Evaluation of Antioxidant and Anti-Inflammatory Effect of Rhododendron brachycarpum Extract Used in Skin Care Product by in Vitro and in Vivo Test

In this study, we aimed to observe the cytotoxicity, antioxidant and anti-inflammatory activity of Rhododendron brachycarpum extract (RbE) in RAW 264.7 macrophage cell in vitro and the antiinflammatory or soothing effect of RbE formulated cream (Multicalm-RHTM) on experimentally induced acute irritated skin in healthy subjects in vivo. In vitro study revealed that RbE significantly inhibited NO and ROS generation in a dose dependent manner without cytotoxicity. In clinical test, Multicalm-RHTM cream siginificantly reduced the SLS-enduced erythema after D5 compared to that of placebo. Overall, it can be concluded that the RbE is a non-cytotoxic, antioxidant and anti-irritant agent which can be used as a potent functional ingredient in cosmetics. Likewise, the in vivo clinical study shows that Multicalm-RHTM cream which contains 3% RbE can be used as an effective skin soothing agent.


Introduction
Envionmental exposure (UV radiation, pollutions, toxic chemicals and other hazardous agents) causes contact dermitites, halogen acne, chemical depigmentation, skin cancer and other acute and chronic skin damages [1].
Recently, to overcome this problem, the cosmetic industries and academic researchers have devoted much research toward the developement of various nautral skin care product from plant parts.
Plants are the natural source of functional ingrediants.Phytochemicals (secondary metabolites) from the plants are characterized for having antioxidant, anti-inflammatory, anti-carcenogenic, anti-aging and anti-bacterail properties [2].The effective plants antioxidant compounds such as tocopherols, flavonoids, phenolic acids, alkaloids, monoterpenes are widely used in cosmetic products to protect the skin from environmental damages [3].
Rhododendron brachycarpum G. Don belongs to the family Ericaceae which has been used in folk medicine for the treatment of diabetes, arthrities, headache and hypertension [4] [5].The plant is reported to have antimicrobial and antioxdant activity [6].Similarly, the plant also possesses anti-inflammatory, anti-tyrosinase and moisturizing effect [7] [8].This plant consists of important flavonoid compounds, in particular, quercetin, avivularin, quercetrin and hyperin [9].Previously, antioxidant and anti-inflammatory activity of Rhododendron brachycarpum extract (RbE) has been researched [10], however, no evidances of clinical effectiveness have been published.Recently, cosmetic company (Natural Solution Co. Ltd., South Korea) is using RbE as an active ingredients in soothing skin care products (Multicalm-RH TM ).Therefore, to examine the efficacy of the extract and its product, we aimed to observe the cytotoxicity, antioxidant and anti-inflammatory activity in RAW 264.7 macrophage cell in vitro and the soothing effect of RbE containing cream (Multicalm-RH TM ) on experimentally induced acute irritated skin in healthy subjects in vivo.

RbE Sample Preparation
Rhododendron brachycarpum G. Don (leaf and stem) was collected from the forest of Taebaek (650 m -700 m from sea level), Kangwon-Do province, South Korea.The samples were dried in oven at 45˚C for 5 days and ground to a fine powder.The powdered sample was extracted with 40% 1, 3-propanediol by ultrasonic extraction method for 3 days.The extracts were filtered and concentrated by evaporation under freeze-dried.The condensed sample was used in the experiment after dilution.

Evaluation of Antioxidant (ROS Inhibition) Activity in Vitro
Antioxidant was evaluated in reactive oxygen species (ROS) inhibition model according to the protocol of Wang et al. [13] with minor modification.Briefly, 1 × 10 4 cells/well were plated into 96-well plates 1 day before the experiments.On the day of the experiments, after removing the medium, different concentrations (0.5%, 1% and 2%) of sample were loaded and incubated for 1 hour in 5% CO 2 /95% air at 37˚C for 30 min.After removing the medium, the cells in the plates were washed with PBS buffer and treated with 20 mM dichlorofluorescin diacetate (DCFH-DA) and 1 mM H 2 O 2 (free radical generator) for 90 min.ROS levels were measured time-dependently (at intervals of 10 min) with an excitation wavelength of 485 nm and emission wavelength of 530 nm using fluorescence spectrophotometer.

Anti-Inflammatory (Soothing Effect) Clinical Test in Vivo
40 subjects (30 women and 10 men) aged 18 -65 years were participated in the research were provided written informed consent.None of the participant had the history of atopy dermatitis, allergies, any skin related diseases, pregnancy, any systemic and topical medication during the last 2 months.The study was a randomized, placebocontrolled which was approved by Guang Dong Light Industry Association Institutional Review Board [14].3% Sodium lauryl sulfate (SLS) was used as an irritant following the protocol of Tupker et al. [15].During the experiment, the patch testing procedure was same as reported previously in our other experiment [14].Briefly, large Finn chambers (+12 mm) with filter disc were fixed to mark test sites on the flexor side of fore arm in the subjects.On D0, normal skin was irritated with occlusive application of 3% SLS on flexor sides of both forearms for 24 hours.The next day (D1), Six hours after removing the patches, approximately 2 mg•cm −2 (40 mg) of cream or placebo was applied to the test site (treated twice a day) for 24 hours for 9 consecutive days.Erythema index was measured using Mexameter MX18 (Cologne, Germany) on D1 (before treatment), D3, D5, D7 and D9.The visual erythema scores were evaluated by dermatologist using the inflammatory severity which contains 10 scales: "0" indicated no erythema, whereas "9" indicated severe erythema.During the investigation, the room temperature and humidity was maintained at 22˚C ± 1˚C and 50% ± 3% respectively.

Statistical Analysis
Statistical analyses were carried out using SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA).The differences among samples were statistically evaluated via one-way analysis of variance (ANOVA) followed by Dunnett's posthoc test or Wilcoxon's test when appropriate.The level of significance was set at P < 0.05.Data are expressed as means ± standard errors.

Determination of Anti-Inflammatory, Antioxidant and Cytotoxicity Effect of RbE in Vitro
The anti-inflammatory effect of RbE tested on macrophage RAW 264.7 is shown in Figure 1.The result revealed that the RbE dose-dependently inhibited LPS-induced NO production.At the concentration of 0.5%, 1% and 2%, RbE inhibited NO up to 5.48%, 13.73% and 21.82% respectively.In ROS inhibition activity, RbE also inhibited in a dose-dependent manner.At the concentration of 1% and 2%, RbE inhibited ROS upto 17.09% and 26.42% respectively.Due to the significant (P < 0.05) inhibition activity of RbE in dose dependent manner on the NO and ROS generation, the cytoprotective effect of extract on LPS-induced cytotoxicity was investigated (Figure 2).According to the data, the extract did not induce cytotoxicity up to 2% concentration used in the experiment.These results suggested that the inhibition of ROS and NO production by RbE might be managed by radical scavenging activity.

Erythema Index and Visual Score
In this study, occlusive application of 3% SLS for 24 hours in the test fields resulted in a strong increase of skin erythema.Application of the cream (MultiCalm-RH) onto the test skin on D1 did not show any significant difference (P < 0.05) with placebo.However, the significant (P < 0.05) reduction of the erythema index compared with placebo were observed on D5 (Figure 3).The visual erythema scores of the sides treated by cream decreased after D3 (Figure 4).On D9, the visual erythema score of the cream was 2.46 lower than that of the placebo, and the erythema value of the cream was 49.75 lower than that of the placebo.

Discussion
It has been reported that ROS plays an important role in an acute skin inflammation [16].To reduce the inflam- * mation, application of moisturizers or ointment often assists in the improvement of irritant contact dermatitis [17].Recently, there is an increasing scientific interest of exploring plant ingredients as anti-inflammatory or soothing agents.Rhododendron barchycarpum plant extract (RbE) has been used as a soothing agent in skin care products.In our study, RbE inhibits LPS-induced NO production in dose-dependent manner.At the concentration of 2% dose, RbE inhibits NO production significantly with 26.42% compared with control.The dose dependent inhibition of NO by RbE can be due to the presence of bioactive compound like Rhododendrin in the extract.In previous research [10], the Rhododendrin isolated from Rb plant showed strong anti-inflammatory activity by inhibiting LPS induced NO production significantly (P < 0.05) and also inhibited iNOS protein and mRNA expression in a concentration dependent manner.According to our data in antioxidant assay, RbE inhibited ROS in dose dependent manner in dichlorofluorescein assay system.In cytotoxic assay, the extract does not induce cytotoxicity up to 2% of concentration used in the experiment.These all observations indicate that RbE is a significant antioxidant and anti-inflammatory agent without toxic effect in vitro.
According to new regulations for cosmetics, manufacturers of skin care products have to prove the efficacy of the product [18].An authentic data in a short time and easy application on the forearms of healthy subjects are highly desirable [19].Therefore, we evaluate the effects of MultiCalm-RH TM cream on forearms of 40 subjects to evaluate the anti-inflammatory efficacy in vivo.In cosmetics, sodium lauryl sulphate (SLS) is most popular model for studying skin irritation and skin barrier damage.It has been reported that the SLS can damages the skin without systematic toxicity or carcinogenic properties [17].Therefore, we have investigated the antiinflammatory effect of cream in the SLS test to facilitate the penetration of creams through the epidermal skin barrier.In this study, 3% SLS is used to induce severe visual inflammation.Otherwise, weaker concentration is known to cause disturbance of barrier function with a single application [20].The resulting irritation is quantified by a visual score or by photometric measurement of the erythema using a Mexameter.The study is designed as a 9-day application, because we have no preliminary information on how long it will take for the skin barrier to start repairing under test condition.The visual erythema scores and the erythema value of cream treatment are significantly (P < 0.05) lower than those of placebo after D5.This result suggests that the cream can reduce the skin inflammation which shows its anti-inflammatory effect in vivo.
Overall, it can be concluded that the RbE is a non-cytotoxic, antioxidant and anti-irritant agent which can be used as a potent functional ingredient in cosmetics.Likewise, the in vivo clinical study shows that Multicalm-RH TM cream which contains 3% RbE can be used as an effective skin soothing agent.

Figure 1 .
Figure 1.The cell viability was determined by the amount of MTT converted to the insoluble formazan salt after the treatment with different concentration of RbE in Macrophage cell.The effect of RbE on the production of NO in lipopolysaccharide-induced RAW 264.7 macrophage cells was measured by using Griess reagent.The data are representative of three different experiments with similar results.* P < 0.05, ** P < 0.01, vs control (LPS-treated, no sample treatment).NO: nitric oxide, LPS: lipopolysaccharide, RbE: Rhododendron brachycarpum extract.

Figure 2 .Figure 3 .
Figure 2. Effect of Rhododendron brachycarpum extract on ROS generation inhibition activity in RAW 264.7 macrophage cells.The data are representative of three different experiments with similar results.Asterisk ( * ) indicates significance at the P < 0.05 level compared to control.

Figure 4 .
Figure 4. Differences in the visual score after the treatment of Multicalm-RH TM cream or the placebo randomly on SLS-induced skin irritation of healthy subjects.Data are expressed as mean ± SD. * P < 0.05, *** P < 0.001 compared to that of placebo.