Cyanobacteria Secondary Metabolites As Biotechnological Ingredients in Natural Anti-Aging Cosmetics

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Abstract: The loss of density and elasticity, the appearance of wrinkles and hyperpigmentation are among the first noticeable signs of skin aging. Beyond UV radiation and oxidative stress, matrix metalloproteinases(MMPs)assume a preponderant role in the process, since their deregulation results in the degradation of most extracellular matrix components. In this survey, four cyanobacteria strains were explored for their capacity to produce secorndary metabolites with biotechnological potential for use in anti-aging formulations. Leptolyngbya boryana LEGE 15486 and Cephalothrix lacustris LEGE 15493 from freshwater ecosystems, and Leptolynglya cf.ectocarpi LEGE 11479 and Nodosilinea nodulosa LEGE 06104 from marine habitats were sequentially extracted with acetone and water, and extracts were analyzed for their toxicity in cell lines with key roles in the skin context (HaCAT,3T3L1, and hCMEC). The non-toxic extracts were chemically characterized in terms of proteins, carotenoids, phenols, and chlorophyll a, and their anti-aging potential was explored through their ability to scavenge the physiological free radical superoxidee anion radical (O2*-), to reduce the activity of the MMPs elastase and hyaluronidase, to inhibit tyrosinase and thus avoid melanin production, and to block UV-B radiation (sun protection factor, SSPF). Leptolyngbya species stood out for anti-aging purposes∶ L. boryana LEGE 15486 presented a remarkable SPF of 19（at 200 μg/mL）， being among the best species regarding O2°~scavenging，（IC50=99..50 μg/mL）and also being able to inhibit tyrosinase （IC25=784 μg/mL），proving to be promising agaiinst UV-induced skin-aging；L.ectocarpi LEGE 11479 was more efficient in inhibiting MMPs （hyaluronic.ase， IC50=863μg/mL；elastase， IC50=391μg/mL），thus being the choice to retard dermal density loss. Principal component analysis (PCA) of the data allowed the grouping of extracts into three groups, according to their chemical composition; the correlation of carotenoids and chlorophyll a with MMPs activity (p<0.01), O2*-scavenging with phenolic compounds (p<0.01),and phycocyanin and allophycocyanin with SPF,pointing to these compounds in particular as responsible for UV-I3 blockage. This original survey explores, for the first time, the biotechnological potential of these cyanobacteria strains in the field of skin aging, demonstrating the promising, innovative, and multifactorial nature of these microorganisms.

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Keywords: melanin; UV-blocker; elastase; hyaluronidase; tyrosinase; oxidative-stress; beta-carotene;phycocyanin; phycoerythrin; allophycocyanin

1. Introduction

Skin is a large and surprisingly complex human organ with a primordial barrier function of protecting internal organs from harmful stressors, such as chemicals, pathogens, cold, heat, and ultraviolet radiation (UVR). However, the importance of the skin goes much further, encompassing an undeniable socio-cultural role. cistanche cholesterol, In fact, skin appearance and shape are of crucial importance to an individuals self-esteem, its care and beautification being part of the daily routine since ancient times [1].

The delay of the skin-aging process has been a main societal demand. cistanche deserticola side effects This slow and complex process is induced by endogenous and exogenous factors that predispose skin to a progressive structural and functional degeneration that, beyond affecting its aesthetic appearance, leaves it prone to the development of a wide variety of diseases. Among exogenous factors, UVR is perhaps the most harmful agent to which the skin is most exposed. Excessive UVR exposure increases free radical generation, triggering a cascade of events that affect a wide variety of cell structures and enzymes, resulting in an immediate inflammatory response,and finally culminating in premature skin aging[2]. A particularly affected target in this theme is the extracellular matrix (ECM), a three-dimensional network of elastin and collagen fibers surrounded by the ground substances, such as hyaluronic acid (HA), that act together to maintain skin filling, elasticity, and flexibility [3]. Under a framework of oxidative stress, elastin and collagen fibers, responsible for the elasticity and resistance of the skin, may lose their structure[4]. This phenomenon, partly due to the deregulated activity of the matrix metalloproteinases (MMPs), such as elastase, collagenase, and hyaluronidase, is at the base of premature skin aging associated with external factors [1]These alterations affect the epidermal thickness, structure, and appearance, resulting in dryness, enlarged pores, fine lines, and wrinkles [5,6]. Therefore, the search for new, effective, multitarget and innovative ingredients for cosmetic formulations able to reach the greatest number of key targets in the skin aging process has been the focus of the cosmetics industry in recent decades.

Cistanche can anti-aging

The growing research on natural sources, more specifically those of marine origin, has provided a countless number of new molecules with promising bioactivities, worthy of further exploitation in the field of skin aging [1]. Among them, cyanobacteria stand out due to their capacity to produce bioactive secondary metabolites with unique structures and mechanisms of action. Beyond representing the only group of prokaryotes that can perform oxygenic photosynthesis, similarly to plants, cyanobacteria are self-renewable, have basic nutritional requirements, require minimal cultivation space,and have a low environmental impact, being a sustainable choice as a green source of cosmetic ingredients [7].

Focusing on skin formulations, cyanobacteria produce a wide array of bioactive metabolites, including phenolic compounds, proteins, pigments, and MMPs inhibitors, that cover the main processes comprising the basis of skin aging, namely antioxidation, photoprotection, and the ability to inhibit crucial enzymes of the ECM [8-12], as recently re-viewed [1,9]. Regarding antioxidant activity,rnumerous strains have been reported to have a representative amount of metabolites with radical scavenging capacity, such as carotenoids, e.g, those from the genera Leptolyngbya, Syneclocystis, and Wollea [9,13], phycobilipro-teins (PBP), such as Arthospira spp. and Spirulina spp. [13,14], and phenolic compounds, such as Nostoc commune [15]. In the field of photoprotection, cyanobacteria also stand out through the production of the well-known UVR-absorbing compounds mycosporine-like amino acids (MAAs) and scytonemin (SCY) [16]. Concerning the potential to inhibit col-lagenases, elastase, hyaluronidases, and tyrosinase, several examples are reported in the literature, such as mycosporine-2-glycine (M2G), isolated from the cyanobacterium Aphan-othece halophytica [17], the cyclic depsipeptides tutuilamides A-C, from Schizothrix spp. and Coleofasciculus spp.[18], a polysaccharide from Nostochopsis lobatus MAC0804NAN [19]and the extract Phormiskin Bioprotech G , from Phormidium persicinum [20], respectively.

Despite the studies presented above, the number of cyanobacteria strains explored in the field of cosmetics is still very low, considering the potentialities of this resource. With this in mind, this survey explores, for the first time, the biotechnological potential of sequential extracts of different polarities, obtained from four cyanobacteria strains,in the field of skin aging, demonstrating the promising, innovative, and multifactorial nature of these microorganisms. 2.Results and

Discussion

In the present study, two freshwater and two marine cyanobacteria strains were cultured, harvest, and subjected to a sequential extraction with acetone and water, with a view to their exploitation for cosmetic purposes. The sequential extraction with acetone followed by water was designed to monetize the biomass, in a more sustainable, environmentally friendly, and economically attractive process.In Table 1, the extraction yields are displayed. The extraction yield was significantly higher with water than with acetone (p<0.05), which has a direct correlation with the affinity of the different compounds to each solvent, their molecular weight, and polarity, as later cdiscussed in this study. It should be noted that Leptolyngbya boryana LEGE 15486 was the strain that showed higher extraction yields, which could be economically interesting when considering possible industrial applications.

2.1.Extracts Cytotoxicity

In order to select the extracts to proceed for biological activities assessment, an in vitro cytotoxicity assay was performed using different cell lines. Cytotoxicity tests are essential during cosmetics production, since they may predict health risks connected with the use of the extracts as bioactive ingredients. Thus,the cytotoxicity of the extracts was evaluated in three different cell lines of key importance regarding products for skin application:keratinocytes (HaCat), fibroblasts (3T3L1),and endothelial cells (hCMEC). Fibroblasts are among the most important cells for skin aging, being responsible for the production of the dermal matrix components essential for the maintenance of skin shape and structure, such as collagen and hyaluronic acid [9,21]. Endothelial cells form a barrier between vessel walls and blood, and were chosen to account for their presence in the dermis which, contrary to the epidermis, is an irrigated layer of the slkin [22].Regarding keratinocytes, they are the key cells of the epidermis, composing about 95% of this layer, but are present in all four layers, and provide structure and defense to the skin [23,24]. None of the extracts presented toxicity for the selected cell lines under the tested concentrations （12.5-200μg dry extract/mL）(Figures S1-S3). cistanche dosage reddit In this regard,all the extracts followed to the next step, where they were subjected to chemical characterization and evaluation of their biological activities. 2.2.Chemical Profile

The acetone and water extracts were chemically characterized in terms of phenols, total proteins,PBPs,total carotenoids,and chlorophyll a, in order to compare the chemical profiles obtained with the different extraction solvents, and to establish a relationship between the chemical composition and the biological activities evaluated.

2.2.1.Total Phenolic Content (TPC)

The total phenolic content (TPC) of both extracts was measured through the Folin-Ciocalteu colorimetric assay. Even considerirg the inherent limitations of the method, this is a standard assay widely used for the quick determination of total phenols, allowing the comparison of different samples, and consequently predicting their antioxidant potential. Table 2 displays the data (expressed in GAEs) for the total phenolic content of the eight extracts explored in the present study. The highest TPC was found in the acetone extract of Leptolyngbya cf.ectopic LEGE 11479,with 17.59 ug GAE/mg ary extract,followed by the water extracts of Leptolyngbya boryana LEGE 15486 and Cephalothrix lacustris LEGE 15493 (Table 2). Considering the yield of the combined extraction, Leptolyngbya cf.exocarp LEGE 11479 was the richest species, totaling about 26 ug GAE/mg dry extract when considering both extraction solvents.

Overall, the water extracts of the freshwater strains presented higher phenolic content than those from marine environments, while the opposite behavior was observed regarding acetone extracts. This observation was also noted for the two strains of the same genera, what leads to the assumption that, beyond species-specific characteristics, marine strains are more likely to produce phenolic compounds of lower polarity, given their higher prevalence in less polar solvents. Phenols have a polar and a nonpolar component in their molecules, possessing a solubility preference to solvents of intermediate polarities such as alcohols and acetone, rather than water. However, the solubility of phenols in different solvents cannot be based only on their polarities, since other parameters such as temperature and pH can have a great influence on their solubility, and thus justify their expressive presence in water extracts [25].

Previous work from our research group also explored TPC in different cyanobacteria extracts. Morone and co-workers [26] used the same methodology for TPC quantification and found that Nodosilinea nodulosa LEGE 06102 had a value of 1.23 mg GAE/g dry biomass which was above the values obtained herein (0.59 mg GAE/g dry biomass, converted according to the extraction yield). However, the authors used 70% ethanol as an extraction solvent, which certainly led to the differences observed. For all the strains analyzed by the authors, the highest value was found in Synechocystis salina LEGE 099 (2.45 mg GAE/g). In another study, with Nodosilinea Antarctica LEGE13457, the TPC content was 19.23 ug GAE/mg (acetone extract) and no phenols were detected in Leptolyngbya-like sp. cistanche extract benefits LEGE 13412, demonstrated the highest variability among similar species. They also reported data for other strains, with Cyanobium gracile LEGE12431 presenting the highest phenol content, with 22.01 ug GAE/mg of acetone extract [27]. Trabelsi and his team [28] reported that the thermophilic cyanobacterium Leptolyngbya sp. possessed 139mg GAE/g, this being among the highest values reported in the literature,and attributed by the authors to the high temperature of the cyanobacterial habitat: to avoid oxidative stress induced by high temperatures, cyanobacteria produce higher amounts of antioxidant compounds such as phenols and flavonoids. Another research group [29]also reported a TPCof 6.24mg GAE/g for Leptolyngbya sp. KC45, sampled from a location with temperatures of approximately 40-45°C.

Despite the differences inherent to the effects of the extraction solvents, and to the cyanobacterial cultivation conditions, such as light conditions, culture medium nutri-ents, and cell density, among others, the TPC values obtained herein are within the same order of magnitude as those reported in the literature. Moreover, Leptolyngbya boryana LEGE 15486 can be pointed out as an interesting species regarding phenolic compounds (3.08 mg GAE/g dry biomass), when compared to the widely known Spirulina spp. (1.78mg GAE/g)[30].

2.2.2.Proteins

Although proteins are generally undervalued, they compose a large fraction of the cyanobacterial biomass[31], with reported antioxidant and immunostimulant properties, as well as the ability to confer moisture retention to the skin, which is essential to prevent skin aging [32]. With this in mind, the extracts explored in the present study were characterized for their protein content, through a general approach focusing on total proteins (acetone and water extracts)(Table 3),and a targeted approach focusing on PBPs(water extracts)(Table4).

Regarding total proteins, it can be concluded that the aqueous extraction resulted in higher values than the acetone extraction, and also, that marine strains were poorer than those from freshwater environments. In terms of dry biomass, Leptolyngbya boryana LEGE 15486 presented the highest content (69.22 mg BSA/g dry biomass),and Nodosilinea nodulosa LEGE 06104 the lowest one(15.23 BSA/g dry biomass). Among other factors, these differences may be due to the different amounts of structural proteins presented by different cyanobacteria strains [32].

A practical example of the use of proteins in cosmetics concerns PBSs, which are naturally present in cyanobacteria. One of the cyanobacteria's defensive mechanisms is the capacity of these macromolecules to absorb light energy without producing reactive oxygen species (ROS), which is possible due to the changes in their content and ratio in phycobilisomes [33]. PBPs are water-soluble proteins that are associated with phycobilins, and divided into three groups according to their structure and light absorption spectra: phycocyanin (PC, 610-625 nm), phycoerythrin (PE, 490-570 nm), and allophycocyanin (APC,650-660 nm). As stated earlier, their interest as cosmetic ingredients is mainly due to their recognized antioxidant potential, thanks to their structural resemblance to bilirubin, which eliminates oxygen derivatives. PC is the most common PBP in cyanobacteria, with interesting antioxidant and radical scavenging properties, as well as the capacity to inhibit cell proliferation [13,14].

Regarding PC, Leptolyngbya boryana LEGE 15486 possessed the highest content （154.07 ug/mg ayy extract）， followed by Cephalothrix lacustris LEGE 15493（115.03 μg/mg dry extrace）which is in accordance with the strong blue color observed in the aqueous extracts of these species. cistanche genghis khan On the other hand, Leptolymgbya cf. ectocarpi LEGE 11479, with an intense pink/purple coloration in the biomass and in the aqueous extract, was the richest in PE （138.73 μg/mg dry extract）. Nodosilinea nodulosa LEGE 06104， with the lightest blue extract， presented visibly lower values of both PBPs(Table 4).

When comparing strains in terms of PBPs, it is extremely important to consider the culture conditions because differences in light, nitrogen, temperature, pH, carbon, and salinity can drastically influence their production [14]. Pumas and co-workers [29]evaluated the PBP content of the thermotolerant cyanobacteria Leptolyngbya sp. KC45,and found a PE content of almost 100mg/g,followed by approximately 40 and 43mg/g of APC and PC, respectively, which is in line with the results obtained herein for Leptolyngbya cf. ectocarpiLEGE11479 (Table 4).The work performed by Pagels and co-workers also explored the content of PBPs in sequential extracts obtained with different solvents. Although exploring a different cyanobacteria strain (Cyanobium sp.), the authors found a total PBP content close to 200 mg/g in a sequential extraction using the same solvents as those used herein, which is in accordance with the values obtained by us [34]. When comparing with the widely known Spirulina sp., with reported total PBPs of about 19mg/g dry biomass [30]it is possible to consider our strains of economic interest. In the same study, the authors also reported values of 127.01 mg/g dry biomass of total PBPs for Lynglya sp. In another study, data were presented from 18 strains, where the highest amount was found in Anabaena NCCU-9, with a value of 91.54 mg/g dry biomass [35]. 2.2.3.Carotenoids and Chlorophyll a

Carotenoids are natural fat-soluble isoprenoids with a wide array of colorations, varying from yellow to red [13]. These compounds have gained increased attention for their association with a decreased risk of several degenerative disorders, and for their recognized antioxidant activity, partly related to the C=C chemical double bonds present in their molecules. Besides these functions, carotenoids act as UV filters by reducing light exposure [36]. Some cyanobacteria such as Wollea vaginicola, Leptolyngbya foveolarum, and Synechocystis salina LEGE 06099 have been highlighted for their significant carotenoid content, enhancing their interest in the pharmacological and cosmetic fields [9,13].

The total carotenoid and chlorophyll contents of the cyanobacteria acetone extracts explored herein are displayed in Table 5. The carotenoid amount ranged from 89 to 159 ug/mg dry extract, Leptolynglya boryana LEGE 15486 being the richest strain. Regarding chlorophyll a, all the strains presented values greater than 100 ug/mg dry extract, with Cephalothrix lacustris LEGE 15493 standing out.

Other cyanobacteria have also been the subject of pigment evaluation. For instance, Lopes and her team 【27】reported 63.9 μg/mg dry extract of total carotenoids and 417.6 μg/mg dry extract of total chlorophylls for Nodosilinea Antarctica leg 13457 acetone extract. The authors also provided data for Leptolyngbya-like sp.LEGE 13412(33.6 ug/mg dry extract), and in both cases, the carotenoid amount was below the values reported herein. It is worth mentioning that, in the work referred to above, carotenoids were profiled by HPLC, and thus, despite species-specific characteristics, the methodology justifies the differences obtained. Regarding chlorophylls, the amount reported by the authors was greater than that obtained herein, which is easily justified by the fact that they present the value of total chlorophylls, whereas the work herein focuses solely on chlorophyll a and its derivatives.

Another study from our research group reported a total of 0.37 mg/g dry biomass of carotenoids for the ethanol extract of Nodosilinea nodulosa LEGE06102[26], which was below the values obtained herein (0.75 mg/g dry biomass, converted based on the extraction yield). This difference is certainly correlated with the solvent used in the extraction since, due to their chemical characteristics, carotenoids have a higher affinity for acetone. Generally speaking, the results obtained herein are within the same order of magnitude as those obtained by other authors, which may be explained by the very similar cultural conditions and methodologies used.

This article is extracted from Mar. Drugs 2022, 20, 183. https://doi.org/10.3390/md20030183 https://www.mdpi.com/journal/marinedrugs