Rhodomyrtone as a potential anti-proliferative and apoptosis inducing agent in HaCaT keratinocyte cells

Author’s Accepted Manuscript Rhodomyrtone as a potential anti-proliferative and apoptosis inducing agent in HaCaT keratinocyte cells Julalak Chorachoo, Dennapa Saeloh, Teerapol Srichana, Thanaporn Amnuaikit, Khadar Syed Musthafa, Somporn Sretrirutchai, Supayang P. Voravuthikunchai

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S0014-2999(15)30402-7 http://dx.doi.org/10.1016/j.ejphar.2015.12.005 EJP70377

To appear in: European Journal of Pharmacology Received date: 15 July 2015 Revised date: 4 December 2015 Accepted date: 4 December 2015 Cite this article as: Julalak Chorachoo, Dennapa Saeloh, Teerapol Srichana, Thanaporn Amnuaikit, Khadar Syed Musthafa, Somporn Sretrirutchai and Supayang P. Voravuthikunchai, Rhodomyrtone as a potential anti-proliferative and apoptosis inducing agent in HaCaT keratinocyte cells, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Rhodomyrtone as a potential anti-proliferative and apoptosis inducing agent in HaCaT keratinocyte cells Julalak Chorachoo1,2, Dennapa Saeloh1,2, Teerapol Srichana3, Thanaporn Amnuaikit1,3, Khadar Syed Musthafa1, Somporn Sretrirutchai4, and Supayang P Voravuthikunchai1,2*

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Excellent Research Laboratory on Natural Products, Faculty of Science and Natural Product

Research Center of Excellence, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 2

Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand 3

Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of

Songkla University, Hat Yai, Songkhla 90112, Thailand 4

Immunology and Virology Unit, Department of Pathology, Faculty of Medicine, Prince of

Songkla University, Hat Yai, Songkhla 90112, Thailand

*Correspondence: [email protected]. Department of Microbiology and Excellent Research Laboratory on Natural Products, Faculty of Science and Natural Product Research Center of Excellence, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

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ABSTRACT Psoriasis is a skin disease associated with hyperproliferation and abnormal differentiation of keratinocytes. Available approaches using synthetic drugs for the treatment of severe psoriasis may cause side effects. Alternatively, plant-derived compounds are now receiving much attention as alternative candidates for the treatment of psoriasis. In this study, the effects of rhodomyrtone, a bioactive plant extract isolated from Rhodomyrtus tomentosa leaves on the proliferation, growth arrest, and apoptosis of HaCaT keratinocytes were investigated. Percentage anti-proliferative activity of rhodomyrtone on HaCaT cells at concentrations of 2-32 µg/ml after 24, 48, and 72 h ranged from 13.62-61.61%, 50.59-80.16%, and 61.82-85.34%, respectively. In a scratch assay, rhodomyrtone at 2 and 4 µg/ml significantly delayed closure of a wound by up to 61.78%, and 71.65%, respectively, after 24 h incubation. HaCaT keratinocytes treated with rhodomyrtone showed chromatin condensation and fragmentation of nuclei when stained with Hoechst 33342. This indicated that rhodomyrtone induced apoptosis in the keratinocytes. In addition, flow cytometric analysis demonstrated an increase in the percentage of apoptosis of keratinocytes after treatment with rhodomyrtone at 2-32 µg/ml from 1.2-10%, 8.2-35.4%, and 21.0-77.8% after 24, 48, and 72 h, respectively, compared with the control. To further develop the compound as a potential anti-psoriasis agent, a rhodomyrtone formulation was prepared and subjected to skin irritation tests in rabbits. The formulation caused no skin irritation including such as erythema and edema. The results indicated that rhodomyrtone had the potential as a promising candidate for further development as a natural anti-psoriasis agent. Keywords Psoriasis, Rhodomyrtone, HaCaT cells, Apoptosis, Anti-proliferative, Anti-psoriasis agent

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1. Introduction Psoriasis is one of the most common chronic autoinflammatory skin diseases. It is characterized by the proliferation of basal layer of epidermis to compensate for skin loss, abnormal keratinocyte differentiation, and massive infiltration of leukocytes. Approximately 2% of the world population is affected (Berneburg et al., 2013). Although, pathogenesis of psoriasis is still not completely understood, there is ample evidence to indicate that genetic inheritance of certain, immune cells in the skin can lead to psoriasis (Jullien, 2012). In addition, environmental factors such as trauma, stress, infections, and drugs may trigger psoriasis (Monteleone et al., 2011). Common psoriasis symptoms include red, scaly, and raised patches on the skin. The condition affects patients’ life, both physically and mentally (Grozdev et al., 2012). Although no treatment measures are available to completely cure psoriasis, a few treatment modalities can be used for relieving psoriatic lesions. Topical treatments such as vitamin D analogues and topical corticosteroids are utilized for mild conditions (Carrascosa et al., 2009). Light therapy or systemic medications such as psoralen plus ultraviolet A therapy, ultraviolet B phototherapy, methotrexate, and cyclosporin are commonly applied for more severe cases (Al‐Hamamy et al., 2014). However, available approaches for the treatment of severe psoriasis may cause side effects for some patients. Recently, much research has focused on investigating the anti-psoriasis potential of some natural products to replace currently available treatment measures and to minimize side-effects. Chinese herbal medicine claims to have some products that can assist healing (Man et al., 2008), tanshinone isolated from Salvia miltiorrhiza (Li et al., 2011), and curcumin (Sun et al., 2013), have been reported to exhibit anti-proliferative activity. Therefore, extracts/compounds obtained from plants could find use as novel therapeutic agents to treat psoriasis. However, there are a number of medicinal plants for which there are ethano-medical

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claims they can cure psoriasis, especially for their anti-proliferative activity have yet to be validated. Rhodomyrtus tomentosa is a medicinal plant belonging to the family Myrtaceae has been used in traditional medicine for the treatment of many diseases including diarrhea (Ong and Nordiana, 1999), urinary tract infections (Wei, 2006), and as an antiseptic wash for wounds (Geetha et al., 2010). In addition, the plant extract possess antioxidant (Lavanya et al., 2012), and antiinflammatory activities (Jeong et al., 2013). Moreover, rhodomyrtone, a compound isolated from Rhodomyrtus tomentosa exhibits profound antibacterial activity (Saising and Voravuthikunchai, 2012). Furthermore, recent study has reported that rhodomyrtone has an immunomodulatory effects on innate immune responses (Srisuwan et al., 2014). It is possible that because of its wide range of biological properties rhodomyrtone might be a good anti-psoriasis agent. However, the anti-proliferative potential of the compound has not been reported. Hence, the aim of this study was to investigate the anti-proliferative property of rhodomyrtone as a potential drug candidate for the treatment of psoriasis.

2. Materials and methods 2.1. Materials Human Skin keratinocyte (HaCaT) cells were obtained from CLS-Cell lines services, Eppelheim, Germany. Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), antibiotics such as streptomycin and penicillin were from Gibco, Grand Island, NY, USA. The chemical, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and Hoechst 33342 stain were from Sigma-Aldrich, USA. Annexin V Alexa

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Fluor 488 and propidium Iodide (PI) Kit was from Invitrogen, Carlsbad, CA, USA. HaCaT cells were cultured in DMEM supplemented with 10% FBS and antibiotics in a 5% CO2 atmosphere at 37 °C. Every second day, the medium in the flask was replaced with fresh medium to allow for the confluent growth of HaCaT cells. When the cells reached confluent growth, they were harvested using 0.05% trypsin-EDTA (Gibco, Grand Island, NY, USA) and fresh culture medium was added to produce a single cell suspension. 2.2. Purification of rhodomyrtone Rhodomyrtone was isolated as described elsewhere (Hiranrat and Mahabusarakam, 2008). The purity of rhodomyrtone was confirmed by nuclear magnetic resonance and mass spectrometry (Salni et al., 2002). The stock solution of rhodomyrtone was prepared by dissolving 100 mg of the compound in 1 ml of 100% DMSO and stored at -20 °C until further use. 2.3. Cell proliferation and viability assay using HaCaT cells Cytotoxicity of rhodomyrtone against HaCaT cells was evaluated by the method previously reported (Mosmann, 1983). HaCaT cells at a concentration of 2 × 104 cells/ml were seeded into a 96-well micro titer plate containing DMEM supplemented with 10% FBS and antibiotics (streptomycin-10 µg/ml and penicillin-10 U/ml) and incubated in an atmosphere of 5% CO2 at 37 °C. After 24 h incubation, the culture medium was replaced with fresh DMEM medium. Rhodomyrtone in DMSO was diluted (2 fold) in the wells containing DMEM to yield a final concentration of 2-32 μg/ml. Cells with DMEM medium and DMSO alone were maintained to check for possible cytotoxicity of the solvent. Cells without added compound were used as a negative control. After incubation at different time intervals (24, 48, and 72 h) at 37 °C in a 5% CO2 atmosphere, the cells were treated with 100 μl of fresh medium along with 50 μl of MTT

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solution (5 mg/ml) and further incubated at 37 °C for 4 h. The medium containing MTT was removed and 100 μl of DMSO was added. The absorbance of the dissolved formazan dye was determined by a microplate reader (Biohit 830, Biohit, Helsinki, Finland) at a wavelength of 570 nm. Percentage cell viability was calculated and compared with the negative control. 2.4. Wound healing property of rhodomyrtone using HaCaT cells An in vitro scratch assay was carried out (Adetutu et al., 2011) using HaCaT cells. Briefly, the cells at 2 × 104 cell/ml were seeded in a 6-well plate containing 2 ml of DMEM. The plate was incubated for 24 h at 37 °C in an atmosphere of 5% CO2. After formation of the confluent monolayer a scratch was created as a straight line using a sterile tip. Cellular debris was removed by washing with PBS and replaced with a medium containing rhodomyrtone (2-4 μg/ml). After incubation at 37 °C at different time intervals, photographs were taken at 40x magnification using a microphotograph and calculated from the following equation: % Migration rate=average distance between scratch sides (0 h) - average distance between scratch sides (12 or 24 h) average distance between scratch sides (0 h)

2.5. Immunofluorescence assay using HaCaT cells An immunofluorescence assay was performed to test the effect of rhodomyrtone on HaCaT cellular morphology. Cells were cultured on a 6-well plate and treated with different concentrations of rhodomyrtone (2-4 μg/ml) for 12 and 24 h. The cells were then fixed with 4% paraformaldehyde for 30 min. Following one more wash with PBS, Hoechst 33342 stain was added to each well and incubated for 15 min at room temperature in the dark. Any morphological

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changes of rhodomyrtone treated and untreated cells were observed using an inverted fluorescence microscope. 2.6. Apoptosis quantification by flow cytometric analysis An apoptosis assay was performed using Annexin V Alexa Fluor 488 & Propidium Iodide (PI) Kit (Invitrogen, Carlsbad, CA, USA). Annexin-V/PI labelling enables the detection of apoptotic cells by externalization of phosphatidylserine (Zhou et al., 2011). Briefly, HaCaT cells at 1 × 105 cell/ml were treated with different concentrations of rhodomyrtone (2-32 μg/ml) and harvested at 24, 48, and 72 h. The cells were trypsinized, pooled together, and stained concomitantly with Annexin-V and PI. The Annexin-V contains a fused green fluorescent protein attached to its N-terminus and Annexin-V staining detected apoptosis in its early stage based on cell membrane alterations and the use of propidium iodide that provided as an indicator of membrane integrity. The stained cells were subsequently analyzed by a Cytomics FC 500 flow cytometry (Beckman Coulter Inc., Fullerton, CA). A minimum of 5,000 events were analyzed for each sample. The Green fluorescence emission was detected through a 525 nm emission filter, and the red fluorescence was detected through a 620 nm emission filter. Analysis was performed with CXP Software. 2.7. Preliminary study on properties of rhodomyrtone formulation 2.7.1. Preparation of rhodomyrtone formulation and evaluation of its stability Rhodomyrtone formulation was prepared by incorporation method. The composition of the formulation was as follows: carbopol ultrez 21 0.2 g, DC RM 2051 2 g, fumed silica 0.5 g, glycerine 20 g, mineral oil 26 g, propylene glycol 30 g, rhodomyrtone 0.01-0.9 g, and distilled water q.s. to 100 g. The raw materials used in the formulation base were accurately weighed and

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the mixture was stirred until congealed at room temperature. The formulation was evaluated for its stability using a freeze-thaw cycle (1 cycle: 4 ºC for 24 h followed by 45 ºC for 24 h) for 6 cycles and again after storage at room temperature for 6 months. Physical appearance of the formulation such as color, smoothness, phase separation, pH value, and viscosity were observed before and after the stability test. 2.7.2. Quantitative determination of rhodomyrtone in the formulation High-performance liquid chromatographic (HPLC) technique for quantification of rhodomyrtone was previously described by Chorachoo et al. (2013). An aliquot of 20 𝜇l of each sample was directly injected into the HPLC system with a mixture of acetonitrile and water (85:15, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed in a Symmetry C8 analytical column (4.6 × 150 mm, particle size, 3 𝜇m) at 30 ºC and the peak was detected at 254 nm. The column was equilibrated for 6 min prior to a subsequent run and was then left saturated with the mobile phase for at least 3 h before each assay. Rhodomyrtone was used as the internal standard for the assay.

2.8. Skin irritation studies The test of acute dermal irritation/corrosion was conducted according to no. 404 of the OECD guidelines for testing of chemicals, 2002. All animal experiments were approved by the Ethics Committee for Animal Experiments of Thailand institute of scientific and technological research (no. TS-59001). Three rabbits were employed and acclimatized to the laboratory environment for one week. One day before experimentation, an area of skin approximately 10 cm × 10 cm on the

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dorso-lumbar region of each rabbit was clipped free of hairs. Two areas of the shaven skin approximately 2.5 cm × 2.5 cm were selected. A volume of 0.5 ml of rhodomyrtone formulation was introduced onto a 2.5 cm × 2.5 cm gauze patch, which served as a treated patch while 0.5 ml of distilled water on another patch served as the control patch. Both patches were applied to the selected skin sites on each rabbit. The patches were then secured to the skin by transpore adhesive tape. The entire trunk of the rabbit was wrapped with elastic cloth to avoid dislocation of the patches for 4 h. At the end of the exposure period, all patches were removed and the treated skin was gently wiped with moistened cotton wool to remove any residual test material. The animals were assessed for evidence of the degree of erythema and oedema at each site 1, 24, 48, and 72 h after removal of the patches. Further observations were made, if necessary, to establish reversibility, if the irritation signs still existed, but this extra time did not exceed 14 days after application. In addition to the observation of irritation, any lesions and other toxic effects were recorded. The skin reactions were independently scored by two inspectors using the numerical scoring system (0 = no erythema or no edema, 1 = barely perceptible erythema or edema, 2 = well-defined erythema or slight edema, 3 = moderate to severe erythema or moderate edema, and 4 = severe erythema or edema). 2.9. Statistical analysis All experiments were performed at least three times. Values are expressed as means ± S.D. or means ± S.E.M. Statistical analysis was performed using a two sample t-test. Differences were considered significant at *P < 0.05 and **P < 0.01. 3. Results 3.1. Inhibitory effects of rhodomyrtone on the growth of keratinocytes

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An anti-proliferative activity of rhodomyrtone on HaCaT cells was observed in concentrationand time-dependent patterns (Fig. 1). After exposure to rhodomyrtone, a delayed growth of the keratinocytes was observed when compared with the control. The data unequivocally confirmed that rhodomyrtone exerted a strong growth inhibitory effect on HaCaT cells in a concentrationand time-dependent manner. Percentage anti-proliferative activity of rhodomyrtone on HaCaT cells at the concentrations of 2-32 µg/ml after 24, 48, and 72 h ranged from 13.62-61.61%, 50.59-80.16%, and 61.82-85.34%, respectively. No cytotoxicity was observed in the cells treated with 0.3% DMSO (control). 3.2. Effects of rhodomyrtone on wound healing of HaCaT cells The results of anti-proliferative assay demonstrated that rhodomyrtone negatively influenced HaCaT cell proliferation. Hence, the impact of the compound on wound healing was observed by scratch assay. The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch towards the center. After 12 h incubation, percentage migration of HaCaT cells, untreated and treated with 2 and 4 µg/ml rhodomyrtone were 27.95%, 20.56%, and 12.71%, respectively. In contrast, a complete wound closure was noticed for the untreated HaCaT cells after 24 h incubation, whereas, the cells treated with 2 and 4 µg/ml rhodomyrtone exhibited migration rates of only 38.22%, and 28.35% (Fig. 2A). The results clearly demonstrated the significant effects of rhodomyrtome in delaying wound closure. Further images of the scratch assay were captured at 40x magnification using a phase-contrast microscope at 0, 12, and 24 h after incubation. As shown in Fig. 2B, rhodomyrtone at the same concentrations delayed the ability of HaCaT cells to regenerate a monolayer which resulted in a delayed wound closure.

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3.3. Effects of rhodomyrtone on HaCaT cellular morphology Hoechst 33342 staining was employed to observe the morphological changes in the cells treated with rhodomyrtone for 12 and 24 h. Live cells appeared with normal nuclei, with a blue pale chromatin having an organized structure using the fluorescence microscope. In contrast, apoptotic cells were identified by the presence of chromatin condensation and intact nuclear boundaries, and the bright blue chromatin was highly condensed. Apoptotic cells with chromatin condensation and /or fragmented nuclei were observed after exposure to rhodomyrtone (Fig. 3C3F). However, no such changes were observed in the control cells (Fig. 3A and 3B). The results indicate that rhodomyrtone induced cellular apoptosis in HaCaT cells. 3.4. Flow cytometric analysis of HaCaT cells by Annexin V-PI (AV-PI) staining The effects of rhodomyrtone in inducing apoptosis of HaCaT cells were further confirmed by flow cytometric analysis. The percentage apoptosis of the cells after treatment with rhodomyrtone at the concentrations of 2-32 µg/ml for 24, 48, and 72 h ranged from 1.2-10%, 8.2-35.4%, and 21.0-77.8%, respectively when compared with the control (Fig. 4). 3.5. Properies of the formulations The average percentage recovery of rhodomyrtone from the formulation ranged from 89 to 101% (Table 1). The viscosity of the formulations varied in the range of 25 × 103 to 55 × 103 cp from 12 rpm as shown in Table 2. The pH of the prepared base and formulation was in the range of 6.34 to 6.49 (Table 2). The formulation-base was a white colour, whereas rhodomyrtone formulations appeared to be a slightly opaque yellow. Stability of the formulations was evaluated using different storage conditions including a freeze-thaw cycle for 6 cycles and after storage at

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room temperature for 6 months. No phase separation was observed during the stability study of the formulations. No liquefaction was observed throughout the study period of 6 months. 3.6. Skin irritation study No signs of acute dermal irritation were observed at any time points. After removal of the patches, the treated skin of each rabbit was observed for any skin reactions at 1, 24, 48, and 72 h. Both the control and treatment groups showed no symptoms of dermal irritation including erythema or edema (Table 3).

4. Discussion Utilization of natural products, especially from plants as anti-psoriasis drugs might reduce the severity of psoriasis with lesser side effects to human. Phytochemicals that exhibited antiproliferative activity along with the potential for inducing keratinocyte apoptosis could be developed as appropriate drug candidates for the treatment of psoriasis. Hence, we attempted to assess the anti-psoriasis activity of rhodomyrtone, a member of acylphloroglucinols derived from Rhodomyrtus tomentosa. It was observed for the first time that rhodomyrtone significantly inhibited the proliferation of HaCaT cells in a dose and time dependent manner. It has been welldocumented that the two transcription factors including STAT3 (Signal transducer and activator of transcription 3) and NF-κB (Nuclear factor kappa B) play a crucial role in the proliferation of keratinocytes in psoriasis patients and leads to the generation of psoriasis-like lesions (Goldminz et al., 2013; Miyoshi et al., 2011). In earlier studies, natural products such as curcumin (Senft et al., 2010), and oak wood vinegar (Lee et al., 2011) have been reported to interfere with the STAT3 signaling pathway, whereas, luteolin (Weng et al., 2014), rottlerin (Valacchi et al.,

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2009), and celastrol (Zhou et al., 2011) exhibit an anti-proliferative effect by interfering with NF-κB signaling pathway. Though our work demonstrated potent anti-proliferatory activity of rhodomyrtone on HaCaT cells, the mechanism of action of the compound on either the STAT3 or NF-κB signaling pathway is yet to be established. Based on these finding of the anti-proliferative activity of rhodomyrtone on HaCaT cells, a wound scratch assay was performed to confirm the effects of the compound in delaying HaCaT keratinocyte proliferation. Wound closure is an important criterion for an effective anti-psoriasis drug as a delay in the abnormal differentiation of keratinocytes demonstrated anti-psoriasis activity in psoriasis patients (Valacchi et al., 2009). To determine the effects of rhodomyrtone on wound closure, the concentrations of the compound at which no considerable cytotoxicity on HaCaT keratinocytes were selected. Rhodomyrtone at the tested concentrations prolonged the ability of keratinocytes to regenerate the monolayer in the scratch area and indicated a delay in wound closure. The observed effects in the present study agreed with previous reports. Rottlerin isolated from Mallotus phillippinensis (Valacchi et al., 2009) and oak wood vinegar derived from Quercus acutissima (Lee et al., 2011) were demonstrated to delay wound closure by suppressing HaCaT cells proliferation. Apoptosis is an active physiological process that maintains keratinocyte proliferation and epidermal growth in human skin. However, in psoriasis patients, impairment of programmed cell death, contributes resistance towards apoptosis which leads to uncontrolled proliferation of keratinocytes (Gabr et al., 2014). Therefore, restoring keratinocytes apoptosis could reduce the severity of psoriasis. In order to verify the effects of the rhodomyrtone to induce apoptosis of HaCaT keratinocytes, nuclei of rhodomyrtone treated cells were stained with Hoechst 33342 and observed under an inverted fluorescence microscope. Hoechst 33342 binds to adenine-thymine-

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rich regions of the minor groove of DNA, apoptotic cells shows bright blue dots in the nuclei, that represents nuclear fragmentation. Apoptosis features including chromatin condensation, shrunken cytoplasm, and nuclear fragmentation in HaCaT keratinocytes all of which were observed following treatment with rhodomyrtone. Daoud et al. (2014) have demonstrated that the compound, B-9-3 exhibited anti-proliferative activity by inducing keratinocytes apoptosis. In a similar way, we propose that the anti-proliferative activity of rhodomyrtone is attributed to its ability to induce apoptosis of HaCaT keratinocytes. In a previous study, the apoptotic effects of celastrol isolated from a chinese herb Celastrus orbiculatus caulis (Zhou et al., 2011) and 1, 4-dihydroxy-2-naphthoic acid from Rubia cordifolia L. (Mok et al., 2013) on HaCaT cells were confirmed by Annexin V and PI staining. Hence, in the present work, the Annexin V and PI staining technique was carried out for the quantitative analysis of induced apoptosis on HaCaT keratinocytes by rhodomyrtone. Treatment of HaCaT keratinocytes with increasing concentrations of the compound resulted in an increase in apoptosis of the cells. At lower concentrations, the compound induced early apoptosis of HaCaT keratinocytes and a maximum absorbance was detected at a wavelength specific to Annexin V stain. However, an increase in the concentrations of the compound and incubation time resulted in late apoptosis as a maximum absorbance was detected at 525 and 620 nm, that is specific to Annexin V and PI stained cells, respectively. Considering the efficiency of rhodomyrtone in reducing hyperproliferation and an abnormal differentiation of HaCaT cell cultures an attempt was made, to prepare a rhodomyrtone formulation to further develop it as a cream for treating psoriasis. The developed rhodomyrtone formulation had good homogeneity with no phase separations after 6 months. Moreover, pH of

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the formulations remained similar to that of the normal human skin. The formulation did not produce any skin irritation including erythema and edema in a rabbit skin model. 5. Conclusions In the present study, rhodomyrtone exhibited potent anti-proliferative activity, it delayed wound closure, and induced apoptosis in HaCaT keratinocytes. Furthermore, rhodomyrtone formulation was found to be stable, caused no erythema or edema in the animal model. The attained results suggest that rhodomyrtone could be a suitable drug candidate for the treatment of psoriasis.

Acknowledgments This work was supported by Research and Researchers for Industries-RRI PhD Program (Grant No. PHD 56I0079) and TRF senior Research Scholar (Grant No. RTA5880005), the Thailand Research Fund. Thanks also to Dr. Brian Hodgson for assistance with the English.

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Figure legends Fig. 1. Percentage proliferative rate of HaCaT cells treated in the absence or presence of different concentrations of rhodomyrtone after 24 h (■), 48 h (□), and 72 h ( ) incubation at 37 °C in 5% CO2 atmosphere. Each value shown is the mean ± S.D. *P < 0.05 and **P < 0.01 compared with the control (n=3).

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Fig. 2. Effects of rhodomyrtone treatment on the regeneration of HaCaT cells observed using the scratch assay. (A) Percentage migration rate of the cells treated with different concentrations of rhodomyrtone after 12 and 24 h. (B) HaCaT cell layer subjected to scratch and treated with rhodomyrtone (2 and 4 µg/ml) and the control without treatment. Images were captured at 40x magnification using a phase-contrast microscope. Each value shown is the mean ± S.D. *P < 0.05, **P < 0.01 compared with the control (n=3). Fig. 3. Cell apoptosis observed by Hoechst 33342 staining. Effects of rhodomyrtone on HaCaT cell morphology as examined by fluorescent microscopy. HaCaT cells were treated with solvent control (A, B), 2 μg/ml (C, D), and 4 μg/ml (E, F) rhodomyrtone for 12 h and 24 h, respectively. Arrows point to the cell with chromatin condensation and/ or fragmentation. Fig. 4. Apoptosis rate measured by flow cytometry. Quadrant dot plots of HaCaT cells were treated with different concentrations of rhodomyrtone after 24 h (A). Percentage apoptosis of the cells treated with different concentrations of rhodomyrtone after 24 h (■), 48 h (□), and 72 h ( ) of incubation at 37 °C in a 5% CO2 atmosphere. Each value shown is the mean ± S.D. *P < 0.01 compared with the control (n=3). Table 1 Quantitative determination of rhodomyrtone present in the formulation by HPLC (n=3). Table 2 Viscosity, pH, and physical appearance of rhodomyrtone formulation before and after the stability test at 25 ºC (n=3). Table 3 Score of the dermal reactions to distilled water (control) and rhodomyrtone formulation on the skin of rabbits (n=3).

Table 1

21

Freeze thaw cycle

Concentration

Recovery (%)

(µg/ml) Before

After

128

100.99±05.90

9,400

101.15±02.34

128

89.72±04.43

9,400

92.11±09.63

Values are mean±S.D.

Table 2

Samples Physical appearance (×103 cp)

Test

Rhodomyrtone formulation 0 slightly opaque yellow and viscous cream

Actual

Viscosity

pH value (months) (µg/ml)

128

28.47±3.03

6.41±0.02

9,400

27.56±2.46

6.45±0.02

-

55.91±4.48

6.45±0.03

Rhodomyrtone formulation 4 128 slightly opaque yellow and viscous cream 9,400 slightly opaque yellow and viscous cream

25.12±5.01

6.34±0.02

25.23±4.36

6.40±0.08

Base white and viscous cream

52.51±7.48

6.36±0.05

slightly opaque yellow and viscous cream Base white and viscous cream

-

22

Rhodomyrtone formulation 6 slightly opaque yellow and viscous cream

128

26.09±3.21

6.35±0.60

9,400 25.46±8.16 6.37±0.21 slightly opaque yellow and viscous cream Base white and viscous cream

-

52.25±8.48

6.49±0.13

Values are mean±S.D.

Table 3

Rabbit

Rhodomyrtone formulation/Control

no.

Scoring time (h) 1

24

48

72

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

100

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

101

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

102

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0