BR102020009660A2 - HERBAL THERAPY OBTAINED FROM THE DRY EXTRACT OF PIPER UMBELLATUM L. LEAVES AND ITS USE FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE - Google Patents
HERBAL THERAPY OBTAINED FROM THE DRY EXTRACT OF PIPER UMBELLATUM L. LEAVES AND ITS USE FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE Download PDFInfo
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Abstract
fitoterápico obtido a partir do extrato seco das folhas de piper umbellatum l. e seu uso para tratamento de doença inflamatória intestinal. a presente invenção se trata do desenvolvimento de um fitoterápico obtido a partir do extrato de folhas secas de piper umbellatum e seu uso para tratamento de doenças inflamatórias intestinais. para tanto, avaliou-se a atividade e os mecanismos envolvidos no efeito anti-inflamatório intestinal do extrato hidroetanólico das folhas de p. umbellatum (ehpu) na colite ulcerativa induzida em ratos. os animais foram pré-tratados via oral (v.o) com ehpu (30, 100 e 300 mg/kg), mesalazina (500 mg/kg) ou veículo (tween 80 a 2%, 10 ml/kg p.c.). os sinais clínicos do desenvolvimento da doença foram analisados pela determinação de perda de peso, danos macroscópicos, área ulcerada e alterações histológicas. as concentrações teciduais de glutationa (gsh) e malonaldeído (mda) e as atividades da superóxido dismutase (sod), mieloperoxidase (mpo) e catalase (cat) foram determinadas. as concentrações dos mediadores pró-inflamatórios como óxido nítrico (no), fator de necrose tumoral alpha (tnf-a) e interleucina 1 beta (il- 1ß) foram quantificadas por imunoensaio de esferas magnéticas de fluorescência codificada. o tratamento com ehpu reduz significativamente a perda de peso, dano macroscópico, área ulcerada e danos histopatológico como edema, infiltrado celular, ulceração e necrose (p < 0.001). além disso, o ehpu (30, 100, e 300 mg/kg, v.o) reduz os níveis de parâmetros oxidativos como mpo (49,37%, 52,85%, e 61,69%, p < 0,001) e mda (75,50%, 83,01%, 70,34%, p < 0.001), e aumenta a atividade antioxidante por meio de sod (208,57%, 192,50%, 64,42%, p < 0.001), gsh (94,66%, 75,13%, 49,11%, p < 0,01), e cat (92,30%, 69,23%, 107,69%, p < 0,01). o extrato reduz os níveis de no (20,53%, 18,93%, 21,93%, p < 0,01) e das citocinas pró-inflamatórias tnf-a (81,40%, 85,35%, 85,20%, p < 0,001) e il-1ß (94,73%, 78,94%, 89,47%, p < 0,001). em conjunto, os resultados revelaram que o ehpu é um promissor fitoterápico para o tratamento de doenças inflamatórias intestinais.herbal medicine obtained from the dry extract of piper umbellatum l leaves. and its use for the treatment of inflammatory bowel disease. The present invention concerns the development of a herbal medicine obtained from the extract of dried leaves of piper umbellatum and its use for the treatment of inflammatory intestinal diseases. to this end, the activity and mechanisms involved in the intestinal anti-inflammatory effect of the hydroethanolic extract of p. umbellatum (ehpu) in induced ulcerative colitis in rats. the animals were pretreated orally (p.o.) with ehpu (30, 100 and 300 mg/kg), mesalazine (500 mg/kg) or vehicle (2% tween 80, 10 ml/kg b.w.). clinical signs of disease development were analyzed by determining weight loss, macroscopic damage, ulcerated area and histological changes. the tissue concentrations of glutathione (gsh) and malonaldehyde (mda) and the activities of superoxide dismutase (sod), myeloperoxidase (mpo) and catalase (cat) were determined. the concentrations of pro-inflammatory mediators such as nitric oxide (no), tumor necrosis factor alpha (tnf-a) and interleukin 1 beta (il-1ß) were quantified by encoded fluorescence magnetic bead immunoassay. treatment with ehpu significantly reduces weight loss, macroscopic damage, ulcerated area and histopathological damage such as edema, cellular infiltrate, ulceration and necrosis (p < 0.001). Furthermore, ehpu (30, 100, and 300 mg/kg, p.o.) reduces the levels of oxidative parameters such as mpo (49.37%, 52.85%, and 61.69%, p < 0.001) and mda ( 75.50%, 83.01%, 70.34%, p < 0.001), and increases antioxidant activity through sod (208.57%, 192.50%, 64.42%, p < 0.001), gsh (94.66%, 75.13%, 49.11%, p < 0.01), and cat (92.30%, 69.23%, 107.69%, p < 0.01). the extract reduces the levels of no (20.53%, 18.93%, 21.93%, p < 0.01) and the pro-inflammatory cytokines tnf-a (81.40%, 85.35%, 85 .20%, p < 0.001) and il-1ß (94.73%, 78.94%, 89.47%, p < 0.001). Taken together, the results revealed that ehpu is a promising herbal medicine for the treatment of inflammatory bowel diseases.
Description
[001] A presente invenção se trata do desenvolvimento de um fitoterápico obtido a partir do extrato de folhas secas de Piper umbellatum e seu uso para tratamento de doenças inflamatórias intestinais.[001] The present invention concerns the development of an herbal medicine obtained from the dry leaves extract of Piper umbellatum and its use for the treatment of inflammatory bowel diseases.
[002] Doenças inflamatórias intestinais (DIIs), como a Doença de Crhon e a colite ulcerativa, são desordens gastrintestinais crônicas caracterizadas por inflamação e danos à mucosa, com incidência até 8/100.000 pessoas no Brasil, encontrando-se em expansão em todo o mundo.[002] Inflammatory bowel diseases (IBDs), such as Crhon's Disease and ulcerative colitis, are chronic gastrointestinal disorders characterized by inflammation and damage to the mucosa, with an incidence of up to 8/100,000 people in Brazil, being found in expansion throughout the country. world.
[003] Todavia, não se encontra descrito na literatura ou publicado previamente em meios de comunicação nenhum dado relacionado ao uso de P. umbellatum para o tratamento de DIIs como ora proposto, apenas seu uso popular para úlceras gástricas, inflamações sistêmicas e infecções, os quais foram comprovados em ensaios farmacológicos pré-clínicos.[003] However, no data related to the use of P. umbellatum for the treatment of IBD as proposed herein is described in the literature or previously published in the media, only its popular use for gastric ulcers, systemic inflammations and infections, the which have been proven in preclinical pharmacological trials.
[004] Por se tratar de patologias de etiologias desconhecidas, com altas taxas de prevalências em países desenvolvidos e em crescimento em países em desenvolvimento, os tratamentos farmacológicos disponíveis para DIIs visam atenuar os sintomas causados pelas doenças, e compreendem os salicilatos, como o ácido-5-aminossalicílico (5-ASA), uma droga anti-inflamatória não esteroidal utilizada em fases médias e moderadas de atividade da doença, os corticoides, as tiopurinas e, mais recentemente, as drogas anti-TNF-α. Com exceção dos 5-ASA, as demais terapias têm função imunossupressora para controlar a inflamação extensa, e, apenas em casos extremos, ocorrem as intervenções cirúrgicas. Entretanto, a eficácia a longo prazo é encontrada em apenas um terço dos pacientes e, ainda assim, é restringida pelos severos efeitos colaterais que incluem perda da função tecidual, aumento do risco de infecções, linfomas e câncer de pele, resultando na diminuição da qualidade de vida dos portadores.[004] As they are pathologies of unknown etiologies, with high prevalence rates in developed countries and growing in developing countries, the pharmacological treatments available for IBDs aim to alleviate the symptoms caused by the diseases, and include salicylates, such as acid -5-aminosalicylic (5-ASA), a non-steroidal anti-inflammatory drug used in medium and moderate phases of disease activity, corticosteroids, thiopurines and, more recently, anti-TNF-α drugs. With the exception of 5-ASA, other therapies have an immunosuppressive function to control extensive inflammation, and only in extreme cases do surgical interventions occur. However, long-term efficacy is found in only a third of patients and yet it is constrained by severe side effects that include loss of tissue function, increased risk of infections, lymphomas and skin cancer, resulting in decreased quality of life of the carriers.
[005] Vale ressaltar que esses são tratamentos de alto custo e não se encontram disponíveis no Sistema Único de Saúde do Brasil, fator de alto impacto na adesão do tratamento.[005] It is noteworthy that these are high-cost treatments and are not available in the Brazilian Unified Health System, a factor with a high impact on treatment adherence.
[006] Nesse sentido, o tratamento com o extrato hidroetanólico seco das folhas de Piper umbellatum L. (EHPu) aqui descrito é capaz de reduzir as lesões macro e microscópicas causadas pelo ácido 2,4,6-trinitrobenzenossulfônico – TNBS (agente lesivo do reto e colon) no tecido intestinal, o que envolveu uma ação anti-inflamatória ao reduzir as concentrações de mediadores inflamatórios como óxido nítrico e citocinas (IL1β e TNF-α), além de um efeito antioxidante ao aumentar os níveis de GSH e as atividades das enzimas CAT e MDA, agentes relacionados à instalação e progressão do processo inflamatório. Esses efeitos do EHPu foram obtidos com potência e eficácia superiores à droga padrão mesalazina, um aminossalicitado de primeira linha no tratamento de DIIs.[006] In this sense, the treatment with the dry hydroethanolic extract of the leaves of Piper umbellatum L. (EHPu) described here is able to reduce the macro and microscopic lesions caused by 2,4,6-trinitrobenzenesulfonic acid - TNBS (harmful agent of the rectum and colon) in the intestinal tissue, which involved an anti-inflammatory action by reducing concentrations of inflammatory mediators such as nitric oxide and cytokines (IL1β and TNF-α), in addition to an antioxidant effect by increasing GSH levels and activities of CAT and MDA enzymes, agents related to the installation and progression of the inflammatory process. These effects of EHPu were obtained with superior potency and efficacy than the standard drug mesalazine, a first-line aminosalicyte in the treatment of IBDs.
[007] O uso de EHPu não apresentou efeitos colaterais, corroborando com dados descritos na literatura sobre a ausência de toxicidade em modelos in vitro e in vivo, considerado assim seguro e eficaz, além de muito menor custo, para o tratamento de patologias que necessitam de uso contínuo.[007] The use of EHPu had no side effects, corroborating data described in the literature on the absence of toxicity in in vitro and in vivo models, considered safe and effective, in addition to being much less costly, for the treatment of pathologies that require of continuous use.
[008] Vale ressaltar que o EHPu pode ter uso adicional ao uso para DIIs, em pacientes acometidos com úlcera péptica, feridas e/ou câncer, já que apresenta propriedades gastroprotetoras, cicatrizante de feridas e anticâncer bem documentadas.[008] It is noteworthy that the EHPu may have additional use to use for IBDs, in patients with peptic ulcer, wounds and / or cancer, as it has gastroprotective, wound healing and anticancer properties well documented.
[009] Desse modo, o desenvolvimento de produto à base das folhas secas de Piper umbelattum adicionaria ao mercado um produto da flora nativa brasileira, o que poderia favorecer o desenvolvimento da cadeia produtiva de plantas medicinais e fitoterápicos no Brasil.[009] Thus, the development of a product based on the dry leaves of Piper umbelattum would add to the market a product from the Brazilian native flora, which could favor the development of the productive chain of medicinal and herbal plants in Brazil.
[010] Amostras contendo material fértil de Piper umbellatum foram coletadas no município de Aripuanã, estado de Mato Grosso, Brasil, e uma exsicata da espécie contendo encontra-se depositada no Herbário da Universidade Federal de Mato Grosso (UFMT), sob o nº. BI-970.[010] Samples containing fertile material from Piper umbellatum were collected in the municipality of Aripuanã, state of Mato Grosso, Brazil, and an exsiccate of the species containing is deposited in the Herbarium of the Federal University of Mato Grosso (UFMT), under nº. BI-970.
[011] Para acesso ao conhecimento tradicional e ao patrimônio genético associado para fins de pesquisa, solicitou-se uma autorização ao Conselho de Gestão de Patrimônio Genético/Ministério do Meio Ambiente (CGen/MMA, autorizações nº 135/2013 e 199/2014).[011] For access to traditional knowledge and associated genetic heritage for research purposes, an authorization was requested from the Genetic Heritage Management Council/Ministry of the Environment (CGen/MMA, authorizations No. 135/2013 and 199/2014) .
[012] As folhas coletadas foram limpas e secas em estufa de circulação de ar forçado a 35±1 ºC até completa eliminação de humidade e pulverizadas em moinho elétrico de facas tâmis 40. A extração foi realizada por maceração em solução hidroetanólica 70% (1:10 p/v) por 7 dias, com agitação periódica duas vezes ao dia. Após filtração, o macerado foi concentrado em evaporador rotativo sob vácuo e pressão reduzida de 600 mmHg, sendo o solvente residual removido em estufa a 38±1 °C. Em seguida, o extrato concentrado foi congelado em Biofreezer a -86° C, liofilizado, e, por fim, o material obtido foi o extrato hidroetanólico das folhas de P. umbellatum (EHPu).[012] The collected leaves were cleaned and dried in a forced air circulation oven at 35±1 ºC until complete elimination of moisture and sprayed in an electric mill with Tamil knives 40. The extraction was performed by maceration in 70% hydroethanolic solution (1 :10 p/v) for 7 days, with periodic shaking twice a day. After filtration, the macerate was concentrated in a rotary evaporator under vacuum and reduced pressure of 600 mmHg, and the residual solvent was removed in an oven at 38±1 °C. Then, the concentrated extract was frozen in a Biofreezer at -86°C, lyophilized, and, finally, the material obtained was the hydroethanolic extract of P. umbellatum leaves (EHPu).
[013] Ratos albinos (Rattus norvegicus), pesando entre 180-200g, foram fornecidos pelo Biotério Central da Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brasil. Os animais foram alojados em gaiolas de polipropileno à 25±2 °C em ciclo claro/escuro de 12 horas e receberam ração padrão e água ad libitum. Os ratos permaneceram em jejum por 12 horas antes do experimento, com o livre acesso à água. Os procedimentos foram realizados em conformidade com as orientações da Comissão Europeia (2010/63/UE) e os animais tratados de acordo com as diretrizes do Comitê de Ética em Uso de Animais da Universidade Federal do Mato Grosso (CEUA-UFMT).[013] Albino rats (Rattus norvegicus), weighing between 180-200g, were provided by the Central Animal Facility of the Federal University of Mato Grosso (UFMT), Cuiabá, Brazil. The animals were housed in polypropylene cages at 25±2 °C on a 12-hour light/dark cycle and received standard chow and water ad libitum. The rats were fasted for 12 hours before the experiment, with free access to water. The procedures were carried out in accordance with the guidelines of the European Commission (2010/63/EU) and the animals were treated in accordance with the guidelines of the Ethics in Animal Use Committee of the Federal University of Mato Grosso (CEUA-UFMT).
[014] A colite ulcerativa (CU) foi induzida por TNBS em ratos distribuídos em seis grupos (n=8) e tratados oralmente com veículo (Tween 20-2%, 1 mL/100 g), EHPu (30, 100 e 300 mg/kg) e mesalazina (500 mg/kg) durante 72h, 48h, 24h e 2h antes da indução da colite. Após o período de tratamento, os animais foram anestesiados via intraperitoneal (ip.) com cetamina/xilazina (60/8 mg/kg) e, em seguida, a CU foi induzida por administração retal de 250 μL de TNBS (30 mg/mL de TNBS em 20% EtOH) com um cateter de poliuretano n° 6 medindo 8 cm, com exceção do grupo sham. Em seguida, os animais foram mantidos por 2 minutos em decúbito ventral para evitar a expulsão do fluido e confirmação da distribuição uniforme do TNBS. Após 24 horas da indução, os animais foram sacrificados sob aprofundamento da anestesia e os segmentos colônicos foram extirpados, abertos e enxaguados completamente em solução salina 0,9% gelada.[014] Ulcerative colitis (UC) was induced by TNBS in rats divided into six groups (n=8) and treated orally with vehicle (20-2% Tween, 1 mL/100 g), EHPu (30, 100 and 300 mg/kg) and mesalazine (500 mg/kg) for 72h, 48h, 24h and 2h before colitis induction. After the treatment period, the animals were anesthetized via intraperitoneal (ip.) with ketamine/xylazine (60/8 mg/kg) and then UC was induced by rectal administration of 250 μL of TNBS (30 mg/mL) of TNBS in 20% EtOH) with a #6 polyurethane catheter measuring 8 cm, with the exception of the sham group. Afterwards, the animals were kept for 2 minutes in the prone position to avoid fluid expulsion and confirmation of the uniform distribution of TNBS. Twenty-four hours after induction, the animals were sacrificed under deep anesthesia and the colonic segments were excised, opened and completely rinsed in ice-cold 0.9% saline solution.
[015] Os tecidos do cólon foram pesados, adicionados a tampão Tris 10 mM, pH 7,4, NaCl 150 mM e Triton X-100 a 1% e armazenados em um biofreezer a -80 ° C. Amostras contendo tiras de cólon foram descongeladas, desintegradas e homogeneizadas em tampão fosfato de potássio (200 mM, pH 6,5), uma alíquota de 50 μL do homogenato resultante foi utilizada para quantificação do teor de glutationa (GSH) e o restante homogeneizado foi centrifugado a 10538 × g por 20 minutos. O sobrenadante resultante foi utilizado para as determinações das atividades das enzimas catalase (CAT), superóxido dismutase (SOD) e malonaldeído (MDA). O sedimento foi utilizado para determinar a atividade da mieloperoxidase (MPO).[015] Colon tissues were weighed, added to 10 mM Tris buffer, pH 7.4, 150 mM NaCl and 1% Triton X-100 and stored in a biofreezer at -80 °C. Samples containing colon strips were thawed, disintegrated and homogenized in potassium phosphate buffer (200 mM, pH 6.5), a 50 μL aliquot of the resulting homogenate was used to quantify the glutathione (GSH) content and the remaining homogenate was centrifuged at 10538 × g per 20 minutes. The resulting supernatant was used for determinations of the activities of catalase (CAT), superoxide dismutase (SOD) and malonaldehyde (MDA) enzymes. The sediment was used to determine myeloperoxidase (MPO) activity.
[016] A avaliação do teor total de nitrato/nitrito (NO3-/NO2-) foi determinada pelo reagente de Griess, que indica a formação de óxido nítrico. Primeiramente, os tecidos foram pesados e homogeneizados em solução tampão de fosfato de potássio (200 mM, pH 6,5), o homogenato foi centrifugado por 15 minutos a 1500 x g para obter o sobrenadante. As amostras foram plaqueadas (placa de 96 poços) em duplicata (80 μL de cada sobrenadante) e incubadas por 10 min com solução reagente (40 μL da enzima nitrato redutase, substrato NADPH, KH2PO4 em água destilada). Após a conversão do nitrato em nitrito, 80 μL da solução de Griess (1% de sulfanilamida em 1% de H3PO4/0,1% NEED/água destilada) foram adicionados a cada poço. A coloração púrpura final obtida pela reação foi lida em espectrofotometria a 540 nm de absorbância e os resultados foram expressos em μM de NO2, utilizando como referência a curva padrão de nitrito para obtenção dos valores.[016] The evaluation of the total content of nitrate/nitrite (NO3-/NO2-) was determined by the Griess reagent, which indicates the formation of nitric oxide. First, the tissues were weighed and homogenized in a potassium phosphate buffer solution (200 mM, pH 6.5), the homogenate was centrifuged for 15 minutes at 1500 x g to obtain the supernatant. The samples were plated (96-well plate) in duplicate (80 μL of each supernatant) and incubated for 10 min with a reagent solution (40 μL of nitrate reductase enzyme, NADPH substrate, KH2PO4 in distilled water). After the conversion of nitrate to nitrite, 80 µL of Griess solution (1% sulfanilamide in 1% H3PO4/0.1% NEED/distilled water) was added to each well. The final purple color obtained by the reaction was read by spectrophotometry at 540 nm of absorbance and the results were expressed in μM of NO2, using the standard curve of nitrite as a reference to obtain the values.
[017] As tiras de cólon foram descongeladas, desintegradas e homogeneizadas em tampão fosfato 0,2 M e as concentrações de interleucina 1β (IL-1β), interleucina-10 (IL10), interleucina-12 IL-12, interleucina-17 (IL-17) e o fator de necrose tumoral α (TNFα) foram determinados seguindo as instruções descritas no kit Genese Multiplex usando um instrumento de detecção baseado em fluxo duplo de laser.[017] The colon strips were thawed, disintegrated and homogenized in 0.2 M phosphate buffer and concentrations of interleukin 1β (IL-1β), interleukin-10 (IL10), interleukin-12 IL-12, interleukin-17 ( IL-17) and tumor necrosis factor α (TNFα) were determined following the instructions described in the Genese Multiplex kit using a dual laser stream-based detection instrument.
[018] Para avaliar o potencial terapêutico de EHPu como possível droga para o tratamento de DIIs, utilizou-se o modelo de colite ulcerativa induzida por TNBS em ratos. O tratamento oral (v.o) com HPu (30, 100 e 300 mg/kg) e mesalazina (500 mg/kg), durante 4 dias reduziu significativamente a perda de peso corporal. Após 24 horas da indução, os ratos tratados com EHPu mostraram uma redução significativa no escore de lesão intestinal e área ulcerada, bem como na manutenção do peso do cólon nas doses de 30, 100 e 300 mg/kg.[018] To assess the therapeutic potential of EHPu as a possible drug for the treatment of IBDs, the TNBS-induced ulcerative colitis model in rats was used. Oral treatment (v.o) with HPu (30, 100 and 300 mg/kg) and mesalazine (500 mg/kg) for 4 days significantly reduced body weight loss. Twenty-four hours after induction, rats treated with EHPu showed a significant reduction in the intestinal lesion score and ulcerated area, as well as in the maintenance of colon weight at the doses of 30, 100 and 300 mg/kg.
[019] Os efeitos do veículo (Veh, Tween-80 a 2%, 10 mL/kg v.o), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg v.o), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6-trinitrobenzenossulfônico (TNBS) são apresentados na Figura 1 (escore de dano colônico), Figura 2 (alteração no peso corporal em dias), Figura 3 (peso do cólon - 8 cm de comprimento de cada cólon) e Figura 4 (área ulcerada do cólon após 4 dias de tratamento de cada grupo).[019] The effects of vehicle (Veh, 2% Tween-80, 10 mL/kg po), hydroethanolic extract of Piper umbellatum (EHPu, 30, 100 or 300 mg/kg po), mesalazine (500 mg/kg) in the development of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis are shown in Figure 1 (colon damage score), Figure 2 (change in body weight in days), Figure 3 (Colon weight - 8 cm of length of each colon) and Figure 4 (ulcerated area of the colon after 4 days of treatment for each group).
[020] Os valores representam média ± erro padrão (E.P.) para 8 animais/grupo. A análise estatística foi realizada utilizando a análise de variância de uma via ANOVA seguida pelo teste Student-Newman-Keuls para comparações múltiplas (Figuras 1 a 12).[020] Values represent mean ± standard error (S.E.) for 8 animals/group. Statistical analysis was performed using one-way ANOVA analysis of variance followed by Student-Newman-Keuls test for multiple comparisons (Figures 1 to 12).
[021] A avaliação do EHPU nos marcadores intestinais de estresse oxidativo EHPu apontou a redução da atividade da mieloperoxidase (MPO). A migração de neutrófilos observada nos tecidos do cólon com úlceras induzidas por TNBS foi determinada indiretamente pela atividade da MPO. Assim, a atividade de MPO foi significativamente aumentada em 107,21% (p<0,01) em animais com lesões intestinais induzidas por TNBS, quando comparado com o grupo sham. A Figura 5 apresenta os efeitos de mieloperoxidase (MPO) do veículo (Veh, Tween-80 a 2%, 10 mL/kg), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg, v.o.), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6- trinitrobenzenossulfônico (TNBS) e grupo de sham (água destilada, 10 mL/kg, v.o.). O pré-tratamento dos animais com EHPu (30, 100 e 300 mg/kg, v.o) impediu significativamente o aumento da atividade da MPO em 49,37% (p<0,01), 52,85% (p<0,01), 61,69% (p<0,01) e p<0,001) respectivamente. A mesalazina (500 mg/kg) também reduziu a atividade da MPO em 59,67% (p <0,001) quando comparada ao grupo controle do veículo.[021] The evaluation of EHPU in intestinal oxidative stress markers EHPu pointed to a reduction in the activity of myeloperoxidase (MPO). The neutrophil migration observed in colon tissues with TNBS-induced ulcers was indirectly determined by MPO activity. Thus, MPO activity was significantly increased by 107.21% (p<0.01) in animals with TNBS-induced intestinal lesions, when compared to the sham group. Figure 5 shows the myeloperoxidase (MPO) effects of vehicle (Veh, 2% Tween-80, 10 mL/kg), Piper umbellatum hydroethanolic extract (EHPu, 30, 100 or 300 mg/kg, po), mesalazine (500 mg/kg) in the development of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and sham group (distilled water, 10 mL/kg, po). Pre-treatment of animals with EHPu (30, 100 and 300 mg/kg, po) significantly prevented the increase in MPO activity by 49.37% (p<0.01), 52.85% (p<0, 01), 61.69% (p<0.01) and p<0.001) respectively. Mesalazine (500 mg/kg) also reduced MPO activity by 59.67% (p<0.001) when compared to the vehicle control group.
[022] Quanto à avaliação do efeito de EHPu nos níveis de malonadeído (MDA), foi realizada a estimativa do nível de MDA no tecido do cólon ulcerado por TNBS por meio do método TBA. O grupo veículo apresentou aumento expressivo da peroxidação lipídica do MDA quando comparado ao grupo sham. Todas as doses testadas para EHPu (30, 100 e 300 mg/kg) reduziram a peroxidação lipídica, respectivamente, em 75,50%, 83,01% e 70,34%,(*** p<0,001). O padrão mesalazina (500 mg/kg) reduziu significativamente o MDA (58,39%, p<0,001) quando comparado ao grupo veículo. A Figura 6 apresenta a concentração de malonadeído (MDA) nos grupos veículo (Veh, Tween-80 a 2%, 10 mL/kg), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg, v.o.), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6- trinitrobenzenossulfônico (TNBS) e grupo de sham (água destilada, 10 mL/kg, v.o.).[022] Regarding the evaluation of the effect of EHPu on malonadeide (MDA) levels, the estimation of the level of MDA in the colon tissue ulcerated by TNBS was performed using the TBA method. The vehicle group showed a significant increase in MDA lipid peroxidation when compared to the sham group. All doses tested for EHPu (30, 100 and 300 mg/kg) reduced lipid peroxidation, respectively, by 75.50%, 83.01% and 70.34%,(*** p<0.001). The mesalazine standard (500 mg/kg) significantly reduced MDA (58.39%, p<0.001) when compared to the vehicle group. Figure 6 shows the concentration of malonadeide (MDA) in the vehicle groups (Veh, 2% Tween-80, 10 mL/kg), hydroethanolic extract of Piper umbellatum (EHPu, 30, 100 or 300 mg/kg, vo), mesalazine (500 mg/kg) in the development of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and sham group (distilled water, 10 mL/kg, po).
[023] O HEPu reverteu a depleção de glutationa (GSH) na colite ulcerativa induzida por TNBS. A exposição do cólon ao TNBS diminuiu o nível de GSH em animais tratados com veículo (solução aquosa Tween 80 a 2%, 10 mL/kg) quando comparado ao grupo sham. O tratamento com HEPu 30, 100 e 300 mg/kg aumentou significativamente o conteúdo de GSH no cólon em 94,66%, 75,13% e 49,11% (p<0,01), respectivamente, em comparação com o grupo veículo. Mesalazina (500 mg/kg), controle positivo para este experimento, apresentou aumento significativo dos níveis de GSH (52,23%, p<0,01) quando comparado ao grupo veículo. A Figura 7 apresenta os níveis de glutationa (GSH) nos grupos veículo (Veh, Tween-80 a 2% 10 mL/kg), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg, v.o.), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6- trinitrobenzenossulfônico (TNBS) e grupo de sham (água destilada, 10 mL/kg, v.o.).[023] HEPu reversed glutathione (GSH) depletion in TNBS-induced ulcerative colitis. Colonic exposure to TNBS decreased the level of GSH in animals treated with vehicle (2% Tween 80 aqueous solution, 10 mL/kg) when compared to the sham group. Treatment with
[024] O EHPu aumenta a atividade da catalase (CAT) na colite ulcerativa induzida por TNBS. O tratamento dos ratos com TNBS diminuiu a atividade de CAT apenas no grupo veículo (Tween-80 a 2%, 10 mg/Kg) em comparação ao grupo sham. O tratamento com EHPu 30, 100 e 300 mg/kg aumentou a atividade da CAT em 92,30% (p<0,05), 69,23% (p<0,01) e 107,69% (p<0,001), respectivamente, em comparação ao grupo veículo. Mesalazina (500 mg / kg) aumentou a atividade da CAT em 92,30% (p<0,001) quando comparado ao grupo controle. A Figura 8 apresenta a atividade da catalase (CAT) nos grupos veículo (Veh, Tween-80 a 2%, 10 mL/kg), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg, v.o.), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6-trinitrobenzenossulfônico (TNBS) e grupo de sham (água destilada, 10 mL/kg, v.o.).[024] EHPu increases catalase (CAT) activity in TNBS-induced ulcerative colitis. Treatment of rats with TNBS decreased CAT activity only in the vehicle group (2% Tween-80, 10 mg/kg) compared to the sham group. Treatment with
[025] O EHPu aumenta a atividade de superóxido (SOD) na colite ulcerativa induzida por TNBS. A atividade da enzima antioxidante SOD foi determinada no tecido intestinal colônico de ratos tratados com TNBS e os resultados mostraram que o EHPu (30, 100 e 300 mg/kg) aumentou significativamente em 208,57%, 192,50% e 64,42% (p<0,001) a atividade enzimática da SOD. A mesalazina (500 mg/kg), padrão para esse experimento, aumentou significativamente (188,92%, p <0,001) a atividade da SOD, enquanto o grupo veículo (10 mL/kg) mostrou uma redução significativa no nível de SOD quando comparado ao grupo sham. A Figura 9 apresenta a atividade da superóxido dismutase (SOD) nos grupos veículo (Veh, Tween-80 a 2%, 10 mL/kg), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg, v.o.), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6- trinitrobenzenossulfônico (TNBS) e grupo de sham (água destilada, 10 mL/kg, v.o.).[025] EHPu increases superoxide (SOD) activity in TNBS-induced ulcerative colitis. The antioxidant enzyme SOD activity was determined in the colonic intestinal tissue of rats treated with TNBS and the results showed that EHPu (30, 100 and 300 mg/kg) significantly increased by 208.57%, 192.50% and 64.42 % (p<0.001) the enzymatic activity of SOD. Mesalazine (500 mg/kg), standard for this experiment, significantly increased (188.92%, p<0.001) SOD activity, whereas the vehicle group (10 mL/kg) showed a significant reduction in the SOD level when compared to the sham group. Figure 9 shows superoxide dismutase (SOD) activity in vehicle groups (Veh, 2% Tween-80, 10 mL/kg), hydroethanolic extract of Piper umbellatum (EHPu, 30, 100 or 300 mg/kg, vo) , mesalazine (500 mg/kg) in the development of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and sham group (distilled water, 10 mL/kg, po).
[026] A quantidade de nitrito no tecido de ratos ulcerados por TNBS está demonstrada na Figura 10. O pré-tratamento com EHPu nas doses de 30, 100 e 300 mg/kg reduziu a concentração de nitrito, em 53%, 18,93% e 21,93% (p<0.01), respectivamente, quando comparados ao grupo veículo. Mesalazina (500 mg/kg) reduziu a concentração de nitrito para 26.73% (p <0.01). A Figura 10 apresenta a redução do éxido nítrico (NO) nos grupos veículo (Veh, Tween-80 a 2%, 10 mL/kg), extrato hidroetanólico de Piper umbellatum (EHPu, 30, 100 ou 300 mg/kg, v.o), mesalazina (500 mg/kg) no desenvolvimento de colite induzida por ácido 2,4,6-trinitrobenzenossulfônico (TNBS) e grupo de sham (água destilada, 10 mL/kg, v.o.).[026] The amount of nitrite in the tissue of rats ulcerated by TNBS is shown in Figure 10. Pretreatment with EHPu at doses of 30, 100 and 300 mg/kg reduced the concentration of nitrite by 53%, 18.93 % and 21.93% (p<0.01), respectively, when compared to the vehicle group. Mesalazine (500 mg/kg) reduced the nitrite concentration to 26.73% (p < 0.01). Figure 10 shows the reduction of nitric oxide (NO) in the vehicle groups (Veh, 2% Tween-80, 10 mL/kg), hydroethanolic extract of Piper umbellatum (EHPu, 30, 100 or 300 mg/kg, vo) , mesalazine (500 mg/kg) in the development of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and sham group (distilled water, 10 mL/kg, po).
[027] A determinação das concentrações de TNF α e IL-1β na colite ulcerativa induzida por TNBS são apresentadas nas Figuras 11 e 12. O tratamento com EHPu reduziu a concentração de TNF-α em todas as doses testadas, respectivamente, em 94,73%, 78,94%, 89,47% (p<0,001), de maneira comparável ao padrão mesalazina (84,21%, p <0,001) em comparação com o grupo de veículo (Figura 11). A Figura 12 demonstra que o HEPu (30, 100 e 300 mg/kg) reduziu a concentração de IL-1β na colite ulcerativa induzida por TNBS em todas as doses testadas, respectivamente, em 81,40%, 85,35% e 85,20% (p<0,001). Enquanto a mesalazina reduziu em 78,75% (p<0,001), quando comparados ao grupo veículo.[027] The determination of the concentrations of TNF α and IL-1β in ulcerative colitis induced by TNBS are shown in Figures 11 and 12. Treatment with EHPu reduced the concentration of TNF-α at all doses tested, respectively, in 94, 73%, 78.94%, 89.47% (p<0.001), in a manner comparable to the mesalazine pattern (84.21%, p<0.001) compared to the vehicle group (Figure 11). Figure 12 demonstrates that HEPu (30, 100 and 300 mg/kg) reduced IL-1β concentration in TNBS-induced ulcerative colitis at all doses tested, respectively, by 81.40%, 85.35% and 85 .20% (p<0.001). While mesalazine reduced by 78.75% (p<0.001) when compared to the vehicle group.
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