Preclinical antitoxic properties of Spirulina (Arthrospira)

Abstract

Context: Spirulina (Arthrospira) exerts a wide spectrum of pharmacological activities which are mainly attributed to its antioxidant effect. However, Spirulina has also been reported (both in preclinical and in clinical scenarios) to exhibit other bioactive effects, including an antitoxic potential.

Objective: We performed a systematic review of the literature, conducted in TOXNET, PubMed/MEDLINE, and Science Direct-Scopus; all available years were included. Searching criteria included the effects of Spirulina on experimental poisonings from arsenic, cadmium, carbon tetrachloride, deltamethrin, fluoride, hexachlorocyclohexane, iron, lead, lindane, and mercury.

Results: In all cases, it was established that the blue-green alga, and its isolated compounds, effectively counteracted these pollutants toxic effects on the exposed organisms. Some molecular mechanisms are proposed, although they have not been fully elucidated yet.

Conclusion: Spirulina could be a useful coadjuvant agent within clinical practice for treatment of these or other pollutants poisonings.

Keywords:

• Antidotes
• environmental pollutants
• occupational pollutants
• poisoning
• therapy
• toxicity

Introduction

In recent years, microalgae have attracted the attention of researchers due to their various chemical, biological and nutritional properties. This has been the case of Chlorella, Dunaliella, Scenedesmus, and cyanobacteria such as Nostoc, Aphanizomenon flosaquae, and Spirulina (Kay, 1991).

Spirulina (Arthrospira), a cyanobacterium generally described as a blue-green algae of the Oscillatoraceae family, grows in highly alkaline waters of tropical and subtropical areas, and is further classified into the well-known species Spirulina platensis, Spirulina fusiformis, and Spirulina maxima (Dillon et al., 1995). The latter has been consumed in Mexico since Aztec civilization age, but nowadays its consumption is globally distributed (Shukla et al., 2009).

Although it grows naturally, it is grown by using open ponds or bioreactors in Asia, Europe, North America, and Latin American countries (Durand-Chastel, 1997) yielding a current production of Spirulina of ≈3000–4000 tons per year worldwide (Belay, 2008), which are used in several ways: as a fertilizer, as feed supplement in poultry and livestock, as a colorant within foods, and as aquafeed.

Spirulina has been reported to be an excellent source of some macro and micronutrients (Hosseini et al., 2013). The protein content of the algae ranges between 60 and 70%, i.e., 10 times higher than soybean and three more than beef; moreover, the amino acid profile is considered to be of “high biologic value” according to the WHO/FAO reference values and some countries legislation (Becker, 1995). In addition, its protein efficiency, digestibility coefficient, and net protein utilization are of substantial nutritional significance for use in humans (Uma et al., 2008).

In terms of micronutrients, Spirulina contains significant amounts of vitamin B₁₂, pro-vitamin A and vitamin E; while regarding mineral content, iron, calcium, magnesium, manganese, potassium, and zinc stand out. It is also one of the few sources of glycolipids and sulfolipids (Belay, 2008).

As a food supplement, it has been widely tested in combination with grains, beans, cottage cheese, bread, pastries, juices, amino acids, and other basic food components, usually at concentrations up to 15%, confirming its usefulness by the positive effects on nutritional quality, which is attributed mainly to its protein content (Becker, 1995; Uma et al., 2008).

Today, Spirulina is also used as a source of natural dyes in pharmaceutical industry and it is included on food, pasta, snacks, chocolates, beverages, and other foodstuffs in food and beverages industries (Iyer et al., 2008).

Furthermore, from preclinical studies in rats fed with 5% Spirulina (Tsuchihashi et al., 1987), it has been reported that animals significantly increased the cecum population of lactobacilli; other studies, for their side, have observed stimulatory effects of extracellular products of the algae on lactic acid bacteria (Parada et al., 1998).

In addition to its nutritional properties, within the last 20 years, other bioactive properties of cyanobacteria have been reported in animal – and other preclinical – models of experimentation, including antioxidant, immunomodulatory, hypolipidemic, hypoglycemic, antiobesity, antiviral, anticarcinogenic, antigenotoxic, antibacterial, hepato-protective, and antiparasitic effects, among others (Khan et al., 2006; Kulshreshtha et al., 2008; Madrigal-Santillan et al., 2014; Muga & Chao, 2014). However, clinical studies in humans have been limited.

Notable among clinical research, is the finding that microalgae has been reported to significantly reduce interleukin-4 (IL-4) concentrations, demonstrating protective effects in allergic rhinitis (Mao et al., 2005). Moreover, Ishi et al. (1993) reported that Spirulina increased the production of immunoglobulin A (IgA) in saliva, indicating a role in secretory immunity. In addition, the increased production of interferon-gamma (IFN-γ) and the cytotoxicity of natural killer cells (NKC) have been suggested as a mechanism to explain the immunological activity of the cyanobacteria (Hirahashi et al., 2002). Related to this activity, it has been also found that when the algae is grown in deep sea water, it increases its in vitro anticancer effect by modulating the activity of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) (Choi et al., 2013).

It has also been shown, in humans, that the cyanobacterium reduces total cholesterol, triglycerides, and low-density cholesterol, while increasing HDL cholesterol levels in patients with ischemic heart disease (Ramaoorthy & Premakumari, 1996). Likewise, a lipid-lowering effect was found together with reduction of systolic and diastolic blood pressure in volunteers (Torres-Duran et al., 2007).

Concerning the anti-toxic activity of Spirulina in humans, it has been shown that the algae, in association with zinc, were useful in the treatment of chronic arsenicosis (Misbahuddin et al., 2006), a condition affecting countries such as Bangladesh, Chile, India, Mexico, and Taiwan (Karkos et al., 2011).

Moreover, Yamani et al. (2009) found that human consumption of ≈10 g/d of Spirulina for 6 months induced no adverse effects. Furthermore, there is evidence that regular consumption in several regions of Africa reaches up to 40 g (Ciferri, 1983) and no adverse effects have been reported.

Many experimental studies have attributed the beneficial effects of Spirulina with its antioxidant capacity (Chu et al., 2010; Kim et al., 2010; Ponce-Canchihuaman et al., 2010), due to its content of phenolic compounds, such as γ-linolenic acid, α-tocopherol, phycobiliproteins, and other phytochemicals (Dartsch, 2007; Kalafati et al., 2010). Consequently, it has been considered as a functional food, because of its ability to provide medical or health benefits, including the prevention and/or treatment of diseases (Baptista, 2008).

For instance, the algae has been proved to counteract colitis in the trinitrobenzenesulfonic acid model (Coskun et al., 2011); it has also been proven to reduce not only the hepato and nephrotoxicity caused by 4-nitroquinoline-1-oxide (Viswanadha et al., 2011) but also the rosiglitazone-induced bone loss in diabetic animals and the teratogenicity produced by hydroxyurea in mice (Vazquez-Sanchez et al., 2009).

Therefore, the main purpose of this review is to analyze the results published in recent years on the preclinical effect of Spirulina and its extracts against specific toxicity induced by environmental and occupational toxic pollutants, in order to extrapolate them to the prevention or treatment of poisoning caused by these contaminants in humans.

Materials and methods

The query for existing literature was conducted in TOXNET, PubMed/MEDLINE, and Science Direct-Scopus search engines and included all available years in each one. To identify research on the nutritional, pharmacological, and toxicological field, the search profiles were Spirulina, Arthrospira, Spirulina toxicity, and Arthrospira toxicity. Only antitoxicity against environmental pollutant studies were selected, other interactions with chemicals such as drugs were excluded. In addition, toxicology books and summaries of symposia concerning algae issues were consulted. Both in vivo and in vitro studies were included.

Results and discussion

Cadmium, mercury, lead, iron, and arsenic (metalloid) are the metals frequently involved in health problems (Goyer & Clarckson, 2001). Among their most common toxic effects are carcinogenesis, teratogenesis, inhibition of immune function, injury to organs including liver and kidney; nervous and respiratory systems, endothelial dysfunction, hypertension, vascular disease, and damage to the intestinal mucosa (Schäfer et al., 1998).

Regardless of the toxicity mechanisms involved in each of the aforementioned agents, their systemic/oral administration, as well as that of carbon tetrachloride or hexachlorocyclohexane, produces oxidative stress. This has been evidenced as increases in lipoperoxidation levels and carbonylated proteins in different tissues; but also as deficits in either non-enzymatic antioxidants such as sulfhydryl groups, reduced glutathione, vitamins C and E, or in antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PD) (Bhadauria et al., 2008; Sinha et al., 2010; Srivastava & Shivanandappa, 2005).

Additionally, metals are capable of interacting with protein sulfhydryl groups, hence inhibiting crucial enzymes, – e.g., ATPases, and thus interfering with key biological processes (Premkumar et al., 2004). Treatment usually includes complexation and chelation therapy. Among the most commonly used chelating agents are 2,3-dimercaptopropanol (BAL), 2,3-dimercaptosuccinic acid, ethylenediaminetetraacetic acid, (EDTA), diethylenetriaminepentaacetic acid (DTPA), deferoxamine, diethyldithiocarbamate (DTC), penicillamine, and N-acetylcysteine (Goyer & Clarckson, 2001).

As shown in Table 1, Spirulina showed a protective effect against arsenic, hexachlorohexane, and carbon tetrachloride, regardless of its dose, route of administration, and animal model. Such beneficial effect of Spirulina has been attributed to its antioxidant capacity, as demonstrated in HgCl₂ (Sharma et al., 2007a,b) and lead (Ponce-Canchihuaman et al., 2010) poisonings.

Table 1. Spirulina protective effects against the toxicity induced by different contaminants.

Pollutant	Test specie	Exposition conditions	Spirulina treatment	Antitoxic effect	References
Arsenic	Duck	100 mg/L in drinking water	30–120 mg/L in drinking water (90 d)	Improved body weight gain and reduced altered hematological parameters	Islam et al. (2009)
Cadmium	Mouse	1.5 mg/kg p.o. (gestation day 7)	62.5–500 mg/kg, p.o. (gestation days 0–17)	Decreased the frequency of exencephaly and other malformations by acting as an antioxidant	Paniagua-Castro et al. (2011)
	Rat	6 mg/kg/d p.o. (30 d)	500 mg/kg/d p.o. (30 d)	Partially prevented reduction of copper, zinc, iron, and selenium serum concentrations. Due to its ability to decrease oxidative stress and structural damage, proved protective capacity against liver and renal damage	Jeyaprakash and Chinnaswamy (2005)
		50 mg/L in tap water (30 d)	300 mg/kg p.o. (30 d)	Protected against liver damage because of its ability to decrease the vacuolar degeneration, fat infiltration, and fibrosis. The effect was related to the reduction of oxidative damage	Karadeniz et al. (2009)
		50 mg/L in tap water (30 d)	300 mg/kg, p.o. (30 d)	Improved hematological parameters affected by cadmium	Simsek et al. (2009)
Carbon tetrachloride	Rat	1:5 v/v 1.5 mL/kg p.o.	50 or 100 mg/kg p.o. (5 weeks)	Ethanol extract prevented oxidative damage; which was explained by its antioxidant and scavenging effects	Kuriakose et al. (2011)
		1 mL/kg i.p.	800 mg/kg p.o. (30 d)	S. platensis enriched in phenolic compounds protected more efficiently against CCl4 hepatotoxicity than that grown in conventional cultured conditions, by increasing its antioxidant defenses	Kepekçi et al. (2013)
Deltamethrin	Rat	30 mg/kg p.o. (5 d)	500–1000 mg/kg p.o. (1 h before the insecticide administration/5 d)	Provided near complete protection in terms of serum and tissues biochemical changes, antioxidant enzymes, and free-scavenging activity	Abdel-Daim et al. (2013)
Fluoride	Rat	20 mg/kg p.o. (gestation day 6 to postnatal 15)	250–500 mg/kg p.o. (gestation day 6 to postnatal 15)	Minimized the risk of neuroendocrinal and neurodevelopmental disorders in offspring of pregnant animals facilitating displacement of the metal, antioxidant formation, and protecting Purkinje cells	Banji et al. (2013)
Iron	Human neuroblastoma  cells	200 μM Fe (30 min)	100–500 μg/mL (30 min before ferrous sulfate)	Scavenged reactive oxygen species and protected the activity of antioxidant enzymes	Bermejo-Bescós et al. (2008)
Lead	Mouse	0.01–1.0 mg/kg i.p.	800 mg/kg p.o. (15 d before and up to 30 after intoxication)	Reduced the affectation on animal and testis weights and tubular diameter, enhancing the survival time	Shastri et al. (1999)
	Rat	2 g/L in drinking water (1 month)	300 mg/kg in drinking water (1 month)	Decreased hematological parameters affectation	Romay et al. (2001)
		100 ppm in drinking water (30 d)	1500 mg/kg in diet (30 d)	Increased SOD, CAT, and glutathione (GSH) in liver, lungs, heart and kidneys; additionally lowered brain metal concentrations and lipoperoxidation (LPO)	Upasani and Balaraman (2003)
		2 g/L in drinking water (1 month)	300 mg/kg in drinking water (1 month)	Reduced the lead-induced increase in mast cells number in the ovarian cortex and medulla during the estrous cycle	Karaca and Simsek (2007)
		25 mg/animal i.p./weekly/thrice	20 g diet 5%/d/rat (30 d)	Prevented body weight decrease and liver impairment; also protected against oxidative damage in both liver and kidney	Ponce-Canchihuaman et al. (2010)
		5 mg/kg in drinking water (5th day of gestation to the 14th day of lactation)	5% + dandelion 2% in diet (5th day of gestation to the 14th day of lactation)	Spirulina or dandelion minimised the lead deposition and lead-induced oxidative stress during gestation and lactation	Gargouri et al. (2012)
Lindane	Rat	1000 ppm free vitamin A diet (7 weeks)	Supplemented diet with 0.0628 – 3.18% (7 weeks)	Normalized both serum and hepatic activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)	Venkataraman et al. (1994)
Mercury	Mouse	5 mg/kg i.p.	800 mg/kg p.o. (before and after HgCl2 exposition)	Reduced activity of both acid and alkaline phosphatases in testicles	Saxena and Kumar (2004)
			800 mg/kg p.o. (10 d before and 30 d after intoxication)	Modulate or modify mercury-induced biochemical alterations in blood in terms of calcium level, ion level, acid and alkaline phosphatase activity and lipid peroxidation and GSH level in blood	Sharma e al. (2005)
			800 mg/kg p.o. (10 d before and 30 d after intoxication)	Protected against renal damage reducing LPO, acid phosphatase activity, tissue degeneration, and increasing ALP and lactate dehydrogenase levels	Sharma et al. (2007a)
			800 mg/kg p.o. (10 d before and 30 d after intoxication)	Decreased oxidative stress reducing LPO and increasing antioxidant enzymes and GSH. Restored ALT and AST activities near normal	Sharma et al. (2007b)
	Rat	5 mg/kg s.c. (3 times/week/60 d)	300 mg/kg p.o. (10 d before and 60 d after)	Attenuated hepatotoxicity as well as altered lipid profile through its antioxidant activity	Bashandy et al. (2011)
			300 mg/kg p.o. (10 d before and 60 d after)	Protected against Hg testicular damage, restoring oxidative stress biomarkers, sperm quality, and histopathological alterations	El-Desoky et al. (2013)

The antioxidant capacity of Spirulina has been proven both in vivo and in vitro experimental conditions, resulting in a decrease of up to 65% of lipid peroxidation, i.e., much higher than that exerted by α-tocopherol, butylated hydroxyanisole (45%) or β-carotene (Manoj et al., 1992). Furthermore, it has been shown that the algae has greater antioxidant capacity than gallic and chlorogenic acids (Chopra et al., 2008). This property has been certainly attributed to its carotene, tocopherol, phenolic compounds, and C-phycocyanin content, which are proposed to play an antioxidant role either alone or together thus resulting in synergistic effects (Miranda et al., 1998).

C-Phycocyanin, one of the main biliproteins in Spirulina, represents 20% of its dry weight and exhibits a strong antioxidant activity as it is capable of scavenging hydroxyl, alkoxy, and peroxyl free radicals, involved in the processes of lipoperoxidation and cytotoxicity (Bhat & Madyastha, 2000); thus, C-phycocyanin, as an antioxidant, is 16 and 20 times more effective than trolox and ascorbic acid, respectively (Romay et al., 2003).

In addition, Spirulina also exerts a modulatory effect on metabolism, detoxification, and antioxidant enzymes. Regarding the latter, the alga has been shown to increase the activity of key antioxidant enzymes such as SOD, CAT, GR, GPx, and GST (Premkumar et al., 2001,2004).

Although the therapeutic utility of antidotes currently used, as those above mentioned, in the treatment of poisoning is irrefutable, they may also cause a variety of adverse effects affecting the central nervous, cardiovascular, neuromuscular, skeletal, and respiratory; moreover, they may also result in liver, kidney, optic, and otic injury, among other alterations (Burda et al., 2008). Unlike current antidotes, Spirulina itself is not toxic, as it has been proven through different preclinical studies of acute toxicity tests, subchronic toxicity, chronic toxicity, fertility and reproduction assessments, teratogenicity, multigenerational tests as well as in vivo mutagenesis assays were used doses much higher than those humans can consume daily (Chamorro et al., 2002).

Within the clinical scenario, human studies have virtually reported no adverse reactions. For this reason, Spirulina has been listed in the FDA-GRAS group (Food and Drug Administration-Generally Recognized As Safe) (Belay, 2008). However, one isolated case of hepatotoxicity (Iwasa et al., 2002) and another one of rhabdomyolysis have been related to the algae consumption (Mazokopakis et al., 2008); C-phycocyanin was reported as the potencially allergenic protein if one case of anaphylaxis to Spirulina (Le et al., 2014; Petrus et al., 2010).

Conclusions

Poisoning by environmental and occupational pollutants or other compounds is, nowadays, treated with drugs or antidotes that may eventually lead to undesirable effects. The use of natural products is an interesting alternative not only because of its safety profile but also because of its efficiency and low cost. Spirulina, due to its high antioxidants content, mainly phycocyanin, its activities on metabolism and detoxification, and virtually none toxicity, has advantages over other products. Thus the alga may be considered as a coadjuvant agent as it may exhibit a synergistic effect together with traditionally drugs. Therefore, further evaluation of Spirulina as antidotal agent and antitoxic against environmental contaminants may lead to establish specific guidelines for its use in humans.

Declaration of interest

The authors report that they have no conflicts of interest. R. P. P. B., M. G. M., and G. A. C. C are fellows of the EDI and COFAA/IPN Programs. E. M. G. is fellow of the EDD and COFAA/IPN Programs. G. C. C. gratefully acknowledges SIP-IPN (Grant no. 20140207).