Antinociceptive and anti-inflammatory effects of cellular and extracellular extracts from microalga Chlamydomonas pumilioniformis on mice

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Inflammation is an important protective biological response to infectious agents, tissue injury and chemical agents, but it also contributes to the pathophysiology of many human diseases. The inflammatory process is initiated by the migration of immune cells from blood vessels and release of mediators. Then, inflammatory cells are recruited and reactive oxygen and nitrogen species and proinflammatory cytokines are produced either to eliminate the pathogen or to repair injured tissue (Libby, 2008;Pan, Lai, & Ho, 2010). Inflammation is clinically characterized by five signs: redness, heat, swelling, pain and loss of function (Ji, Chamessian, & Zhang, 2016). Pain, which is one of the most important symptoms of inflammatory diseases, impacts quality of life and is frequently the reason why patients seek pharmacological treatments (Uritu et al., 2018).

Non-steroidal anti-inflammatory drugs have been mostly prescribed to relieve pain and reduce inflammation. Although these drugs have good anti-inflammatory and analgesic properties, they have a wide range of side effects specially when used for long periods (Wongrakpanich, Wongrakpanich, Melhado, & Rangaswami, 2018). Thus, the search for new anti-inflammatory and analgesic substances derived from natural sources could contribute to the development of new drugs with fewer adverse effects (Rodrigues et al., 2012;Hädrich et al., 2016;Uritu et al., 2018).

Microalgae are a particularly interesting group of aquatic organisms which constitute a valuable source of compounds with positive effects on human and animal health (Mimouni et al., 2012;Ulmann et al., 2017). Their constituents are also of great interest as long-chain fatty acids that could be used as dietary supplements, as well as proteins and pigments, which have interesting properties to the pharmaceutical industry. Polysaccharides, which are produced by many species of microalgae, have several applications to the pharmaceutical field (Raposo, Morais, & Morais, 2013).

Some studies of the pharmacological activity of microalgae have centered on antimicrobial (Raposo, Morais, & Morais, 2014; Dantas et al., 2019), antiviral (Kim et al., 2012), antitumoral (Koníčková et al., 2014; Trabelsi, Chaieb, Mnari, Abid-Essafi, & Aleya, 2016), antioxidant (Tobón-Velasco et al., 2013;Silva et al., 2020), anti-inflammatory (Pak et al., 2012; Hwang, Chen, & Chan, 2013) and immunomodulatory properties (Park et al.2011;Abdel-Daim, Farouk, Madkour, & Azab, 2015). For example, hydrosoluble extracts from Chlorella stigmatophora and Phaeodactylum tricornutum showed anti-inflammatory and antinocicpetive activities in acute experimental rat models (Guzmán, Gato, & Calleja, 2001). In another study, Abdel-Daim et al. (2015) verified the anti-inflammatory activity of extracts from two microalga species, Spirulina platensis and Dunaliella salina, in experimental colitis in rats. Moreover, some studies have shown that microalga-derived polysaccharides play an important role in anti-inflammatory activity in preclinical trials (Levy-Ontman, Huleihel, Hamias, Wolak, & Paran, 2017;Barboríková et al., 2019).

Microalga species of the genus Chlamydomonas (Chlorophyceae) have attracted interest in studies of the effects of their constituents. The genus includes unicellular chlorophyte algae that have a distinct cell wall, a nucleus, two anterior flagella, a single basal chloroplast and an eyespot (Harris, 2001). Some studies have shown that microalgae from this genus, Chlamydomonas debaryana and Chlamydomonas hedleyi, exhibit anti-inflammatory and antioxidant effects in experimental models in vitro and in vivo (Ávila-Román et al., 2014;Reyes et al., 2014;Suh et al., 2014). In the study carried out by Ávila-Román et al. (2014), the authors showed that oxylipin-containing biomass of microalga Chlamydomonas debaryana decreases inflammatory processes by reducing polymorphonuclear infiltration and levels of tumor necrosis factor-α and by suppressing the expression of cycloxigenase-2 (COX-2) and nitric oxide synthase (iNOS) in induced colitis in rats. This research group isolated and characterized oxylipins from Chlamydomonas debaryana that inhibited TNF-α production in lipopolysaccharide-stimulated macrophages (Reyes et al., 2014). Furthermore, mycosporine-like amino acids obtained from extracts from Chlamydomonas hedleyi decreased mRNA levels of COX-2, which increase in response to inflammation in cell culture of keratinocytes (Suh et al., 2014).

Although some studies report pharmacological activities of microalgae of the genus Chlamydomonas, there are no studies of the anti-inflammatory and analgesic properties of the species Chlamydomonas pumilioniformis. Therefore, considering that this microalga species could be a potential source of bioactive compounds, this study aimed to characterize carbohydrate contents of two different extracts (cellular and extracellular) from C. pumilioniformis and investigate their antinociceptive and anti-inflammatory activities in mice models.

Total carbohydrate contents of cellular and extracellular extracts from C. pumilioniformis were 90 and 20 µg mL-1, respectively. The former was mainly composed of glucose, but with significant proportions of arabinose, galactose, mannose and/or xylose and minor ones of fucose, rhamnose, amino sugars and uronic acids. The latter were composed of a similar proportion of glucose, galactose and mannose/xylose, besides significant proportions of arabinose, fucose and galacturonic acid. Minor proportions of rhamnose and amino sugars were also detected (Table 1).

The writhing test was used to investigate antinociceptive effects of C. pumilioniformis extracts. Results in Table 2 show that both cellular and extracellular extracts were able to inhibit acetic acid-induced abdominal constrictions in mice (78.3 and 26.7%, respectively). However, the cellular extract was more effective than the extracellular one. Moreover, the cellular extract was more effective than diclofenac, a standard non-steroidal anti-inflammatory drug. Neurogenic and inflammatory nociception was evaluated by the formalin test. In the formalin test (Table 3), only the extracellular extract from C. pumilioniformis significantly inhibited the first phase of the test (43.7%). On the other hand, both extracts were effective in the late phase with 94.8 and 85.7% of inhibition for cellular and extracellular extracts, respectively.

Table 1.
Monosaccharide composition of cellular and extracellular extracts from Chlamydomonas pumilioniformis.

Table 2.
Effect of Chlamydomonas pumilioniformis extracts on acetic acid-induced writhing behavior in mice.

Table 3.
Effect of Chlamydomonas pumilioniformis extracts on formalin-induced nociception in mice.

The intraperitoneal pretreatment with both cellular and extracellular extracts from C. pumilioniformis reduced the formalin-induced paw edema only 24 hours after the treatment (Figure 1). Diclofenac, used as a positive control, was effective from 1 hours after the treatment up to 24 hours.

Figure 1
Effect of Chlamydomonas pumilioniformis extracts on formalin-induced paw edema in mice. Results are expressed as mean ± SEM of the percentage of increase in paw edema (n = 9-10, diclofenac n = 4). Volume of the paw at time zero was considered as 100%. *p < 0.05 indicates statistical difference between diclofenac and the control group; #p < 0.05 indicates statistical difference between cellular extract and the control group; §§p < 0.01 indicates statistical difference between extracellular extract and the control group (one-way ANOVA followed by Bonferroni post hoc test).

Among metabolites produced by microalgae, polysaccharides have emerged as an important class of bioactive natural products with benefits to human health (Raposo et al., 2013;Talero et al. 2015)). In this study, we investigated the carbohydrate composition of cellular and extracellular extracts from C. pumilioniformis and their relations with pharmacological activities in inflammation and nociception.

Cellular extract, which contains the reserve and structural polysaccharides, is composed of arabinose, galactose, mannose and/or xylose and a high proportion of glucose. A previous study revealed arabinose associated with galactose in Chlamydomonas sp (Maria, Vieira, & Giroldo, 2011). This composition indicates the presence of pherophorins, which are cell wall glycoproteins rich in hydroxiprolin, arabinose and galactose, typically found in Chlamydomonadales (Morita et al., 1999;Hallmann, 2006). Pherophorins show similar composition to some pectins produced by vascular plants, which are associated with several biological activities, such as anti-inflammatory and immunostimulatory ones (Paulsen, 2001;Maria et al., 2016).

There are some differences in the composition of extracellular extract, by comparison with the cellular one. This extract has high proportion of fucose, that is the fundamental subunit of sulfated polysaccharides, such as fucoidan (Garcia-Vaquero, Rajauria, O’Doherty, & Sweeney, 2017). Moreover, galacturonic acid, which is a component of pectic polysaccharides with arabinose, galactose and rhamnose (Paulsen, 2001), has high content in this extract. Arabinose and galactose can also be found in extracellular medium because of the cell division pattern of this microalga, i. e., the old cell wall is released and a new one is synthetized by daughter cells (Maria et al., 2011).

Taking into account the biological potential of previously described polysaccharides, we studied the antinociceptive potential of C. pumilioniformis extracts. The writhing response to an intraperitoneal injection of acetic acid is used to screen peripherally and central analgesic drugs (Couto et al., 2011; Hort et al., 2018). Acetic acid induces the release of endogenous mediators, such as substance P, bradykinins, prostaglandins, as well as pro-inflammatory cytokines, such as interleukins (IL-1, IL-6 and IL-8) and TNF-α, that stimulate peripheral nociceptors which are sensitive to non-steroidal anti-inflammatory drugs and opioids (Verma, Joharapurkar, Chatpalliwar, & Asnani, 2005; Pinheiro et al., 2011). Our findings showed significant decrease in the number of acetic acid-induced constrictions in both cellular and extracellular aqueous extracts from C. pumilioniformis, indicating their promising analgesic properties. Studies demonstrating antinociceptive effects of microalgae are quite restricted in the literature. In the study carried out by Guzmán et al. (2001), the authors showed the antinociceptive effect of extracts from the species Chlorela stigmata and Phaeodactylum tricornutum on mice. To date, this is the first evidence of the antinociceptive effect of C. pumilioniformis. In agreement with our data, previous studies showed that polysaccharide-rich extracts obtained from vegetal sources have analgesic properties (Nascimento et al., 2015; Nascimento, Baggio, Werner, Iacomini, & Cordeiro, 2016;Silva-Leite et al., 2018).

To further study analgesic properties of these extracts, the formalin test was performed. In this assay, initial nociception (early phase) is caused by a direct stimulation of nociceptors and reflects centrally mediated nociception, whereas late phases are secondary to the inflammatory process, as a response to inflammatory mediators, such as prostaglandins, serotonin, histamine and bradykinin (Hunskaar & Hole, 1987). Drugs that act primarily on the central nervous system, such as morphine, inhibit both phases of this test, while drugs that act peripherally, such as cyclooxygenase inhibitors (ex: diclofenac), inhibit only the second phase (Yaksh et al., 2001). In our study, extracts from C. pumilioniformis showed different response profiles in this test and some differences in the chemical composition may explain the antinociceptive activity.

The extracellular extract inhibited both phases of formalin and implied central and peripheral effects of this extract, similar to opioids. Higher amounts of galacturonic acid, galactose and fucose may explain the effect of extracellular extract on the first phase of the formalin test. A previous study showed that Ximenia americana fraction of heteropolysaccharides, with a high content of uronic acids and galactose, ameliorates visceral hypernociception in mice, through a mechanism involving cannabinoid receptors (Silva-Leite et al., 2018). Moreover, fucose in the extract could indicate that sulfated polysaccharides, such as fucoidan, can be found in C. pumilioniformis. Similar to our results, a sulfated polysaccharide, composed mainly of α-L-fucose, isolated from brown marine alga Spatoglossum schroederi, reduced both phases of formalin test in mice, suggesting a mechanism similar to morphine (Farias et al., 2011). Rodrigues et al. (2012) showed the antinociceptive effect of a sulfated polysaccharide fraction isolated from the green seaweed Caulerpa cupressoides. This fraction inhibited both phases of the formalin test and caused antinociceptive effect in hot plate test, indicating that mechanisms of analgesia occur via central acting mechanisms and suggesting the involvement of the opioid system.

On the other hand, the cellular extract was effective to inhibit only the second phase of the formalin test. It suggests that peripheral mechanism is involved in its effects and may also exhibit an associated anti-inflammatory effect. Anti-inflammatory effects of microalgae are a little more explored that analgesic activity in the literature. Studies have already shown anti-inflammatory activity of microalgae Chlorella stigmatophora, Phaeodactylum tricornutum (Guzmán et al., 2001), Spirulina platensis, Dunaliella salina (Abdel-Daim et al., 2015), Chlorella vulgaris (Barboríková et al., 2019) and Chloromonas reticulate (Suh et al., 2019). Oxylipin-containing biomass of microalga Chlamydomonas debaryana demonstrated in vitro (Reyes et al., 2014) and in vivo (Ávila-Román et al., 2014) anti-inflammatory activity by inhibiting production of proinflammatory cytokines. Mycosporine-like amino acids found in Chlamydomonas hedleyi microalgae showed protection by anti-inflammatory and antioxidant activity against UV-induced damage (Suh et al., 2014).

Since the reduction of the second phase of the formalin test indicates an effect on inflammation mediators, we evaluated the anti-edematogenic effect of the extracts. The formalin stimulus produce an acute inflammatory response triggered by different mediators: bradykinin, cytokines (IL-1β, IL-6, IL-8 and TNF-α), eicosanoids and nitric oxide (Hunskaar & Hole, 1987;Moore, Oluyomi, Babbedge, Wallace, & Hart, 1991; Muley, Krustev, & McDougall, 2016). Both cellular and extracellular extracts reduced the edema only after 24 hours of treatment. Polysaccharides, specially sulfated forms, in the microalga, have already been found to contribute to anti-inflammatory activity of microalga (Raposo et al., 2013). Some mechanisms have been previously described in the anti-inflammatory activity of these compounds: reduction in myeloperoxidase activity (Carneiro et al., 2014) and neutrophil infiltration (Silva-Leite et al., 2018), decrease in proinflammatory cytokines, such as TNF-α, IL1-β and IL-17 (Brito et al., 2013; Miao et al., 2015), increase in nitric oxide (Brito et al., 2013) and inhibition in NF-κB activation (Miao et al., 2015). Antioxidant activity of polysaccharides, especially by modulating antioxidant defenses (Subash, Veeraraghavan, Sali, Bhardwaj, & Vasanthi, 2016), can also contribute to anti-inflammatory activity of C. pumilioniformis. This study has some limitations that should be highlighted. Only one dose of each extract was evaluated to assess anti-inflammatory and antinociceptive activity, which limits the establishment of the dose-effect relationship. Extracts were characterized as to their carbohydrate content, which can contribute to their pharmacological activity. However, other classes of chemical compounds must be studied in the future and their participation in the biological effect cannot be ruled out. Finally, although our studies indicate a possible central analgesic effect of the extracellular extract, further experimental tests are needed to confirm this effect.

Conclusion In sum, results of this study demonstrate that cellular and extracellular extracts from microalga C. pumilioniformis exhibit antinociceptive and anti-inflammatory effects on some classical models of nociception and inflammation in mice. Moreover, the pharmacological activity of this microalga could be directly influenced by its carbohydrate composition. These findings evidenced the relevant potential of C. pumilioniformis as a source of bioactive compounds and encourage further pharmacological studies to highlight the mechanism of its pharmacological action.

This study was partially funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) - Finance Code 001.