Potent pigmentation inhibitory activity of incensole‐enriched frankincense volatile oil‐identification, efficacy and mechanism

Frankincense volatile oil (FVO) has long been considered a side product in pharmaceutical industry since frankincense of large molecular weight is the prime target. However, the volatile oil recycled in the extract process might contain a series of functional actives, serving as promising ingredients in the cosmetic field.

and terpenoids. 6Due to its complicated composition and rich functionalities, frankincense extract has attracted great attention from researchers in different disease treatments.
Apart from its pharmaceutical uses, more and more dermatological applications of frankincense have been discovered.
Considering itsantibacterial functions, frankincense might serve as a promising candidate for oily skin and acne inhibition by balancing the skin microbiome. 7On the other hand, frankincense also exhibits its unique anti-inflammation function on skin.In vitro studies reveals that frankincense could reduce the level of interferon gamma-induced protein 10 (IP-10) and intracellular cell adhesion molecule 1 (ICAM1), 8 both of which are important inflammatory biomarkers leading to unpredicted skin sensitivity.In vivo tests also consolidate its anti-inflammation effect, since frankincensecontaining formula can improve itch and erythema conditions in erythematous eczema and psoriasis patients. 9In addition, frankincense, in the form of volatile oil, can even accelerate the transdermal absorption of other actives, by enhancing the ingredients transfer from epidermis to dermal capillaries through skin blood flow, which makes it a great transdermal permeation agent in the dermatologic field. 10ough frankincense is a promising and valuable ingredient, there exists tremendous resource waste in extracting frankincensebased medicines.In pharmaceutical field, gums and resins with higher molecular weight are the major extraction targets, while small molecules residing in volatile oil are considered as side products and are deserted.As indicated in various reports, frankincense is of high value and currently some of its species are facing the threat of extinction. 11At the same time, these small molecules usually excel more in transdermal absorption and provide skincare functions.Therefore, it is of great necessity to take full advantage of frankincense resources and explore the potential of frankincense volatile oil (FVO) in dermatological field, converting the "waste" into high-value cosmetic actives.
Given that, in this study, we collected the FVO and provide a comprehensive analysis of its composition, efficacy and possible mechanisms (Scheme 1).The FVO was achieved after the traditional Chinese herbal medicine processing procedure under 230°C for 15-20 min.The qualitative and quantitative analysis was first performed by GC-MS to identify the exact composition of FVO.About 40 actives molecules were identified, accounting for 92.16% of the total content, where incensole, acetate incensole, and acetate incensole oxide were the top three abundant actives in FVO.Based on the knowledge of its composition, efficacy evaluations were further carried out.The FVO exhibited unique whitening effect by suppressing melanin synthesis, in a dose-dependent way.In vitro test, volatile oil could reduce neutrophil aggregates by 33% and inhibit ROS generation by 54% in zebrafish models and scavenge free radicals in DPPH model, suggesting the anti-oxidative and anti-inflammatory effect.
To reveal its underlying mechanism, we performed network pharmacology to construct the interaction between FVO and skin whitening.One hundred ninety-two hub gene targets were screened out and the primary molecular function, biological process and signal pathways were identified through enrichment analysis.To sum up, this work systematically evaluated the possible skincare possibility of FVO, offering a new solution to topical application.

| Extraction of FVO
According to "Chinese Pharmacopoeia General Rules 0213", the Frankincense first went through a cooking process under 230°C for about 15-20 min, with the extracting solution of vinegar.The surface of Frankincense raw material gradually turned into bright color, and the evaporated oil was thus collected.

| Isolation and identification of FVO
FVO were analyzed using an Agilent 8890 equipped with an CTC PAL RSI 85 autosampler (CTC Analytics, Switzerland), and the split/splitless injector was operated in splitless mode.The apparatus was coupled to an Agilent 5977B mass spectrometer (Agilent Technologies, Inc).The analytical column was an HP-5MS Agilent column (30 m × 0.25 mm × 0.25 μm).The chromatograph was programmed for an initial temperature of 60°C for 2 min.The temperature was increased to 220°C at a rate of 10°C/min held for 1 min, and then increased to 280°C at a rate of 5°C/min, held for 10 min.The total run time was 41 min.Helium was used as the carrier gas (flow rate: 1 mL/min), split ratio 1:50.The mass spectrometer was operated in the electron impact (70 eV) and SCAN mode for qualitative analysis, with a solvent delay of 3 min.
The compounds were identified by calculating the retention index of each peak and comparing the mass spectra fragmentation with those from the National Institute of Standards and Technology (NIST) Library.The relative content was determined by the peak area normalization method.

| Inhibition of melanin synthesis
Six test groups were set up in the test, including the control group with mere zebrafish embryo, 100% inhibition group treated by phenylthiocarbamide, positive control group treated by kojic acid solution and the tested group treated by FVO of different concentrations.Danio rerio of wild type AB was selected as the model, with 12 pieces for each group in 96-well plate.The embryo was treated by 200 μL water, 30 mg/L phenylthiocarbamide solution, 2.5 g/L kojic acid solution and FVO solution (5%, 50%, and 100%) for 48 h at 28°C.After incubation, the intensity of melanin was measured by the microscope (AZ100, Nikon) and analyzed through ImageJ.The melanin inhibition rate could be calculated by the equation: In the equation, T represents the average melanin intensity in sample group, C represents the average intensity in control group and P represents the average intensity in 100% inhibited group.Two-tailed t-test to obtain p-value.(Significance levels were denoted as: *p < 0.05, **p < 0.01 and ***p < 0.001).

| Inhibition of inflammation
Twenty-four pieces of zebrafish embryo were selected and pretreated with copper sulfate to induce the nerve colliculus cell damage and subsequent neutrophil aggregation.In detail, 24 pieces of zebrafish were exposed to 0.16 mg/L copper sulfate and FVO solution with different concentrations (5%, 50%, and 100%), to achieve a constant volume of 5 mL.After 40 min incubation, the zebrafish embryo was fixed by 4% paraformaldehyde for 1 h, and washed with 0.04% Triton X-100 (dissolved in PBS) and 50% ethanol.The Sudan black staining solution, composed of 900 μL 70% ethanol, 100 μL 0.4% Sudan black ethanol solution and 1 μL 0.12% phenol water-ethanol solution, was used to staining the zebrafish embryo for 1 h under room temperature, followed by repeat washing with Triton X-100 and ethanol solution.The number of neutrophil was counted and the microscopic (AZ100, Nikon) photo was also taken and analyzed.The inhibition rate of inflammation could be calculated by the equation:

S C H E M E 1 Illustration of the study process: Collection of FVO extract, analysis of its active ingredients, efficacy tests on zebrafish models, and bioinformatics analysis on the ingredients interaction
In the equation, T represents the mean value of neutrophil number in sample group, and C represents the mean value of neutrophil number in control group.Two-tailed t-test to obtain p-value.

| Elimination of reactive oxygen species (ROS)
Twenty-four pieces of zebrafish embryos were selected and exposed

| DPPH scavenging activity
DPPH scavenging activity of FVO was evaluated according to the method reported by Omoboyowa et al 12 with slight modification.

| Bioinformatics analysis
The canonical SMILES (Simplified Molecular Input Line Entry System) structures of the main ingredients in FVO were searched and downloaded from the PubChem database.Then, we predicted the putative targets of these chemicals using the online tool Swiss TargetPrediction. 13Only the targets with a p > 0.1% were kept for the downstream analysis.The names of targets were normalized into UniProt ID.Finally, the active component-target network was visualized using Gephi software. 14en, we searched the keywords "pigmentation, sunspots, and whitening" against the Genecards, OMIM, and DisGeNet databases to collect the disease-related targets.After de-duplication and gene name normalization, we found the potential action targets of FVO on skin pigmentation by constructing the Venn diagram.Further, the function of these genes was annotated by Gene Ontology (GO) database. 15And the top 30 pathways were significantly enriched using the ggplot2 package in R platform. 16e Protein-Protein Interaction (PPI) analysis of common genes was performed by Search Tool for the Retrieval of Interacting Genes (STRING) database. 17The related network was further reconstructed using the Cytoscape software. 18Then the top 20 genes were screened with a high degree of connectivity using the cyto-Hubba plugin. 19| RE SULTS

| The identification and chemical composition of FVO by GC-MS analysis
The chromatographic analysis of FVO is shown in Figure 1, and its composition is listed in Table 1.Around 40 compounds were identified and quantified, with the relative percentage content accounting for 92.16% of the total content.The FVO con-

| The skincare whitening effect of FVO
The whitening effect of FVO was first consolidated by the inhibition rate of melanin synthesis.As seen in Figure 2, both the microscopic observation and quantification of melanin intensity were evaluated.
Phenltiocarbamide is a highly-effective melanin inhibition agent, the introduction of which could block the synthesis of melanin in zebrafish embryo.As a result, the melanin content in phenylthiocarbamidepretreated group is defined as 100% inhibited.Other groups were thus compared with it to quantify the relative inhibition rate.It could be inferred that the melanin synthesis was prominently reduced after the incorporation of FVO in a dose-dependent manner.When co-cultured with 5% FVO, the inhibition rate was about 9% (p < 0.05).However, when the concentration of volatile oil reached 100%, the melanin inhibition reached 91%.It was also worth mentioning that no obvious toxicity was observed within our testing range.In conclusion, FVO exhibited a great whitening effect by suppressing melanin synthesis.

| The anti-inflammatory effect of FVO
Since inflammation is always involved in the pigmentation process, the ability of FVO in reducing local inflammation is evaluated in zebrafish model by observing neutrophil aggregation.Neutrophils are the first white cells to appear at the site of injury or invasion of bacteria, and remove harmful substances from infection.By counting the number of aggregated neutrophils, the soothing effect and the inhibition of inflammation could be calculated.As shown in Figure 3, there were severe neutrophil aggregation in control group treated by copper sulfate, which meant significant inflammation after chemical inducement.
However, with the incorporation of FVO, the number of neutrophil aggregates prominently reduced.At the concentration of 5%, the inflammation was improved by 21%.By further increasing the concentration to 100%, the inhibition rate could reach 33%.These results indicated that FVO could play important roles in reducing inflammation and contribute to the control of melanin synthesis.

TA B L E 1
Frankincense volatile oil (FVO)main composition.

| The anti-oxidative effect of FVO
Oxidative environment would also facilitate the conversion of melanin.Thus, the anti-oxidation ability was tested in the study.The intracellular ROS content was selected as the marker to reflect the anti-oxidative ability of FVO.Due to the similarity of ROS regulation system in human and zebrafish, the zebrafish embryo was stained with H2DCFDA to evaluate the fluorescence intensity, which served as an indicator of intracellular ROS content.It could be inferred from

| The potential skin whitening mechanism of FVO
Only the ingredient with >1% amount in FVO was selected as the active compound and we finally got 250 predicted targets.
And according to multiple disease databases, a total of 7604 skin whitening-related genes were acquired.The network of main ingredients and skin whitening targets were shown in Figure 6.
One hundred ninety-six genes were commonly found in both groups, which were considered as the putative action targets.

| DISCUSS ION
It has been reported that there are abundant alkaloids, flavonoids, terpenoids, tannins and phenols in the methanol extract of frankincense, where the content of mastic acid accounts for 37%, showing anti-inflammatory and anti-aging effects. 20In another case, the main components of frankincense oil by steam distillation contains mono-, sesqui-, di-, and tri-terpenoids, with anti-inflammatory, cytotoxic, hepatoprotective, antibacterial, and antifungal activities. 21,22wever, the main components of FVO in this study are cembrane- and cembrene C, with a total content of about 61%.Cembrane-type diterpenoids are a class of terpene with a 14-membered oxygenated macrocycle consisting of four isoprenoid units, with higher bioactivities.Thus, it is of great significance to systematically investigate its effect in dermatology.As consolidated by our tests (Figure 2-5), FVO exerts anti-oxidation, depigmentation and anti-inflammation functions.
To understand its action mechanism, the top 20 hub genes were screened out by network pharmacology (Table S1).The most genes were enriched in MAPK-pathway (mitogen-activated protein kinase), including MAP2K1, MAPK1, MAPK3, MAPK14, and MAPK8.MAPK1 and MAPK3 genes were also named ERK1 and ERK2 (Extracellular signal-regulated kinase). 23ERK2 demonstrates critical role in adjusting MITF (microphthalmia-associated transcription factor) expression. 24MITF activity can be regulated by the phosphorylation of specific serine positions and it is also the most frequently studied target gene on melanogenesis.The downregulation of MAP2K can probably increase MITF expression and further inhibit the synthetic pathway. 25Also, the MAPK8 gene can modulate toll-like receptor signaling in skin psoriasis, inducing and its related receptor MC1R in keratinocytes. 28In conclusion, the above gene targets discovered by our bioinformatics analysis confirmed that FVO could directly target at MITF and modulate melanin synthesis.
It is interesting to find that hub targets are also strongly related to cellular oxidation and inflammation.For example, STAT3 could be activated by α-MSH in melanocytes, and the phosphorylation of STAT3 greatly reduces the transcription of TYR genes, leading to the inhibition of melanin. 29,30Apart from this direct approach, the stimulation of STAT3 in miR-7/STAT3 pathway could provoke the paracrine axis to indirectly affect the secretion of melanin.Meanwhile, by targeting JAK/STAT (Janus kinase-signal transducer and activator of transcription) pathway, the melanogenesis in epidermal melanocytes can be modulated by interleukin 4 (IL-4) through this pathway. 31JAK/STAT pathway is commonly found in immune-mediated inflammatory skin diseases and can impact cell proliferation and inflammatory cellular environment. 32PTGS2 is closely related with the expression of TYR, TRP-1, TRP 2, gp100, and MITF, 33 which are also core targets in skin pigmentation.By inhibiting COX2, the reflection of PTGS2, the melanin generation is greatly reduced.It is worth mentioning that PTGS2 has interconnections with STAT3.
As reported before, melatonin can target JAK2/STAT3 signal pathway, 34 thus causing a sharp decrease of ET-1 and PTGS2 in HaCaTs.
Through the synergistic way, they contribute to the skin whitening effect in FVO.HSP90AA1, a stress inducible gene within heat shock protein 90 alpha family class A, is commonly found in the core network of tyrosinase-associated melanogenesis process.It can adjust the cellular ROS cytoprotective pathway via pro-apoptotic repairing mechanisms. 35HIF-1α could activate NF-κB pathways, therefore participating in endogenous anti-oxidative system.To sum up, the FVO boosts anti-oxidative and inflammatory effects by targeting different genes, contributing to its skin whitening function.
Skin pigmentation is a complicated bioprocess.The generation of melanin is affected by a series of melanosome-related proteins and enzymes, adjusted by various signaling pathways,  during melanin synthesis to achieve skin whitening effect.It could be speculated that the unique skin whitening function of FVO is dated back to the activation of multiple gene targets and signal pathways as mentioned (Figure 8).
Apart from the above skin whitening approaches, we also find some interesting genes and potential mechanisms.NR3C1 has the ability to tune HPA axis.The glucocorticoid receptors (GR) coded by NR3C1 can bind to cortisol, leading to a negative loop in HPA axis and provoking the neuroendocrine response. 38The methylation of NR3C1 would weaken the expression of GR, which bring significant change to the skin.As a result, chemokines and biomarkers of allergy are up regulated, causing an inflammatory local environment, aggravating skin pigmentation.In future studies, HPA-associated approach could be further investigated to consolidate its correlation with skin whitening.

| LI M ITATI O N S
In spite of the inspiring results of FVO as skin whitening agent, there still exists a few limitations.The solubility (only soluble in polyols) of FVO might bring extra difficulty in formulation design.
To cope with this problem, delivery systems such as liposomes or microemulsions could be introduced to encapsulate FVO and achieve a higher loading content.The dark color of FVO also limits its usage as cosmetic ingredient.Therefore, the after our extraction process, a post-treatment of decolorization and refining is required and the manufacturing parameters needs to be optimized.Apart from the ingredient itself, further evaluations on both safety and function are required to consolidate our findings.
To begin with, acute and chronic toxicity in vitro and in vivo should be tested to confirm the safety of FVO.Secondly, the enriched pathways and targets in bioinformatics analysis needs to be investigated through multi-omics, to link the theoretical results with experimental outcomes.Moreover, to further exploit the potential of FVO, the function of FVO-containing formula should be examined clinically.In conclusion, the current study is a pioneering study on FVO, yet still tremendous work needs to be done in the future.
to 5% FVO solution, with a total volume of 200 μL.Meanwhile, the control group with pure culture media and positive group with 200 μL 0.1 g/L glutathione solution were set up, respectively.H2DCFDA solution (0.1 g H2DCFDA dissolved in 10 mL DMSO) was employed to stain the fish embryos after 24 h of exposure.After 2 h of incubation at 28°C, the fluorescence intensity of ROS was measured by fluorescence microscope (AZ100, Nikon) and analyzed by ImageJ.The ROS elimination rate could be calculated by the equation: In the equation, T represents the mean value of ROS intensity in sample group, and C represents the mean value of ROS intensity in control group.Two-tailed t-test to obtain p-value.

FVO was added to 4
mL of DPPH solution (50 mg of DPPH in 100 mL of Ethanol).The above ingredients were mixed thoroughly to achieve a homogeneous solution and incubated in the dark for 30 min.The absorbance was measured at 517 nm.The following equation was used to calculate the radical scavenging activity: In the equation, A 0 represents the absorbance of control, and A S represents absorbance of sample group of volatile oil.Error bars represent standard error of the mean (n = 5 per group).

Figure 4 F I G U R E 3
Figure4that there existed ROS signal in control group.However, when antioxidants were introduced, the intensity of ROS greatly reduced.FVO could efficiently eliminate ROS as shown in Figure4C,D, reaching 54% ROS elimination rate at the concentration of 5%, higher than the commonly-used antioxidant glutathione (0.1 g/L).
type diterpenoids.It mainly includes incensole, incensole oxide, acetate incensole, acetate incensole oxide, cembrene, cembrene A F I G U R E 4 The ROS elimination ability of FVO.(A-C): Elimination of ROS in zebrafish under fluorescence microscope; (D) Quantification of the inflammation inhibition rate of FVO with different concentrations.Significance: *p < 0.05.F I G U R E 5 DPPH scavenging activity of FVO at different concentrations.Data are presented as mean ± SD (n = 5).

F I G U R E 6
The ingredients-target interactions and functional analysis.(A) Network analysis of main FVO ingredients and their targets.(B) The potential targets of FVO ingredients on skin whitening.(C) The KEGG pathway enrichment of common genes.pathological pigmentation. 26Both acute and chronic inflammatory skin reactions can increase the risk of skin pigmentation, resulting in the hyperpigmentation or hypopigmentation.Apart from MAPK family, other genes such as PPARG or MDM2 could also directly inhibit melanin synthesis.The protein coded by PPARG could modulate the differentiation and metabolism of lipids and glucose.It has been found that compounds with PPARα and PPARγ activities could hamper the expression of TRY mRNA.MDM2 gene, encoding the murine double minute 2, is the important inhibiter of p53, which can function as the E3 ubiquitin ligase. 27As a tumor suppressor protein, p53 had a strong relationship with ultraviolet B-induced skin pigmentation and might be impacted by various stress conditions.It can stimulate the transcription of melanogenic paracrine cytokines, POMC (pro-opiomelanocortin)

F I G U R E 7
PPI network and key subnetworks by Cytoscape.(A) PPI network exported from the Cytoscape.(B) The top 20 critical genes analyzed by CytoHubba.