**Antioxidant and Photoprotection Networking in the Coastal Diatom** *Skeletonema marinoi*

**Arianna Smerilli 1, Sergio Balzano 1, Maira Maselli 1,2, Martina Blasio 1, Ida Orefice 1, Christian Galasso 1, Clementina Sansone 1,\* and Christophe Brunet <sup>1</sup>**


Received: 7 May 2019; Accepted: 29 May 2019; Published: 1 June 2019

**Abstract:** Little is known on the antioxidant activity modulation in microalgae, even less in diatoms. Antioxidant molecule concentrations and their modulation in microalgae has received little attention and the interconnection between light, photosynthesis, photoprotection, and antioxidant network in microalgae is still unclear. To fill this gap, we selected light as external forcing to drive physiological regulation and acclimation in the costal diatom *Skeletonema marinoi*. We investigated the role of light regime on the concentration of ascorbic acid, phenolic compounds and among them flavonoids and their connection with photoprotective mechanisms. We compared three high light conditions, differing in either light intensity or wave distribution, with two low light conditions, differing in photoperiod, and a prolonged darkness. The change in light distribution, from sinusoidal to square wave distribution was also investigated. Results revealed a strong link between photoprotection, mainly relied on xanthophyll cycle operation, and the antioxidant molecules and activity modulation. This study paves the way for further investigation on the antioxidant capacity of diatoms, which resulted to be strongly forced by light conditions, also in the view of their potential utilization in nutraceuticals or new functional cosmetic products.

**Keywords:** light; ascorbic acid; phenolic compounds; flavonoids; photoprotection

#### **1. Introduction**

Aerobic organisms need to deal with reactive oxygen species (ROS) which are harmful to their metabolism since high ROS concentrations can damage cellular machinery ultimately threatening cell survival; simultaneously ROS play also a role as secondary messengers. In all cells, mitochondria, NADPH oxidase (NOX) complexes and the enzyme lipoxygenase are major ROS sources. Photosynthetic eukaryotic cells possess, in addition, the chloroplasts, in which ROS are formed via energy transfer from chlorophyll or via electron transfer. Indeed, ROS intracellular concentration controls the photosystem II (PSII) activity and therefore photosynthesis and defense strategies [1]. The balance between toxicity, when ROS are in excess, and the signaling action requires cells to finely tune the ROS concentration [2–4], thanks to an efficient intracellular network composed by antioxidant molecules and enzymes. Antioxidants include molecules such as ascorbic acid (AsA), carotenoids, glutathione (GSH), tocopherols as well as phenolic compounds. AsA is a strong antioxidant component of the cell plasma [5]. AsA is also substrate of antioxidant enzymes such as peroxidases and violaxanthin de-epoxidase, thus contributing to dissipate excess energy [6]. Carotenoids occur in the chloroplast membranes interacting directly where photosynthesis-derived ROS are generated. Besides their role as photosynthetic pigments, carotenoids can efficiently quench peroxides and singlet oxygen thus

preventing the formation of ROS [5,7–9]. GSH buffers the redox equilibrium of the cell by undergoing oxidation or reduction reactions, according to the redox potential of the cell. Specifically, GSH can act as an electron donor inactivating free radicals. Moreover, GSH is also a cofactor of several antioxidant enzymes [10]. Tocopherols are only produced by photoautotrophic taxa, they are lipophilic and can use resonance energy transfer to scavenge singlet oxygen, thus protecting PSII [11]. Phenolic compounds are present in all plants and derive from the shikimate-phenylpropanoids-flavonoids pathways [12]. They include a wide range of molecules with several phenol structural units. The most important phenolic compounds are flavonoids, which can donate both electrons or hydrogen atoms directly to ROS [13]. A wide range of flavonoids are present in photosynthetic microorganisms [14]. A recent study highlighted the diversity of flavonoids in phytoplankton and found that ferulic acid and apigenin are the dominant flavonoids in both cyanobacteria and eukaryotic microalgae [14]. In contrast with higher plants, their distribution and functions in microalgae are not fully clear [14,15]. Antioxidant enzymes are located in various cell compartments and include catalase (CAT), superoxide dismutase (SOD), and several peroxidases. Ascorbate peroxidase (APX) and glutathione peroxidase (GPX) accept, as substrates, AsA and GSH, respectively, in order to detoxify ROS. Furthermore, three additional enzymes, the monodehydroascorbate reductase (MDHAR), the dehydroascorbate reductase (DHAR), and the glutathione reductase (GR) contribute to regenerate the antioxidant substrates.

With the exception of carotenoids [16–18], the knowledge on antioxidant molecules and enzymes from marine microalgae is still scarce. Few studies focused on the concentration and composition of antioxidants in marine microalgae [17,19–24], and even fewer on the mechanisms for antioxidant defense in these microorganisms [24]. Indeed, both the photoprotective and antioxidant network appeared strongly controlled by light spectral composition and intensity, resulting in a complex regulation system, which allows planktonic diatoms to survive in their highly fluctuating light environment they naturally inhabit [24].

While the photoprotective mechanisms have been investigated in diatoms [25–29], only few studies investigated the role of the antioxidant network as a second defense line able to reduce the light stress [30,31]. Being light a crucial ecological axis in ruling the metabolism of photosynthetic organisms, and thus modulating the growth, the objective of this study was to investigate the impact of light intensity, photoperiod, and wave light distribution on the cellular concentrations of antioxidant molecules such as AsA and flavonoids, and total phenolic content. This knowledge can thus to be exploited to improve microalgal culturing with a productivity-driven purpose. Indeed, marine microalgae are gaining increasing attention for ecofriendly production of new antioxidant compounds. The ultimate aim of this study is to clarify the role of microalgal antioxidants in modulating light stress.

#### **2. Materials and Methods**

#### *2.1. Skeletonema marinoi and Culture Conditions*

The experiments were conducted on the diatom *Skeletonema marinoi* Sarno and Zingone (CCMP 2092); we selected *S. marinoi* since it is a cosmopolitan centric diatom broadly used in aquaculture [32] that can be cultured in different media [33] under different conditions of light intensity [34] and salinity [35].

This strain was grown at 20 ◦C in 4.5 L glass tanks in autoclaved seawater, pre-filtered through a 0.7 μm GF/F glass-fiber filter under water movement using an aquarium wave maker pump (Sunsun, JVP-110, Sunsun manufacturer, Zhoushan, China). A modified f/2 medium, in which the concentrations of phosphate, dissolved silica, vitamins, and trace metals are twice compared to those typically present in f/2 [36], was used.

Cells were pre-acclimated to a sinusoidal light distribution with a midday peak of 150 μmol photons m−<sup>2</sup> s−<sup>1</sup> and with a photoperiod equal to 12:12 dark:light, following the results obtained by [24,34,37].

The white light was composed by Red:Green:Blue (RGB) with a ratio of 10:40:50 and provided by a custom-built LED illumination system (European patent registration number: EP13196793.7), allowing to modulate the spectral composition and light intensity [38]. The three colors were provided at wavelengths of 460 nm (±36 nm, blue), 530 nm (±50 nm, green) and 626 nm (±36 nm, red).

Light intensity (Photosynthetically Active Radiation, PAR) was measured inside each tank by using a laboratory PAR 4 π sensor (QSL 2101, Biospherical Instruments Inc., San Diego, CA, USA).

#### *2.2. Experimental Strategy*

All experiments were run in triplicate and consisted in monitoring the biological responses of *S. marinoi* after the shift from pre-acclimation light condition to different light conditions, spanning from prolonged darkness to very high light climate (Table 1). The light shift started after the 12 h dark period of the previous day. The experimental conditions are presented in the Figure 1 and described in Table 1.


**Table 1.** Experimental strategy.

**Figure 1.** Details of the experimental setting used here. The different light wave distributions are explained in the inset. Treatments based on sinusoidal, quadratic or continuous light wave are shown in green, blue, and red, respectively. Refer to Table 1 for abbreviations.

The condition-oriented sampling strategy was carried out as follows. All the cultures were sampled at predawn (in the dark before the new condition operation), at 6 h (midday) and at 24 h (at the end of the dark period). Since phytoplankton cells are known to operate a rapid photoprotective mechanism when exposed to high (>200 μmol photons m<sup>−</sup>2s <sup>−</sup>1) light conditions [39] an additional sampling after 2 h from the light shift was performed in all the high light experiments (Sin 600, Quad 300 and Quad 600). Furthermore, since cultures incubated to square light wave experienced a sharp shift from 0 to 300 or 600 μmol photons m−<sup>2</sup> s−1, samples were harvested from these cultures also 10 and 30 min after the light shift in order to evaluate the short-time response of *S. marinoi* to cope with this unnatural and drastic enhancement of light.

#### *2.3. Ancillary Data*

#### 2.3.1. Cell Concentration and Growth Rate

To assess cell density, 2 mL of cell suspension were collected from each tank and fixed with Lugol's iodine solution (1.5% *v*/*v*). Then, 1 mL of this solution was used to fill a Sedgewick Rafter counting cell chamber. Cells were then counted using a Zeiss Axioskop 2 Plus light microscope (Carl Zeiss, Göttingen, Germany).

The growth rate was estimated from cell concentration measurements using the following equation:

$$\mu \text{ (d ${}^{-1}$ )} = \ln(\text{C}\_{\text{n-1}}/\text{C}\_{\text{n}})/(\text{t}\_{\text{n}} - \text{t}\_{\text{n-1}}) \text{.}$$

where <sup>μ</sup> is the growth rate, Cn−<sup>1</sup> and Cn are cell concentrations (mL<sup>−</sup>1) at day n <sup>−</sup> 1 (tn−1) and day n (tn).

2.3.2. Photochemical Efficiency of the Photosystem II and Non-Photochemical Quenching Measurements

To assess the photosynthetic capacities and the photophysiological state of phytoplankton cells, active chlorophyll *a* (Chl-*a*) fluorescence was measured using a DUAL-PAM fluorometer (Heinz Walz GmbH, Effeltrich, Germany). The photochemical efficiency of the PSII (Fv/Fm) was estimated by:

$$
\Phi\_{\rm P} = (\rm F\_{m} - F\_{0})/\rm F\_{m} = \rm F\_{v}/\rm F\_{m\nu}
$$

where Fv is the variable fluorescence (Fv = Fm − Fo).

The measurement of Fo was done using light of low intensity (1 μmol photons m−<sup>2</sup> s<sup>−</sup>1) and low frequency (approximately 25 Hz). Fm was measured by applying a short and intense flash of actinic light which completely reduces QA. In our case, the saturation flash of bright red light (655 nm) were applied at an intensity of 2400 μmol photons m−<sup>2</sup> s−<sup>1</sup> for a duration of 450 ms. Fv/Fm corresponds to the maximal photochemical efficiency of the PSII (or the maximal light utilization efficiency of PSII) and dark acclimation for 15 min allows the recovery of photosystems II, this leading to reliable measurements of Fo [38].

Estimation of the non-photochemical quenching is calculated by the Stern–Volmer expression [40]:

$$\text{NIPQ} = (\text{F}\_{\text{m}} - \text{F}'\_{\text{m}}) \text{/F}'\_{\text{m}} = \text{F}\_{\text{m}} / \text{F}'\_{\text{m}} - 1.$$

The estimation of NPQ consisted of measuring Fo and Fm on 15 min dark-acclimated samples and then measuring Fm' and F0 every minute on the same sample illuminated by an actinic light (setup at 399 μmol photons m−<sup>2</sup> s<sup>−</sup>1) for 10 min.

#### 2.3.3. Electron Transport Rate-Light Curves Determination

The electron transport rate (ETR) vs. irradiance (E) curves were determined on 15-min dark-acclimated samples by applying a series of 10 increasing intensity actinic lights (composed by 2/3 of blue and 1/3 of red light, lasting 1.5 min each, ranging from 1 to 1222 μmol photons m−<sup>2</sup> s<sup>−</sup>1). The photochemical efficiency of the PSII was measured on the 15-min dark-acclimated sample, while the light utilization efficiency of the PSII (ΔΦ) was measured after each actinic light level.

The relative ETR, taking into account the part of incident light energy effectively absorbed by the photosystem, was calculated as follows:

$$\text{relBERTR} = \mathbf{F'}\_{\text{v}} / \mathbf{F'}\_{\text{m}} \cdot \mathbf{E} \cdot \mathbf{0}.\mathbf{5} \cdot \mathbf{a} \text{ \*},$$

where E is irradiance, and *a* \* is the cell specific absorption coefficient expressed in m2 cell−<sup>1</sup> [24]. A factor of 0.5 was applied since it is assumed that half of the incident light is absorbed by the PSI and half by the PSII. The relative ETR is expressed in nmol e<sup>−</sup> h−<sup>1</sup> cell<sup>−</sup>1.

Determination of relETRmax was retrieved according to the equation of Eilers and Peeters (1988) [41].

#### *2.4. Pigment Analysis*

Pigment analysis was conducted by High Performance Liquid Chromatography (HPLC) as described by Smerilli et al. [24]. An aliquot of algal culture (10 mL) was filtered on GF/F glass-fiber filter (25 mm, Whatman, Maidstone, UK) and immediately stored in liquid nitrogen until further analysis. Pigments were extracted by mechanical grounding for 3 min in 2 mL of absolute methanol. The homogenate was then filtered onto Whatman 25-mm GF/F filters and the volume of the extract accurately measured. Prior to injection into the HPLC, 250 μL of 1 M ammonium acetate were added to 500 μL of the pigment extract and incubated for 5 min in darkness at 4 ◦C. This extract was then injected in the 50 μL loop of the Hewlett Packard series 1100 HPLC (Hewlett-Packard, Wilmington, NC, USA). The reversed-phase column (2.6 mm diameter C8 Kinetex column; 50 <sup>×</sup> 4.6 mm; Phenomenex®, Torrance, CA, USA) corresponded to an apolar stationary phase composed of silica beads possessing aliphatic chains of eight carbon atoms (C8). The temperature of the column was steadily maintained at 20 ◦C and the flow rate of the mobile phase was set up at 1.7 mL min<sup>−</sup>1.

The mobile phase was composed of two solvents mixtures: A, methanol:0.5 N aqueous ammonium acetate (70:30, *v*/*v*) and B, absolute methanol. During the 12-min elution, the gradient between the solvents was programmed: 75% A (0 min), 50% A (1 min), 0% A (8 min), 0% A (11 min), 75% A (12 min).

Pigments were detected at 440 nm using a Hewlett Packard photodiode array detector model DAD series 1100 which gives the 400–700 nm spectrum for each detected pigment. A fluorometer (Hewlett Packard standard FLD cell series 1100) with excitation at 410 nm and emission at 665 nm allowed the detection of fluorescent molecules (chlorophylls and their degraded products). Pigments were identified based on their retention time and quantified based on pure standards from the D.H.I. Water and Environment (Hørsholm, Denmark) as described previously [38].

#### *2.5. Antioxidant Molecules and Antioxidant Activity Analysis*

#### 2.5.1. Ascorbic Acid Content Determination

To assess the ascorbic acid (AsA) content in cells, the procedure modified from [42] was the following. A 150 mL volume of culture was centrifuged at 3600 g for 15 min at 4 ◦C (DR15P centrifuge, B. Braun Biotech International, Melsungen, Germany), the pellet was weighed, flash frozen in liquid nitrogen, and stored at −20 ◦C. Pellets were resuspended in 5% trichloroacetic acid (TCA) and sonicated for 1 min with a microtip at 20% output on ice (S-250A Branson Ultrasonic). Cell debris were precipitated by centrifugation at 5000× *g* for 5 min at 4 ◦C. The supernatant was then used for spectrophotometric analysis after mixing it with a reagent. The reagent consisted of a 0.5% solution of 2,2 -dipyridyl mixed with an 8.3 mM ferric ammonium sulfate solution in 15% (*v*/*v*) o-phosphoric acid in a ratio 4 to 1. Supernatant and reagent were mixed (1:5) immediately before use. After 1 h the absorbance was read at 520 nm and AsA concentration was calculated thanks to factor calibration retrieved from calibration curves using AsA standards. AsA concentration is reported in fg AsA cell<sup>−</sup>1.

#### 2.5.2. Preparation of the Methanolic Extracts

For the determination of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, total phenolic content (TPC) and total flavonoid content (TFC), the pellets were prepared as follows. Cells were re-suspended in methanol and sonicated for 1 min with a microtip at 20% output on ice (S-250A Branson Ultrasonic). The suspension was left for 30 min at room temperature in the dark, and then was centrifuged at 3600× *g* for 10 min at 4 ◦C. Supernatant was collected and the pellet was re-suspended in an equal volume of methanol and left other 30 min at room temperature in the dark. The suspension was centrifuged again in the same conditions, and the two supernatants were combined.

#### 2.5.3. Total Phenolic Content

Polyphenols in plant extracts react with specific redox reagents (Folin-Ciocalteu reagent) to form a blue complex that can be quantified by visible-light spectrophotometry. Total phenolic content (TPC) was estimated by the Folin–Ciocalteu method [43] as described by Li and collaborators [44].

Briefly, 200 μL of the sample was mixed with 1 mL of Folin-Ciocalteu's phenol reagent, pre-diluted in distilled water 1:10 *v*/*v*. After 4 min, 800 μL 75 g/L Na2CO3 were added to the mixture, shacked vigorously and stored at room temperature for 2 h. The absorbance was read at 765 nm. Gallic acid was used for the standard calibration curve. The results were expressed in fg of gallic acid equivalents (GAEq) cell<sup>−</sup>1.

#### 2.5.4. Total Flavonoid Content

The total flavonoid content was estimated by aluminum chloride colorimetric method [45]. Briefly, 600 μL of sample were pre-diluted 1:2 *v*/*v* in methanol 80% *v*/*v* and mixed with an equal volume of AlCl3 2%. The mix was shaken and incubated at room temperature for 1 h. The absorbance was measured at 410 nm and quercetin was used for the standard calibration curve. The results were expressed in fg of quercetin equivalents (QEq) cell<sup>−</sup>1.

#### 2.5.5. ABTS Radical Scavenging Activity

The antioxidant activity was assessed by the 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity assay. The scavenging activity of ABTS radical was measured following [16]. The ABTS free radical was generated by mixing 7 mM ABTS diammonium salt with 2.45 mM potassium persulfate and stored overnight at room temperature. The solution was diluted with methanol till the absorbance at 734 nm reached 0.70 ± 0.01 units. Then one part of sample was mixed with three parts of ABTS radical solution. The mix was shaken and left 1 h at room temperature in the dark. The absorbance was read at 734 nm. Ascorbic acid was used for the standard calibration curve. The results were expressed in fg of ascorbic acid equivalents (AEq) cell<sup>−</sup>1.

#### *2.6. Statistical Analysis*

Calculations of mean, standard deviation, variance, coefficient of variation (CV), Student's *t*-test for mean comparison, Spearman rank correlation, analysis of variance (ANOVA) and Tukey test for multiple comparisons were performed using the PAST software package, version 3.10 [46].

#### **3. Results**

#### *3.1. Growth Rate and Photosynthesis*

After 24 h from the light shifts, in all the experimental conditions the growth rate of *S. marinoi* decreased (Table 2), in contrast with what observed in the control condition, revealing a physiological stress induced by the variations of light environment. The highest decrease in cell abundance was observed for cultures shifted to square wave light distribution at 600 μmol photons m−<sup>2</sup> s−<sup>1</sup> and to continuous light (Table 2).


**Table 2.** Cell concentration and growth rate at time To and after 24 h from the start of the experiment.

Photosynthetic electron transport rate (ETR) did not vary significantly over time under Sin 150, dark conditions, and Sin 10. Significant increases were observed under continuous light (Con 10), stationary conditions (Sin 150 Stat), and for cultures incubated under high light conditions (Table 3). The photochemical efficiency of photosystem II (Fv/Fm, Table 3) decreased significantly after 6 h for cultures exposed to square-wave light distribution type. Fv/Fm restored after 24 h in Quad 600, whereas it did not reach its initial value in Quad 300 probably because of photoinhibition. Fv/Fm decreased over time in Sin 10, Con 10 and Sin 600 treatments, whereas it did not change significantly for the control and the dark treatments.

**Table 3.** Electron photosynthetic rate (ETR, nmol e<sup>−</sup> h−<sup>1</sup> cell<sup>−</sup>1), Fv/Fm, Chlorophyll-*a* concentration (Chl-*a,* fg cell<sup>−</sup>1), and Fucoxanthin concentration (Fuco, fg cell−1) measured under the different light conditions.


#### *3.2. Photosynthetic Pigments*

The cellular concentration of Chl-*a* varied differently depending on the light climate cells were exposed to. Decreases were observed in the Sin 150 Stat (6 h), whereas sharp increases occurred in the dark treatment (6 h) and in the Con 10 (24 h). In the Quad 300 the concentration of Chl-*a* decreased (24 <sup>±</sup> 1 to 15.1 <sup>±</sup> 6.0 fg cell<sup>−</sup>1) after 2 h and then increased again in the following 4 h (22.2 <sup>±</sup> 2.1 fg cell<sup>−</sup>1). The cellular concentration of fucoxanthin (fuco) increased in the cultures incubated under low light conditions (Con 10 and Sin 10) and did not change significantly in the other cultures except in Quad 300, where it decreased after 2 h and then increased again during the following 22 h (Table 3). Under control condition (Sin 150), the increase in Chl-*a* content per cell at midday was attributed to cell cycle progression, and its decrease at 24 h was the result of cell division occurring during night (Table 3). Conversely, under Sin 600 or Quad 600 and 300, cell pigment content did not change significantly with time, probably because of the arrest of cell cycle progression or related to the two antagonist effects of high light regulation (lowering Chl-*a* concentration) and cell cycle progression (increasing it).

Prolonged darkness induced an increase of Chl-*a* with time revealing an acclimation strategy in the reaction centers of photosystems (Table 3). Under Sin 10, after 6 h Chl-*a* decreased significantly while Fuco tended to increase.

Under Con 10, both Fuco and Chl-*a* were strongly enhanced the second day of experiment (Table 3).

#### *3.3. Photoprotection: NPQ and Xanthophyll Cycle*

Major changes in NPQ as well as the pigments related to the xanthophyll cycle were mostly observed in the cultures exposed to high light. Indeed, the NPQ was found to be more variable in cultures incubated under Sin 600 and square light wave conditions compared to the other treatments (Figure 2). Under continuous light (Con 10) the cellular concentration of Dt increased and the NPQ decreased over time. While the increase in Dt (Figure 2A–C) mostly occurred in the last 18 h, the NPQ decreased during the first 6 h (Figure 2D–F). Cells incubated under sinusoidal conditions in both exponential (Sin 150) and stationary (Sin 150 stat) phases did not exhibit significant infradiel variations in Dt and NPQ (Figure 2B,D). Under Sin 600, NPQ increased rapidly in the first 2 h (*p* < 0.05), coming back to the pre-dawn values at midday (Figure 2F). At 24 h NPQ was significantly higher (*p* < 0.001) with respect to the previous predawn day value (Figure 2F). Under Quad 300, NPQ slightly increased after 10 min and later on decreased progressively reaching the lowest value after 6 h (*p* < 0.05, Figure 2C). Conversely, Dt progressively increased reaching its maximal concentration at 6 h (*p* < 0.05, Figure 2C) and the DES significantly increased after 30 min (data not shown). Under Quad 600, the cellular concentration of Dt increased after 2 h and then decreased again; the NPQ was fairly constant within the first 2 h while it decreased significantly after6h(*p* < 0.05, Figure 2F).

The cellular concentrations of Dd and β-car did not change significantly over time in most cases (Figure 3). A small increase in both Dd and β-car was only observed in the Con 10 treatment after 24 h since the beginning of the experiments (Figure 3A,D). NPQ and the concentration of the photoprotective pigment Dt were thus not correlated (Table 4), as previously observed under sinusoidal light distribution [24,47].

**Figure 2.** Temporal changes in the cellular concentrations of diatoxanthin ((**A**): low light climates, (**B**): moderate light, (**C**): high light climates) and non-photochemical quenching (NPQ; (**D**): low light climates, (**E**): moderate light, (**F**): high light climates) in the different culturing treatments of *Skeletonema marinoi* CCMP 2092. Refer to Table 1 for abbreviations. "\*" means significantly different from time 0 (*p* < 0.05); "\*\*" means significantly different from time 0 (*p* < 0.01).

**Figure 3.** Temporal changes in the cellular concentrations of diadinoxanthin ((**A**): low light climates, (**B**): moderate light, (**C**): high light climates) and β-carotene ((**D**): low light climates, (**E**): moderate light, (**F**): high light climates)in the different culturing treatments of *S. marinoi* CCMP 2092. Refer to Table 1 for abbreviations. "\*" means significantly different from time 0 (*p* < 0.05); "\*\*" means significantly different from time 0 (*p* < 0.01).


**Table 4.** Spearman correlation matrix between the antioxidant capacity, antioxidant molecules, and the photoprotection-related parameters evaluated under control (Sin150) and stationary phase (Stat) conditions 1,2.

<sup>1</sup> Abbreviations and units used for the correlation are as follows: Ascorbic acid (AsA in fg/cell); Phenolics (in fg GAEq/cell); Flavonoids (in fg QEq/cell); ABTS test (in fg AEQ/cell); Diatoxanthin (Dt in fg/cell); Diadinoxanthin (Dd in fg/cell); β-carotene (β-car in fg/cell); Light (in μmol photons m−<sup>2</sup> s<sup>−</sup>1). <sup>2</sup> n.s. = non-significant (*p*-value > 0.01).

#### *3.4. Antioxidant Molecules and Activity*

Similarly to the NPQ and the xanthophyll-cycle related pigments, most changes in the cellular concentrations of antioxidant molecules occurred in the cultures incubated under high light (Figures 4 and 5). Under the control condition (Sin 150), the cellular concentration of ascorbic acid (AsA) increased at midday (Figure 4). The phenolic content followed the same trend, with higher values found at midday compared to pre-dawn (Figure 5A–C). As AsA and phenolic content, flavonoids increased at midday compared to pre-dawn (Figure 5D–F). In contrast when cells entered stationary phase, these daily variations disappeared and the flavonoids, phenolics, and AsA concentrations stabilized on higher values with respect of those recorded in the exponential phase (Figures 4 and 5).

Consistent with this observation, the ABTS test reflected the trend observed for the antioxidant molecules, following infradiel variations, with an enhancement at midday, and further increase during the stationary phase (Figure 6).

**Figure 4.** Temporal changes in the cellular concentrations of ascorbic acid ((**A**): low light climates, (**B**): moderate light, (**C**): high light climates) in the different culturing treatments of *S. marinoi* CCMP 2002. Refer to Table 1 for abbreviations. "\*" means significantly different from time 0 (*p* < 0.05); "\*\*" means significantly different from time 0 (*p* < 0.01).

**Figure 5.** Temporal changes in the cellular concentrations of phenolic compounds ((**A**): low light climates, (**B**): moderate light, (**C**): high light climates) and flavonoids ((**D**): low light climates, (**E**): moderate light, (**F**): high light climates) in the different culturing treatments of *S. marinoi* CCMP 2002. Refer to Table 1 for abbreviations. "\*" means significantly different from time 0 (*p* < 0.05); "\*\*" means significantly different from time 0 (*p* < 0.01).

**Figure 6.** Radical scavenging activity of the different culturing treatment based on the assay of 2,2 -azino-bis (3 ethylbenzthiazoline-6-sulfonic acid, ABTS; (**A**): low light climates, (**B**): moderate light, (**C**): high light climates). Data are indicated in fg of ascorbic acid equivalent per cell (fg AEq cell<sup>−</sup>1). Refer to Table 1 for abbreviations. "\*" means significantly different from time 0 (*p* < 0.05); "\*\*" means significantly different from time 0 (*p* < 0.01).

Antioxidant activity (ABTS) and antioxidant compounds (AsA, phenolics, and flavonoids content) were significantly correlated with all the parameters related to photoprotection (Dt and its two precursors Dd and β-car) except NPQ as well as light intensity (Table 4). The situation changed a little when cells entered into the stationary phase, in which light was no longer the only parameter inducing antioxidant response (lack of correlation, Table 4). ABTS activity was linked to phenolic content, AsA, and Dt, these three parameters being linked between them (Table 4). In this condition, a negative correlation between Dt and β-car was noticed on the opposite to the exponentially grown cells, revealing that Dt might be enhanced from β-car pool that was not fully replenished.

Under Sin 600, AsA conserved the infradiel variation previously observed (Figure 4C). AsA concentration doubled already at 2 h, keeping its concentration fairly constant towards the end of

the experiments. The total phenolic content and flavonoids concentration followed a trend similar to AsA in Sin 600, although the increase was observed after 6 h since the beginning of the experiments (Figure 5C,F). Under Quad 600 the infradiel trend of AsA previously observed was still present (Figure 4C). Intriguingly, AsA concentration was halved in 10 min revealing its very fast consumption, then AsA was recycled and newly synthesized, reaching the highest values at 2 and 6 h (*p* < 0.05 and *p* < 0.01, respectively, Figure 4C). The increase between the time 0 and 2–6 h was greater than the increase observed in the control condition, being of circa three times against 2.4 in Sin 150 and 1.8 in Sin 600 (Figure 4B,C).

The ABTS test paralleled the increase of the antioxidants' concentration, remaining high at 6 h (Figure 6). After one day since the light shift, the antioxidant capacity was still higher than T0 (*p* < 0.01), while the antioxidant molecules decreased or were stable with respect to the previous day (Figures 4–6).

Under both square light wave conditions AsA content (Figure 4C) did not vary within the first minutes of the experiment, increasing at 2 and 6 h (*p* < 0.05) and restoring the starting values after 24 h. The increase in AsA found in Sin 600 was lower than the one observed in Quad 300 and Quad 600 (Figure 4C). The phenolic content in both Quad 300 and Quad 600 peaked at 2 h (*p* < 0.05), then decreased restoring the initial values at 24 h while flavonoids did not show any significant variation (Figure 5C,F). The ABTS followed the same trend of AsA, increasing at 2 h and keeping high values at 6h(*p* < 0.05, Figure 6).

During the shift to Sin 600, the ABTS activity was correlated with light distribution as well as with phenolic content and the pigments Dt and β-car (Table 5). On the difference to the Sin 150 condition, AsA and flavonoids seemed to do not be involved in ABTS activity (lack of correlation, Table 5), conversely to what was observed in Sin 150. When cells coped with high light square wave distribution, the correlations changed again. Under Quad 600, ABTS activity was only related to flavonoids content (Table 5) while phenolic content was linked to Dt. Under Quad 300, i.e., a square wave distribution with a daily light dose similar to Sin 600, the ABTS activity was related to AsA, with the latter negatively correlated to NPQ (Table 5).

Under prolonged darkness, phenolic compounds, flavonoids, AsA, and ABTS did not vary significantly over time (Figures 4–6). Under continuous light the cellular concentration of AsA did not change over time while that of both phenolic compounds and flavonoids doubled and quadrupled, respectively, after 24 h (Figure 4). The increase of ABTS by five fold after 24 h in the Con 10 treatment was coupled to the antioxidant molecules increase (Figures 5 and 6).

Changes in the cellular concentration of antioxidants, as well as ABTS over time were not significant for both Sin 10 and dark treatments (Figure 5A–D and Figure 6A). In continuous dark, ABTS scavenging activity was significantly related to the Dt and β-car while none of the antioxidant molecules was correlated to ABTS (Table 6). Conversely, under Con 10, the unique parameter significantly involved in the ABTS activity was the flavonoids concentration. Under Sin 10, ABTS activity was correlated to phenolic compounds (but not flavonoids, Table 6). These results highlighted the diversity of the responses of the cells when copying with different low light treatments.




Abbreviationsand measurementunitsasinTable

 4.

1

#### **4. Discussion**

Current results highlighted that *Skeletonema marinoi* turned out to be rich in phenolic compounds, which are the most widespread antioxidant substances in photosynthetic organisms [48,49]. Phenolic compounds are able to act directly against radical species as well as indirectly via the inhibition of pro-oxidant enzymes such as lipoxygenase or through metal chelation, preventing the occurrence of the Haber–Weiss and the Fenton reactions, which are important sources of radical species [50]. Although some studies reported that phenolic compounds are the main contributors to microalgal antioxidant capacity [17,24,48,51], the microalgal phenolic content is studied little [14,17,20]. The most abundant phenolic compounds in phytoplankton are phloroglucinol, *p*-coumaric acid as well as flavonoids such as ferulic acid and apigenin [14]. Among them, few studies explored their modulation in response to environmental forcing changes [44,49,52,53]. As reported previously [17,24], the content of phenolic compounds in microalgae is higher than in macroalgae and many higher plants. Assuming a dry weight per cell equivalent to 55 pg as in *Skeletonema costatum* [54], we estimate an average phenolic content <sup>≈</sup> 5.5 mg GAE g−<sup>1</sup> DW, with values up to 12.7 mg GAE g−<sup>1</sup> DW in some conditions. These values are in the range of previous estimations on the same species object of the present study [24] and in the higher range of results from different studies [17,44,48,51]. Yet, another study [20] reported high phenolic content (8–17.5 mg GAE g−<sup>1</sup> DW) in four microalgae from different taxa: *Nannochloropsis oceanica* (Eustigmatophyceae), *Chaetoceros calcitrans* (diatom), *Skeletonema costatum* (diatom), and *Chroococcus turgidus* (cyanophyte).

Among the phenolic compounds family, recent findings demonstrated diatoms' ability to produce flavonoids [49], which display relevant antioxidant activity and act as signaling molecules able to up-regulate the defense strategies [13,49]. In most of the light conditions tested in our study, flavonoids' concentration generally shows the same trend observed for the phenolic compounds. Flavonoids are located in different organelles, including chloroplasts where they play a key photoprotective role [55–57]; in particular, they can scavenge radical species and stabilize membranes containing non-bilayer lipids, such as monogalactosyldiacylglycerol (MGDG) [58].

Our study shows that flavonoids are strictly related to ABTS scavenging activity under unnatural light stress, such as continuous (0:24 h, light: dark) and very high light (600 μmol photons m−<sup>2</sup> s−1; Quad 600), conversely to AsA. This might confirm the powerful capacity of flavonoids to act as defense against stress as photoprotector [59]. Their concentration ranges from circa 50 to 400 fg quercetin equivalent (QEq) cell<sup>−</sup>1, corresponding to <sup>≈</sup> 1 to 8 mg quercetin equivalent (QEq) g−<sup>1</sup> DW. Interestingly, these values correspond to concentrations reported in a wide range of vegetables, fruits or higher plants [60–62].

AsA concentration in *S. marinoi* is also high, with values spanning from 10 to 300 fg AsA cell−<sup>1</sup> (≈1.8–5.5 mg AsA g−<sup>1</sup> DW) in the range of the values previously reported for the same species [24], as well as other phytoplankters [32,63,64]. The latter study highlighted the high variability of AsA concentration among different groups and between exponential and stationary growth phases, with concentrations up to 16 mg AsA g−<sup>1</sup> DW. Our results highlight the huge potential of *S. marinoi*, the diatom model used in this study, as alternative source of antioxidant molecules. This study also shows the relevance of light driven-modulation on the intracellular concentrations of these molecules.

Current results highlight a substantial infradiel variability in the cellular concentrations of antioxidants. The increase in antioxidants observed at midday confirms the role of light in controlling antioxidant synthesis; antioxidants counteract the detrimental effect of the ROS which are produced as consequence of light exposure, as already observed in higher plants [65,66]. In the absence of light or under an extremely low sinusoidal light, infradiel variations of protective or antioxidant responses disappear, highlighting a direct light stimulus control, excluding an internal circadian clock, of these variations. A circadian clock synchronized with predictable daily environmental cyclic variations generally represents an evolutionary adaptation able to increase the fitness of the organism [67]. Instead, under the highly fluctuating light environment naturally experienced by diatoms, which frequently move along the water column, the presence of a rigid scheme ruling cell physiology could

be a disadvantage. A better strategy might consist of promptly modifying the metabolism following the external stimuli, resulting in a great plasticity, which is a known feature attributed to diatoms.

In contrast with what was reported under sinusoidal light distribution, square wave distribution does not induce cyclical infradiel variability. The sinusoidal high light distribution, although slowing microalgal growth, is well tolerated, thanks to the activation and functioning of the antioxidant-photoprotective network. By contrast, square wave distribution with high light intensity strongly affects cell performance impairing the normal functioning of the defense processes network.

Light climate changes experienced by cells induce an uncoupling of the regulative responses (photoprotection vs. AsA, phenol and flavonoid contents) compared to the synergy of these photoresponses observed under pre-acclimation light (Sin 150). Sinusoidal high light exposition leads ABTS scavenging activity to be related to phenolic content as well as Dt and β-car while a non-significant role of flavonoids or AsA content is observed. Parallel responses of Dt and phenolic compounds' concentrations have been already reported [24], along with no significant relationship between Dt and NPQ ([24,37,47]; this study) confirming an alternative role of this pigment, which is likely to have an additional antioxidant function. Under sinusoidal light distribution, with either moderate or high intensity, significant contribution of Dt in ROS scavenging activity is detected. Intriguingly, the relationship between Dt and ABTS is always accompanied by the significant correlation between β-car and ABTS (except when cells enter the stationary phase) that might reveal a similar role of these two pigments in ROS scavenging. The discrepancy between NPQ and Dt is related to an earlier NPQ response compared to Dt as observed under Sin 600, with the highest NPQ recorded after 2 h and subsequently decreasing. This uncoupling between NPQ and Dt confirms the role of NPQ as first defense strategy against light-related stress and that of Dt as a less quick ROS quencher [47].

In Quad 600 the significant role of flavonoids into the ABTS scavenging activity, by contrast to the other phenolic compounds, agree with the fact that flavonoids are known to have strong antioxidant activity [68–70], together with a relevant role in photoprotection [58] that relies on their enhanced concentration in chloroplasts, sites of light-driven ROS production [71,72].

The peculiar response of AsA under Quad 600, with a decrease recorded after 10 min of light exposure, might be due to its fast consumption to counteract the oxidative process induced by abrupt and strong high light exposure.

By contrast in lower light square wave distribution (Quad 300) AsA seems to control ABTS scavenging activity since they are both significantly correlated.

Under low light conditions, different bioactive compounds families with respect to the light climate modulate ABTS scavenging activity.

Under prolonged darkness the increased concentration of Dt is induced by the chlororespirationdependent trans-thylakoid ΔpH [39,73,74], and significantly linked to ABTS scavenging activity. Under very low light conditions (Sin 10), ABTS only significantly relies on phenolic content, as it was also observed—together with Dt—in Sin 600. This very low light intensity does not determine any increase in Dt, probably because of the absence of chlororespiratory pathway development as observed in prolonged darkness.

By contrast, the continuous low light causes a strong impairment of the normal cell functioning inducing high cell mortality. Under this condition, such as under Quad 600 ABTS scavenging activity is only related to flavonoids content.

Not only light distribution and/or intensity, but also culture age changes dramatically the photoresponses of the cells. All the antioxidant molecules as well as Dt increase during cell senescence. The accumulation AsA has been already observed in the senescent diatom *S. marinoi* [64]. The infradiel variations observed during the active growth phase were disrupted during the stationary phase, vouching for the drastic changes to which the cells were subjected [75]. Conversely to exponentially grown cells, NPQ remains high at midday together with the antioxidant capacity and molecule concentration. The integrated defense strategy development suggests the high level of ROS produced in senescent cultures. In higher plants, the early event of cell senescence is the inactivation of

the enzyme Rubisco [76,77] not paralleled by a loss of the thylakoid proteins, which happens at a later time [77]. Therefore, the potential exposure to increasing light induces the development of the first defense mechanism represented by NPQ and, subsequently, the antioxidant network is involved in the scavenging of the ROS, which are produced by the accumulation of electrons from the photosynthetic process.

#### **5. Conclusions**

In conclusion, phenols do account for scavenging activity in the case of natural gradual light variations (moderate, high, or extremely low light), while flavonoids are the family of compounds "selected" in the case of un-natural and very stressful change of light (Con 10, Quad 600).

This study provides evidence of the interconnection between xanthophyll-cycle-relied photoprotection and synthesis of antioxidant molecules. This study highlights the great potential of diatoms as alternative source of natural antioxidant molecules such as carotenoids, phenolic compounds, flavonoids, and ascorbic acid—as well as on the role of light manipulation as an effective tool for enhancing antioxidant molecules synthesis in diatoms.

**Author Contributions:** C.B. supervised the work. A.S., M.M., I.O. and C.B. carried out the experiments and analyzed the samples. A.S., S.B., M.M., M.B., C.G., C.S., C.B. contributed to data interpretation, analyzed the results and drafted the manuscript. All authors approved the submitted version and agreed to be personally accountable for the authors' own contributions.

**Funding:** This research received no external funding. Arianna Smerilli was funded by a PhD grant from the Stazione Zoologica Anton Dohrn.

**Acknowledgments:** The authors thank Federico Corato for the light system device realization. We acknowledge the three reviewers for their comments on the previous version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Red Light Control of** β**-Carotene Isomerisation to** *9***-cis** β**-Carotene and Carotenoid Accumulation in** *Dunaliella salina*

#### **Yanan Xu and Patricia J. Harvey \***

Faculty of Engineering and Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK; y.xu@greenwich.ac.uk

**\*** Correspondence: p.j.harvey@greenwich.ac.uk

Received: 14 May 2019; Accepted: 26 May 2019; Published: 27 May 2019

**Abstract:** *Dunaliella salina* is a rich source of *9-cis* β-carotene, which has been identified as an important biomolecule in the treatment of retinal dystrophies and other diseases. We previously showed that chlorophyll absorption of red light photons in *D. salina* is coupled with oxygen reduction and phytoene desaturation, and that it increases the pool size of β-carotene. Here, we show for the first time that growth under red light also controls the conversion of extant *all-trans* β-carotene to *9-cis* β-carotene by β-carotene isomerases. Cells illuminated with red light from a light emitting diode (LED) during cultivation contained a higher *9-cis* β-carotene content compared to cells illuminated with white or blue LED light. The *9-cis*/*all-trans* β-carotene ratio in red light treated cultures reached >2.5 within 48 h, and was independent of light intensity. Illumination using red light filters that eliminated blue wavelength light also increased the *9-cis*/*all-trans* β-carotene ratio. With norflurazon, a phytoene desaturase inhibitor which blocked downstream biosynthesis of β-carotene, extant *all-trans* β-carotene was converted to *9-cis* β-carotene during growth with red light and the *9-cis*/*all-trans* β-carotene ratio was ~2. With blue light under the same conditions, *9-cis* β-carotene was likely destroyed at a greater rate than *all-trans* β-carotene (*9-cis*/*all-trans* ratio 0.5). Red light perception by the red light photoreceptor, phytochrome, may increase the pool size of anti-oxidant, specifically *9-cis* β-carotene, both by upregulating phytoene synthase to increase the rate of biosynthesis of β-carotene and to reduce the rate of formation of reactive oxygen species (ROS), and by upregulating β-carotene isomerases to convert extant *all-trans* β-carotene to *9-cis* β-carotene.

**Keywords:** *9-cis* β-carotene; *all-trans* β-carotene; *Dunaliella salina*; red LED; blue LED; growth; light intensity; carotenoids; isomerisation

#### **1. Introduction**

Carotenoids are synthesized by photosynthetic organisms for light-harvesting and for photo-protection of the pigment-protein light-harvesting complexes and photosynthetic reaction centres in the thylakoid membrane [1–4]. *Dunaliella salina*, a halotolerant chlorophyte, is one of the richest sources of natural carotenoids, and accumulates up to 10% of the dry biomass as β-carotene under conditions that are sub-optimal for growth, i.e., high light intensity, sub-optimal temperatures, nutrient limitation and high salt concentrations [5–8]. Two pools of β-carotene have been identified, which may be distinguished on the basis of geometric isomer configuration, *cis* or *trans* (*Z*/*E*), and enzyme complement. Thylakoid β-carotene consists principally of *all-trans* β-carotene (*all-trans* βC), and may be constitutively expressed; the 'accumulated' β-carotene, which is found in globules of lipid and proline-rich, β-carotene globule protein (the βC-plastoglobuli) in the inter-thylakoid spaces of the chloroplast, appears in high concentration of both *cis*/*trans* (*Z*/*E*) configurations, ratio ~1 [5,9–11].

The occurrence of such high concentrations of *9-cis* βC in *D. salina* is of great pharmaceutical interest. *9-cis* βC has a higher antioxidant activity than *all-trans* βC, and may also be more efficient than *all-trans* βC in vivo [12,13]. *9-cis* βC has been proposed in treatments for retinal dystrophies, chronic plaque psoriasis and atherosclerosis and as an anti-ageing therapy [14–18]. Importantly, a synthetic pure preparation of *9-cis* βC has recently been shown to inhibit photoreceptor degeneration of eye cups from mice with a retinoid cycle genetic defect [19].

However, the mechanism and regulation of the biosynthesis of *9-cis* βC in *D. salina* is unclear. Using different inhibitors of β-carotene biosynthesis, Shaish et al. [20] found that all the intermediates between phytoene and β-carotene in cultures maintained under low light intensity and N-starvation contained similar ratios of *9-cis*/*all-trans* stereoisomers. They concluded that the isomerisation step must occur at or before phytoene, and that no further isomerisation was likely to occur during the further transformation of phytoene to β-carotene. On the other hand, in cultures maintained under light stress, *9-cis*/*all-trans* βC isomerases were identified in high concentrations in plastidic globules, and were shown in vitro to catalyse conversion of *all-trans* βC to *9-cis* βC, whilst the expression of the corresponding genes was enhanced under stress conditions [21]. The *9-cis*/*all-trans* βC ratio has been shown to increase four-fold and the β-carotene content two-fold when the culture temperature decreased from 30 °C to 10 °C [22], and to increase with increased light intensity [21,23,24], but to be independent of light wavelength within the photosynthetically active range [7]. There have also been reports of a higher *9-cis*/*all-trans* βC ratio in *D. salina* cultivated under low light intensities [25,26].

Recently, we showed that growth of *D. salina* under high intensity red light was associated with carotenoid accumulation and a high rate of oxygen uptake [1]. We proposed a mechanism for carotenoid synthesis under red light, which involved absorption of red light photons by chlorophyll to reduce plastoquinone in photosystem II, coupled with phytoene desaturation by a plastoquinol:oxygen oxidoreductase, with oxygen as electron acceptor. Partitioning of electrons between photosynthesis and carotenoid biosynthesis would depend on both red photon flux intensity and phytoene synthase upregulation by the red light photoreceptor, phytochrome.

In this paper, the effects of red, white and blue light on the β-carotene isomeric composition in *D. salina* were investigated. Isomerisation between *all-trans* and *9-cis* βC in *D. salina* was regulated by light wavelength but not light intensity, with red light shifting the equilibrium in the direction of *9-cis* βC production. In blue light, *9-cis* βC was more rapidly destroyed than *all-trans* βC.

#### **2. Materials and Methods**

#### *2.1. Strains and Cultivation*

*D. salina* strain CCAP 19/41 (PLY DF15) was isolated from a salt pond in Israel and obtained from the Marine Biological Association (MBA, Plymouth, UK). Algae were cultured in Modified Johnsons Medium [27] in an ALGEM Environmental Modeling Labscale Photobioreactor (Algenuity, Bedfordshire, UK) and growth was monitored as described previously [1]. For initial experiments described by Figure 1, *D. salina* cells were grown under 12/12 light/dark (L/D) with 200 μmol photons m−<sup>2</sup> s−<sup>1</sup> supplied by white light emitting diode (LED) light (Figure A1a) to exponential growth phase, then dark-adapted for 36 h. After dark adaptation, they were transferred to continuous white, blue or red LED light at light intensities of 200, 500, or 1000 μmol photons m−<sup>2</sup> s−<sup>1</sup> for 48 h. Samples were taken at 0, 24 and 48 h for carotenoids analysis. For experiments with norflurazon described by Figure 5, cultures were grown for 24 h under white LED light then norflurazon as added to cultures to a working concentration of 5 μM and maintained for a further 48 h under red, blue or a mix of red and blue LED light at 200 μmol m−<sup>2</sup> s−<sup>1</sup> or kept in the dark. Red filters (Lee filter 26 Bright red, 27 Medium red, and 787 Marius red (Figure A1b–d)) when used, were purchased from Lee Filters Andover (Hampshire, UK) and placed over the LED lights. The cultures were shaken for 10 min at 100 rpm every hour before taking samples to monitor cell growth in order to minimise sheer stress to the cells which have no cell wall.

#### *2.2. Carotenoids Analysis*

The composition of pigments was analysed by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) (Agilent Technologies 1200 series, Agilent, Santa Clara, United States). Biomass was harvested and extracted for HPLC analysis as described previously [1], and analysed at least in triplicate. A carotene standard for *all-trans* βC was obtained from Sigma-Aldrich Inc. (Merck KGaA, Darmstadt, Germany); a carotene standard for *9-cis* βC was obtained from Dynamic Extractions (Tredegar, Gwent, UK). The *all-trans* and *9-cis* βC contents were quantified from their absorption at 450 nm.

#### *2.3. Statistical Analysis*

Each experiment was carried out at least in triplicate. The collected data were analyzed in R by one way analysis of variance (ANOVA) with posterior Dunnett's test and Turkey multiple pairwise-comparisons. A *p* < 0.05 value was considered significant.

#### **3. Results**

9-*cis* βC and *all-trans* βC were the major carotenoids that accumulated in *D. salina* biomass after 48 h exposure to red or blue LED light, but the relative pool sizes of each depended on the concentration of red and blue photons of light received. Under blue light, the contents of both *9-cis*- and *all-trans* βC per cell increased with time (Figure 1a,b), and the ratio of *cis*/*trans* βC isomers remained approximately the same at all light intensities (Figure 1c). The concentration of *9-cis* βC was ~half as much as *all-trans* βC. Under red light, by contrast, the concentration of *9-cis* βC and total pool of carotenoids increased massively compared to that in blue in all light intensities and the content of *9-cis* βC was ~twice as much as *all-trans* βC (Figure 1a,b). With increasing light intensity, the relative pool sizes of the isomers changed; that of *all-trans* βC decreased and that of *9-cis* βC increased. Furthermore *9-cis* βC increased with time to >60% of total β-carotene under red light (Figure 1d). HPLC profiles of the carotenoid extracts showed 9-*cis* βC and *all-trans* βC were the major carotenoids that accumulated in *D. salina* biomass, and that the ratios of the two isomers were different under different wavelengths (Figure 1e).

To test the effect of blue light exposure on carotene isomers that had accumulated in red light and vice versa, dark-adapted cultures of *D. salina* were cultivated in red or blue LED high intensity light for 24 h (T0), and then cultivated for a further 24 h in red, blue, or a mixture of red and blue LED light (1:1) with the same light intensity, or the dark. As before, red-shifted cells maintained in red light produced the greatest amount of carotenoids with ~twice as much as *9-cis* βC as *all-trans* βC (Figure 2). On the other hand, *9-cis* βC decreased when red-shifted cells were transferred to blue light (Figure 2), to the same level as for blue-shifted cells maintained continuously in blue (Figure 3); the pool size of carotenoids for both conditions was about the same and the concentration of *9-cis* βC was ~half as much as *all-trans* βC. Conversely, blue-shifted cells when transferred to red LED produced more carotenoids (28% greater content), principally as *9-cis* βC (Figure 3).

Since red light increased the net content of *9-cis* βC, the effects of red light/dark cycles of increasing red light duration during cultivation were tested. Increasing red light duration increased the total amount of β-carotene, in particular the amount of *9-cis* βC (Figure 4). With a red light/dark cycle of 10 min/110 min, the ratio of *9-cis*/*all-trans* βC was 1.1, but in a red light/dark cycle of 30 min/30 min, this increased to 2.2, similar to that in continuous red (2.3). However, in continuous red light, the total pool size β-carotene was nearly 25% greater.

**Figure 1.** *Cont*.

**Figure 1.** Cultivation of *D. salina* under continuous blue or red LED light at three different light intensities of 200, 500 and 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 48 h. (**a**) Cellular content of *9-cis* βC, (**b**) cellular content of *all-trans* βC; (**c**) *9-cis*/*all-trans* βC ratio. (**d**) Percentage of *9-cis* and *all-trans* βC in total βC. (**e**) HPLC profiles at 450 nm of carotenoid extracts from *D. salina* cultivated under continuous white light, red light or blue light, each at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 48 h. Peak 1: *all-trans* β-carotene; peak 2: *9-cis* β-carotene. Biomass was collected at 48 h illumination and carotenoids extracted for HPLC analysis. Each culture condition was set up at least in triplicate. mAU: milli-absorbance unit.

**Figure 2.** (**a**) Cellular content of *9-cis* βC and *all-trans* βC and (**b**) *9-cis*/*all-trans* βC ratio in *D. salina* cultures exposed to continuous red LED light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 24 h followed by 24 h under either red light, a mix of 1:1 red and blue light, blue light at the same light intensity of 1000 μmol m−<sup>2</sup> s−<sup>1</sup> or dark. Each culture condition was set up at least in triplicate. Results were analysed by one way analysis of variance (ANOVA) with posterior Dunnett's test compared to T0 and Tukey multiple pairwise-comparisons. Asterisks represent different levels of significance (\*\*\* 0 < *p* ≤ 0.001, \*\* 0.001 < *p* ≤ 0.01, \* 0.01 < *p* ≤ 0.05).

**Figure 3.** (**a**) Cellular content of *9-cis* βC and *all-trans* βC and (**b**) *9-cis*/*all-trans* βC ratio in *D. salina* cultures exposed to continuous blue LED light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 24 h followed by 24 h under either red light, a mix of 1:1 red and blue light, blue light at the same light intensity of 1000 μmol m−<sup>2</sup> s−<sup>1</sup> or dark. Each culture condition was set up at least in triplicate. Results were analysed by one way ANOVA with posterior Dunnett's test compared to T0 and Tukey multiple pairwise-comparisons. Asterisks represent different levels of significance (\*\*\* 0 < *p* ≤ 0.001, \*\* 0.001 < *p* ≤ 0.01, \* 0.01 < *p* ≤ 0.05).

**Figure 4.** Effect of cultivating D. salina under different red light/dark cycles. (**a**) Cellular content of *9-cis* βC, *all-trans* βC and total βC and (**b**) *9-cis*/*all-trans* βC ratio and (**c**) specific growth rate of *D. salina* cultures grown under different light/dark cycles of red LED light supplied at 500 μmol m−<sup>2</sup> s<sup>−</sup>1. Cultures of *D. salina* were grown to a cell density of ~0.2 million cells mL−<sup>1</sup> under white LED light and then transferred into red LED light growth cycles of different duration. Carotenoids were analysed after 6 days growth.

The accumulation of carotenoids under red light has previously been shown to involve upregulation of phytoene synthase to increase the pool size of phytoene in *D. salina* cultures [1]. In order to test the effect of blue and red light on the β-carotene isomer composition, but without interference of de novo synthesis of β-carotene from phytoene, norflurazon, a phytoene desaturase inhibitor, was applied to the *D. salina* cultures (Figure 5). After 48 h without light, the total pool size of carotenoids was the same as that at the outset of the experiment (T0) before light treatment i.e., norflurazon blocked any further downstream synthesis of β-carotene. Under these conditions, the β-carotene isomer composition, *9-cis*/*all-trans* βC, was 1.1, the same as that recorded for growth in a red light/dark cycle of 10 min/110 min. Both red and blue light treatments lowered the total pool size of total β-carotene, blue more than red: ~31–32% total β-carotene was destroyed under red light and under the 1:1 red/ blue light mix, and ~41% under blue light. Carotenoids absorb photons in the range 400–550 nm, exactly overlapping the emission spectrum of the blue LED (440–500 nm) therefore the greater loss in blue light compared to red was to be anticipated. Furthermore, although both *all-trans* βC and *9-cis* βC were destroyed under blue light, the loss of *9-cis* βC was very much greater: only ~40% of the content of *9-cis* βC recorded in dark-treated cultures remained, compared to 78% for *all-trans* βC. Since *9-cis* βC has a higher antioxidant activity than *all-trans* βC, this result might also be anticipated. Somewhat surprisingly, however, loss of *9-cis* βC under red light compared to blue was much smaller and the ratio of *9-cis*/*all-trans* βC was 3-fold greater than under blue light. Since the emission spectrum of the red LED (625–680 nm) emits photons that are not absorbed by β-carotene, these data imply isomerisation of extant *all-trans* βC to *9-cis* βC to increase the content of *9-cis* βC at the expense of *all-trans* βC during growth.

**Figure 5.** Production of carotenes in *D. salina* cultures treated with 5 μM norflurazon. (**a**) cellular content of *9-cis* βC, *all-trans* βC and total βC and (**b**) *9-cis*/*all-trans* βC ratio. Cultures were grown for 24 h under white LED light then treated with norflurazon and maintained for a further 48 h under red, blue or a mix of red and blue LED light at 200 μmol photons m−<sup>2</sup> s−<sup>1</sup> or kept in the dark. T0: time point after growth for 24h under white LED light only, before addition of norflurazon. Results were analysed by one way ANOVA with posterior Dunnett's test compared to T0 and Tukey multiple pairwise-comparisons. Asterisks represent different levels of significance (\*\*\* 0 < *p* ≤ 0.001, \*\* 0.001 < *p* ≤ 0.01, \* 0.01 < *p* ≤ 0.05).

A similarly greater loss of *all-trans* βC compared to *9-cis* βC in red light was obtained using Lee Bright Red, Medium Red or 787 Marius Red filters: these transmitted only a fraction (8.6%, 3.6% and 1.0%) of the light intensity applied with a red LED (1000 μmol m−<sup>2</sup> s−1), but importantly excluded light wavelengths below 550 nm (Figure A1b–d). Each increased the total β-carotene pool size and the *9-cis*/*all-trans* βC ratio was higher (Figure 6). With the 787 Marius Red filter, cells received only approximately 10–17 μmol m−<sup>2</sup> s−<sup>1</sup> light intensity of the red wavelength but this was still sufficient to increase the ratio of *9-cis*/*all-trans* βC ratio, the amount of *9-cis* βC per cell and total β-carotene to values approaching those found using white light at 1000 μmol m−<sup>2</sup> s<sup>−</sup>1.

**Figure 6.** Cultivation of *D. salina* using red light filters. *D. salina* was cultivated under white light to early orange phase (cell density of ~0.5 <sup>×</sup> 10<sup>6</sup> cells mL<sup>−</sup>1; carotenoid: chlorophyll ratio ~3), and then cultures were diluted with fresh medium to a cell density of ~0.2 <sup>×</sup> 106 cells mL−<sup>1</sup> (no nutrient stress) (T0) and then further cultivated for 48 h under white, red or blue LED light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> or under white LED light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> covered with one of three different red filters (Lee filter 26 Bright red; Lee filter 27 Medium red; or Lee filter 787 Marius red). (**a**) Cellular content of *9-cis*, *all-trans* and total β-carotene. (**b**) *9-cis*/*all-trans* β-carotene ratio. Results were analysed by one way ANOVA and Tukey multiple pairwise-comparisons. Asterisks represent different levels of significance (\*\*\* 0 < *p* ≤ 0.001, \*\* 0.001 < *p* ≤ 0.01, \* 0.01 < *p* ≤ 0.05).

The co-regulation by light and temperature on the β-carotene production and isomeric composition in *D. salina* is shown in Figure 7. Cultivation at 15 ◦C compared to 25 ◦C increased the *9-cis*/*all-trans* βC ratio, especially under red light, but decreased the pool size of β-carotene measured over the same time frame (48 h).

**Figure 7.** *D. salina* cultivated under red or blue light at either 15 °C or 25 °C. (**a**) Cellular content of *9-cis* βC and *all-trans* βC (**b**) *9-cis*/*all-trans* βC ratio. Cells were cultured under a light:dark 12h:12h white light growth regime to mid-log phase of the growth cycle (0.1–0.2 <sup>×</sup> 106 cells mL<sup>−</sup>1) then transferred to the dark for 24 h before treatment for 48 h at either 15 ◦C or 25 ◦C under continuous blue or red LED light at 1000 μmol m−<sup>2</sup> s<sup>−</sup>1. Each culture condition was set up at least in triplicate.

Finally, the effects of blue and red light on the destruction of *all-trans* βC were evaluated. No reaction of *all-trans* βC solutions was detected under red light in nitrogen (Figure 8a). Under red light in air, (Figure 8b), 40% destruction of *all-trans* βC was recorded, whereas in blue light (Figure 8c), *all-trans* βC was fully destroyed within the same time frame. These data show that blue light is more damaging to *all-trans* βC than red light.

**Figure 8.** Effect of red or blue LED light on the photo-destruction of *all-trans* βC. *All-trans* βC was dissolved in chloroform to a final concentration of 2.4 μM and vials were thoroughly flushed with either nitrogen or air, sealed and incubated for 24 h at 25 ◦C under different LED lights at 200 μmol m−<sup>2</sup> s<sup>−</sup>1. (**a**) Red light under nitrogen; (**b**) Red light in air; (**c**) Blue light in air.

#### **4. Discussion**

In the present work, we found that under high intensity red LED light (up to 1000 μmol m−<sup>2</sup> s<sup>−</sup>1) but in conditions of nutrient sufficiency, *D. salina* accumulated carotenoids rapidly within 48 h. Surprisingly, the major accumulated isomer was *9-cis* βC, ~twice as much as *all-trans* βC. In vitro, *9-cis* βC is a better scavenger of free radicals than *all-trans* βC [12], and reportedly degrades more rapidly compared to *all-trans* βC under both light and dark conditions [28]. Furthermore, chlorophyll absorbs photons in the range of the emission spectrum of the red LED used here (625–680 nm) and therefore in *D. salina* cultures in high intensity red light, a high rate of photo-oxidation of *9-cis* βC might have been anticipated. Carotenoids are known antioxidants synthesized by many microalgae to prevent photoinhibition caused by photo-oxidation of photosynthetic reaction centres. Photooxidative damage occurs when species such as singlet oxygen (1O2) are formed under saturating light conditions as a result of transfer of energy from chlorophyll in the triplet excited state (3Chl\*) to the ground state of O2. 1O2 react readily with fatty acids to form lipid peroxides and will set up a chain of oxygen activation events that may eventually lead to a hyperoxidant state and cell death [29]. Carotenoids protect the photosystems in the following ways: (i) by reacting with lipid peroxidation products and terminating free radical chain reactions as a result of the presence of the polyene chain; (ii) by scavenging 1O2 and dissipating the energy as heat; and (iii) by reacting with triplet excited chlorophyll 3Chl\* to prevent formation of 1O2 or by dissipation of excess excitation energy through the xanthophyll cycle [3,30,31].

The simplest explanation to resolve the seeming anomaly, namely accumulation of the more readily degraded *9-cis* βC under high intensity red light conditions that should be associated with high rates of photo-oxidation, invokes the activity of β-carotene isomerases, the gene transcripts of which are increased in light stress [21]. Davidi et al. [11] showed that all the enzymes in the biosynthetic pathway from phytoene to β-carotene were present in the plastidic lipid globules and included enriched concentrations β-carotene isomerases; two of these, *9-cis*-βC-ISO*1* and *9-cis*-βC-ISO*2*, were shown to be responsible for the catalytic conversion of *all-trans* βC to *9-cis* βC. Based on the data presented here we propose that the expression of gene transcripts of β-carotene isomerases may be triggered by specific light sensing, possibly through phytochrome.

In red light compared to blue, the apparent loss of *9-cis* βC with norflurazon was surprisingly small and the ratio of *9-cis*/*all-trans* βC was 3-fold greater than in blue light (Figure 5). Accumulation of *9-cis* βC by phytoene synthase (PSY) gene activation, whose expression has been shown to be greatly increased 6–48 h following stress [11] was precluded by the presence of norflurazon, which blocked phytoene desaturation and consequent carotene synthesis. Under these conditions, the relative increase in pool size of *9-cis* βC in red light implies a much higher rate of *9-cis* βC formation from extant *all-trans* βC, caused by increased isomerase activity, than the rate of *9-cis* βC destruction (see Figure 5). Carotenes absorb photons in the range 400–550 nm, exactly overlapping the emission spectrum of the blue LED (440–500 nm). However blue light catalysed a much more rapid rate of destruction of carotenes than red light (Figure 8). In blue LED light, *9-cis* βC would be destroyed more rapidly than could be replenished by adjustment of the *9-cis*/*all-trans* βC equilibrium position because increased β-carotene isomerase activity from red-light activated gene expression for β-carotene isomerases is not possible in blue light (see Figure 5).

Red light stimulation of the expression of gene transcripts of β-carotene isomerases by a phytochrome to increase the rate of accumulation of *9-cis* βC by β-carotene isomerases is also supported by the increase in pool size of *9-cis* βC under low intensity red light (Figure 6). Each of the Lee red light filters increased the total β-carotene pool size and the *9-cis*/*all-trans* βC ratio was higher despite the much lower light intensity of the red wavelength compared to the red LED light. The effects of low temperature on *9-cis* βC-accumulation in *D. salina* are also noteworthy, since enzyme catalysis typically shows a Q10 (temperature coefficient) ~2, yet in the present work, formation of *9-cis* βC in low temperature compared to high was increased under red light, and had little effect in blue. In higher plants, the activated phytochrome B, a red light photoreceptor, is considered to function as the thermal sensor to sense environmental temperature [32]. Mutants with no phytochromes showed a constitutive warm temperature transcriptome even at low temperatures [33]. Red light sensing to increase the concentration of β-carotene isomerases and catalyse conversion of *all-trans* βC at low temperatures, as well as high, may play a significant role in photoprotection in *D. salina*.

We recently proposed that red light enhanced the production of carotenoids in a mechanism dependent on both photon flux density as well as upregulation of phytoene synthase by the red light photoreceptor phytochrome and that chlorophyll absorption of red light photons and subsequent plastoquinone reduction in photosystem II was coupled with oxygen reduction and phytoene desaturation by plastoquinol:oxygen oxidoreductase [1]. According to the findings in the previous work [1], the partitioning electron flux between photosynthesis and carotenoid biosynthesis could be augmented by addition of the regulation of the pool size of *9-cis* βC, as seen in the Scheme 1.

Red light sensing by phytochrome to increase the pool size of phytoene by phytoene synthase has been reported in higher plants [34]. Red light control of carotenoid biosynthesis coupled with the accumulation of the more readily oxidized *9-cis* βC as a consequence of isomerisation from *all-trans* βC reserves would therefore rapidly increase the pool size of anti-oxidant to reduce the rate of formation of ROS under stress (See Scheme 1).

**Scheme 1.** Regulation of the pool size of *9-cis* βC. Red photon flux intensity controls the partitioning of electrons either for carotenoid biosynthesis or for photosynthesis, via energy absorption by chlorophyll and the PQ pool [1]. Red photon flux also controls phytochrome regulation of the production of gene transcripts for phytoene synthase and β-carotene isomerases. CHL A: chlorophyll a; P680: chlorophyll a, primary electron donor of Photosystem II; PQox: plastoquinone, oxidised form; PQred: plastoquinone, reduced form; Cyt b6ox: cytochrome b6f complex, oxidised form; NADP<sup>+</sup>: NADP oxidised form; NADPH: NADP reduced form; PSY: phytoene synthase; 9-*cis*-βC-ISO: *9-cis* βC isomerase.

#### **5. Conclusions**

Red light availability regulates the isomerisation of *all-trans* β-carotene to *9-cis* β-carotene and upregulates carotenoid biosynthesis in the halotolerant microalga *Dunaliella salina*. In red light *9-cis* βC accumulated, caused by increase in the rate of isomerisation of *all-trans* βC to *9-cis* βC relative to the rate of its destruction. Red light may have industrial value as an energy-efficient light source for production of natural *9-cis* βC from *D. salina*.

**Author Contributions:** Y.X. and P.J.H. conceived the work, analysed the data, and wrote the article; Y.X. conducted experiments and curated data; P.J.H. agrees to serve as the author responsible for contact and ensures communication.

**Funding:** This work was supported by EU KBBE.2013.3.2-02 programme (D-Factory: 368 613870) and by the Interreg 2 Seas programme 2014-2020 co-funded by the European Regional Development Fund under subsidy contract No ValgOrize 2S05017.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Figure A1.** *Cont*.

**Figure A1.** (**a**) Typical relative spectral power distribution of white, blue and red LED lights in the Algem bioreactor; (**b**–**d**) The light transmission (Y%) for each wavelength (nm) of filters that were used to transmit red light. (**b**) Lee Filters 026 Bright red (Transmission 8.6%), (**c**) 027 Medium Red (Transmission 3.6%), (**d**) 787 Marius Red (Transmission 1.0%).

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Optimization of Microwave-Assisted Extraction of Polysaccharides from** *Ulva pertusa* **and Evaluation of Their Antioxidant Activity**

#### **Bao Le 1, Kirill S. Golokhvast 2, Seung Hwan Yang 1,\* and Sangmi Sun 1,\***


Received: 22 April 2019; Accepted: 11 May 2019; Published: 14 May 2019

**Abstract:** The use of green marine seaweed *Ulva* spp. as foods, feed supplements, and functional ingredients has gained increasing interest. Microwave-assisted extraction technology was employed to improve the extraction yield and composition of *Ulva pertusa* polysaccharides. The antioxidant activity of ulvan was also evaluated. The impacts of four independent variables, i.e., extraction time (X1, 30 to 60 min), power (X2, 500 to 700 W), water-to-raw-material ratio (X3, 40 to 70), and pH (X4, 5 to 7) were evaluated. The chemical structure of different polysaccharides fractions was investigated via FT-IR and the determination of their antioxidant activities. A response surface methodology based on a Box–Behnken design (BBD) was used to optimize the extraction conditions as follows: extraction time of 43.63 min, power level of 600 W, water-to-raw-material ratio of 55.45, pH of 6.57, and maximum yield of 41.91%, with a desired value of 0.381. Ulvan exerted a strong antioxidant effect against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and showed reducing power in vitro. Ulvan protected RAW 264.7 cells against H2O2-induced oxidative stress by upregulating the expression and enhancing the activity of oxidative enzymes such as superoxide dismutase (SOD) and superoxide dismutase (CAT). The results suggest that the polysaccharides from *U. pertusa* might be promising bioactive compounds for commercial use.

**Keywords:** antioxidant activities; Box–Behnken design; microwave-assisted extraction; polysaccharide; *Ulva pertusa*; seaweed

#### **1. Introduction**

The genus *Ulva* (Chlorophyta) is a cosmopolitan, abundant, and fast-growing green macroalgae forming natural beds in shallow waters throughout the world [1]. *Ulva* is widely distributed, grows rapidly, and causes "green tides" in response to elevated levels of nitrogenous and phosphorus materials in coastal areas [2]. *Ulva* spp. are a relatively rich source of different bioactive compounds, in particular, polyphenols and dietary fiber [3].

*Ulva* contains a polysaccharide present in high amounts in the cell wall (38% to 54% in dry weight) and commonly known as ulvan [4], which belongs to a group of sulfated hetero-polysaccharides comprising glucose, glucuronic acid, rhamnose, xylose, and galactose [5]. Ulvan has been demonstrated to play an important role as an antitumor [6] and antihyperlipidemic [7] substance in living organisms and induces a defense mechanism in crops [8]. It is also one of the important antioxidant compounds, whose antioxidant properties are mainly attributed to its scavenging activity against superoxide and hydroxyl radicals, its chelating ability, singlet and triplet oxygen quenching activity, and reducing

power [9]. However, the understanding of the structural characteristics of polysaccharides extracted from *Ulva pertusa* is limited, which affects their application.

Hot water or aqueous organic solvents are the most common and conventional methods for extracting water-soluble polysaccharides from *Ulva* sp. [7]. However, such methods are time-consuming and have a low extraction efficiency owing to the complex polymers of the algae cell wall [10]. Therefore, additional methods are being used to improve the extraction process, such as microwaving [11], ultrasonication [5], and enzymatic reduction [12]. Microwave-assisted extraction (MAE) methods have demonstrated better performances with the advantages of a short operation time, simplicity, low cost, and high efficiency [13]. MAE is a "green" extraction process based on the use of electromagnetic waves with high frequencies. The high temperature produced by molecular motions increases the solubility of the extracted compounds and the solvent diffusion rate, thereby enhancing their quality and yield [14]. Although this extraction method has been proved efficient in fucoidan and carrageenan isolation from brown and red seaweed [15,16], only a few studies have been carried out on green seaweed such as *Ulva meridional*, *Ulva ohnoi*, and *Monostroma latissimum* [11].

The main aim of this study was to optimize the operational parameters (power, time, water-to-raw-material ratio, and pH) of MAE to obtain the maximum yield of ulvan extracted from *U. pertusa*. Furthermore, the antioxidant activity of ulvan was evaluated in hydrogen peroxide (H2O2)-treated RAW 264.7 cells through in vitro assays.

#### **2. Materials and Methods**

#### *2.1. Seaweeds and Chemicals*

*U. pertusa* gametophytes were collected from June to July 2018 in Dolsan, Yeosu, Korea (34◦40 N, 127◦46 E), put into sterilized plastic bags containing seawater, placed in an ice box, and transferred to the laboratory immediately. The vegetative materials were rinsed several times to clear their surface, oven-dried at 40 ◦C, and maintained at −80 ◦C until use. All chemicals and reagents applied were of analytical grade.

#### *2.2. Microwave Extraction of Ulvan*

The dried *U. pertusa* thallus (100 g) was ground in a high-speed disintegrator to make a fine powder. The powder was pretreated with 80% ethanol (400 mL) in a water bath at 85 ◦C for 2 h to remove pigments and low-molecular-weight compounds. After incubation, the precipitate was collected through centrifugation at 4000× *g* for 10 min and was then dried in an oven at 50 ◦C. The pretreated sample (1 g) was extracted using MAE based on specific extraction time, amount of microwave power, water-to-raw-material ratio, and pH (Table 1). The aqueous extract was separated from the insoluble residue through centrifugation (6000× *g*, 20 min). The solution was precipitated with the addition of ethanol to a final concentration of 85% and maintained at 4 ◦C overnight. The crude polysaccharide was separated through centrifugation (6000× *g* for 20 min) and air-dried for 12 h. Ulvan was weighed and stored at −20 ◦C until analyzed. The total content of the polysaccharides was measured using a phenol–sulfuric acid method [17]. The yield of ulvan (%) was calculated as follows:

Yield of ulvan (%) = polysaccharides content of the extract (g)/weight of the pretreated sample (g) (1)

#### *2.3. Single-Factor MAE Experiments*

The influence of the process parameters including extraction time, microwave power, water-to-raw-material ratio, and pH on the extraction yield and identify the independent variables as well as on the optimum ranges of the Box–Behnken Design (BBD) was determined using a series of single-factor experiments. The effects of each factor were evaluated by determining the ulvan yield.


**Table 1.** Box–Behnken (BBD) matrix of the four variables, levels for response surface methodology (RSM), experimental data, and predicted values of ulvan extraction.

#### *2.4. Experimental Design*

A response surface methodology (RSM) was used to optimize the effects of the independent variables on the extraction yield of ulvan polysaccharide. Four processing variables, i.e., time (X1), power (X2), water-to-raw-material ratio (X3), and pH (X4) were chosen on the basis of the results of single-factor experiments and were then investigated using BBD (Table 1). The yield was taken as the response to the design experiments. The selected variables were coded using the following equation:

$$\mathbf{x}\_{i} = (\mathbf{X}\_{i} - \mathbf{X}\_{o}) / \Delta \mathbf{X}\_{i} \tag{2}$$

where xi is a variable, Xo and Xi are the actual values for the ith independent variable at the center point, and ΔX is the value of the step change.

A second-order regression analysis of the data was defined using the response function (Y) including the linear, quadratic, and interactive components and the proposed model, as follows:

$$\mathbf{Y} = \beta\_{\rm o} + \sum \beta\_{\rm i} \mathbf{x}\_{\rm i} + \sum \beta\_{\rm ii} \mathbf{x}\_{\rm i}^{\rm 2} + \sum \beta\_{\rm ii\backslash} \mathbf{x}\_{\rm i} \mathbf{x}\_{\rm j} \tag{3}$$

where Y is a dependent variable, β<sup>o</sup> is a constant coefficient, and βi, βii, and βij are the regression coefficients for the intercept, linear, quadratic, and two-factor interaction variables, respectively.

#### *2.5. FT-IR Spectrometric Analysis*

The ulvan extract was ground with potassium bromide (KBr) powder before measurement. IR spectra were acquired on an FT-IR spectrophotometer (VERTEX 70, Bruker, Germany) in the frequency range of 4000–400 cm<sup>−</sup>1.

#### *2.6. Determination of the Antioxidant Activity of Ulvan Extracts in Vitro*

#### 2.6.1. DPPH Radical-Scavenging Activity

The free scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) was investigated using the method mentioned by Bondet et al. [18]. The reaction mixtures consisted of 2 mL of ulvan extracted under optimal conditions (0 to 0.8 mg/mL) and 2 mL DPPH (0.05 mM in ethanol). The reaction tubes were incubated in darkness at 25 ◦C for 30 min. The absorbance of the mixture was measured at 517 nm using ascorbic acid as a positive control. The scavenging DPPH activity was calculated according to Equation (4):

$$\text{Scavenging activity} \left(\% \right) = \left(1 - (\text{A}\_1 - \text{A}\_2) / \text{A}\_0 \right) \times 100 \tag{4}$$

where A0, A1, A2 are the absorbance of the DPPH solution used as a negative control, of the sample with the DPPH solution, and of the sample without the DPPH solution, respectively.

#### 2.6.2. ABTS Radical-Scavenging Activity

The assay was carried out using the procedure described by Hromadkova et al. [19]. The working solution was prepared by mixing 25 mL of a 7 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solution and 12.5 mL of 2.4 mM potassium persulfate. The mixture was kept in the dark at room temperature for 12 to 16 h prior to use. The ABTS solution was adjusted to an absorbance of 0.7. For the assays, 0.1 mL of extract was allowed to react with 1 mL of the ABTS solution, and the absorbance was recorded at 734 nm after 7 min using a spectrophotometer. The scavenging ABTS activity was calculated according to Equation (5):

$$\text{Scavenging activity} \left(\% \right) = \left(1 - (\text{A}\_1 - \text{A}\_2) / \text{A}\_0 \right) \times 100 \tag{5}$$

where A0, A1, A2 are the absorbance of the ABTS solution used as a negative control, of the sample with the ABTS solution, and of the sample without the ABTS solution, respectively.

#### 2.6.3. Determination of the Reducing Power

The reducing power was evaluated using a method by Dahmoun et al. [20]. The reaction mixture consisted of 2.5 mL of a 0.2 M phosphate solution, 2.5 mL of 1% (*w*/*v*) potassium ferricyanide, and 2.4 mL of varying concentrations of the ulvan extracts. After the mixture was incubated at 50 ◦C for 20 min, 2.5 mL of 10% (*w*/*v*) trichloroacetic acid was added, and the mixture was centrifuged at 900× *g* for 10 min. The supernatant (5 mL) was mixed with 5 mL of distilled water and 1 mL of 0.1% (*w*/*v*) ferric chloride. The absorbance of the resulting solution was measured for 2 min at 700 nm.

#### *2.7. In Vitro Antioxidant Activity*

The RAW 264.7 murine macrophage cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) (Wellgen, Daegu, Korea) containing 4.5 g/L glucose, 4 mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 15 mg/L phenol red, 3.7 g/L sodium bicarbonate, 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 50 ug/mL streptomycin in a humidified atmosphere at 37◦C, 5% CO2. RAW 264.7 cells were split and seeded in 96-well cell culture plates (2.0 <sup>×</sup> 104 cells/well) and incubated in the same culture conditions overnight. The medium was replaced with fresh DMEM medium containing various concentration of ulvan or 600 μM hydrogen peroxide (H2O2) for 24 h. After incubation, the cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) (Sigma Aldrich, St. Louis, MO, USA) assay [21].

Oxidative damage of the cells was induced using hydrogen peroxide [22]. RAW 264.7 cells (2.0 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well) were seeded in 96-well cell culture plates and incubated overnight. The cells were then washed with 0.1 PBS (pH 7.2) and pretreated with fresh DEME medium containing various concentrations of ulvan for 2 h. To stimulate oxidative stress, the cells were then incubated with 600 μM H2O2 for 24 h under the same conditions. Ascorbic acid was used as a positive control. After incubation, the cells were collected, suspended in 0.1 M cold PBS buffer, and lysed using ultrasonic decomposition in an ice-water bath. The cell-free supernatant was used for analysis of superoxide dismutase (SOD; cat. no. STA-340, Cell Biolabs Inc., San Diego, CA, USA) and catalase (CAT; cat. no. STA-340, Cell Biolabs Inc., San Diego, CA, USA) activities using commercial kits following the manufacturers' instructions.

To determine the expression levels of antioxidant-related genes, total RNA from the treated RAW 264.7 cells was purified by using the Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. Total RNA (1 μg) was reverse-transcribed into cDNA using the ImProm-II™ Reverse Transcription System (Promega, USA), and the target cDNA was amplified using the following primers: β-actin, forward 5 -AAG ACC TCT ATG CCA ACA CAG T-3 , reverse 5 -CAT CGT ACT CCT GCT TGC TGA T-3 ; glutathione S-transferases (GST), forward 5 -TGA GAG GAA CCA AGT GTT TGA G-3 , reverse 5 -CAG GGG GAC TTT AGC TTT AGA A-3 ; catalase (CAT), forward 5 -GGG ATT CCC GAT GGT-3 , reverse 5 -GCC AAA CCT TGG TCA G-3 ; MnSOD, forward 5 -TCC CAGACC TGC CTT ACG A-3 , reverse 5 -TCG GTG GCG TTG AGA TTG-3 ; GPx, forward 5 -CTC GGT TTC CCG TGC AAT CAG-3 , reverse 5 -GTG CAG CCA GTA ATC ACC AAG-3 [23].

#### *2.8. Statistical Analysis*

The experimental design and graphical and statistical analysis for the RSM were conducted using Minitab 17 (Minitab Inc., State College, Pennsylvania). All trials were conducted in triplicate. Data differences between two groups were analyzed using the Student's t test (*p* < 0.05) by the SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).

#### **3. Results**

#### *3.1. E*ff*ect of Process Parameters on Microwave Extraction E*ffi*ciency*

As shown in Figure 1a, the yield of the polysaccharides increased significantly with increasing extraction times ranging from 15 to 45 min; the highest extraction yield was obtained at 45 min. To study the effect of microwave powers on the yield of the polysaccharides, the extraction processes were carried out at 300, 400, 500, 600, 700, and 800 W for 45 min. The results shown in Figure 1b indicate that the maximum ulvan yield (35.14%) occurred when the power was 600 W. The yield of ulvan affected by the different ratios between water and raw materials is shown in Figure 1c, whereas the other extraction variables were as follows: 600 W power, pH of 6, and extraction time of 45 min. The extraction yields increased as the water-to-raw-material ratio ascended slightly from 25 to 85 mL/g and reached the maximum value (25.23%) when the ratio was 70 mL/g. To evaluate the pH effects on the yield, the extraction process was conducted at different pH values, and the results are shown in Figure 1d. As shown in the figure, the polysaccharide yield increased with an increase in the pH level and significantly decreased when the pH was higher than 7.

#### *3.2. Optimization of the Procedure Using RSM*

In the present study, the ulvan extraction yield was investigated according to BBD (27 batch experiments), and the corresponding results are shown in Table 1. The experimental data were then investigated using a multiple regression analysis and an analysis of variance, and the adequacy and fitness of the models are summarized in Table 2. As the results demonstrated, the fitness of the model was highly significant (*p* < 0.0001). According to the multiple regression analysis, the independent variables were related on the basis of a mathematical model describing the ulvan extraction yield (Y) and following a second-order polynomial equation:

$$\begin{array}{l} \text{Y} = 40.84 - 1.076 \text{X}\_1 + 0.697 \text{X}\_2 - 0.207 \text{X}\_3 + 2.283 \text{X}\_4 - 0.84 \text{X}\_1 \text{X}\_2 - 1.335 \text{X}\_1 \text{X}\_3 + 1.238 \text{X}\_1 \text{X}\_4 \\ + 1.575 \text{X}\_2 \text{X}\_3 + 2.505 \text{X}\_2 \text{X}\_4 - 2.055 \text{X}\_3 \text{X}\_4 - 6.333 \text{X}\_1^2 - 1.111 \text{X}\_2^2 - 4.526 \text{X}\_3^2 - 4.032 \text{X}\_4^2 \end{array} \tag{6}$$

where X1, X2, X3, and X4 are the time, power, water-to-raw-material ratio, and pH, respectively.

**Figure 1.** Effect of different times (**a**), powers (**b**), water-to-raw material ratios (**c**), and pH (**d**) on the extraction yield of ulvan. Different letters show statistically significant differences among the groups (*p* < 0.05).


**Table 2.** ANOVA of the RSM model for the prediction of ulvan yield.

\* Significant coefficient (*p* < 0.05). \*\* Highly significant coefficient (*p* < 0.01).

Among the four independent variables studied, only the power and pH exerted a positive linear effect on ulvan extraction. However, the quadratic effects of all parameters negatively affected the extraction process (Table 2). In this study, ulvan yield was significantly influenced by nitrate, time, power, water-to-raw-material ratio, and pH.

The coefficient of determination (*R*<sup>2</sup> = 0.9830) and the adjusted determination coefficient (adj. *R*<sup>2</sup> = 0.9631) for the model exhibited a high correlation between the experimental and theoretical values [24]. In this study, the coefficient of variation (*C.V.*, 1.15%) was no greater than 10%, indicating a high precision and strong reliability of the experimental values. A smaller *C.V.* is a better expression of low variance than the percentage of the mean [25].

The optimal extraction conditions used to obtain the maximum ulvan extraction yield were determined according to Derringer's desired function methodology [26], with extraction time of 43.63 min, power level of 600 W, water-to-raw-material ratio of 55.45, pH of 6.57, and maximum yield of 41.91%, with a desired value of 0.381. The verification experiments were carried out under the optimized conditions, and the mean values (42.12 ± 0.674%, *n* = 3) demonstrated the validity of the optimized conditions.

Response surface plots were generated to understand the significant interaction between the variables. Figures 2a and 3a, which show the polysaccharide extraction yield as a function of extraction time and power, showed that the extraction yield increased rapidly with the increase of the extraction time from 30 to 45 min, while it only slowly increased with the increase of power from 500 to 600 W. Time affected the yield more than power. The interaction effects of different extraction times and water-to-raw-material ratios are illustrated in Figures 2b and 3b. Ulvan yield increased linearly at first with the increase of time from 30 to 45 min and of the water-to-raw-material ratio from 40 to 55 but then decreased for further increases of these variables. Figures 2c and 3c show the relationship between extraction time and pH. The yield initially increased quickly reaching its maximum as both time and pH increased and decreased thereafter. Moreover, the interaction between power and water-to-raw-material ratio (Figures 2d and 3d, and power and pH (Figures 2e and 3e) on the yield were shown to be both positive and significant. In Figures 2f and 3f, the yield improved significantly with the increase of pH from 6 to 6.5. However, the interaction between pH and water-to-raw-material ratio on the yield was characterized by a negative coefficient in the fitting equation. In summary, extraction time and pH were the major factors causing significant effects on the yield of polysaccharides.

**Figure 2.** Response surface (3D) showing the effects of variables on the yield of ulvan. Effects of (**a**) extraction time (X1) and power (X2), (**b**) extraction time and water-to-raw-material ratio (X3), (**c**) extraction time and pH (X4), (**d**) power and water-to-raw-material ratio, (**e**) power and pH, (**f**) water-to-raw-material ratio and pH on ulvan yield (Y, %).

**Figure 3.** Contour plots showing the effects of the above-mentioned variables on the yield of ulvan. Effects of (**a**) extraction time (X1) and power (X2), (**b**) extraction time and water-to-raw-material ratio (X3), (**c**) extraction time and pH (X4), (**d**) power and water-to-raw-material ratio, (**e**) power and pH, (**f**) water-to-raw-material ratio and pH on ulvan yield (Y, %).

#### *3.3. FT-IR Spectral Analysis*

The FT-IR spectrum of the ulvan extract is shown in Figure 4a. The high absorptions at 874 and 1623 cm−<sup>1</sup> were attributed to the bending vibration of sulfate in axial position in C–O–S [27]. The specific intense peaks at 3353, 2926, and 1034 cm−<sup>1</sup> were due to O–H, C–H, C–O stretching vibrations, respectively. The absorption at 1623 cm−<sup>1</sup> was indicative of C=O [28]. The signal at approximately 1414 cm−<sup>1</sup> may suggest the presence of uronic acid [16]. In addition, the spectra of ulvan extract obtained by MAE were quite similar to those obtained after autoclaving [27]. Overall, these results showed that the ulvan extract exhibited the typical absorption peaks of a polysaccharide.

**Figure 4.** FT-IR spectra and antioxidant activity of the polysaccharide extracts from *Ulva pertusa* by the microwave-assisted extraction (MAE) method (mean ± SD, *n* = 3). (**a**) FT-IR spectra, (**b**) ABTS free radical scavenging assay, (**c**) DPPH free radical scavenging assay and (**d**) Reducing power assay. ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), DPPH: 1,1-diphenyl-2-picrylhydrazyl.

#### *3.4. In Vitro Antioxidant Activities of Ulvan*

In this study, the scavenging capabilities of different ulvan extracts for ABTS radicals were measured and are shown in Figure 4b. The scavenging capability of ulvan for ABTS radicals was 20.15% at 0.5 mg/L, with a 1.5-fold increase in activity at 0.8 mg/mL. As shown in Figure 4c, ulvan showed a dose-dependent DPPH scavenging effect weaker than that of ascorbic acid at each concentration. The scavenging capability of the polysaccharide increased from 5.61% to 46.51% as the concentration of the polysaccharide increased from 0.025 to 0.800 mg/mL. The reducing power of ulvan is depicted in Figure 4d. The reducing power of ulvan increased with increasing concentrations (0.5–3 mg/mL).

#### *3.5. E*ff*ect of Ulvan on RAW 264.7 Macrophage Cell Viability and SOD and CAT Activities*

The toxicity of ulvan in RAW 264.7 macrophage cells was evaluated in Figure 5A. An MTT assay demonstrated that ulvan did not significantly affect cell viability at concentrations below 200 μg/mL compared with untreated cells. At the concentration of 400 μg/mLof ulvan, cell viability reduced significantly (*p* < 0.05) (Figure 5A). Ulvan at concentration from 50 to 200 μg/mL showed no cytotoxic effects, thus concentrations in this range were selected for further study.

**Figure 5.** Effects of ulvan (μg/mL) on cell viability (**a**) and production of superoxide dismutase (SOD) (**b**) and catalase (CAT) (**c**) in RAW 264.7 cells. The results are presented as means ± SD (*n* = 3); \* *p* < 0.05 and \*\* *p* < 0.01 vs H2O2 treatment; # *p* < 0.05 compared with control group. AA, ascorbic acid at concentration of 100 μg/mL.

The induction of SOD and CAT was determined to evaluate the antioxidant activity of ulvan in RAW 264.7 cells stimulated by H2O2. As shown in Figure 5B, compared with the control group, treatment with 600 μM of H2O2 significantly decreased SOD activity (*p* < 0.05). SOD activity was significantly increased after treatment with 100 and 200 μg/mL of ulvan. At 200 μg/mL of ulvan, SOD activity was close to that measured in cells treated with ascorbic acid (positive control) at 100 μg/mL. In addition, CAT activity showed a similar trend to that of SOD activity in cells treated with H2O2. However, CAT activity increased upon ulvan treatment at 200 μg/mL. We found that the reduction of SOD and CAT activities in RAW 264.7 cells stimulated by H2O2 could be prevented by a high concentration of ulvan (≥200 μg/mL).

#### *3.6. E*ff*ects of Ulvan on the Expression of Antioxidant Genes*

We examined whether ulvan affected the transcriptional profiles of genes associated with the antioxidant system, such as *GST*, *CAT*, *MnSOD*, and *GPx*, in RAW 264.7 cells (Figure 6). The results showed the downregulation of mRNA expression for these genes compared with the control group (*p* < 0.05) upon treatment with H2O2. In contrast, ulvan significantly increased the expression of *GST*, *CAT*, *MnSOD*, and *GPx* compared in the presence of H2O2 in macrophage RAW 264.7 cells in a dose-dependent manner.

**Figure 6.** Effects of ulvan (μg/mL) on the expression of the antioxidant genes *GST* (**a**), *CAT* (**b**), *MnSOD* (**c**), *GPx* (**d**) in 264.7 cells treated with H2O2. The results are presented as means ± SD (*n* = 3); \* *p* < 0.05 and \*\* *p* < 0.01 vs H2O2 treatment; # *p* < 0.05 compared with the control group.

#### **4. Discussion**

In this study, we developed an extraction process that allows to obtain high yields of polysaccharides from *U. pertusa* while maintaining their antioxidant effects, as confirmed through functional and molecular experiments. To our knowledge, this is the first study to describe the mechanism of the antioxidant activity of ulvan on RAW 264.7 cells.

A single-factor experimental analysis was applied to select the appropriate conditions and enhance ulvan extraction yields. The microwave power controls the extraction temperature, which is the main parameter influencing water physicochemical properties, thereby increasing the solubility of lowly polar compounds in water [29]. Therefore, four extraction parameters including extraction time, microwave power, water-to-raw-material ratio, and pH were investigated separately. After 45 min of extraction, the extraction efficiency decreased slightly owing to the degradation of the polysaccharides [30]. The diffusion coefficient and solubility of the polysaccharides increases at high temperatures [31] which were achieved using the microwave power control. Moreover, high power causes the disruption of the vegetable cell, which allows the target compounds to dissolve more quickly. However, the structure of the target compound is degraded at high values of microwave power [19]. The degradation of polysaccharides by temperature was reported in different materials such as the roots of valerian [19], *Polygonatum sibiricum* [32], and *Eucommia ulmoides* Oliver leaves [33]. As the water-to-raw-materials ratio continued to increase, the yield tended to decrease. Many studies have reported that a high water-to-raw-material ratio is beneficial for the enhancement of the solvent diffusivity and polysaccharide desorption [26]. However, excess water can absorb the energy in the extraction process, resulting in a lower ulvan extraction yield [34]. The optimal water-to-raw-material

ratio to ensure homogeneous and effective heating was determined to be from 40 to 70 mL/g. A possible reason for this phenomenon is that the increase in pH enhances the dissociation of the acidic groups of the polysaccharide, thereby leading to an increase in the polysaccharide solubility in water [35], whereas a decrease of the solubility of the polysaccharide occurs in alkaline solutions [36]. On the basis of the result of single-factor experiments, time, power, water-to-raw-material ratio, and pH were further optimized by RSM using the BBD method to increase the extraction yield of ulvan.

ABTS and DPPH radical scavenging activity and reducing power were determined as reference indicators to evaluate the potential antioxidant activities of the polysaccharide [37]. The antioxidant activity of ulvan depend on many factors, such as the sulfate group, the contribution of the monosaccharides with a variable content of hydroxy and carboxyl groups, as well as the hydrogen donation capability [37]. Ulvan reducing power was weaker than that of ascorbic acid at all concentrations tested. However, it was relatively higher than at the absorption of no more than 0.15 reducing power of *Laminaria japonica* at 3 mg/mL [38]. The antioxidant activity of polysaccharides has been confirmed in other species including *Ulva linza* and *Ulva intestinalis* [39,40]. The antioxidant activity of polysaccharides depends on the degree of substitutions, monosaccharides, and glycosidic linkages [41]. These relations are not always described by linear regression. Wang, Hu, Nie, Yu, and Xie [41] found that DPPH radical scavenging ability of polysaccharides from *Pleurotus eryngii* was improved by an increasing degree of sulfation. Another study also revealed a unlinear regression between the polysaccharides from pumpkin (*Cucurbita moschata*) and the scavenging effects [42]. Lo et al. [43] investigated the relationship between the antioxidant properties of polysaccharides and monosaccharides or glycosyl linkages, using four conventional antioxidant models (conjugated diene, reducing power, DPPH scavenging activity, and ferrous ions chelation) by multiple linear regression analysis (MLRA).

Macrophages are usually employed to evaluate the response to oxidative stress for host defense. Hydrogen peroxide (H2O2) is commonly used for inducing oxidative stress-mediated cell injury in various kinds of cells [44,45]. We confirmed that 600 μM H2O2 was sufficient to induce oxidative injury in RAW 264.7 macrophages. The present study was designed to investigate whether treatment with ulvan decreased the cytotoxicity caused by H2O2 in RAW 264.7 cells and, thus, if ulvan could be proposed as an antioxidant agent. In order to evaluate the protective mechanism of ulvan against H2O2 stress in RAW 264.7 cells, we analyzed SOD and CAT enzymatic activities and the mRNA expression of *GST*, *CAT*, *MnSOD*, and *GPx*. In our study, SOD and CAT activities were found to be significantly increased after 24 h of ulvan treatment in RAW 264.7 cells. These results are in line with those of Yan et al. [46], who reported that polysaccharides from green tea could decrease H2O2-induced cell death and increase the levels of SOD and CAT in human ARPE-19 cells. Although many studies have shown a strong protective effect of polysaccharides from green and other seaweed [47] and have suggested a correlation between the antioxidant activity of polysaccharides and the expression of antioxidant gene, they did not provide any experimental evidence proving these observations. In this study, the expression of antioxidant genes was also found to increase in a dose-dependent manner. These results indicate that ulvan may upregulate antioxidant enzymes and enhance their enzymatic activity.

#### **5. Conclusions**

This study provides an efficient extraction process leading to a high yield of polysaccharides from *U. pertusa* according to an RSM model. An analysis of variance showed that the optimal extraction conditions leading to a yield of 41.91% were 43.63 min with 600 W of power, water-to-raw-material ratio of 55.45, and pH of 6.57. Ulvan extracted from *U. pertusa* showed a strong in vitro antioxidant capacity by increasing the activity of anti-oxidant enzymes. Ulvan provided a protective effect against cytotoxicity induced by H2O2 in macrophage cells. This effect was related to the upregulation of SOD and CAT.

Ulvan can be useful as a potential supplement food and reduce the problems in utilizing waste algae from "green bloom". Further studies are needed to understand the relationship between the chemical properties of ulvan and its antioxidant activity.

**Author Contributions:** Conceptualization, S.S.; Methodology, B.L.; Software, B.L. and K.S.G.; Formal analysis, K.S.G.; Investigation, S.S.; Data curation, K.S.G.; writing—original draft preparation, B.L.; Writing—review and editing, S.H.Y.; Supervision, S.H.Y. and S.S.; Project administration, S.S.; Funding acquisition, S.S.

**Funding:** This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education(NRF-2017R1D1A1B03035600).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Carotenoid Production by** *Dunaliella salina* **under Red Light**

#### **Yanan Xu and Patricia J. Harvey \***

Faculty of Engineering and Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK; y.xu@greenwich.ac.uk

**\*** Correspondence: p.j.harvey@greenwich.ac.uk; Tel.: +44-20-8331-9972

Received: 18 April 2019; Accepted: 5 May 2019; Published: 7 May 2019

**Abstract:** The halotolerant photoautotrophic marine microalga *Dunaliella salina* is one of the richest sources of natural carotenoids. Here we investigated the effects of high intensity blue, red and white light from light emitting diodes (LED) on the production of carotenoids by strains of *D. salina* under nutrient sufficiency and strict temperature control favouring growth. Growth in high intensity red light was associated with carotenoid accumulation and a high rate of oxygen uptake. On transfer to blue light, a massive drop in carotenoid content was recorded along with very high rates of photo-oxidation. In high intensity blue light, growth was maintained at the same rate as in red or white light, but without carotenoid accumulation; transfer to red light stimulated a small increase in carotenoid content. The data support chlorophyll absorption of red light photons to reduce plastoquinone in photosystem II, coupled to phytoene desaturation by plastoquinol:oxygen oxidoreductase, with oxygen as electron acceptor. Partitioning of electrons between photosynthesis and carotenoid biosynthesis would depend on both red photon flux intensity and phytoene synthase upregulation by the red light photoreceptor, phytochrome. Red light control of carotenoid biosynthesis and accumulation reduces the rate of formation of reactive oxygen species (ROS) as well as increases the pool size of anti-oxidant.

**Keywords:** *Dunaliella salina*; microalgae; red LED; blue LED; growth; carotenoids; plastoquinol:oxygen oxidoreductase; photosynthesis

#### **1. Introduction**

Carotenoids are orange, yellow or red pigments which are synthesized by all photosynthetic organisms for light-harvesting and for photo-protection, and for stabilising the pigment–protein light-harvesting complexes and photosynthetic reaction centres in the thylakoid membrane. They may also be accumulated by some non-photosynthetic archaea, bacteria, fungi and animals for pigmentation [1–3]. Carotenoids are also the precursors of a range apocarotenoids of biological and commercial importance, such as the phytohormone abscisic acid, the visual and signalling molecules retinal and retinoic acid, and the aromatic or volatile beta-ionone [4]. Increasingly sought after as natural colorants, there is accumulating evidence that carotenoids protect humans against ageing and diseases that are caused by harmful free radicals and may also reduce the risks of cataract, macular degeneration, neurodegeneration and some cancers [5,6]. They have also been implicated as the actives for treating diseases associated with retinoids [4].

In most plants and algae containing chlorophyll a (λmax ~680 nm) and b (λmax ~660 nm), photons with a wavelength of 660–680 nm yield the highest quantum efficiencies. However the solar spectrum at the surface of the Earth is at its maximum intensity in the blue and green regions of the visible spectrum (400–550 nm), which is where carotenoids have strong absorption. In photosynthetic organisms in the light, carotenoids drive photosynthesis by transferring absorbed excitation energy to chlorophylls, which have poor absorption in this range. Carotenoids are also able to protect photosynthetic organisms

from the harmful effects of excess exposure to light by permitting triplet–triplet energy transfer from chlorophyll to carotenoid and by quenching reactive oxygen species (ROS) [2].

*Dunaliella salina*, a halotolerant chlorophyte, is one of the richest sources of natural carotenoids and, similar to various members of the Chlorophyceae, accumulates a high content (up to 10% of the dry biomass) of carotenoids under conditions that are sub-optimal for growth i.e., high light intensity, sub-optimal temperatures, nutrient limitation and high salt concentrations. In *D. salina*, the major accumulated carotenoid is β-carotene, which is stored in globules of lipid and proline-rich, carotene globule protein in the inter-thylakoid spaces of the chloroplast (βC-plastoglobuli) [7–10]. The pathway for β-carotene synthesis and accumulation in *D. salina* has been partly mapped out [11,12], but the physiological role and signals triggering its accumulation are not well established. In other members of the Chlorophyceae, such as *Haematococcus pluvialis* and *Chlorella zofingiensis*, high levels of oxygen-rich, secondary ketocarotenoids, astaxanthin and canthaxanthin, also accumulate under high light stress or nutrient stress, often in lipid bodies located outside the chloroplast in the cytoplasm. Accumulation of these may also be accompanied by cell encystment. Lemoine and Schoefs [13] proposed that these carotenoids accumulate as a metabolic means of lowering ROS levels by lowering cellular oxygen concentration, as well as serving as a convenient way to store energy and carbon for further synthesis under less stressful conditions [13,14]. Chemically generated ROS will trigger astaxanthin accumulation [15] and recently Sharma et al. [16] showed that a small dose (up to 50 mJ cm2) of short wavelength ultraviolet C (UV-C ) light (100–280 nm) in cultures of either *D. salina* or *H. pluvialis* massively increased carotenoids accumulation as well as detached the flagellae to increase cell settling, 24 h after exposure: UV-light exposure is typically accompanied by ROS formation.

However in *D. salina* there may be additional mechanisms leading to carotene accumulation. Jahnke [17] for example found that whilst supplements to visible radiation of long wavelength ultraviolet A (UV-A) radiation (320–400 nm) specifically increased carotenoid levels and the ratio of carotenoids to chlorophylls in the closely related *D. bardawil,* neither blue light nor medium wavelength ultraviolet B (UV-B) light (290–320 nm) supplements were similarly effective. In blue light, Loeblich [8] found that green cells of *D. salina* with a low carotenoid to chlorophyll ratio had a relatively depressed photosynthetic activity, which was even more exaggerated in red cells with a high carotenoid to chlorophyll ratio. They proposed that blue light, which was absorbed by the accumulated β-carotene, was not available for photosynthetic oxygen evolution. Amotz et al. [18] on the other hand found a marked photo-inhibition for both red and green cells under high intensity red light, which is absorbed by chlorophylls, but red cells, when transferred to high intensity blue light were seemingly photoprotected. Since the accumulated carotenoids were physically distant from chlorophylls located in thylakoid membranes, Amotz et al. [18] proposed that in high intensity red light, the carotenoids were unable to provide photoprotection against chlorophyll-generated ROS or quench chlorophyll excited states, supporting the argument that carotene globules may function as a screen against high irradiation in blue light to protect photosynthetic reaction centres in *D. salina*. Fu et al. [19] examined the effects of different light intensities of red LED light on carotenoid production in *D. salina,* and showed that the major carotenoids changed in parallel to the chlorophyll b content and that both carotenoids and chlorophyll b decreased with increasing red light intensity and increased with nitrogen starvation.

Light-emitting diodes (LEDs) can be applied to adjust the biochemical composition of the biomass produced by microalgae via single wavelengths at different light intensities [20–23] and recently Han et al. [20] successfully used a low light intensity blue-red LED wavelength-shifting system to increase carotenoid productivity in *D. salina*. In this paper we explore the effects of red, blue and white LEDs on the growth and content of carotenoids and chlorophyll in four different *D. salina* strains under nutrient-sufficient conditions using a temperature-controlled photobioreactor (PBR) favouring growth. We show that in this system, cultivation using red LED was particularly effective in supporting a high rate of carotenoid productivity. We suggest that in strains of *Dunaliella salina*, accumulating carotenoids may be synthesised principally as a mechanism for maintaining cellular homeostasis under conditions

which might otherwise lead to over-reduction of electron transport chains, formation ROS and of a hyperoxidant state and ultimately lead to cell death.

#### **2. Materials and Methods**

#### *2.1. Strains and Cultivation*

Strains *D. salina rubeus* CCAP 19/41 and *D. salina salina* PLY DF17 were isolated from a salt pan in Eilat, Israel. *D. salina* CCAP 19/40 was isolated from a salt pond in Monzon, Spain. Strain UTEX 2538 (*D. salina bardawil*) was purchased from the Culture Collection of Algae and Protozoa (CCAP), Scotland, UK.

Algae were cultured in 500 mL Modified Johnsons Medium [24] containing 1.5 M NaCl and 10 mM NaHCO3 in Erlenmeyer flasks (Fisher Scientific, UK) in an ALGEM Environmental Modelling Labscale Photobioreactor (Algenuity, Bedfordshire, UK) at 25 °C. The cultures were shaken for 10 min at 100 rpm every hour before taking samples to monitor cell growth. Cells were grown under 12/12 light/dark (L/D) with 200 μmol photons m−<sup>2</sup> s−<sup>1</sup> supplied by white LED light to exponential growth phase and then dark-adapted for 36 h. After dark adaption, cultures were exposed continuously to blue, red or white LED light at light intensities of 200, 500, or 1000 μmol photons m−<sup>2</sup> s<sup>−</sup>1. Cultures acclimated to white, red or blue LED light for 24 h were used to monitor the changes in cellular carotenoids after further growth for 24 h in white, red or blue LED light. Cell density of the cultures was determined by counting the cell number of cultures using a haemocytometer after fixing with 2% formalin.

#### *2.2. Pigment Analysis*

The composition of pigments was analysed by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) (Agilent Technologies 1200 series, Agilent, Santa Clara, United States), using a YMC30 250 × 4.9 mm I.D S-5μ HPLC column (YMC, Europe GmbH) at 25 °C with an isocratic solvent system of 80% methanol: 20% methyl tert-butyl ether (MTBE) and flow rate of 1 mL min−<sup>1</sup> at a pressure of 78 bar. Carotenoid standards of β-carotene, α-carotene, lutein, zeaxanthin and phytoene were obtained from Sigma-Aldrich Inc. (Merck KGaA, Darmstadt, Germany) and dissolved in methanol or acetone to generate standard curves and DAD scans analysed at wavelengths of 280 nm (phytoene), 355 nm (phytofluene), 450 nm (β-carotene, α-carotene, lutein and zeaxanthin), and 663 nm (chlorophylls). Pigments were extracted from the biomass of 15 mL samples of culture. Samples were harvested by centrifugation at 3000× *g* for 10 min and pigments extracted after sonication for 20 s with 10 mL MTBE–MeOH (20:80). Samples were clarified at the centrifuge then filtered (0.45 μm filter) into amber HPLC vials before analysis.

Total carotenoids and total chlorophyll in the cultures were measured using a Jenway 6715 UV/Vis spectrophotometer (Cole-Parmer, Staffordshire, UK). Pigments were extracted from the harvested algal biomass of 1 mL culture using 1 mL of 80% (*v*/*v*) acetone, then clarified at 10,000× *g* for 10 min. The content of total carotenoids was calculated from absorbance values at 480 nm according to Strickland & Parsons [25]. Chlorophyll a, b and total chlorophyll content was measured at 664 nm and 647 nm according to Porra et al. [26].

#### *2.3. Oxygen Evolution and Dark Respiration*

Samples of cultures exposed to white, red or blue LED light were collected and the rates of O2 evolution and dark respiration were measured as described by Brindley at al. [27] at 25 °C using a Clark-type electrode (Chlorolab 2, Hansatech Instruments Ltd, Norfolk, UK). O2 evolution/uptake was induced by white, red, or blue LED light supplied by the manufacturer at a light intensity of 1000 μmol m−<sup>2</sup> s−1. After an initial period of 30 min of dark adaption of 1.5 mL of each culture, the rate of O2 evolution/uptake was measured for 20 min followed by dark respiration for 20 min. The average rate of photosynthesis was determined from the linear rate of oxygen evolution during 5–15 min of the light period. Dark respiration was determined by following the same procedure, except that the rate was calculated using the data from the last 15 min of the measurement. Air saturated water and nitrogen were used to calibrate the electrode.

#### **3. Results**

#### *3.1. Cell Growth and Carotenoids Production in Acclimated Cultures*

Figure 1a shows that in high intensity blue, white or red LED light, the growth rate recorded as cell density for *D. salina* CCAP 19/41 was the same. There was no significant difference in cell size (data not shown). However the contents of total carotenoids and total chlorophyll depended on the relative proportions of blue or red light supplied. The initial phase of growth in all high intensity light conditions, apart from blue, caused an initial sharp drop in chlorophyll content; the drop was greatest in high intensity red LED light but decreased depending on the relative proportions of red: blue light supplied. On the other hand, cultures maintained in red LED accumulated carotenoids at the highest rate; in blue, the content declined depending again on the relative proportions of red and blue light supplied (Figure 1b,c). The carotenoids/chlorophyll ratio is often used to evaluate carotenogenesis in *D. salina*. As shown in Figure 1d, the ratio increased rapidly with the increasing proportion of red light supplied but remained the same for blue light.

**Figure 1.** (**a**) Cell growth; (**b**) Cellular content of total carotenoids; (**c**) cellular content of total chlorophyll; (**d**) Carotenoids/Chlorophyll ratio in *D. salina* CCAP 19/41 grown under different ratios of red and blue light (Red/Blue 1/0, 2/1, 1/1, 1/2, 0/1) or white light with a total light intensity of 1000 μmol photons m−<sup>2</sup> s−<sup>1</sup> after dark-acclimation. Each culture condition was set up at least in triplicate.

Different *Dunaliella* strains responded differently to cultivation in high intensity blue or white LED (see Figure 2). All showed a decline in chlorophyll content in white LED compared to cultivation in high intensity blue but only strain CCAP 19/41 showed a significant increase in carotenoid content in white compared to blue light.

**Figure 2.** Cellular content of total carotenoids and of total chlorophyll for different *D. salina* strains cultivated each to the mid log phase under either white (**a**) or blue (**b**) light with a total light intensity of 1000 μmol photons m−<sup>2</sup> s−<sup>1</sup> after dark-acclimation. Each culture condition was set up at least in triplicate. Results were analysed by one way ANOVA comparing blue light to white light, \*\*: 0.001 < *p* ≤ 0.01; \*: 0.01 < *p* ≤ 0.05.

Carotenoids in *D. salina* CCAP 19/41 cultures exposed to different light conditions: white, red or blue LED light at 1000 μmol photons m−<sup>2</sup> s<sup>−</sup>1, a mixture of white and red (1:1) or a mixture of white and blue (1:1) with a total intensity of 1000 μmol photons m−<sup>2</sup> s−<sup>1</sup> for 48 h were extracted and the major carotenoids were identified and quantified by HPLC. Cultures exposed to continuous red LED light had the highest contents of all the identified carotenoids, while cultures maintained under blue LED light showed the lowest content. The difference was mainly due to variation in β-carotene content between treatments: there was no significant difference in relative content of all other carotenoids (Figure 3).

**Figure 3.** (**a**) Cellular content of total carotenoids; (**b**) Relative composition of major carotenoids characterised by HPLC in total carotenoids in *D. salina* CCAP19/41 cells exposed to continuous LED of different wavelength distribution (red, blue, white, white/red 1:1, and white/blue 1:1) for 48 h. The total light intensity for all conditions was the same at 1000 μmol photons m−<sup>2</sup> s<sup>−</sup>1. Results were analysed by one way ANOVA.

The total carotenoids and total chlorophyll contents after 48 h exposure to red or blue LED at different light intensities are shown in Figure 4. Carotenoids accumulated with increasing blue LED intensity between 200 μmol m−<sup>2</sup> s−<sup>1</sup> and 1000 μmol m−<sup>2</sup> s<sup>−</sup>1. In red LED light, cultures contained high amounts of carotenoids even under low light intensity and the content increased with increasing red LED intensity up to 500 μmol m−<sup>2</sup> s−1. With further increase in light intensity to 1000 μmol m−<sup>2</sup> s<sup>−</sup>1, carotenoids declined slightly (<10% of the value recorded at 500 μmol m−<sup>2</sup> s−1), but chlorophylls declined 34%. The carotenoids/chlorophyll ratio increased with the increase of light intensity both red and blue LED light, however, under red LED light a much higher carotenoids/chlorophyll ratio was recorded than under blue. Cellular content of β-carotene and phytoene showed a similar trend to that of total carotenoids, except that the highest β-carotene content under red light was achieved at 500 μmol m−<sup>2</sup> s<sup>−</sup>1, while the highest phytoene content under red light was achieved at 1000 μmol m−<sup>2</sup> s<sup>−</sup>1.

**Figure 4.** Cellular content of total carotenoids and total chlorophyll (**a**) and carotenoids/chlorophyll ratio (**b**) in *D. salina* CCAP19/41 grown under continuous red (R200; R500; R1000) or blue (B200; B500; B1000) LED light at three different light intensities of 200, 500 and 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 48 h. Each culture condition was set up at least in triplicate.

A phytoene desaturase inhibitor norflurazon known to cause accumulation of phytoene was used to treat *D. salina* cultures maintained under red, blue or white LED light. Figure 5 shows that under these conditions the cellular content of phytoene increased, as expected, but cultures maintained under red LED accumulated a significantly higher amount of phytoene compared to cultures maintained under white or blue light.

**Figure 5.** Cellular content of phytoene in cultures treated with no inhibitors (control) or with 5 μM norflurazon. Cultures were maintained under red, blue and white LED light at 200 μmol m−<sup>2</sup> s−<sup>1</sup> for 48 h.

#### *3.2. Acclimation and Carotenoids Production in Response to Wavelength Switching*

Dark-adapted cultures of *D. salina* CCAP19/41 were cultivated in red, white or blue LED light for 24 h (T0), and then cultivated for a further 24 h in red, blue, or a mixture of red and blue LED light (1:1), or the dark. Blue-acclimated cells produced slightly more carotenoids (14% greater content) when transferred to red LED but chlorophyll content declined from that at the start of the experiment to an

amount only 62% of that in continuous blue (Figure 6a). On the other hand the chlorophyll content increased when red-acclimated cells were exposed to blue light, but the total carotenoids content declined sharply, approximately in proportion to the amount of blue LED supplied (see Figure 6b). Red LED cultures maintained for a further 24 h in red LED accumulated 24.5 ± 1.3 pg carotenoid cell<sup>−</sup>1, but after 24 h in blue LED instead of red, the carotenoid content was 50% lower (11.4 <sup>±</sup> 0.4 pg carotenoid cell<sup>−</sup>1) and less than if they had been transferred to the dark (12.3 <sup>±</sup> 0.5 pg carotenoid cell<sup>−</sup>1).

**Figure 6.** Cellular content of total carotenoids and chlorophyll under continuous blue (**a**) or red (**b**) LED light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 24 h followed by 24 h growth under either red light, a mix of 1:1 red and blue light, blue light at the same light intensity of 1000 μmol m−<sup>2</sup> s−<sup>1</sup> or dark. Each culture condition was set up at least in triplicate. T0: time point after growth for 24h only, in either blue (a) or red (b) light at 1000 μmol m−<sup>2</sup> s<sup>−</sup>1.

Dark-adapted cultures were cultivated in red, blue or white light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 48 h before measuring the rates of oxygen evolution/uptake over a 20 min period with illumination supplied once more by white, red or blue LED lights at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> (Figure 7a). Dark respiration was also recorded (Figure 7b). The rate profiles of oxygen uptake/evolution are shown in Figure 8. Red LED supported net oxygen evolution (55 <sup>±</sup> 15 nmol O2 h−<sup>1</sup> <sup>μ</sup>g chlorophyll<sup>−</sup>1) but on transfer to blue light in the Clark-type electrode, photo-oxidation massively exceeded the rate of oxygen evolution and oxygen was consumed at an exponentially increasing rate (Figure 7a; 222 <sup>±</sup> 32 nmol O2 h−<sup>1</sup> μg chlorophyll−1). Significantly cultures grown in red LED also supported the highest rate of dark respiration (320 <sup>±</sup> 17 nmol O2 <sup>h</sup>−<sup>1</sup> <sup>μ</sup>g chlorophyll<sup>−</sup>1, ~2.6-fold greater than that for cultures maintained in either blue or white LED light), but this also declined when cultures were transferred to blue light in the Clark-type electrode. By contrast, cultures maintained in blue LED supported ~3-fold higher rate of net oxygen evolution (141 nmol O2 h−<sup>1</sup> μg chlorophyll−1) in the Clark-type electrode in blue light, compared to those in maintained in red LED. On transfer of blue light cultures to red light in the Clark-type electrode, the rate of oxygen evolution doubled to 280 nmol O2 h-1 μg chlorophyll−<sup>1</sup> (Figure 7b), and was maintained at a linear rate during the period of measurement. The rate of dark respiration also increased slightly from 123 <sup>±</sup> 2.7 nmol O2 h−<sup>1</sup> <sup>μ</sup>g chlorophyll−<sup>1</sup> to 175 <sup>±</sup> 5.0 nmol O2 h−<sup>1</sup> μg chlorophyll<sup>−</sup>1.

**Figure 7.** Oxygen evolution/uptake by *D. salina* cultures in different white, red or blue LED light sources (**a**) and in the dark (**b**). Cultures were grown under continuous red, blue or white light at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> for 48 h before measurement.

**Figure 8.** Rate profiles for oxygen uptake/evolution measured with different wavelengths of LED lights measured using a Clark-type electrode for 1.5 mL cultures of *D. salina* CCAP19/41 maintained at 25 °C. (**a**) Red light acclimated cultures measured under blue light. (**b**) Blue light acclimated cultures measured under red light; (**c**) White light acclimated cultures measured under red light; (**d**) White light acclimated cultures measured under blue light.

#### **4. Discussion**

LEDs with different wavelengths have been increasingly used to study the wavelength effects on the growth and productivity of photoautotrophic microalgae, and much effort is being invested to understand the most energy-efficient way to incorporate their use for large-scale algal cultivation [20–23,28,29]. In the present work we explored the effects of using red, white, blue and mixtures of red and blue LEDs at different intensities to evaluate the basis for carotenoid accumulation in strains of *Dunaliella salina*.

The emission spectrum of the red LED used in the present work (625–680 nm) emits photons with the exact range required by molecules of chlorophyll a and chlorophyll b to initiate photosynthesis [30]. In *D. salina,* action spectra of O2 evolution rates show maximum photosynthetic activity within the red absorption bands of the chlorophylls [8]. Photosystems I and II (PSI, PSII), which both contain chlorophyll a, work together in a series of more than 40 steps that proceed with the efficiency of nearly 100% to transfer electrons from water to nicotinamide adenine dinucleotide phosphate in its oxidised form (NADP+) [2]. Consequently the wavelength range of the red LED should be the most efficient emission required for photosynthesis in this alga and deliver the highest specific growth rate. However this also depends on the rate at which the absorbed light energy from any given applied photon flux density is converted to chemical energy: with increasing photon flux density, photosynthesis eventually achieves a light-saturated maximum rate that is limited by the rate of carbon fixation in the Calvin cycle. *Spirulina platensis* for example exhibited the highest specific growth rate using high intensity red LED [28]. *C. reinhardtii* however, showed unstable growth in high intensity orange-red and deep red LED, which ceased completely after a few days and was accompanied by cell agglomeration [22]: agglomeration is typical of oxidant stress and formation of a hyperoxidant state [31].

In the present work, we found that in high intensity red light in conditions of nutrient sufficiency, *D. salina* strains maintained a growth rate at least equal to that in white light or blue LED light, seemingly in contrast with the work of others [8,18,19]. However we also found that some but not all strains accumulated carotenoids rapidly, within 48 h of exposure. Carotenoids are known antioxidants that are synthesized by many microalgae as part of the battery of photoprotective mechanisms necessary to prevent photo-inhibition caused by photo-oxidation of photosynthetic reaction centres [2,32,33]. Photo-oxidation may occur in photon flux density levels that result in absorption of more light than is required to saturate photosynthesis. At the molecular level, when a photon is absorbed by a chlorophyll molecule, it enters a short-lived singlet excited state (1Chl\*): the longer the excitation of 1Chl\* lasts, which increases under saturating light conditions, the greater the chance that the molecule will enter the triplet excited state (3Chl\*) via intersystem crossing. 3Chl\* has a longer excitation lifetime and can transfer energy to the ground state of O2 to form singlet oxygen, 1O2, predominantly at the reaction centre of PSII and, to a lesser extent, in the light-harvesting complexes. Photo-oxidative damage occurs to the photosynthetic apparatus when species such as 1O2 react with fatty acids form lipid peroxides, setting up a chain of oxygen activation events that may eventually lead to a hyperoxidant state and cell death. Carotenoids may protect the photosystems by reacting with lipid peroxidation products to terminate these chain reactions; by scavenging 1O2 and dissipating the energy as heat; by reacting with 3Chl\* to prevent formation of 1O2 or by dissipation of excess excitation energy through the xanthophyll cycle. It is tempting therefore to suppose that the differences observed by different workers simply reflects differences in carotenoids content between different strains, but this does not explain what triggered the differences in carotenoids content.

In the *D. salina* strain CCAP 19/41, accumulation of carotenoids was accompanied by the highest rate of O2 consumption and a low rate of net O2 evolution, which might imply 1O2 formation and ROS accumulation. In the non-photosynthetic, astaxanthin-accumulating yeast, *Pha*ffi*a rhodozyma*, artificially generated 1O2 was proposed to degraded astaxanthin to relieve feedback inhibition of carotenoid biosynthesis and also to induce carotenoid synthesis by gene activation [34]. However these authors also found that carotenoid biosynthesis was linked to O2 consumption by a cyanide-insensitive alternative oxidase, serving to consume oxygen without chemiosmotic synthesis of adenosine triphosphate (ATP). In *C. reinhardtii,* a specific thylakoid-associated, terminal plastoquinol:oxygen oxidoreductase has been identified with homology to the mitochondrial alternative oxidase [35]. The smaller rate of oxygen uptake compared to mitochondrial respiration suggested a function in directly coupling oxygen uptake and the exergonic reaction of plastoquinol oxidation with plastoquinone reduction by a phytoene/phytoene desaturase couple, to permit endergonic carotene desaturation without ATP involvement [35]. In *D. bardawil*, a decrease in oxygen consumption rate coupled to phytoene

accumulation caused by norflurazon inhibition of phytoene desaturase also suggests a connection between direct desaturation of phytoene and chloroplastic oxygen dissipation [36].

In the present work, high intensity red light in conditions of nutrient sufficiency maintained growth at the same rate as in blue or white light, and red light also led to carotenoid accumulation albeit to different extents in different strains. These data support involvement of a plastoquinol:oxygen oxidoreductase as originally proposed for *C. reinhardtii* [35], but controlled by red photon flux intensity (see Scheme 1). In this scheme, chlorophyll absorption of red light photons is coupled to plastoquinone reduction in photosystem II, and oxygen reduction is coupled to phytoene desaturation by plastoquinol:oxygen oxidoreductase leading to carotenoid accumulation. Partitioning of electrons between photosynthesis and carotenoid biosynthesis would depend on both red photon flux intensity as well as upregulation of phytoene synthase. The observed increase in O2 consumption coupled to accumulation of carotenoids via the carotenoid biosynthetic pathway would reduce the tendency for 1O2 formation under high photon flux and maintain cytosolic redox potential.

**Scheme 1.** Partitioning electron flux between photosynthesis and carotenoid biosynthesis. Red photon flux intensity controls the partitioning of electrons either for carotenoid biosynthesis or for photosynthesis, via energy absorption by chlorophyll and the plastoquinone (PQ) pool. Red photon flux density also controls phytochrome regulation of phytoene synthase. CHL A: chlorophyll a; P680: chlorophyll a, primary electron donor of Photosystem II; PQox: plastoquinone, oxidised form; PQred: plastoquinone, reduced form; Cyt b6ox: cytochrome b6f complex, oxidised form; NADP<sup>+</sup>: NADP oxidised form; NADPH: NADP reduced form.

The coupling of reduction of the plastoquinone pool to carotenoid synthesis driven by chlorophyll absorption of red light may involve the red light photoreceptor phytochrome. Photosynthetic organisms are known to perceive red light signals via phytochrome. The synthesis of phytoene by phytoene synthase is under phytochrome regulation [37–39] and is upregulated by both red and far-red light [37]. Red light also lowers the concentration of the transcription factor PIF1, a repressor of carotenoid biosynthesis [40]. In those strains which do not accumulate carotenoids, alternative mechanisms may serve to consume energy e.g., via NAD(P)H reduction of dihydroxyacetone phosphate to form glycerol [41].

In support of this model, transfer from high intensity red light to blue with higher energy content caused a massive drop in the accumulated carotenoid content, very high rates of photo-oxidation and low respiratory rates. Carotenoids in both the accumulated pool and in the light harvesting antenna, but not chlorophyll, absorb photons in the range 400–550 nm, exactly overlapping the emission spectrum of the blue LED (440–500 nm). Failure of chlorophyll molecules to use the absorbed energy to reduce the plastoquinone pool would be expected to reduce the rate of electron flux through the plastoquinol:oxygen oxidoreductase as well as uncouple carotenoid synthesis and consequently increase cellular O2 concentration. This would lead in turn to increased ROS formation by reaction

of O2 with reduced electron transport chains, initiating further oxygen radical chain reactions, and carotenoid oxidation. Furthermore, in continuous high intensity blue LED, growth was maintained without carotenoid accumulation, but transfer to high intensity red LED light stimulated a small increase in carotenoid content, once again putting red light absorption by chlorophylls and transfer of absorbed energy to the plastoquinone pool at the centre of carotenoid biosynthesis. Transfer from blue to red light in the Clark-type electrode would cause absorption of more light than was required to saturate photosynthesis; if upregulation of the carotenoid biosynthetic pathway via phytochrome perception was required before coupling with O2 uptake via the plastoquinol:oxygen oxidoreductase, this would result in initial increase in the rate of photoinhibition and O2 uptake in the Clark-type electrode, and consequent loss in chlorophyll content, as was observed.

β-carotene accumulation in βC-plastoglobuli has parallels with that for astaxanthin accumulation, serving both as a carbon sink and end-product of an alternative oxygen-consuming biosynthetic pathway that on the one hand, controls over-reduction of photosynthetic (and respiratory) electron transport chains at the same time as removes oxygen from the plastid to limit formation of ROS. It is also able to quench any ROS that form. In blue light it may serve as a screen to absorb excess irradiation [7,18] but clearly offers photoprotection in red light as well. These functions are seen as distinct from its role as an accessory pigment in light-harvesting antennae systems.

Recently Davidi et al. [10] showed that the formation of cytoplasmic triacylglyceride (TAG) under N deprivation preceded that of βC-plastoglobuli, reaching a maximum after 48h of N deprivation and then decreasing. They suggested that βC-plastoglobuli are made in part from hydrolysis of chloroplast membrane lipids and in part by a continual transfer of TAG or of fatty acids derived from cytoplasmic lipid droplets. TAG synthesis represents a pathway for restricting over-reduction of electron transport chains [42] and its recruitment in formation of βC-plastoglobuli is entirely consistent with steps to dissipate excessive energy absorbed by chlorophyll in high intensity red light.

Overall, cultivation with red light may hold potential to enhance carotenoids production in carotenoid-accumulating strains of *D. salina*. Red light treatment has also been reported as an effective way to accelerate ripening of tomato fruit and increase the content of carotenoids [43]. Compared to other commonly used approaches to induce carotenogenesis, such as high light stress, high salt stress and addition of hydrogen peroxide or sodium hypochlorite, the use of red light provides a clean, convenient and economic alternative to promote carotenoids production from *D. salina* in a short time.

#### **5. Conclusions**

This study shows that under conditions of nutrient sufficiency, high intensity red light enhanced the production of carotenoids, mostly β-carotene, by upregulating the entire biosynthetic pathway of carotenoids, and that accumulation of carotenoids was accompanied by the highest rate of O2 consumption and a low rate of net O2 evolution. The data support a model of flexible co-operation between photosynthesis and carotenoid production via the plastoquinone pool. Chlorophyll absorption of red light photons and plastoquinone reduction in photosystem II is coupled to oxygen reduction and phytoene desaturation by plastoquinol:oxygen oxidoreductase. Partitioning of electrons between photosynthesis and carotenoid biosynthesis depends on photon flux intensity as well as upregulation of phytoene synthase by the red light photoreceptor phytochrome. Red light control of carotenoid biosynthesis and accumulation reduces the rate of formation of ROS as well as increases the pool size of anti-oxidant.

Red light may have industrial value as an energy-efficient light source for carotenoid production by *D. salina*.

**Author Contributions:** Conceptualization, Y.X., P.J.H.; methodology, Y.X.; formal analysis, Y.X., P.J.H.; data curation, Y.X.; writing—original draft preparation, Y.X., P.J.H; writing—review and editing, Y.X., P.J.H.; supervision, P.J.H.; project management, P.J.H.

**Funding:** This research received funding from EU KBBE.2013.3.2-02 programme (D-Factory: 368 613870) and from the Interreg 2 Seas programme 2014-2020 co-funded by the European Regional Development Fund under subsidy contract No ValgOrize 2S05017.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **The Long-Term Algae Extract (***Chlorella and Fucus sp***) and Aminosulphurate Supplementation Modulate SOD-1 Activity and Decrease Heavy Metals (Hg**++**, Sn) Levels in Patients with Long-Term Dental Titanium Implants and Amalgam Fillings Restorations**

#### **José Joaquín Merino 1,\*, José María Parmigiani-Izquierdo 1, Adolfo Toledano Gasca <sup>2</sup> and María Eugenia Cabaña-Muñoz <sup>1</sup>**


Received: 16 February 2019; Accepted: 9 April 2019; Published: 16 April 2019

**Abstract:** The toxicity of heavy metals such as Hg++ is a serious risk for human health. We evaluated whether 90 days of nutritional supplementation (d90, *n* = 16) with *Chlorella vulgaris* (CV) and *Fucus sp* extracts in conjunction with aminosulphurate (nutraceuticals) supplementation could detox heavy metal levels in patients with long-term titanium dental implants (average: three, average: 12 years in mouth) and/or amalgam fillings (average: four, average: 15 years) compared to baseline levels (d0: before any supplementation, *n* = 16) and untreated controls (without dental materials) of similar age (control, *n* = 21). In this study, we compared levels of several heavy metals/oligoelements in these patients after 90 days (*n* = 16) of nutritional supplementation with CV and aminozuphrates extract with their own baseline levels (d0, *n* = 16) and untreated controls (*n* = 21); 16 patients averaging 44 age years old with long-term dental amalgams and titanium implants for at least 10 years (average: 12 years) were recruited, as well as 21 non-supplemented controls (without dental materials) of similar age. The following heavy metals were quantified in hair samples as index of chronic heavy metal exposure before and after 90 days supplementation using inductively coupled plasma-mass spectrometry (ICP-MS) and expressed as μg/g of hair (Al, Hg++, Ba, Ag, Sb, As, Be, Bi, Cd, Pb, Pt, Tl, Th, U, Ni, Sn, and Ti). We also measured several oligoelements (Ca++, Mg++, Na+, K+, Cu++, Zn++, Mn++, Cr, V, Mo, B, I, P, Se, Sr, P, Co, Fe++, Ge, Rb, and Zr). The algae and nutraceutical supplementation during 90 consecutive days decreased Hg++, Ag, Sn, and Pb at 90 days as compared to baseline levels. The mercury levels at 90 days decreased as compared with the untreated controls. The supplementation contributed to reducing heavy metal levels. There were increased lithium (Li) and germanium (Ge) levels after supplementation in patients with long-term dental titanium implants and amalgams. They also (d90) increased manganesum (Mn++), phosphorum (P), and iron (Fe++) levels as compared with their own basal levels (d0) and the untreated controls. Finally, decreased SuperOxide Dismutase-1 (SOD-1) activity (saliva) was observed after 90 days of supplementation as compared with basal levels (before any supplementation, d0), suggesting antioxidant effects. Conversely, we detected increased SOD-1 activity after 90 days as compared with untreated controls. This SOD-1 regulation could induce antioxidant effects in these patients. The long-term treatment with algae extract and aminosulphurates for 90 consecutive days decreased certain heavy metal levels (Hg++, Ag, Sn, Pb, and U) as compared with basal levels. However, Hg++ and Sn reductions were observed after 90 days as compared with untreated controls (without dental materials). The dental amalgam restoration using activated nasal filters in conjunction with long-term nutritional supplementation enhanced heavy metals removal. Finally, the long-term supplementation

with these algae and aminoazuphrates was safe and non-toxic in patients. These supplements prevented certain deficits in oligoelements without affecting their Na+/K<sup>+</sup> ratios after long-term nutraceutical supplementation.

**Keywords:** algae; Chlorella; Fucus; detoxification; environmental pollution; antioxidants; heavy metals; selenium; SOD-1; neurotoxicology; aminoazuphrates; clinical medicine; nutrition; neuropathology

#### **1. Introduction**

Humans are exposed to pollutants, xenobiotics, and heavy metals that can be accumulated in the body when detox mechanisms are defective. Heavy metals can affect metallothionein and glutathione levels (its reduced form labeled herein as GSH) as well as SuperOxide Dismutase-1 (SOD-1) enzymatic activity [1–4]. Selenium (Se) is a crucial element for heavy metal removal by conjugation with GSH [2]. These xenobiotics can provoke hypertension and other clinical alterations in patients [5,6]. Mercury may cause neurodevelopmental disorders as autism spectrum disorders. Dental amalgams contain 50% of mercury (Hg), 41% of silver (Ag), Tin (Sn: 5–8%), Zn++, and Cu++ as minority oligoelements; titanium dental implants contain Ti-6Al-4V alloy [7]. Levels of heavy metals/oligoelements can be measured by inductively coupled plasma-mass spectrometry (ICP-MS) [8] in human samples such as urine, plasma, or hair [9,10]. The toxicity of heavy metals (e.g., mercury, cadmium) depends on the route, the concentration [11], and the exposure time and mixtures of heavy metals [12]. The function of oligoelements in odontology is still little studied (to review its functions, consult reference number [13].

The increasing concern of health problems associated with environmental pollutants is a serious one in humans because aluminum (Al) [14], lead (Pb), mercury (Hg++), cadmium (Cd), arsenic (As), nickel (Ni), copper (Cu++), iron (Fe++), chromium (Cr), and cobalt (Co) could provoke health problems in the case of heavy metal accumulation [14–16], which could be reduced by microalgae [17]. The occupational exposure to Cd and Hg++ are associated with antropometric activities, cremation, plastics, glass, and metal alloys. These heavy metals are also present in electrode material, nickel-cadmium batteries, water, and cigarette smoke [18,19].

Detoxification is the ability to remove drugs, mutagens, and other harmful agents from the body. The detoxification takes place in the intestinal tract, the liver, and the kidneys by microbiota able to chelate several heavy metals [20,21]. For instance, increased blood lead, mercury, and zinc levels were associated with Sarcopenia in the elderly population [6]. In addition, increased hair mercury levels (but not urinary levels) were correlated with the elevated title for the Lupus Eritematose marker in women (nuclear antigen: ANA) [10]. Several metabolic pathways in food-derived compounds are involved in detoxification [21]. Thus, clinical protocols able to prevent heavy metal accumulation are necessary in patients in conjunction with long-term nutritional supplementation. Antioxidants contribute to chelating reactive oxygen species (ROS) by removing heavy metals; thus, the screening of new antioxidants from plants is important from a clinical view point.

Some microalgae can remove heavy metals from wastewater. *Chlorella vulgaris* (CV) is a unicellular marine algae rich in chlorophyll (1–4%) that contains 55–67% protein, 9–18% dietary fiber, minerals, vitamins, and several oligoelements [17,22]. The CV algae are considered to be highly resistant to heavy metals and are widely used as a food supplement in Japan [17,23]. The *Chlorella sorokiniana* can promote antioxidant responses under zinc tolerance by increasing antioxidant enzymatic activities and increasing flavonoids, polyphenols, tocopherols, glutathione, and ascorbate (ASC) levels [24]. The CV extract can also excrete dioxin [25] and remove Cd levels by inducing metallotionein-like proteins. The biosorption of Pb2<sup>+</sup> and Cd2<sup>+</sup> have been described on a fixed bed column with immobilized Chlorella algae biomass [26]. *Chlorella protothecoides* algae promote heavy metal detoxification in chlordecone poisoned-treated rats by reducing the half-life of the toxin from 40 to 19 days. In addition, the *Fucus spiralis* is a marine brown alga (spiral wrack) that contains phlorotannins (antioxidant) [22]. The phytochelatins are short produced peptides from plants, algae, and fungi in response to heavy metal exposure, which detoxificate heavy metals by its high cysteine-content. *Fucus versiculosus* also may chelate Zn++ [22,27]. Phytochelatins are a natural source of novel angiotensin-I converting enzyme (ACE) inhibitors [28].

Our hypothesis is that the use of nasal filters (active carbon) in conjunction with long-term algae extract (*Chlorella and Fucus sp*) and aminosulphurates supplementation for 90 consecutive days contributes to the removal of heavy metals (Hg++, Ag, Sn, Pb) in patients with long-term dental titanium implants and amalgam fillings restorations.

#### **2. Aim**

We evaluated whether dietary chronic supplementation with CV and aminoazuphrates during 90 consecutive days could contribute to detoxificating heavy metals and/or prevent certain oligoelement deficits in patients with long-term dental titanium implants and amalgam fillings restorations. Therefore, the study was conducted to investigate whether long-term dietary CV contributed to the prevention of heavy metal accumulation after 90 days of supplementation (d90) in patients with long-term dental titanium implants and amalgam fillings restorations as compared with their own baseline levels (before any nutritional supplementation: d0) as well as untreated controls (without dental materials).

#### **3. Materials and Methods**

#### *3.1. Patients*

All selected patients were 49–68 years old (average: 58.5 years). The percentage of smokers was 7%, and their sociocultural states were medium-high levels (higher school education: 70%). Similar untreated (non-supplemented) control patients were included in this study. Their average age was similar to the rest of the patients. These untreated controls did not receive nutritional supplementation with the formulations. They did not have dental materials in their mouths (*n* = 21 controls).

The average number of dental amalgam fillings was 4, and there were 3 dental titanium implant alloys on average. All selected patients had a dental filling at least 10 years in their mouths (average: 15 years) and long-term titanium dental alloys for at least 10 years as well (average: 12 years). We selected 16 patients who had at least two or more long-term dental amalgams.

The number of enrolled patients suitable according to inclusion criteria was 21 untreated controls as well as 16 patients.

They received nutritional algae extracts (*Chlorella*/*Fucus sp*) and aminoazuphrates supplementation during 90 consecutive days (d90). Their heavy metal/oligoelement levels were compared at day 90 (d90, *n* = 16) with their basal levels (d0: before any supplementation, baseline, *n* = 16) as well as untreated controls (without dental materials in mouth, *n* = 21). These controls did not receive supplementation.

Their dental amalgams fillings were progressively replaced by composites (bisphenol A free) every 20 days following a clinical safe protocol by using active carbon filters (@InspiraHealth, Barcelona, Spain) and nutritional supplementation [29]. There are four quadrants in the mouth, and these amalgams were progressively replaced by each quadrant (each session within 20 days). Thus, some patients still had dental fillings during the 90 consecutive days of supplementation before their complete removal. The patients took supplements by oral intake (formulations) from the beginning (day 0, baseline levels) until the end of supplementation (d90: 90 consecutive days). We also compared levels of heavy metals/oligoelements after 90 days of supplementation with untreated controls (control, *n* = 21) and baseline levels (day zero, d0: before any supplementation). The nutritional supplementation took place during the time of dental amalgam restoration by composites. It is noteworthy that all dental materials were progressively replaced by composites at the time of collecting hair samples (90 days of supplementation). The fish consumption was 1–2 times per week in all recruited patients, including the controls. The basal heavy metals/oligoelements levels were taken at the initial visit to the dental clinic (CIROM: Centro de Implantología y Rehabilitación Oral Multidisciplinaría, https://clinicacirom.com/) before taking any nutritional supplementation, which were termed d0 (day 0) patients in the present study.

All supplemented patients received nutritional treatment by oral intake during 90 consecutive days with the following formulations: GREEN-FLOR (2-0-2; 4 capsules/day: *Chlorella and Fucus* algae extract), ERGYTAURINE (1-0-1; 2 capsule/day), and ERGYLIXIR formulations (Laboratorios Nutergia) during 90 consecutive days [from the initial day that patients visited the dental clinic (day 0) until the end of nutritional supplementation (d90: day 90)]. Controls without dental materials did not receive these treatments.

All ICP-MS heavy metal or oligoelements data were evaluated as percentiles (median, 25% and 75%) for non-parametric data (Kruskal-Wallis) and expressed as μg/g of hair in all cases (except vadanium). The ANOVA (analysis of variance) evaluated differences for vanadium levels, which were expressed as mean values ± standard error media (S.E.M). S.E.M was the variance divided by root square, and *n* was the size simple. The size sample was *n* = 16 patients at d90 and d0 and *n* = 21 untreated controls; the following heavy metals and oligoelements, respectively, were quantified by ICP-MS in the hair samples (Al, Hg++, Ba, Sn, Ag, Sb, As, Be, Bi, Cd, Pb, Pt, Tl, Th, U, Ni, Sn, Ti); (Ca++, Mg++, Na+, K+, Cu++, Zn++, Mn++, Cr, V, Mo, B, I, P, Se, Sr, P, Co, Fe, Ge, Rb, Zr).

These heavy metal/oligoelements were compared after 90 days of nutritional supplementation (d90) with their own basal levels (d0: before any supplementation) as well as untreated controls without dental materials (control, *n* = 21, non-supplemented). These untreated controls did not receive supplements and they did not carry dental materials. Saliva samples were taken at these study times for SOD-1 determination.

The limitation of the present study was the size sample (pilot study) and the absence of a placebo group. However, we included untreated controls (without dental materials and non-supplemented). This placebo group in patients with long-term dental implants and amalgam fillings could be not ethically justifiable since it is not possible to keep dental amalgam fillings, which release mercury in patients [9]. The implementation of this protocol in the Caucasian population (Spaniards) was the other limitation.

A dentist assessed 152 interviews or call phones to select potentially eligible patients. There were 35 patients who declined to participate and 30 patients who did not meet the inclusion criteria. We also excluded patients with fish consumption higher than 2 times by week. We did not enroll patients with periodontal disease or metabolic alterations. Thus, we selected 40 patients, and 37 participated in the end, which comprised the untreated controls (*n* = 21) and the baseline patients [before taking supplements (d0, *n* = 16) and after 90 days of nutritional supplementation (d90, *n* = 16), see study groups in Figure 1]. All statistical analyses were evaluated in 37 patients and 53 hair samples for each heavy/metal oligoelement determination here.

#### *3.2. Inclusion Criteria*

This study followed the Declaration of Helsinki (1974, updated 2000), and it was approved by the Institutional Review Board from CIROM (Murcia, #2016/014). All subjects were properly instructed by signing the appropriate consent paperwork. In addition, all efforts were made to protect patient privacy and anonymity. The CIROM Center was approved and certified by AENOR Spain (Spain; CIROM CERTIFICATE for dentist services, CD-2014-001 number; ER-0569/2014 following UNE-EN ISO 9001: 2008 as well as UNE 179001-2001 Directive from Spain). We selected 16 patients who had at least two or more long-term dental amalgams. They had long-term dental amalgam fillings for at least 10 years in their mouths (average: 15 years) and long-term titanium dental implants for at least 10 years (average: 12 years). The fish consumption was 1-2 times per week in all recruited patients. The average number of dental amalgam fillings was 4, and the average number of dental titanium implant alloys was 3.

Controls were selected after clinical examination. They did not have dental materials in their mouths, nor did they show signs of periodontal diseases. We excluded patients who had fish consumption higher than 2 times by week.

**Figure 1.** Study groups.

#### *3.3. Exclusion Criteria*

Physically handicapped patients who had metabolic diseases [diabetes, metabolic syndrome, liver/kidney disease, systemic inflammation, lupus/autoimmune disease, thyroid disease, adrenal disease, or neuropsychiatry disorders Diagnostic and Statistical manual of Mental disorders (4th Edition, DSM IV)] [30], were excluded in the present study. Patients taking regular medication or stimulants, anticonvulsants, atypical antipsychotic drugs, or those who had history of liver/kidney disease or DMSA (dimercaptosuccinic acid) prescribed (or chelators) patients were also excluded. Particularly, hypertensive patients and those who had periodontal disease tattoos or were taking nutritional supplements were excluded in the present study. Finally, patients who had orthodontic devices were not included here. The correct diagnostic of periodontal disease was based on several parameters, such as visual exploration (palpation), presence of dental calculus, radiographic evaluation, dental mobility, and oclusal exploration (pathological eroding facets). Periodontal disease was also a cause of exclusion, which was identified by following several criteria by an expert dentist, such as a deep dental probe higher than 3 mm, loss of bone (radiography), possible bleeding, and dental mobility [31].

#### *3.4. Composition of Nutritional Supplementation (Algae and Other Bioactive Phytomolecules)*

All patients took the following nutritional supplementation during 90 consecutive days (oral intake): GREEN-FLOR (2-0-2), ERGYTAURINE (1-0-1), and ERGYLIXIR formulation (Nutergia, 1 bottle/month) following the patterns of their antioxidants properties (see Table 1). The controls of the intakes were registered by dentists every 20 days, and we administered the following dosages according our previous clinical experience.



#### *3.5. Inductible Coupled Mass Spectromery Analysis (ICP-MS)*

In the weight of the dental amalgam fillings, mercury (Hg) was 50%, and silver (Ag) was 41%, Sn was approximately 5–8%, and Cu++ and Zn++ levels were in the minority. Hair samples close to the scalp were taken from all subjects (0.25 g from the occipital area) to measure a plethora of heavy metals/oligoelements by ICP-MS (Doctor's Data, USA). Doctor's Data is a pioneer laboratory specializing in the toxicology of heavy metals with over 35 years of experience, and they provide analytical tests for healthcare practitioners. ICP-MS values for heavy metals were expressed in μg/g of hair.

#### *3.6. Super Oxide Dismutase-1 (SOD-1 Activity)*

The saliva SOD-1 activity was measured following a modified protocol by [9] Cabaña-Muñoz et al., 2015. Briefly, the buffer assay contained 0.1 mM EDTA (Ethylenediaminetetraacetic acid), 50 mM sodium carbonate, and 96 mM of nitro blue tetrazolium (NBT). Then, 470 μL of the above mixture was added to 100 μL of saliva, and the auto-oxidation of hydroxylamine was observed by adding 0.05 mL of

hydroxylamine hydrochloride (pH 6.0). Finally, SOD-1 activity was measured by the change in optical density at 560 nm for 2 min at 30/60 s intervals and normalized as optical density (D.O) by protein [9].

#### *3.7. Statistical Analysis*

All data were analyzed by SPSS software (v17.0) (U.C.M: Universidad Complutense, Madrid, Spain) and Sigma Plot (v11.0, U.C.M, Madrid, Spain). Mean 25%, 75%, and median values (μg/g of hair) were estimated for heavy metals/oligoelements in the hair samples. Non-parametric tests were applied in cases without homogeneity of variance (Mann Whitney/Kruskal Wallis). The Bonferroni tests were applied for multiple comparisons when there was homogeneity of variance (e.g., vanadium, V). All results were expressed as percentiles 25%, 75%, and median (μg/g of hair) according to Kruskal Wallis values (H value) and Mann Whitney (MW) and Dunn's post hoc test in the case of non-parametric data between (d0: *n* = 16, d90: *n* = 16) and controls (control: *n* = 21). The Levene test identified whether or not there was homogeneity of variance depending on its significance. Correlations between variables were performed by Spearman's rank correlation. Differences were considered statistically significant if *p* < 0.05 and highly significant when *p* < 0.01.

#### **4. Results**

*4.1. SOD-1 Activation Reflects Antioxidants Responses in Patients after Long-Term Supplementation with Algae Extract and Aminoazuphrates Compared with Untreated Controls*

There was a statistically significant effect for SOD-1 activity in the Kruskal Wallis analysis (H = 45.1, *p* ≤ 0.001) for SOD-1 activity (saliva). The parametric Dunn's non-analysis revealed decreased SOD-1 activity after 90 days of supplementation (d90) compared to their basal levels (d0: before any supplementation, *p* < 0.05); Conversely, increased activity SOD-1 activity was detected before any supplementation as compared with untreated controls (without dental materials, *p* < 0.05, Table 2).


**Table 2.** Regulation of SuperOxide Dismutase-1 (SOD-1) by long-term algae and aminosulphurate supplementation and SOD-1 activity.

\* *p* < 0.05 vs. control, # *p* < 0.05 d90 vs. d0; control: controls without dental materials and non-supplemented (*n* = 21); d0: patients with long-term titanium implants and dental amalgam fillings restorations before any nutritional supplementation (d0, *n* = 16); d90: patients with long-term titanium implants and dental amalgam fillings restorations after 90 days of supplementation (d90, *n* = 16).

*4.2. Reduced Mercury (Hg*++*) and Silver (Ag) Levels after 90 Days of Nutritional Supplementation (d90) as Compared with Their Baseline Levels (d0) as ell as Untreated Controls (Without Dental Materials and non Supplemented, cont)*

We compared levels of heavy metals (Hg++, Ag, Sn) as well as titanium alloys (Ti-6Al-4V) in patients with long-term dental titanium implants and amalgams fillings after 90 days of nutritional supplementation as compared with their own basal levels (before any supplementation, d0) as well with non-supplemented controls (without dental materials, controls). The Kruskal Wallis and Mann Whitney post hoc analyses revealed mercury (Hg++) and tin (Sn) reductions after 90 days of supplementation as compared with their own basal levels (d0) without reaching a significant effect in Zn++, Co, Ni, or Cu++ levels (Table 3).

Finally, Ag levels decreased after 90 days as compared to their basal levels (d0, before any supplementation) without reaching a significant effect as compared to untreated the controls.

The aluminium (Al) levels decreased after 90 days of supplementation (d90) as compared with untreated controls (*p* < 0.05); increased d90 vanadium (V) levels were observed as compared with basal levels (d0, *p* < 0.05). There were no effects in Ti or Co levels by treatment (*p* > 0.05, non-supplemented, Table 3).


**Table 3.** Heavy metals/oligoelements of dental materials.


**Table 3.** *Cont.*

Percentiles analysis for heavy metals/oligoelement levels in Kruskal-Wallis (H) between patients with long-term titanium implant and dental fillings after 90 days of supplementation (d90, *n* = 16) as compared with their own basal levels (d0: before any supplementation, *n* = 16) and untreated (non-supplemented) controls without dental materials (control, *n* = 21). All heavy metals and oligoelements were expressed as μg/g of hair. H is the Krukal-Wallis analysis and F is ANOVA data. MW = Mann Whitney, S.E.M = standard error media; Control: controls without dental materials and non-supplemented (*n* = 21); d0: patients with long-term titanium implants and dental amalgam fillings restorations before any supplementation (d0, *n* = 16); d90: patients with long-term titanium implants and amalgams after 90 days of supplementation (*n* = 16); n.s: not significant effect, *p* > 0.05. \* *p* < 0.05 vs. Control; # *p* < 0.05 d90 vs. d0.

*4.3. Levels of Oligoelements Involved in Metabolic Functions (Se, Mn*++*, Li, Mg*++*, Ge, S, P, I, Ca2*<sup>+</sup>*, Sr, Na*+*, K*+*)*

Patients with long-term titanium implants and amalgam fillings increased germanium (Ge), manganesum (Mn++), chromium (Cr), vanadium (V), phosphorum (P), and lithium levels (Li) after 90 days of supplementation (day 90) as compared with untreated controls (control, *n* = 21). In addition, after 90 days (d90, *n* = 16), their selenium (Se) levels decreased in comparison to their basal levels (d0, *n* = 16, *p* < 0.05, μg/g of hair); however, they were higher than the control values (Table 4, *p* < 0.05). These supplements could promote antihypertensive effects by rising certain oligoelements. Finally, there were no effects in other oligoelements (Ca2+, Mg2<sup>+</sup>, I, Sr, B, Rb) or for Be, Bi, Tl, To (data not shown, *p* > 0.05, n.s).

**Table 4.** Percentiles for oligoelements involved in metabolic functions in patients with long-term titanium implant and dental fillings after 90 days of supplementation (d90, *n* = 16) and their basal levels (d0: before any supplementation, *n* = 16) and non-supplemented controls (control: without dental materials and non-supplemented, *n* = 21). All heavy oligoelements were expressed as μg g/g of hair.



**Table 4.** *Cont.*


\* *p* < 0.05 vs. control; # *p* < 0.05 d90 vs. d0; controls (without dental materials and non-supplement; control, *n* = 21); d0: patients with long-term titanium implants and dental amalgam fillings restorations before any supplementation (d0, *n* = 16); d90: patients with long-term titanium implants and dental amalgams after 90 days of supplementation (d90, *n* = 16); (n.s: not significant effect, *p* > 0.05; \* *p* < 0.05 vs. Control; # *p* < 0.05 d90 vs. d0).

#### *4.4. Metals of Environmental Exposure*

The algae extract and aminoazuphrates supplements decreased lead (Pb) levels after 90 days of supplementation (day 90) as compared with baseline levels (d0: before any supplementation); the aluminium (Al) levels were reduced after 90 days in comparison to untreated controls (see Table 5).

There was a lack of effect in several heavy metals (As, Ti, Pt, Sb, Tl, To, Cd, Be, Bi, Zr, *p* > 0.05, n.s) and oligoelements by treatment (Zn++, Cu++, Ca++, Sr, B, I, K+, Mg++, Rb) after 90 days of supplementation as compared with their baseline (d0) and control levels.

**Table 5.** Decreased lead (Pb) levels after 90 days of supplementation as compared with baseline levels.


These tables show percentile values (median, 25%, and 75%) in Kruskal Wallis analysis for several heavy metals/oligoelements (Hg++, Ag, Sn, Zn++, Cu++, Al, Cr, V, Co, and Ni), metabolic oligoelements (Se, Mo, Mn++, Li, Ge, S, P, I, Ca++, Sr, B, Na+, K+, Mg++, Rb, B, and Fe++), and metals of environmental exposure in Table 6 (Ba, As, Pt, Sb, Tl, To, Cd, Be, Bi, Zr, Pb, Cd, As, and U). The ANOVA data for V are shown by mean values ± S.E.M [the root square divided by n; n was the size sample; *n* = 16 (d90), *n* = 16 (d0), *n* = 21 controls]. Post hoc differences were evaluated by the Mann Whitney or Dunn's method; Control: controls without dental materials and non-supplemented (*n* = 21); d0: patients with long-term titanium implants and dental amalgam fillings restorations (d0, *n* = 16); d90: patients with long-term titanium implants and dental amalgams after 90 days of supplementation (*n* = 16); n.s: not significant effect, *p* > 0.05). \* *p* < 0.05 vs. Control; # *p* < 0.05 d90 vs. d0).

#### *4.5. E*ff*ects on Selenium (Se) Ratios and Heavy Metals after 90 Days of Nutritional Supplementation*

For example, we found decreased Se/Hg++, and increased Se/Al, and Mo/Hg++ ratios after 90 days of supplementation (d90) compared to their respective basal levels (before any treatment, d0, *p* < 0.05); However, these Se/Hg++, Se/Ag, and Mo/Hg++, and Na+/K<sup>+</sup> ratios decreased before any treatment (d0) as compared with non-supplemented patients (controls, *p* < 0.05, Table 6).


**Table 6.** Effects on Se/Hg++, Se/Ag, Se/Al, Se/Pb, Mo/Hg++, Na+/K+ before/after nutritional supplementation and untreated controls.

Control: controls without dental materials and non-supplemented (*n* = 21); d0: patients with long-term titanium implants and dental amalgam fillings restorations (d0, *n* = 16); d90: patients with long-term titanium implants and dental amalgams after 90 days of supplementation (*n* = 16); n.s: not significant effect, *p* > 0.05; \* *p* < 0.05 vs. Control; # *p* < 0.05 d90 vs. d0.

#### *4.6. Correlations between Selenium (Se) and Heavy Metals Ratios after 90 Days of Nutritional Supplementation*

The *r* Spearman correlations between selenium and heavy metal ratios are shown in Table S1. For example, there was a strong correlation between the Se/Hg++ (d90) ratio and Se levels after 90 days of supplementation [Se (d90), *r* = <sup>−</sup>0.76, *p* = 0.004] as well as with Mo/Hg++ (d90) ratio after 90 days (d90, *r* = 0.6, *p* = 0.02). Two outlier values were excluded for statistical analysis herein (Table S1, see Supplementary Materials). Table S2 showed other correlations between heavy metals and oligoelements (see Supplementary Materials).

#### **5. Discussion**

This section discusses the effects of dental amalgam restoration in mercury reduction in patients with long-term titanium implants and dental amalgam restorations using carbon active (nasal filters) and long-term algae and aminoazuphrates supplementation.

The exposure derived from amalgam fillings exceeds that from food, air, or beverages. Chronic nutritional supplementation contributes to preventing mercury release peaks caused by dental amalgam restoration (replacement by biocompatible materials like Bisphenol A free composites). A study of 12 patients demonstrated that the long-term presence of dental amalgam (at least five years) did not result in any remarkable changes in mercury or tin levels in the pulp tissue after comparing 12 restored amalgams and 12 non-restored patients. However, elevated blood mercury levels were observed even five years after the placement of the restoration [32]. These data suggest that mercury release is important even after complete dental amalgam restoration with composites, because five years after its restoration, mercury is still present in the blood [32]. Bergerow et al. reported that within 12 months after removing dental amalgam fillings (restoration by composites), patients showed substantially lower urinary mercury levels [33]. In the present study, the period of supplementation was shorter (three months: 90 days), which minimized mercury release by using carbon active (nasal filters) during dental restorations [29]. The synergic algae and aminoazuphrates treatment contributed to activating the detoxification because the mercury reached peaks shortly at 24 h after replacement with composites until 3–7 days later [34].

#### *5.1. Detoxification of Heavy Metals in Patients with Long-Term Amalgam Fillings and Titanium Dental Implants*

We determined that chronic nutritional *Chlorella* and *Fucus* algae extract supplementation in conjunction with aminosulphurates lowered certain heavy metal levels in patients with long-term titanium implants and dental amalgams restoration using activated carbon active nasal filter as well as the nutraceuticals. Preclinical findings suggest a role of *Chlorella vulgaris* as a heavy chelator in preventing toxicity of certain xenobiotics and accelerating dioxin excretion in rats [25,35]. The mercury and tin reduction after 90 days in patients agreed with enhanced heavy metal removal by *Chlorella sp* [36–38]. However, the exact mechanism by which chronic algae consumption removes heavy metals has not been tested yet in humans. Our aim is to develop a clinical and practical protocol to chelate heavy metals with a mixture of bioactive nutraceuticals such as algae extracts and aminosulphurates that could act independently of signaling pathways involved in detoxification.

Supplementation with *Chlorella sp* promoted detoxification of heterocyclic amines (carcinogenic chemical) in six young Korean adults [39]. This randomized, double blind, placebo-controlled crossover study was performed in six female supplemented-patients; the nutritional period of three months in our study was longer than in the Korean study. Our findings also reflected enhanced removal of certain heavy metals, including lead (a metal of environmental exposure). Our patients' Hg++, Sn, and Pb accumulations were strongly reduced after 90 days of consecutive nutritional supplementation as compared with basal levels (before any supplementation). Interestingly, mercury and Sn levels reductions were observed after 90 days as compared with untreated controls (without dental materials and non-supplemented).

#### *5.2. SOD-1 Activity in Patients with Long-Term Dental Titanium Implants and Amalgams Restorations*

Although it was not possible to elucidate the exact nutraceutical involved in SOD-1 activation here, we must consider that SOD-1 activation decreased after 90 days as compared with their basal levels (d0, before any supplementation). Conversely, higher SOD-1 activity was observed after long-term supplementation (day 90) compared with untreated controls; this suggests algae and aminoazuphates treatment may activate SOD-1. In addition, increased Mn++ levels could suggest enhanced antioxidant responses after 90 days of supplementation. In fact, Ala16Val MnSOD-2 polymorphism has been described in cells exposed to methylmercury [40]. We have previously observed higher SOD-1 activation

in women with long-term dental amalgams only (without titanium dental alloys) as compared with controls (without dental materials) [9]. Our clinical findings were in consonance with the detoxification induced by Ag nanoparticles through inducing SOD, peroxidase, catalase, and glutamine synthetase enzymatic activities [41,42]. The silver (Ag) reduction after 90 days of supplementation as compared with the patients' baseline levels (before any supplementation) agreed with the enhanced removal of heavy metals. However, silver levels after 90 days did not differ with controls.

Heavy metals detox requires (i) a healthy gut microbiome state [20], (ii) the induced-activation of endogenous hepatic I-II-enzyme, which can be activated by phytonaturals in these formulations [43], and (iii) the chelation and excretion of these heavy metals [44]. Steps (i) and (ii) are activated by natural products from these formulations. The ERGYLIXIR formulation contains synergic depurative bioactive compounds from extracts such as *Cinara scolymus* (artichoke) [45], *Raphanus niger* [46], *Taraxacum o*ffi*cinale* [47], *Arctium lappa* (dandelion root) [48], *Vaccinium macrocarpo* [49], *Solidago virgaurea* (quercitin, afzelin) [50], *Rosmarinus o*ffi*cinallis* [51], *Scolymus hispanicus* [52], and *Sambucus nigra* (elderberry with antocianines) [53]. In addition, sulfur-rich extracts such as garlic acid (*Allium sativa* in the ERGYTAURINE formulation) may enhance heavy metal removal by inducing antioxidant activities [54–56]. Apple pectin [56] and acerole (very rich in vitamin C) also contribute to heavy metals removal [57]. In addition, *Sambucus nigra* (elderberry) contains antocyanines that supply 87% of the daily vitamin C levels necessary for humans [57]. Vitamins B6, B-9, and B-12, as well as Se, Zn++, and Mg++ (ERGYTAURINE formulation) [58] are necessary for certain enzymatic activities.

#### *5.3. Possible Role of Selenium (Se) in Detox after Long-Term Chlorella CV Supplementation in Patients*

Because Se levels decreased after 90 days of supplementation, we cannot exclude the possibility that selenomercurials reflect the Se-heavy metal complex formation in order to prevent mercury toxicity (or other metals) in patients with long-term dental amalgams and titanium alloys. As the Na+/K<sup>+</sup> ratio did not differ after 90 days as compared with their basal levels (before any supplementation), we can confirm that chronic algae and aminoazuphrates supplementation are safe and non-toxic for humans. The increased Se/Hg++ ratios suggest enhanced detoxification after 90 days compared to their basal levels (before any supplementation) as well as untreated controls. Surprisingly, a toxic effect has been demonstrated in autistic children who had elevated hair selenium levels [59]. These lower Se levels observed in conjunction with the lack of effect on the Na+/K<sup>+</sup> and Se/Pb ratios could prevent mercury accumulation at 90 consecutive days of supplementation. In fact, antagonistic interaction between selenomethionine enantiomers and methylmercury toxicity was described with *Chlorella sorokiniana* [60]. Mercury loss with *Chlorella vulgaris* is largely influenced by amino acids, cysteine being the most effective in promoting the detoxification of mercury (Hg2<sup>+</sup>) − in *Chlorella sp* exposed to this metal [61]. The amino acid taurine (ERGYTAURINE formulation) is derived from cysteine [62] and also contributes to heavy metal detoxification. In fact, increased oxidative stress and low systemic taurine levels were demonstrated in patients with long-term dental amalgam fillings and/or titanium alloys [63]. This indirect evidence agreed with a study in which selenocystine (SeCys2) reduced MeHg cytotoxicity in Hepatic HepG2 cells by inducing MeHg-glutathione (GSH) and also formed MeHg-cysteine (Cys) complex in vitro [64]. These indirect findings suggest that selenium contributed to detoxification in the present clinical study. Uchikawa et al. (2011) described the enhanced removal of tissue methylmercury in (BP) *Parachlorella beijerinckii*-fed mice; this continuous BP intake (10%) accelerated MeHg excretion and subsequently decreased tissue mercury accumulation by inducing the GSH metabolism [65].

Other metals such as Pb, Cd, and U that are associated with occupational exposure were significantly decreased after three consecutive months of supplementation compared with their basal levels (before any supplementation) without affecting the untreated controls (without dental materials). The biosorption of Pb2<sup>+</sup> and Cd2<sup>+</sup> was detected using a fixed bed column analysis with immobilized Chlorella algae biomass [66]. Pb levels decreased after 90 days of supplementation, agreeing with the 56% Pb reduction at four days of algae *Chlorella sp* supplementation, 69% at eight days, and

77% at 12 days of treatment [26]. Although U levels were within the normal detection range in our patients, their decrease after 90 days of supplementation was crucial. As selenium-enriched spirulina formulation reduces the development radiation that is *pneumonitis*-induced [67], the lower U levels after chronic algae supplementation are important from a clinical view point. In addition, a glutathione-dependent detoxification pathway has been described in *Chlorella algae* exposed to U [68–70].

#### *5.4. The Nutritional Supplementation after 90 Days Prevented Certain Oligoelements Deficit in Patients with Long-Term Titanium Implants and Dental Amalgam Restorations*

These polyphenols from Azorean brown algae (*Fucus spiralis or Fucus vesiculosus in* GREEN-FLOR formulation) may enhance heavy metal removal in patients with long-term dental fillings and titanium alloys. In fact, the marine algae *Ulva lactuca* and *Fucus vesiculosus* can sequester Cd and Cu++ [70], which explained the induced-detoxification here. The phlorotannins have potential impact on public health, particularly in hypertensive patients [71,72]. The in situ determination of trace elements in fucoids by field-portable-X-ray fluorescence (FP-XRF) provides a rapid monitoring environmental contamination [73]. Increased mercury levels can provoke hypertension, and Se may exhibit a protective effect against cardiovascular disease [6]. Long-term nutritional supplementation could increase germanium (Ge) levels in patients with long-term dental amalgam fillings and titanium implants, seemingly by reflecting antihypertensive effects. However, a direct causal relationship between antihypertensive effects and Cr and Ge elevations was not conclusive in the present study. The Sn-Se correlation observed in conjunction with Ge, Li, Cr, P and I elevations after 90 days of supplementation could be explained by the high oligoelement content (10–15%) in the supplement, resulting from its marine origin [74]. The detoxification of Hg++ and Cd levels here agreed with the enhanced Hg++, Cd++, and Pb removal by *Fucus* from contaminated salt waters exposed to heavy metals for seven days [74]. The *Fucus sp* algae is also traditionally used to prevent obesity or gastrointestinal diseases. As *Fucus vesiculosus* extracts reduced the blood glucose peak in mice fed with a normal diet [75], the possibility that chromium Cr and Ge elevation could contribute to these antihypertensive effects should not be excluded here. These oligoelements also increased after 90 days of supplementation as compared with untreated controls. The increased Li levels suggest a better regulation of gut microbiota after treatment with these formulations, since the host serotonine biosynthesis is regulated by intestinal microbiota [76]. In fact, the strong r Spearman correlation together with the Se/Li ratio and Li correlation suggest a better state of gut microbiota in treated patients at 90 days of supplementation as compared with their basal and control levels. Finally, the augmented phosphorous (P) levels described here may have been a consequence of chronic spirulina supplementation (GREEN-FLOR). Since undernourished children receiving *Spirulina platensis* plus Misola extract treatment have a better hematocrite that those taking Misola alone [77], the chronic algae-supplementation could prevent iron deficit. These synergic supplementations contribute to heavy metal removal in these patients. Moreover, increased systemic malondialdehyde levels and lower Mo/Co and Mo/Fe2<sup>+</sup> ratios have been described in patients with long-term dental titanium implants and dental amalgams [74]. Further studies should evaluate detoxification pathways by which long-term supplementation *Chlorella or Fucus vesiculosus* treatment contribute to the removal of heavy metals in patients with long-term dental amalgam fillings and titanium implants. The absence of placebo, the non-RCT (randomised controlled trials), the size sample (pilot study), as well as the Caucasian population (Spaniards) are limitations in this study.

#### **6. Conclusions**

The aminosulphurates and *Chlorella and Fucus sp* algae supplementation enhanced detoxification of heavy metals by reducing Hg++, Ag, Sn, and Pb levels in patients with long-term dental amalgam filling and titanium implants. The chronic nutritional supplementation with algae extract reduced Hg++ and Sn levels in patients with long-term titanium implants and dental amalgam restorations as compared with untreated controls (without dental materials). In addition, increased Mn++, Li, Ge, Cr and lower U levels, and decreased Se levels were observed after 90 days of supplementation as compared to their basal levels (before any supplementation). These findings suggest that these nutraceuticals promote beneficial effects in patients. The safety of long-term algae and aminoazuphrates supplementation were confirmed by the lack of effect in Ka+/K<sup>+</sup> and Se/Pb ratios after 90 days compared to their basal levels (before any supplementation) and untreated controls. The SOD-1 activity could explain antioxidant and enhanced detoxification of certain heavy metals by nutritional supplementation in the present study.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/8/4/101/s1, Table S1: correlations between Se, Mo and heavy metal ratios, Table S2: correlations between heavy metals and oligoelements (r Spearman).

**Author Contributions:** J.J.M., M.E.C.-M.: writing the manuscript; M.E.C.-M., J.M.P.-I.: performed the samples collection and clinical data; J.J.M., J.M.P.-I., A.T.G., M.E.C.-M.: statistical analysis and experimental design; J.J.M., J.M.P.-I., A.T.G., M.E.C.-M. planned and supervised and the final revision.

**Funding:** Funds # 20151602 from CIROM (Murcia, Spain), Article processing charge (APC) supported by Nutergia Laboratories (San Sebastian).

**Acknowledgments:** We thank Laboratorios Nutergia (Basque Country) the support of this research project to Jose Joaquin Merino. We also thank InspiraHealth® (Barcelona) for the supplying of nasal filters. GREEN-FLOR (Nutergia), ERGYLIXIR (Nutergia), ERGYTAURINE (Nutergia) were supplied by Nutergia Laboratories. We also thank all enrolled patients from CIROM (Murcia, Spain) in the present study. The principal researcher of this project Jose Joaquin Merino thanks the support of Nutergia laboratories and CIROM Clinic (Murcia, Spain). IP Research project: ¨Detoxification of heavy metals by long-term algae and aminoazuphrate supplementation in patients with long-term dental amalgam fillings and titanium implants to Jose Joaquin Merino.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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