Abstracts
PURPOSE: The use of medicinal plants for the treatment of human diseases has increased worldwide. Many of them are used by oral administration and, after absorption, may affect many organs. Therefore, this study aimed at assessing the effects of the aqueous extract of Sida cordifolia leaves, popularly known in Brazil as "malva-branca", on liver regeneration. METHODS: Twenty rats were divided into four groups: control, Sida100, Sida200 and Sida400 groups. All animals were submitted to oral administration of distilled water, 100, 200 and 400 mg/kg of the aqueous extract of Sida cordifolia, respectively. Immediately after this, they underwent 67% partial hepatectomy. Twenty four hours later, their livers were removed. Hepatic regeneration was assessed by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) using the PC-10 monoclonal antibody. RESULTS: Sida100 and Sida200 groups disclosed higher liver regeneration indices than control group (p<0.001 and p<0.05, respectively). CONCLUSION: The aqueous extract of Sida cordifolia stimulates liver regeneration after 67% partial hepatectomy in rats.
Partial Hepatectomy; Liver Regeneration; Sida cordifolia; Medicinal Plant
INTRODUÇÃO: O uso de plantas medicinais para o tratamento de patologias humanas tem aumentado em todo mundo. Muitas delas são usadas por administração oral, e após a absorção podem afetar muitos órgãos. OBJETIVO: Esse estudo, tem como objetivo verificar o efeito do extrato aquoso de Sida cordifolia, popularmente conhecida no Brasil como "malva-branca", na regeneração hepática. MÉTODOS: Vinte ratos foram divididos em 4 grupos: controle, Sida 100, Sida 200 e Sida 400. Os animais foram submetidos a administração oral de água destilada, 100, 200 e 400 mg/kg de extrato aquoso de Sida cordifolia, respectivamente. Imediatamente após, foi realizada hepatectomia parcial 67%. Vinte quatro horas após, os fígados foram removidos. A regeneração hepática foi avaliada por imunohistoquímica (PCNA), usando o anticorpo monoclonal PC-10. RESULTADOS: Os grupos Sida100 e Sida200 mostraram índices de regeneração hepática maiores que o grupo controle (p<0.001 e p<0.05, respectivamente). CONCLUSÃO: O extrato aquoso de Sida cordifolia estimula a regeneração hepática após hepatectomia parcial a 67% em ratos.
Hepatectomia Parcial; Regeneração Hepática; Sida cordifolia; Planta Medicinal
ORIGINAL ARTICLE
Effect of the aqueous extract of Sida cordifolia on liver regeneration after partial hepatectomy1 1 Study performed in the Laboratory of Biochemistry of Liver Transplantation Unit of Division of Gastroenterology of the Departament of Surgery and Anatomy of Ribeirão Preto Faculty of Medicine-University of São Paulo (FMRP USP), Brazil.
Efeito do extrato aquoso de Sida cordifolia na regeneração hepática após hepatectomia parcial
Renata Lemos SilvaI; Gustavo Barreto de MeloI; Valdinaldo Aragão de MeloII; Ângelo Roberto AntoniolliIII; Paulo Roberto Teixeira MichelloneIV; Sérgio ZucolotoV; Maria Aparecida Neves Cardoso PicinatoVI; Clarice Fleury Fina FrancoVI; Gustavo de Assis MotaVII; Orlando de Castro e SilvaVIII
IGraduate student of Federal University of Sergipe, Brazil
IIAssociate Professor of the Department of Medicine, Federal University of Sergipe, Brazil
IIIAssociate Professor of the Department of Physiology, Federal University of Sergipe, Brazil
IVAssociate Professor of the Marília Faculty of Medicine (FAMEMA), São Paulo, Brazil
VFull Professor of the Department of Pathology, (FMRP-USP), Brazil
VIBiomedical of Laboratory of Biochemistry of Liver Transplantation Unit of the Departament of Surgery and Anatomy, (FMRP-USP), Brazil
VIIMD, Assistant of the Department of Surgery and Anatomy, (FMRP-USP), Brazil
VIIIFull Professor and Head of Division of Gastroenterology of the Department of Surgery and Anatomy, (FMRP-USP), Brazil
Correspondence Correspondence: Orlando de Castro e Silva Jr. Rua Campos Salles, 809 - 9º andar CEP: 14015-110 Centro, Ribeirão Preto - SP - Brazil. Email: orlando@fmrp.usp.br
ABSTRACT
PURPOSE: The use of medicinal plants for the treatment of human diseases has increased worldwide. Many of them are used by oral administration and, after absorption, may affect many organs. Therefore, this study aimed at assessing the effects of the aqueous extract of Sida cordifolia leaves, popularly known in Brazil as "malva-branca", on liver regeneration.
METHODS: Twenty rats were divided into four groups: control, Sida100, Sida200 and Sida400 groups. All animals were submitted to oral administration of distilled water, 100, 200 and 400 mg/kg of the aqueous extract of Sida cordifolia, respectively. Immediately after this, they underwent 67% partial hepatectomy. Twenty four hours later, their livers were removed. Hepatic regeneration was assessed by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) using the PC-10 monoclonal antibody.
RESULTS: Sida100 and Sida200 groups disclosed higher liver regeneration indices than control group (p<0.001 and p<0.05, respectively).
CONCLUSION: The aqueous extract of Sida cordifolia stimulates liver regeneration after 67% partial hepatectomy in rats.
Key words: Partial Hepatectomy. Liver Regeneration. Sida cordifolia. Medicinal Plant.
RESUMO
INTRODUÇÃO: O uso de plantas medicinais para o tratamento de patologias humanas tem aumentado em todo mundo. Muitas delas são usadas por administração oral, e após a absorção podem afetar muitos órgãos.
OBJETIVO: Esse estudo, tem como objetivo verificar o efeito do extrato aquoso de Sida cordifolia, popularmente conhecida no Brasil como "malva-branca", na regeneração hepática.
MÉTODOS: Vinte ratos foram divididos em 4 grupos: controle, Sida 100, Sida 200 e Sida 400. Os animais foram submetidos a administração oral de água destilada, 100, 200 e 400 mg/kg de extrato aquoso de Sida cordifolia, respectivamente. Imediatamente após, foi realizada hepatectomia parcial 67%. Vinte quatro horas após, os fígados foram removidos. A regeneração hepática foi avaliada por imunohistoquímica (PCNA), usando o anticorpo monoclonal PC-10.
RESULTADOS: Os grupos Sida100 e Sida200 mostraram índices de regeneração hepática maiores que o grupo controle (p<0.001 e p<0.05, respectivamente).
CONCLUSÃO: O extrato aquoso de Sida cordifolia estimula a regeneração hepática após hepatectomia parcial a 67% em ratos.
Descritores: Hepatectomia Parcial. Regeneração Hepática. Sida cordifolia. Planta Medicinal.
Introduction
Sida cordifolia L. (Malvaceae) is popularly known as "malva-branca" (white mallow) or "malva-branca-sedosa" (silky white mallow) in Brazil. It grows as a bush of up to 2 m in height. The leaves are light green, cordiform, serrated and oval-elongated, and the pedunculated flowers are arranged in axillary or terminal racemes. In traditional medicine, the plant is used for the treatment of stomatitis, blenorrhea, asthmatic bronchitis and nasal congestion1. In Brazil, it is also used due to its anti-inflammatory properties. Some studies have demonstrated the presence in the leaves of sympathomimetic amines, ephedrine and pseudoephedrine, vasocinone 2 and vasicine as major alkaloids 3. Other studies also showed the presence of fatty oils, steroids, resin, resin acids, mucin and potassium nitrate in the plant 4. The effectiveness of the extract of Sida cordifolia leaves as analgesic, anti-inflammatory (inhibition of prostaglandin synthesis) and hypoglycaemic has been reported 4,1. Considering that there are no studies defining the effects of this plant on liver, this research aimed at assessing the effects of Sida cordifolia leaves on liver regeneration after 67% partial hepatectomy in rats.
Methods
Plant material and preparation of the aqueous extract - Sida cordifolia leaves (identified by Dr C. Dias Silva Jr.; voucher no. 30171, deposited in the Department of Biology at Universidade Federal de Sergipe) were collected outside the blossoming period from the Live Pharmacy of Aracaju, Brazil. The leaves were dried in an oven with air renewal and circulation (model MA-037) at 37° C until complete dehydration. Dried Sida cordifolia leaves were triturated in a blender until a finely granulated powder was obtained. The extract was got from this powder (100 g) by adding distilled water (3:10 w/v) under constant shaking for 4 h at 35° C, followed by filtration (pH 6.0). The filtrate was lyophilized (aqueous extract) and stored at 5° C, yielding 16.2 g (16.2%) of lyophilized active material. At the time of use, the extract was resuspended in distilled water at the desired concentrations.
Animals - The study was carried out on 20 male Wistar albino rats (180 to 230 g). They were fed with a standard pellet diet and water ad libitum. All animals were randomly assigned to 4 groups, which consisted of 5 rats each. Control group animals were submitted to oral administration of distilled water at the time of surgery. Rats of Sida100, Sida200 and Sida400 groups underwent partial hepatectomy and oral administration of 100, 200 and 400 mg/kg of the aqueous extract of Sida cordifolia.
Surgical procedure - All operations were performed under ether anesthesia as it is not hepatotoxic. They always happened at the same time in order to avoid the influence of the changes in circadian cycle. The median and left lateral lobes of the liver (corresponding to 67% of the organ) were excised 5. After surgery, the rats were on a normal diet for the whole experimental period. Twenty four hours later, they underwent a new operation to remove the remaining liver. Then, they were sacrificed.
Liver regeneration analysis - Liver regeneration was evaluated by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) using monoclonal primary anti-PCNA antibody (PC-10; DAKO A/S, Glostrup, Denmark) on formalin-fixed and paraffin-embedded liver tissues 6. Sections were cut at 4 µm, mounted on poly-L-lysine-coated glass slides, deparaffinized, rehydrated in an increasing alcohol series, placed in PBS, and treated with 2% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. Nonspecific protein binding was blocked by preincubation with 5% normal horse serum diluted in PBS for 30 min. This was followed by incubation with PC-10 monoclonal primary antibody, diluted 1:40 in PBS for 120 min at 25ºC. The sections were then incubated for 35 min with a biotinylated horse antimouse immunoglobulin. The reaction product was detected with an avidin-biotin-peroxidase complex and diaminobenzidine was used as a chromogen substrate. Positive and negative controls were used to assess and control the staining procedure. Sections were examined blindly at high power (400X), and 20 fields were chosen at random. Nuclear labeling indices for PCNA (positive nuclei/total number of counted nuclei) were determined by evaluation of at least 1,000 hepatocyte nuclei.
Statistical analysis of data - Mann-Whitney's test was used to compare the results between the groups, with the level of significance set to 5%.
Results
The results are shown in Table 1.
Discussion
Hepatocyte regeneration index was significantly increased in Sida100 group when compared to control group (p<0.001) and all other groups. Sida200 group also disclosed a statistically significant increase in regeneration rate in comparison to control group (p<0.05). After many studies, some regulation factors that trigger or control the liver regeneration phenomenon had their effect demonstrated on hepatocyte proliferation. The elucidation of the mechanisms involved in this phenomenon has been possible through cultured rat hepatocytes and gene expression pattern after partial hepatectomy. Among these substances, there are the ones that act directly or indirectly on DNA synthesis. They are called mitogens and co-mitogens, respectively. The main mitogens are: hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFá) and acidic fibroblast growth factor (aFGF). The co-mitogens are: hepatic stimulatory substance (HSS), norepinephrine and alpha-1 adrenoreceptors, vasopressin, angiotensin I and II, insulin, glucagon, estrogens, testosterone, immunosuppressive agents, bradykinin, angiotensinconverting enzyme inhibitors, tumor necrosis factor alpha (TNFá) and prostaglandins 6,7. In 1999, it was disclosed that the extract of Sida cordifolia leaves, which was proved to be anti-inflammatory, analgesic and hypoglycaemic, augments insulin release after its administration4. The alcoholic extract of the plant, at 150, 300 and 600 mg/kg, was orally administered to Wistar rats. Then it was noticed a significant decrease in glucose levels. These findings suggested that Sida cordifolia stimulates insulin release by pancreatic â cells, acting like sulphonylureas. Knowing that insulin acts as an important co-mitogen 8,9 it can be raised the hypothesis that the augmented release of this hormone, stimulated by Sida cordifolia, can exert a permissive role in the hepatocellular synthesis of DNA and, consequently, in liver regeneration process. It is important to mention that this is only one idea to explain how the aqueous extract of Sida cordifolia leaves causes stimulation of liver regeneration. Due to the fact that hepatocyte proliferation involves many different factors, it is important that more studies are carried out in order to establish other mechanisms to explain these results. These results show that the aqueous extract of Sida cordifolia leaves at single oral doses of 100 and 200 mg/kg causes a high liver regeneration rate in rats submitted to partial hepatectomy.
Conflict of interest: none
Financial source: CNPq and FAPESP
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Publication Dates
-
Publication in this collection
20 Sept 2006 -
Date of issue
2006