The Effect of Alkanna Tincturia and Peganum Harmala Extracts on Leishmania Major ( MRHO/IR/75/ER) in Vitro

Iranian J Parasitol: Vol. 4, No.1, 2009, pp. 40-47

Original Article

The Effect of Alkanna tincturia and Peganum harmala Extracts on Leishmania major ( MRHO/IR/75/ER) in vitro

Ruhallah Yousefi, *Fatemeh Ghaffarifar, Abdolhosein Dalimi Asl

Dept. of Parasitology, School of Medicine, Tarbiat Modarres University, Tehran, Iran

(Received 6 Aug 2008; accepted 9 Jan 2009)

Abstract Background: Cutaneous leishmaniasis is an important health problem caused by Leishmania spp . As there is no vaccine, drug treatment is the only way to tackle leishmaniasis. In the present study, inhibitory and killing effects of Peganum harmala and Alkana tinctoria extracts on amastigotes and promastigotes forms of Leishmania were evaluated in-vitro. Methods: The seeds of Peganum harmala, Stems and roots of Alkanna tictoria were collected and crude extraction carried out. In this experimental study, Leishmania major promastigotes were cultured in RPMI- 1640 with 10% FBS at 22-26°C, and infected macrophages with amastigotes were cultured in RPMI-1640 with 10% FBS at 37°C in 5% CO2. Then the extracts of each plant were added to cultivated parasites and incubated for 3 days. Promastigote and amastigote assay was carried out using counting assay based on growth inhibition. Results: The results indicated that both extractions can inhibit the growth of promastigotes, and in concentrations of 40µg/ml of P. harmala , 200µg/ml of A. tincturia, and 20 µg/ml of equal combination of P. hamala and A. tincturia are Inhibitory Concentration (IC 50 ) for parasites growth. By adding these concentrations of the extracts to the infected macrophages in the culture, their effects were separately evaluated. The mean of amastigotes number in macrophages in the culture with P. harmala, A. ticturia , combination and control groups were 0.7, 0.7, 0.6, 2.3 amastigotes per macrophage, respectively. Conclusion: By this method, inhibition of intracellular and extracellular growth of L. major was demon- strated suggesting that, plant drugs with efficacy and safe products can be applied as new treatment for cutaneous leishmaniasis .

Key words : Leishmania major, Peganum harmala , Alkanna tincturia , In-vitro

Introduction

eishmaniasis has been identified choices against leishmaniasis are sodium as a major public health problem, stibogluconate and meglumine anti- L particularly in Africa, Asia and monite (both pentavalent antimony der- Latin America. About 1.5 to 2 million ivatives). Pentavalent antimony and people are affected annually by this meglumine antimonate are characteris- parasitic infection (1). As there is not tically moderately toxic and there are any applied vaccine, therefore the drug risks of recurrence and unsatisfactory treatment is the topic way against side effects. Amphotericin B is rec- leishmaniasis. The chemical drugs of ommended as a second-line treatment.

*Corresponding author . Tel, : +982182883566 E-mail: [email protected] 40 Yousefi et al: The Effect of Alkanna tincturia …

The most dangerous side effect of tan and India, but later Introduced and a amphotericin B is kidney damage. The few areas of Southwest USA, South Af- lowest incidences were in the generic sti- rica and New South Wales in Australia. bogluconate group. The efficacy and tol- It now grows wild in Eurasia and has re- erance of inexpensive generic stiboglu- cently been spread to Texas, Nevada, conate appears comparable to the branded New Mexico and Southern California. formulations for the treatment of cuta- The main products of Peganum har- neous leishmaniasis in the endemic reg- mala are alkaloids (6). P. harmala alka- ions. loids (1) are known for their antimicro- Topical application of paromomycin in bial (7-8) and hypothermic (9). In an ointment led to protozoal clearance in Moroccan traditional medicine, seeds 76% to 86.3% of the patients with of P. harmala have been used for the cutaneous leishmaniasis caused by L. empiric treatment of cancers (10). It had major (1-3). However, in another study, usages in traditional medicine as an an- the efficacy of paromomycin remains thelmintic, lactogenic, antispasmodic controversial (3). Recently photodynamic and emetic (1, 11). therapy was introduced as an attractive Alkanna tinctoria perennial grows to antiparasitic therapeutic option that 0.2m by 0.25m. It is in flower in June. offers rapid localized destruction of the The flowers are hermaphrodite (have lesions without affecting the adjacent both male and female organs). The plant normal tissues. Furthermore, in contrast prefers sandy and loamy soils, requires to systemic treatments, photodynamic well-drained soil and can grow in nutri- therapy has no risk of toxicity, but it is tionally poor soil. The plant prefers neu- an expensive method (4). tral and alkaline soils. It can grow in Unfortunately, no ideal therapy for the semi-shade or no shade. It requires dry cutaneous leishmaniasis has yet been or moist soil and can tolerate drought. identified (1, 3, 5). The plant can tolerate maritime exposure. Finding healing powers in plants is an Habitats and possible locations are ancient idea. The use of and search for woodland, cultivated beds and dappled drugs and dietary supplements derived shade (12). from plants have been accelerated in re- It helps old ulcers, hot inflammation and cent years. Traditional healers have long burnings by common fire. For these used plants to prevent or cure infectious uses, the best way is to make it into an conditions. Plants are rich in a wide vari- ointment. Also if you make a vinegar of ety of secondary metabolites such as tan- it, it helps the morphy and leprosy. It nins, terpenoids, alkaloids and flavono- also cures the yellow jaundice, spleen, ids, which have been found in vitro to and gravel in the kidneys. It also kills have antimicrobial properties (5). worms, bruises and falls, and is as gal- The aim of the present study was to lant a remedy to drive out the smallpox investigate the inhibitory and killing ef- and measles as any is; an ointment made fects of plant extractions ( P.harmala and of it is excellent for green wounds, A.tincturia ) on promastigotes and amas- pricks or thrusts (8, 13). tigotes forms of L. major in-vitro. P. The root is antibacterial, antipruritic, as- harmala or Syrian Rue grows in semi- tringent and vulnerary (12). It is used ex- arid conditions in parts of North Africa, ternally in the treatment of varicose Mediterranean, the Middle East, Pakis- veins, indolent ulcers, bedsores and itch-

41 Iranian J Parasitol: Vol. 4, No.1, 2009, pp. 40-47 ing rashes. The root is harvested in the early log phase (2 10 5 promasti- autumn and can be dried for later uses gotes/ml) and allowed to multiply for 60 (12). hrs in the medium alone (control group) or in the presence of serial dilutions of Material and Methods the drug until late log phase (10 7 parasites/ml). Plant extract susceptibility Peganum harmala experiments were performed in RPMI The seeds of Peganum harmala were 1640 (Gibco) media with 20% FBS collected, dried and finely ground in a (Gibco). The parasites were counted spice mill. Fifty grams of the seeds pow- daily for 3 days in a haemocytometer der were dissolved in 500 ml of water with a light microscope, and the results shook overnight, then centrifuged and were compared with those from the con- the supernatants were collected. The su- trols. Each assay was performed in pernatants were then filtered, evaporated duplicate and repeated in separate experi- under vacuum (at 100ºC) and lyophi- ments. lized. The powder resulting from lyophilisation was termed the aqueous Amastigote/ Macrophage Assay crude extract and the residue was dis- Drug susceptibilities of the amastigotes solved. The aqueous crude extraction in the macrophage BALB/c mice were was autoclaved, aliquot and froze at -20° determined by following a modification C (11). of the method of Chang (14). Briefly, peritoneal macrophages were collected Alkanna tinctoria and infected in-vitro with promastigotes Stems and roots of Alkanna tictoria were in RPMI medium with 10% heat-inacti- vated fetal bovine serum to yield 10 6 collected, dried and ground in a spice 7 mill, then 50g of the powder was dis- cells and 10 promastigotes per ml. The solved in 500ml of chloroform shook cultures were incubated for about 3 days overnight, then centrifuged and the at 37°C in 5% CO2 to allow phagocyto- supernatants were collected. The super- sis of the promastigotes and adhesion to natants were filtered and evaporated un- the surface. The number of parasites were calculated in 100 macrophages, der vacuum (11). 5 then 2 ×10 macrophages/well were cul- Leishmania culture tured in 24-well plates with RPMI and L. major (MRHO/IR/75/ER) was cul- 10% FBS. The extracts of plant with the tured in RPMI 1640 (Gibco) with 20% concentration that obtained by IC 50; were FCS (Gibco) for preparation of adequate added to the wells after 24 h The plates promastigotes. incubated in 37°C and 5% CO2 incubator and the amastigotes in 100 macro- Promastigote assay phages were counted in 24, 48 and 60 h Promastigote assay was carried out using after incubation. a previously described direct counting assay based on growth inhibition (6). Cytotoxicity assay against macrophages The effects of the crude extracts were The cytotoxic effect of the plant, ex- evaluated in 24-well microtitre plates. pressed as cell viability, was assessed on The promastigotes were seeded at an ini- the mice macrophages. The test was car- tial concentration equivalent to that of ried out in 24-well microtitre plates. A

42 Yousefi et al: The Effect of Alkanna tincturia …

suspension of 5 × 10 5 macrophages was added to each well, and then incubated Amastigote-macrophage assay in a 37°C and 5% CO2 incubator. The mean of the amastigotes/mac- Viability of the macrophages was evalu- rophages before adding the extracts was ated after 60 h (15). 1.5 and after adding the extracts Pega- num harmala , Alkanna tinctoria and Results combination of two extracts after 60 h was 0.7, 0.7 0.6, respectively whereas it Promastigote assay was 2.6 for control group. The complete Growth inhibition of promastigotes was results for 24 and 48 h are shown in evaluated by four concentrations of each Table 1 and Fig. 2. plant extraction (20, 40,100 and 200µg/ml) after 60 h (Fig. 1). Following Cytotoxicity assay against macrophages the application of Peganum harmala , Al- For checking the effect of cytotoxicity of kanna tinctoria and equal combination these extracts on macrophages, the of the two plants, promastigote IC 50 were viability of macrophages after 60h was measured as 40 µg/ml, 200µg/ml and 10 checked and the results are shown in Ta- µg/ml, respectively. ble 2.

Table 1 : Mean number of amastigote/macrophage in 24, 48 and 60 h after adding extracts

Mean number of amastigote/macrophage during Drugs 24 h 48 h 60 h P. harmala 40 µg/ml 1.2 0.9 0.7 A. tincturia 200 µg/ml 1.3 0.9 0.7 P. harmala 10 µg/ml 1.4 1 0.6 A. tincturia 10 µg/ml Control group 1.8 2 2.3

Table 2 : Percentage of Macrophages viability after 60 h of adding extracts

Drug concentration ( µg/ml)

Drugs 20 40 100 200 Macrophage viability (%) P. harmala 90 85 83 80

A. tincturia 90 86 82 80

P. harmala and 90 85 82 79 A. tincturia Control groups 95 95 95 95

43 Iranian J Parasitol: Vol. 4, No.1, 2009, pp. 40-47

3.5

3.0

2.5

2.0

1.5

1.0 H

.5 A

0.0 HA number of promastigote after 60 hour hour 60 after promastigote of number 0 20 40 100 200

Concentration of Drugs

Fig. 1 : Number of promastigotes after 60 h use of different concentration of Peganum harmala (H) Alkanna tincturia (A) and equal combination of Peganum harmala and Alkanna tincturia (HA)

2.5

P.harmala 1.5 A.tincturia p.har&A.tin 1 control

0.5

Amastigote/Macrophages 0 0 24 48 60 hrs

Fig. 2 : Mean number of amastigotes in macrophages affected by Peganum harmala , Alkanna tinctoria and combination of two extracts during 60 h

44 Yousefi et al: The Effect of Alkanna tincturia …

Discussion

The recommended drugs for both visceral Leishmania ssp. at100 µg/ml. This drug and cutaneous leishmaniasis are the penta- reduced the parasite loads by 91% in the valent antimonials sodium stibogluconate lesion (23). (Pentostam) and meglumine antimoniate These results confirmed the effect of (Glucantime). Both drugs, used for over these extracts for the treatment of 50 years, require long courses of paren- cutaneous leishmaniasis in-vivo (24). teral administration and have toxic side ef- Mirzaie et al. (2007) found P. harmala fects (16). extract inhibited the growth of promas- Treatment of cutaneous leishmaniasis is tigote forms of L. major in vitro after 72 directed toward the eradication of amas- h. of incubation and had an IC 50 of tigotes and the reduction of the size of the 1832.65 ± 89.72 µg/mL (25) . lesions to promote healing and achieve The results of the present study showed maximum efficacy with minimal scarring that the plant drugs with efficacy and and toxicity (5). However, no treatment safe products can be applied as new has proved to be completely satisfactory. treatment for cutaneous leishmaniasis. Pharmaceutical research in natural prod- Alkaloids are one of the most important ucts represents a major strategy for discov- classes of natural products providing ering and developing new drugs. The use drugs for humans since ancient times. of medicinal plants for the treatment of Most alkaloids are well known because parasitic diseases is well known and has of their toxicity, but many alkaloids have been documented since ancient times by had a deep impact on the treatment of the use of Cinchona succiruba (Rubiaceae) parasitic infections. Peganum harmala as an antimalarial (17). had better effect than Alkanna tincturia Alkaloids are one of the most important ( IC 50 for P. harmala 40 µg/ml and for classes of natural products providing A. tincturia 200), whereas the cytotoxic- drugs for humans since ancient times. ity for both of them in the same Most alkaloids are well known because concentration is alike. Combination of of their toxicity, but many alkaloids have two extracts had a good effect with low had a deep impact on the treatment of doses (IC50 for the equal combination of parasitic infections (18). P. harmala and A. tincturia was 10 On the other hand, alkaloids have been µg/ml from each one) and low toxicity in

found to have microbiocidal effects in- the same IC 50 . The results also indicated cluding against Giardia and Entamoeba that the effects of two combined ex- species (19). Berberine is an important tracts were better than the separated ex- representative of the alkaloid group. It is tracts. potentially effective against trypanosomes and plasmodia (20). The mecha- Acknowledgements nism of action of highly aromatic planar quaternary alkaloids such as berberine This study was supported by Tarbiat and harmane (21) is attributed to their Modares University through pecuniary ability to intercalate with DNA (22). aid. The authors wish to thank Miss The crude alkaloidal extract of Zanthox- Ghasemi for kind helping. The authors ylum chiloperone stem bark exhibited in declare that there is no conflict of inter- vitro activity against various strains of ests.

45 Iranian J Parasitol: Vol. 4, No.1, 2009, pp. 40-47

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