Biologically active compounds from cyanobacteria extracts: in vivo and in vitro aspects

Biologically active compounds from cyanobacteria extracts: in vivo and in vitro aspects

Luciana R. Carvalho^I; Adriana Costa-Neves^II; Geanne A. A. Conserva^I; Rafael L. Brunetti^III; Guilherme S. Hentschke^I; Camila F. S. Malone^I; Luce M. B. Torres^I; Célia L. Sant'Anna^I; Marisa Rangel^III

^IInstituto de Botânica, Núcleo de Pesquisa em Ficologia, SP, Brazil

^IIInstituto Butantan, Laboratório de Biologia Celular, Brazil

^IIIInstituto Butantan, Laboratório de Imunopatologia, Brazil

^{Correspondence}

ABSTRACT

An investigation was directed towards the antiacetylcholinesterase activity of the acid aqueous and methanolic extracts of five cyanobacterial taxa, which encompasses an enzymatic inhibition essay and the evaluation of the physiological responses of mice to cyanobacterial extracts along with toxicological observations. The strains Calothrix sp. CCIBt 3320, Tolypothrix sp. CCIBt 3321, Phormidium cf. amoenum CCIBt 3412, Phormidium sp. CCIBt 3265, and Geitlerinema splendidum CCIBt 3223 were from the São Paulo Botanical Institute Cyanobacterial Culture Collection and all of them showed inhibitory effect on acetylcholinesterase activity (in vitro) and caused systemic effects similar to those described for anticholinesterase drugs (in vivo). With the exception of G. splendidum and Tolypothrix sp. strains, all extracts produced reversible antiacetylcolinesterase effects in mice. Complementary histopathological studies were carried out on tissues from animals administered with Phormidium sp. and P. cf. amoenum.

Keywords: acetylcholinesterase activity; Calothrix sp.; Geitlerinema splendidum; Phormidium cf. amoenum; Phormidium sp.; Tolypothrix sp.

Introduction

Cyanobacteria are common members of microscopic populations of freshwater lakes and reservoirs worldwide. They are capable of forming blooms and producing potent toxins, which can present serious human and animal health problems (Van Apeldoorn et al., 2007, Pearson et al., 2010). Besides cyanotoxins, these organisms have the ability to synthesize considerable amounts of structurally distinct compounds that can be used as food and feed, fuel, dyes, sunscreen agents, as well as therapeutic drugs (Abed et al., 2009).

In scientific literature there is an appreciable amount of studies on the potential use of cyanobacterial compounds as medication, along with cyanotoxin poisoning cases in humans by ingesting contaminated water and food or by accidental administration during dialysis treatment. Some of these compounds are already being employed in anal fissures and common fistula treatment, as well as anti-HIV drugs (Botos & Swlodawer, 2003; Garrido et al., 2007), however, one of the most interesting set of activities displayed by cyanobacterial metabolites is their inhibitory action on certain enzymes (Grainger et al., 1989; Chen et al., 2007; Zelik et al., 2009). The cyanotoxins microcystins and anatoxin-a(S) have anti-phosphatase and anticholinesterase effects, respectively (Van Apeldoorn et al., 2007). However, there are other antienzymatic activities described for compounds synthesized by these organisms (Radau, 2000; Sisay et al., 2009; Matthew et al., 2010).

Compounds bearing antiacetylcholinesterase action play a very important role in the search for potential drug candidates against Alzheimer disease (AD); this neurodegenerative condition is associated with brain neurotransmitter deficits and its symptomatic treatment is the restoration of cholinergic function by inhibiting acetylcholinesterase (Francis et al., 1999; Trevisan et al., 2003).

Therefore, the effect of an antiacetylcholinesterase drug is a long-lasting and more effective stimulation of the cholinergic system, which results in responses from autonomic effector organs, autonomic ganglia, as well as skeletal muscles, and from cholinergic receptors in the Central Nervous System. According to its molecular structure, each antiacetylcholinesterase compound has its own chemical characteristics that determine its reactivity. As a consequence, the binding between the antiacetylcholinesterase compound and the enzyme shall be either a short, medium (reversible), or long-term (irreversible) association, being considered as potential therapeutic drugs only the ones which form reversible bonds (Nair et al., 2004).

There is an ongoing search for new bioactive compounds in cyanobacterial extracts from the São Paulo Institute of Botany Cyanobacterial Culture Collection strains, where some caused similar physiological responses in the mouse bioassays, which relate to responses demonstrated by antiacetylcholinesterase compounds. Such extracts were evaluated for AChE inhibition and short-term toxicity to mammals; a first step for further studies.

Materials and Methods

The organisms and extract preparation

Five different cyanobacterial strains were studied: Calothrix sp. CCIBt 3320, Tolypothrix sp. CCIBt 3321, and Phormidium cf. amoenum CCIBt 3412, isolated from soil samples in the Atlantic Rainforest, State Park of the Serra do Mar, SP (23º24' S and 45º11'06'' W); Phormidium sp. CCIBt 3265, isolated from an alkaline lake from the Pantanal, MS (18º57'42'' and 56º37'26''); and Geitlerinema splendidum CCIBt 3223, from Guarapiranga Reservoir waters, SP (23º43' S and 46º32') (Figure 1). The strains were cultured under the following conditions: ASM-1 medium, temperature 23±1 ºC, and continuous irradiance 40-50 Mmol/m^-2 s^-1 (Azevedo & Sant'Anna, 2003).

For each strain, the biomass obtained was freeze-dried, divided into two halves: one of them subjected to ultrasound-assisted extraction (5x, 30 s, 100 W) with 0.1 M aqueous acetic acid (AAE) and the other, with methanol 100% (ME). After centrifugation (1,750 x g, 50 min), the aqueous supernatants were lyophilized and the methanolic ones were dried via speed-vac centrifugation. The dried samples were stored in hermetically sealed vials, at -20 ºC, until analysis (Conserva et al., 2011).

In vitro assay: qualitative evaluation of acetylcholinesterase inhibitory activity

This in vitro assay was accomplished following Rhee et al. (2001) TLC autographic protocol: aliquots of 100 µg of each dried extract were dissolved and spotted on a pre-coated plate (Silica gel 60 F₂₅₄, 10x 10 cm, layer thickness 0.2 mm, E. Merck, Germany). The chromatogram was developed with mobile phase CHCl₃: MeOH:H₂O (64:36:8, v/v/v), dried and sprayed with the enzyme solution (6,66 U mL), thoroughly dried and incubated in a humid atmosphere, at 37 ºC, for 20 min. Subsequently, the plate was sprayed with a 0.25% 1-naphthylacetate in ethanol plus 0.25% aqueous Fast Blue B salt solution. The spots corresponding to potential acetylcholinesterase inhibitors were unambiguously identified as clear zones against a purple background. The Electric eel AChE type V (Product no C 2888, 1000 U) was purchased from Sigma as well all analytical grade reagents.

The retention factors (R_f) of the compounds that positively reacted against the enzyme were also calculated.

In vivo assay: acute toxicity study (i.p.)

Toxicological assays were performed in triplicate on each crude extract by using mice of the same sex, which simultaneously allowed the achievement of reliable data and the use of a minimum number of animals (Rangel et al., 2012). The procedures were carried out according to the WHO guidelines (Harada et al., 1999) and a single dose of 1,000 mg dried cells/kg body weight was used, which enabled extracts to be ranked as low toxicity, if animal death was caused (Lawton et al., 1994). Ethical clearance was obtained from the Ethical Committee for Animal Research of Butantan Institute - Protocol No. 385/07.

Male Swiss mice (19-21 g, 50 days) were intraperitoneally (i.p.) treated with dried aqueous acetic acid or methanolic cyanobacterial extracts dissolved in Milli-Q water; the control animals received only the vehicle (Milli-Q water), according to WHO protocol. Any changes in the skin, fur, eyes and respiratory, autonomic and central nervous system, somamotor activity and behavior pattern were observed, and signs of tremors, convulsions, salivation, diarrhea and lethargy and coma were noted as well. The animals were observed for 8 days following administration because, after an extended observation time, notable findings on tissue lesions can be observed (Rangel et al., 2012). Surviving animals were euthanized with CO₂, necropsy findings were recorded and tissue samples were taken.

Results and Discussion

Extract preparation and qualitative evaluation of AChE inhibitory activity

The freeze-dried cyanobacterial biomass and the dried extract weights are displayed in Table 1, along with the qualitative results of the antiacetylcholinesterase bioautographic assay (Figure 2) and the R_f (retention factors) of spots corresponding to antiacetylcholinesterase compounds.

All extracts were previously analyzed for the presence of microcystins (Conserva et al., 2011).

In vivo assays: acute toxicity study (i.p.)

In acute toxicity testing, among the treated animals, the ones that received the AAE G. splendidum CCIBt 3223 and only one administered with AAE Tolypothrix CCIBt 3321 died, while all the others showed mild and transient physiological effects, which can be associated with the intrinsic side effects induced by anti-AChE drugs (McGleenon et al., 1999; Xavier et al., 2007; 2008).

The physiological responses, time to death, and gross changes seen post-mortem are compiled in Chart 1.

Similar responses were observed in animals treated with extracts of all studied strains, which are also very similar to those observed in the mouse studies on anti-AChE drugs (McGleenon et al., 1999; Xavier, 2008) and such responses are related to muscarinic and nicotinic actions as well as on the central nervous system.

Among the muscarinic manifestations are dyspnea, abdominal cramps and diarrhea; among those resulting from overstimulation of the nicotinic receptors are muscle cramps, motor weakness, paralysis, tachycardia, and piloerection, and among those due to Central Nervous System are tremors, ataxia, and walking difficulty. The extent, the progression and the persistence of clinical observations depend on administration route, bioactive compound structure, and exposure magnitude (Andrade Filho & Romano, 2001). The compound structure defines the bond nature to the enzyme, which can be reversible, or irreversible, being the intermediate compound short, medium (reversible), or long acting (irreversible), respectively. The long acting intermediates are considered to be toxic (Nair et al., 2004).

The clinical signs complete regression ranged between two to three hours in three out of five groups of mice tested; that is, AAE G. splendidum CCIBt 3223 caused acute intoxication and only one animal that received AAE Tolypothrix sp. CCIBt 3321 died after four days from administration.

The results also showed that the AAE of the strain G. splendidum CCIBt 3223 possessed a lethal anti-AChE activity, most likely by the presence of long acting inhibitory substances to the enzyme (Nair et al., 2004), but there is insufficient information on the AE Tolypothrix sp. effects.

Anatoxin-a(S), anticholinesterase with potent lethal effect, is the only organophosphate produced by Cyanobacteria (Van Apeldoorn et al., 2007); meanwhile, great part of the compounds considered highly toxic are synthetic, such as carbamate Aldicarb (Cazenave et al., 2005) and of the organophosphate Parathion (Bardin et al., 1994).

Our results of the in vitro anti-AChE assay and clinical observations indicate that the methanolic and aqueous extracts of Calothrix sp. CCIBt 3320, Tolypothrix sp. CCIBt 3321, Phormidium sp. CCIBt 3265, Phormidium cf. amoenum CCIBt 3412, and G. splendidum CCIBt 3223 contain compounds which inhibit the enzyme acetylcholinesterase in a transient or reversible way.

Compounds which act as transient anticholinesterase drugs should be evaluated as potential therapeutic drugs because the current cholinesterase inhibitors prescribed for the treatment of AD are tacrine, donepezil, rivastigmine, galantamine, and serine which present several adverse side-effects such as hepatotoxicity, gastrointestinal disturbance and depression (Yoon et al., 2008). Due to these limiting factors for their use, new anticholinesterase drugs will be well received.

Aside from allowing a detailed observation of the biological responses to active compounds, an important outcome of the mouse bioassay is to determine the nature and extense of the adverse effects to a single dose or an overdose of a toxic or a therapeutic compound (Xavier, 2008). In our studies, with the exception of the acetic acid extract from G. splendidum CCIBt 3223 and Tolypothrix sp. CCIBt 3321, all other extracts are not lethal and caused physiological responses associated with the pharmacological actions of antiacetylcholinesterase compounds.

Complementary histopathological studies were carried out on liver, kidney, and lung tissues of animals intoxicated with extracts of Phormidium sp CCIBt 3265 and P. cf. amoenum CCIBt 3412.

Previous studies have described and illustrated the microscopic lesions caused by CCIBt strain 3223 (Rangel et al., 2012). The mice lungs were severely affected, presenting hemorrhage focuses, edema, alveolar collapse, and hyperplasia, due to an increase in the number of immune system cells (macrophages). Disorganization of the hepatic parenchyma, necrosis, loss of vein endothelim, and presence of giant multinuclear cells and polymorphonuclear cells in the liver were observed. Finally, the kidneys of mice intoxicated with AAE CCIBt 3223 (G. splendidum) presented alterations in the convoluted tubules and necrotic areas (Rangel et al., 2012).

Histological sections from control animal are shown in Figure 3. The main alterations observed in the organs of animals tested with AAE CCIBt 3265 were: in the lungs, edema, hemorrhage, and alveolar collapse and in the liver, areas of necrosis and steatosis (Figure 4).

In the animals that received ME CCIBt 3265, necrosis, steatosis, damage in endothelial cells of central lobular veins, enlarged sinusoids, inflammatory infiltrate close to bile duct, and giant multinuclear cells were observed in the liver; in the lungs, hemorrhage and alveolar collapse; and in the kidneys, the interstitial space between tubules was greater than normal and there was increase in light of convoluted tubules (Figure 5).

The microscopic lesions provoked by AAE CCIBt 3412 were shown in the Figure 6: granulomatous foci, hemorrhage, and alveolar collapse were seen in the lungs, large amounts of inflammatory infiltration close or not to bile duct cells were seen in the liver, and enlarged convoluted tubules were seen in the kidneys.

In the only study concerning antiacetylcholinesterase effects on mice, the microscopic lesions observed were: in the lungs, hemorrhage, edema and congestion; in the liver, vacuolar degeneration and in the kidneys, hemorrhage, congestion and tubular degeneration (Xavier, 2008). Comparison between the findings on AAE and ME CCIBt 3265 microscopic lesions showed similarity with the results obtained by Xavier (2008). Other histological findings could be attributed to unknown substances in the extracts.

In conclusion, the present study identifies G. splendidum CCIBt 3223 as a producer of a toxin, which administered as a single dose of 1,000 mg dried cells/kg body weight, causes animal death within 2 h and indicates ME- Tolypothrix sp. CCIBt 3321 as a candidate for further studies for potential anticholinesterase drugs.

Acknowledgments

The authors thank the Instituto Nacional em Ciência e Tecnologia em Toxinas; Conselho Nacional de Desenvolvimento Científico e Tecnológico; Fundação de Amparo à Pesquisa do Estado de São Paulo.

Author's contributions

LRC designed the study, supervised the laboratory work, contributed to analysis of the data and drafted the paper and wrote the final manuscript; ACN contributed to histological studies and to critical reading of the manuscript; GAAC contributed in running the laboratory work and to chemical and biological studies; RLB contributed to toxicological analysis; GSH and CFSM contributed to cyanobacterial collection, identification and culture. LMBT contributed to biological analysis and to critical reading of the manuscript; CLS contributed to cyanobacterial identification and to critical reading of the manuscript and MR contributed to toxicological studies and to critical reading of the manuscript. All the authors have read the final manuscript and approve the submission.

Received 26 Nov 2012

Accepted 16 Apr 2013

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Publication Dates

• Publication in this collection
  14 May 2013
• Date of issue
  June 2013

History

• Received
  26 Nov 2012
• Accepted
  16 Apr 2013