LC–MS profiling‐based non‐targeted secondary metabolite screening for deciphering cosmeceutical potential of Malaysian algae

Application of natural resources from the marine environment in the cosmeceutical industry is gaining great attention.


| INTRODUC TI ON
Natural ingredients have been used in skincare products and are gaining more attention nowadays due to concerns about the side effects of synthetic ingredients.2][3] Metabolites are organic and inorganic chemicals with low molecular weight which act as intermediates, reactants, or products from biochemical reactions that are enzyme-mediated. 4The biometabolites are categorized into two types: the primary metabolites that involve in the growth, development, and reproduction, while the secondary metabolites are those that have specific roles in providing protection from harsh environments. 5Secondary metabolites can be distinguished from primary metabolites through their differences in chemical structure, synthetic pathways, and their precursor molecules.A set of primary and secondary metabolites present within an organism is called a metabolome and to understand the complex networks of metabolites to their corresponding biological systems, metabolomics is the all-inclusive analysis of metabolites in detailed characterization phenotypes. 6,7smeceutical potential of marine resources has gained significant attention nowadays due to the side effects and low absorption rate of synthetic cosmetics. 8Marine life comprises a vast source of functional materials such as polyunsaturated fatty acids (PUFA), vitamins, antioxidants, bioactive peptides, polysaccharides, enzymes, and essential minerals. 9][10] Recent studies have shown that metabolites of marine algae possess cosmeceutical potentials.For instance, Laurencia snackeyi is found to have antiinflammatory properties 11 ; fucoxanthin, an abundant carotenoid found in most brown algae, has the properties of anti-inflammation and antioxidant; 12 Fucus vesiculosus possess anti-aging properties 13 and the Porphyra umbilicalis 14 and Padina australis 15 are effective against UV sunlight.Therapeutic and pharmaceutical properties of a few Malaysian algae have been well documented such as neuritogenic and anti-cholinesterase activities of Gracilaria manilaensis, 16,17 alleviation of bisphenol A-induced adverse reproductive abnormalities in mice using Gracilaria changii 18 ; antidepressant activities of Padina australis, 19 and anti-neuroinflammation properties of Kappaphycus malesianus. 20However, their cosmeceutical potential has yet to be explored and harnessed for its use as a substitute in the cosmeceutical industry.Therefore, this study aimed to decode the secondary metabolites of two Malaysian algae, brown alga Sargassum sp. and red alga Kappaphycus sp., using LC-MS analysis to assess their cosmeceutical potential.Alongside the metabolite profiling, in vitro antioxidant capacity of both species was also determined.Upon collection, the specimens were washed with saltwater to remove debris and mud by successive washing with the final rinse in distilled water (dH 2 O) and frozen.Frozen samples were freeze dried (LaboGene) and ground into fine powder using food grinder, then kept at −20°C prior to use.

| Preparation of ethanolic algae extracts
A 5-g of algae powder was dissolved in 25 mL of distilled ethanol and sonicated for 30 min and then incubated in an incubator shaker for 24 h.The mixture was centrifuged at 2000 rpm for 5 min.Supernatant was concentrated using rotary evaporator and then vacuum concentrator.Crude extracts were kept at 4°C until use.

| LC-MS profiling of metabolites
The crude algae extracts were diluted to 10 mg mL −1 using distilled ethanol, then filtered through a 0.45 μm membrane and followed by a 0.2 μm membrane filters.The purified extracts were diluted into 2 mL of 1 mg mL −1 concentration by mixing 200 μL of 10 mg mL −1 purified algae extract with 1.8 mL of double distilled water (ddH 2 O).The secondary and primary metabolite analysis was done by using Agilent 1290 Infinity LC system coupled with Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with  The initial absorbance of the ABTS working solution at 734 nm was recorded.The samples or the positive control (100 μL) was allowed to react with 1 mL of ABTS working solution in the dark for 6 min.
The absorbance was then measured at 734 nm using UV-Vis spectrophotometer microplate reader (Infinite 200 Pro, Tecan).All assays were performed in triplicate.The ABTS scavenging activity or the AA% value was calculated using the formula: The EC 50 was determined through the linear regression equation of the scatter plot of AA% value against the concentration of extracts.The EC 50 values were determined using regression equation of the scatter plot of absorbance reading against sample or positive control concentrations to calculate the concentration needed to achieve the set point at absorbance of 0.5. 22,23All assays were performed in triplicate.

| Statistical analysis
The data were subjected to one-way analysis of variance (ANOVA) with post hoc tests (Tukey for p > 0.05 and Games-Howell for p < 0.05) using SPSS ver.23, to calculate for mean and standard deviation as well as test for significant differences between extracts.

| LC-MS profiling of metabolites
A total of 132 compounds from Kappaphycus sp. were detected while only 47 are identified, and Sargassum sp. has 110 identified compounds from 268 detected compounds (Figure 1).The identification and generation of compound list are through the METLIN database. 21The functions of the identified compounds were referred to PubChem, 24   Sargassum sp. also possesses 5 anti-microbial and 3 antiinflammatory metabolites, while Kappaphycus sp. was found to also possess 2 anti-microbial and 3 anti-inflammatory metabolites.It is Absorbance of samples at 518 nm Absorbance of negative control at 518 nm × 100 (2) Absorbance of samples at 734 nm Initial absorbance of ABTS at 734 nm × 100 also observed that there are same compounds found in both which are 3 antioxidants namely 3-tert-Butyl-5-methylcatechol, (−)-Isoimijiol, and (6S)-dehydrofoliol; 2 anti-microbials (alpha-Kosin and phytosphingosine) as well as one anti-inflammatory metabolite (phytosphingosine).

| Antioxidant assay results
Antioxidant scavenging activity of Sargassum sp. and Kappaphycus sp. was determined through DPPH, ABTS and Reducing power assays.The EC 50 values were recorded from each assay done in triplicates and analyzed for mean, standard deviation, and significance through one-way ANOVA with post hoc test.All samples were significant to each other as their p values were <0.05 in all three assays.Sargassum sp.possess lower EC 50 values than Kappaphycus sp. in all three assays, which informs that Sargassum sp. has a higher free radical scavenging and reducing activities than Kapppahycus sp.(Table 1).

| DISCUSS ION
There are a variety of commonly found metabolites between the two species.Antioxidant, such as 3-tert-butyl-5-methycatechol, is an anti-pigmentation with definite but weak depigmentation effect as studied by Zhai and Maibach; 26 however, it is suitable to be used in cosmeceuticals for the treatment of acne scars and provide certain levels of whitening effect.A natural occurring antioxidant, (−)-Isoamijiol and (6S)-dehydrofoliol were also found.Antioxidants like fucoxanthin, enzacamene, and 2-hydroxyl-hexadecanoic acid found in Sargassum sp. have the effects to reduce redness and irritation, as well as anti-aging properties whereby it can help to reduce the ROS generation when exposed to UVB radiation which can also prevent wrinkles. 27 have been found from Malaysian Padina austalis, which ited UV-protective effects against UVB-induced cytotoxicity in HaCaT human keratinocytes. 15As for Kappaphycus sp., the antioxidant idebenone metabolite acts to restore succinate oxidation.
Tanacetol A is a natural occurring antioxidant.while 2-fluoro palmitic acid is involved in the inhibition of sphingosine biosynthesis where sphingosine signals for cell apoptosis.These natural antioxidants found in marine algae can be applied into skincare products for anti-aging, hence replace the use of the synthetic chemical hydroquinone, a skin-bleaching agent with harmful side effects especially to sensitive skin which may even cause ochronosis. 28ti-microbial compounds such as α-Kosin and phytosphingosine are found in both species.They are found to be able to treat bacteria-infected acne. 29,30While pseudolaric acid B found in Sargassum sp. was studied for its anti-fungal properties in the treatment of fungal infections on skin. 31Anti-inflammatory metabolites are also found in both species, which can be used as soothing agents in skincare products for treatment of easily irritated skin.
Other than the metabolites with cosmeceutical potentials, compounds like the 8E-tetradecenyl acetate, N-undecylbenzenesulfonic acid and 2-dodecylbenzenesulfonic acid were also observed in both algae species.These three compounds are pollutants or hazards, where 8E-tetradecenyl acetate is an ingredient of insecticides, while the other two are detergents and additives used in food processing.
This shows that either the environment where the algae samples originated from have nearby farm and factories, or that the marine environment is polluted.This also determines that the LC-MS analysis may help to indicate pollutants and hazards.

| CON CLUS IONS
This study reveals that both Sargassum sp. and Kappaphycus sp. are potent agents in cosmeceutical applications as there are natural antioxidants and other secondary metabolites with cosmeceutical potential.These features make both algae species as potential ingredients in cosmeceutical products.We found that Sargassum sp.

2 | 2 . 1 |
MATERIAL S AND ME THODS Algae specimens processingFresh specimens of Sargassum sp. and Kappaphycus sp. were collected from Port Dickson (Negeri Sembilan, March 2019) and Semporna (Sabah, December 2018), respectively.The specimens were identified morphologically and deposited in the university.
dual electron spray ionization (ESI) source.The column used is the Agilent Zorbax Eclipse XDB-C18, Narrow-Bore column with diameter of 2.1 mm, 150 mm length and pore size of 3.5 mm The column temperature and autosampler temperature were set at 25 and 4°C, respectively, using water and acetonitrile (60:40) as the mobile phase with a flowrate of 0.5 mL min −1 .The algae samples were injected 3.0 μL into the system and run in positive and negative polarity with applied fragmentor voltage at 125 V and use of nitrogen as collision gas.The extracts were analyzed in positiveion ESI mode with scanning mass range set to 100-3200 m/z, with acquisition rate of 1.03 s.For the peak integration, the Agilent MassHunter Qualitative Analysis (version B.05.00) software was used.The identification of metabolites was achieved by comparing the LC-MS peaks to the METLIN database. 21LCMS analyses were performed in triplicate.The LC-MS compound lists of Kappaphycus sp. and Sargassum sp. were tabulated in Data S1 (Appendix A and B).

2. 4 |
Antioxidant capacity 2.4.1 | 2,2-Diphenyl-1-pircryhydrazyl (DPPH) scavenging activity assay The positive control used was ascorbic acid (Sigma).50 μL of the positive control or samples was allowed to react with 1 mL of DPPH (0.1 mM, Alfa Aesar) in the dark for 30 min.The blank set (negative control) was done by mixing 50 μL of positive control or samples with 1 mL of methanol.The absorbance values were taken at 518 nm using UV-Vis spectrophotometer microplate reader (Infinite 200 Pro, Tecan, Switzerland).All assays were performed in triplicate.DPPH scavenging activity or the percentage antioxidant activity (AA%) was calculated using the formula:The value of half-maximum effective concentration (EC 50 ) was determined through the linear regression equation of the scatter plot of AA% value against the concentration of extracts.2.4.2 | 2,2′-Azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) scavenging activity assayAscorbic acid (Sigma) was used as the positive control.The stock solution was made by mixing 15 μL of 7 mM of ABTS (Roche, Germany) with 15 mL of 2.45 mM of potassium persulfate and allowed to react in the dark for 12-16 h.The activated ABTS stock solution was diluted by ethanol at a ratio of approximately 1:10 (v/v) to achieve the ABTS working solution at the absorbance of 0.7 ± 0.02 at 734 nm.
Positive control used is ascorbic acid (Sigma).Two hundred and fifty microlitres of 0.2 M phosphate buffer and 250 μL of 1% potassium ferricyanide was added to mix with 100 μL of samples or positive control and incubated for 20 min at 50°C.After the incubation, 250 μL of 10% trichloroacetic acid was added into each sample or positive control mixtures and centrifuged the mixture at 3000 rpm for 10 min.Supernatant were aliquoted 250 μL to mix with 250 μL of 0.1% iron (III) chloride and 250 μL ddH 2 O. Absorbance of the sample or positive control mixtures were read at 700 nm using UV-Vis spectrophotometer microplate reader (Infinite 200 Pro, Tecan).
ChemSpider 25 and journal articles.A complete compound lists of Kappaphycus sp. and Sargassum sp.showing the compounds' name, class, chemical structure, and their functions were tabulated in Data S1 (Appendix A and B).
has more secondary metabolites than Kappahycus sp., which links to the antioxidant assay results proving that Sargassum sp. has a better antioxidant property than Kappaphycus sp.Future studies would suggest further analysis of the metabolite with cosmeceutical potential to undergo a LCMS/MS analysis to elucidate the detailed function of each metabolite to ensure for its application in cosmeceutical industries.Furthermore, more studies on cosmeceutical potential of both species using cell-based in vitro methods, in vivo animal models, and clinical trials have to be carried out to determine skin absorption, irritation, allergens contents, and their underlying mechanisms.Moreover, some challenges such as biomass culturing techniques, metabolites extraction methods, and quality assurance and regulations of using algae in the cosmeceutical industry are awaiting to be solved.ACK N OWLED G M ENTSThis research was supported by Department of Biological Sciences, School of Medical and Life Sciences, Sunway University, Malaysia.