IN VITRO ANTI-LEISHMANIAL ACTIVITY  AGAINST CUTANEOUS LEISHMANIA PARASITES AND PRELIMINARY PHYTOCHEMICAL ANALYSIS OF FOUR YEMENI MEDICINAL PLANTS

Manal Mutahar Ali Al- Hajj¹, Hassan A Al-Shamahy², Bushra Y Alkhatib³, Bushra A Moharram⁴

¹Biology Department, Faculty of Sciences, Sana'a University, Republic of Yemen.

²Medical Microbiology and Clinical Immunology, Faculty of Medicine and Health Sciences, Sana’a University, Republic of Yemen.

³Unit of Immunology and Physiology - Biology Department, Faculty of Sciences, Sana’a University, Republic of Yemen.

⁴Department of Pharmacognosy, Faculty of Pharmacy, Sana'a University, Republic of Yemen.

DOI: https://doi.org/10.22270/ujpr.v3i4.183

INTRODUCTION

Cutaneous leishmaniasis (CL), one clinical form of leishmaniasis, a term referred to skin clinical symptoms caused by several species of obligate intra-monocular phagocytic cells protozoan parasites produce a skin ulcer that heals spontaneously in most cases leaving an unsightly scar . These parasites belong to the genus Leishmania, that transmitted by the bite of a female phlebotomine sand fly^1-4. Cutaneous leishmaniasis is still one of the world’s most neglected disease that significant morbidity worldwide and shows a worrying increasing trend. CL is endemic in large areas of the tropics, subtropics, and the Mediterranean basin⁵. In Yemen, this disease is endemic and the most prevalent skin infectious diseases^6,7. To date progression in developing an effective vaccine against CL has not been successes and chemotherapy is the only effective way to treat the disease. However, current therapy is toxic, expensive, have severe side effects, as well as it emerges a resistance to drugs. Therefore, there is a great and urgent need for developing a new and safe anti-leishmanial drug^8-11. Investigation bioactive compounds from plants that used medicinally are regarded as one of the strategies to discover new drugs for leishmaniasis¹².  The World Health Organization (WHO) has estimated that approximately 80% of individuals rely on traditional medicines for their primary health care needs^13,14.  In different cultures and countries, many plants are used in the form of powders, crude extracts or infusion to treat several diseases including parasitic diseases without any scientific evidence of efficacy. In Yemen, there is a rich tradition of the use of herbal medicine for the treatment of various diseases, including inflammations, infections and other diseases^15-18.

Despite the worldwide spread of Cutaneous leishmaniasis and the significant morbidity that caused by this disease in the world as well as in Yemen, its current drugs have limitations. So the investigation of plants that are used in folk medicine may have prognostic value to discover new and safe cutaneous leishmanicidal drug. The present study conducted to offer a scientific basis for the traditional use of Euphorbia cactus Ehrenb, Euphorbia ammak Forssk, Euphorbia inarticulate Schweinf, stems and leaves of Pergularia tomentosa L as cutaneous leishmanicidal, this may be regarded as future promising phytotherapeutics in the treatment Cutaneous leishmaniasis. The objectives of this study were to evaluate the in vitro cutaneous anti-leishmanial activity of Euphorbia cactus Ehrenb, Euphorbia ammak Forssk, Euphorbia inarticulate Schweinf, stems and leaves of Pergularia tomentosa L extracts. Also carrying out preliminary phytochemical screening of those plant extracts.

SUBJECTS AND METHODS

Plant materials

Four selected plants (Euphorbia cactus Ehrenb, Euphorbia ammak  Forssk, Euphorbia inarticulate  Schweinf, stem and leaf of Pergularia tomentosa L (table 1) commonly used in Yemen by traditional healers   for the treatment of Cutaneous leishmaniasis  and other skin diseases  were collected  from  Taiz governorate of Yemen in September 2016, and botanical identification was by Dr. Hassan Ibrahim botanist at the Botany section, Biology department, Faculty of Science,  Sana^'a university, Sana'a (Yemen).

Plant preparation

Stems and leaves of Pergularia  tomentosa L and phylloclade of Euphorbia inarticulate  Schweinf were collected, washed, sliced, weighed and sundried under the shade at room temperature.  After complete drying , they grinded to a coarse powder  in electrical blender.  The dried crude plants were maintained in dark vials  and stored at  -20 °C until used^19,20.  The latex of Euphorbia cactus Ehrenb and Euphorbia ammak  Forssk were collected in clean glass bottles and were kept in the refrigerator (4°C – 8°C)  until extraction²⁰.

Plants extraction

The dried plants were extracted successively with MeOH (80%) by using a Soxhlet extractor.  The obtained extracts were filtered through a Whatman-1 filter paper. The filtrates were dried by evaporation on a rotary evaporator below 45°C and freeze dryer to give the crude dried extract.  All extracts were stored at        -20^oC until used, the yield obtained are shown in Table 2. Latex was extracted by maceration using MeOH (80%). The latex was soaked (1 ml) in 10 ml of solvent in a stoppered container with frequent agitation at room temperature. After maceration the soaked latex were filtrated through a Whatman-1 filter paper. The filtration were dried by a rotary evaporation and freeze drier to give the crude dried extract. The crude dried extract of latex  were stored at – 20, the yield obtained are shown in Table 2.

Anti-promastigote assay

Patient selection and Leishmania spp. isolation

 Following clinical diagnosis by dermatologist, and confirmed by laboratory. Leishmania spp.  were isolated from the patient with Cutaneous leishmaniasis infection. Skin lesions of the patients were cleansed with 70% ethanol before sample scraping. These preparations were stained with Giemsa and examined under a light microscope with magnification (×1000),  and it was inoculated in Nicolle-Novy-McNeal (NNN) culture medium. The culture tubes were kept in an incubator at 25⁰C for 5-10 days. Leishmania spp.  Promastigote that observed during microscopy transferred to RPMI-1640 and incubated at 25 °C for mass cultivation medium. Patient who included in this study were: (1) patient with infection not exceed 6 months, (2) patient did not used drugs or herbs, and (3) Heavy infection.

Determination of 50% promastigote growth inhibitory concentration (IC₅₀) of the plant extracts

 Stock solution of crude extracts was prepared in 2 % (DMSO)/deionized water at 10 mg/10mL.  RPMI-1640 medium were distributed in each well of a 96 well  plate. The extract solutions (100µl) were serially diluted down each lane of 96-well plate with medium. Then, the growing cells at 10⁶ promastigotes/ml  were added to each well to give final six concentration of plant extracts 15.6µgml^-1, 31.25 µgml^-1, 62.56 µgml^-1, 125 µgml^-1, 250 µgml^-1, 500 µgml^{-1 (}two folds dilutions). The final concentration of DMSO was not be higher than 1% (v/v)  as this concentration did not affect the parasite growth rate, mobility, morphology²¹  in all experiments. A Pentostam (Albert David Limited-India) was used   as positive control at 10µgml^-1, untreated media were used as negative control, and DMSO alone was used as solvent control.  96-well micro-plates were incubated at 26⁰C.  After the incubation,  the effect of the isolated extracts on cell viability was assessed using the 3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which measures the metabolic activity of mitochondria²².   In which, 20μL of MTT  solution (5mgmL^-1)  (SIGMA) was added to each well and incubated at 26^°C for 4 h at 26°C. The test principle "Tetrazolium salts are cleaved to formazan dye by cellular enzyme mitochondrial succinate dehydrogenase (only in the viable promastigites)”. Finally, the MTT solubilization solution   was added to each well to dissolve the insoluble purple formazan product into coloration solution and incubated for another 10 min. The absorbance was measured at 570 nm for each well,  using an ELISA reader.^23,24 The live promastigotes percentage inhibition ratio were calculated as described by Bansal et al. ²⁵.

Inhibition Rate (I%)

Data were presented as mean±SD of triplicate experiments for each well.  Mediam inhibitory concentration value (IC₅₀ value₎, the concentration that decreased parasite growth by 50% was calculated using linear regression analysis (dose –response analysis) associated with 95% confidence interval. Lower IC50 value indicates greater anti-leishmanial activity.

Leishmania spp. counting

 20 µl of cells from the culture were taken and placed in 1.5 ml Eppendorf tube containing 20 µl of Eosin (1%) then mixed. After that 10µl of cells were placed on haemocytometer (Thoma slide).  5 squares were counted then the result numbers were multiply by 100000 so we can get the number of cells in 1 ml of culture.

Preliminary Qualitative Phytochemical Screening

 The dried extracts were tested to identify alkaloids, carbohydrates, glycosides, fixed oil and fats, anthraquinones, phenolic compounds and tannins, phytosterols, proteins, saponins, gum and mucilage. A preliminary phytochemical analysis was carried out using tube-test reaction and thin-layer chromatography (TLC) as described by Banu and Cathrine¹⁹.

Statistical Analysis

 Data are presented as mean±SD of triplicate experiments, and the plant extract  concentration required for 50% inhibition in vitro (IC₅₀) calculated  by dose-response  analysis associated with 95% confidence interval by Graph pad prism 7 Demo.

Ethical Consideration

Ethical clearance for the study was taken from the Faculty of Medicine and Health Sciences Research Review Committee. Informed Consent was taken from the patients before the collecting specimens.

RESULTS

The percentages of extraction yield for each investigated plant were summarized in Table 2. The results of anti-Promastigote assay (in vitro test) for the five  plant extracts against promastigotes stages  of  Leishmania spp. are summarized in Table 3,4, 5, 6,7. Data in Table 8 elucidate that the highest antileishmanial activity was obtained from Euphorbia cactus Ehrenb and Euphorbia ammak Forssk  with IC₅₀ value < 15.6 µgml^-1. The leaves and stems  of Pergularia tomentosa L  and Euphorbia innerticulata  Schweinf extracts showed  inactive antileishmanial activity (IC₅₀ value > 500 µgml^-1₎.

Phytochemical analysis and thin layer chromatography results-

Phytochemical analysis of extracts indicated the presence of various bioactive components. Compounds, such as alkaloids were found in the five plant extracts. However, fixed oils, fats and Anthraquinones were absent in all extracts,  Phytosterols were found in the four plant extract and  in Euphorbia ammak Forssk extract was absent.  Glycosides, Gum and Mucilage were found only in Euphorbia cactus Ehrenb and Euphorbia ammak Forssk, Protins were found only in Euphorbia cactus Ehrenb and Euphorbia inarticulate Schweinf. However, Saponines were found only in Euphorbia cactus Ehernb, Euphorbia ammak Forssk, stem and leaf of Pergularia tomentosa L, and carbohydrates were found in Euphorbia inerticulata schweinf, stem and leaf of Pregularia tomentosa L (Table 9). The TLC analysis showed spots of determined constitutes in each extract (Table 10).

DISCUSSION

The first choice drug for treatment of cutaneous leshmaniasis  is Pentostam which has toxic side effects. Also, no vaccine is available to cure this disease.  Most of the studies directed towards plants that is used traditionally as potential source of new alternative medicines. Some of the drugs obtained from plants used in the treatment of diseases caused by protozoan include alkaloids quinine obtained from the plant genus  Cinchora and  artemisinin  obtained from  the plant genus Artemisia  annua and  both of  them used in the treatment of malaria.  As well as, emetin obtained from the plant genus Cephaelis used in treatment of ameobiasis²⁶.  Our findings showed that the methanol latex extract of Euophobia cactus Ehrenb (Euophrbiaceae) had choice antileshmanial activity (IC₅₀<15.6 µgml^-1 against promastigotes).  There was no previous studies reported about its biological activities against Leishmania spp. to compare with, so our results appear to be one of the first that studied its activity.  The methanol latex extract of Euphorbia ammak Forssk showed a good antileishmanial activity (IC₅₀<15.6µgml^-1 against promastigotes). Our data is compatible with data  reported by  Abdel-Sattar et al. (2010) against Leishmania infantum which found to inhibit the growth of  Leishmania infantum with IC₅₀ value < 24.05 µg/ml²⁷. These findings confirmed that latex of Euophobia cactus Ehrenb and Euphorbia ammak Forssk could be considered as having promising antileishmanial activity.

The methanol extract of Euphorbia inarticulate   Schweinf phylloclades showed no antileishmanial activity (IC₅₀>500 µgml^-1).  Although, there was no previous studies reported about its biological activities against Leishmania spp.  to compare with, so our results appear to be  one of the first that study its antileishmanial activity. The leaves and stems extracts of  Pergularia tomentosa L and Euphorbia inarticulate  Schweinf showed no antileshmanial activity (IC₅₀>500 µgml^-1 ), while several studies reported good activity of these plants against fungi^28,29. Flavnoids may be the active compound in Euphorbia ammak Forssk against Leishmania spp. in our study. Our findings can be confirmed by the finding of Das et al.³⁰  in which they tested the antileishmanial activity of flavonoids  against topoisomerase I of Leishmania donovani. They illustrated that these compounds inhibited topoisomerase I which subsequently inhibit the relegation step in parasite growth³⁰. Phenolic, saponins, and alkaloids that we detected in our study may be they are the active compounds responsible for antileishmanial activity in Euphorbia ammak Forssk and Eiphorbia cactus Ehrenb.   Kayser et al.³¹  in their comprehensive review of antiparasitic drug development view  phenolic, saponins, and alkaloids  have inhibition activity against Leishmania parasite^{31, 32}.

Phytochemical results of leaves and stems of Pergularia tomentosa L indicated the presence of saponins, phytosterols, phenolic compounds, tannins, carbohydrates, alkaloids, and flavonoids, except coumarins only present in stems. The phytochemical results of leaves and stems of Pergularia tomentosa L in our study is in agreement with  Hassan et al. and Shinkafi in Nigeria, except glycosides, and anthraquainins were not present in our extracts  of Pergularia tomentosa L^{28, 29}.  The absent of glycosides, and anthraquainins in our plant Pergularia tomentosa L might be due to the environmental factors, such as altitude, temperature, illumination, precipitation, humidity, soils and locations as described by Zidorn and Stuppner in which they referred these differences in components is due  to the environmental factors³³.  

CONCLUSION

In conclusion, the methanolic extract of  Euphorbia cactus Ehrenb and Euphorbia ammak Forssk  showed a good antileishmanial activity with IC₅₀ value>15.6 µgml^-1 relative to negative control. The preliminary phytochemical investigation reveal the extracts contain secondary metabolites, that indicate these plants may be highly promising candidate drugs. Furthermore the results offer a scientific basis for the traditional use of investigated plants.  This is a preliminary evaluation using promastigotes must be complemented with an evaluation using intracellular amastigotes in macrophages. At the same time, an evaluation of the possible cytotoxicity of the tested plants is important. Further screening for Yemeni plants species especially that are used in traditional medicine must be done for searching of potential anti-leishmanial active constituents and record ethno-botanical data. 

ACKNOWLEDGMENTS

The  authors would like  to acknowledge  Tropical Disease Research Centre at Technology and information university, Faculty of Pharmacy, Sana'a  University , and  the Microbiology Department of the National Center of  Public Health Laboratories (NCPHL) and Yemen Lab, Sana'a, Yemen  which provided working space. Thanks go to Musned  Education fund for financial supporting.

CONFLICT OF INTEREST 

"No conflict of interest associated with this work”.

AUTHOR’S CONTRIBUTION

This research work is part of A M.Sc. thesis. The candidate is the first author (MMA) who conducted experiments and wrote up the thesis. The corresponding author (HAA) supervised the experimental work, revised, and draft the manuscript. (BYA) was co-advisor of the work, and (BAM) helped in chemical analysis. 

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Table 1: List of the selected plants that used in the investigation

   a- S. :Stem,  Pha. : phylloclades,  L:Leaves ,  Lat.: Latex. 

Table 2: Percentage  extraction yield of plant extracts. 

Table 3: In vitro l activity of Euphorbia cactus Ehrenb methanolic extracts on  Leishmania spp.   after  24h

Mean ±SD is OD values of 3 wells. * Average ± standard division, ^a No significant difference compared to each other (P>0.05). ^b Significant difference compared to untreated wells (P<0.05)

Table 4: In vitro activity of Euphorbia ammak Forssk methanolic extracts against  Leishmania spp. after 24h.

Mean ±SD is OD values of 3 wells. * Average ± standard division,  ^b Significant difference compared to untreated wells (P<0.05). ^c significant difference compared to 15.6, 31.25, and 62.5µg/ml  (P<0.05).

Table 5: In vitro activity of Euphorbia inerticulata Shweinf extracts against  Leishmania spp. for 24h

Mean ±SD is OD values of 3 wells. * Average ± standard division, ^b Significant difference compared to untreated wells (P<0.05). ^c significant difference compared to 15.6µg/ml  (P<0.05).

Table 6: In vitro activity of Pergularia tomentosa L. leaves methanolic extracts against Leishmania spp. for 24h.

Mean ±SD is OD values of 3 wells. * Average ± standard division,  ^a No significant difference compared to each other (P>0.05). the ^b Significant difference compared to untreated wells (P<0.05)

Table 7: In vitro activity of Pergularia tomentosa L. stems methanolic extracts against Leishmania spp. for 24h

Mean ±SD is OD values of 3 wells. * Average ± standard division,  ^b Significant difference compared to untreated wells (P<0.05). c significant difference compared to 15.6 and 31.25 µg/ml  (P<0.05).

Table 8: IC₅₀ values (µg/ml) for promastigotes growth inhibition of the methanol extracts of  five plant.

IC₅₀=Inhibitory concentration 50%, L= leaves, S= stems.

Table 9:  Qualitative phytochemical analysis of plant extracts by chemical method.

+= signify presence , -= signify Absence,  E.C= Euphorbia cactus, E.I= Euphorbia inerticulata, E.A= Euphorbia ammak,  P.L= Pergularia tomentoa L leaves, P.S= Pergularia tomentoa L stems

Table 10:  TLC investigation of methanol extract of five plant extracts.

TLC: thin layer chromatography, EtoAc: ethyle acetate, MeOH: methanol, CHCL₃:chloroform, a: acid, RF:   retention factor,---:absent,            EC: Euphorbia cactus Ehrenb, EA:Euphorbia ammak, EI=Euphorbia inerticulata shwenf , PL:pergularia tomentosa  L.leaves, PS:Pergularia tomentosa L.  stem

TLC: (a)  anth (anthraquinones) after sprayed  with 10% ethanolic KOH agent, (b) BIT (bitter principles) after sprayed with vanillin sulpharic acid, (c) Fl (flavonoids) after sprayed with polythlene glycol, (d)  saponins   after sprayed with vanillin supharic acid ), and(e)   coumarins after sprayed with 10 % ethanolic KOH. Abbreviations at the end of plates in each plateare:PL, Pergularia tomentosa L leaves, PS: Pergularia tomentosa L stems, EA: Euphorbia ammak, EI: Euphorbia inerticulta, EC:Euphorbia cactus