] Optionally the mangiferin extract is present at an effective concentration for protecting against DNA damage.

[0052] Optionally the mangiferin extract is present at an effective concentration of 1000 μg/ml or greater.

[0053] Optionally the mangiferin extract is present at an effective concentration of between 1000 μg/ml and 5000 μg/ml.

[0054] Preferably the method is for preventing mitochondrial DNA damage in humans.

[0055] More preferably the method is for preventing mitochondrial DNA damage in human skin cells. Yet more preferably the method is for preventing mitochondrial DNA damage in human dermal fibroblast cells.

[0056] According to another aspect of the present invention there is provided an in vitro method of inhibiting mitochondrial DNA damage in living cells comprising providing a test sample of living cells and providing a protective composition between the test sample and source of potential damage, the protective composition comprising a compound having the following general formula I:

##STR00009##

wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 is selected from the group consisting of —H, —OH and a glucosyl radical, or of one of the esters or its pharmaceutically acceptable salts, for the prevention of mitochondrial DNA damage.

[0057] Preferably the compound is mangiferin, having the formula:

##STR00010##

or isomers or derivatives or an ester thereof or one of its pharmaceutically acceptable salts.

[0058] Preferably the compound is a naturally occurring plant extract.

[0059] Most preferably the compound is naturally occurring mangiferin extract.

[0060] Optionally the mangiferin extract is present at an effective concentration for protecting against DNA damage.

[0061] Optionally the mangiferin extract is present at an effective concentration of 1000 μg/ml or greater.

[0062] Optionally the mangiferin extract is present at an effective concentration of between 1000 μg/ml and 5000 μg/ml.

[0063] Optionally the method comprises contacting said compound with said test sample.

[0064] The invention also relates to an additive for a skin care composition, wherein the additive is a complex of mangiferin with β-cyclodextrin or a β-cyclodextrin derivative.

[0065] Mangiferin (MGN) is a naturally-occurring xhantone, (1,3,6,7-tetrahydroxy-2-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]xanthen-9-one) mainly found in higher plants such as Mangifera indica L (commonly known as mango). The inventors have advantageously determined that mangiferin shows good UV absorbance, while protecting against mtDNA damage. However, mangiferin has poor solubility in water, limiting its use, particularly in cosmetic products which often require aqueous compositions or phases.

[0066] The mangiferin can be a naturally-occurring mangiferin extract, or a synthetic analog thereof.

[0067] Advantageously, the additive complexes according to the invention show good solubility in water, and can be formulated into aqueous cosmetic compositions and into emulsions at functionally-relevant and economically-viable concentrations. Skin care compositions to which the additives can be incorporated include moisturisers, tinted moisturisers, primers, foundations, fake tan products, toners and serums, for example, as well as more specific sun-care products such as sunscreens.

[0068] According to the present invention, the additive comprises mangiferin complexed with β-cyclodextrin or a β-cyclodextrin derivative.

[0069] In an embodiment, the β-cyclodextrin derivative is hydroxypropyl-β-cyclodextrin, sulfo-butyl-ether β-cyclodextrin or mono (6-ethylene-diamino-6-deoxy)-β-cyclodextrin.

[0070] In an embodiment, the stoichiometric ratio of mangiferin:β-cyclodextrin/β-cyclodextrin derivative is 1:1.

[0071] The additive may be used for the prevention of mitochondrial DNA damage.

[0072] The additive may consist of the mangiferin/β-cyclodextrin or mangiferin/β-cyclodextrin derivative complex or a mixture of these, or may comprise additional components.

[0073] The invention also relates to a skin care composition, comprising the mangiferin complex additive described above. The additive can be included in the skin care composition at an effective concentration to protect against mtDNA damage. In an embodiment, the additive can be included in the skin care composition such that the concentration of the mangiferin is from 1 to 10% (w/w).

[0074] Advantageously, the inventors have determined that when the mangiferin-βcyclodextrin complex is incorporated into a formulation within this concentration range, good UV absorbance can be achieved, while the mtDNA damaging-preventing functionality of the mangiferin is retained.

[0075] The skin care composition may be a pharmaceutical skin care composition or may be a cosmetic skin care composition.

[0076] The composition may comprise at least one additional ingredient selected from an organic UV filter, an inorganic UV filter, an emulsifier, a suspending agent, a film former and a skin conditioner.

[0077] The skin care composition may be an aqueous composition.

[0078] Alternatively, the skin care composition may be an emulsion. The emulsion may comprise the mangiferin complex in an aqueous phase.

[0079] As would be understood by one skilled in the art, the term “emulsion” is intended to encompass oil-in-water emulsions as well as water-in-oil emulsions, and multiple emulsions (such as water-in-oil-in water etc.).

[0080] According to an aspect of the invention, there is provided a method of using the skin care compositions described above to protect against UV-induced mtDNA damage. Such methods involve topically administering the skin care compositions to the skin. In an embodiment, the skin is mammalian skin, such as human skin.

[0081] The skin care composition may be a topical composition.

[0082] The skin care composition may be in the form of a cream, oil, ointment, lotion, gel or spray.

[0083] The skin care composition may comprise additional UV protection or UV filtering additives which can work in conjunction with the additive of the invention to provide broad UV protection. Advantageously, mangiferin has been demonstrated to show good absorbance in the UVA spectrum, where traditionally, suncream ingredients have shown poor protection.

[0084] The invention also relates to a method of preparing an additive for a skin care composition, the method comprising:

[0085] preparing a solution of β-cyclodextrin or a β-cyclodextrin derivative in water;

[0086] adding mangiferin to the solution;

[0087] allowing the complex to form at a temperature between 30° C. and 50° C. for between 3 to 6 hours; and

[0088] removing solids by filtration.

[0089] Surprisingly, the inventors have determined that a longer reaction/complexation time, does not lead to any increase in solubility for the resultant complex. Accordingly, when the complexation reaction is allowed to take place over a 3 to 6 hour period, this increases the efficiency of the process, particularly from a commercial perspective.

[0090] In an embodiment, the step of preparing a solution of β-cyclodextrin or a β-cyclodextrin derivative in water comprises preparing a solution comprising β-cyclodextrin or a β-cyclodextrin derivative at approximately its solubility limit. In an embodiment, the β-cyclodextrin or a β-cyclodextrin derivative is added at a concentration of from 1 to 3% (w/w).

[0091] In an embodiment, the step of adding mangiferin to the solution comprises adding mangiferin at, or approximately at, its solubility limit in water. In an embodiment, mangiferin is added at a concentration of from 0.1-0.3% (w/w).

[0092] Adding the β-cyclodextrin/β-cyclodextrin derivative and/or the mangiferin at, or approximately at their solubility limits, reduces the level of unreacted compounds in the mixture after complexation and results in a more efficient process. This is particularly useful for mangiferin, which is an expensive product, and further increases its viability as a cosmetic ingredient.

[0093] In an embodiment, the β-cyclodextrin or β-cyclodextrin derivative is β-cyclodextrin.

[0094] In an embodiment, the β-cyclodextrin or β-cyclodextrin derivative is a β-cyclodextrin derivative. The β-cyclodextrin derivative can be selected from hydroxypropyl-β-cyclodextrin, sulfobutylether β-cyclodextrin and mono (6-ethylene-diamino-6-deoxy)-β-cyclodextrin.

[0095] In an embodiment, the step of allowing the complex to form includes stirring of the solution. Preferably the stirring is continuous. Optionally, the stirring takes place at intervals.

[0096] The invention also relates to a method of preparing a skin care composition comprising: [0097] preparing a water phase comprising the additive described above; [0098] preparing an oil phase; [0099] adding the water phase to the oil phase to form an initial water-in-oil emulsion and subsequently an oil-in-water emulsion.

[0100] The emulsion may be prepared using an “inversion” method. In this method, a water-in-oil emulsion is created initially after a small amount of the water phase has been added to the oil phase. As further water is added, the emulsion inverts to an oil-in-water emulsion. This method is preferable for achieving a small droplet size. The small droplets are formed at the inversion point, and therefore the water should be added relatively slowly, preferably dropwise, until this point is passed.

[0101] In an embodiment, the emulsion is subjected to at least one microfluidisation step.

[0102] Microfluidisation is a technique used to reduce particle sizes in emulsions. During the microfluidisation step, high pressures are applied to force a liquid through a chamber of microchannels.

[0103] The microfluidisation step may be performed at a pressure of from 5,000 to 15,000 psi.

[0104] The emulsion may have a droplet size of from 100-120 nm.

[0105] Droplet size may be measured using a laser diffraction particle sizer.

EXAMPLES

Materials

[0106] Mangiferin (Sigma Aldrich)

[0107] β-cyclodextrin (Sigma Aldrich);

[0108] Isohexadecane emollient oil (Arlamol HD™ from Croda™);

[0109] Polysorbate 60 non-ionic surfactant (Tween 60™ from Croda™);

[0110] Polysorbate 61 non-ionic surfactant (Tween 61™ from Croda™);

[0111] Sodium stearoyl glutamate ionic surfactant (Eumulgin SG™ from Cornelius (distributed by BASF UK);

[0112] PEG-8 solvent (Pluracare E400 SG™ from Cornelius);

[0113] Octocrylene organic sunscreen (MfSorb 104™ from Aston Chemicals or Surfacare PCE597™ from Surfachem™);

[0114] Avobenzone organic sunscreen (Mfsorb 502™ from Aston Chemicals);

[0115] Phenoxyethanol preservative (Preserve PCG 50% phenoxyethanol in Caprylyl glycol from Gracefruit);

[0116] Vitamin E antioxidant (from Gracefruit);

[0117] Titanium dioxide inorganic sunscreen 40wt % dispersed in water (Solaveil™ CT12W from Croda™)

[0118] The following examples are included by way of illustration only, and are not intended to be limiting on the invention, the scope of which is defined by the appended claims.

Example 1: Cell Viability Assay for Varying Concentrations of Mangiferin Extract

[0119] Stock solutions of mangiferin were made by dissolving mangiferin extract in water, and diluting to the required concentration. The stock solutions were then added to HDFn cells at a range of concentrations from 50 μg/ml to 50,000 μg/ml, and incubated for 24 hours under standard culture conditions. Viability was then analysed using an MTS cell proliferation assay, and the results are shown in FIG. 2. The differences in viability between the control (no extract) and the different extract concentrations were compared to determine significance (FIG. 2 shows n=2 biological repeats, of 8 technical repeats each).

[0120] The results illustrate cell viability up to concentrations of .sup.˜5,000 μg/ml, above which concentration cell death was observed.

[0121] While the results shown are for unfiltered extract with HDFn cells, similar results were observed when the mangiferin was filtered, and also when the mangiferin extract was tested on primary human fibroblast cells grown directly from the skin of donors.

Example 2: Analysis of mtDNA Damage in Human Skin Fibroblasts and HDFn Cells Following UV Radiation

[0122] mtDNA damage in primary human skin fibroblasts and HDFn cells was determined by qPCR in primary human skin cells and in a human dermal cell line (HDFn) following UV irradiation with a Cleo Performance lamp (iSOLde, Germany). mtDNA damage was found to be induced the most in HDFn. As illustrated in FIG. 3, primary fibroblasts showed an increase in mtDNA damage of 1.6 Cts following UV irradiation at 2 standard erythemal doses (SED) compared to the foil-covered control cells. HDFn cells showed a difference in mtDNA damage of 3.1 Cts following irradiation. Since a 1 Ct difference is equivalent to a 2-fold difference in damage, the primary fibroblast cells showed .sup.˜3-fold more damage (2{circumflex over ( )}1.6), whereas the HDFn cells showed a much larger increase in damage of .sup.˜8-fold (2{circumflex over ( )}3.1). HDFn cells were therefore chosen to assess the protective effect of mangiferin extract in further studies.

Example 3: Analysis of Protective Effect of Mangiferin Extract on mtDNA Damage in HDFn Cells

[0123] 1,000 μg/ml solutions of mangiferin extract were prepared as outlined in Example 1 above. 3 ml of the mangiferin solution was added to a 45 mm diameter quartz glass petri dish. Quartz was chosen as it is known to permit both UVA and UVB rays to pass through. The quartz glass dish was then placed on top of a slightly smaller dish containing the cultured HDFn cells. The cells were then irradiated through the quartz glass petri dish, using either a Cleo Performance (iSOLde, Germany) or Aramid B (Cosmedico, Germany) lamp at 2 standard erythemal doses (SED). In order to assess damage caused by irradiation, controls were carried out with water in the quartz glass dish, and by providing non-irradiated cell dishes wrapped in foil to protect against UV-induced DNA damage.

[0124] Following irradiation, total DNA was extracted and mtDNA damage analysed using a 1 kb real-time qPCR assay. mtDNA damage is expressed as a Ct value (where a 1 Ct difference is equivalent to a 2-fold difference in damage), and is based on the observation that damaged mtDNA takes longer to be amplified via qPCR. To find the acellular UV transmission of extracts, the dish containing the diluted extract was placed on top of a spectroradiometer and the amount of UV which passes through the dish was measured. All statistical differences were determined using one-way ANOVA with Dunnett's test to compare to a control column (0.05*, P<0.01**, P<0.001***).

[0125] As illustrated in FIG. 4, mangiferin extract showed significant prevention of mtDNA damage. The difference between the foil-covered cells and the water control was 3.1 Cts, or an approximately 8-fold increase in damage. In the presence of mangiferin, this increase in damage was reduced to 1.6 Cts, or an approximately 3-fold increase in damage compared with the foil-wrapped samples (i.e. no irradiation). This study therefore indicates that mangiferin extract may have excellent UV preventative properties.

Example 4: Comparison of Protective Effect of Mangiferin Extract on mtDNA Damage With That Provided by SPF15 and SPF30 Products

[0126] To determine the efficacy of mangiferin extract in comparison with known UV-protection compounds, the experiments of Example 3 were repeated using SPF0, SPF15 and SPF30 cream products. The results of this study are shown in FIG. 5 and suggest that mangiferin extract may provide similar protective effects to known commercial products.

Example 5: UV Absorbance of Mangiferin Extract

[0127] A UV absorbance scan was performed on mangiferin extract at a concentration of 1,000 μg/ml, and the results are shown in FIG. 6. The spectrometer was set up using a quartz cell with a 1.05 ms integration time (the minimum for the spectrometer) and the spectra was based on an average of 100 samples. Mangiferin shows peak absorbance in the UVA region, with absorbance peaks at .sup.˜320 nm and .sup.˜370 nm. This strongly suggests utility in UV protection of human skin, since the majority (96%) of UV rays that reach the earth's surface are within the UVA spectrum, compared with UVB (only 4%).

[0128] Example 6: Solubility of Mangiferin

[0129] In order to assess the solubility of mangiferin in aqueous solution, 0.18 g of mangiferin was added to 100 mls of deionised water. The solution was heated to 40° C. and stirred at 500 rpm for 4 hours or for 12 hours. The solution was then Buchner filtered, and the mass of the undissolved material weighed. Solubility was calculated based on the difference between the initial mass of mangiferin and that of the undissolved material. The results are shown in Table 1, and indicate that mangiferin is very insoluble in water, even after stirring for 24 hours.

TABLE-US-00001 TABLE 1 Solubility of mangiferin Stirring Initial Mass of Mass of Solubility time mass of undissolved mangiferin of (40° C., mangiferin material in solution mangiferin Precipitate 500 rpm) (g) (g) (g) (g/L) formed  4 hours 0.1826 0.1783 0.0043 0.043 Very small amount of yellow precipitate (<01%) 24 hours 0.1831 0.1692 0.0139 0.139 Yellow precipitate (<0.1%)

Example 7: Preparation of Mangiferin-β-Cyclodextrin Complex

[0130] A mangiferin-β-cyclodextrin complex was prepared as follows. 1.85 g of β-cyclodextrin was added to 100 ml of deionised water and stirred at room temperature until dissolution of all of the solids had occurred. 0.18 g of mangiferin was added to the β-cyclodextrin solution and the solution was stirred at 500 rpm for 4 hours. The solution was filtered using Buchner filtration. Formation of the mangiferin-β-cyclodextrin complex was confirmed by FTIR.

Example 8: Solubility of Mangiferin-β-Cyclodextrin Complex

[0131] In order to assess the solubility of the mangiferin-β-cyclodextrin complex in aqueous solution, 1.85 g of β-cyclodextrin was dissolved in 100 ml of deionised water at 40° C. .sup.˜0.18 g of mangiferin was added to the β-cyclodextrin solution, and the solution was stirred at 500 rpm for 4 or 24 hours at 40° C. The solution was then Buchner filtered at 40° C. (Examples 1, 2, 6 and 7), hot filtered (Example 3), cold filtered (Example 4), or unfiltered (Example 5). Example 6 used β-cyclodextrin hydrate, and Example 7 studied the effect of using a co-solvent. Solubility was calculated based on the difference between the initial mass of mangiferin and β-cyclodextrin and that of the undissolved material. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Solubility of mangiferin-β-cyclodextrin complex Stirring time Initial Mass of Volume (500 Stirring mass of residual of Solubility Ex. rpm) temp. Filtration MGN MGN water of MGN Precipitate No. hours ° C. temp (g) (g) (ml) (g/l) formed 1 4 40 40 0.1837 0.1064 100 0.773 Clear/white precipitate (<1%) 2 24 40 40 0.1850 0.1153 100 0.697 Clear/white precipitate (<1%) 3 4 60 Hot 0.0912 0.0575  50 0.674 Clear precipitate (<1%) 4 4 60 cold 0.0912 0.0503  50 0.818 Very narrow supernatant yellow insoluble (<1%) 5 4 40 NONE 0.0695 n/a 100 0.0695 Dark solids suspended in solution (impurities) small amount of clear precipitate (<1%) 6* 4 40 40 0.1814 0.1195 100 0.619 Clear precipitate (<1%) 7** 4 40 40 0.1850 0.1233 100 0.617 None *cyclodextrin hydrate used instead of cyclodextrin **10:90 Ethanol:water used instead of water

[0132] These results indicate that the complexed mangiferin is .sup.˜18-fold more soluble than mangiferin on its own over a 4-hour complexation period (i.e. from 0.043 g/l to 0.773 g/l). A .sup.˜5.6 fold increase was observed over the 24 hour solubility of mangiferin (i.e. from 0.139 g/l to 0.773 g/l). An increased complexation time did not result in an increase in solubility. An increase in temperature (i.e. from 40° C. to 60° C.) also did not result in increased solubility (in fact a minor insignificant decrease was observed).

Example 9: UV Absorbance of Mangiferin-β-Cyclodextrin Complex

[0133] UV absorbance scans were performed on stock solutions of the mangiferin-complexes prepared in example 8 above and the results are shown in FIG. 7. The spectrometer was set up using a quartz cell with a 1.05 ms integration time (the minimum for the spectrometer) and the spectra was based on an average of 100 samples. The mangiferin-only solution required to be diluted to 25% of its original concentration to reach the point at which it no longer saturated the spectrometer, while mangiferin-complex solutions were diluted to 2.5%. However, good absorbance was then observed for all of the samples, with the characteristic peaks at .sup.˜320 nm and .sup.˜370 nm clearly visible.

Example 10: Preparation of Mangiferin-Containing Sunscreen Formulation

10.1 Mangiferin Encapsulated in β-Cyclodextrin

[0134] A water bath was set up on a hot plate on a lab jack with a temperature probe controlling the bath temperature to 40° C. A stirrer bar was placed into the water bath and set to 400 rpm.

[0135] A 1,000 ml beaker was filled with deionised water (500 ml) and clamped above the water bath. A stirrer bar was added to the beaker. The hotplate and bath were then raised up to the beaker to submerge it to the bath close to the fill line. The stirring speed was kept at 400 rpm. β-cyclodextrin (9.00 g) was added to the beaker, and allowed to fully dissolve.

[0136] Once the β-cyclodextrin was fully dissolved, mangiferin (0.36 g) was added to the beaker. The beaker was then capped with aluminium foil and left to stir at 40° C. for 4 hours.

[0137] The beaker was then removed from the water bath, decanted into a 500 ml Duran bottle for use in the full formulation sunscreen, and left to cool to room temperature before being stored in the fridge.

10.1.1. Creation of the Oil Phase

[0138] The following were weighed out into a 500 ml beaker:

[0139] Octocrylene (66.75 g), Avobenzone (13.33 g), Isohexadecane (19.73 g), Polysorbate 60 (5.91 g),

[0140] Polysorbate 61 (10.52 g), Eumulgin SG (1.04 g), PCG (8.05 g) and Vitamin E (0.48 g).

[0141] The Polysorbates needed pre-warming to enable aliquots to be taken from their containers. The beaker was then placed into a stirrer water bath at 50° C. An overhead stirrer was then placed into the beaker and stirred at ca.200 rpm.

10.1.2. Creation of the Water Phase

[0142] The Mangiferin complex solution was filtered through a Whatman No1 filter using a Buchner filter. Mangiferin complex solution (154.52 g) was weighed out into a 200 ml beaker and PEG-8 (20.02 g) added. The beaker was then placed into a stirred water bath at 50° C. A magnetic stirrer bar was added to the beaker and stirred at 290 rpm.

10.1.3. Creation of the Emulsion

[0143] The emulsion was created using an ‘inversion’ method, i.e. a water-in-oil emulsion was created initially after a small amount of water had been added to the oil phase, with further water addition the emulsion inverted to an oil-in-water emulsion. The method is preferred over the ‘direct’ emulsion process (adding the oil phase to the water phase) in order to achieve small oil droplets. The small droplets are formed at the inversion point thus the water should be added relatively slowly until this point is passed (often indicated by an increase in the formulation viscosity). Hence the water phase was added to the oil phase (stirred at 433 rpm) in a steady stream of drops using a disposable pipette. After the addition was finished the emulsion was allowed to stir for 10 minutes. The emulsion was then transferred to the Silverson high shear mixer and mixed at 4,440 rpm for 15 minutes. A sample (.sup.˜15 ml) was then taken to determine the effect of subsequent micro-fluidisation on particle size.

[0144] The emulsion was then transferred to the high pressure homogeniser (Microfluidizer from Microfluidics Corp.) where it passed through the equipment at 10,000 psi. 3 samples of approx. 50 ml were collected before the equipment blocked during collection of the 4th sample. These samples, S1 to S3, were subjected to particle size analysis in Example 11 below. It was visually observed that after being passed through the micro fluidizer the white emulsion of the mangiferin-containing sunscreen formulation gained a blue tinge around the edges of the solution. This was an indication that the microfluidizer had reduced the particle size to be comparable with visible wavelengths.

10.2 Water-based Sunscreen Formulation (Mangiferin-Free, Reference Formulation)

[0145] A reference formulation, in which the mangiferin complex was replaced with deionised water, was prepared as follows:

10.2.1. Oil Phase

[0146] The following were weighed out into a 500 ml beaker:

[0147] Octocrylene (66.71 g), Avobenzone (13.37 g), Isohexadecane (19.90 g), Polysorbate 60 (6.03 g),

[0148] Polysorbate 61 (10.03 g), Eumulgin SG (1.05 g), PCG (7.88 g), and Vitamin E (0.49 g).

10.2.2. Water Phase

[0149] Water (154.52 g) was weighed out into a 200 ml beaker and PEG-8 (20.03 g) added.

10.2.3. Creation of the Emulsion

[0150] The same process as described in Example 10.1 above was adopted. In this case no Microfluidizer blockage occurred, enabling 6 samples of approximately 50 ml each to be collected (Samples S5 to S10). Samples S5, S6 and S7 were passed through the microfluidizer a second time at 10,000 psi., and collected as 3 samples (numbered S11 to S13).

10.2.4. Addition of TiO.SUB.2

[0151] Sample 8 from the microfluidizer (50.66 g) was taken and weighed into a clean glass jar. Solaveil™ CT 12 W (5.63 g) was weighed out into the formulated sunscreen. The two were then mixed at 4500 rpm for 2 minutes on the Silverson high shear mixer.

Example 11: Particle Size Analysis

[0152] The particle size of the droplets prepared in Example 10.1.3 (i.e. S1 to S3) was analysed using a Horiba LA950 laser diffraction particle sizer. The refractive index used was 1.564: this is a weighted average of Octocrylene (1.567) and Avobenzone (1.546) based upon their mass within the formulation.

[0153] The microfluidisation was shown to reduce particle size to between 100-120 nm. This compares favourably with the average particle size in leading commercial products, which is typically of the order of ca. 130 nm. The reduction in particle size resulted in the edges of the white emulsion having a blue tint to them. Reduced particle size is typically associated with improved sensorial feel and a semi-transparent to transparent look on a user's skin, both of which are highly advantageous properties for sunscreens or cosmetic formulations, and therefore the reduced particle size of the mangiferin-containing sunscreen formulation is highly beneficial.

[0154] The particle size analysis was repeated for the water-based sunscreen formulation (mangiferin-free, reference formulation) prepared in Example 10.2 above. The results were similar to those obtained for the mangiferin-containing formulation. The addition of titanium dioxide in Example 10.2.4 did not change the particle size distribution. A second pass on the micro fluidizer did not reduce the droplet size any further than what had already occurred after the first pass.

Example 12: Stability Testing

[0155] Stability testing was done at 40° C. and assessed both visually and instrumentally. Samples were placed into the ‘Turbiscan’ (Formulation TAGS) ageing station at 40° C. A spectrum of the transmitted and backscattered light was taken every 3 hours for 31 days. Some samples were left longer than the test period at 40° C. but no further data was collected. Table 3 below shows the tested samples, the time they were tested for and the results of the test.

TABLE-US-00003 TABLE 3 Results of stability testing at 40° C. Time of Stability Test Sample (Days) Visual observations Mangiferin-based sunscreen pre- 46 60% creamed emulsion microfluidiser Mangiferin-based sunscreen after 49 ca. 2% coalesced oil layer 1 pass on the micro fluidiser on top of water Mangiferin-free sunscreen pre- 32 75% creamed emulsion microfluidiser Mangiferin-free sunscreen after 1 32 No separation pass on the micro fluidiser Mangiferin-free sunscreen after 2 32 A good emulsion but starting passes on the micro fluidiser to form a coalesced oil droplet on top Mangiferin-free sunscreen after 1 32 A good emulsion but starting pass on the micro fluidizer and to form oil droplets on top addition of TiO.sub.2

[0156] The improvement in stability resulting from the microfluidisation was evident from a visual observation, with the larger droplets of the pre-microfluidised samples showing extensive creaming. Thus, the microfluidised samples were shown to exhibit generally good stability after prolonged periods at elevated temperatures. The mangiferin-based sunscreen formulations showed good initial colloidal stability although some coalescence was observed after a month at elevated temperatures.

Example 11: Seahorse Analyser

[0157] The Seahorse XFe96 analyser is utilised to monitor oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells in real time. OCR predominantly represents mitochondrial function with oxygen consumed as the final acceptor of electrons, vital in formation of cellular energy (ATP) via oxidative phosphorylation (Oxphos). ECAR represents the complimentary energy pathway of glycolysis, which has a significantly lower yield of ATP from glucose, in the absence of oxygen.

[0158] The aim of this experiment was to test the protective potential of mangiferin-based sunscreen formulations against solar radiation. Human dermal fibroblasts (HDFn) were plated in the Seahorse 96 well culture plates. Cells were irradiated with a solar simulator, delivering UV, visible and infrared light, replicative of the solar spectrum. A dose of 4.32 standard erythemal doses (SED) was given. Formulations in cream form were spread equally across semi-porous tape at a mass to area of 50 mg/cm.sup.2. Formulations were placed between light source and cells. Following irradiation, cells were bioenergetically analysed using a mitochondrial stress test assay monitored with the Seahorse analyser. The formulations tested were the mangiferin-based sunscreen formulation prepared in Example 10.1 (which was run as two separate tape preparations, as MGN-1 and MGN-2), the water-based sunscreen formulation (mangiferin-free, reference formulation) prepared in Example 10.2 (as AQ-1), and a leading commercial product (LCP-1), which comprised the following ingredients: Aqua/Water, Diisopropyl Sebacate, Alcohol Denat, Glycerin, Dimethicone, Isohexadecane, Butyl Methoxydibenzoylmethane, Octocrylene, Silica, Drometrizole Trisiloxane, Isononyl Isononanoate, Zea Mays Starch/Corn Starch, C12-C15 Alkyl Benzoate, Styrene/Acrylates Copolymer, Ethylhexyl Triazone, PEG-30 Dipolyhydroxystearate, Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine, Isododecane, Phenoxyethanol, Isopropyl Lauroyl Sarcosinate, Terephthalylidene Dicamphor Sulfonic Acid, Silica Silylate, Lauryl PEG/Ppg-18/18 Methicone, PEG-8 Laurate, Caprylyl Glycol, Triethanolamine, Disteardimonium Hectorite, Tocopherol, Disodium EDTA, Dodecene, Propylene Carbonate, Poloxamer 407, Zinc Gluconate, Perlite.

11.1 Basal Oxygen Consumption

[0159] The results of the basal oxygen consumption test are shown in FIG. 8. Whole cell total oxygen consumption is significantly reduced with no protection (tape control) (p=0.0003). All sunscreen formulations tested did not show a reduction in total oxygen consumption, indication solar protection. Data represents a single biological repeat (N=1) with multiple experimental repeats (n=4-6). The mangiferin sunscreen was tested as two separate tape preparations

11.2 Mitochondrial Respiration

[0160] Oxygen consumption is influenced by multiple cellular processes; however, mitochondria respiration is the predominant consumer. The level of decline in mitochondrial population can therefore be used as a marker of mitochondrial DNA damage. The results shown in FIG. 9 illustrate that all of the tested formulations showed moderate levels of decline. While mitochondrial specific respiration was not significantly reduced by 4.32 SED when mangiferin-based sunscreen was applied for one group (MGN-1), the second group (MGN-2) showed a significant level of mitochondrial OCR decline (p=0.0343). However, the leading commercial product (LCP) also displayed mitochondrial decline with a higher statistical significance value (p=0.0056).

11.3 Glycolytic Index

[0161] Glycolytic index represents the percentage of energy demands met by glycolysis for cells. As mitochondria are damaged, cells become reliant on glycolysis to meet energy demands, and therefore an increase in glycolysis can be used as an indicator of mitochondrial damage. The results of this experiment are shown in FIG. 10. The control (tape only) showed the most damage to mitochondria as cells become more glycolytic (p=0.0001). The only population with protection to show significant increase in glycolysis was the leading commercial product (p=0.0326). All other tested formulations suggest they have significantly blocked solar radiation to prevent mitochondrial damage and halt reliance on glycolysis.

[0162] The results of the various tests set out above confirm mangiferin as a useful extract in UV protection for human skin. The results further, and advantageously demonstrate that complexes of mangiferin with β-cyclodextrin or a β-cyclodextrin derivative protect against mtDNA damage, and as such, can be used as additives in skin care products, to protect against UV radiation and to provide positive effects such as anti-aging effects associated with a reduction or prevention of mtDNA damage. The complexation of the mangiferin with the β-cyclodextrin/β-cyclodextrin derivative, beneficially increases the availability of the mangiferin in the aqueous composition/aqueous phase, which retaining the mtDNA damage-preventing effects. Comparable particles sizes (i.e. 100-120 nm) compared with leading competitor products (typically .sup.˜130 nm), suggest ease of application to the skin with transparent/near transparent properties. These properties confirm that mangiferin can be used in the preparation of an economically-viable skin care product, such as a suncream, or other cosmetic product in which mtDNA damage prevention is desirable.

[0163] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.