Negev, Dead Sea and Arava Studies 13 (3), 75–81 (2021) 81–75 ,(3) 13 מחקרי הנגב, ים המלח והערבה

Negev, Dead Sea and Arava Studies מחקרי הנגב, ים המלח והערבה

Research articleמאמר מחקר

Combination of Haloferax volcanii and Dunaliella salina reduces air pollution damage and enhances wound closure in vitro

O. Raz1, N. Ogen-Shtern1, 2, N. Kuchina3, G. Cohen1, 2*

1 The Skin Research Institute, The Dead-Sea & Arava Science Center, Masada, Israel2 Ben Gurion University of the Negev, Eilat Campus, Eilat, Israel3 Clinic Lenom LTD, Rishon Lezion, Israel

* Correspondending author: guy@adssc.org + 972 8 9448739 Fax + 972 8 9448734

Natural products have long been used for skincare and dermo-cosmetic topical formulations. Of particular interest are the unique phytochemical and properties of marine-based extracts and compounds. Both Dunaliella salina and Haloferax volcanii have been shown to possess several health-promoting properties. However, the impact of the extracts on air-pollution-induced damage and wound healing had not been elucidated, as well as their compatibility and possible synergistic actions. Thus, HaCaT human keratinocytes cells were treated without or with increasing concentrations of Dunaliella salina extract, Haloferax volcanii extract, and a combination of both. Their ability to prevent induction of radical formation and skin inflammation by air pollution (diesel particulate matter, DPM) was monitored. In addition, their impact on wound closure and the scavenging capacity of the extracts were determined. DPM markedly enhanced cellular reactive oxygen species (ROS) formation and secretion of the inflammatory cytokine interleukin-8 (IL-8). Treatment with Haloferax volcanii extract reduced ROS in a dose-dependent manner, whereas Dunaliella salina had no significant effect. Importantly, the combination of both showed synergistic activity. Similar results were obtained in the quantification of IL-8 secretion. In the wound healing model, both extracts were ineffective as individual treatments, and their combination markedly enhanced wound closure. Finally, the acellular scavenging results correlate with the in vitro data, suggesting that the synergistic action is due to the antioxidant properties of the combinations. The results suggest that a combination of Dunaliella salina and Haloferax volcanii can be used as an effective skincare complex with several applications. Thorough phytochemical analysis should be performed in order to pinpoint the active medicinal compounds underlining these in vitro activities, as well as a clinical validation.

A B S T R A C T

Keywords:HaCaT Keratinocytes Air pollution Antioxidant Wound healing Dunaliella salina Haloferax volcanii

1. IntroductionAmong its many functions, the skin acts as a physical, chemical, and biological barrier to maintain homeostasis and prevent foreign molecules from permeating through the skin (Chambers and Vukmanovic-Stejic, 2020). As the interface with the surrounding, it is constantly bombarded with environmental stress caused by ultraviolet radiation, dry air, dust, air pollution,

and more (Parrado, Mercado-Saenz, Perez-Davo et al., 2019). These may cause disruption in the barrier function of the skin, cellular and molecular damage, DNA mutagenesis, induction of ROS, and improper immune responses. Collectively, these can result in premature extrinsic aging and increase the prevalence of skin cancer formation (Manisalidis, Stavropoulou, Stavropoulos et al., 2020).

O. Raz, N. Ogen-Shtern, N. Kuchina, G. Cohen 76

Marine-based compounds have been suggested as a renewable source of rich and unique active medicinal ingredients. The recent need for natural, safe, and effective compounds has driven both academia and industry to explore the exceptional biological resources present in the seas and oceans. Others and we have also suggested that extreme environmental conditions, such as hypersalinity and high temperature, may drive the production of novel compounds with improved qualities that can be harnessed as health-promoting agents (Borges, Minatel, Gomez-Gomez et al., 2017; Cohen, Raz, Fahham et al., 2015).

Dunaliella salina, a halotolerant chlorophyte, is one of the richest sources of natural carotenoids and their production is augmented upon stress conditions such as oxidative environment and ROS (Xu and Harvey, 2019). In these conditions, β-carotene, the main active ingredient molecule of this microalga, can constituent up to 10% of its dry weight (Paniagua-Michel, Capa-Robles, Olmos-Soto et al., 2009). The biosynthesis of carotenoids, typically C40 isoprenoids and composed of eight isoprene units, is well characterized in Dunaliella salina, and the genes responsible for carotenogenesis processes, with emphasis on β-carotene synthesis, are known. The synthesis pathway can be divided into three steps, geranylgeranyl pyrophosphate biosynthesis, lycopene biosynthesis, and generation of carotenoids with cyclohexene rings (Ye, Jiang and Wu, 2008). This natural synthesis pathway will generate both trans and cis isomers of β-carotene as well as other pigments. Of interest, Dunaliella salina has been found to bloom even in the Dead Sea indicative of its unique chemical composition that can withstand such harsh conditions (Oren, 2005).

Apart from being the precursor in vitamin-A synthesis, the antioxidant activity of carotenoids is well known and frequently utilized in cosmetics. In addition, carotenoids per se, or their metabolites like retinal, apocarotenoids, ketones, aldehydes, and epoxides can modulate several biochemical pathways in the body (Murthy, Vanitha, Rajesha et al., 2005). Haloferax volcanii is a halophilic archaeon that thrives in high salt environments such as the Dead Sea (Pohlschroder and Schulze, 2019). Less is known about its medicinal and dermo-cosmetic properties, yet it was introduced in several commercial Dead-Sea cosmetic products.

We had recently shown that a combination of Dunaliella salina and Haloferax volcanii could recuse skin cancer growth and regeneration in vitro (Raz, Fahham, Kuchina et al., 2019). Here, we explore the possibility of exploiting their synergistic activities for dermatological and skincare usage, in particular, their ability to reduce air pollution-induced damage and expedite wound healing in vitro.

2. Materials and methods 2.1 Materials Cell culture, media, and supplementation were purchased from Biological Industries. Unless specified, all other chemicals were from Sigma-Aldrich, including DPM. Dunaliella salina extract (in jojoba oil, IBR-CLC) was generously given by Clinic Lenom LTD. DPM stock solution (50 mg/ml) was freshly prepared in ethanol on the day of each experiment and mixed vigorously.

2.2 Methods2.2.1 Cell culture The human keratinocyte cell line (HaCaT) was purchase from CLS Cell Lines Service GmbH. The cells were grown in DMEM high glucose supplemented with 10% fetal bovine serum, and 1% (v/v) penicillin/streptomycin and maintained at 37°C in a humidified 5% CO2 incubator. Haloferax volcanii (ATCC) were cultured in broth media, according to the manufacturer’s instructions. Before harvesting, it was grown at 1000 ml and collected by centrifugation. Active compounds [carotene-enriched extract] were eluted in acetone-hexane-dichloromethane solution (1:1:1) at a final volume of 500 ml (added in a stepwise manner, 100 ml every 5 min) for 30 min, filtered, evaporated, and resuspended at 1g/l.

2.2.2 Cell viability assayIn order to verify that the extracts were non-toxic, the MTT assay was employed. Briefly, following treatments, the cell culture media was aspirated and 0.5 mg/ml MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were added in PBS. The cells were incubated for 1 hr. at 37°C, the solution was aspirated, and isopropanol was added to solubilize the colored crystals. The absorbance at 570 nm was measured in an ELISA reader.

2.2.3 Intracellular Reactive Oxygen Species (ROS) determinationThe cells were seeded in 96 well plates at 2.5*105 cells/ml in a final volume of 170 µl of complete growth media and incubated for 24 hr. After 24 hr., the media was aspirated and the cells were loaded with 50 µM of DC-FDA (dye 5, (and 6) -carboxy-2' 7'-dichlorodihydrofluoresceine diacetate) ROS indicator. Then, the cells were incubated with DPM at a final concentration of 100 µg/ml in the absence or presence of the indicated concentrations of the compounds. Fluorescence was determined (ex. 485 nm, em. 538 nm) after 45 min of incubation. Blank (cells not mounted with DC-FDA) readings were subtracted from all treatments.

Synergy of Haloferax volcanii & Dunaliella salina 77

2.2.4 IL-8 quantificationFollowing treatment, the spent media was collected, cleared by centrifugation (1500 rpm, 5 min), aliquoted, and stored at -80°c until used. Human IL-8 quantification was performed by ELISA kits (sensitivity of 8 pg/mL), according to the manufacturer’s instructions and using standard curves. Absorbance was measured at the wavelength 540 nm using a microplate reader (Infinite f200, TECAN).

2.2.5 Acellular (ROS) quantificationThe direct scavenging activity of the extracts was determined by the DPPH method. DPPH reagent (2,2-Diphenyl-1-picrylhydrazyl) was dissolved in ethanol to yield 290 µM working solution that was freshly prepared on the day of the experiment. The extracts and combination (final volume of 20 µl) were added to 380 µL of DPPH working solution. Concomitantly, Trolox was added similarly to the DPPH working solution in order to generate the calibration curve. After 30 min incubation period at RT in reduced light condition, the absorbance of 100 µL aliquots was measured by spectrophotometer (517 nm). The scavenging efficacy will be determined in the linear range of the curve, as written below:

Scavenging efficacy =(O. D. vehicle − O. D. treatment) ∗ 100

O. D. vehicle

2.2.6 In vitro wound-healing assayHaCaT cells were seeded in 12 well plates at 2.5*105 cells/well in a final volume of 1 ml of complete growth media and incubated for 24 hr. Then, the scratch assay technique was used, and in vitro wound was prepared by gently scratched across the center of the well with a sterile 200 microliter tip. Next, the medium was removed and the wells were washed once with PBS and the cells were incubated without or with the indicated concentrations of the compounds in serum-free conditions. After 48 hr., the cells were stained with crystal violet solution, and images were taken. Ten randomized measurements in the central area of each well were taken and wound closure was quantified by imageJ program.

3. Results and discussionHaCaT keratinocyte cells are a well-established in vitro model system to explore the impact of phytochemicals on the skin (Colombo, Sangiovanni, Maggio et al., 2017; Kahremany, Babaev, Gvirtz et al., 2019; Tyagi, Bhardwaj, Srivastava et al., 2015). Being rich in antioxidants, Dunaliella salina and Haloferax volcanii extracts are likely to affect processes regulated by radical balancing. Therefore, we selected to use HaCaT cells in two separate models mimicking such processes.

In order to demonstrate the wound-healing abilities of the extracts, we performed a scratch assay. In addition, an air pollution model was used to evaluate the protective capacity of Dunaliella salina and Haloferax volcanii extracts against the deleterious action of the polluted environment. Thus, HaCaT cells were stimulated by diesel particulate matter (DPM, NIST), which are well-characterized pollution particles, in the absence or presence of the extracts. The major pathways suspected to mediate the toxicity of pollutants to cells are the induction of oxidative stress , and initiation of an inflammatory response (Kim, Cho and Park, 2016). Hence, cellular ROS production and IL-8 secretion were targeted.

As expected, exposure to DPM enhanced dramatically ROS generation by 20-folds (Figure 1). In addition, the powerful antioxidant, N-acetyl cysteine, serving here as a positive control for a reduction in oxidative stress, inhibited ROS levels by 60%. Dunaliella salina extract had no significant influence on air pollution-induced ROS generation by itself whereas Haloferax volcanii (HB) dose-dependently reduced ROS generation by approx. 30%. Of note, Figure 1D depicts that a combination of HB and Dunaliella salina in a ratio of 1:10 synergistically inhibited DPM-induced ROS formation.

Figure 1: Dunaliella salina and Haloferax volcanii synergistically reduce Air

pollution induced-ROS generation. HaCaT cells were treated w/o

or with 200 µg/ml diesel particulate matter in the absence or

presence of the Test items. (A) The cells were mounted with DC-

FDA for 30 min., and 1 hr. post DPM exposure ROS were quantified

by fluorimeter measurement. (B & C) Similar tests were performed

with increasing concentrations of Dunaliella salina and Haloferax

volcanii. (D) Combination of Haloferax volcanii and Dunaliella

salina increase synergistically their ability to attenuate ROS

generation. n= 4. */#p < 0.05 from the untreated or stimulated

control; $p < 0.05 synergistic action above the theoretical additive

combination. DPM – diesel particulate matter; NAC – N-acetyl

cysteine; HB – Haloferax volcanii ; DS – Dunaliella salina

O. Raz, N. Ogen-Shtern, N. Kuchina, G. Cohen 78

Another key aspect of air pollution-induced damage is the increase in inflammation. Thus, to validate whether Dunaliella salina and Haloferax volcanii may synergistically reduce DMP-induced damage, the secreted levels of interleukin-8 (IL-8) were monitored. The results shown in Figure 2 confirm the hypothesis. As can be seen, IL-8 levels were significantly increased by DPM while dexamethasone, the commercial steroid used as a positive control, reversed this effect. Importantly, the combination of HB and Dunaliella salina also reduced IL-8 hypersecretion by approx. 40%. In addition, no toxic effect was found in the viability evaluation assessed by the MTT assay (Figure 2, lower panels).

Figure 2: Dunaliella salina and Haloferax volcanii synergistically reduce

inflammation caused by air pollution. HaCaT cells were treated

w/o or with 200 µg/ml of DPM in the absence or presence of

the tested extracts. (A) Media were taken, and IL-8 levels were

quantified by ELISA. (B) Combination of Haloferax volcanii and

Dunaliella salina (1:10 v/v) synergistically increases their ability to

attenuate cytokine secretion. (C & D) Cell viability, determined by

MTT. n= 4; */#p < 0.05 from the untreated or stimulated control;

$p < 0.05 synergistic action above the theoretical additive

combination. DPM – diesel particulate matter; HB – Haloferax

volcanii ; DS – Dunaliella salina

Air pollution has been recognized as a risk factor in skin diseases, as it increases both their occurrence rate and severity. Previous studies have linked both indoor and outdoor air pollution in the induction or aggravate atopic dermatitis (Ahn, 2014). In addition, clinical findings suggest that exposure to pollutants is linked to premature skin aging (Dijkhoff, Drasler, Karakocaket et al., 2020; Vierkötter, Schikowski, Ranft et al., 2010). The ability of the pollutants to cross the skin barrier and

the increasing levels of exposure are the main cause of their damage to the skin. Particulate matter consists of a mixture of organic and inorganic compounds. Due to the inherent differences between sores, the use of the standardized NIST mixture is widely used, as in this current study. In vitro data have linked their action to enhanced production of ROS and inflammation (Li, Kang, Jiang et al., 2017; Lin, Lee, Tsai et al., 2016; Piao, Ahn, Kanget al., 2018).

Our results are in line with the current knowledge and validate the finding on the ameliorating action of the complex. Surprisingly, when tested individually, commercial preparations of Dunaliella salina presented low and non-significant activity, whereas Haloferax volcanii enriched carotene extracts had a significant influence on ROS generation. This may be the result of low carotene levels of the commercial preparation of Dunaliella salina. Results from the inflammatory IL-8 reinforce these findings.

As mentioned earlier, an additional mechanism known to be modulated by an oxidative environment is wound healing (Dunnill, Patton, Brennan et al., 2017). Therefore, the ability of Dunaliella salina and Haloferax volcanii extracts to regulate this process was examined by scratch assay. As shown in Figure 3, a bell shape curve was obtained, showing a marked efficacy of the combined extracts and their superiority on the individual extracts that were ineffective.

Figure 3: Dunaliella salina and Haloferax volcanii synergistically promote

wound closure in vitro. The ability of the compounds to induce

wound closure was assessed by the scratch assay. (A) Representative

images were taken after 48 hr. and wound width quantification are

presented. n= 3; $p < 0.05 synergistic action above the theoretical

additive combination. HB – Haloferax volcanii ; DS – Dunaliella salina

Synergy of Haloferax volcanii & Dunaliella salina 79

Among the various molecular mechanisms underlying wound healing, the impact of a suitable oxidative environment had been emphasized (Dunnill, Patton, Brennan et al., 2017; Sanchez, Lancel, Boulanger et al., 2018). The current results suggest that combination of Dunaliella salina and Haloferax volcanii extracts can improve wound closure. Antioxidants are suggested to counteract wound oxidative stress and thereby accelerate its healing (Addis, Cruciani, Santaniello et al., 2020; El-Ferjani, Ahmad, Dhiyaaldeen et al., 2016; Pessoa, Florim, Rodrigues et al., 2016). Although many over-the-counter antioxidants are available, only one was approved by the FDA for the treatment of wound healing (Fitzmaurice, Sivamani and Isseroff, 2011). This can be due to conflicting results suggesting a balance in the oxidative environment is of need for proper wound healing and other physiological processes. (Auf Dem Keller, Kümin, Braunet et al., 2006; Dunnill, Patton, Brennan et al., 2017; Loo and Halliwell, 2012).

To investigate whether the synergistic properties of Dunaliella salina and Haloferax volcanii are due to their antioxidant capacity, the acellular ROS scavenging capacity of the extracts were evaluated by the DPPH method. As depicted in Figure 4, Dunaliella salina extract exhibited low, but significant antioxidant strength (maximal value of 4.5% obtained in the highest concentration tested) and reached a steady-state at concentrations above 0.02 mg/l. whereas Haloferax volcanii showed high efficacy, reaching 100% at concentrations as little as 0.01 mg/l. In order to evaluate the compatibility and possible synergistic action of the extracts, several ratios of Dunaliella salina and Haloferax volcanii were investigated in the same acellular model system. The amount of HB was restricted to its EC50 and the maximal concentration of Dunaliella salina was used. As can be seen, the combinations were more effective than the predicted additive values (shown in the dashed line) at the various range, picking approximately at 10:90 ratio.

An extensive evaluation is of need to investigate the molecule mechanism of this improvement. However, it is reasonable to assume that the same antioxidant stability is behind this phenomenon. The shift in the optimal ratio between the results of the air pollution and the scratch assay can be due to the changes in cellular response after the inflicting of the wounds. In addition, the impact of different extraction methodologies and growth conditions should be evaluated for both Dunaliella salina and Haloferax volcanii synergistic activities. The use of a natural source of Dunaliella salina extract and not only the commercial material should also be evaluated as the composition and concentration of antioxidant and carotenoids

may differ significantly and the low ROS scavenging effect of the extract, as well as the inability to reduced DPM-induced ROS generation shown here, may change.

Figure 4: Dunaliella salina and Haloferax volcanii synergistically reduce

ROS in vitro: The antioxidant capacity of the two Test items was

evaluated according to the acellular DPPH method. (A) Trolox

calibration curve was performed vs. its scavenging capacity value.

(B & C) The scavenging capacity of Dunaliella salina and Haloferax

volcanii were determined. (D) Combination of Haloferax volcanii

and Dunaliella salina increase synergistically their capacity. n= 4;

*p < 0.05 from the untreated control; $p < 0.05 synergistic action

above the theoretical additive combination

To conclude, the current study aimed to support possible usages of Dunaliella salina and Haloferax volcanii in dermatology. The results clearly show synergistic action in two independent models – reducing air pollution-induced damage and enhanced wound healing. The acellular DPPH results suggest that novel chemical interaction influences the antioxidant capacity of the complex.

AcknowledgmentsThis study was supported by the Ministry of Science and Technology (Israel) and Clinic Lenom donations.

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