Development and Preliminary Cosmetic Potential Evaluation of Melaleuca alternifolia cheel ( Myrtaceae ) Oil and Resveratrol for Oily Skin

Background: Oily skin presents shine in excess, as well as increased pores and acne. For this reason, people with oily skin have more difficulty using cosmetics in general. This is the first report in literature to evaluate amulti-purpose dermatological emulsion containing Melaleuca alternifolia Cheel (Myrtaceae) (tea tree) oil and resveratrol for oily skin. Objectives: This study aimed to develop and evaluate the potential of a cosmetic sunscreen formulation for acne-prone skin containing tea tree oil and resveratrol in volunteers living in Zona da Mata Mineira. Methods: For photoprotection, in vitro determination tests were performed for Sun Protection Factor (SPF) and UVA Protection Factor (UVAPF). In addition, in vivo tests were carried out for oiliness, hydration, porphyrins, pores and flaking in human volunteers. Results: Most of the volunteers showed improvement in oiliness, porphyrins, hydratation and desquamation by the use of resveratrol and tea tree oil. From the analysis, we obtained the SPF = 15.23 ± 5 76, UVAPF = 9.0 ± 1.6, critical wavelength = 379 nm, and the UVA/UVB ratios were: initial = 0.82 and final = 0.81. Conclusion: The developed product presented moderate UVA and UVB photoprotection (4-stars by Boots Star rating). It also seems to act as an element in the reduction of P. acnes by multiple mechanisms, envolving reduction of oiliness, hydration, desquamation and pores sizes.

show signs as excess shine, enlarged pores and acne lesions.For this reason, people with this type of skin have more difficulty using cosmetic products, such as sunscreens [1].
As sunscreens play an important role in the prevention of skin cancer and sun protection, their use in all ages and all types of skin is essential.The development of a sunscreen with high UVA/UVB protection for oily skin is a challenge, since its composition must be specific to this type of skin [2].
We glimpsed an opportunity to carry out this study and aimed to evaluate the effectiveness of an innovative sunscreen for oily skin.In this regard, we selected the raw material of natural origin resveratrol, whose photoprotection had already been described in prior work, besides being an important antioxidant, which has a variable anticancer activity, it also suppresses, slows or reverses the deleterious effects of ultraviolet radiation [3,4].

Introduction
Among the types of skin, oily skin needs special care, due to the amount of sebum, mainly on the face.Oily skin may sometimes Check for updates acquisition of the second UV spectrum, and then the final UVAPF, the UVA/UVB Ratio and the Critical Wavelength were calculated.Theoretical background can be found in Polonini, et al. [3].

In vivo determination of cosmetic activities
Volunteers: The present study investigated five volunteers residing in the Zona da Mata of Minas Gerais.Inclusion criteria were: a) healthy subjects; b) healthy skin in the test region; c) agreement to adhere to the procedures and requirements of the study; d) capacity to consent their participation in writing; e) individuals of both sexes and all ethnicities; f) aged between 18 and 40.
Exclusion criteria were: a) individuals who were using any skin product to control oiliness; b) individuals who have been making use of sunscreen; c) individuals who have declared allergic reaction to components of the formula to be used; d) individuals who had been treated by topical or systemic medication for acne in the 30 days preceding the start of the research.
All study participants used the sunscreen with best results in the in vitro assay for 30 consecutive days (12h/12h regimen, at 7 am and 19h).The overnight use is justified due to the importance of protection to visible light that are emitted by TVs, mobile phones and laptops.
This study was approved by the Ethics Committee of the Faculdade de CiênciasMédicas da Saúde de Juiz deFora (Suprema) under the number: 1,424.117.

Oiliness, desquamation and pores:
In order to determine the oiliness, a Visioscope PC 35 ® (CK Electronic, Germany) that has the function of making visual inspection of any part of the skin was used.To analyze the pores and to quantify oiliness of the skin, we used special Sebufix ® adhesives; to quantify the desquamation, Corneofix ® adhesives.We visualized regions of the right cheek and forehead, and the quantification was then performed with the aid of the software Complete Skin Investigation (CSI) (CK Electronic, Germany).
Porphyrins: For the determination of porphyrins, we used Visiopor ® N PP34 equipment (CK Electronic, Germany).We investigated the forehead, cheeks (left and right) and nose.The device was placed in contact with the skin, the image was captured, and then the analysis was made by the Visiopor software (CK Electronic, Germany).

Hydration:
The determination of skin hydration was made using a VapoMeter (Delfin, Finland) after allowing its contact with the skin for 10 seconds.The regions evaluated were the forehead and the right and left cheeks.

Results and Discussion
The oiliness of the skin is caused by excessive production of sebum by the sebaceous glands, which are usually found in association with a hair follicle -which, together with the gland is referred to as a pilosebaceous unit [1].The oily skin causes clinical signs that bother and greatly influence the lives of people, and the excess of skin oil can cause from psychological discomforts to skin problems such as acne [8].

Formulation
The proposed formulation is a gel-cream, with composition described in table 1.
The compounding processes for the gel-creams were similar to all formulations: 1.The components of Phase 3 were weighed crushed in a mortar and then homogenized.
2. The components of Phase 2 were weighed and mixed to homogeneize.
3. The tea tree oil was added to Phase 3 and mixed to homogenize. 4. Phase 2 was poured into Phase 3.
5. The Sepigel ® was weighed and added to a mortar.
6.The mixture of Phases 2 and 3 was poured slowly and gradually over Sepigel ® , under constant agitation.7. Chemynol ® was added to Step 6, and then the product was homogenized once again.
8. The product was packaged in aluminum tubes.

In vitro determination of the photoprotective potential
The photoprotection assay followed the protocol described by Colipaand recommended by Brazilian guidelines [7,8].The samples were accurately and quickly weighed (to reduce product evaporation and dryness) to satisfy the application rate of 1.3 mg cm -2 in each PMMA plate (actual quantity applied: 32.5 mg, determined by weighing the plates before and immediately after applying the products).They were directly weighed on the plate surface, applied as a large number of small droplets of approximately equal mass, and distributed in an even manner on the roughened surface of the plate.The products were then spread over the whole surface with a fingertip covered with a vinyl glove and pre-saturated with the product to prevent possible losses of the amount weighed.For each product, three plates were kept protected from light exposure in a dark chamber at room temperature (≈20°C) for 15 minutes.After this period, the plates containing the product were placed in the light-path of the transmittance analyzer.The transmission of UV radiation through the sample was measured from 290 to 450 nm at 1 nm intervals at 9 different sites of each plate (total measurement area = 2.0 cm 2 ).The blank was prepared using the HD6 plates covered with 15-μL of glycerin, because of its non-fluorescence and UV transparency.For UVAPF, the plates were inserted into the UV irradiation source (temperature maintained below 40°C) and then exposed to a calculated UV dose.After that, new transmission measurements of the sunscreen samples were conducted for inflammatory response, which creates the classic acne comedones [10].Knowing how this bacterium is involved in the pathogenesis of acne has clinical implications in the treatment of the disease, as it provides a specific direction in order to reach the best results [9].
In the acne treatment, patients should use special products, in order to minimize the problem or solve it [9].One of the traditional products used is the tea tree oil that is obtained from the leaves of Melaleuca alternifolia, popularly known as tea tree, which can be found in Europe and South America [5].This oil has antiseptic, antifungal and natural parasiticide properties; in addition, it is extremely effective in combating a wide variety of micro-organisms, including P. acnes [5,11].The responsible compounds for this action are their chemical constituents, namely: 1,8-cineole, 1-terpinen-4ol, rho-cymene, linalool, alpha-terpinene, gamma-terpinene, alphaterpineol and terpinolene.The antimicrobial activity is directly related to terpinen-4-ol [5,11,12].
In this study, we used tea tree associated with resveratrol, which is a natural compound produced by some spermatophytes such as grape [10].It is a powerful antioxidant and anti-inflammatory compound and it has been shown to possess anti-neoplastic activity, as well as capacity to assist in the healing of wounds [13].It has recently been proven its inhibitory ability to some bacteria, including P.acnes [10,13].In this work, we used the oil as it was received from the manufacturer, so we could mimic the routine of a compounding pharmacy that would prepare the product containing it.This action is of great importance, since it is known that this material frequently suffers adulteration.
Finally, 10% of micronized zinc oxide was also used, as it has the ability to reflect and disperse the UVB, UVA and visible radiations through an opaque barrier which is formed by particles film on the skin [14].In a research conducted in MedLine, Scielo and Lilacs, we found no scientific evidence that it influences on skin oiliness.
From the SPF obtained (15.23 ± 5.76), it is suggested that the photoprotection obtained arises from the synergistic action of F4 components, because of the results found for the other formulations that contained isolated active constituents.For F3, the result is corroborated by Sunscreen Simulator tool (BASF Pharmaceuticals, www.sunscreensimulator.com), which states that this substance in this concentration possesses an SPF = 4.7.For its turn, the isolated resveratrol in formulation F2 shows SPF = 7, which is in accordance to a previous study [3].It was also expected that the resveratrol did not suffer UV-degradation, as the results in F4 (pre-and post-UV irradiation) did not considerably varied.UVA protection was acceptable, when compared to SPF, as the results were higher than 1/3 of the SPF [15].The critical wavelength (λc) is another parameter used to evaluate the total capacity of the UVA protection of the product, and it should be greater than 370 nm [15].The obtained value was 379 nm, which allows one to affirm that the formulation has broad spectrum protection [7].
The UVA / UVB ratio can also be used to provide the so-called Boots Star Rating, which classifies the level of photoprotection of a product, from 0 to 5 stars.Since it takes into account the results of photoprotection before and after exposure to a radiation source, this classification is important to determine whether the photoprotection values generated are stable, since the components in the formulation can degrade over time and then changes mayoccurin the photoprotective action after irradiation [16].By Boots Star criteria, the sunscreen received a rating of 4 out of 5 stars.
In the article of Polonini, et al. [3] the results obtained for resveratrol, alone in a formulation, were different from the results found in this study, though the resveratrol was not tested by itself in the formulation.The result in all parameters (critical wavelength, SPF, PFUVA and UVA/UVB ratio) obtained in this formulation were higher than the values found by these authors.When compared to the study of Mercurio [1], which used Anacardiumoccidentale in the formulation and performed the same tests, our result were very different but better, since they were higher.Therefore, it means that the formulation developed in this study offers more protection to the skin.

In vivo assays
Subclinical presence of P.acnes bacteria is responsible for producing inflammatory mediators, free fatty acids and porphyrins.These porphyrns, when exposed to UV light, emit strong orange-red fluorescence in the follicle openings, thus permitting a visualization and evaluation [17].The result of the evaluation of porphyrins for    each volunteer can be seen in table 3. Figure 1 shows the images of the nose area of a porphyrin of volunteers on days 0 and 30 to illustrate typical test results.
The results were conflicting depending on the analyzed area.The nose showed decreased amounts with treatment (e.g., 101 to 58, or 42.57% in the volunteer 4).For the cheeks, from 50 to 30, corresponding to 40% decrease on the left cheek, and from 43 to 30, a 30.23%decrease on the right one).The forehead porphyrins decreased up to 25.45% (in the volunteer 4).However, the volunteer 3 had a significant increase (an average of 48.58%) in all the analyzed areas.
A smaller number of P.acnes can result in a lower probability of developing acne since it is a causative factor of the disease, but it is not the sole cause.One of the factors for the development of acne is the oiliness, which was the second parameter to be analyzed.The results found during 30 days of continuous use of the sunscreen formulation of each subject are presented in table 4. In figure 2, a graphical result can be seen.
Volunteer 3 decreased by 14.29% his oiliness on the forehead, and 78.05% in the cheek.Volunteers 1, 2 and 5 maintained their oiliness values in at least one of the analyzed areas, suggesting that the product is likely not to increase the production of sebum of the skin even in the presence of oil (tea tree oil) in the formulation.
However, an increase in sebum content in the regions analyzed on volunteers 4 and 5 was observed during the period of 30 days.This effect may have occurred because of a change in temperature conditions on the weeks of the original measures and final measures during the study, since the increased production of sebum may be due to seasonal temperature variations [18].
Quantification of pores was also carried out, since these are directly related to the sebum, which is a very common feature in patients that have oily skin.The results of individual volunteers are shown in table 5. Figure 3 shows the images of the pores of a volunteer on day 0 and on day 30 the area of the right cheek.
After application of the product for 30 days, we could notice a decrease in the values (size) of the pores, it was significantly in at least one of the two face area of all the volunteers (for instance, volunteer 3 showed 43.9% smaller pores in the left cheek after treatment), which demonstrates the benefit of the application of the formulation during the day.
Regarding the quantification of skin desquamation level, it was   These values show that most of the patients showed a reduction in skin desquamation.In volunteer 3, the difference was 13.22% and 0.82% on the forehead on the right cheek, respectively; in volunteer 4, this difference was 0.47% and 9.38%; and in volunteer 5, 10.94% and 9.21%.For his turn, the volunteer 2 showed increments of 5.59% and 11.29% in the same areas.In an overview, this result proves to ) = Percentage of the area covered with the fluorescence of porphyrins (red / orange) and other substances (green or yellow color), Quantity: Number of fluorescent spots, porphyrins, in the analyzed area.

Figure 1 :
Figure 1: Porphyrins determination.A) Porphyrins on the nose on day 0; B) Porphyrins on the nose on day 30 (both on the volunteer 4).

Figure 2 :
Figure 2: Typical graphic result for determination of oiliness.A) Test on day 0; B) Test on day 30; C) Right cheek on day 0; D) Right Cheek on 30.All images are from volunteer 3.

Table 4 :
Results of oiliness evaluation in human volunteers.

Figure 3 :
Figure 3: Results typical graph for determining the pores.A) Right Cheek on day 0; B) right cheek 30 on both the voluntary 3. The pores can be displayed by the green marking.

Table 1 :
Components used in the formulations.

Table 3 :
Results of the evaluation of porphyrins in human volunteers.

Table 5 :
Pore evaluation results in human volunteers.
observed a decrease in this feature in the volunteers, as it is shown in table 6 and figure4.

Table 6 :
Results of evaluation of desquamation on human volunteers.