Skin lightening properties of zerumbone cream: A placebo‐controlled study

Despite the demonstrated anti‐melanogenic and UV protective effects of Zerumbone (ZER) in vitro, there is a lack of clinical trials that have been done to assess these properties. The primary objective of this study was to assess the effectiveness of ZER in lightening the skin tone of human participants with a single‐blind approach.

skin pigmentation in individuals, whereas a greater production of eumelanin results in darker skin pigmentation.The practice of skin lightening has a longstanding history, and the cosmetics industry has witnessed substantial expansion in recent years.This phenomenon might be attributed to the societal perception that pale skin is correlated with notions of attractiveness, good health and elevated levels of self-assurance. 1,2ny compounds demonstrated both safety and efficacy in terms of skin-lightening effects such as ascorbyl glucoside, 3 hexylresorcinol, 4 bakuchiol, 5 resveratrol 6 and niacinamide. 7A significant outcome emerges from a recent comparative clinical study that evaluated the effectiveness of hexylresorcinol and hydroquinone lotions for the purpose of skin lightening.The findings of the study indicated that the utilization of a 1% hexylresorcinol lotion yields skin-lightening outcomes that are comparable to those obtained with a 2% hydroquinone lotion. 4The most significant transformative effects often arise when these components combine in synergistic patterns.Numerous noteworthy combinations have demonstrated exceptional effectiveness in the domain of skin lightening.Several combinations, including hexylresorcinol + niacinamide, 8 hexylresorcinol + ethyl linoleate/oleate, 9 and ascorbyl glucoside + hexylresorcinol, 10 have exhibited promising results.
Zerumbone (ZER) is a phytochemical compound isolated from the rhizome of Zingiber zerumbet, a ginger plant often known as Lempoyang in Malaysia. 11,12[15][16][17][18] In addition to the aforementioned actions, Oh et al. (2018) have documented that ZER possesses the capacity to inhibit the synthesis of melanin pigments using stem cell factors (SCF)-induced modulation of the MITF and MC1R/α-MSH signaling pathway. 19 was found that a concentration of 10 μM of ZER had a greater inhibitory effect on melanogenesis compared to two commonly utilized natural skin lightening chemicals: kojic acid and arbutin, which were administered at concentrations of 1 mM and 0.2 mM, respectively.This observation was made in B16F10 mouse melanoma cells over 3 days. 19Despite the demonstrated antimelanogenic and UV protective effects of ZER in vitro, there is a lack of clinical trials that have been done to assess these properties.Hence, the present study aimed to investigate the lightening effects of ZER in human subjects.
Bhd. Glyceryl monostearate, glycerine, xanthan gum and potassium sorbate were purchased from local stores.

| Isolation of zerumbone
Zerumbone was provided and characterized as previously described.The authentication of the plant (Zingiber zerumbet) was conducted at with reference to voucher specimen number SK3327/18.Briefly, the rhizomes were subjected to crushing using a Clevenger-type device.Hydrodistillation was then carried out over 4 h, with three repetitions, to extract the essential oil.The distillate containing volatile oil was collected during distillation, followed by adding n-hexane drop by drop to obtain ZER crystals which were then kept at 4°C.The purity of Zerumbone was at least 95% based on High-performance liquid chromatography analysis. 20

| Formulation of placebo and ZER cream
The composition of the creams was formulated as shown in Table 1.
Both placebo and ZER creams were formulated using similar procedure.The cream formulation process involves the combination of excipients from the oil and aqueous phases in two distinct beakers under heat application.In the preparation of the ZER cream, ZER was included in the oil phase at a temperature of 75°C.The mixture was then agitated using a digital overhead stirrer (IKA® RW20 digital), operating at 500 rpm for 5 min.In the subsequent stages, the aqueous phase was introduced into the oil phase after heating both phases to a temperature of 75°C.The water-in-oil emulsion was continually blended in a unidirectional manner.The addition of potassium sorbate occurred after the cream reached a temperature of 40°C, and the emulsion was subjected to continuous mixing until it reached ambient temperature.
The concentration of ZER cream [0.5% (w/w)] utilized in this study was obtained from the investigation completed by Yang's research group. 21The creams were subjected to microbial and heavy metal testing at the HAVEC Laboratories in order to ascertain their safety for utilization in the study.

| Qualitative evaluation of cream
Participants were provided with a fresh supply of cream every week for their follow-up period.A qualitative assessment was conducted on the placebo and ZER cream for each of the four batches regarding appearance, aroma, pH, spreadability, afterfeel, and removal.Upon completion of the studies, participants were requested to complete a questionnaire to assess the cream's texture based on a 5-point scale, ranging from "very poor" (1) to "very good" (5).The factors assessed were odor, spreadability, skin hydration, skin smoothness, cream glossiness, and overall satisfaction with the cream. 22

| Inclusion and exclusion criteria
Volunteers were recruited based on the following criteria: (iii) Disease-free participants, those with no dermatological or systemic disorders.
(iv) Available throughout the study period.
A volunteer who meets any of the criteria as follows was excluded from the study: (i) Those with a history of allergic reactions to cosmetics in general.
(ii) Any skin cancer or disease that could interfere with the test results.
(iii) Diagnosed with chronic skin disease.
(iv) Excessive hair on the test sites.
(v) Females who are pregnant or breastfeeding (vi) Non-compliant volunteer (vii) Outliers in data analysis

| Study protocol
Participants who satisfied the predetermined criteria for inclusion and exclusion were enlisted as volunteers after a thorough review of the patient information sheet and subsequent signature of the consent form.The participants were assigned to groups A and B in a random manner, as depicted in Figure 1.Participants in Group A were administered ZER cream on their right volar forearm while a placebo cream was applied to their left volar forearm and vice versa for Group B. The volar side was selected as the test site, considering it is more protected against UV radiation than the dorsal side, which would provide greater data accuracy. 22,23ior to the commencement of the study, a one-week washout period was implemented.The participants were instructed to refrain from direct exposure to sunlight and cease the application of any skin-lightening agents and skincare products at the designated test areas, as well as any oral supplements that may enhance skin conditions, for the duration of the study. 24xt, participants were administered a weekly dosage of 1 g of placebo and ZER cream over 4 weeks.The participants were provided with instructions and reminders to administer approximately 0.07 g twice daily, specifically in the morning and evening, at the same time each day.The participants underwent training to develop the ability to estimate the quantity of 0.07 g that should be administered to their volar forearms.To ensure compliance, the participants were informed to fully apply the remaining placebo and ZER cream on the test side before their follow-up appointment.
The participants' melanin level baseline was measured with Mexameter MX 18 (CK Electronic GmbH, Cologne, Germany).
Subsequently, measurements were taken every week for 4 weeks.
The measurement was repeated three times to calculate the mean value.The change in melanin level compared to baseline in the placebo and ZER group was analyzed individually for each participant.
The mean and standard deviation of the changes were calculated.

F I G U R E 1
Flowchart of the study protocol.

| Allergic reactions assessment
The assessment of allergic reactions was conducted at the initiation of the trial.Erythema levels were assessed, and visual inspections were conducted before and 30 min after the initial administration of both placebo and ZER cream.In addition, participants were instructed to communicate with the researcher regarding any sensations of burning, itching, or discomfort induced by the cream, if experienced, during the study.

| Melanin Measurement
The participants were instructed to rest in an environment with a temperature range of 24 ± 3°C and a relative humidity range of 65% ± 15% for 15 min before measuring their melanin levels.The Mexameter MX 18 probe has a high level of sensitivity, such that even a minor deviation in the positioning of the probe can lead to fluctuations in melanin levels, thereby impacting the precision and reliability of the obtained measurements.Hence, during the measurement, the arms of the participant were positioned on a counter in a state of relaxation.
The probe was positioned perpendicular to the test site during the measurement process.To maintain data accuracy, the readings were consistently collected at a single place throughout the study.At the end of the study, participants were required to complete a questionnaire to collect data regarding factors affecting the study results, such as their daily exposure to sunlight.The change rate of the melanin was calculated using the equation as follows 25 :

| Statistical analysis
The

| ZER cream received high satisfactory scores
According to the findings obtained from the questionnaire, the mean overall satisfaction rating for the cream was 4.2 ± 0.83, as depicted in Figure 2. The subjects expressed satisfaction with the spreadability and skin hydration, achieving mean scores of 3.8 ± 1.01 and 3.75 ± 0.97, respectively.Furthermore, the skin smoothness and cream glossiness measurements yielded mean scores of 3.65 ± 1.14 and 3.65 ± 0.99, respectively.
Both creams exhibited a whitish appearance, with the ZER cream emitting a subtle herbal fragrance, but the placebo cream was devoid of any discernible aroma.However, a participant mentioned that both creams possessed the characteristic scent commonly associated with traditional medicine.A total of 8 participants described the aroma of the cream as reminiscent of ginger, coconut, and herbal that both creams exhibit no discernible scent.The mean pH value of the placebo and ZER cream was 6.5 and 6.4, respectively.
The observed texture of both creams exhibits a smooth consistency, which stays consistent even after more than 5 weeks under refrigeration.Nevertheless, the cream transformed consistency, resembling an ointment, after being stored for 14 days at ambient temperature while being shielded from direct exposure to sunlight.Both creams exhibited a moderate level of spreadability and produced a moisturizing and lubricating effect upon application.The applied cream can be removed with ease as well.(p = 0.047), with the test group having a melanin reduction rate of 6.41% and 7.80%, respectively, more than the control group.

| ZER cream did not induce erythema
None of the participants showed signs of redness on site of application for both creams.According to the erythema level measured using Mexameter MX 18, the mean baseline erythema for the control and test group was 233.21 ± 51.40 and 231.33 ± 50.57, respectively, as shown in Table 5.There was an increment in the erythema level 30 minutes after the application of the cream for the control group TA B L E 3 Parameters that may influence results.
b A total of 1 g or more of cream taken in a week.
c A total of 0.86 to 0.99 g of cream was taken in a week.
d A total of less than 0.86 g of cream taken in a week.
e An average daily exposure to sunlight per week for 4 weeks.However, these changes were insignificant.Similarly, when comparing the rate of change of erythema for both groups, the results were also shown to be not significant (p = 0.379).

Group
Based on the adverse effects reported by the participants, it was observed that none exhibited symptoms such as itchiness, stinging, burning, or redness upon using ZER cream.Nevertheless, it was observed that on certain occasions, one of the subjects reported the sensation of itchiness after applying the placebo cream.The sensation of itchiness persisted for a prolonged duration and manifested intermittently.It should be noted that the subject did not experience this phenomenon during the initial 30-min period following the initial application of the cream.

| DISCUSS ION
This study is the first documentation of the efficacy and safety of ZER cream when topically administered to human skin.No statistically significant alterations were observed in melanin levels within the control group.Nevertheless, a decrease in melanin levels was observed during both week 1 and week 2 in comparison to the first baseline measurement.The observed phenomenon could be attributed to glycerine use within the recipe, which has been found to possess hydrating and softening properties on the epidermis, hence facilitating the exfoliation process.As a direct outcome, it enhances the skin's complexion, resulting in a brighter appearance. 25 the other hand, there was a significant reduction in melanin levels for the test group.This is due to the presence of ZER in the that ZER increases the expression of heme-oxygenase-1 (HO-1) protein, an antioxidative enzyme, in a time and concentration-dependent manner through activation of Nrf2 signaling in both in vitro and in vivo studies. 21,26,27This antioxidative property may help to reduce oxidative stress-induced melanogenesis.According to Costin and Hearing (2007), UV radiation causes an increase in reactive oxygen species in keratinocytes and melanocytes.This leads to an increase in tyrosinase mRNA concentration, ultimately increasing melanin production. 28e melanin level in both the control and test group increased in week 3 and week 4 compared to week 2.This trend may be due to increased outdoor activities and sunlight exposure, considering there were public holidays during week 3.The UV index in Malaysia is at an all-time high throughout the year, with a UV index >11 in Malaysia during the study period.This UV index level is extremely harmful to unprotected sun exposure.Besides, it takes a maximum of 15 minutes to be sunburned.Despite the increase in melanin trend, the presence of ZER in the ZER cream can still suppress the melanin level more than the control product due to its anti-melanogenic properties.
The observed lack of significance between the placebo and ZER TA B L E 5 The erythema index (mean ± SD) of the test site.
(i) Healthy individuals aged between 18 to 60 years old with Fitzpatrick skin type II, III, and IV (Melanin level: more than 100) (ii) Not taking medication or under the care of a doctor 1 month before the commencement and throughout the entire study period.
statistical analysis was conducted with Statistical Package for the Social Sciences (SPSS) version 28.0.The data collected was presented in mean ± standard deviation (SD) format.The changes in erythema level 30 min after applying both placebo and ZER cream compared to their respective baseline were analyzed with paired t-tests.Besides, the time-dependent changes of melanin compared to baseline for both control and treatment groups and the differences in melanin between the groups at each time point were analyzed with Repeated Measures ANOVA and independent t-test, respectively.Analyzed data was considered statistically significant when the p-value was <0.05. 3 | RE SULTS 3.1 | Demographic data A total of 20 participants were included in the data analysis, while 6 individuals were excluded due to non-compliance.Table 2 displays the demographic characteristics of the 20 participants in the data analysis.The participants consisted of persons of both genders, ranging in age from 19 to 30 years.The majority of the individuals' volar forearms were categorized as Fitzpatrick skin type II (55%-60%).

TA B L E 2 2
fragrances without explicitly indicating which cream they were referring to.Notwithstanding this, most participants (n = 12) indicated The change rate of the melanin (%) = (value after treatment − baseline value) baseline value x 100% Demographic characteristics of participants in the data analysis.Participants' satisfactory score toward the creams.

dose and 2
participants missing doses weekly.Regarding sunlight exposure, 14 participants reported having an average of less than 2 h of daily exposure to sunlight per week for 4 weeks.The remaining 6 participants reported having an average daily exposure to sunlight of 2 to 4 h every day per week throughout the study.The rate of change in melanin level for the control group showed no significant reduction compared to the baseline throughout the study, as shown in Table 4.The placebo cream reduced the melanin level from ∑ = 152.84± 53.68 to 148.85 ± 52.59 in week 2.However, the melanin value increased up ∑ = 154.83± 55.82 and was maintained at approximately ∑ = 154.65 ± 51.93 in weeks 3 and 4. Likewise, the mean of the melanin for the test group shows a similar trend as the control group.The melanin value decreased from baseline ∑ = 156.41± 49.24 to ∑ = 142.38 ± 49.60 in week 2 and increased to ∑ = 149.54± 48.26 in week 4.However, unlike the control group, the rate of change of the melanin level compared to baseline in week 1 (p = 0.018) and 2 (p = 0.003) was significant.Comparing the rate of change of melanin value from baseline at each time point of the control and test groups, the test group consistently showed a more significant reduction level than the control group.Despite that, the changes were only significant in week 2 (p = 0.042) and week 3 ZER cream.Following the study ofOh et al. (2018), ZER phosphorylates the ERK 1/2 protein kinase and suppresses SCF-induced MITF and tyrosinase protein expression.19In addition, ZER reduces α-MSH production through the MC1R/α-MSH signaling pathway in mouse B16F10 and human G361 melanoma cells, affecting the downstream biochemical process required for melanin production.Apart from that, Shin et al. (2011), Yang et al. (2018) and Hseu et al. (2019) reported groups after the fourth week of the study prompts consideration of several potential factors influencing these findings.We can reasonably exclude treatment adherence as a factor since our participants adhered to the treatment plans.However, plausible explanations include the possibility that the cream's efficacy diminishes or reaches a plateau after a certain duration, warranting further studies for confirmation.Moreover, unaccounted confounding variables such as stress levels and dietary habits might have influenced the cream's effectiveness or participants' outdoor activity levels.Additionally, limitations in sample size and participant variability may have hindered the detection of smaller differences accurately.Biological factors, such as variations in skin types, individual biological responses, or metabolic rates, could also contribute to the observed diminishing effect of the cream.To comprehensively address these potential issues and attain a more profound understanding, conducting a longer-term study with expanded sample size, meticulous control of confounding variables, and potential consideration of alternative study designs, like a randomized controlled trial, are imperative.These measures are crucial to eliminate uncertainties and accurately identify the underlying cause behind the observed phenomenon.The primary irritation test shows that both creams did not induce an allergic reaction.However, comparing the change rate of erythema from baseline for both placebo and ZER cream, the presence of ZER in the ZER cream decreased the erythema level.This may be due to the ability of ZER to inhibit the pro-inflammatory bioactive protein.Haque et al. (2018) reported that ZER suppressed the production of cytokines, prostaglandin E2 (PGE 2 ), tumor necrosis 0.05 versus the baseline of each group.# p < 0.05 versus the control group at each time point.Paired t-test and independent t-test were used to analyze the p-value of the second reading vs the respective group baseline and p-value between the two groups at each time point, respectively.

Table 3
displays the data indicating that, over a period of 4 weeks, 15 individuals in both the control and test groups fully complied with the recommended dosages of cream application.It was observed that both groups consistently had participants experiencing under- The melanin index (mean ± SD) of the test site.(242.79 ± 61.78) but otherwise for the test group (228.89 ± 40.83).
*p < 0.05 versus the baseline of each group.#p < 0.05 versus the control group at each time point.Repeated measure ANOVA and independent t-test were used to analyze the p-value of each time point vs the respective group baseline and p-value between two groups at each time point, respectively.TA B L E 4