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Antiprotozoal and Antimicrobial Activity Of Shimaa WO etR alL.D J O U R N A L O FWorld PHA JournalRMAC ofY Pharmacy AND PH andAR PharmaceuticalMACEUTIC SciencesAL SCIENCES SJIF Impac t Factor 5.210 Volume 4, Issue 05, 1720-1740. Research Article ISSN 2278 – 4357 ANTIPROTOZOAL AND ANTIMICROBIAL ACTIVITY OF SELECTED MEDICINAL PLANTS GROWING IN UPPER EGYPT, BENI-SUEF REGION Shimaa Mohammed Abdel Gawad1, 2*, Mona Hafez Hetta1, Samir Anis Ross2 and Farid Abd El- Reheim Badria3 1Pharmacognosy Department, Faculty of Pharmacy, Beni-Suef University, 62514, Egypt. 2National Center for Natural Products Research, and Department of Bio Molecular Sciences, School of Pharmacy, University of Mississippi, MS 38677, USA. 3Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, 35516, Egypt. Article Received on ABSTRACT 15 March 2015, Alcoholic extracts of fifty six plants cultivated in Beni-Suef Revised on 06 April 2015, Governorate (Egypt) were screened in vitro for their antiprotozoal and Accepted on 27 April 2015 antimicrobial activities. Emblica officinalis, Quercus infectoria and Punica granatum were the most active as antimalarial with IC50 4.92, *Correspondence for 2.51, 10.61µg/mL respectively against the chloroquine sensitive strain Author Shimaa Mohammed of Plasmodium falciparum and IC50 3.1, 2, 7.4µg/mL respectively Abdel Gawad against chloroquine resistant strain of Plasmodium falciparum. The Pharmacognosy extracts of Ricinus communi, Corchorus olitorius and Psidium guajava Department, Faculty of were the most active as antileishmanial and the percentage of Pharmacy, Beni-Suef inhibition were 91.4%, 90.9% and 90.3% respectively. The extract of University, 62514, Egypt. Emblica officinalis, Punica granatum, Quercus infectoria, Ricinus communi, Tamarix nilotica, Camellia sinensis and Curcuma aromatic were active against Candida glabrata with IC50 <8, <8, <8, 52.25, 17.12, 45.3, 26.91 µg/mL respectively while the extract of Emblica officinalis, Quercus infectoria galls and Curcuma aromatica were the most active against Cryptococcus neoformans with IC50 10.8, <8, 50.6 µg/mL respectively. A good antibacterial activity against MRSA was shown by the ethanol extracts of Spinacia oleracea, Corchorus olitorius, Cyperus alopecuroids, and Sesamum indicum with IC50 13.5, 45.31, 18.73 and 19.32µg/mL respectively. Tannins and phenolic acids constituents of these plants proposed to be responsible for the activity through carbonic anhydrase inhibition. www.wjpps.com Vol 4, Issue 05, 2015 1720 Shimaa et al. World Journal of Pharmacy and Pharmaceutical Sciences KEYWORDS: Medicinal plants; Beni-Suef; Egypt; Antiprotozoal; Antimicrobial; Carbonic anhydrase inhibitors. INTRODUCTION Natural products were used by humans in many countries such as Egypt, China and India as drugs for thousands of years.[1] It was reported in a recent study conducted by the information and decision support centre in Egypt that 23% of the Egyptian use medicinal plants as a remedy.[2] There are more than 250,000 species of higher plants in the world among them only a small percentage of them have been investigated for their potential value as drugs.[3] There are several approaches for selecting plants as candidate for drug discovery such as random selection followed by chemical or biological screening, follow up of ethnomedical uses of plants and biological activity reports.[4,5] There is no previous study could be traced on plants growing in Beni-Suef Governorate as antiprotozoal and antimicrobial agents, therefore the present study focused on a systemic evaluation of a selection of commonly growing plant species in this area. Infectious diseases either protozoal such as malaria and leishmania or microbial infections are considered as the major killing factors in the third world countries and the most important causes of premature death.[6] Difficulty of controlling the sources of infection, the high cost of treatment/prevention, poor compliance, low efficacy, poor safety and drug resistance are the major factors that may retard the treatment of these diseases. The drug resistance has further complicated the treatment of infectious diseases in immune-compromised AIDS and cancer patients. Therefore, there is always need for the development of new and more effective drugs. In this respect, natural products offer good sources for new drug discovery.[6] Various antiparasitic drugs have been developed from natural sources, including Quinine, Artemisinine and Atovaquone as antimalarials and Amphotericin B as antileishmanial drug.[7] The Centers for Disease Control and Prevention[8] received information that 19 locally transmitted malaria cases have been reported in one village in Aswan Governorate in Egypt. These are the first malaria cases seen in Egypt since 1998. www.wjpps.com Vol 4, Issue 05, 2015 1721 Shimaa et al. World Journal of Pharmacy and Pharmaceutical Sciences MATERIALS AND METHODS Plant material The fifty six plants used in this study (Table 1) were collected at the period from September to December 2010 from different regions: Beni-Suef fields and herbal markets. The plants were kindly authenticated by Prof. Dr. Lotfy Boules, Prof. Dr. Mohammed El Gibali, (senior botanists, Faculty of Science, Cairo University), Dr. Mahmoud Omar (lecturer at plant taxonomy Department, Faculty of Science, Beni-Suef University) and Mrs Thérèz Labib (botanist specialist at Orman garden, Giza, Egypt). Voucher specimens (BUPD-35: BUPD- 90) were kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University. Preparation of extracts The air-dried powder of each plant material (100 g) was extracted with 75% ethanol (500 mL ×3). Each extract was filtered off and concentrated using a rotatory evaporator. Each residue was weighed and stored in a refrigerator at −4 °C until used. Microbial strains used Antimalarial activity was tested in vitro against Chloroquine sensitive (D6, Sierraleon) and Chloroquine resistant (W2, Indo-China) strains of Plasmodium falciparum. Antileishmanial activity was tested against Leishmania donovani promastigotes. The used fungal strains were Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), and Aspergillus fumigates (ATCC 90906). The used bacterial strains were methicillin-resistant Staphylococcus aureus (ATCC 33591), Cryptococcus neoformans (ATTC 90113), Staphylococcus aureus (ATTC 29213), Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853), and Mycobacterium intracellulare (ATCC 23068). Standards used For the antimalarial activity, Chloroquine and Artemisine were used as positive controls while Pentamidine and Amphotericin B were used as positive controls in the antileishmanial testing. Amphotericin B was used also as a positive control in the antifungal activity and Ciprofloxacine in the antibacterial. All reference drugs were obtained from (ICN Biomedicals, Ohio). www.wjpps.com Vol 4, Issue 05, 2015 1722 Shimaa et al. World Journal of Pharmacy and Pharmaceutical Sciences Biological Assays Antimalarial screening[9] Crude extracts were initially tested in duplicate against D6 Plasmodium falciparum strain as a primary screen at 15.867 µg/mL and percent inhibition (% inhibition) was calculated relative to negative and positive controls. The extracts which showed more than 50% inhibition proceeded to the secondary assay. In the Secondary antimalarial assay, the samples were dissolved to 20 mg/mL and tested at 47.6, 15.867, and 5.289 µg/mL and IC50s in µg/mL were calculated against both D6 and W2 strains. In addition to P. falciparum strains, samples were tested in the VERO mammalian cell line as an indicator of general cytotoxicity. The selectivity indices (SI) – ratio of VERO IC50 to D6 or W2 IC50- were calculated. All IC50 were calculated using XLFit fit curve fitting software. Antileishmanial screening[10, 11] A culture of Leishmania donovani promastigotes was grown in RPMI 1640 medium supplemented with 10% GIBCO fetal calf serum at 26°C. Growth of leishmanial promastigotes was determined by the Alamar Blue assay (BioSource International, Camarillo, CA). Standard fluorescence was measured by a Fluostar Galaxy plate reader (excitation wavelength, 544 nm; emission wavelength, 590 nm). Crude extracts were initially tested in duplicate as a primary screen at 80µg/ mL and % inhibitions were calculated. Antifungal and antibacterial screening[9] Crude extracts were initially tested in duplicate as a primary screen at 50µg/ mL and % inhibitions were calculated relative to negative and positive controls. The extracts which showed more than 50% inhibition were proceeded to a secondary assay. In the latter, the samples were dissolved to 20mg/ mL and tested at 200, 40, 8µg/mL and then IC50 were determined. RESULTS Antimalarial activity (Table 1) Results showed full inhibition with Emblica officinalis and Quercus infectoria (100% inhibition) followed by Punica granatum (96 % inhibition) with IC50 values of 4.92, 2.51, 10.61µg/mL respectively against chloroquine-sensitive (D6) strain of P. falciparum and IC50 values of 3.1, 2.0, 7.4 µg/mL respectively against chloroquine- resistant (W2) strain of P. falciparum compared to Artemisine (IC50 value of 0.0269 µg/mL ; D6, 0.0165; W2) and www.wjpps.com Vol 4, Issue 05, 2015 1723 Shimaa et al. World Journal of Pharmacy and Pharmaceutical Sciences Chloroquine (IC50 of 0.0098 µg/mL ; D6, 0.187; W2) without showing any cytotoxicity to the mammalian cells (Table 2). Antileishmanial activity (Table1) Ricinus communi, Corchorus olitorius and Psidium guajava showed the best
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