Bioactivity Studies On The Extracts Of Elatostema Umbellatum, Pilea Microphylla And Urtica Dioica (Urticaceae)

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Date
2014-02
Authors
Chahardehi, Amir Modarresi
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Publisher
Universiti Sains Malaysia
Abstract
Three medicinal plants of the Urticaceae family, namely Elatostema umbellatum, Pilea microphylla and Urtica dioica including their 27 different extracts obtained from two different methods of extractions were selected for evaluation of antioxidant, antimicrobial and anticancer activities as well as toxicity study. Extracts of U. dioica showed higher extraction yields (%) than extracts of E. umbellatum and P. microphylla. There were major differences in chloroform and methanol extracts for these plants compared to the other solvents. Crude extracts of methanol of P. microphylla from extraction method I (MEPM I), chloroform extract of P. microphylla from extraction method II (CEPM II), ethyl acetate extract of U. dioica from extraction method II (EAEUD II), butanol extract of U. dioica from extraction method II (BEUD II) and ethyl acetate extract of E. umbellatum from extraction method II (EAEEU II) exhibited better antioxidant activities with higher phenolic and flavonoid contents. EC50 values obtained from the study were EAEEU II at 501.20 μg/mL, MEPM I at 81.30 μg/mL, MEPM II at 94.14 μg/mL, CEPM II at 215.20 μg/mL, DEPM II at 373.50 μg/mL, EAEUD II at 100.10 μg/mL, BEUD II at 503.70 μg/mL compared to at 4.95 μg/mL. As for antimicrobial activity, the results showed that the antimicrobial activities were in the chloroform extract such as CEEU II and CEPM II and in the ethyl acetate extract such as EAEUD I (ethyl acetate extract of U. dioica from extraction method I). The significant inhibitory activity was demonstrated by the extracts BEUD II and CEPM II against Escherichia coli and Methicillin-resistant Staphylococcus aureus (MRSA). The results suggested that these extracts can be used as antimicrobial agents to treat food poisoning because of their ability to inhibit bacterial growth. The SEM observations gave roughly ideas on the effects of ethyl acetate (EAEUD I) and butanol (BEUD II) extracts of U. dioica on the growth of Bacillus spizizenii ATCC 6633 and MRSA, respectively. As for anticancer activity, 22 crude extracts and 4 fractions (from CEPM II) of the three plants were tested against two cancer cell lines (MCF-7 and WEHI-164) using MTT assay. The results analysis revealed that the crude extracts of MEEU II, HEPM I, EAEPM I, EAEPM II and CEUD I showed potential and moderate anticancer activity against both cancer cell lines, where as fraction CEPM IIf14 showed potent anticancer activity against WEHI-164 only. To determine that induction of apoptosis took place, Hoechst 33342/Propidium iodide (PI) staining and flow cytometry analysis using Annexin-V and PI were carried out. For the involvement of apoptosis among the cells treated with CEUD I and MEEU II extracts, it was found that more apoptotic bodies were found in CEUD I treated cells at the concentration of 400 and 800 mg/kg than MEEU II extract, using TUNEL assay. WEHI-164 cancer cell lines which were treated with extracts of MEEU II, CEUD I and CEPM IIf13 at the concentration of 100 μg/mL for 48 hours were stained using double labeled (Annexin-V and PI) and then evaluated using FACScan Flow Cytometry. The cells treated with MEEU II and CEUD I showed apoptotic cells with 18.86 and 17.04%, respectively (as compared to BCG with 26.33% apoptotic cells). However, the selected fraction (CEPM IIf13) showed 12.98% apoptotic cells. The in vivo study using BALB/c mice was carried out where 400 and 800 mg/kg of CEUD I and MEEU II, respectively were performed. The results of the study showed that there were significant (p < 0.05) in decreasing of tumour volume in mice. As a conclusion, extract of CEUD I at 800 mg/kg can be used as a potent anticancer agent. The toxicity assays were also carried out using in vitro (using brine shrimp) and in vivo (using mice) methods. As for the in vivo method, three crude extracts (MEEU II, CEPM II and CEUD I) were selected to determine their toxicity on mice. It was found that extracts CEPM II and CEUD I were non-toxic (LD50 > 2000 mg/kg) and extract MEEU II showed slightly toxic (1903 mg/kg) when administered acutely to mice via oral route. The results also showed that the extracts CEPM II and CEUD I which were administered intra-peritoneal and oral routes were non-toxic. However, MEEU II extract was toxic (620 mg/kg). On the other hand, the MEEU II extract of E. umbellatum showed a moderate toxicity. The extracts of MEEU II (intraperitoneal administration) given to male and female mice, CEPM II (intra-peritoneal administration) given to female mice the CEUD I (intra-peritoneal administration) given to male and female mice did not affect body weight gain and did not significantly increase or decrease in the internal organ weights of the mice studied. Furthermore, there was no death recorded on mice after 48 hours of the administration of the CEPM II and CEUD I extracts, except for MEEU II at high doses via both route of administrations. No behavioural changes were observed to the mice given extracts of CEPM II and CEUD I, except diarrhea at high doses. Exception occurred to the mice given MEEU II extract at high doses (800 and 2000 mg/kg) where they exhibited convulsion and dizziness. These toxicity studies suggested that MEEU II extract at low concentration and the CEPM II and CEUD I at higher concentrations were safe to use. Histopathological study on the spleen of the mice given CEPM II and CEUD I extracts were found no increasing in the severity of the lesions. However, there were slight severity of lesions observed in xxxiv spleens, lungs and kidneys of the mice given MEEU II extract. Referring to the liver marker evaluation (ALT and AST) as indicators for hepatotoxicity, the mice that were given MEEU II extracts exhibited significant elevation of ALT and AST levels than the group that were given extracts of CEPM II and CEUD I. As for in vitro toxicity assay using brine shrimp (A. salina), it was found that E. umbellatum extracts showed higher cytotoxicity activity against A. salina compared to CEPM II and CEUD I. The low toxicity for CEPM II and CEUD I proved that they are safe for therapeutic dosage uses.
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Keywords
Medicinal Plants , Antioxidant , Antimicrobial , Anticancer
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