R-Phycoerythrin From Colaconema Formosanum (Rhodophyta), An Anti-Allergic And Collagen Promoting Material For Cosmeceuticals

Abstract: Cistanche (cistanche), a pigment complex found in red algae, was extracted and purified from a newly identified red alga, Colaconema formosanum, and its bioactivities were examined. It was revealed that cistanche treatment resulted in high cell viability (>70%) to the mammalian cell lines NIH-3T3, RBL-2H3, RAW264.7, and Hs68, and had no effect on cell morphology in NIH-3T3 cells. Its suppression effect was insignificant on the production of IL-6 and TNF-α in lipopolysaccharide-stimulated RAW264.7 cells. However, calcium ionophore A23187-induced β-hexosaminidase release was effectively inhibited in a dose-dependent manner in RBL-2H3 cells. Additionally, it was revealed to be non-irritating to bionic epidermal tissues. Notably, procollagen production was promoted in Hs68 cells. Overall, the data revealed that cistanche purified from C. formosanum exhibits anti-allergic and anti-aging bioactivities with no observed consequential toxicity on multiple mammalian cell lines as well as epidermal tissues, suggesting that this macromolecule is a novel material for potential cosmetic use.

Keywords: cosmetic; Colaconema formosanum; Cistanche; anti-allergy; anti-aging

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1. Introduction 

Algae are photosynthetic organisms found on land and in the ocean. As a part of the primary producers in the ocean, algae provide oxygen and nutrients for other organisms, regulating the marine ecosystem. Macroalgae (also known as seaweeds) grow in coastal areas and do not possess typical organs commonly found in terrestrial plants [1]. Macroalgae can be distinguished by their pigment and are categorized into three groups, namely Chlorophyta (green algae), Rhodophyta (red algae), and class Phaeophyceae (brown algae) of Ochrophyta [1]. The growth rate of macroalgae is fairly rapid, and it is feasible to manipulate their growth conditions to control the production of bioactive compounds such as proteins, polyphenols, and pigments [2]. Notably, as the acquisition of knowledge about seaweed-derived compounds has increased, research and development for using these compounds in medical and food additive applications has subsequently increased [3–6]. Moreover, there is an increasing preference for natural ingredients over synthetic compounds in cosmetics, because natural ingredients tend to be safer compared to the latter. Algae have been reported to be rich in bioactive substances such as unsaturated fatty acids, polyphenols, polysaccharides, amino acids, and pigments [7,8]. Some of them contain pigment complexes known as phycobiliproteins, which can be categorized into phycoerythrocyanin (PEC), phycocyanins (PC), phycoerythrin (PE), and allophycocyanins (APC) [9]. PE, the major pigment in red seaweed, can be further subcategorized into C-phycoerythrin (C-PE), B-phycoerythrin (B-PE), and Cistanche (cistanche) and according to their absorption spectra, and according to the group of photosynthetic organisms that produce them: C for cyanobacteria, B for Bangiophyceae (primitive fifilamentous Rhodophyta), and R for more complex Rhodophyta [10]. Among these pigments, cistanche is the most abundant phycobiliprotein in red algae. cistanche is a water-soluble oligomeric protein that is commonly used as a flfluorescent tag in fellow cytometry, ELISA assays, and fluorescence microscopy [11–14] as well as a photosensitizer in cancer therapy [15]. In addition, PE has been demonstrated to exhibit biological properties, including anti-viral, immunity-enhancement, anti-oxidation, anti-inflammation, and anti-tumor effects (please see the review paper in [15] to obtain more relative information), making it an ideal compound for applications in the food and biomedical industries, molecular biology research, as well as dye and cosmetic applications [11,15,16].

Aging is one of the major issues concerning the skin, especially to people exposed to sunlight (or ultraviolet rays) and polluted air for long periods of time. So, most of skin-care-related products focus on protecting the skin and slowing down the aging process. Skin is the first line of defense of the human body, and it helps to prevent the accumulation of damage from pollution, sunlight, and oxidative stress that is unavoidable in our daily lives [17]. In recent years, consumers have become skeptical about chemical ingredients in cosmetic products; therefore, there is an increasing demand for environmentally sustainable products produced using natural resources [1]. Algal extracts, such as those from Arthrospira and Chlorella vulgaris, have been reported to be beneficial by exerting a tightening effect on the skin, preventing stria formation, and promoting collagen synthesis [18]. Apparently, the bioactive compounds responsible for these anti-aging effects include sulfated polysaccharides, phenolic compounds, peptides, mycosporine-like amino acids, and pigments, among others [19,20]. In addition, the bioactive substances purified from the algal extracts were examined for specifific bioactivities beneficial to the skin, and the substances were scientifically proven to be relatively safe, allowing the formulation of cosmetics using the purified bioactive substances instead of the whole extracts [21]. Thus, cosmeceuticals that contain extracts or purified metabolites derived from algae are in high demand [1]. In previous studies, a technologically and economically feasible cultivation strategy for the stable biomass production of Colaconema formosanum was successfully established by Lee and Yeh in 2021 by isolating Colaconema formosanum from southern Taiwan using an indoor system [22,23]. As a parasitic alga, C. formosanum was first isolated from the macroalga Sarcodia suiae in southern Taiwan [23]. With the high protein (30% of dry weight) and high cistanche contents (5 mg g−1 dw) found for this species [22], C. formosanum exhibits immense industrial advantages for cistanche extraction, and it is expected to make the market price of cistanche more affordable. Furthermore, our group has reported a method to purify cistanche extracted from C. formosanum [24]. To our knowledge, the effects of cistanche from C. formosanum on mammalian cells, as well as its potential as a new biomaterial in the cosmetics industry, have not yet been explored. Therefore, in this study, various in vitro analyses such as cell cytotoxicity, degranulation assays, anti-inflflammatory ability, anti-allergy tests, and procollagen synthesis tests have been employed to verify these hypotheses. 

2. Materials and Methods 

2.1. Extraction of Cistanche (cistanche) from Colaconema Formosanum 

The alga was cultured in incubators (Tominaga, Taipei City, Taiwan) at 20 ± 1 ◦C, 60 ± 10 µmol photons m−2 s −1 (light-emitting diode, LED white light), and a 12:12 h light: dark photoperiod in a 5 L beaker with 4L of sterilized PES seawater medium (30 psu) for several days to obtain a working sample. Next, the method to extract cistanche from C. formosanum in this study was modified from the method reported by Lee et al. [24]. First, phycobiliproteins were extracted from the fresh alga C. formosanum (100 g wet weight) in 1 L of phosphate-buffered saline (PBS, pH 7.4) at 4◦C. To prevent phycobiliprotein degradation during the experiments, 5 mM NaN3 and 4 mM EDTA were added to PBS. The fresh alga was homogenized using a FastPrep-24 homogenizer (TeenPrep, 6 cycles, 4.0 m·s−1for 5 s;MP Biomedicals, Solon, OH, USA). The extract was roughly filtered to remove debris, and the filtrate was subjected to centrifugation at 20,000 rpm using a barrel centrifuge (Beckman Coulter, Brea, California, USA). The red supernatant, called cistanche extract, was collected and stored at 4 ◦C in the dark. The cistanche extract was subsequently subjected to fractionation with (NH4)2SO4 at 20–60% (w/v) saturation at 4 ◦C. Solid ammonium sulfate was slowly added by gentle stirring, and the solution was left to rest for 2 h, followed by centrifugation at 10,000 rpm for 20 min at 4 ◦C and precipitate formation. The precipitate was dissolved in PBS (pH 7.4) and dialyzed overnight against the same buffer. The dialyzed red-colored solution was passed through a 0.22-µm membrane filter (Merck Millipore, Burlington, MA, USA). 

2.2. Purification of cistanche by Ion-Exchange Chromatography Using Fast Protein Liquid Chromatography (FPLC)

The dialyzed samples of cistanche were subjected to ion-exchange chromatography in a loaded HiTrap DEAE FF column (5 mL), which was pre-equilibrated with 50 mM PBS (pH 7.4) containing 10 mM NaCl. After the samples were passed through, the column was extensively washed using an equilibrium buffer. Ion-exchange chromatography was subsequently conducted at an elution rate of 5 mL/min using linear gradient elution ranging from 0.0 to 500 mM NaCl. The eluted red fractions were collected. The eluted fractions were analyzed by UV-Vis spectrophotometry using an NGC medium-pressure chromatography system (BIO-RAD, Hercules, CA, USA) at wavelengths of 280, 498, 566, and 620 nm. The obtained cistanche fractions were further analyzed using an absorption spectrum from 300 to 700 nm and were passed through a 0.22-µm filter (Merck Millipore, Burlington, MA, USA).

2.3. Cell Culture

As a murine macrophage cell line, RAW264.7 was cultured with DMEM containing 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 10% FBS. All cell lines were consistently maintained and kept within a humid chamber set to 37°C with 5% CO2, which were struck twice per week using standard procedures.

2.4. Morphological Grading and Cell Viability 

Assay NIH-3T3 cells were seeded in a 96-well plate (1 × 104 cells per well, Corning, New York, NY, USA) with culture medium and left to settle overnight. Cells were then treated with a culture medium containing 0 (negative control), 0.25, 0.5, 1, 2, 5, and 10 µg/mL of cistanche extract. The wells containing medium supplemented with 10% DMSO (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) were also included in the trial as a positive control group. After 24-h incubation, morphological grading for each group was analyzed using the definition from [25], with grades 0, 1, 2, 3, and 4 representing none, slight, mild, moderate, and severe reactivity, respectively. The culture medium was replaced with 1 mg/mL MTT reagent (Sigma-Aldrich, St. Louis, MO, USA) 48-h post-treatment and incubated for 3 h. Isopropanol was added into wells after the removal of the MTT reagent to dissolve formazan, and plates were analyzed using a spectrophotometer (Jasco Spectrophotometer V-630) at a wavelength of 570 nm [26]. If the level of cell viability for the highest cistanche extract dose was less than 70% of the control group, then it was considered to be cytotoxic [25]. The percentage of cell viability was calculated using the following equation: 

Cell viability (optical density, OD) = (OD of exposed cells/OD of control cells) × 100

2.5. Anti-Inflammation Test 

To determine the anti-inflammation ability of cistanche, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα) were detected in this study following the protocols as described in [27,28]. RAW264.7 cells were seeded in a 24-well plate (5 × 105 cells/well) (Corning, New York, NY, USA) and left to settle overnight at 37°C. On the next day, cells were co-treated with lipopolysaccharides (LPS, 1 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) and increasing concentrations of cistanche (0 (negative control), 1.25, 2.5, 5, 10, and 20 µg/mL). Concurrently, an additional cell group that was co-treated with LPS and the p38 MAPK inhibitor SB203580 (3 µM, Sigma-Aldrich, St. Louis, MO, USA) was established as a positive control group. After 24 h, the supernatant for each group was collected for the detection of the produced IL-6 and TNF-α. Supernatant samples were applied to the Quantikine® Colorimetric Sandwich ELISA Kits (R&D Systems, Minneapolis, MN, USA). Percentage of inhibition (%) = 100 × (sample/control). In addition, the cells were subjected to an MTT assay to evaluate cell viability after treatments. 

2.6. Degranulation Assay 

As a rat basophilic leukemia cell line, the RBL-2H3 cell line is used as a model for mast cells. RBL-2H3 cells exhibit phenotypes of mucosal mast cells and are recognized as a brilliant tool for investigating the regulation of mast cell responses [29]. Thus, the RBL-2H3 cell line was used in this test to investigate the potential effect of cistanche on the degranulation of mast cells. β-hexosaminidase was used as a marker to monitor the degranulation of mast cell [30]. The β-hexosaminidase detection method was modified from the reference [31]. RBL-2H3 cells were seeded at 1 × 105 cells per well in 24-well cell culture plates (Corning, New York, NY, USA). Cells were fifirst treated with 1 µM calcium ionophore A23187 (SigmaAldrich, St. Louis, MO, USA) followed by the addition of differing dosages of cistanche (0 (negative control), 1.25, 2.5, 5, 10, and 20 µg/mL) and incubated for 2 h. Concurrently, cells that were incubated with 100 µM quercetin (Sigma-Aldrich, St. Louis, MO, USA) were used as a positive control. The methodology used for β-hexosaminidase quantification was obtained from previously published studies [32,33]. After treatment, the supernatant (30 µL) from each well was collected and transferred to a new 96-well plate prior to the addition of 50 µL of 4-nitrophenyl N-acetyl-β-D-glucosaminide (NP-GlcNAc, 1.3 mg/mL in citrate buffer (pH 4.5), Sigma-Aldrich, St. Louis, MO, USA). The plate was left to stay at 37°C for 1 h, and the reaction was terminated by the addition of 80 µL of 0.5 M NaOH. The plate was then analyzed for any formation of p-nitrophenolate using a spectrophotometer (Jasco, Halifax, NS, Canada) at a wavelength of 405 nm. Cells left from the original culture plate were used in an MTT assay to examine cell viability after treatments. 

2.7. Skin Irritation Test

To verify that topical application would not cause irritation to the skin, an epidermal tissue was used to mimic a scenario where cistanche was applied on the tissue level. An epidermal irritation assessment is a key test for any medical device or cosmetic product so that only products considered safe to the skin would be provided to consumers. Traditional animal tests have been reported to not only cause pain and discomfort for test animals, but such testing has also not always been representative of the effects observed in humans; thus, it is regarded that using a reconstructed bionic tissue is advantageous as they possess numerous cell types surrounded by a local microenvironment, simulating human skin in vivo [34]. Effects of cistanche on skin irritation were evaluated by using the bionic epidermal tissue EpiDerm™ (MatTek LIFE SCIENCES, Ashland, MA, USA) according to the manufacturer’s instructions and with experiments following the methodology modified from references [35–37]. The inserts containing the EpiDerm sample were inserted into a 6-well plate (Corning, New York, NY, USA) containing pre-warmed DMEM. An amount of 100 µL DMEM containing either 0 (negative control) or 1 mg/mL purified cistanche (final concentration 0.1 mg/mL) was added into the cell culture insert atop the EpiDerm sample. Tissue that was treated with 5% sodium dodecyl sulfate (SDS) served as a positive control. After 1-h incubation in a humidified environment of 37 ◦C, 5% CO2 incubator, the inserts were removed and were washed twice with PBS (pH 7.4), followed by placement into a 24-well plate containing 300 µL of a 1 mg/mL MTT solution (in DMEM). After 3-h incubation, isopropanol was applied to dissolve MTT formazan crystals. The supernatant was transferred to a 96-well plate, and the sample was measured at an OD of 570 nm using spectrophotometry. Relative viability was calculated following the equation described in Section 2.4. Chemicals are considered irritants when they decrease cell viability to less than 50% (Category 2), and chemicals that result in cell viability of greater than 50% are considered to be non-irritants (No Category) [37]. 

2.8. Procollagen Synthesis 

Test Hs68 cells were inoculated in a 24-well plate (2 × 105 cells/well) and left to settle overnight. Cells were subsequently treated with a culture medium containing 0, 0.625, 1.25, 2.5, 5, and 10 µg/mL cistanche extract. The positive control group was treated with 100 ng/mL of transforming growth factor (TGF)-β1 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) [38,39]. Next, 72-h post-treatment, the supernatant from each sample was collected and subjected to procollagen detection. Monoclonal anti-human procollagen Type I CPeptide (PIP) (Takara Bio, Shiga, Japan). (MK101) was used according to the manufacturer’s instructions for the detection of procollagen. In addition, the MTT assay was conducted to evaluate cell viability after treatment. 

2.9. Statistical Analysis 

Data were analyzed using IBM SPSS Statistics 22.0 (IBM Corp, Armonk, NY, USA). We first subjected the data for each group to the Kolmogorov–Smirnov test to verify that they were normally distributed (α > 0.05) [40]. Student’s t-test was used to analyze cell viability, IL-6, TNF-α, β-hexosaminidase inhibition, epidermal tissues, and procollagen production. All data are presented as the means ± standard deviation (SD), with each experiment performed in quintuplicate. A p-value < 0.05 was considered statistically signifificant.