|Year : 2019 | Volume
| Issue : 62 | Page : 34-37
In vitro antileishmanial activity of methanolic extracts for some selected medicinal plants
Ahmed Gomaa Gomaa Darwish1, Mamdouh Nabil Samy2, Sachiko Sugimoto3, Katsuyoshi Matsunami3, Hideaki Otsuka4
1 Department of Biochemistry, Faculty of Agriculture, Minia University, Minia, Egypt; Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
2 Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt
3 Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
4 Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University; Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan
|Date of Submission||16-Nov-2018|
|Date of Decision||17-Dec-2018|
|Date of Web Publication||26-Apr-2019|
Ahmed Gomaa Gomaa Darwish
Department of Biochemistry, Faculty of Agriculture, Minia University, Minia 61519
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The aim of this study is to evaluate the antileishmanial activity of selected medicinal plants; ten well-known medicinal plants cultivated and growing under African environmental conditions were studied. Materials and Methods: The methanolic extracts of these plants were screened for their antileishmanial activity against Leishmania major using 3-(4.5-dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide assay. Results: The methanol extract of Colchicum autumnale and Alpinia officinarum showed potent antileishmanial activity at inhibition% value of 98.29% ± 0.75% and 97.25 ± 1.63%, respectively, while Silybum marianum exhibited inhibition% value of 90.97% ± 1.13%, compared with the standard amphotericin B (89.31% ± 2.08%). Conclusion: Considering these results, medicinal plants from African environment could constitute a developer source for antileishmanial compounds.
Keywords: 3-(4.5-dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide assay, Alpinia officinarum, amphotericin B, antileishmanial, Rosa damascene, Silybum marianum
|How to cite this article:|
Gomaa Darwish AG, Samy MN, Sugimoto S, Matsunami K, Otsuka H. In vitro antileishmanial activity of methanolic extracts for some selected medicinal plants. Phcog Mag 2019;15, Suppl S1:34-7
|How to cite this URL:|
Gomaa Darwish AG, Samy MN, Sugimoto S, Matsunami K, Otsuka H. In vitro antileishmanial activity of methanolic extracts for some selected medicinal plants. Phcog Mag [serial online] 2019 [cited 2019 Oct 22];15, Suppl S1:34-7. Available from: http://www.phcog.com/text.asp?2019/15/62/34/257280
- The medicinal plants from African environment such as Colchicum autumnale and Alpinia officinarum could establish a developer source for antileishmanial compounds.
Abbreviations used: FBS: Fetal bovine serum; MTT: 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium; DMEM: Dulbecco's modified Eagle's medium; DMSO: Dimethyl sulfoxide.
| Introduction|| |
Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. It spreads through sandflies. Their primary hosts are vertebrates; Leishmania commonly infects hyraxes, Canidae, rodents, and humans and currently affects more than 12 million people. Leishmaniasis is a disease with a prolonged worldwide distribution in 98 countries. Leishmaniasis is considered as a serious health problem worldwide, especially in Africa, where it is significant morbidity and mortality., Chemotherapy of leishmaniasis is still inspiring, due to the limitation of the efficiency of the drug, for example, miltefosine was the first oral antileishmanial drug that is considered for the treatment of visceral leishmaniasis in India and Germany and for cutaneous leishmaniasis in Colombia. In vitro Leishmania promastigotes resistant to miltefosine concentrations of up to 40 μM were easily produced and resistance was conferred to the intracellular amastigote stage., Amphotericin B was originally extracted from Streptomyces nodosus. Amphotericin B deoxycholate (Fungizone®), a micellar formulation, is highly effective. It is used as first-line treatment in zones with high rates of unresponsiveness to antimonials and second-line treatment elsewhere. In addition to many other drugs such as pentavalent antimonials, paromomycin, sitamaquine, 2-substituted quinoline alkaloids, buparvaquone, and 8-aminoquinolines, solid nanoparticles of amphotericin B deoxycholate have shown activity against Leishmania donovani. However, many parasites are resistant to these drugs. Medicinal plants are a good source of bioactive phytochemicals that showed several pharmacological properties such as antibacterial,, antioxidant, antitumor, antifungal, anti-litholytic,, and antileishmanial activities., These secondary metabolites are complex molecules with various functional structures such as polyphenols, flavonoids, terpenoids, and coumarins. In this way, recent studies that focused on antileishmanial activities of medicinal plant products showed the success of these products in the inhibition of growth of several Leishmania species such as Leishmania major (cutaneous leishmaniasis) and Leishmania infantum (visceral leishmaniasis). However, there is an under exploitation of the explored medicinal plants such as Alpinia officinarum, Achillea millefolium, Colchicum autumnale, Chrysanthemum morifolium, Humulus lupulus, Matricaria chamomilla, Tilia tomentosa, Rosa damascena, Silybum marianum, and Vitex agnus-castus. These plants have important charges of phenolic and flavonoid contents and possess significant antibacterial and antioxidant effects.,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The recent studies suggested that the following in vitro and in vivo models, respectively, are the most suitable for the assessment of antileishmanial drugs: L. major–C57BL/6 mice (or–vervet monkey, or–rhesus monkeys), Leishmania tropica–CsS-16 mice, Leishmania amazonensis–CBA mice, Leishmania braziliensis–golden hamster (or–rhesus monkey). The aim of our study was the screening of the antileishmanial activity of some selected plant extracts against L. major.
| Materials and Methods|| |
The plants were collected in February 2014 from Botanical Garden, Giza, Egypt. The plant was kindly identified by Eng. Esraa Mohamed, Department of Agricultural Chemistry, Faculty of Agriculture, Minia University, Egypt. A voucher specimen of the plant was deposited in the Herbarium of the Department of Agricultural Chemistry, Faculty of Agriculture, Minia University, Egypt (Mn-Agri-1-10). The collected parts of plants [Table 1] were separated, cleaned from dust, and placed in the shade inside a well-ventilated room until were completely dried and weight was obtained. Dried parts of plants were grounded to a fine powder.
Solvents and chemicals
Dulbecco's modified Eagle's medium (SIGMA), penicillin-streptomycin (WAKO 119-00703), fetal bovine serum (FBS), and 3-(4,5 Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) was obtained from Nacalai Tesque.
Preparation of plant extracts
The air-dried powdered parts (250 g) of A. officinarum, A. millefolium, C. autumnale, C. morifolium, H. lupulus, M. chamomilla, T. tomentosa, R. damascene, S. Marianum, V. agnus-castus were extracted with 70% methanol (3 L × 3) till exhaustion and concentrated under reduced pressure at 50°C using a rotary evaporator to yield viscous gummy materials, and then, they subjected to drying in vacuum desiccators (oil pump) to yield 84.58, 68.67, 91.46, 56.34, 102.78, 62.56, 78.74, 86.60, 82.45, and 96.21 g, respectively. All the extracts kept in the dark bottles at 4°C (cold room).
Determination of the antileishmanial activity
The leishmanial activities of methanolic extracts were performed using the colorimetric MTT assay. Medium 199 supplemented with 10% heat-inactivated FBS and 100 μg/ml of kanamycin was used as the cell culture medium. The methanolic extracts were dissolved in dimethyl sulfoxide (DMSO) and added to each well of the 96-well micro-titration plates at 1% as the final concentration. L. major cells (2 × 105 cells/well) were cultured in a CO2 incubator at 25°C for 72 h, and then, MTT solution was added to each well and the plates were incubated overnight at 25°C. The absorbance was measured at 540 nm using a Molecular Device Versamex tunable microplate reader. Amphotericin B was used as a positive control. The inhibition% was calculated using the following equation:
%Inhibition = [1− (Asample− Ablank)/(Acontrol− Ablank)] × 100
Where Acontrol is the absorbance of the control reaction mixture (containing DMSO and all reagents except for the methanolic extracts). IC50 was determined as the concentration of the sample required to inhibit the formation of MTT formazan by 50%.
The analysis was performed using data analysis and statistical application available for Microsoft Excel (XLSTAT 2018.3.16, Florida, USA).
| Results|| |
The aim of this study was to evaluate the antileishmanial activity for ten methanolic plant extracts.
Antileishmanial activity of the plant extracts
The results are shown in [Table 2] and [Figure 1]. The methanolic extract of C. autumnale and A. officinarum with concentration 100 μM showed antileishmanial activity at inhibition% value of 98.29% ±0.75% and 97.25% ±1.63%, respectively, compared to the standard amphotericin B (99.13% ±2.08%), while the remaining tested extracts had no antileishmanial activity.
|Table 2: Antileishmanial activity percentage and IC50 values for the selected plants|
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|Figure 1: The antileishmanial activity at a concentration (100 μM) of the plant extracts. Inhibition percentages are expressed as mean values ± standard deviation of triplicates|
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| Discussion|| |
A. officinarum, A. millefolium, C. autumnale, C. morifolium, H. lupulus, M. chamomilla, Tilia tomentosa, R. damascena, S. marianum, and V. agnus-castus were found to have multiple biological activities and broad traditional uses against infectious and non-infectious diseases. A. officinarum is used in folk medicine as anticancer, antioxidant, antifungal, and antimicrobial.,,, Conventionally, A. millefolium is used as antiseptic, antispasmodic, astringent, carminative, diaphoretic, digestive emmenagogue, stimulant, tonics, vasodilator, and vulnerary and also used against colds, cramps, fevers, and kidney disorders., C. autumnale is used as anti-inflammatory, antimitotic, and antifibrotic activity and involved in the inhibition of microtubule formation., C. morifolium possesses antihepatotoxic and antigenotoxic effects. It exhibits an allelopathic activity and has anti-inflammatory, immunomodulatory, humoral, and cellular and mononuclear phagocytic activities. H. lupulus is traditionally used to relieve insomnia, anxiety, excitability, restlessness associated with tension, headache, and gastrointestinal spasms., M. chamomilla showed different pharmacological activities such as anti-inflammatory, anticancer, anti-allergic activities and is used in the treatment of stress and depression., T. tomentosa has been used as diuretic, diaphoretic, antispasmodic, stomachic, and sedative activities and has been taken for the treatment of flu, cough, migraine, nervous tension, ingestion problems, various types of spasms, and liver disorders.,, R. damascena has been used as cardiotonic, mild laxative, anti-inflammatory, cough suppressant, anti-HIV, antibacterial, and antitussive.,,,,, S. marianum has hepatoprotective and antidepressant activities and is used in the treatment of diabetes, varicose veins, selenic congestions, amenorrhea, and uterine hemorrhage.,, The essential oils of V. agnus-castus have antifungal and antimicrobial activities.
The therapeutic targets and the mode of action for some chemotherapeutic agents such as miltefosine suggested that uptake of miltefosine into L. donovani is mediated by a plasma membrane P-type ATPase aminophospholipid translocase. The proposed targets of miltefosine in Leishmania include perturbation of ether-lipid metabolism, glycosylphosphatidylinositol anchor biosynthesis and signal transduction as well as inhibition of the glycosomal located alkyl-specific acyl-Co-A acyltransferase, an enzyme involved in lipid–remodeling. Recently, mitochondria and specifically the cytochrome c oxidase have been implicated as a target of miltefosine in L. donovani promastigotes. Effects on lipid metabolism, specifically phospholipid content, fatty acid, and sterol content, have also been described in L. donovani promastigotes. However, paromomycin as a chemotherapeutic agent in Leishmania spp. has implicated mitochondrial membrane depolarization, ribosomes, and respiratory dysfunction in the mode of action of this molecule. In the present study, the antileishmanial activity of ten plants was evaluated for the first time. The methanol extract of C. autumnale and A. officinarum showed potent antileishmanial activity at inhibition% value of 98.29% ± 0.75 and 97.25% ± 1.63%, respectively, at concentration 100 μg/ml with IC50 60.09 ± 0.81 and 65.16 ± 2.71 μg/ml, respectively, while S. marianum exhibited inhibition % value of 90.97% ± 1.13% with IC50 77.34 ± 3.01 μg/ml, compared with the standard amphotericin B (89.31% ±2.08%).
The remaining plant extracts did not show any antileishmanial activity at a concentration of 100 μg/ml.
| Conclusion|| |
The results demonstrate that the medicinal plants are a good source of new antileishmanial drugs. Future studies will be conducted to study the different fractions of the most effective extracts to identify the main phenolic components responsible for the antileishmanial and anticancer activities.
This work was supported by the Ministry of Higher Education, Egypt through the Scientific Mission System and the authors are grateful for the Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan, Department of Biochemistry, Faculty of Agriculture and Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Egypt.
Financial support and sponsorship
The experimental study was sponsored by the Ministry of Higher Education, Egypt and the Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan.
Conflicts of interest
The authors declare no conflict of interest
| References|| |
Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012;7:e35671.
Chiheb S, Guessous-Idrissi N, Hamdani A, Riyad M, Bichichi M, Hamdani S, et al. Leishmania tropica
cutaneous leishmaniasis in an emerging focus in North Morocco: New clinical forms. Ann Dermatol Venereol 1999;126:419-22.
El Aasri A, El Madhi Y, Najy M, El Rhaouat O, Belghyti D. Epidemiology of Cutaneous leishmaniasis in Sidi Kacem province, Northwestern Morocco (2006-2014). Asian Pac J Trop Dis 2016;6:783-6.
Seifert K, Matu S, Javier Pérez-Victoria F, Castanys S, Gamarro F, Croft SL, et al.
Characterisation of Leishmania donovani
promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 2003;22:380-7.
Seifert K, Pérez-Victoria FJ, Stettler M, Sánchez-Cañete MP, Castanys S, Gamarro F, et al.
Inactivation of the miltefosine transporter, LdMT, causes miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani
and persists in vivo
. Int J Antimicrob Agents 2007;30:229-35.
Alvar J, Croft S, Olliaro P. Chemotherapy in the treatment and control of leishmaniasis. Adv Parasitol 2006;61:223-74.
Bouyahya A, Bakri Y, Khay EO, Edaoudi F, Talbaoui A, Et-Touys A, et al
. Antibacterial, antioxidant and antitumor properties of Moroccan medicinal plants: A review. Asian Pac Trop Dis 2017;7:57-64.
Bouyahya A, Abrini J, Talbaoui A, Et-Touys A, Chatoui K, Harhar H, et al
. Phytochemical screening, antiradical and antibacterial activities of Cistus crispus
from Morocco. J Mater Environ Sci 2017;8:1560-6.
Bouyahya A, Bensaid M, Bakri Y, Dakka N. Phytochemistry and ethnopharmacology of Ficus carica
. Int J Biochem Res Rev 2016;14:1-12.
Aneb M, Talbaoui A, Bouyahya A, El Boury H, Amzazi S, Benjouad A, et al
cytotoxic effects and antibacterial activity of Moroccan medicinal plants Aristolochia longa
and Lavandula multifida
. Eur J Med Plant 2016;6:1-13.
Fadel F, Ben Hmamou D, Salghi R, Chebli B, Benali O, Zarrouk A, et al
. Antifungal activity and anti-corrosion inhibition of Origanum compactum
extracts. Int J Electrochem Sci 2013;8:11019-32.
Khouchlaa A, Ait BA, Lahcen S, Youssef B, Nadia D, Tijane M. Phytochemical screening, evaluation of antioxidant activity and litholytic effect of Zizyphus lotus
L. extracts. World J Pharm Res 2017;6:1354-67.
Khouchlaa A, El-Yahyaoui A, Bouyahya A, Tijane M. Determination of phenol content and evaluation of in vitro
litholytic effects on urolithiasis of Moroccan Zizyphus lotus
L. extract. Phytotherapie 2017;6:14-9.
Et-Touys A, Fellah H, Mniouil M, Bouyahya A, Dakka N, Abdennebi EH, et al
. Screening of antioxidant, antibacterial and antileishmanial activities of Salvia officinalis
L, extracts from Morocco. Br Microbiol Res J 2016;6:1-10.
Et-Touys A, Fellah H, Sebti F, Mniouil M, Aneb M, Elboury H, et al
antileishmanial activity of extracts from endemic Moroccan medicinal plant Salvia verbenaca
(L.) briq. Spp verbenaca Maire (S. clandestina
Batt. non L). Eur J Med Plants 2016;16:1-8.
Lahlou M. The success of natural products in drug discovery. Pharmacol Pharm 2013;4:17-31.
Essid R, Rahali FZ, Msaada K, Sghair I, Hammami M, Bouratbine A, et al
. Antileishmanial and cytotoxic potential of essential oils from medicinal plants in Northern Tunisia. Indus Crop Prod 2015;77:795-802.
Ray PG, Majamadar SK. New anti-fungal substance from Alpinia officinarum
hance. Indian J Exp Biol 1976;13:409.
Asolkar LV, Kakkar KK, Chakre OJ, Chopra RN, Nayar SL. Glossary of Indian Medicinal Plants. (A-K). New Delhi, India: Publications and Information Directorate; 1992. p. 1-13.
An N, Zou ZM, Tian Z, Luo XZ, Yang SL, Xu LZ, et al.
Diarylheptanoids from the rhizomes of Alpinia officinarum
and their anticancer activity. Fitoterapia 2008;79:27-31.
Srividya AR, Dhanabal SP, Misra VK, Suja G. Antioxidant and antimicrobial activity of Alpinia officinarum
. Indian J Pharm Sci 2010;72:145-8.
] [Full text]
Stojanović G, Radulović N, Hashimoto T, Palić R.In vitro
antimicrobial activity of extracts of four Achillea
species: The composition of Achillea clavennae
L. (Asteraceae) extract. J Ethnopharmacol 2005;101:185-90.
Yaeesh S, Jamal Q, Khan AU, Gilani AH. Studies on hepatoprotective, antispasmodic and calcium antagonist activities of the aqueous-methanol extract of Achillea millefolium
. Phytother Res 2006;20:546-51.
Boye O, Brossi A. Tropolonic colchicum alkaloids and allocongeners. In: Brossi A, Cordell GA, editors. The Alkaloids. Vol. 41. New York: Academic Press; 1992. p. 125.
Katzung BG. Basic and Clinical Pharmacology. 9th
ed. New York: Lange Medical Books, McGraw-Hill; 2004. p. 527.
Lee HJ, Hwang Y, Park E, Choi SU. Antihepatoxic and antigenotoxic effects of herb tea composed of Chrysanthemum morifolium
ramat. J Korean Soc Food Sci Nutr 2011;40:78-83.
Beninger CW, Hall JC. Allelopathic activity of luteolin 7-O-glucuronide isolated from Chrysanthemum morifolium
L. Biochem Syst Ecol 2005;33:103-11.
Cheng W, Li J, You T, Hu C. Anti-inflammatory and immunomodulatory activities of the extracts from the inflorescence of Chrysanthemum indicum
linné. J Ethnopharmacol 2005;101:334-7.
Newall CA, Anderson LA, Phillipson JD. Herbal Medicines. A Guide for Health. Care Professionals. London: The Pharmaceutical Press; 1996. p. 162-3.
Schulz V, Hansel R, Tyler VE. Rational Phytotherapy. 4th
ed. Germany: Heidelberg, Springer-Verlag; 2001. p. 87-8.
Shipochliev T, Dimitrov A, Aleksandrova E. Anti-inflammatory action of a group of plant extracts. Vet Med Nauki 1981;18:87-94.
Al-Hindawi MK, Al-Deen IH, Nabi MH, Ismail MA. Anti-inflammatory activity of some Iraqi plants using intact rats. J Ethnopharmacol 1989;26:163-8.
Baytop T. Treatment with Plants in Turkey. Vol. 40. Turkey: İstanbul University; 1984.
Weiss RF. What is herbal medicine? Translated by Meuss A. Herbal Medicine. 6th
ed. Beaconsfield, UK: Beaconsfield Publishers; 1988.
Toker G. Biological activities and usage of linden flowers and barks. FABAD J Pharm Sci 1995;20:75-9.
Yassa N, Masoomi F, Rohani Rankouhi SE, Hadjiakhoondi A. Chemical composition and antioxidant activity of the extract and essential oil of Rosa damascena
from Iran, population of Gulian. DARU J Pharm Sci 2009;17:175-80.
Zargari A. Medicinal Plants. 5th
ed., Vol. 2. Tehran: Tehran University Publications; 1992. p. 281-4.
Loghmani-Khozani H, Sabzi Fini O, Safari J. Essential oil composition of Rosa damascena
mill. cultivated in central Iran. Sci Iran 2007;14:316-9.
Shafei MN, Rakhshandah H, Boskabady MH. Antitussive effect of Rosa damascena
in guinea pigs. Iran J Pharm Res 2003;2:231-4.
Mahmood N, Piacente S, Pizza C, Burke A, Khan AI, Hay AJ, et al.
The anti-HIV activity and mechanisms of action of pure compounds isolated from Rosa damascena
. Biochem Biophys Res Commun 1996;229:73-9.
Basim E, Basim H. Antibacterial activity of Rosa damascena
essential oil. Fitoterapia 2003;74:394-6.
Der Marderosian A. The Reviews of Natural Products. 2nd
ed. St. Louis, Missouri: Facts and 1st
Evans WC. Trease and Evans Pharmacognosy. 15th
ed. New Delhi: Reed Elsevier India Pvt., Ltd.; 2002.
Barceloux DG. Medical Toxicology of Natural Substances. New Jersey: John Wiley and Sons, Inc.; 2008.
Hoffaman D. Medical Herbalism: The Science and Practice of Herbal Medicine. Rochester, VT: Healing Arts Press; 2003. p. 62-120.
Mears ER, Modabber F, Don R, Johnson GE. A review: The current in vivo
models for the discovery and utility of new anti-leishmanial drugs targeting cutaneous leishmaniasis. PLoS Negl Trop Dis 2015;9:e0003889.
Samy MN, Sugimoto S, Matsunami K, Otsuka H, Kamel MS. One new flavonoid xyloside and one new natural triterpene rhamnoside from the leaves of Syzygium grande.
Phytochemistry Lett 2014;10:86-90.
Pérez-Victoria FJ, Gamarro F, Ouellette M, Castanys S. Functional cloning of the miltefosine transporter. A novel P-type phospholipid translocase from Leishmania
involved in drug resistance. J Biol Chem 2003;278:49965-71.
Lux H, Hart DT, Parker PJ, Klenner T. Ether lipid metabolism, GPI anchor biosynthesis, and signal transduction are putative targets for anti-leishmanial alkyl phospholipid analogues. Adv Exp Med Biol 1996;416:201-11.
Lux H, Heise N, Klenner T, Hart D, Opperdoes FR. Ether – Lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether – Lipid analogues in Leishmania
. Mol Biochem Parasitol 2000;111:1-4.
Luque-Ortega JR, Rivas L. Miltefosine (hexadecylphosphocholine) inhibits cytochrome c oxidase in Leishmania donovani
promastigotes. Antimicrob Agents Chemother 2007;51:1327-32.
Rakotomanga M, Blanc S, Gaudin K, Chaminade P, Loiseau PM. Miltefosine affects lipid metabolism in Leishmania donovani
promastigotes. Antimicrob Agents Chemother 2007;51:1425-30.
Maarouf M, Lawrence F, Croft SL, Robert-Gero M. Ribosomes of Leishmania
are a target for the aminoglycosides. Parasitol Res 1995;81:421-5.
[Table 1], [Table 2]