Jasmine rice panicle: A safe and efficient natural ingredient for skin aging treatments

Author’s Accepted Manuscript Jasmine rice panicle: a safe and efficient natural ingredient for skin aging treatments Mayuree Kanlayavattanakul, Nattaya Lourith, Puxvadee Chaikul www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)31165-5 http://dx.doi.org/10.1016/j.jep.2016.10.013 JEP10471

To appear in: Journal of Ethnopharmacology Received date: 4 November 2015 Revised date: 28 July 2016 Accepted date: 5 October 2016 Cite this article as: Mayuree Kanlayavattanakul, Nattaya Lourith and Puxvadee Chaikul, Jasmine rice panicle: a safe and efficient natural ingredient for skin aging treatments, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.10.013 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.

Jasmine rice panicle: a safe and efficient natural ingredient for skin aging treatments Mayuree Kanlayavattanakul*, Nattaya Lourith, Puxvadee Chaikul School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand *

Corresponding author: M. Kanlayavattanakul

School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand. Tel.: +66-53-916-834, Fax; +66-53-916-831, E-mail: [email protected] Summary Ethnopharmacological relevance: While rice is one of the most important global staple food sources its extracts have found many uses as the bases of herbal remedies. Rice extracts contain high levels of phenolic compounds which are known to be bioactive, some of which show cutaneous benefits and activity towards skin disorders. This study highlights an assessment of the cellular activity and clinical efficacy of rice panicle extract, providing necessary information relevant to the development of new cosmetic products. Materials and methods: Jasmine rice panicle extract was standardized, and the level of phenolics present was determined. In vitro anti-aging, and extract activity towards melanogenesis was conducted in B16F10 melanoma cells, and antioxidant activity was assessed in human skin fibroblast cell cultures. Topical product creams containing the extract were developed, and skin irritation testing using a single application closed patch test method was done using 20 Thai volunteers. Randomized double-blind, placebo-controlled efficacy evaluation was undertaken in 24 volunteers over an 84 day period, with the results monitored by Corneometer ® CM 825, Cutometer® MPA 580, Mexameter® MX 18 and Visioscan® VC 98. Results: Jasmine rice panicle extract was shown to have a high content of p-coumaric, ferulic and caffeic acids, and was not cytotoxic to the cell lines used in this study. Cells treated with

1

extract suppressed melanogenesis via tyrosinase and TRP-2 inhibitory effects, which protect the cell from oxidative stress at doses of 0.1 mg/ml or lower. The jasmine rice panicle preparations (0.1-0.2%) were safe (MII = 0), and significantly (p < 0.05) increased skin hydration levels relative to the placebo. Skin lightening, and anti-wrinkle effects related to skin firmness and smoothness were observed, in addition to a reduction in skin wrinkling. Improvements in skin biophysics of both 0.1 and 0.2% extracts were showed to be comparable (p > 0.05). Conclusions: Jasmine rice panicle extract having high levels of phenolics shows cutaneous benefits as the basis for skin aging treatments, as indicated through in vitro cytotoxicity assessments and skin testing in human subjects. Keywords: jasmine rice panicle, skin aging, in vitro activities, cellular activities, clinical trials Graphical abstract 1. Introduction Rice [Oryza sativa cv. indica (Oryzeae)] extracts have been used in many Asian traditional medicines for the treatment of diabetes, inflammation, gastrointestinal disorders, cardiovascular disease, and in diuretics. Furthermore, eye lotions developed from rice are a traditional Malaysian remedy and Ayurveda prescriptions also make note of the use of a rice ointment to cure skin ailments (Ahuja et al., 2008; Umadevi et al., 2012), and to combat skin aging (Burlando and Cornara, 2014). Rice based skin remedies are not limited to Asia: the use of a traditional Italian remedy for skin smoothing and lightening (Pieroni et al., 2004; Saikia et al., 2006) and subsequent marketing has highlighted the aesthetic benefits of using rice in skin rejuvenation treatments (Costin and Hearing, 2007; Currais, 2015; Jenkins, 2002; Kammeyer and Luiten, 2015). However, detailed scientific evidence linking beneficial effects to particular components in these traditional remedies has, to this point, been limited.

2

Skin aging is one of the main dermatological concerns with clinical signs being irregular dryness, skin hyperpigmentation, furrowing, and loss of elasticity. Cutaneous aging is exacerbated by the presence of free radicals which cause inflammation, and disorganization of proteins in the dermal matrix, particularly collagen and elastin. Additionally, melanocytes are up-regulated in tandem with fibroblast down-regulation, resulting in the formation of dark spots, loss of skin hydration, and wrinkling (Costin and Hearing, 2007; Currais, 2015; Jenkins, 2002; Kammeyer and Luiten, 2015). Development of new anti-aging products is therefore a major endeavor for cosmetics companies, with promising treatments based on herbal extracts being particularly attractive (Mukherjee et al., 2011). Phytonutrients in rice have recently been touted as being beneficial for human health, and are attracting attention as a specialty raw material for nutraceutical and pharmaceutical product development (Kanlayavattanakul et al., 2015). Phenolic compounds are a key component of phytonutrients, with some proving active in the treatment of skin disorders and protection against aging, in particular the hydroxycinnamic acids (Mukherjee et al., 2011; Nichols et al., 2010). Constituent profiles of biologically active rice phenolics undergo changes during grain development, with the activity and total phenolic content being highest during the flowering state where the panicles are still green. p-Coumaric acid, the cellular melanogenesis inhibitor (Jun et al., 2012), and ferulic and caffeic acids which are important therapeutic agents for skin disorder treatments showing photoprotective properties (Kumar and Pruthi, 2014; Mancuso and Santangelo, 2014; Saija et al., 2000) were found to be the major phenolic constituents in rice panicles (Kanlayavattanakul et al., 2015). In this context, the dermatologic potential of rice panicle extract of jasmine rice (Khao Dawk Mali 105 variety, ML) was assessed in terms of phenolic composition, and in vitro biological activity. Thereafter, the jasmine rice panicle extract was developed into a prototype topical product to treat or prevent mild skin abnormalities (Elsner and Maibach, 2000) and

3

the results assessed in terms of skin hydration, coloring, smoothness and wrinkle formation, the dermatological signs of cutaneous aging (Kanlayavattanakul and Lourith, 2015).

2. Materials and methods 2.1 Preparation and standardization of jasmine rice panicle extract Rice panicle extract of jasmine rice or Khao Dawk Mali 105 variety (ML) cultivated in Chiang Rai was harvested. The plant material was identified by botanist Dr. Nijsiri Ruangrungsri, Faculty of Pharmaceutical Sciences, Department of Pharmacognosy, Chulalongkorn University, Bangkok, Thailand. The voucher specimen (MKOS 05ML14,) was deposited for further reference at our laboratory herbarium at Mae Fah University, Chiang Rai. The harvested rice panicles were separately cleaned, dried and ground into powder (Retsch, Germany; < 1 mm) and hydrolyzed using 2 M NaOH. This soluble was neutralized with 2 M HCl, partitioned with ethyl acetate and concentrated in vacuo to give the high phenolics rice panicle extract (Kanlayavattanakul et al., 2015), and named ML extract. The preparation of ML extract was undertaken for more 2 times, and the extractive yield was calculated.

2.1.1 Total phenolics content (TPC) TPC was determined using Folin-Ciocalteu assay. The reagent was mixed with Na2CO3 and absorbance measured using the microplate reader (ASYS, UVM340, UK). The TPC was compared with the gallic acid and expressed as g of gallic acid equivalents (gGAE) per 100 g of ML extract. The procedure was repeated in triplicate (Kanlayavattanakul et al., 2015).

2.1.2 Phenolics analysis with UPLC

4

UPLC analysis was carried out on an ACQUITY H-Class system equipped with an ACQUITY UPLC PDA eλ detector using a BEH C18 1.7 µm column (2.1 × 100 mm). Gallic (GA), protocatechuic (PA), chlorogenic (ChA), caffeic (CA), syringic (SyA), p-coumaric (pCA), ferulic (FA), sinapic (SiA) and rosmarinic (RA) acids, vanillin (V) and quercetin (Q) at various concentrations in AcCN were used to prepare calibration curves. The samples were successively separated by a gradient mobile phase consisting of AcCN (A) and 3 % aq. AcOH (B). The eluent was programmed as follows: 0 min 100 % B, 1.5 min 95 % B, 3 min 85 % B, 5 min 80 % B and 8 min 70 % B at a flow rate of 0.6 ml/min. Validation of the analytical method in terms of linearity, sensitivity, accuracy and precision was undertaken according to AOAC guidelines (2002). Characterization of phenolics in the ML extract (1 mg/ml) was performed in three cycles (Kanlayavattanakul et al., 2015).

2.2 In vitro activities 2.2.1 Antioxidant activity Ferric reducing ability of plasma (FRAP) of the ML extract was examined. A FRAP reagent was prepared in a 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ) solution with HCl, FeCl3, and acetate buffer. The extract (3 mg/ml) was reacted with the FRAP reagent, and absorbance recorded at 595 nm. The reducing power was determined in triplicate and expressed as an equivalent concentration (EC) to that of FeSO4 (Kanlayavattanakul et al., 2015). pCA was examined as the standard. 2.2.2 Enzyme inhibitory effects Tyrosinase The activity was determined using a modified DOPAchrome method. The ML extract (0.4 mg/ml) in phosphate buffer and mushroom tyrosinase were incubated with L-DOPA. The

5

absorbance was measured at 490 nm, using kojic acid (0.2 mg/ml) as a positive control (Kanlayavattanakul et al., 2015). Elastase The mixture containing Tris-HCl buffer, Porcine pancreatic elastase and N-succinyl-AlaAla-Ala-p-nitroaniline (AAAPVN) was prepared. The absorbance of the mixture was monitored at 410 nm throughout the reaction, and after incubating with the ML extract (0.08 mg/ ml). The enzyme inhibitory effect was compared with that of ursolic, p-coumaric and ferulic acids (0.04 mg/ml, each) (Thring et al., 2009). Collagenase Tricine buffer was mixed with collagenase and N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). The absorbance was recorded at 340 nm before and after incubation of the ML extract (0.1 mg/ml) with enzyme and FALGPA. The anti-collagenase activity was compared with epigallocatechin gallate (0.05 mg/ml) (Thring et al., 2009). 2.3 Cellular activity assessments in B16F10 melanoma cells B16F10 melanoma cells (ATCC® CRL 6475, USA) were cultured in 75-cm2 flask in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. Cells were grown to semiconfluence and harvested by 0.25%, w/v trypsin and 0.06 mM EDTA in phosphate buffer saline. 2.3.1 Cytotoxicity assays The sulforhodamine B (SRB) assay was used for cell cytotoxicity determination (Papazisis et al., 1997). Briefly, cells were seeded at the density of 1 ™ 104 cells/well in 96-well plate and incubated for 24 h. Cells were then treated with different concentrations (0.0001 – 1 mg/ml) of the samples for 72 h. The adherent cells were fixed, washed and dyed prior to the absorbance measurement at 540 nm with the microplate reader. The cell viability was compared with the control treated with absolute ethanol. 6

2.3.2 Melanogenesis assays Melanin content measurement The melanin content was measured according to the literature method (Oka et al., 1996) with a slight modification. Cells with a similar density as cytotoxicity assay were plated in 6well plate and incubated for 24 h in the incubator. Samples at different concentrations, kojic acid (positive control), theophylline (negative control) and absolute ethanol were separately added and incubated for 72 h under the same condition. Melanin content was measured at 450 nm and total protein content was measured by Bradford method (Bradford, 1976). The relative ratio of melanin content (%) was calculated. Tyrosinase activity assessment B16F10 melanoma 5™ 105 cells/well in 6-well plate was incubated for 24 h, treated with the samples and incubated for 72 h. The treated cells were washed, lysed with RIPA buffer containing protease inhibitor and then incubated at 4 °C for 30 min before a centrifugation at 14,000´g for 10 min. The supernatants were mixed with 0.05% L-DOPA in 50 mM phosphate buffer (pH 6.8) and incubated for 2 h at 37 °C. Tyrosinase activity was determined by means of DOPAchrome formation at 490 nm. The enzyme activity was compared with the standard mushroom tyrosinase. The relative ratio of the enzyme activity (%) was then calculated (Ohguchi et al., 2005). Tyrosinase related proteins-2 (TRP-2) activity assessment The supernatant obtained from the lysis treated cells was mixed with 1 mM phenylthiourea, 2 mM EDTA and 10 mM sodium phosphate buffer (pH 6.8). DOPAchrome solution containing 1 mM L-DOPA and 2 mM NaIO4, was added into the mixture and incubated at 37 ºC for 2 h. The reduction of DOPAchrome was measured at 490 nm. The reaction mixture with bovine serum albumin instead of the cell supernatant was used as a

7

negative control. The TRP-2 activity was compared with the control and expressed as the relative ratio (%) (Barber et al., 1984). 2.4 Cellular activity assessments in human skin fibroblasts Human skin fibroblasts (ATCC® CRL 2097, USA) at 6 - 13th passage were cultured in 75cm2 flask in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C under 5% CO2. Cells were grown and harvested by 0.25% w/v trypsin and 0.06 mM EDTA in phosphate buffer saline. 2.4.1 Cell cytotoxicity assays The SRB assay was used for cell cytotoxicity determination (Papazisis et al., 1997) as above. 2.4.2 Antioxidant activity assays Fibroblasts were seeded in 96-well plate (1™ 104 cells/well) and incubated for 48 h. Cells were treated with various concentrations of the samples and the solvents (absolute ethanol or culture medium) for 24 h prior to treat with the fresh medium containing 150 mM H2O2 and further incubated for 3 h. The cells were fixed, washed, dyed with SRB, and solubilized with 10 mM Tris base and recorded the absorbance at 540 nm for cell viability calculation (Viriyaroj et al., 2009). 2.5 Physicochemical property and stability of the extract Solubility of the ML extract was examined in propylene glycol and glycerin, separately. The physicochemical properties were observed in terms of color, precipitation and pH (Satorius Docu-pH+ Meter, Germany). The ML extract was chemical and physical stability tested under the storage at 4 ± 2 °C for 24 h and switched to 45 ± 2 °C for 24 h (counted as 1 cycle). The heating-cooling cycle was repeated for more 6 times (totally 7 cycles). The physicochemical properties and TPC were re-assessed (Arabshashi et al. 2007).

8

2.6 Topical product formulations All of the ingredients were of cosmetic grade except those for TPC were of analytical grade. Creams containing the ingredients as shown in Table 1 were formulated. Base cream and creams containing the ML extract (ML creams) were physicochemically evaluated in terms of color, odor, pH and viscosity. The base and ML creams were preliminary stability tested by means of a centrifugation assay and 7 heat-cool cycles. Consequently, stability in terms of appearance, homogeneity, pH, viscosity and TPC was examined (Antignac et al., 2011; Kanlayavattanakul et al., 2015). 2.7 Clinical study 2.7.1 Ethical committee of Mae Fah Luang University Prior to clinical study, the study protocol was filed for the institutional human experimentation committee approval. All of the investigation methods in human subjects filed in the study protocol following guidelines of the Declaration of Helsinki and Tokyo for humans was approved by the ethical committee of Mae Fah Luang University with the registered approval no. REH-57058. 2.7.2 Volunteer recruitment Healthy Thai volunteers aged between 25-50 years old having none of skin disease were enrolled this study. Those of hypersensitive skin as well as allergy history were excluded including who were pregnant or lactating or dieting. All recruited subjects were informed about the study both in writing and verbally and signed a written consent form which was approved by the ethical committee of the Mae Fah Luang University prior to enrolment. 2.7.3 Patch test A single application closed patch test was preliminary performed in 20 subjects for 24 h. Finn chambers (8 mm) obtained from Smartpractice (USA) were used for an observation of skin irritation (20 µl) on the volar forearms of the subjects. Skin irritation severity was graded

9

0-4 for MII (the Mean Irritation Index) calculation. The MII < 0.2 was interpreted as none of irritation. Of which the test samples were; 1) sodium lauryl sulfate 0.5% in water as the positive control, 2) water as the negative control, 3) cream base, 4) 0.1% ML and 5) 0.2% ML creams (Futrakul et al., 2010). 2.7.4 Clinical evaluation Twenty-four healthy females who were allergy-free for 1 week and had not used steroids for 4 weeks prior to study enrollment were included. Any skin treatments on the tested area were prohibited as were smoking and liquor drinking. In addition, the treated areas were protected against strong sun light and UV exposure including any skin insulting during the study which lasted 84 days. The measurement room was controlled at 20 ± 1 °C and 40 - 60% relative humidity. The randomized double-blind placebo controlled was conducted in the volunteers who applied the samples twice daily (morning and evening) on the inner forearm. The treated skin was evaluated by Corneometer® CM 825, Cutometer® MPA 580, Mexameter® MX 18 and Visioscan® VC 98 (Courage and Khazaka, Germany) at the base line and after 14, 28, 56 and 84 days of the application. The measurement was installed in the above acclimated condition (Churienthong et al., 2010; Futrakul et al., 2010). 2.8 Sensory evaluation Preference towards the developed ML cream was carried out by 30 female volunteers. The cream was scored by the hedonic system from 1 – 5 (dislike – most prefer) using the interview questionnaires. 2.9 Statistical analysis Data are presented as the mean ± SD. The results from the clinical study are expressed as the mean ± SEM. The parameters were compared and analyzed using one sample t test and ANOVA test with a significance level of p < 0.05 using the SPSS program version 16.0.

10

3. Results and Discussion 3.1 Preparation and standardization of ML extract with in vitro anti-aging efficacy ML extract (0.32 ± 0.03% yield) with high TPC was prepared by the previously published extraction protocol (Kanlayavattanakul et al., 2015). The antioxidant extract possessed inhibitory effects against skin abnormalities (Elsner and Maibach 2000; Thring et al., 2009) and tyrosinase, elastase and collagenase as shown in Table 1. Inhibitory activity against tyrosinase was lower than the control (kojic acid), but anti-elastase activity was comparable. However, the ML extract possessed higher anti-collagenase activity (47% inhibition) than that of green tea (10% inhibition) when tested at the same concentration (Thring et al., 2009). These results demonstrate the potential feasibility of rice panicle extract as a candidate for treatment of cutaneous disorders. None of interferences were observed for any analysis of 10 standard phenolics at different concentrations. The chromatographic peak of each standard was symmetry and pure due to a higher peak purity threshold than peak purity angle indicating specificity of the analytical condition as shown in Fig. 1A. Of which, GA was firstly eluted followed by PA, ChA, SyA, V, pCA, FA, SiA, RA and Q, respectively. Sensitivity was determined by limit of quantification (LOQ) and limit of detection (LOD) (LOQ = 3.3 × SD, LOQ = 10 × SD; SD is standard deviation from 6 replicates of the lowest concentration of standard solution or the standard deviation of the response/the slope of the calibration curve) based on laboratory fortified blank. The presenting UPLC method has LOD and LOQ of each phenolic as shown in Table 1. In addition, linearity in the range of 5 – 500 mg/ml with a coefficient (r) of more than 0.995 was consistent with the good and acceptable linearity analytical method in AOAC (2000) and ICH (2005) guidelines. The accuracy of the method was determined by spiking sample with the known amount of the standard phenolics for 3 concentrations (10, 50 and 500 mg/ml). The assay was evaluated in 6 replicates and the recovery (%) of the majorly

11

determined phenolics was calculated (Table 2). Intra-day precision was evaluated from RSD of 6 replicates determination of the standard solutions. Inter-day precision assay was obtained from 12 replicates determination from 2 different days of analysis (6 replicates/ day). The RSD of intra-day and inter-day precisions confirmed the precision of this developed UPLC method according to USFDA criteria (2013). Thus, the validated method was therefore used for rice panicle phenolics analysis. pCA, FA and CA were determined as the major extract components through UPLC analysis, along the presence of a further nine minor phenolics (Fig. 1B). A comparison with the Phitsanulok 2 (PL) variety (Kanlayavattanakul et al., 2015) indicates that the ML variety should show greater potential for cutaneous benefits due to the higher level of active phenolics being present, as shown in Fig. 2A. This variety was therefore chosen for further study in this context. The extract was examined for its stability by means of accelerated stability testing (HC), and was found to be stable in relation to total phenolic content variations over time. UPLC analysis indicated that was 5.37 ± 0.24% reduction of phenolics of which pCA, the major constituent was 7.33 ± 0.17% (Fig. 2B) reduced that in accordance with the 7.62% reduction of TPC assessed by means of Folin-Cioclateu assay (Table 1). Thus, TPC analysis can be used as a feasible method for quality control of the ML extract (Antignac et al., 2011). pCA, an antioxidant (Killiç and Yeşloğlu, 2013) showing angiogenic effects (Kong et al., 2013) suppressing melanogenesis in murine melanoma cells via tyrosinase inhibition, can also prevent UV induced oxidation in the cells by down-regulating melanin production (An et al., 2010). The biological activity of this compound is related to its disrupting cellular phosphorylation (Jun et al., 2012). Other phenolics derived from the same biosynthesis pathway are FA and CA, which similarly inhibit melanogenesis (Choi et al., 2007).

12

Accordingly, the activity of the ML extract towards melanogenesis (Costin and Hearing, 2007) in melanoma cells was investigated. 3.2 Cellular activity assessments in B16F10 melanoma cells 3.2.1 Cytotoxicity assays Cytotoxicity profiles of the ML extract towards B16F10 melanoma cells were obtained through SRB assay to determine the optimal concentrations for melanin synthesis and tyrosinase activity measurements. These were compared with those of major phenolics, pCA and FA, and the results shown in Fig. 3A. ML extract can be considered non-cytotoxic (cell viabilities > 80%), similar to those obtained on testing with pCA and FA. These results concur with previous data obtained for pCA with melanocytes (0.05 - 0.16 mg/ml) (Izushi et al., 2000; Song et al., 2011), however cell viability decreased to 50% at higher test concentrations (1 mg/ml) in line with previously documented toxicity profiles for hydroxycinnamic acids (Liu et al., 1995; Sharma, 2011). Therefore, the optimized ML extract concentration for further assessment (melanin content measurements and enzyme activity) was chosen to be 0.1 mg/ml.

3.2.2 Melanogenesis assays Theophylline induced melanogenesis in B16F10 melanoma cells was carried out in the absence of UV exposure. The inhibitory activity of the ML extract was assessed in comparison with those of pCA and FA, against the standard (kojic acid; KA) at an identical concentration (0.1 mg/ml). Theophylline (T) induces melanogenesis through the cAMPdependent signaling pathway, with induction of gamma-glutamyl transpeptidase-, and tyrosinase-reactive cells (Busca and Ballotti, 2000; Chang, 2012; Hu, 1982) enhancing the melanin content by 156.97 ± 10.38%. By comparison with KA, a melanogenesis inhibitor, chelates with Cu2+ in tyrosinase to prevent tautomerization of DOPAchrome to 5,6-

13

dihydroxyindole-2-carboxylic acid (an intermediate of the melanin synthesis pathway and common skin lightening agent) (Gillbro and Olsson, 2011; Kim et al., 2012) resulting in 79.28 ± 1.87% melanin production (Fig. 3B). Surprisingly, the ML extract and its major constituents, pCA and FA were shown to be more potent, reducing melanin content levels to 38.34 ± 0.12, 25.73 ± 6.75 and 47.66 ± 7.08%, respectively. Relative melanin content ratios are further expressed in Fig. 3C. Melanogenesis inhibition by ML extract (61.66 ± 0.12%) was similar (p > 0.05) to that induced by pCA and FA (74.27 ± 6.75 and 52.34 ± 7.08%). In contrast, KA resulted in significantly lower suppression (20.72 ± 1.87%) levels (p < 0.001). These results would indicate that pCA, the major phenolic constituent, is primarily responsible for the ML extract activity. pCA proved significantly (p = 0.01) more potent than FA against melanogenesis, a factor related to its structural similarity to tyrosine, the precursor of tyrosinase, and subsequent action by competitive inhibition (An et al., 2010). Examination of melanogenesis suppression at lower the ML extract concentrations (0.0001 - 0.1 mg/ml) as shown in Fig. 3D indicated that inhibition levels followed a dose dependent profile. Melanin production is regulated by tyrosinase, and related proteins TRP-1 and TRP-2. Tyrosinase catalyzes the hydroxylation of tyrosine to produce 3,4-dihydrozyphenylalanine (DOPA), and further oxidation of DOPA to form DOPAquinone. Thereafter, TRP-2 functions as a DOPAchrome tautomerase catalyzing the rearrangement of DOPAchrome to 5,6dihydroxy-indole-2-carboxylic acid or DHICA, which is further oxidized by TRP-1 to a carboxylated indole-quinone. Thus inhibition of melanogenesis regulating enzymes, particularly tyrosinase and TRP-2, is a focal point for research into prevention of skin hyperpigmentation, one of the clinical signs of cutaneous aging (Costin and Hearing, 2007; Kammeyer and Luiten, 2015). Tyrosinase inhibition activity of the ML extract in B16F10 melanoma cells was then determined at the optimized concentration (0.1 mg/ml). T significantly (p = 0.022) increased enzyme activity (109.52 ± 1.72%) in contrast to KA, ML

14

extract, pCA and FA which significantly suppressed tyrosinase activity (Fig. 4A). Relative tyrosinase inhibitory effects are shown in Fig. 4B, with the ML extract showing significant (p < 0.001) inhibition over pCA and FA (92.80 ± 0.05, 59.56 ± 3.93 and 20.45 ± 4.11%). Moreover, the ML extract proved more potent than a previously reported Chinese herbal formula composed of four herbs showing tyrosinase inhibition in B16 cells (Tsang et al., 2012). The potent activity of the extract is proposed to arise from synergistic effects relating to the total pool of active phenolic constituents (Fig. 2A). In addition, tyrosinase inhibition proved concentration dependent as shown in Fig. 4C, in accordance with melanin content as exhibited in Fig. 3D. The activity of the ML extract on inhibition of TRP-2 was also examined, with the results shown in Fig. 5. Clear evidence of TRP-2 inhibition was found, with pCA, the major constituent, being responsible for protein inhibition (Fig. 5A). Furthermore, the antienzymatic activity from the ML extract proved higher than that of the previously reported Chinese remedy, which only exhibited 44% TRP-2 inhibition at the same test concentration (Tsang et al., 2012). As in the cases of melanin content and tyrosinase activity, TRP-2 inhibition followed a dose dependent profile, as shown in Fig. 5B. 3.3 Cellular activity assessments in human skin fibroblasts An assessment of the safety of the ML extract, including its major phenolic constituents, was undertaken through testing in human skin fibroblast cells at a concentration of 0.1 mg/ml with vitamin C (Vit. C) as a positive control (Fig. 6A). The ML extract, including pCA and FA, were found to be safe and caused no cytotoxic effects in epidermal cells. Cellular antioxidant activity was assessed by means of H2O2-induced oxidative prevention (Fig. 6B). Cell viability of the control groups receiving media and treated with the solvent were 73.72 ± 3.36 and 76.50 ± 0.92%, consistent with levels in a previous report using human foreskin fibroblast tissue (Ruktanonchai et al., 2009). Interestingly, fibroblast viabilities on treatment

15

with the ML extract, pCA, FA and Vit. C were shown to be concentration dependent and all significantly prevented cellular oxidation (p = 0.037, 0.001, 0.018 and 0.039). 3.4 Formulation of stable topical products After identification of the therapeutic agents in the ML extract and investigating their cellular and biological activities, the standardized extract was further developed into a prototype topical product for clinical testing. The ML extract was dissolved in propylene glycol, or glycerin, at 10 and 8.5% (w/w) giving a clear solution free of precipitates, accordingly propylene glycol was chosen as the co-solvent in subsequent emulsions. Physicochemical properties of the ML extract solution were determined, and accelerated stability tests indicated that the ML extract solutions in propylene glycol were stable (Table 3), Oil in water emulsions of the ML extract were developed for application studies. The ingredients with the ML extract in propylene glycol are listed in Table 4. All creams proved stable as shown by accelerated testing (centrifugation assay). The cream base, and ML creams that passed the centrifugation assay were subjected to 7 heating-cooling cycles, and product appearance, pH and viscosity were monitored over time and compared with the as freshly prepared emulsions (Table 5). Varying the amount of the ML extract in the emulsion resulted in different physicochemical properties, with higher extract content resulting in less viscous, lower pH formulations. Following accelerated tests, formulations remained homogenous with only slight changes in viscosity but acidity levels increased, particularly for the 0.3% ML cream. However, the magnitudes of these changes are in the acceptable range for cosmetic formulations. ML creams containing 0.1 and 0.2% extract were assessed for chemical stability based on TPC, which did not show significant (p > 0.05) reduction over time as shown in Fig. 7.

16

3.5 Preliminary skin irritation evaluation in human subjects Preliminary skin irritation testing was conducted in 25 healthy subjects using the closed patch method. All subjects showed no signs of skin irritation on exposure to either base cream, ML creams, or water as control (MII = 0). 3.6 Clinical efficacy evaluation in human subjects The subjects (7 male and 17 female) aged 37.46 ± 6.66 years were subjected to a randomized, double-blind placebo controlled trial using the 0.1 and 0.2% ML creams on either their left or right hand (total 3 creams for 3 application spots). Non-invasive instruments were used to track the anti-aging efficacy of the products at the base line, and post-treatment (Kanlayavattanakul and Lourith, 2015). All subjects reported no adverse effects over the study period, confirming the safety of the ML creams. 3.6.1 Skin hydrating efficacy ML creams were shown to enhance skin hydration relative to the cream base at all points during the study period. Application of the ML creams over a longer period (28 - 84 days) significantly (p < 0.05) improved skin moisture levels over those measured after the initial treatment time (14 days). Moreover, skin moisture levels after 84 days of application were significantly higher than those at 56 days (p < 0.05), as shown in Table 6. 3.6.2 Skin lightening efficacy ML cream application resulted in lightened skin color tone (Table 6), of which the efficacy was significant (p < 0.001) following 28 days of treatment. However, the efficacies of the 0.1 and 0.2% ML creams were comparable over this period (p ≥ 0.622). 3.6.3 Skin firming efficacy Following 28 days of treatment, the ML creams significantly (p ≤ 0.045) increased skin firmness (Ur/Uf) relative to the cream base, whose application did not result in any

17

significant skin firmness enhancement (p ≥ 0.953). Skin firmness was enhanced by longer periods of treatment, with 84 days of the application giving significantly higher results than for shorter (56 and 28 days) application times (p < 0.05). However, efficacy of treatment proved to be dose independent (Table 6). 3.6.4 Skin wrinkle reduction and smoothing efficacies The ML creams were shown to significantly reduce the degree of skin wrinkling and induce smoothness (p ≤ 0.004 and ≤ 0.038) following 56 days of treatment (Fig. 8 and Table 6). In addition, both active creams (0.1 and 0.2%) similarly suppressed wrinkling and improved smoothness (p ≥ 0.774 and ≥ 0.979) at each monitored time interval. The ability of rice panicle extract to moisturize the skin was superior to that of Lithospermum erythrorthizon root extract, which required higher concentrations (1.0, 2.5 and 5.0%) to provide meaningful effects (10.72, 11.58 and 11.77% after 28 days) (Chang et al., 2008). Furthermore, the skin lightening efficacy of rice phenolics was higher than resveratyl triacetate, which was employed at a higher concentration (0.4%) to obtain high lightening efficacies (8.84 and 17.60% reduced tanning of skin forearm following 28 and 56 days, respectively) (Ryu et al., 2015). The level of skin abnormalities, in terms of anti-wrinkle efficacy, was greatly improved by ML cream application, as assessed by Cutometer® MPA 580 and Visicoscan® VC98. Skin elasticity showed noticeable improvements after application for 56, and 84 consecutive days (Table 6). Moreover, the anti-aging efficacy of the rice panicle extract creams was confirmed by means of skin surface analysis. Skin surface improvements, in particular wrinkle reduction and smoothness, were more pronounced for rice extract than for green and red teas (9.9%), or gingko (4.32%) cosmetics after 28 days (Churienthong et al., 2010). ML creams also proved more effective in promoting skin smoothness than higher content (2.5%) lotus cosmetics, which only showed large improvements (19.33%) after 56 days of treatment (Mahmood et al., 2013).

18

3.7 Preference of the 0.1% ML cream The opinions of thirty subjects aged between 20-60 years old towards the ML creams were obtained by means of a questionnaire. The subjects clearly (> 80%) preferred the 0.1% ML cream over the 0.2% cream in terms of its viscosity, color, odor, spreadability, skin adsorption, greasiness and skin moisturizing effect. 4. Conclusions Jasmine rice panicle extract has been shown to exhibit a variety of properties giving its potential as a base for treatment of skin disorders. The active constituents of the extracts were identified, and the biological properties of the extract examined in vitro, ex vivo and in vivo to determine the optimal concentration for use in cosmetic formulations. Therapeutic phenolics in the jasmine rice panicle are thought responsible for the extract activity. Standardized jasmine rice panicle extract was used for topical product development, with stable emulsions being obtained which were shown to effectively increase skin hydration, firmness and smoothness as well as having benefits against dark spot formation and wrinkle. This study provides a scientific basis to traditional remedies highlighting the use of rice in skin therapies. Acknowledgements This study was financial supports by Mae Fah Luang University fiscal year 2013 and 2014 with grant no. 5620800514 and 5720800505, the Agricultural Research Development Agency of Thailand for fiscal year 2014 with grant no. CRP5705020310 and Technology Research Grant of Thailand Toray Science Foundation of 2013. References Ahuja, U. Ahuja, S.C., Thakrar, K., Singh, R.K., 2008. Rice – a nutraceutical 12, 93-108.

19

An, S.M., Koh, J.S., Boo, Y.C., 2010. p-Coumaric acid not only inhibits human tyrosinase activity in vitro but also melanogenesis in cells exposed to UVB. Phytotherapy Research 24, 1175-1180. Antignac, E., Nohynek, G.J., Re, T., Clouzeau, J., Toutain, H., 2011. Safety of botanical ingredients in personal care products/cosmetics. Food and Chemical Toxicology 49, 324341. AOAC, 2002. Guidelines for single laboratory validation of chemical methods for dietary supplements

and

botanicals.

http://www.aoac.org/imis15_prod/AOAC_Docs/Standards

Development/ SLV_Guidelines_ Dietary_ Supplements.pdf (accessed 12.05.16).

Barber, J.I., Townsend, D., Olds, D.P., King, R.A., 1984. Dopachrome oxidoreductase: a new enzyme in the pigment pathway. Journal of Investigative Dermatology 83, 145-149.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Burlando, B., Cornara, L., 2014. Therapeutic properties of rice constituents and derivatives (Oryza sativa L.): a review update. Trends in Food Science & Technology 40, 82-98.

Busca, R., Ballotti, R., 2000. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell & Melanoma Research 13, 60-69.

20

Chang, M.J., Huang, H.C., Chang, H.C., Chang, T.M., 2008. Cosmetic formulations containing Lithospermum erythorohizon root extract show moisturizing effects on human skin. Archives of Dermatological Research 300, 317-323.

Chang, T.S., 2012. Natural melanogenesis inhibitors acting through the down-regulation of tyrosinase activity. Materials 5, 1661-1685.

Choi, S.W., Lee, S.K., Kim, E.O., Oh, J.H., Yoon, K.S., Parris, N., Hucks, K.B., Moreau, R.A., 2007. Antioxidant and antimelanogenic activities of polyamine conjugates from corn bran and related hydroxycinnamic acids. Journal of Agricultural and Food Chemistry

55,

3920-3925.

Churienthong, P., Lourith, N., Leelapornpisid, P., 2010. Clinical efficacy comparison of antiwrinkle cosmetics containing herbal flavonoids. International Journal of Cosmetic Science 32, 99-106.

Costin, G.-E., Hearing, V.J., 2007. Human skin pigmentation: melanocytes modulate skin color in response to stress. The FASEB Journal 21, 976-994.

Currais, A., 2015. Ageing and inflammation – a central role for mitochondria in brain health and disease. Ageing Research Reviews 21, 30-42.

Dipti, S.S., Bergman, C., Indrasari, S.D., Herath, T., Hall, R., Lee, H., Habibi, F., Bassinello, P.Z., Graterol, E., Ferraz, J.P., Fitzgerald, M., 2012. The potential of rice to offer solutions for malnutrition and chronic diseases. Rice 5, 16.

21

Elsner, P., Maibach, H.I., 2000. Cosmeceuticals: drugs vs. cosmetics. Marcel Dekker, New York.

Futrakul, B., Kanlayavatanakul, M., Krisdaphong, P., 2010. Biophysics evaluation of polysaccharide gel from durian’s fruit hulls for skin moisturizer. International Journal of Cosmetic Science 32, 211-215.

Gillbro, J.M., Olsson, M.J., 2011. The melanogenesis and mechanisms of skin-lightening agents-existing and new approaches. International Journal of Cosmetic Science 33, 210-221.

Hu, F., 1982. Theophylline and melanocyte-stimulating hormone effects on gamma-glutamyl transpeptidase and DOPA reactions in cultured melanoma cells. Journal of Investigative Dermatology 79, 57-62. ICH, 2005. Validation of Analytical Procedures: text and methodology Q2(R1). http://www. ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1_ Guideline.pdf (accessed 12.05.16). Izuishi. K., Kato. K., Ogura, T., Kinoshita, T., Esumi, H., 2000. Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy. Cancer Research 60, 6201-6207. Jenkins, G., 2002. Molecular mechanisms of skin ageing. Mechanisms of Ageing and Development 123, 801-810. Jun, H.-J., Lee, J.H., Cho, B.-R., Seo, W.-D., Kim, D.-W., Cho, K.-J., Lee S,-J., 2012. pCoumaric acid inhibition of CREB phosphorylation reduces cellular melanogenesis. European Food Research and Technology 235, 1207-1211.

22

Kammeyer, A., Luiten, R.M., 2015. Oxidation events and skin aging. Ageing Research Reviews 21, 16-29.

Kanlayavattanakul, M., Lourith, N., Tadtong, S., Jongrungruangchok, S., 2015. Rice panicles: new promising unconventional cereal product for health benefits. Journal of Cereal Science 66, 10-17.

Kanlayavattanakul, M., Lourith,. N., 2015. An update on cutaneous aging treatment usingherbs. Journal of Cosmetic and Laser Therapy 17, 343-352.

Killiç, I., Yeşloğlu, Y., 2013. Spectroscopic studies on the antioxidant activity of p-coumaric acid. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy115, 719-724.

Kim, H., Choi, H.-R., Kim, D.-S., Park, K.-C., 2012. Topical hypopigmenting agents for pigmentary disorders and their mechanisms of action. Annals of Dermatology 24, 1-6. Kong, C.-S., Jeong, C.-H., Choi, J.-S., Kim, K.-J., Jeong, J.-W., 2013. Antiangiogenic effects of p-coumaric acid in human endothelial cells. Phytotherapy Research 27, 317-323.

Kumar, N., Pruthi, V., 2014. Potential application of ferulic acid from natural sources. Plant Biotechnology Reports 4, 86-93. Liu, L., Hudgins, W.R., Shack, S., Yin, M.Q., Samid, D., 1995. Cinnamic acid: A natural product with potential use in cancer intervention. International Journal of Cancer 62, 345350. Mahmood, T., Akhtar, N., 2013. Combined topical application of lotus and green tea improves facial skin surface parameters. Rejuvenation Research 16, 91-97.

23

Mancuso, C., Santangelo, R., 2014. Ferulic acid: pharmacological and toxicological aspects. Food and Chemical Toxicology 65, 185-195.

Mukherjee, P.K., Maity, N., Nema, N.K., Sarkar, B.K., 2011. Bioactive compounds from natural resources against skin aging. Phytomedicine 19, 64-73. Nichols, J.A., Katiyar, S.K., 2010.

Skin photoprotection by natural polyphenols: anti-

inflammatory, antioxidant and DNA repair mechanisms. Archives of Dermatological Research 302, 71-53.

Ohguchi, K., Banno, Y., Nakagawa, Y., Akao, Y., Yoshinori, N., 2005. Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells. Journal of Cellular Physiology 205, 444-451.

Oka, M., Ilzuka, N., Yamanoto, K., Gondo, T., Abe, T., Hazama, S., Akitomi, Y., Koishihara, Y., Ohsugi, Y., Ooba, Y., Ishihara, T., Suzuki, T., 1996. The Influence of interleukin-6 on the growth of human esophageal cancer cell lines. Journal of Interferon & Cytokine Research 16, 1001-1006. Papazisis, K.T., Geromichalos, G.D., Dimitriadis, K.A., Kortsaris, A.H., 1997. Optimization of the sulforhodamine B colorimetric assay. Journal of Immunological Methods 208, 151158. Pieroni, A., Quave, C.L., Villanelli, M.L., Mangino, P., Sabbatini, G., Santini, L., Boccetti, T., Profili, M., Ciccioli, T., Rampa, L.G., Antonini, G., Girolamini, C., Cecchi, M., Tomasi, M. 2004. Ethnopharmacognostic survey on the natural ingredients used in folk cosmetics, cosmeceuticals and remedies for healing skin diseases in the inland Marches, Central-Eastern Italy. Journal of Ethnopharmacology 91, 331-344.

24

Ruktanonchai, U., Bejrapha, P., Sakulkhu, U., Opanasopit, P., Bunyapraphatsara, N., Junyaprasert, V., Puttipipatkhachorn, S., 2009. Physicochemical characteristics, cytotoxicity, and antioxidant activity of three lipid nanoparticulate formulations of alphalipoic acid. AAPS PharmSciTech 10, 227-234. Ryu, J.H., Seok, J.K., An, S.M., Baek, J.H., Koh, J.S., Boo, Y.C., 2015. A study of the human skin-whitening effects of resveratryl triacetate. Archives of Dermatological Research 307, 239-247. Saija, A., Tomaino, A., Trombetta, D., de Pasquale, A., Uccella, N., Barbuzzi, T., Paolino, D., Bonina, F., 2000. In vitro and in vivo evaluation of caffeic and ferulic acids as topical photoprotective agents. International Journal of Pharmaceutics 199, 39-47. Saikia, A.P., Ryakala, V.K., Sharma, P., Goswami, P., Bora, U., 2006. Ethnobotany of medicinal plants used by Assamese people for various skin ailments and cosmetics. Journal of Ethnopharmacology 106, 149-157. Sharma, P., 2011. Cinnamic acid derivatives: a new chapter of various pharmacological activities. Journal of Chemical and Pharmaceutical Research 3, 403-423. Song, K., An, S.M., Kim, M., Koh, J.-S., Boo, Y.C., 2011. Comparison of the antimelanogenic effects of p-coumaric acid and its methyl ester and their skin permeabilities. Journal of Dermatological Science 63, 17-22. Thring, T.S.A., Hili, P., Naughton, D.P., 2009. Anti-collagenase, anti-elastase and antioxidant activities of extracts from 21 plants. BMC Complementary and Alternative Medicine 9, 27. Tsang, T.-F., Ye, Y., Tai, W.C.-S., Chou, G.-X., Leung, A.K.-M., Yu, Z.-L., Hsiao, W.-L.W., 2012. Inhibition of the p38 and PKA signaling pathway is associated with the antimelanogenic activity of Qian-wan-hong-bai-san, a Chinese herbal formula, in B16 cells. Journal of Ethnopharmacology 141, 622-628.

25

Umadevi. M., Pushpa, R., Sampathkumar, K.P., Bhowmik, D., 2012. Rice – traditional medicinal plant in India. Journal of Pharmacognosy and Phytochemistry 1, 6-12.

USFDA. 2013. Guidance for industry: bioanalytical method validation. http://www. fda.gov/downloads/Drugs/Guidance/ucm070107.pdf (accessed 12.05.16).

Viriyaroj, A., Ngawhirunpat, T., Sukma, M., Akkaramongkolporn, P., Ruktanonchai, U., Opanasopit, P., 2009. Physicochemical properties and antioxidant activity of gammaoryzanol-loaded liposome formulations for topical use. Pharmaceutical Development and Technology 14, 665-671.

Fig. 1 UPLC chromatogram of the standard phenolics (A) and the ML extract analysis using UPLC method. Fig. 2 Phenolics of the ML extract (A), and chemical stability of the ML extract (B). Fig. 3 Safety of the ML extract in B16F10 melanoma cells and its activity against melanin production.* p < 0.05, ** p < 0.001 Fig. 4 Effects of the ML extract against tyrosinase activity in B16F10 melanoma cells * p < 0.05, ** p < 0.001 Fig. 5 Effects of the ML extract against TRP-2 activity in B16F10 melanoma cells.* p < 0.05, ** p < 0.001 Fig. 6 Safety (A) and prevention of oxidative stress (B) in human skin fibroblasts of the ML extract* p < 0.05, ** p < 0.001. Fig. 7 Total phenolics content of the 0.1 and 0.2% ML creams before (Int) and after 7 heating-cooling cycles (HC). Fig. 8 Skin surfaces at the base line and following the treatment for 84 days.

26

Table 1 Accuracy of the assayed UPLC method for phenolics determination Paramet Phenolic er

GA

PA

ChA

CA

SyA

pCA

FA

SiA

RA

V

Q

r

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

96

82

75

80

80

73

85

71

61

81

74

2.05

2.92

6.90

0.76

3.56

1.36

4.85

3.14

2.65

1.55

4.06

0.64

0.92

2.17

0.24

1.12

0.43

1.52

0.99

0.83

0.49

1.25

LOD (mg/ml) LOQ (mg/ml)

Table 2 Intra- and Inter-day precisions of the major constituent phenolics analyzed by UPLC method Phenolic

Concentration (mg/ml)

pCA

10 50 500

Intraday Recovery (%) RSD (%) 172.56 2.61 97.30 4.24 98.86 2.42 27

Interday Recovery (%) RSD (%) 174.32 99.53 100.70

2.05 3.77 2.97

FA

CA

10 50 500 10 50 500

176.31 94.86 96.58 173.05 89.99 101.89

8.24 2.57 3.36 3.15 1.66 3.05

167.38 95.62 100.53 171.73 92.76 100.61

8.39 2.82 5.05 2.56 4.37 2.97

Table 3 Stability of the ML extract before (Int) and after the accelerated test (HC) Parameter Standard* ML extract Before (Int) After (HC)** TPC (g GAE/100 g) 22.48 ± 1.20 20.78 ± 0.61 EC (µg FeSO4 /1 mg) pCA; 1164.53 ± 19.27 515.2 ± 22.06 nd Anti-tyrosinase (%) KA; 78.65 ± 1.51 62.40 ± 2.32 nd Anti-elastase (%) UA; 66.72 ± 4.88 pCA; 25.62 ± 6.30 48.74 ± 4.67 nd FA; 45.95 ± 3.55 Ant-collagenase (%) EGCG; 78.64 ± 7.65 79.27 ± 5.24 nd Color Brownish yellow Brownish yellow pH 4.73 ± 0.23 4.68 ± 0.31 * KA; kojic acid, pCA; p-coumaric acid, FA; ferulic acid, UA; ursolic acid, EGCG; epigallocatechin gallate ** nd; not determined

Table 4 Ingredients of base and ML creams % (w/w) Ingredients Base 0.1% ML cream Carbopol Ultrez-21 DI water 87.14 87.04 Propylene glycol 4 Na EDTA Cyclomethicone Dub BOIS Shea butter 12.0 12.0 Stearic acid Rice bran oil TEA 99% Vitamin E acetate 0.86 0.86 Liquid germal plus Rice panicle extract 0.1

28

0.2% ML cream

0.3% ML cream

86.92

86.79

12.0

12.0

0.88

0.91

0.2

0.3

Table 5 Accelerated stability test of the creams Condition Int. HC

pH

Base 6.39 ± 0.03

Cream 0.1% ML 5.99 ± 0.01

0.2% ML 5.93 ± 0.01

0.3% ML 5.93 ± 0.01

viscosity* (cps) pH

3,051 ± 91.5 6.32 ± 0.01

2,912.0 ± 87.4 5.92 ± 0.50

2,094.7 ± 62.8 5.91 ± 0.20

2,076.0 ± 62.3 5.78 ± 0.03

viscosity* (cps)

4,159.0 ± 30.7

2,920.3 ± 64.0

2,063.3 ± 61.3

1,553.7 ± 15.9

* spindle no. 5, 120 rpm, % torque > 60

Table 6 Clinical efficacies of the ML creams Efficacy (%)

Time interval

Base cream

0.1% ML cream

0.2% ML cream

D14-D0

9.17 ± 3.11

11.42 ± 2.42

12.46 ± 2.88

D28-D0

18.06 ± 3.22

19.27 ± 1.76

20.51 ± 2.93

D56-D0

18.77 ± 2.81

20.13 ± 3.11

21.57 ± 3.21

D84-D0

20.20 ± 2.88

22.71 ± 2.85*

22.89 ± 2.33*

D14-D0

2.56 ± 0.44

3.24 ± 0.51

3.85 ± 0.40*

D28-D0

2.61 ± 0.68

13.53 ± 0.84*

14.10 ± 1.55*

D56-D0

2.85 ± 0.81

20.21 ± 1.17*

21.60 ± 1.12*

D84-D0

2.91 ± 0.98

23.59 ± 1.23*

23.97 ± 1.37*

D14-D0

1.61 ± 0.44

1.43 ± 0.51

1.45 ± 0.40

D28-D0

1.55 ± 0.68

3.63 ± 0.84*

3.06 ± 1.55*

D56-D0

2.81 ± 0.81

6.75 ± 1.17*

6.77 ± 1.13*

D84-D0

3.32 ± 0.98

8.75 ± 1.23*

8.33 ± 1.37*

D14-D0

1.97 ± 0.36

3.00 ± 0.88*

2.94 ± 0.75*

D28-D0

3.27 ± 0.47

4.78 ± 0.91

4.84 ± 0.79

D56-D0

4.73 ± 0.57

10.74 ± 1.68*

10.88 ± 1.22*

Skin hydrating

Skin lightening

Skin firming

Skin wrinkle reducing

29

D84-D0

5.78 ± 0.59

12.70 ± 1.62*

11.53 ± 1.20*

D14-D0

6.77 ± 1.86

6.25 ± 2.23

6.70 ± 1.21

D28-D0

16.23 ± 3.14

17.45 ± 3.18

17.10 ± 2.57

D56-D0

17.58 ± 3.17

31.75 ± 3.71*

30.64 ± 4.89*

D84-D0

17.89 ± 3.19

39.44 ± 4.30*

39.46 ± 4.81*

Skin smoothing

* p < 0.05

Graphical-abstract

30

1

(A)

2 3

4

(B)

5 6

Fig. 1

1

4000

Content (mg/ml)

3500

ML

3000 2500

PL

2000 1500 1000 500 0 CA

pCA

FA

Phenolics 1

(A)

2 7000

6000 Int

Content (mg/ml)

5000 HC 4000

3000

2000

1000

0 CA

pCA

FA

Phenolics 3

(B)

4 5

Fig. 2 2

Total

0

20

40

60

80

100

120

140

Fig. 3

Cell viability (%)

160

Melanin content ratio (%)

0

20

40

60

80

100

**

**

*

ML

**

ML

*

**

pCA

*

(C)

pCA

**

**

Sample

Sample

**

(A)

**

FA

**

** *

FA

**

KA

**

**

1 mg/ml

0.1 mg/ml

0.01 mg/ml

0.001 mg/ml

0.0001 mg/ml

3

0

20

40

60

80

100

Melanin content (%) Melanin content ratio (%)

0

20

40

60

80

100

120

140

160

180

0.0001 mg/ml

ML

**

FA

T

0.01 mg/ml

(B)

Sample

(D)

ML concentration

0.001 mg/ml

pCA

**

**

*

0.1 mg/ml

KA

**

Tyrosinase activity ratio (%)

-20

0

20

40

60

80

100

0

Fig. 4

Tyrosinase inhibition (%)

50

100

150

0.0001 mg/ml

ML

**

**

**

4

ML

pCA

(C)

0.1 mg/ml

*

(B)

0

**

(A)

KA

20

40

60

80

Sample

T

**

**

FA Sample

0.001 mg/ml 0.01 mg/ml ML concentration

pCA

**

**

*

100 Tyrosinase inhibition (%)

FA

**

KA

**

Relative ratio of TRP-2 activity (%)

140 120 * 100

**

80 60

**

40 20 0 ML

pCA

FA Sample

T

KA

(A)

Relative ratio of TRP-2 activity (%)

100

**

80

60 ** 40

**

20

0 0.0001 mg/ml -20

0.001 mg/ml

0.01 mg/ml

ML concentraction

(B)

Fig. 5

5

0.1 mg/ml

150

Cell viability (%)

0.0001 mg/ml

0.001 mg/ml

0.01 mg/ml

0.1 mg/ml ** **

100

**

** **

**

**

** ** **

*

50

0 ML

pCA

FA

Vit. C

Sample

(A) 100 * ** **

**

**

** ** *

80

Cell viability (%)

**

**

**

*

60 0.0001 mg/ml 0.001 mg/ml

40

0.01 mg/ml 0.1 mg/ml

20

0 ML

pCA

FA

Sample

(B) Fig. 6

6

Vit. C

7 Int 6

HC

g GAE

5 4 3 2 1 0 0.1% ML cream

0.2% ML cream

Fig. 7

Fig. 8

7