Srikant Kumar Puspaa Sinha Akhtar Parwez Birendra Kumar Kumar Lav Kush Tarun Sudhanshu Kumar Bharti


Leishmania donovani is an obligatory intracellular digenetic parasite transmitted by insects, causing serious global health problems as it is endemic to most developing countries. Extensive use of antimony compounds as drugs poses high toxicity and cost; therefore, herbal medicine has identified a position. This study explored the developmental and biochemical characteristics of L. donovani promastigote and the effect of ethanolic extract of Achyranthes aspera Linn (Amaranthaceae) plant on it. The parasites were incubated at 2.5×106 cells/well for 72 h at 23 °C in the presence of various concentrations of extract (µg/mL) dissolved in 1% dimethyl sulfoxide (DMSO) with sterile phosphate-buffered saline and 1% DMSO as negative controls and meglumine antimoniate as positive control. Friedman’s repeated measures analysis showed that 96hr of development is the junction point in promastigotes ontogeny. Post 96hr, it grows with a long stationary phase with higher enzymatic activities of acid phosphatase, superoxide dismutase and glutathione (oxidized and reduced). Total protein estimated showed a linear relationship (R2 = 0.999). Phytochemical screening of the plant extract showed the presence of alkaloid, flavonoid, fixed oil and fats, saponin, tannin and phenolic compounds. It showed an effectual free radical scavenging in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with an inhibitory concentration IC50 value of 61.70 µg/ml. At a concentration of 250 µg/mL, the plant extract completely inhibited the promastigotes in vitro while at 50 µg/mL and 100 µg/mL, the survival level declined by 25-50%. These findings corroborate the ethnopharmacological use of this plant for the treatment of leishmaniasis caused by L. donovani.




Achyranthes aspera Linn, Inhibitory concentration, Leishmania donovani, Phytochemicals, Promastigote

Alvar, J., Vélez, I. D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J. & den Boer, M. (2012). WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One, 7(5), e35671. doi:10.1371/journal.pone.0035671.
Anonymous. (2016). The Indian Pharmacopoeia. 1st edition. Government of India, New Delhi. Ministry of Health and family welfare. https://cdn.ayush.gov.in/wp-content/uploads/2021/03/Ayurvedic-Pharmacopoeia-of-India-part-1-volume-IX.pdf.
Aragon, V., Kurtz, S. & Cianciotto, N. P. (2001). Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infection and Immunity, 69(1), 177-185. doi: 10.1128/IAI.69.1.177-185.2001.
Bakri, I.M. & Douglas, C.W.I. (2005). Inhibitory effect of garlic extracts on oral bacteria. Archives of Oral Biology, 50(7), 645–651. doi:10.1016/j.archoralbio.2004.12.002.
Bharti, S.K., Krishnan, S. & Gupta, A.K. Herbal Formulation to Combat Type 2 Diabetes Mellitus. LAP LAMBERT Academic Publishing (2013-09-01). ISBN-13:978-3-659-43204-0. (https://www.morebooks.de/shop-ui/shop/product/978-3-659-43204-0).
Bharti, S.K., Krishnan, S., Kumar, A. & Kumar, A. (2018). Antidiabetic phytoconstituents and their mode of action on metabolic pathways. Therapeutic Advances in Endocrinology and Metabolism, 9(3), 81–100. doi:10.1177/2042018818755019.
Bharti, S.K., Krishnan, S., Kumar, A., Rajak, K.K., Murari, K., Bharti, B.K. & Gupta, A.K. (2013). Antidiabetic activity and molecular docking of fructooligosaccharides produced by Aureobasidium pullulans in poloxamer-407-induced T2DM rats. Food Chemistry, 136, 813–821. doi: 10.1016/j.foodchem.2012.08.083.
Bharti, S.K., Krishnan, S., Sharma, N.K., Kumar, A., Prakash, O., Gupta, A.K. & Kumar A. (2015). In vivo and in silico investigation of antidiabetic activity of fruit of Withania coagulans Dunal. Current Hypertension Reviews, 11(2):143- 58. doi: 10.2174/157340211102150731120254.
Bharti, S.K., Sharma, N.K., Gupta, A.K. & Padamdeo, S.R. (2012). Antidiabetic effect of aqueous extract of Withania coagulans flower in Poloxamer-407 induced type 2 diabetic rats. Journal of Medicinal Plants Research, 6(45), 5706-5713. doi: 10.5897/JMPR12.646.
Bharti, S.K., Krishnan, S. & Kumar, A. (2016). Phytotherapy for diabetes mellitus: back to nature. Minerva Endocrinoligica, 41, 143–146. PMID: 26878565.
Bharti, S.K., Krishnan, S. & Kumar, A. (2012). Antihyperglycemic activity with DPP-IV inhibition of alkaloids from seed extract of Castanospermum australe: investigation by experimental validation and molecular docking. Phytomedicine, 20, 24–31. doi: 10.1016/j.phymed.2012.09.009.
Bharti, S.K., Kumar, A. & Sharma, N.K. (2013). Tocopherol from seeds of Cucurbita pepo against diabetes: validation by in vivo experiments supported by computational docking. Journal of the Formosan Medical Association, 112, 676–690. doi: 10.1016/j.jfma.2013.08.003.
Bhowmick, S. & Ali, N. (2009). Identification of novel Leishmania donovani antigens that help define correlates of vaccine-mediated protection in visceral leishmaniasis. PLoS One, 4(6):e5820. doi: 10.1371/journal.pone.0005820.
Colotti, G. & Ilari, A. (2011). Polyamine metabolism in Leishmania: from arginine to trypanothione. Amino Acids, 40(2), 269–285. doi: 10.1007/s00726-010-0630-3.
Corrêa, D.S., Tempone, A.G., Reimão, J.Q., Taniwaki, N.N., Romoff, P., Fávero, O.A., Sartorelli, P., Mecchi, M.C., Lago, Jão. & Henrique. G. (2011). Anti-leishmanial and anti-trypanosomal potential of polygodial isolated from stem barks of Drimys brasiliensis Miers (Winteraceae). Parasitology Research, 109(1), 231-236. doi: 10.1007/s00436-010-2229-8.
Costa, T.L., Ribeiro-Dias, F., Oliveira, M.A.P., Bezerra, J.C.B. & Vinaud, M.C. (2011). Energetic metabolism of axenic promastigotes of Leishmania (Viannia) braziliensis. Experimental Parasitology, 128, 438-443. doi: 10.1016/j.exppara.2011.05.018.
da Silva, E, R., da Silva, M.F., Fischer, H., Mortara, R.A., Mayer, M.G., Framesqui, K., Silber, A.M. & Floeter-Winter, L.M. (2008). Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme. Molecular and Biochemical Parasitology, 159(2), 104-111. doi: 10.1016/j.molbiopara.2008.02.011.
Dangi, A.S., Sharma, M. & Aparna, J.P. (2012). Antimicrobial activity of Achyranthus aspera Linn. International Journal of Pharmacy and Biological Sciences, 1, 1–6.
Deepachandi, B., Weerasinghe, S., Andrahennadi, T.P., Karunaweera, N.D., Wickramarachchi, N., Soysa, P. & Siriwardana, Y. (2020). Quantification of soluble or insoluble fractions of Leishmania parasite proteins in microvolume applications: A simplification to standard Lowry assay. International Journal of Analytical Chemistry, 6129-6132. doi: 10.1155/2020/6129132.
Dutta, A., Bandyopadhyay, S., Mandal, C. & Chatterjee, M. (2005). Development of a modified MTT assay for screening antimonial resistant field isolates of Indian visceral leishmaniasis. International Journal for Parasitology, 54,119–122. doi: 10.1016/j.parint.2005.01.001.
Essid, R., Rahali, F.Z., Msaada, K., Sghair, I., Hammami, M., Bouratbine, A., Aoun, K. & Limam, F. (2015). Antileishmanial and cytotoxic potential of essential oils from medicinal plants in Northern Tunisia. Industrial Crops and Products, 77, 795-802. doi: 10.1016/j.indcrop.2015.09.049.
Et-Touys, A., Bouyahyal, A., Fellah, H., Mniouil, M., El-Bouryl, H., Dakka, N., Sadak, A. & Bakri, Y. (2017), Antileishmanial activity of medicinal plants from Africa: a review. Asian Pacific Journal of Tropical Disease, 7, 826-840. doi: 10.12980/apjtd.7.2017D7-215.
Field, A. (2005). Discovering Statistics using SPSS, 2nd ed. Sage, London.
Ghosh, S., Goswami, S. & Adhya, S. (2003). Role of superoxide dismutase in survival of Leishmania within macrophage. Biochemical Journal, 369, 447-452. doi: 10.1042/BJ20021684.
Jaiswal, S, K., Sharma, N.K., Bharti, S.K., Krishnan, S., Kumar, A. & Prakash, O. (2018). Phytochemicals as uropathognic Escherichia coli FimH antagonist: in vitro and in silico approach. Current Molecular Medicine, 18(9), 640–653. doi: 10.2174/1566524019666190104104507.
Kar, K. (1997). Folic acid, the essential supplement to brain-heart infusion broth for cultivation and cloning of Leishmania donovani promastigotes. Parasitology, M5, 231-235. doi: 10.1017/s0031182097001340.
Kopec, W., Telenius, J. & Khandelia, H. (2013). Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes. FEBS Journal, 280, 2785–2805. doi: 10.1111/febs.12286.
Kumar, S., Sinha, P., Parwez, A., Kumar, B., Tarun, LKK. & Bharti, SK. (2022). The developmental and biochemical features of Leishmania donovani promastigote and their repression by Datura stramonium plant extract. International Journal of Scientific Research, 11(9), 1-5. doi: 10.36106/ijsr.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193(1), 265–275. PMID: 14907713.
McDonald, S., Prenzler, P.D., Autolovich, M. & Robards, K. (2001). Phenolic content and antioxidant activity of olive extracts. Food Chemistry, 73, 73–84. doi:10.1016/S0308-8146(00)00288-0.
Mukherjee, S., Bandyapadhyay, R. & Basu, M.K. (1988). Leishmania donovani: superoxide dismutase level in infected macrophages. Bioscience Reports, 8(2), 131-137. doi:10.1007/BF01116457.
Mukhopadhyay, D., Das, N.K., De-Sarkar, S., Manna, A., Ganguly, D.N., Barbhuiya, J.N., Maitra, A.K., Hazra, A. & Chatterjee, M. (2012). Evaluation of serological markers to monitor the disease status of Indian post kala-azar dermal leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 106(11), 668–676. doi:10.1016/j.trstmh.2012.07.005.
Murari, K., Sharma, N.K. & Bharti, S.K. (2016). The coming age of future of medicine: Next Frontier. Biochemistry & Analytical Biochemistry, 5(297). Doi: 10.4172/2161-1009.1000297. doi: 10.4172/2161-1009.1000297.
Mutoro, C.N., Kinyua, J., Ng'ang'a, J., Kariuki, D., Ingonga J., Christopher, M. & Anjili O. (2018). Efficacy of the combination of crude extracts of Solanum nigrum and Plumbago capensis on Leishmania major. F1000Research. 7: 1556. doi:10.12688/f1000research.15955.1.
Nikmehr, B., Ghaznavi, H., Rahbar, A., Sadr, S. & Mehrzadi, S. (2014). In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves. Chinese Journal of Natural Medicines, 12(6), 423-427. doi: 10.1016/S1875-5364(14)60066-2.
Pan American Health Organization. Leishmaniasis: Epidemiological Report in the Americas. Number 9, December 2020. Washington, D.C.: PAHO; 2020. URL: https://iris.paho.org/handle/10665.2/53090.
Papadaki, A. & Boleti, H. (2019). Measurement of acid ecto-phosphatase activity in live Leishmania donovani parasites. Bio-protocol, 9(19):e3384. doi: 10.21769/BioProtoc.3384.
Patil, V, S. & Malpathak, N.P. (2016). Micro-morphoanatomical approach for comparative analysis of Tinospora cordifolia (Willd.) Miers and its adulterant plant using SEM and Cryostat. Pharmacognosy Journal, 9(1), 39–45. doi:10.5530/pj.2017.1.8.
Pratihast, K., Kumar, R. & Bharti, S, K. (2019). Comparative antimicrobial activity of ethanolic and aqueous extract of Tinospora cordifolia. The Pharma Innovation Journal, 8(3), 129-136.
Rashid, U., Khan, M.R., Jan, S., Bokhari, J. & Shah, N, A. (2013). Assessment of phytochemicals, antimicrobial and cytotoxic activities of extract and fractions from Fagonia olivieri (Zygophyllaceae). BMC Complementary and Alternative Medicine, 13, 167. doi:10.1186/1472-6882-13-167.
Saeed, N., Khan, M.R. & Shabbir, M. (2012). Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complementary and Alternative Medicine, 12, 221. doi:10.1186/1472-6882-12-221.
Santarém, N., Cunha, J., Silvestre, R., Silva, C., Moreira, D., Ouellette, M. & Cordeiro-DA-Silva, A. (2014). The impact of distinct culture media in Leishmania infantum biology and infectivity. Parasitology, 141(2),192-205. doi: 10.1017/S0031182013001388.
Shendkar, C, D., Chandrachood, P, S., Pawar, A.B., Lavate, S, M. & Deshpande, N, R. (2011). Quantitative estimation of macro, micro nutrients and trace elements by X-ray fluorescence spectroscopy (XRF) from Achyranthes aspera Linn. International Journal of ChemTech Research, 3, 610–613.
Silveira, E, S., Rodrigues, N, L, D, C., Machado, N, J., Fonseca, F, R, M., Teixeira, M, J. & Leal, L, K, A, M. (2019). Medicinal plants containing coumarin or essential oils from the Brazilian biome may be new option for treating Leishmaniasis? Pharmacognosy Review, 14(27), 53-61.
Sinan, K, I., Zengin, G., Zheleva-Dimitrova, D., Etienne, O, K, Fawzi., Mahomoodally, M., Bouyahya, A., Lobine, D., Chiavaroli, A., Ferrante, C., Menghini, L., Recinella, L., Brunetti, L., Leone, S. & Orlando, G. (2020). Qualitative Phytochemical Fingerprint and Network Pharmacology Investigation of Achyranthes aspera Linn. Extracts. Molecules, 25, 1973. doi:10.3390/molecules25081973.
Sundar, S., Rai, M., Chakravarty, J., Agarwal, D., Agrawal, N., Vaillant, M., Olliaro, P. & Murray, H, W. (2008). New treatment approach in Indian visceral leishmaniasis: single-dose liposomal amphotericin B followed by short-course oral miltefosine. Clinical Infectious Diseases, 47(8), 1000-1006. doi: 10.1086/591972.
Thakur, S., Joshi, J. & Kaur, S. (2020). Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods. Journal of parasitic diseases: official organ of the Indian Society for Parasitology, 44(2), 253–272. doi: 10.1007/s12639-020-01212-w.
Wabwoba, B.W., Anjili, C, O., Ngeiywa, M, M., Ngure, P, K., Kigondu, E, M., Ingonga, J. & Makwali, J. (2010). Experimental chemotherapy with Allium sativum (Liliaceae) methanolic extract in rodents infected with Leishmania major and Leishmania donovani. Journal of Vector Borne Diseases, 47(3), 160–167. PMID: 20834086.
Warburg, A., Gelman, S. & Deutsch, J. (2008). Xanthine in urine stimulates growth of Leishmania promastigotes in vitro. Journal of Medical Microbiology, 57, 136-138. doi: 10.1099/jmm.0.47419-0.
Zar, J, H. (1999). Biostatistical Analysis, 4th ed. Pearson, London.
Research Articles

How to Cite

Study on developmental biochemical characteristics of Leishmania donovani promastigote and inhibitory effect of Achyranthes aspera Linn plant extract on it. (2022). Journal of Applied and Natural Science, 14(4), 1542-1551. https://doi.org/10.31018/jans.v14i4.3944