The use of some nanoemulsions based on aqueous propolis and lycopene extract in the skin's protective mechanisms against UVA radiation
© Butnariu and Giuchici; licensee BioMed Central Ltd. 2011
Received: 23 November 2010
Accepted: 4 February 2011
Published: 4 February 2011
The use of natural products based on aqueous extract of propolis and lycopene in the skin's protective mechanisms against UVA radiation was evaluated by means of experimental acute inflammation on rat paw edema. The aim of the present study was to evaluate the harmlessness of propolis - lycopene system through evaluation of skin level changes and anti-inflammatory action. The regenerative and protective effect of the aqueous propolis and lycopene extract is based on its richness in biologically active substances such as: tocopherols, flavonoids, amino acids, polyunsaturated fatty acids, the chlorophyll pigment, all substances with strong antioxidant activity, that modify the oxidative stress, mainly by reducing the prooxidant processes and enhancing the antioxidant ones. These substances participate in the synthesis of prostaglandins and phospholipids components of cell membrane thus enhancing skin protection mechanisms.
The experimental systems offered a sustained release of the drug, in vitro, for aim eight hours. The prepared formulations aim did not reveal a deteriorating effect on tissues. They proved a better therapeutic efficiency Compared to standard suspension, they provided a better therapeutic efficiency coupled with extended time interval of tested parameters (24 hours). Preliminary examination of tissues showed that the experimental formulations did not irritate. Local application of propolis and lycopene aqueous extract nanoemulsion has a high potential both regarding its efficiency (the analgesic effect) and therapeutic safety.
This study demonstrates that propolis and lycopene extract nanoemulsions, preparations contains active substances, can confer better therapeutic effects than those of the conventional formulations, based on local control-release of dozed form, for a longer period of time, which probably improve its efficiency and skin acceptance, meaning a better compliance. The information obtained in the present study suggests that administration of propolis and lycopene aqueous extract nanoemulsion is safe. The preparation can be useful for further preclinical studies lycopene embedded in aqueous propolis extract to be used in pharmaceuticals (targeted medical therapy).
In recent years, it has been noticed that the incidence of skin cancer has increased alarmingly. Exposure to UV irradiation has instantaneous effects (erythema and pigmentation) and delayed effects (premature skin ageing and different forms of cancer) . UVB radiation has a stronger energy compared to UVA radiation and is absorbed directly by a series of cellular constituents, such as nucleic acids, proteins and urocanic acid. UVB radiation has also mutational effect . UVA radiation penetrates easily through epidermis and acts on its basal proliferative layer and even on blood components of the dermis [3, 4]. It acts indirectly on the cellular constituents, through oxidative mechanisms that forma reactive oxygen species [5, 6]. Reactive oxygen species have a relative short lifespan, nevertheless are highly reactive with the vast majority of cellular components: nucleic acids, proteins, lipids, polysaccharides. Frequently their action induces irreversible modifications [7, 8]. UVA radiation acts upon biological environments through oxidative mechanisms, correlated with the formation of reactive oxygen species: singlet oxygen, hydroxyl radicals, superoxide anions, hydrogen peroxide . Nucleic acids and proteins adsorb poorly radiation however but the initial event triggering biological effects is made up of absorption of UVA photons by different chromophores in the cellular environment such as: quinones, steroids, porphyrins, proteins with flavin coenzymes and heme group (cytochrome, peroxidase, catalase) . Many cellular components are targed by reactive oxygen species generated by UVA irradiation [11, 12].
Hydroxyl radicals react with almost all cell molecules types: carbohydrates, phospholipids, nucleotides, organic acids and amino acids. On enzymes, the effect of reactive oxygen species results in catalytic capacity reduction, often determined by sulphhydryl oxidation and modification of amino groups by malonylation . Organisms are protected against reactive oxygen species attack in several ways: cellular compartmentalization, protection afforded by antioxidant compounds and enzyme systems, their ability to develop adaptive responses inducible under oxidative stress conditions. Repair and turnover processes help to minimize these [14, 15]. Under normal circumstances there is a balance between antioxidant systems and reactive oxygen generative systems. Lack of balance in favor of prooxidant systems causes the apparition of oxidative stress, with pathological implications . Skin is the organ most exposed to solar radiation . Skin presents a series of structures with a protective role, such as stratum corneum and melanin. Superficial corneum layer functions as optical barrier by reflection, scattering and absorption of incident radiation. Larger part of UVA radiation penetrates deeply into the skin, to dermis . UVA radiation can be absorbed by different components of the blood, at the level of blood vessels. UVA radiation acts as inducer of enzymes responsible for polyamine synthesis. An additional mechanism of epidermis protection is to stimulate skin pigmentation with melanin . The protection mechanisms are established and inducible protections at the skin level. Inducible defence mechanisms were not identified at epidermis , but was identified in dermis, where increased heme oxygenase, which are correlated with an increase in ferritin levels . Pharmaceutical and cosmetics industries have launched a wide range of substances that act as filters capable of absorbing UV photons . Photoprotection products are characterized by the protection factor. An accurate assessment of the effectiveness of photoprotection products should be based on their ability to inhibit the isomerisation reaction of urocanic acid and prevent accumulation of the protein . "Quantum dot" nanostructures have been used (nanoparticles with quantum properties and ability to change size according to light emission). Another reason for the use of these products is their ability to "connect" many substances, thanks to a large surface area, and easy transport due to their small sizes (10 to 100 nanometers). These substances can also remain and accumulate preferentially at skin level, facilitated by surface drainage . Currently, nanomedicine is seen not only as a possible and promising path to an early and effective treatment, but also a possible way to prevent certain types of diseases .
Characterization of lycopene extract
Data correlation coefficients regarding the release of active constituents of propolis lycopene systems
k (% h-1/2)
k (% h-1)
formulation with 20% lycopene, 27% propolis, 53% water vol./vol (nanoemulsion 1)
After three weeks
formulation with 35% lycopene, 35% propolis, 30% water vol./vol (nanoemulsion 2)
After one week
After two weeks
After three weeks
The properties of the two experimental formulations
Micrometric properties of the two formulations tested
35.60 ± 1.33
51.94 ± 1.68
Density of the nanoemulsion (merged)
0.38 ± 0.03
0.41 ± 0.02
Density of the nanoemulsion
0.53 ± 0.07
0.42 ± 0.02
42 ± 1.3
68 ± 2.4
Each value represents the average (± SD) of three independent determinations.
The higher the SPF values, the more efficient the photoprotection
UVA radiation, the UVA/UVB ratio and nanoemulsion SPF with and without UVR-absorber
Nanoemulsion 1+absorber-UVR (Saliform)1:1 (v/v)
Nanoemulsion 2 +absorber-UVR (Saliform)1:1 (v/v)
Enzymatic activity of collagenase in the presence of nanoemulsions
Enzymatic activity (Units/mg of protein)
1.205 ± 0.001
1.033 ± 0.003
1.648 ± 0.007
Reaction conditions: T = 25°C, pH 7.5, λ = 345 nm, t = 5 min.
Activity of aqueous extract of propolis and lycopene assessed on induced mouse paw edema
Mean ± SE difference in right and left paw volumes (ml)
Reduction of edema (%)
0.147 ± 0.02
0.186 ± 0.11
0.246 ± 0.03
0.165 ± 0.01
0.039 ± 0.03
0.026 ± 0.10
0.020 ± 0.14
0.015 ± 0.004
0.019 ± 0.03
0.014 ± 0.01
0.011 ± 0.02
0.007 ± 0.005
0.013 ± 0.04
0.008 ± 0.02
0.003 ± 0.02
0.001 ± 0.00
Emulsions are a mixture of molecules in a combination of two liquids that keep their properties unaltered. This feature has been used in the delivery of poorly soluble drugs . Nanoemulsions have a greater capacity for micellar solubilisation compared to simple solutions and offer advantages in thermodynamic stability to unstable dispersions (suspensions), as can be produced with less energy input and have a greater shelf life . The nanoemulsions are systems with droplet sizes of approximately 45 μm, having surfactant ratios of 47/53 and respectively 70/30 of aqueous extract of propolis-lycopene. UVA absorbance with relative parameter 5 is manifested by nanoemulsion 2 +absorber-UVR (Saliform) 1:1 (v/v), in the case of the UVA/UVB ratio with relative parameter 0.82 by the same nanoemulsion and in the case of SPF with absolute parameter 10.9 by the nanoemulsion 2. Highest absorbance parameters can be assigned to nanoemulsion 2. Prepared formulations showed a pseudo-plastic rheology, under the influence of shear stress. It appears that viscosity is directly dependent on the propolis content of the formulation. Lycopene is insoluble in water; it can be dissolved only in organic solvents and oils [28–30]. Researchers have correlated the antioxidant function of lycopene (ability to protect cells and other body structures caused by oxidative damage) with the protection of DNA (our genetic material) inside the white blood cells .
White blood cells (WBC) are mediators of inflammation and the immune response. Unlike other food phytonutrients, whose effects have only been studied in animals, lycopene from tomatoes has been repeatedly studied in humans, where research has shown additional protection against many types of diseases [32, 33].
The experimental nanoemulsions in a high kinetic stability, and reduction in collagenase activity by 37.14% for a 70/30 surfactant ratio and respectively 26.81% for a 47/53 ratio. These nanoemulsions provided a sustained drug release in vitro for a period of 8 hours. Lycopene antioxidant as a nanoemulsion component beside its moisturizer characteristic improves the ability of the skin to defend against sunlight.
Aqueous extract of propolis
Aqueous extract of propolis was obtained by refluxing 100 g of propolis powder and 250 ml of double distilled water. It was concentrated in a water bath then filtered resulting in 1.5 cm3 of extract with 95% of dry substance.
Determination of lycopene
Lycopene was obtained from ripe tomatoes (Lycopersicon esculentum) by solvent extraction. Samples were homogenized in a laboratory homogenizer. 5ml 0.05% BHT in acetone, 5 ml of ethanol and 10 ml of hexane were added to 0.6 g homogenated sample. The supplemented homogenate was kept on ice and stirred with a magnetic stirrer for 15 minutes. Then 3 ml of deionized water were added and samples were mixed for additional 5 minutes. Samples were then left at room temperature for 5 minutes to allow phase separation. The absorption of the hexane layer (upper layer) was measured in a 1cm quartz cuvette at a wavelength of 503 nm, against hexane as blank. Lycopene was measured quantitatively by UV-VIS spectrophotometer T60U, PG Instruments Limited, UV WIN®version 5.05; detection was performed at 503 nm and calculated using the following formula:
Absorbance at 503 nm (A503) =
[Lycopene concentration (M)]
The measuring conditions were: scan speed 90 nm/min and an interval of 1 nm. After extraction, was hexane evaporated to dryness in a vacuum evaporator, under a nitrogen stream . All substances were purchased from Sigma Chemical.
Preparation of nanoemulsions
Nanoemulsions were prepared by adding lycopene to aqueous solution of propolis (50 mg propolis in 10 ml D.W.), using a magnetic stirrer at ~ 2000 rpm. The mixture was introduced for in an ultrasonic bath at 20 kHz 20 minutes. The nanoparticles that are formed have a lycopene-propolis loaded shape. After ultrasonic treatment the solution is brought at room temperature (22°C).
The excess organic solvent in excess was evaporated using a rotary evaporator and samples were kept for further analysis by lyophilisation. Independent of preparation temperature, samples were kept at 25°C .
Fourier transforms infrared spectrometry (FTIR)
For spectral characterization (FTIR spectrometry), samples were prepared as follows: compounds obtained after heat treatment were mixed at a temperature of 1300°C with potassium bromide powder, previously dried for 24 hours at a temperature of 120°C, at a mass ratio of 0.04:1. After a vigorous mix to obtain uniformisation, pills with a thickness of 0.5-0.75 mm and 13 mm in diameter at a pressure of 0.3 GPa in normal atmosphere were prepared. The pills were analyzed using the JASCO 660 PLUS spectrophotometer, which recorded the IR absorption spectra in the area 4000 cm-1 100-400 cm-1. For in vitro characterization of the experimental nanoemulsions, permeability studies and rheological measurements were carried out .
Membrane permeability of the experimental nanoemulsions was investigated by filling the donor compartment of a diffusion cell with a 2 g test mixture. All other experimental conditions identical to those described for permeability studies of solid systems .
Apparent viscosity of the experimental nanoemulsions was determined using a viscometer Brookfield Rheostress DV-III + Rheometer. Measurements were performed 3 times at 25°C using SC4 spindle. To determine the influence of shear stress applied to the microstructure of the prepared nanoemulsions, measurements were made at a rotation speed of 1 and 10 rpm.
Apparent viscosity of the controlled product (2% HPMC dispersion in water), were examined under similar conditions .
Enzyme activity measurements
Enzyme activity is determined by a continuous spectrophotometric method, using as substrate 2-L-leucylglycyl furanacryloyl-L-prolyl-L-alanine (FALGPA, a specific collagenase substrate), as it is preferentially hydrolyzed much faster than other synthetic substrates.
Measurement of the substrate absorbance decrease was done at 345 nm.
Pharmacological evaluation of formulations
Identification and quantification are done according to the methods described in the European Directorate for the Quality of Medicines .
Preparation: adult, young, healthy animals of the species of guinea pigs, breed albino were used. The animals were acclimatized to laboratory conditions for at least five days before test. Animals were divided randomly into treatment and control groups before test. Their skin was cleaned by clipping, shaving or, if possible, by chemical depilation without excoriation (cleaning method is based on the test method used). Animals have been weighed before and after test.
Superficial skin burns were induced using a UV radiation lamp.
Testing the anti-inflammatory action
Assessment of anti-inflammatory action was achieved by evaluating the inhibition of rat paw edema induced by a 2% solution of carrageenan.
The percentage inhibition of edema was calculated using the following equation:
The edema inhibition rate of each group was calculated as follows:
Monitoring and staging: approximately 21 hours after the patch removal, hair is cleared off the surface exposed to challenge concentration. After 3 hours (about 30 hours after the challenge patch application) skin reactions were observed and recorded. After an additional time of 24 hours (54 hours) skin reactions were observed and recorded again. "Blind" reading is recommended for tested and control animals. All skin reactions and any unusual results, including systemic reactions caused by induction and challenge procedures were observed and recorded in accordance with the Magnusson/Kligman staging. If any of the reactions are difficult to interpret, other procedures can be taken into account, for instance histopathological examination or measurements of the skin fold.
Staging Magnusson/Kligman scale for assessing the post-challenge responses: 0 = no visible change, 1 = erythema or discrete form of spot, 2 = moderate and confluent erythema, 3 = intense erythema and swelling 
Determination of in vitro sun protection factor (SPF)
Determination of SPF (sun protection factor) was performed using a spectrophotometer, equipped with an integrating sphere, with appropriate software and a TRANSPOR 3 TM support, with a composition similar to that of natural skin, on which the amount of 2 mg/cm2 of nanoemulsion was applied .
Values were expressed as mean ± S.D. Statistical significance was evaluated by Students-„t‟ test at 5% level of significance (p < 0.05).
The authors would like to thank the European regional development fund (ERDF) to finance project "Environment-Biochemical Cooperation for prognosis of natural water and soil pollution in the Hungarian and Romanian cross-border region to Shun Catastrophe" acronym "R & D SZTE, BAÁE, no. HURO/0801/038".
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