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Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi
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ARTICLE IN PRESS Journal of Infection and Public Health xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph
Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi Dipti Ranjan Behera, Sunita Bhatnagar ∗ Medicinal and Aromatic Plants, Regional Plant Resource Centre, Bhubaneswar, Odisha, India
a r t i c l e
i n f o
Article history: Received 16 May 2017 Received in revised form 3 January 2018 Accepted 9 January 2018 Keywords: Setaria cervi DEC (diethylcarbamazine) macrofilaricidal MTT reduction GST (glutathione-S-transferase) inhibition
a b s t r a c t Antifilarial potential of three medicinal plants namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa was explored using Setaria cervi, a bovine filarial parasite at concentrations of 2.5, 5 and 10 mg/ml. Amongst all the extracts, methanol extract of T. bellerica showed highest macrofilaricidal activity i.e. 84.63 ± 1.11 at 10 mg/ml in MTT reduction assay with IC50 value of 2.7 mg/ml. which was better than the standard DEC i.e. 79.22 ± 3.1% at 10 mg/ml with IC50 value 2.84 mg/ml. Other plant extracts showed mild in vitro macrofilaricidal activity. T. bellerica methanol extract exhibited significant GST activity of 18.86 ± 0.21 and 12.83 ± 0.03 M/ml/min at 5 and 10 mg/ml with percentage inhibition value of 73.96% and 82.29% respectively. DEC showed GST activity value of 40.03 ± 4.14 and 21.48 ± 6.44 M/ml/min with percentage inhibition value of 21.76% and 58.01% at 5 and 10 mg/ml respectively. Thus, methanol extract of leaves of T. bellerica exhibited highly significant antifilarial potential and needs detailed analysis. © 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Filariasis is a major public health problem affecting over 120 million people in Central Africa, Central and South Africa and large regions of Asia including China and India [1]. Currently, 1.10 million people in 55 countries are living in areas that require preventive chemotherapy to stop the spread of infection. The current drug regime for lymphatic filariasis include diethylcarbamazine (DEC), albendazole and ivermectin. These drugs have been consistently used in the MDA (Mass Drug Administration) programme to block the disease transmission in the endemic areas. These drugs are active against blood infesting microfilariae (mf) thus efficient in interrupting transmission of the disease; however, they are less effective against the adult worm [2,3]. These drugs have some side effects as well, such as nausea, vomiting, gastric disturbances and giddiness [4]. Owing to this still there is a need for safe, non toxic and macrofilaricidal drug. Secondary metabolites from plants have always served as potential source of alternative medicines. In this study, three plants of Terminalia genus namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa were explored for their antifilarial activities using Setaria cervi, a bovine filarial parasite as target. Terminalia sp., belongs to family Combretaceae, consisting of approximately 150 species of trees and shrubs [5]. The leaves
and bark are well known for their medicinal potential [6] whereas fruits of T. bellerica and T. chebula are used in Ayurvedic medicine “Triphala”, which is a powerful antioxidant and detoxifier [7]. T. catappa leaves contain several bioactive compounds responsible for antiviral, antibacterial, antifungal and anticancerous activities [8]. Keeping in view of medicinal potential of all the three species same were explored for their antifilarial potential for the development of a safe and non toxic alternative for present day synthetic drugs. Materials and methods Collection and processing of plant material The leaves of the medicinal trees namely T. bellerica, T. chebula and T. catappa were collected from Regional Plant Resource Centre, Bhubaneswar, Odisha, India. Solvent extracts were prepared using the standard protocols [9]. Collection of Setaria worms The bovine S. cervi adult parasites were collected from the peritoneal cavity of the freshly slaughtered cattles in the slaughter house of Nandankanan Zoological Park, Bhubaneswar, Odisha, India. Worm motility inhibition assay
∗ Corresponding author. E-mail address: [email protected] (S. Bhatnagar).
In vitro worm motility inhibition assay was performed by the Standard method [10]. The worms were transferred to RPMI-
https://doi.org/10.1016/j.jiph.2018.01.006 1876-0341/© 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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1640 medium and supplemented with 5% (v/v) heat-inactivated Fetal bovine serum. Stock solution of strength 200 mg/ml was prepared of solvent leaf extracts and standard DEC in DMSO (Dimethyl sulphoxide). Two adult worms were introduced into each vial and were subjected to solvent extracts at concentrations of 2.5–10 mg/ml. A simultaneous positive control was kept without the test solution but with equal volume of DMSO as in test vials. Experimental and controls were incubated at 37 ◦ C for 24 h. The motility readings were taken in each hour interval up to four hours to examine the drug activity on worms. After 24 h exposure, the worms were transferred to fresh PBS (1×) to check the motility of the worms. If the worms did not regain their motility, then activity was considered irreversible and showed that the drugs used were active against filarial worms. Motility assay was followed by MTT reduction assay. MTT reduction assay Effect of solvent extracts on adult female Setaria worm was studied by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] formazan reduction assay [11]. After worm motility inhibition study, the treated worms were washed in fresh PBS (1×) pH 7.4. Then the parasites were transferred to 24 well culture plates containing 0.01% MTT prepared in PBS (1×) and incubated at 37 ◦ C for 1h duration. After incubation the treated worms were trans-
ferred to chilled PBS (1×). PBS was removed by drying the worm on blotting paper. Worms were carefully transferred to 24 well culture plates containing 2 ml DMSO and incubated at 37 ◦ C for 2 h. The absorbance of the resulting formazan solution was then determined at 510 nm in a microplate reader. Percent inhibition was calculated as follows: Percentage inhibition = Control − Sample/Control × 100 IC50 value of standard drug and extracts were compared for effective extracts. Glutathione S-transferase (GST) inhibition assay GST inhibition study was carried out by the standard method [12]. For GST assay, worms were washed thoroughly with phosphate-buffered saline (PBS), pH 7.4, prepared a 10% homogenate, centrifuged at 10,000 × g for 30 min at 4 ◦ C, supernatant containing the cytosolic fraction was used for GST assay. GST activity was measured according to the method of Habig [12] using 1-chloro-2,4-ditnitrobenzene (CDNB) and GSH as substrates. The protein content in the crude homogenate of the worm extract was determined [13] using bovine serum albumin as standard protein. The cytosolic fraction was incubated with concentrations 5 and 10 mg/ml of each extract/standard drug for 10 min at room temperature in the reaction mixture containing 465 l Assay buffer
Table 1 In vitro macrofilaricidal activity of plant extracts/DEC against adult Setaria cervi in terms of motility inhibition. Plants used
Extracts
Dose (mg/ml)
1h
2h
3h
4h
24 h
Terminalia bellerica
Hexane
2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 3+ 3+
4+ 4+ 4+ 4+ 4+ 3+ 3+ 3+ 3+ 4+ 2+ 1+
4+ 4+ 4+ 4+ 3+ 2+ 3+ 2+ 2+ 1+ 1+ 1+
2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 1+ 3+ 1+ 1+ 4+ 4+ 4+ 4+ 4+ 4+
3+ 1+ 1+ 2+ 1+ 1+ 4+ 4+ 4+ 4+ 4+ 2+
2+ 1+ 1+ 1+ 1+ 1+ 4+ 4+ 4+ 4+ 4+ 1+
1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 3+ 1+ 1+ 1+ 1+ 4+ 4+ 3+ 4+ 4+ 4+
3+ 1+ 1+ 1+ 1+ 1+ 4+ 3+ 1+ 4+ 4+ 3+
2+ 1+ 1+ 1+ 1+ 1+ 2+ 2+ 1+ 4+ 3+ 3+
2+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
2.5 5 10
4+ 4+ 4+
4+ 4+ 1+
4+ 4+ 1+
4+ 4+ 1+
1+ 1+ 1+
Chloroform
Acetone
Methanol
Terminalia catappa
Hexane
Chloroform
Acetone
Methanol
Terminalia chebula
Hexane
Chloroform
Acetone
Methanol
DEC (standard)
Motility score of adult worms (in h)
Motility readings: 4+ (highly motile), 3+ (motile), 2+ (sluggish), 1+ (non-motile).
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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Fig. 1. MTT reduction assay of solvent leaf extracts of Terminalia bellerica (TBL), Terminalia chebula (TCHL) and Terminalia catappa (TCL).
(1×), 10 l GSH reduced (100 mM), 5 l CDNB (100 mM) and 20 l enzyme (Standard/Sample). Absorbance was recorded at 340 nm. IC50 (concentration giving 50% enzyme inhibition) value of each compound was determined. The reagent mixture without enzyme (standard/sample) was taken as blank. The experiment was repeated three times to assess the enzyme inhibition activity. Statistical analysis All the data were presented as Mean ± SD using three replicates and differences were considered statistically significant at P < 0.05. Results Worm motility assay Worm motility assay exhibited that all the plant extracts such as hexane, chloroform, acetone and methanol of T. bellerica, T. chebula and T. catappa showed decline in motility of the worms at higher doses of 5 and 10 mg/ml after 4 h of incubation, whereas DEC (diethylcarbamazine) worms were active at all the doses upto 4 h and further after 24 h followed by MTT reduction assay (Table 1). MTT reduction assay In order to ascertain the degree of mortality in solvent extracts, MTT reduction assay was conducted for complete set of experi-
ments as above with DEC (diethylcarbamazine) as standard drug. The result showed that methanol extracts of all the three plants namely T. bellerica, T. chebula and T. catappa was better than other extracts. The methanol extract exhibited more than 80% activity at the highest dose level of 10 mg/ml. The activity were 84.63 ± 1.11, 82.67 ± 0.16 and 84.52 ± 0.21 at highest dose of 10 mg/ml for T. bellerica, T. chebula and T. catappa plants respectively. The IC50 thus obtained was comparatively better in methanol extracts than other three extracts such as 2.7, 1.96 and 2.58 mg/ml for T. bellerica, T. chebula and T. catappa respectively. DEC (standard) showed percentage inhibition value of 43.96 ± 3.05, 62.84 ± 2.05 and 79.22 ± 3.1 at 2.5, 5 and 10 mg/ml respectively with IC50 value of 2.84 mg/ml, which was comparatively higher than IC50 values of methanol extracts. Remaining extracts showed less than 80% inhibition (Fig. 1).
GST inhibition assay GST inhibition of the most active extract i.e., methanol extract of three plants T. bellerica, T. chebula and T. catappa was conducted. Highest GST activity of 18.86 ± 0.21 and 12.83 ± 0.03 M/ml/min at 5 and 10 mg/ml respectively was found in the methanol extract of T. bellerica. The percentage of inhibition of GST enzyme was also highest in the same extract i.e. 73.96% and 82.29% for 5 and 10 mg/ml, which was more than other two extracts including the standard drug DEC (Table 2).
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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Table 2 GST activity with protein content after treatment with active plant extracts. Name of the plants
Dose (mg/ml)
GST specific activity (M/ml/min)
% of Inhibition (C − S/C × 100)
Protein content(mg/ml)
T. bellerica
5 10
18.86 ± 0.21* 12.83 ± 0.03*
73.96 82.29
1.26 ± 0.05* 1.35 ± 0.37*
T. chebula
5 10
38.56 ± 0.35* 29.02 ± 0.48*
30.22 47.48
1.53 ± 0.28* 1.06 ± 0.15*
T. catappa
5 10
39.06 ± 1.28* 21.48 ± 1.11*
4.75 47.62
1.93 ± 0.18* 1.91 ± 0.05*
DEC (standard)
5 10
40.03 ± 4.14 21.48 ± 6.44
21.76 58.01
1.51 ± 0.6* 1.42 ± 0.19*
*Highly significant as per p value.
Discussion Filariasis is a menacing health problem in tropical countries; a large number of manpower is lost due to swelling of limbs resulting into most chronic elephantiasis [14]. Although Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia malayi, but both the models are difficult to procure, so work was conducted on S. cervi, which is similar to the human parasite in nocturnal periodicity and response to standard drug DEC [15]. Major drug in filariasis is DEC, has been reported to have side effects known as Mazzotti reaction, a complex, acute inflammatory response characterized by fever, tachycardia, hypotension, adenitis, and an ocular inflammatory response in some cases [16]. Thus, there is still a need for the development of safe and non toxic alternatives. Secondary metabolites produced by plants constitute a wide variety of bioactive chemical substances that act as lead for many synthetic drugs used against an array of diseases. For example, Quinine, an alkaloid isolated from bark of Cinchona became the lead for chloroquine, artemisinin isolated from Artemisia annua showed good antimalarial activity [17]. These findings demonstrate that natural products can provide efficient antiparasitic agents. Genus Terminalia is well known for its medicinal potential. T. chebula has been reported to have antiviral, anti-arthritic, wound healing, antioxidant, antimutagenic, antidiabetic, antiulcerogenic properties [18]. T. bellerica is known for its analgesic and antihypertensive, antidiarrhoeal, antioxidant, wound healing, anticancer, anti-mutagenic and antipyretic activities [19]. Similarly T. catappa has various pharmacological activities that are explored by researchers across the globe. This plant has antimicrobial, anti-inflammatory, analgesic and immuno modulatory activity, wound healing, antidiabetic, antioxidant, hepatoprotective, anticancer and anti-aging activities [20]. In the present study three parameters were taken up these were worm motility assay for assessing short term effect of the extracts, MTT reduction assay for understanding the persistence/irreversible effect of extracts on adult worms and finally GST reduction assay for exploring role of extracts on targeting the GST of parasites which make them survive in host systems.
Worm motility assay Adult worms of S. cervi has been used in preliminary screening for antifilarials. Mobility assay indicates towards the apoptosis caused by the tested agents on adult worms as well as on the micorfilarae inside the gravid female worms [21]. As observed in the results, Upto 4 h, methanol extracts of all the Terminalia sps. were better than the standard compound DEC, but after 24 h mobility was reduced in most of the extracts, which could be due to insufficient amount of nutrients remaining in the medium. Thus, to validate the activity of extracts further test is required. Hence worm motil-
ity assay is followed by MTT reduction assay in order to find out the actual effect of test material. MTT reduction assay The IC50 of methanol extracts was 2.7, 1.96 and 2.58 mg/ml for T. bellerica, T. chebula and T. catappa respectively at the highest dose. DEC (Standard) showed IC50 value of 2.84 mg/ml, which was comparatively higher than IC50 values of methanol extracts. This clearly indicated that Terminalia extracts exhibited better activity in comparison to the DEC. Higher activity of extracts has indeed provided a lead for the isolation of active principles from the Terminalia sp. GST inhibition assay GST plays an important role in detoxification of xenobiotics by catalysing the conjugation of reduced form of glutathione (GSH) to endogenous and exogenous substrates for the purpose of their easy removal by making them water soluble. GST is present in prokaryotic as well as eukaryotic body system as their defence mechanism against xenobiotics. The ability of helminth GSTs to effectively neutralize the cytotoxic products arising from reactive oxygen species provides evidence that GSTs have potential to protect the parasite against the host immune response. The inhibition of parasite GST(s) thus deprives the parasites its major defense against oxidative stress and makes them unable to survive [22,23]. GST has been considered as one of the biochemical targets for antifilarial drug development [24]. Methanol extract of T. bellerica exhibited significant activity (80%) and was clearly way ahead in comparison to DEC. T. bellerica is endowed with a number of pharmacological properties [19] and its antifilarial potential has further added to its importance as a valuable medicinal plant. Further it can be concluded that all the three plants selected exhibited potential macrofilaricidal activity against S. cervi parasite. Methanol extract of leaves of T. bellerica exhibited significant activity in all the parameters like worm motility, MTT reduction and GST inhibition assays and was better than the standard drug DEC. Thus, study has provided lead for the development of a potent macrofilaricidal molecule. Conflict of interest None declared. Acknowledgements The authors are grateful to the Forest and Environment Dept., Govt. of Odisha for the funding support and also to the authorities of Nandankanan Zoological Park, Odisha for their permission and support for the collection of Setaria worms.
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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[14] Vanamail P, Subramanian S, Das PK, Pani SP, Rajagopalan PK. Estimation of fecundic life span of Wuchereria bancrofti from longitudinal study of human infection in an endemic area of Pondicherry (South India). Indian J Med Res 1990;91:293–7. [15] Ahmad R, Srivastava AK. Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents. J Helminthol 2007;81:261–80. [16] Francis H, Awadzi K, Ottesen EA. The Mazzotti reaction following treatment of onchocerciasis with diethylcarbamazine: clinical severity as a function of infection intensity. Am J Trop Med Hyg 1985;34(3):529–36. [17] Haynes RK, Vonwiller SC. Qinghao marvelous herb of antiquity to the antimalarial trioxane Qinghaosu. Some remarkable new chemistry. Acta Chem Res 1997;30:73–9. [18] Gupta PC. Biological and pharmacological properties of Terminalia chebula Retz. (Haritaki) — an overview. Int J Pharm Pharm Sci 2012;4:62–8. [19] Deb A, Barua S, Das B. Pharmacological activities of Baheda (Terminalia bellerica): a review. J Pharmacogn Phytochem 2016;5(1):194–7. [20] Anand AV, Divya N, Kotti PP. An updated review of Terminalia catappa. Pharmacogn Rev 2015;9(18):93–8. [21] Mukherjee N, Mukherjee S, Saini P, Roy P, Sinhababu SP. Antifilarial effects of polyphenol rich ethanolic extract from the leaves of Azadirachta indica through molecular and biochemical approaches describing reactive oxygen species (ROS) mediated apoptosis of Setaria cervi. Exp Parasitol 2014;136:40–58. [22] Nathan ST, Nisha M, Kalyanasundaram M, Balaraman K. Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti — a target for drug development against adult worm. J Mol Model 2005;1:194–9. [23] Brophy PM, Pritchard DI. Parasitic helminth glutathione-S-transferases: update on their potential for immuno and chemotherapy. Exp Parasitol 1994;79:89–96. [24] Lakshmy S, Nisha M, Kalyanasundaram M. In vitro antifilarial activity of glutathione S-transferaseinhibitors. Parasitol Res 2009;105(4):1179–82.
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
ARTICLE IN PRESS Journal of Infection and Public Health xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph
Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi Dipti Ranjan Behera, Sunita Bhatnagar ∗ Medicinal and Aromatic Plants, Regional Plant Resource Centre, Bhubaneswar, Odisha, India
a r t i c l e
i n f o
Article history: Received 16 May 2017 Received in revised form 3 January 2018 Accepted 9 January 2018 Keywords: Setaria cervi DEC (diethylcarbamazine) macrofilaricidal MTT reduction GST (glutathione-S-transferase) inhibition
a b s t r a c t Antifilarial potential of three medicinal plants namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa was explored using Setaria cervi, a bovine filarial parasite at concentrations of 2.5, 5 and 10 mg/ml. Amongst all the extracts, methanol extract of T. bellerica showed highest macrofilaricidal activity i.e. 84.63 ± 1.11 at 10 mg/ml in MTT reduction assay with IC50 value of 2.7 mg/ml. which was better than the standard DEC i.e. 79.22 ± 3.1% at 10 mg/ml with IC50 value 2.84 mg/ml. Other plant extracts showed mild in vitro macrofilaricidal activity. T. bellerica methanol extract exhibited significant GST activity of 18.86 ± 0.21 and 12.83 ± 0.03 M/ml/min at 5 and 10 mg/ml with percentage inhibition value of 73.96% and 82.29% respectively. DEC showed GST activity value of 40.03 ± 4.14 and 21.48 ± 6.44 M/ml/min with percentage inhibition value of 21.76% and 58.01% at 5 and 10 mg/ml respectively. Thus, methanol extract of leaves of T. bellerica exhibited highly significant antifilarial potential and needs detailed analysis. © 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Filariasis is a major public health problem affecting over 120 million people in Central Africa, Central and South Africa and large regions of Asia including China and India [1]. Currently, 1.10 million people in 55 countries are living in areas that require preventive chemotherapy to stop the spread of infection. The current drug regime for lymphatic filariasis include diethylcarbamazine (DEC), albendazole and ivermectin. These drugs have been consistently used in the MDA (Mass Drug Administration) programme to block the disease transmission in the endemic areas. These drugs are active against blood infesting microfilariae (mf) thus efficient in interrupting transmission of the disease; however, they are less effective against the adult worm [2,3]. These drugs have some side effects as well, such as nausea, vomiting, gastric disturbances and giddiness [4]. Owing to this still there is a need for safe, non toxic and macrofilaricidal drug. Secondary metabolites from plants have always served as potential source of alternative medicines. In this study, three plants of Terminalia genus namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa were explored for their antifilarial activities using Setaria cervi, a bovine filarial parasite as target. Terminalia sp., belongs to family Combretaceae, consisting of approximately 150 species of trees and shrubs [5]. The leaves
and bark are well known for their medicinal potential [6] whereas fruits of T. bellerica and T. chebula are used in Ayurvedic medicine “Triphala”, which is a powerful antioxidant and detoxifier [7]. T. catappa leaves contain several bioactive compounds responsible for antiviral, antibacterial, antifungal and anticancerous activities [8]. Keeping in view of medicinal potential of all the three species same were explored for their antifilarial potential for the development of a safe and non toxic alternative for present day synthetic drugs. Materials and methods Collection and processing of plant material The leaves of the medicinal trees namely T. bellerica, T. chebula and T. catappa were collected from Regional Plant Resource Centre, Bhubaneswar, Odisha, India. Solvent extracts were prepared using the standard protocols [9]. Collection of Setaria worms The bovine S. cervi adult parasites were collected from the peritoneal cavity of the freshly slaughtered cattles in the slaughter house of Nandankanan Zoological Park, Bhubaneswar, Odisha, India. Worm motility inhibition assay
∗ Corresponding author. E-mail address: [email protected] (S. Bhatnagar).
In vitro worm motility inhibition assay was performed by the Standard method [10]. The worms were transferred to RPMI-
https://doi.org/10.1016/j.jiph.2018.01.006 1876-0341/© 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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1640 medium and supplemented with 5% (v/v) heat-inactivated Fetal bovine serum. Stock solution of strength 200 mg/ml was prepared of solvent leaf extracts and standard DEC in DMSO (Dimethyl sulphoxide). Two adult worms were introduced into each vial and were subjected to solvent extracts at concentrations of 2.5–10 mg/ml. A simultaneous positive control was kept without the test solution but with equal volume of DMSO as in test vials. Experimental and controls were incubated at 37 ◦ C for 24 h. The motility readings were taken in each hour interval up to four hours to examine the drug activity on worms. After 24 h exposure, the worms were transferred to fresh PBS (1×) to check the motility of the worms. If the worms did not regain their motility, then activity was considered irreversible and showed that the drugs used were active against filarial worms. Motility assay was followed by MTT reduction assay. MTT reduction assay Effect of solvent extracts on adult female Setaria worm was studied by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] formazan reduction assay [11]. After worm motility inhibition study, the treated worms were washed in fresh PBS (1×) pH 7.4. Then the parasites were transferred to 24 well culture plates containing 0.01% MTT prepared in PBS (1×) and incubated at 37 ◦ C for 1h duration. After incubation the treated worms were trans-
ferred to chilled PBS (1×). PBS was removed by drying the worm on blotting paper. Worms were carefully transferred to 24 well culture plates containing 2 ml DMSO and incubated at 37 ◦ C for 2 h. The absorbance of the resulting formazan solution was then determined at 510 nm in a microplate reader. Percent inhibition was calculated as follows: Percentage inhibition = Control − Sample/Control × 100 IC50 value of standard drug and extracts were compared for effective extracts. Glutathione S-transferase (GST) inhibition assay GST inhibition study was carried out by the standard method [12]. For GST assay, worms were washed thoroughly with phosphate-buffered saline (PBS), pH 7.4, prepared a 10% homogenate, centrifuged at 10,000 × g for 30 min at 4 ◦ C, supernatant containing the cytosolic fraction was used for GST assay. GST activity was measured according to the method of Habig [12] using 1-chloro-2,4-ditnitrobenzene (CDNB) and GSH as substrates. The protein content in the crude homogenate of the worm extract was determined [13] using bovine serum albumin as standard protein. The cytosolic fraction was incubated with concentrations 5 and 10 mg/ml of each extract/standard drug for 10 min at room temperature in the reaction mixture containing 465 l Assay buffer
Table 1 In vitro macrofilaricidal activity of plant extracts/DEC against adult Setaria cervi in terms of motility inhibition. Plants used
Extracts
Dose (mg/ml)
1h
2h
3h
4h
24 h
Terminalia bellerica
Hexane
2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 3+ 3+
4+ 4+ 4+ 4+ 4+ 3+ 3+ 3+ 3+ 4+ 2+ 1+
4+ 4+ 4+ 4+ 3+ 2+ 3+ 2+ 2+ 1+ 1+ 1+
2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 1+ 3+ 1+ 1+ 4+ 4+ 4+ 4+ 4+ 4+
3+ 1+ 1+ 2+ 1+ 1+ 4+ 4+ 4+ 4+ 4+ 2+
2+ 1+ 1+ 1+ 1+ 1+ 4+ 4+ 4+ 4+ 4+ 1+
1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
2.5 5 10 2.5 5 10 2.5 5 10 2.5 5 10
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 3+ 1+ 1+ 1+ 1+ 4+ 4+ 3+ 4+ 4+ 4+
3+ 1+ 1+ 1+ 1+ 1+ 4+ 3+ 1+ 4+ 4+ 3+
2+ 1+ 1+ 1+ 1+ 1+ 2+ 2+ 1+ 4+ 3+ 3+
2+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
2.5 5 10
4+ 4+ 4+
4+ 4+ 1+
4+ 4+ 1+
4+ 4+ 1+
1+ 1+ 1+
Chloroform
Acetone
Methanol
Terminalia catappa
Hexane
Chloroform
Acetone
Methanol
Terminalia chebula
Hexane
Chloroform
Acetone
Methanol
DEC (standard)
Motility score of adult worms (in h)
Motility readings: 4+ (highly motile), 3+ (motile), 2+ (sluggish), 1+ (non-motile).
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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Fig. 1. MTT reduction assay of solvent leaf extracts of Terminalia bellerica (TBL), Terminalia chebula (TCHL) and Terminalia catappa (TCL).
(1×), 10 l GSH reduced (100 mM), 5 l CDNB (100 mM) and 20 l enzyme (Standard/Sample). Absorbance was recorded at 340 nm. IC50 (concentration giving 50% enzyme inhibition) value of each compound was determined. The reagent mixture without enzyme (standard/sample) was taken as blank. The experiment was repeated three times to assess the enzyme inhibition activity. Statistical analysis All the data were presented as Mean ± SD using three replicates and differences were considered statistically significant at P < 0.05. Results Worm motility assay Worm motility assay exhibited that all the plant extracts such as hexane, chloroform, acetone and methanol of T. bellerica, T. chebula and T. catappa showed decline in motility of the worms at higher doses of 5 and 10 mg/ml after 4 h of incubation, whereas DEC (diethylcarbamazine) worms were active at all the doses upto 4 h and further after 24 h followed by MTT reduction assay (Table 1). MTT reduction assay In order to ascertain the degree of mortality in solvent extracts, MTT reduction assay was conducted for complete set of experi-
ments as above with DEC (diethylcarbamazine) as standard drug. The result showed that methanol extracts of all the three plants namely T. bellerica, T. chebula and T. catappa was better than other extracts. The methanol extract exhibited more than 80% activity at the highest dose level of 10 mg/ml. The activity were 84.63 ± 1.11, 82.67 ± 0.16 and 84.52 ± 0.21 at highest dose of 10 mg/ml for T. bellerica, T. chebula and T. catappa plants respectively. The IC50 thus obtained was comparatively better in methanol extracts than other three extracts such as 2.7, 1.96 and 2.58 mg/ml for T. bellerica, T. chebula and T. catappa respectively. DEC (standard) showed percentage inhibition value of 43.96 ± 3.05, 62.84 ± 2.05 and 79.22 ± 3.1 at 2.5, 5 and 10 mg/ml respectively with IC50 value of 2.84 mg/ml, which was comparatively higher than IC50 values of methanol extracts. Remaining extracts showed less than 80% inhibition (Fig. 1).
GST inhibition assay GST inhibition of the most active extract i.e., methanol extract of three plants T. bellerica, T. chebula and T. catappa was conducted. Highest GST activity of 18.86 ± 0.21 and 12.83 ± 0.03 M/ml/min at 5 and 10 mg/ml respectively was found in the methanol extract of T. bellerica. The percentage of inhibition of GST enzyme was also highest in the same extract i.e. 73.96% and 82.29% for 5 and 10 mg/ml, which was more than other two extracts including the standard drug DEC (Table 2).
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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Table 2 GST activity with protein content after treatment with active plant extracts. Name of the plants
Dose (mg/ml)
GST specific activity (M/ml/min)
% of Inhibition (C − S/C × 100)
Protein content(mg/ml)
T. bellerica
5 10
18.86 ± 0.21* 12.83 ± 0.03*
73.96 82.29
1.26 ± 0.05* 1.35 ± 0.37*
T. chebula
5 10
38.56 ± 0.35* 29.02 ± 0.48*
30.22 47.48
1.53 ± 0.28* 1.06 ± 0.15*
T. catappa
5 10
39.06 ± 1.28* 21.48 ± 1.11*
4.75 47.62
1.93 ± 0.18* 1.91 ± 0.05*
DEC (standard)
5 10
40.03 ± 4.14 21.48 ± 6.44
21.76 58.01
1.51 ± 0.6* 1.42 ± 0.19*
*Highly significant as per p value.
Discussion Filariasis is a menacing health problem in tropical countries; a large number of manpower is lost due to swelling of limbs resulting into most chronic elephantiasis [14]. Although Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia malayi, but both the models are difficult to procure, so work was conducted on S. cervi, which is similar to the human parasite in nocturnal periodicity and response to standard drug DEC [15]. Major drug in filariasis is DEC, has been reported to have side effects known as Mazzotti reaction, a complex, acute inflammatory response characterized by fever, tachycardia, hypotension, adenitis, and an ocular inflammatory response in some cases [16]. Thus, there is still a need for the development of safe and non toxic alternatives. Secondary metabolites produced by plants constitute a wide variety of bioactive chemical substances that act as lead for many synthetic drugs used against an array of diseases. For example, Quinine, an alkaloid isolated from bark of Cinchona became the lead for chloroquine, artemisinin isolated from Artemisia annua showed good antimalarial activity [17]. These findings demonstrate that natural products can provide efficient antiparasitic agents. Genus Terminalia is well known for its medicinal potential. T. chebula has been reported to have antiviral, anti-arthritic, wound healing, antioxidant, antimutagenic, antidiabetic, antiulcerogenic properties [18]. T. bellerica is known for its analgesic and antihypertensive, antidiarrhoeal, antioxidant, wound healing, anticancer, anti-mutagenic and antipyretic activities [19]. Similarly T. catappa has various pharmacological activities that are explored by researchers across the globe. This plant has antimicrobial, anti-inflammatory, analgesic and immuno modulatory activity, wound healing, antidiabetic, antioxidant, hepatoprotective, anticancer and anti-aging activities [20]. In the present study three parameters were taken up these were worm motility assay for assessing short term effect of the extracts, MTT reduction assay for understanding the persistence/irreversible effect of extracts on adult worms and finally GST reduction assay for exploring role of extracts on targeting the GST of parasites which make them survive in host systems.
Worm motility assay Adult worms of S. cervi has been used in preliminary screening for antifilarials. Mobility assay indicates towards the apoptosis caused by the tested agents on adult worms as well as on the micorfilarae inside the gravid female worms [21]. As observed in the results, Upto 4 h, methanol extracts of all the Terminalia sps. were better than the standard compound DEC, but after 24 h mobility was reduced in most of the extracts, which could be due to insufficient amount of nutrients remaining in the medium. Thus, to validate the activity of extracts further test is required. Hence worm motil-
ity assay is followed by MTT reduction assay in order to find out the actual effect of test material. MTT reduction assay The IC50 of methanol extracts was 2.7, 1.96 and 2.58 mg/ml for T. bellerica, T. chebula and T. catappa respectively at the highest dose. DEC (Standard) showed IC50 value of 2.84 mg/ml, which was comparatively higher than IC50 values of methanol extracts. This clearly indicated that Terminalia extracts exhibited better activity in comparison to the DEC. Higher activity of extracts has indeed provided a lead for the isolation of active principles from the Terminalia sp. GST inhibition assay GST plays an important role in detoxification of xenobiotics by catalysing the conjugation of reduced form of glutathione (GSH) to endogenous and exogenous substrates for the purpose of their easy removal by making them water soluble. GST is present in prokaryotic as well as eukaryotic body system as their defence mechanism against xenobiotics. The ability of helminth GSTs to effectively neutralize the cytotoxic products arising from reactive oxygen species provides evidence that GSTs have potential to protect the parasite against the host immune response. The inhibition of parasite GST(s) thus deprives the parasites its major defense against oxidative stress and makes them unable to survive [22,23]. GST has been considered as one of the biochemical targets for antifilarial drug development [24]. Methanol extract of T. bellerica exhibited significant activity (80%) and was clearly way ahead in comparison to DEC. T. bellerica is endowed with a number of pharmacological properties [19] and its antifilarial potential has further added to its importance as a valuable medicinal plant. Further it can be concluded that all the three plants selected exhibited potential macrofilaricidal activity against S. cervi parasite. Methanol extract of leaves of T. bellerica exhibited significant activity in all the parameters like worm motility, MTT reduction and GST inhibition assays and was better than the standard drug DEC. Thus, study has provided lead for the development of a potent macrofilaricidal molecule. Conflict of interest None declared. Acknowledgements The authors are grateful to the Forest and Environment Dept., Govt. of Odisha for the funding support and also to the authorities of Nandankanan Zoological Park, Odisha for their permission and support for the collection of Setaria worms.
Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006
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Please cite this article in press as: Behera DR, Bhatnagar S. Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.01.006