Neste ensaio, a substância de teste é administrada por via oral diariamente em doses graduadas para vários grupos de animais experimentais, um nível de dose por grupo por um período de 28 dias. Durante o período de administração, os animais são observados quanto a sinais de toxicidade, diariamente. Os animais que morrem ou são eutanizados durante o teste são necropsiados e, no final, os animais sobreviventes são eutanizados e necropsiados. As doses utilizadas na determinação da toxicidade oral com doses repetidas são determinados através de informações obtidas no teste de toxicidade oral aguda (OECD, 2008; VALADARES, 2006).
Um estudo de 28 dias fornece informações sobre os efeitos da exposição oral repetida e pode indicar à necessidade de novos estudos de prazo mais longo, e fornecer informações sobre a seleção de deses para estes estudos. Além de permitir a caracterização da toxicidade da substância testada, fornece uma indicação da relação dose-resposta e a determinação do Nível de Efeito Adverso Sem Observações (NOAEL) (OECD 2008; CAMPOS; AMARAL, 2009).
A diretriz original do teste 407 foi adotada em 1981, revisada em 1995, 1998 e em 2008, para obter uma versão capaz de gerar informações sobre neurotoxicidade, imunotoxicidade e disturbios endócrinos provocadas pelas substâncias teste. Este ensaio utiliza roedores de ambos os sexos, preferencialmente ratos, adultos jovens, com cinco animais por grupo das doses a serem testadas e ainda recomenda-se a utilização de grupos satélite, no grupo controle e no grupo da maior dose testada, que permanecerão em observação por mais 14 dias após o 28º dia, para observação da persistência, reversibilidade ou efeitos posteriores (OECD 2008).
A diretriz 407 destina-se a investigar os efeitos sobre uma variedade muito ampla de potenciais alvos de toxicidade e seus resultados devem ser utilizados para identificação de
perigos e avaliação de risco, fornecendo informações sobre os possíveis riscos para a saúde que podem surgir de exposição repetida durante um período de tempo relativamente limitado, incluindo efeitos nos sistemas nervoso, imune e endócrino (OECD 2008).
O método compreende o estudo básico de toxicidade de dose repetida que pode ser usado para substâncias químicas nas quais um estudo de 90 dias não está indicado ou, como preliminar para um estudo de longo prazo (VALADARES, 2006; OECD 2008; CAMPOS; AMARAL, 2009).
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APÊNDICE A – GASTROPROTECTIVE EFFECTS OF SULPHATED POLYSACCHARIDES FROM THE ALGA CAULERPA MEXICANA REDUCING
Pharmaceuticals 2017, 10, 92; doi:10.3390/ph10040092 www.mdpi.com/journal/pharmaceuticals
Gastroprotective Effects of Sulphated
Polysaccharides from the Alga Caulerpa mexicana
Reducing Ethanol-Induced Gastric Damage
José Gerardo Carneiro 1,2, Ticiana de Brito Lima Holanda 1, Ana Luíza Gomes Quinderé 1,
Annyta Fernandes Frota 1, Vitória Virgínia Magalhães Soares 1, Rayane Siqueira de Sousa 1,
Manuela Araújo Carneiro 1, Dainesy Santos Martins 3, Antoniella Souza Gomes Duarte 3 and
Norma Maria Barros Benevides 1,*
1 Department of Biochemistry and Molecular Biology, Federal University of Ceará, s/n Humberto Monte Avenue, Pici Campus, 60455-760 Fortaleza, Brazil; [email protected] (J.G.C.);
[email protected] (T.d.B.L.H.); [email protected] (A.L.G.Q.); [email protected] (A.F.F.); [email protected] (V.V.M.S.);
[email protected] (R.S.d.S.); [email protected] (M.A.C.)
2 Federal Institute of Education, Science and Technology of Ceará, Armando Sales Louzada Street, 62580-000 Acaraú, Brazil
3 Department of Morphoology, Faculty of Medicine, Federal University of Ceará, s/n Delmiro de Farias Street, Porangabuçu Campus, 60416-030 Fortaleza, Brazil; [email protected] (D.S.M.);
[email protected] (A.S.G.D.)
* Correspondence: [email protected]; Tel.: +55-85-3366-9402
Received: 11 October 2017; Accepted: 19 November 2017; Published: 21 November 2017
Abstract: The development of the gastric lesion is complex and the result of the imbalance between
aggressive and protective factors, involving the generation of free radicals and disturbance in nitric oxide (NO) production. Sulphated polysaccharides (SP), from marine algae, are widely used in biotechnological and pharmaceutical areas. In this study, we evaluated the effects of SP from the green marine alga Caulerpa mexicana (Cm-SP) in ethanol-induced gastric damage models in mice. Cm-SP (2, 20, or 200 mg/kg), administered p.o., significantly reduced gastric damage, and these effects were inhibited through pretreatment with indomethacin. Cm-SP (200 mg/kg) prevented the ethanol-induced decline in glutathione and restored its normal level. Moreover, it was able to normalize the elevated thiobarbituric acid reactive substance levels. However, Cm-SP did not show any significant effects on NO2/NO3 level, when compared to the ethanol group. The pretreatment
with L- NAME induced gastric mucosal damage and did not inhibit the gastroprotective effect of Cm-SP (200 mg/kg). In conclusion, the gastroprotective effects of Cm-SP in mice involve prostaglandins and reduction in the oxidative stress and are independent of NO.
Keywords: marine alga; gastric ulcer; gastroprotection; cytoprotection
1. Introduction
Gastrointestinal diseases are an important public health problem affecting many people worldwide. Stress, nutritional disorders, alcohol consumption, prolonged use of non-steroidal anti- inflammatory drugs (NSAID), and glucocorticoids are followed by gastric complications, including stomach ulcers [1–4]. Their development is complex and the result of the imbalance between aggressive and protective factors, involving the generation of free radicals and disturbance in nitric oxide (NO) production [5–7]. In the model of ethanol-induced gastric damage, the administration of absolute ethanol caused macroscopic and microscopic mucosa gastric damage, which is characterized
widely used to study gastroprotective effects of new bioactive compounds [1,6].
The discovery of new bioactive compounds with potential pharmaceutical activity, presenting minimal adverse effects, is of great importance. Thus, study groups are currently researching new natural compounds and their wide range of applications. Seaweeds are sources of sulphated polysaccharides, and the chemical structure of these polymers varies according to the species [8–10]. Sulfated polysaccharides (SP) comprise a group of heterogeneous macromolecules (as alginates, agar, agarose, carrageenans, fucoidan, laminaran, ulvan) presenting various biological effects, with biotechnical potential, and already in use in the food, cosmetics, and pharmaceutical industries [8– 11]. Our group has demonstrated various biological activities of SP, such as anticoagulant [12], antithrombotic [13], neuroprotective [14], antinociceptive, and anti-inflammatory [15–18].
Caulerpa mexicana Sonder ex Kützing is a marine green alga of the Cauleparceae family, which is widely encountered along the coast of Brazil. SP obtained from this alga species have presented antinociceptive and anti-inflammatory effects in a model of ulcerative colitis [19], in oedema caused by histamine and neutrophils migration [18]. However, no study has described the effects of SP from C. mexicanain in gastric lesions models. In the present study, we investigated the effects of SP from the green seaweed C. mexicana on ethanol-induced gastric damage in mice and the involvement of endogenous prostaglandins (PG) and NO pathway.
2. Results
2.1. Effect of Cm-SP on Ethanol-Induced Gastric Damage
Oral administration of ethanol absolute (0.2 mL) induced gastric mucosal damage in mice (24.57 ± 2.23 mm2). Administration of Cm-SP (2, 20 or 200 mg/kg, p.o.) reduced, in a dose-dependent
manner, the gastric damage by 49% (12.41 ± 1.71 mm2), 87% (3.09 ± 0.82 mm2) and 92% (2.03 ± 0.54
mm2), respectively, when compared with the ethanol group (p < 0.05). Moreover, the groups that
received Cm-SP (20 and 200 mg/kg) did not show any significant difference when compared to the control group saline. The treatment with indomethacin reversed the gastroprotective effects that are promoted by Cm-SP (200 mg/kg). Furthermore, the pretreatment with L-NAME induced gastric mucosal damage (29.12 ± 3.48) and did not inhibit the gastroprotective effect of Cm-SP (200 mg/kg; 85%; 4.34 ± 0.93) (Figure 1).
Figure 1. Effect of Caulerpa mexicana (Cm-SP) on ethanol-induced gastric damage. Mice were treated
with Cm-SP (2, 20, or 200 mg/kg) or saline (sal) by gavage. Another groups received L-NAME (20 mg/kg) or indomethacin (10 mg/kg) (indo). After 1 h, groups were treated with saline (sal) or Cm-SP (200 mg/kg). After 1 h, the gastric damage was induced by administration of ethanol absolute (0.2 mL, p.o.). After 1 h, mice were sacrificed and the total area of macroscopic gastric lesions was determined. The results are expressed as mean ± SEM of a minimum of 6 animals per group. (A) p < 0.05 vs. saline group; (B) p < 0.05 vs. group ethanol; ANOVA and Newman-Keuls test.
Administration of ethanol causes mucosal epithelium damage with disrupted glandular structure, oedema of submucosa, and excessive inflammatory infiltrate (Figure 2).
Figure 2. Histological evaluation of the ethanol-induced gastric damage in mice. (A) Control stomach:
intact gastric epithelium with organized glandular structure and normal submucosa could be seen; (B–E) ethanol-induced damage; (B) mice pre-treated with vehicle: * indicates damaged mucosal epithelium with disrupted glandular structure and arrow depicts oedema of submucosa and inflammatory infiltrate of mucosa; (C) Cm-SP 2 mg/kg; (D) Cm-SP 20 mg/kg; (E) Cm-SP 200 mg/kg. (C–E) depict a recovery in mucosa epithelium and reorganized glandular structure, as well as improvement of oedema by Cm-SP. (H&E staining; magnification 100×).
However, the administration of Cm-SP (2, 20, and 200 mg/kg) maintained the integrity of the mucosa with uninjured epithelium and organized glandular structure, as well as an improvement of oedema of submucosa and of inflammatory infiltrate, suggesting that Cm-SP exerts a gastroprotective effect. Histological analyses showed that Cm-SP (200 mg/kg) decreased hemorrhagic lesion, oedema, and erosion (loss of epithelial cell architecture), induced by ethanol (Table 1).
Table 1. Protective effect of Cm-SP in ethanol-induced microscopic gastric damage.
Experimental Group (N = 6) Hemorrhagic Lesion (Score 0–4) Oedema (Score 0–4) Erosion (Loss of Cell Architecture) (Score 0–3) Cell Infiltrate (Score 0–3) Total (Scores 14) Saline 0 0 0 0 0 Ethanol 4 (3–4) A 2 (2–4)A 2.5 (2–3)A 2 (2–3) A 10.5 (9–14) A Cm-SP (2 mg/kg) 1 (0–1) B 2 (0–3) 1 (0–2) 0 (0–1) B 4 (0–7) Cm-SP (20 mg/kg) 0 (0–1) B 0 (0–3) 0 (0–3) 0 (0–2) 0 (0–9) Cm-SP (200 kg/mg) 0 (0–0) B 0 (0–2) B 0 (0–1) B 0 (0–2) 0 (0–5) B Values denote median with minimum and maximum, respectively. Test of Kruskal-Wallis. A p < 0.05 vs. group Saline; B p < 0.05 vs. group Ethanol.
2.3. Effect of Cm-SP on Malondialdehyde (MDA), Glutathione (GSH), and NO2/NO3 Levels
Administration of ethanol decreased GSH levels (20.96 ± 1.87), and elevated lipid peroxidation (TBARS content) (391.42 ± 23.06) in gastric tissues when compared to the saline group. Cm-SP (200 mg/kg) prevented the ethanol-induced decline in GSH content and restored its normal level (38.26 ± 0.57). Moreover, Cm-SP (200 mg/kg) normalized the elevated TBARS levels (153.3 ± 8.28). However, the administration of Cm-SP (200 mg/kg) did not show any significant effects on NO2/NO3 level, as
Experimental Groups MDA GSH NO2/NO3 Saline 152.30 ± 9.91 31.81 ± 8.48 16.05 ± 3.24 Ethanol 391.42 ± 23.06 A 20.96 ± 1.87 A 28.02 ± 2.74 A Cm-SP (200 mg/kg) 153.30 ± 8.28 B 38.26 ± 0.57 B 23.14 ± 3.52 L-NAME 541.60 ± 49.02 19.28 ± 3.74 7.52 ± 1.68 L-NAME + Cm-SP (200 mg/kg) 383.0 ± 23.97 C 32.35 ± 1.56 C 8.66 ± 1.62
Data are expressed as the mean ± SEM (n = 6). ANOVA and Newman-Keuls test. A p < 0.05 vs. group