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  Int. J. Med.Arom. Plants, ISSN 2249  –  4340 RESEARCH ARTICLE Vol.4, No.1, pp.16-25,March2014*Corresponding author: (E-mail)gkinuthia<@> © 2014Copyright by the Authors, licensee Open Access Science Research Publisher. ijmap@openaccessscience.comThis is an openaccess article distributed under the termsof theCreative CommonsAttribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0)License(   ) Efficacy of crude methanolic extracts of   Allium sativum L. and  Moringa stenopetala (Baker f.) Cufod. against  Leishmania major Geoffrey K. KINUTHIA 1 , Ephantus W. KABIRU 2 , Christopher O. ANJILI 3 , Elizabeth M.KIGONDU 4 , Veronica N. NGURE 5 , Johnstone M. INGONGA 3 , Nicholas K. GIKONYO 6 1  Department of Science & Engineering, Daystar University, PO BOX 44400, Nairobi, Kenya 2 School of Public Health, Kenyatta University, PO BOX 43844, Nairobi, Kenya 3 Center for Biotechnology Research and Development, Kenya Medical Research Institute, PO BOX 54840, Nairobi, Kenya. 4 Center for Traditional Medicine & Drug Research, Kenya Medical Research Institute, PO BOX 54840, Nai-robi, Kenya. 5  Directorate of Research, Extension & Consultancy, Laikipia University, Po BOX 1100, Nyahururu, Post Code 20300, Kenya. 6   Department of Pharmacy and Complementary/Alternative Medicine, Kenyatta University, PO BOX 43850, Nairobi, Kenya. Article History : Received14 th February2014, Revised21 st March2014, Accepted24 th March2014. Abstract :  Leishmania major  is a protozoan parasite responsible for cutaneous leishmaniasis (CL) in humans. CL istransmitted via a bite by infected female phlebotomine sand fly. Research on herbal therapy for leishmaniases is increas-ing globally because conventional drugsare costly, toxic and require a prolonged administration.  In vitro and in vivo antileishmanial activities of dried  Allium sativum (garlic) and  Moringa stenopetala methanolic extracts against  L. major  were studied. Minimum inhibitory concentrations (MICs)of methanolic extracts of   A. sativum (A) and  M. stenopetala (M) against  L. major  were 3 and 5 mg/ml and IC 50 of 863.12 and 1752.92  µ g/ml respectively. The blend AM (1:1) hadIC 50 of 372.1  µ g/ml and promastigotes’ viability of 71.03% compared to IC 50 of 0.26 and 0.82  µ g/ml and promastigotes’ viability of 18.41% and 12.22% for Pentostam and Liposomal amphotericin B respectively. Multiplication indices (MIs)of   L. major  amastigotes ranged from 43.67%to45.93% after treatment with extracts A or M or blend AM at125  µ g/mland were significantly different (P < 0.05) from Liposomal amphotericin B at 12.5  µ g/ml.Oral extract A reduced signifi-cantly (P > 0.05)  L. major  caused foot pad lesions in BALB/c mice while oral extract M did not. Blend AM (ip) reducedthe lesionsizes and its efficacy was close to Pentostam and Liposomal amphotericin B. Oral extract A had a high parasitereduction rate of 60.70% and average LDU of 0.22±0.15 compared to Pentostam at 66.40% and LDU of 0.18±0.08. Inconclusion, methanolic extract of   A. sativum showed anti-leishmanial activity both in vitro and in vivo and it decreased  L.major  caused foot pad lesions in BALB/c mice. Methanolic extracts of   M. stenopetala (ip) reduced the amastigotes bur-den in spleens of BALB/c mice. A blend of garlicand moringa methanolic extracts (AM at 1:1) were active against  L.major. The active ingredients in crude methanolic extracts of garlic and moringa plants should be established and testedagainst  L. major  when blended. Keywords :  A. sativum ;  M. stenopetala ;methanolic extracts; blend; antileishmanial;  Leishmania major  . Introduction Cutaneous leishmaniasis (CL) is one of theneglected tropical protozoan diseases and it istransmitted by a bite of an infected femalephlebotomine sand fly (Diptera: Psychodidae).The protozoan responsible for this disease be-longs to the genus  Leishmania (Kinetoplastida:Trypanosomatida)and it is endemic in 98 coun-tries worldwide (WHO, 2010).An estimatedyearly incidence of about 1.2 million cases of CL occur each year worldwide (Alvar et al .,2012). The main clinical manifestations of CLare painful skin lesions that can elicit socialstigmatization and significant morbidity in thesufferers (Bailey, 2013).  17Int. J. Med. Arom. Plants  Antileishmanial activity of A. sativum and M. stenopetala methanol extracts Kinuthiaet al. According to Nilforoushzadeh et al . (2007),there is no effective andsafe treatment forleishmaniases.The CL drugs which includePentavalent antimonials, Amphotericin B andPentamidine among others are toxic, expensiveand are often associated with several healthcomplications. Drug resistances by  Leishmania parasites andtherapeutic failure have also beenobserved in leishmaniasis patients.  Leishmaniamajor  (Kinetoplastida: Trypanomastidae) is oneof the species that cause CL in Kenya and it isendemic in Baringo County, in the great RiftValley.Herbal products are potential sources of anti-leishmanial compounds since they have a widechemistry, remarkable diversity and a high ac-cessibility in nature (Monzote, 2009). Increasedresearch on plant derived anti-leishmanial com-pounds has identified herbal products that areeffective against  Leishmania parasites.  Alliumsativum L. (garlic) is a perennial plant that be-longs to the family Amaryllidaceae and ithasbeen used as food, spice and medicine for thou-sands of years in different parts of the world(Singh & Singh, 2008). The medicinal proper-ties of   A. sativum range from antimicrobial,hypolipidemic, antithrombotic to antitumouractivities (Augusti, 1996).Previous studieshave indicated that garlic extracts or their frac-tions augment parasite engulfment and destruc-tion ofintracellular  Leishmania amastigotes byBALB/c mice peritoneal macrophages(Ghazanfari et al ., 2006). Similarly, McClure et al . (1996) reported that Allicin, the active anti-microbial compound in  A. sativum , demon-strates a significant inhibitory effect onleishmanial cell growth.  Moringa stenopetala (Baker f.) Cufod. is asmooth barked, deciduous flowering plant wide-ly distributed in southern parts of Ethiopia andnorthern Kenya (Mekonnen et al ., 1999) particu-larly in Lake Baringo islands.  M. stenopetala belongs to the monogeneric family Moringaceae(Order Capparales). In Ethiopia, the leaves andfruits of   M. stenopetala are eaten as vegetableswhich are rich in proteins, calcium, phospho-rous, iron plus vitamins A and C. The leaves,roots, fruits and the barks of the  M. stenopetala are used to treat different ailments includingstomach problems, malaria, hypertension, diabe-tes, asthma and expelling retained placenta(Mekonnen et al ., 1999). Fresh root wood etha-nol extracts and dried leaves acetone extracts of   M stenopetala have been reported to possessantitrypanosomal property (Mekonnen et al .,1999). In our previous study, dried leaves crudeaqueous extract of   M. stenopetala was demon-strated to possess in vitro inhibitory effectagainst  L. major  promastigotesat a concentra-tion of 3mg/ml (Kinuthia et al ., 2013).It is against this background that the presentstudy was designed to investigate the in vitro and in vivo activity of crude methanolic extractsof A . sativum L. and  M. stenopetala (Baker f.)Cufod. against  L. major  . The crude methanolicextracts were studied singly or as blends. Materials andmethods Plant materials Bulbs of   A. sativum were purchased fromNakumat super market in Nairobi, Kenya whileyoung leaves of   M. stenopetala were pickedfrom trees growing on the slopes of LakeBaringo islands in Kenya. The study plants wereauthenticated at University of Nairobi Herbari-um, in the department of Botany, ChiromoCampus. The young leaves of   M. stenopetala and thin slices of   A. sativum cloves were dried atroom temperature at Kenya Medical ResearchInstitute (KEMRI),  Leishmania unit, until theyattained a constant weight. The dried plant mate-rials were labeled appropriately and then trans-ferred to the Center of Traditional Medicine &Drug Research (CTMDR) at KEMRI, wherethey were ground into a powder using an electricmill and then stored at minus 20 o C until requiredfor extraction. Crude Extracts The crude methanolic extracts were preparedas described by Cock (2008). Briefly, a 100g of ground plant material was soaked in 500 ml of analytical grade methanol for 72 hours at roomtemperature with gentle shaking, then filteredand concentrated using a rotary evaporator toobtain the crude methanolic extracts. The dryextracts were weighed and storedat 4 o C untilrequired for the bioassays. The methanolic crude  18Int. J. Med. Arom. Plants  Antileishmanial activity of A. sativum and M. stenopetala methanol extracts Kinuthiaet al. extracts of   A. sativum was coded as extract Awhile that of   M. stenopetala was coded as ex-tract M. With an initial weight of 100g groundplant material, the methanolic crude extracts of   A. sativum (A) was 5.698g (5.70%) while that of   M. stenopetala (M) was 9.217g (9.22%).  Leishmania parasites The  Leishmania major  strain(IDUB/KE/94=NLB-144) used in this study wasacquired from Institute of Primate Research(IPR) Kenya, where it had been maintained bycryopreservation in liquid nitrogen. The para-sites were grown to stationary phase at 25 o C in Schneider’s insect medium su pplemented with20% heat inactivated fetal bovine serum, 100U/ml penicillin and 500  μ g/ml streptomycin(Hendricks & Wright, 1979), and 250  μ g/ml of 5-fluorocytosine arabinoside (Kimber et al .,1981). The stationary-phase metacyclic stagepromastigotes were then harvested by centrifu-gation at 1500 rpm for 15 minutes at 4 o C. Themetacyclic promastigotes were then used for the in vitro and in vivo assays.  Experimental animals Eight week old male inbred BALB/c micewere used for in vivo macrophage assay for theplant extracts. The inbred BALB/c mice wereobtained from International LivestockResearchInstitute (ILRI), Kenya. They were then housedat the KEMRI animal house at 23 o C to 25 o C andwere fed on standard commercial diet in theform of mice pencils and were given tap water ad libitum . The mice were handled in accord-ance with the regulations that have been set by KEMRI’s Animal Care and Use Committee (ACUC).  Evaluation of minimum inhibitory concentration(MIC) The MIC was determined as described byWabwoba et al . (2010). Promastigotes at a con-centration of 1×10 6 metacyclic promastigotesper ml of culture medium were exposed to sev-eral concentrations of the test individual plantextracts A and M. The lowest concentration of the test plant extracts that inhibitedpromastigotes growth was taken to be the MIC.  In vitro anti-promastigote assay This was evaluated as described byWabwoba et al . (2010). The metacyclicpromastigotes at a concentration of 1x10 6 promastigotes per ml were incubated in culturemedium in 24-well plates in presence of differ-ent concentrations of plant extracts for five daysat 25 o C. The aliquots of parasites were thentransferred into 96-well micro-titer plates, andincubated at 27 o C for 24 hours. Two hundredmicro liters of test extracts A and M sampleswere then added to the parasite cultures at con-centrations ranging from 5 mg/ml to 0.5 mg/mlof extracts. The plates were then incubated fur-ther at 27 o C for 48 hours. The control wells con-tained culture alone. Ten micro liters of 2, 5-diphenyltetrazolium bromide (MTT) reagentwas added into all the wells and incubatedfor 4hours. The medium together with MTT wereaspirated off, followed by addition of 100  μ l of dimethyl sulfoxide (DMSO) per well and shakenfor 5 minutes. Absorbance was measured foreach well at 562 nm using a micro-titer reader.The absorbance readings were used to generatethe 50% inhibitory concentration (IC 50 ) valuesfor extracts A and M . Percentage promastigotes’ viability (%) was determined using the formuladescribed by Mosmann (1983), in which, viablepromastigotes (%) = ( at   –  ab ) / ( ac ) × 100,where at  was the absorbance of treated samplesand ab was the absorbance of the blank wellsand ac was the absorbance of the control wells.  In vitro anti-amastigote assay This was carried out as described byDelorenzi et al . (2001). Peritoneal macrophageswere obtained from clean BALB/c mice. Tenmilliliters of sterile cold phosphate-bufferedsa-line (PBS) was injected into the peritoneum of anaesthetized BALB/c mice whose body surfacehad been disinfected with 70% ethanol. ThePBS containing the macrophages was washedthrough centrifugation at 2,000 rpm for 10minutes and the macrophages were adsorbed insterile 24-well plates for 4 hours at 37 o C in 5%  19Int. J. Med. Arom. Plants  Antileishmanial activity of A. sativum and M. stenopetala methanol extracts Kinuthiaet al. CO 2 . Adherent macrophages were infected withpromastigotes and further incubated at 37 o C in5% CO 2 for 4 hours and then washed with ster-ile PBS to remove the free promastigotes. Thiswas followedby further incubation of the in-fected macrophages for 24 hours in RPMI 1640culture medium. The infected macrophages werethen treated with extracts A and M. Pentostamand Liposomal amphotericin B were used aspositive control drugs. The medium, test extractsand control drugs were replenished daily for 3days. After 5 days, the macrophages werewashed with PBS at 37 o C, fixed in methanol andstained with 10% Giemsa. The number of amastigotes was determined by counting at least100 macrophages in duplicate cultures, and theresults was expressed as infection rate (IR) andmultiplication index (MI) as described by Ber-man & Lee (1984) .  Infection and treatment of BALB/c mice The BALB/c mice were placed into 10groups caged separately, where each group wascomprised of at least 5 mice. All the mice ineach group were infected with  L. major  promastigotes in the foot pads as described byWabwoba et al ., 2010. Briefly, the thickness of the hind legs footpads was measured using areading vernier caliper prior to infection. Theleft hind footpads of the mice were then subcu-taneously inoculated with 1×10 6 stationaryphase infective metacyclic promastigotes of   L.major  in 40  μ l sterile PBS. Lesion developmentwas monitored for four weeks after whichtreatment commenced. Afour week treatmentwas given to the infected mice that had devel-oped footpad lesions. Groups of mice weretreated with individual methanolic extracts A,extracts M, blend of A and M in a ratio of 1:1,and with positive and negative controls. Treat-ment was administered orally daily using a can-nula for each extract or intra-peritoneally usinga fine 1ml 30 gauge insulin needles (BD Micro-Fine Plus ® , USA) at a dose of 20 mg/kg dailyfor each extract. The positive control groups of mice were treated intra-peritoneally withPentostam and Liposomal amphotericin Bleishmaniases drugs at a dose of 20 mg/kg perday. Two groups of negative control mice weretreated with phosphate buffered saline (PBS),one group orally and the other intraperitoneally.The progression of the foot pads lesions sizeswas monitored weekly using a vernier caliper tomeasure the thickness of the infected left hindfoot pad and comparing it with that of non in-fected right hind foot pad as described by Nolan& Farrel (1987). Post treatmentparasite burden in BALB/c micespleens After a four week treatment period, the micewere sacrificed using 100  μ l pentobarbitone so-dium (Sagatal ® ). At necropsy, the spleens wereweighed and spleen impression smears weremade as described by Chulay & Bryceson(1983). The impression smears were fixed inmethanol and stained with Giemsa. The smearswere examined under a microscope to enumer-ate the number of amastigotes per 1000 nucleat-ed spleen cells. The relative and total numbersof parasites in the spleenwere estimated by cal-culating the spleen index (%), Leishman-Donovani Units (LDUs) and total Leishman-Donovani Units (total LDUs) as described byBradley & Kirkley (1977). Spleen index (%)was determined using the formula: Spleenweight (g) divided by bodyweight (g) × 100%.LDU was the number of amastigote per 1000nucleated splenocytes while the total LDU wascalculated using the formula: LDU × spleenweight (g) × (2 × 10 5 ).  Data analysis Data was analyzed using SPSS version 17.0for windows at 5% level of significance. Oneway ANOVA (F test) was used to comparepromastigotes viabilities (%) after differenttreatments. Other variables compared using Ftest were infection rates (IRs) and multiplicationindices (MIs) of amastigotes in peritoneal mac-rophages and also the lesion sizes in differentgroups of BALB/c mice under different treat-ments. Cases in which homogeneity test of vari- ance (Levene’s test) were significant, robust tests of equality of means that included Brown-forsythe and Welch tests were carried out as al-ternative versions of the F-statistics. Multiplecomparisons of the individual treatments were
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