ORIGINAL PAPER

Effect of root bark extract of Berberis vulgarisL. on Leishmania major on BALB/c mice

Alireza Salehabadi & Mahdi Karamian &

Motevalli Haghi Farzad & Mohammad Hasan Namaei

Received: 21 October 2013 /Accepted: 27 November 2013 /Published online: 12 December 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Leishmaniasis is one of the most important diseasestransmitted by arthropod. This disease is present in 88 coun-tries. Approximately 400 million people are at risk, and 12million are involved. We aimed to examine the application ofethanolic extract of the root bark of Berberis vulgaris L. fortreatment of mice infected with cutaneous leishmaniasis. Atfirst, 40 BALB/c mice were infected to Leishmania majorpromastigotes and were divided in two groups A and B. Then,each of A and B groups were divided to two subgroups. Micefrom subgroup A1 were treated with 10 % root bark alcoholicextract, and mice from subgroup A2 were treated with onlyalcohol (control). Mice from subgroup B1 were treated with20 % root bark alcoholic extract, and mice from subgroup B2were treated with only alcohol (control). The 90 % recoverywas found in the mice treated with 20 % root bark extract, and55 % recovery was found with 10 % root bark extract, but inthe control group, 0 % recovery was found. The results of ourstudy showed that the lotion of root bark extract has goodsuppression effects on parasites. Therefore, it might be a profor developing new antileishmanial drugs.

Introduction

Leishmaniasis is one of the most important vector-borne dis-eases. Leishmania major is the causative agent of zoonoticcutaneous leishmaniasis (ZCL) in which gerbils is the reser-voir host. ZCL is of great public health importance in Iran(Parvizi et al. 2008).

It is a public health problem throughout. The preva-lence of these diseases is more in the tropical and sub-tropical world and is a growing concern in war-torncountries (Shokri et al. 2011).

Leishmaniasis is endemic in 88 countries. There are 350million people at risk, and 12million are involved. Every year,an incidence of new visceral leishmaniasis (VL) and cutane-ous leishmaniasis (CL) is 0.5 million and 1.5 million, respec-tively (Sharma et al. 2009).

Leishmaniasis is globally a widespread disease and pre-sents a wide clinical spectrum (beginning from cutaneouslesion to fatal visceral disease). Over 90 % of cutaneousleishmaniasis occurs in Afghanistan, Algeria, Iran, Iraq,Saudi Arabia, Syria (Old World), Brazil, and Peru (NewWorld) (Kheiri et al. 2012).

After malaria, it is the most important of parasitic diseases.CL is a major health problem in Iran, with the rate of prevalentinfection between 1.8 and 37.9 % (Alavinia et al. 2009).

Materials and methods

Collection of the plant material

The roots of Berberis vulgaris L. were obtained by diggingthe soil and uprooting in March or November in Birjand, Iran(Zargari 1992). The experimental material was authenticatedin a herbarium of Mashhad, Razavi Khorasan Province, Iran.The barks of roots were separated and after washing, shade

A. SalehabadiDepartment of Microbiology, Birjand University of MedicalSciences, Birjand, Irane-mail: salehabadia@gmail.com

M. Karamian :M. H. Namaei (*)Birjand Hepatitis Research Center, Birjand University of MedicalSciences, Ghafary Ave., Birjand, Irane-mail: mhnamaei@hotmail.com

M. Karamiane-mail: Karamianm@yahoo.Com

M. H. FarzadFaculty of Health, Mazandaran University of Medical Sciences, Sari,Mazandaran, Irane-mail: Haghi77@yahoo.Com

Parasitol Res (2014) 113:953–957DOI 10.1007/s00436-013-3727-2

dried. The barks of roots were ground separately and convert-ed to powder and, after sieving the powder by sieve number50, stored them in glass bottles (Fig. 1).

Extraction

Ten grams from dried powdered material (roots bark) wasmixed with 100 ml of 70 % aqueous ethanol with initialwarming at 30 °C followed by keeping at room temperature(23 to 25 °C) for 48 h. This mixture was filtered through

Fig. 1 B. vulgaris L. a Shrubs ofB. vulgaris L. b Dry root ofB. vulgarisL. c Dry root bark powder of B. vulgaris L.

Fig. 2 Flow chart of in vivoexperiment

Fig. 3 Impression smears from mice lesions: a before treatment, b aftertreatment

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Whatman filter paper no. 1. The filtrate was concentratedusing the rotary evaporator. The concentrated filtrate (10 and20 % of the alcoholic extract of roots) was mixed in Vaselinebase. This Vaseline product was prepared for topical applica-tion (Harborne 1998).

Culture of L. major

L. major promastigotes, MHOM/IR/75/ER, was kindly pro-vided by Dr. M. Mohebali (professor of the School of PublicHealth, Tehran University of Medical sciences, Iran) for theseexperiments. They were grown in Novy-MacNeal-Nicole(N.N.N) medium and then transferred to a RPMI 1640Medium (Sigma Germany) supplemented with 100 μg/ml ofstreptomycin and 100 μg/ml of penicillin G, 50 μM of 2-mercaptoethanol, 1 μM of sodium pyruvate, 20 μM ofHEPES, and 10 % heat-inactivated fetal calf serum (FCS) bykeeping in 23 to 25 °C. FCS was heat inactivated in a waterbath at 60 ° C for 30min. It was liquated and kept at −20 °C till

further use. The parasite L. major promastigotes were main-tained in the RPMI 1640 Medium, supplemented with 10 %heat-inactivated FCS in vitro, and the virulence was main-tained by regular passage through susceptible BALB/c mice.Stationary-phase (after 120 h) promastigotes were harvestedand centrifuged at 3,000 rpm for 10 min at 4 °C. The pelletwas washed three times in phosphate-buffered saline (PBS)(Esmaeili et al. 2008).

One hundred parasites were inoculated in 2.5 ml of culturegrowth medium (CGM) or RPMI 1640. After growth, thesewere kept in plates (24 wells). Its multiplication was observedafter 5 days (120 h).

After day 5, the medium was saturated with the flagellateforms of the parasite (promastigotes). There were 2×107

promastigotes in 1.0 ml.The multiplication of leishmanias was observed every 24 h,

and the number of parasites was calculated by counting theparasites in drop (10 μL) by using a Neubauer slide.

Incubation

The parasites were incubated at a temperature of 23 to 25 °C.

Infecting of BALB/c mice by injecting of L. major

Forty female BALB/c mice (6–8 weeks old) were obtainedfrom the Animal Breeding Stock Facility of Razi Institute ofIran, Karaj, Iran. They were kept in polypropylene cagesprovided with saw dust, which was changed twice in a week.Food and water were provided free. They were exposed in 12-h light and 12-h dark conditions. The temperature was fixed at21±2 °C and humidity of 60±10 % (Tuli et al. 1995).

Table 1 Healing rate of lesions in study groups and control group aftertreatment with root bark extract of B. vulgaris L.

P value No healinga Partialhealingb

Completehealingc

Root barkextractconcentration

<0.001 1 case (11 %) 3 cases (33 %) 5 cases, 55 % 10 %

<0.001 0 case 1 case (10 %) 9 cases, 90 % 20 %

<0.001 8 cases (80 %) 2 cases (20 %) 0 case Control group

aNo healing = 4+: 1–10 parasites per fieldb Partial healing = 1+: 1–10 parasites per 1,000 fieldsc Complete healing: 0 parasite per 1,000 fields

Fig. 4 Healing rate of lesions instudy groups and control groupafter treatment with root barkextract of B. vulgaris L.

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Female BALB/c mice were infected subcutaneously with2×106 (0.1 ml) L. major promastigotes (MHOM/IR/75/ER)at the base of the tail.

The experimental mice were divided in two groups, andthen every group was divided into two subgroups; so, everysubgroup has ten mice (Fig. 2).

Application of plant extracts for the treatment of infected mice

After 5 weeks when leishmanial lesions appeared first, thediameter of lesions was measured before treatment. Then,every subgroup mice was marked with picric acid, and treat-ment started. Lotion (alcoholic extract) was applied three timesa day for 30 days. For the control subgroup mice, only ethanoland distilled water were applied. Before and after treatment, thediameter of lesions was measured, and the number of parasiteswas counted. Impression smears were prepared from lesions,methanol fixed, and stained with 10 % aqueous Giemsa stain.

Statistical analysis

Statistical significance between groups was analyzed byStudent's t test using SPSS version 10. Values of P <0.05were considered statistically significant.

Result

In cases treated with 20 % root bark extract, the number ofparasites decreased the most, while 10 % of root bark extractdecreased intermediate but, in control group, did not decrease(Fig. 3).

Ninety percent recovery was observed in the cases treatedwith 20% of root bark extract, whereas with 10% of root barkextract, 55 % of recovery was observed (Table 1, Fig. 4).

The mean of the diameter of lesions after treatment of thesubgroup with 10 % root bark alcoholic extract concentration,0.07 cm decreased and of the subgroup with 20 % rootalcoholic extract concentration, 0.38 cm decreased but, inthe control group, 0.31 cm increased (Table 2, Fig. 5).

Discussion

Leishmaniasis is a globally widespread disease still treatedwith expensive compounds that present severe side effects andare frequently ineffective, emphasizing the importance tosearch new compounds against this disease. The standardagents for leishmaniasis are pentavalent antimonials, pentam-idine, and amphotericin B (Esmaeili et al. 2008).

These compounds have some disadvantages. First isthe method of administration: daily intramuscular or in-travenous injection, one each for 20 to 28 days. Secondis toxicity: sometimes, intravenous injection of pentava-lent antimony can result in more serious side effectsincluding cardiotoxicity and sudden death (Konecnyand Stark 2007). Third is resistance of the parasites tothese drugs (Hadighi et al. 2007).

About 10 to 15 % CL patients are resistant to meglumineantimoniate (Esmaeili et al. 2008), and 50 to 60%VL patients

Table 2 Variation of lesion diameter in infected BALB/c mice to cuta-neous Leishmaniasis in study groups and control group before and aftertreatment with root bark extracts of B. vulgaris L.

Root bark extractconcentration

Diameter of lesions(cm) before treatment

Diameter of lesions(cm) after treatment

P value

10 % 1.15±0.25 1.08±0.24 <0.001

20 % 1.18±0.20 0.8±0.22 <0.001

Control group 1.16±0.21 1.47±0.24 <0.001

Fig. 5 Variation of lesiondiameter in infected BALB/c miceto cutaneous Leishmaniasis instudy groups and control groupbefore and after treatment withroot bark extracts of B. vulgaris L.

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in India are resistant to sodium stibogluconate (Bhattacharyaet al. 2007).

Last is the cost of the drug: Treatment for every patient is85 US dollars (WHO 2007).

Because of a lot of side effects of pentavalent antimonials,there have been efforts to use herbal drugs. Among them, wedecided to assess the efficacy of an herbal drug obtained fromB. vulgaris L., the most significant representative ofBerberidaceae (Arayne et al. 2007).

Lotion medications have an obvious request for its ease ofapplication, domiciliary treatment, no hospital costs, and nolimitation by bed capacity. Thus, the quest for an effectivelotion of antileishmanial drug has been ongoing for a longperiod.

The results of our study shows that 20 % root barkextract has a good suppression effects on leishmaniasis(90 % recovery).

Decrease in the size of lesions after treatment with increas-ing concentration of ethanolic extracts of root, stem, and leafof B. vulgaris L. in BALB/c mice infected with L. majorpromastigotes has been reported; the alcoholic extract ofB. vulgaris L. root was more effective than that of the stemand leaf extracts (Fata et al. 2006).

Decrease in (a) size of lesion, (b) number of parasites and(c) increase in the weight of BALB/c mice first infected withL. major promastigotes (MHO/IR/75/ER standard variety)followed by treatment with 20, 40, and 80 % of ethanolicstem bark extract has been studied. It was found that the 20 %extract was effective, but 80 % extract was harmful (Kazemiet al. 2007).

Alkaloid fraction of both stem and bark ethanolic extract ofBerberis boliviana Lechl, alkaloid fraction of bark ethanolicextracts of Berberis bumeliaefolia Schum, and alkaloid frac-tion of stem ethanolic extract of Berberis laurina Epl havebeen shown to have antileishmanial activity againstLeishmania amazonensis , Leishmania braziliensis andLeishmania donovani .

The alkaloid fraction of stem bark ethanolic extract ofBerberis paucidentata Rusby has been shown to haveantileishmanial activity against Leishmania amazonensisand L. braziliensis (Rocha et al. 2005).

The results of our study show that the lotion root barkextract has good suppression effects on parasites. Therefore,it might be a pro for developing new antileishmanial drugs.

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