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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2022 Nov 27;47(1):152–160. doi: 10.1007/s12639-022-01550-x

In vitro efficacy of plumbagin and thymol against Theileria annulata

E Eben Titus 1, Azhahianambi Palavesam 1,✉, Srinivasan Morkonda Rajaram 2, Pandikumar Perumal 3, Santhaanam Sylvester Darwin 4, Nagul Kumar Sanmugapriya 5, Ganesh Janarthanam 1, Raman Muthusamy 1,6
PMCID: PMC9998759  PMID: 36910313

Abstract

Phytochemical compounds, plumbagin and thymol were evaluated for their efficacy against Theileria annulata using MTT cell viability assay. Plumbagin and thymol were found to be effective in preventing the proliferation of Theileria annulata infected bovine lymphocytes. The IC50 values of plumbagin and thymol were 0.019 µM and 0.009 µM, respectively. Plumbagin and thymol were found to be non-cytotoxic to the bovine peripheral blood mononuclear cells. However, both the compounds were found to have inhibitory effect on vero cell proliferation. Plumbagin had primarily anti-theilerial activity but thymol had primarily anti-mitotic activity. The in vitro efficacy and cell toxicity studies indicate the potential application of plumbagin, purified from Plumbago indica as a lead therapeutic molecule against T. annulata infection in cattle.

Graphical Abstract

graphic file with name 12639_2022_1550_Figa_HTML.jpg

Supplementary Information

The online version contains supplementary material available at 10.1007/s12639-022-01550-x.

Keywords: Plumbago indica, Thymus vulgaris, Tropical bovine theileriosis, Plumbagin, Thymol, Anti-theilerial drug

Introduction

Tropical Bovine Theileriosis (TBT) is one of the most important tick-borne haemoprotozoan diseases caused by Theileria annulata (Viseras et al.1999). TBT is prevalent over a broad geographic region globally ranging from Asia, Middle East, southern Europe and northern Africa (Bilgic et al. 2010; Purnell 1978). The most susceptible population for bovine theileriosis is the young indigenous calves, exotic and crossbred cattle (Muleya et al. 2012). The annual economic loss due to TBT in India was estimated to be US$ 384.3 million per annum (Minjauw and McLeod 2003). As per the recent estimate, the economic loss due to TBT in India is US$ 1,295 million (Rs 8,426.7 crore) annually (Narladkar 2018). The economic losses due to TBT were estimated to be US $598,000, $133,000 and $130,000 in the endemic stable zones of Turkey (Cicek et al. 2009). It was also reported that 30% to 60% crossbred cattle in India were seropositive for T. annulata (Kumar et al. 2018) and the mortality rate was found to be 40% to 90% in newly introduced cross breed cattle (Campbel and Spooner 1999). A recent meta-analysis-based study estimated the prevalence of theileriosis in India to be 20% (95% CI, 16%–25%) (Krishnamoorthy et al. 2021). Rakshavac-T® (Indian Immunologicals Ltd, Hyderabad, India) is the only live attenuated commercial vaccine available in India for immunoprophylaxis (Grewal et al. 1997), but liquid nitrogen storage and transportation of the vaccine are the major limitations. Tayledoll® (Dollyet, Turkey) and Teylovac® (Vetal, Turkey) are the two live attenuated vaccines against T. annulata available in Turkey. The drug of choice available in the field for the treatment of TBT (Wilkie et al. 1998) is buparvaquone which is expensive and not affordable to small and marginal dairy farmers. Also, buparvaquone resistant strains of T. annulata had been reported in the field which further adds to the concern (Mhadhbi et al. 2015). Thus, an effective and affordable alternative drug is the need of the hour for the treatment of T. annulata infection in cattle.

A detailed literature review was done to shortlist plant derived compounds having anti-protozoal activity. Plumbagin derived from Plumbago indica and thymol from Thymus vulgaris were reported to have anti-parasitic and antiprotozoal activity (Tasdemir et al. 2006; Sharma et al. 2012; Metwaly et al. 2020; Dominguez-Uscanga et al. 2021) apart from its anti-cancer, antibacterial and anti-inflammatory actions. Plumbagin (5-hydroxy 2-methyl 1,4 naphthoquinone) isolated from the leaves and roots of P. indica (Chauhan 2014) which is a perennial herb/shrub distributed all along south east Asia including Sri Lanka and various parts of India (Devi et al. 1994). It has a widespread usage against rheumatism, colic, cough and bronchitis. Structural similarity scores were computed by ChemMine Tools (https://chemminetools.ucr.edu/) which predicted 12 maximum common substructures between plumbagin and buparvaquone (Chen et al. 2002) indicating the structural similarity between two naphthoquinone molecules. Because of the structural similarity of plumbagin with buparvaquone, plumbagin was selected for in vitro screening to determine its activity against T. annulata.

Thymol is a monoterpene phenol obtained from the oil of T. vulgaris. It is a tiny perennial shrub growing up to 15–30 cm tall and widely distributed in European countries including France, Italy, Portuguese and Bulgaria. It has antimicrobial, antioxidative, antispasmodic, antiparasitic and antiprotozoal properties (Hoferi et al. 2009). Thymol is present in higher concentration in its oil (24.7 mg/g) and in other parts such as leaves. Thymol was reported to be active against Cryptosporidium spp. (Dominguez-Uscanga et al. 2021), murine toxoplasmosis (Oliveira et al. 2016) and Leishmania spp. (Youssefi et al. 2019). Due to its activity against apicomplexan and kinetoplastid parasites, thymol was selected for analyzing its inhibitory activity against T. annulata, an apicomplexan parasite in our study.

Plumbagin and thymol showed activity against apicomplexan parasites such as Plasmodium spp. Plumbagin, a vitamin K3 analogue ameliorated malaria pathogenesis by reducing parasitemia and increasing mean survival time of Plasmodium berghei infected mice (Gupta et al. 2018). Plumbagin exhibited in vitro efficacy against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum with relatively low toxicity in vivo (Sumsakul et al. 2014). Thymol based pyrazolines showed anti-malarial activity with IC50 less than 2 µM in vitro and it showed decreased parasitemia, increased mean survival time and hemoglobin content in vivo (Raghuvanshi et al. 2019). The current study is first of its kind in screening the laboratory purified plumbagin and thymol for their activity against T. annulata, an apicomplexan tick borne haemoprotozoan parasite of cattle.

Materials and methods

Parasite source

T. annulata schizont infected bovine lymphocyte culture T399 (80th passage) maintained at Translational Research Platform for Veterinary Biologicals (TRPVB), TANUVAS, Chennai, Tamil Nadu, India, has been used in this study (Buvanesvaragurunathan et al. 2022). Field isolated T. annulata schizont infected bovine lymphocytes were maintained in RPMI 1640 medium (Gibco) with 20% Fetal Bovine serum (South American origin).

Test molecule and drug source

Plumbagin was extracted from roots of P. indica and purified. The compound was purified by vapor distillation and High-Performance Liquid Chromatography (HPLC). It was characterized by Thin Layer Chromatography (TLC) and Spectroscopy. Thymol and Buparvaquone were purchased from SigmaⓇ. Solvent used for Plumbagin and Thymol was DMSO. Diethyl ether at analytical grade was purchased from Avra chemicals (Chennai, Tamil Nadu, India) and used without further purification. 1H and 13C NMR spectra were taken on Bruker AV-500 (500 and 125 MHz) with CDCl3 with TMS as the internal standard. Chemical shift values were given in δ scale.

Evaluation of anti-theilerial activity (MTT cell viability assay)

T. annulata schizont infected bovine lymphocyte culture maintained in RPMI 1640 with 20% FBS were used in this assay. Cells were counted using hemocytometer and live and dead count was performed by trypan blue dye exclusion method (Saravanan et al. 2003). Live T. annulata schizont infected bovine lymphocytes were seeded at the concentration of 1X105 cells/well in the 96 well cell culture plates (Thermofisher scientificⓇ). A volume of 30 µl containing 1X105 live cells was added into each well and activity of plumbagin and thymol was tested in a series of concentrations between 1 µM to 0.01 µM. Plumbagin and thymol were prepared in two different stock concentrations of 100 µM and 10 µM. Volumes of 3 µl for 1 µM and 0.9 µl for 0.3 µM were used from 100 µM stock in MTT assay. Volumes of 3 µl for 0.1 µM, 0.9 µl for 0.03 µM and 0.3 µl for 0.01 µM were used from 10 µM stock in MTT assay. The total volume of the assay was made up to 300 µl by adding cell culture medium. T. annulata schizont infected cells without plumbagin and thymol and media only controls were maintained in the assay. T. annulata schizont infected cells with anti-theilerial drug buparvaquone was maintained in the assay as a positive control. The culture plates were incubated at 37 °C with 5% CO2 in a CO2 incubator for 24 h.

After 24 h of incubation, 270 µl of culture supernatant was taken out. MTT dye stock solution was prepared as 10 mg/ml concentration. The final working concentration was 5 mg/ml after 1:2 dilutions with sterile PBS (Saravanan et al. 2003). A volume of 10 µl of MTT working solution was added in each well which was followed by incubation for 4 h at 37 °C with 5% CO2 in a CO2 incubator. Then 100 µl of DMSO was added and incubated for 20 min at 37 °C and then the readings were taken in the microplate reader (Tecan Multimode reader, Switzerland) at 570 nm. All the assays were performed in triplicates.

The efficacy assay was also performed with the anti-theilerial drug, buparvaquone from 0.2 mM to 50 mM concentrations to determine the in vitro anti-theilerial activity.

Cytotoxicity assay with bovine PBMC cells

Bovine peripheral blood mononuclear cells (PBMC) were isolated from cattle blood received from university veterinary hospital. Bovine PBMC were separated using Histopaque 1077 by centrifugation at 3000 rpm for 3 min. The separated cell pellet was washed with RPMI 1640 medium and counted using a hemocytometer (1X105 cells/7 µl).

A volume of 7 µl of bovine PBMC (1X105cells) and 293 µl of cell culture medium were added in to each well of the 96 well cell culture plate (Saravanan et al. 2003). Plumbagin and thymol with 1 mM stock solution were used in the cytotoxicity study. A volume of 3 µl was added for 10 µM drug concentration and 0.3 µl was added for 1 µM drug concentration. The plate was incubated in a CO2 incubator for 24 h at 37 °C. MTT dye stock solution (10 mg/ml) was diluted 1:2 and the working concentration of 5 mg/ml was prepared. After 24 h, 270 µl of supernatant was taken out from each well, 10 µl of dye was added to each well and the plates were incubated for 4 h at 37 °C in CO2 incubator. Then, DMSO was added to each well and incubated for 20 min at 37 °C in CO2 incubator. The reading was taken in a microplate reader (Tecan Multimode reader, Switzerland) at 570 nm. All the assays were performed in triplicates.

Cytotoxicity assay with Vero cells

Vero cells (1X105 cells) were seeded in the 96 well cell culture plates along with Dulbecco Minimal Essential Medium (Gibco) with a total volume of 300 µl. Plumbagin and thymol at 10 µM and 1 µM, respectively the concentrations used in the vero cell cytotoxicity study. A volume of 3 µl and 0.3 µl was taken for 10 µM and 1 µM concentrations, respectively. The plate was incubated at 37 °C for 24 h in a humidified CO2 incubator (5% CO2). After 24 h, 270 µl of supernatant were taken out from each well. The MTT dye was prepared to the working concentration of 5 mg/ml from the stock solution, added 10 µl to every well and incubated for 4 h at 37 °C in CO2 incubator (Saravanan et al. 2003). Then 100 µl of DMSO was added to every well and incubated for 20 min at 37 °C in CO2 incubator. The plate was then read in the microplate reader (Tecan Multimode reader, Switzerland) at 570 nm. All the assays were performed in triplicates.

IC50 value determination

Probit analysis was done to determine IC50 value in vitro (Finney & John, 1952).

Results

Isolation and purification of Plumbagin from Plumbago indica L.

Roots of P. indica (2 kg) were collected from Srivilliputhur, Tamil Nadu, India (9.5121° N, 77.6340° E) and authenticated by Dr. P. Pandikumar, St Xavier’s college, Palayamkottai, Tamil Nadu, India. The roots were shade dried and coarsely powdered with an electric blender. The powder was hydro-distilled using a Clevenger apparatus. The distillate was then extracted with diethyl ether; the organic layer was dried over anhydrous sodium sulphate and concentrated using a rotary evaporator. It was then crystallized in methanol to yield pure plumbagin (4.59 g) and the yield was 0.23%.

Structural elucidation of the compounds isolated from Plumbago indica L.

Plumbagin was isolated as yellowish red crystals and it was analyzed for C11H8O3. 1H NMR (δH CDCl3, 400 MHz): 2.18 (3H, d, Me-2), 6.8 (1H, d, H-3), 7.23 (1H, m, H-6), 7.60 (2H, m, H-7, 8), 11.95 (OH)and 13C NMR (δC CDCl3, 125 MHz): 16.50 (2-CH3), 115.07 (C-4a), 119.07 (C-8), 124.13 (C-6), 132.01 (C-8a), 135.41 (C-3), 136.07 (C-7), 149.58 (C-2), 161.13 (C-5), 184.71 (C-1), 190.22 (C-4) (Supplementary file). The spectroscopic data were in accordance with the values reported in the literature (Bothiraja et al. 2011; Pradeepa et al. 2016).

Anti-theilerial activity of plumbagin and thymol

Plumbagin and thymol had a dose dependent effect on the viability of T. annulata schizont infected lymphocyte cells. Negative correlation between drug concentration and % cell viability of T. annulata infected cells was found (R = −0.578) (Table 1). Further, IC50 values (the minimal concentration of the drug that is required for the inhibition of 50% cells in vitro) of plumbagin and thymol were calculated by using probit analysis. The IC50 values of plumbagin and thymol compounds were determined as 0.019 µM and 0.009 µM, respectively. The values of % mean cell viability along with standard deviation and IC50 values for different concentrations of plumbagin and thymol was shown in Table 1.

Table 1.

Different concentrations of plumbagin and thymol and % mean cell viability of MTT assay with standard deviation and IC50 values

S. No Concentration Plumbagin Thymol
Mean % cell viability ± SD IC50 value and correlation coefficient (R) Mean % cell viability ± SD IC50 value and correlation coefficient (R)
1 Positive control (Cells + DMSO) 100 ± 0.0 0.019 µM R =  − 0.579 100 ± 0.0 0.009 µM R =  − 0.579
2 1 µM 27.8315 ± 4.66 20.2195 ± 0.078
3 0.3 µM 32.4595 ± 6.04 21.8800 ± 1.41
4 0.1 µM 39.3920 ± 3.79 30.0740 ± 6.29
5 0.03 µM 46.5340 ± 0.84 41.6665 ± 3.2
6 0.01 µM 59.0360 ± 2.53 61.0075 ± 1.498
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Buparvaquone also had a dose dependent effect on the viability of T. annulata schizont infected bovine lymphocyte cells. Negative correlation between buparvaquone and % cell viability of T. annulata infected cells was found (R = −0.89). The IC50 value of buparvaquone was 7.2 mM (Table 2).

Table 2.

Different concentrations of buparvaquone and % mean cell viability of MTT assay with standard deviation and IC50 values

S. No Concentration Buparvaquone
Mean % cell viability ± SD IC50 value and correlation coefficient (R)
1 Positive control (Cells + DMSO) 100 ± 0.0 7.2 mM R =  − 0.89
2 0.2 mM 76.698 ± 1.67
3 2 mM 68.2155 ± 2.04
4 10 mM 63.7035 ± 1.14
5 12.5 mM 56.2925 ± 1.84
6 25 mM 24.882 ± 1.59
7 50 mM 19.7285 ± 0.39
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The test molecules plumbagin and thymol were found to have very low IC50 values than the standard anti-theilerial drug buparvaquone in vitro which indicates the higher potency of test molecules in inhibiting T. annulata schizont infected lymphocyte cells. The values of % mean cell viability along with standard deviation and IC50 value for different concentrations of buparvaquone was shown in Table 2.

Cytotoxicity of plumbagin and thymol

Cytotoxicity assay using bovine PBMC cells

Plumbagin and thymol were not cytotoxic to the bovine PBMC cells even at 10 µM concentration which is 500 to 1000 times higher than the IC50 values of plumbagin and thymol. Cell viability was found to be significantly higher (P < 0.05) at 10 µM concentration of plumbagin and thymol. The values of % mean cell viability along with standard deviation for different concentrations (1 µM and 10 µM) of plumbagin and thymol was shown in Table 3.

Table 3.

Cytotoxicity assay in bovine PBMC and Vero cells with 1 µM and 10 µM concentrations of plumbagin and thymol

Bovine PBMC Vero cells
S. No Concentration Plumbagin Thymol Plumbagin Thymol
Mean % cell viability ± SD Mean % cell viability ± SD Mean % cell viability ± SD Mean % cell viability ± SD
1 Positive control (Cells + Con A) 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0
2 1 µM 109.38 ± 0.99 115.00 ± 0.47 51.87 ± 0.87 18.07 ± 0.61
3 10 µM 141.86 ± 1.4 126.32 ± 0.95 18.80 ± 0.48 16.21 ± 0.16
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Cytotoxicity assay using vero cells

Plumbagin at 1 µM concentration inhibited only 48% of vero cells indicating its major activity is against T. annulata schizont stage. However, at 1 µM concentration, plumbagin inhibited 72% of T. annulata infected lymphocyte cells. Thymol at 1 µM concentration inhibited only 82% of vero cells indicating its major activity is against proliferating vero cells. However, at 1 µM concentration, thymol inhibited 79.8% of T. annulata infected lymphocyte cells. Plumbagin and thymol significantly (P < 0.01) inhibited the viability of proliferating vero cells at 1 µM and 10 µM concentration. The values of % mean cell viability along with standard deviation for different concentrations (1 µM and 10 µM) of plumbagin and thymol was shown in Table 3.

Discussion

Buparvaquone is a commercial drug available for the treatment of T. annulata infection in animals (Wilkie et al.1998; Azhahianambi et al., 2021). However, several drug resistant strains of T. annulata were reported globally (Mhadhbi et al 2010, 2015; Sharifiyazdi et al. 2012) which threatens livestock farming in disease endemic area. Multiple point mutations in the cytochrome b gene of T. annulata had been attributed to the drug resistance (Sharifiyazdi et al. 2012; Chatanga et al. 2019). As an effective alternate control strategy, crude herbal extracts and purified phytochemicals were tested against Theileria spp. Methanolic extracts of Capsicum annum (Rizk et al. 2022) and Curcuma longa (Rizk et al. 2021) showed a dose dependent inhibition of Theileria equi in vitro. Aqueous extracts of Kigelia africana (Farah et al. 2015) and Gardenia ternifolia fruit (Farah et al. 2012) has anti-theilerial activity against T. lestoquardi in vitro. Flower extracts of Calotropis procera showed an increased efficacy against Theileria infection compared to buparvaquone in cattle (Siddiqui et al. 2017). However, majority of the anti-theilerial drug efficacy experiments were done using crude plant extracts and not as purified phytochemical compounds. This study is first of its kind in determining the efficacy and safety of plumbagin and thymol as purified phytochemical compounds against T. annulata in vitro.

The purified phytochemical compounds such as plumbagin and thymol showed a dose dependent inhibition against T. annulata schizont infected bovine lymphocytes. A pentacyclic triterpenoid, 18 β-Glycyrrhetinic acid (18β-GA) and its isopropyl esters showed anti-theilerial effect against T. annulata schizont infected bovine lymphocyte but comparatively less potent than plumbagin and thymol in vitro (Buvanesvaragurunathan et al. 2022). The GI50 value of isopropyl esters of 18 β-GA against T. annulata was estimated as 1.63 µM, which is higher than the plumbagin and thymol. Plumbagin and thymol compounds showed potent in vitro efficacy against the kinetoplastid and apicomplexan parasites of man and animals. Plumbagin was effective in inhibiting growth of promastigotes in Leishmania spp in vitro (Hazra et al. 2002; Sharma et al. 2012) and 3D7 chloroquine sensitive P. falciparum and K1 chloroquine-resistant P. falciparum clones in vitro (Sumsakul et al. 2014). Thymol was also found to have a profound inhibitory activity against Leishmania amazonensis in vitro along with its other ester derivatives (Silva et al. 2017). Thymol and its derivatives had extensive inhibitory activity against Cryptosporidium parvum (Dominguez-Uscanga et al. 2021) and NF-54 sensitive strains, K1 resistant strains of Plasmodium spp. in vitro (Kumar et al. 2021). The inhibitory activity of plumbagin and thymol against T. annulata, an apicomplexan bovine parasite is in similar lines with their activity against kinetoplastid and apicomplexan parasites of man and animals.

Plumbagin and thymol showed no cytotoxicity even up to 100 µM in human PBMC cells (Subramaniya et al. 2011; Deb et al. 2011). These results were in congruence with bovine PBMC where it was non-cytotoxic and % cell viability is significantly (P < 0.05) higher up to 10 µM concentration.

Plumbagin and thymol showed a significant inhibition (P < 0.01) of vero cell proliferation. Plumbagin at 1 µM and 10 µM showed 48% and 82% inhibition in vero cell proliferation. However, at 1 µM concentration, plumbagin inhibits 72% T. annulata schizont infected bovine lymphocytes which suggests its explicit and prominent anti-theilerial effect. On the contrary, thymol at 1 µM and 10 µM shows 82% and 84% inhibition of vero cell proliferation. It inhibited 79.8% of the T. annulata schizont infected bovine lymphocytes at 1 µM showing its anti-proliferative property.

Plumbagin as a purified compound was reported to have an inhibitory activity against vero cells (Lin et al. 2003) which is in agreement with our results. However, the derivatives of plumbagin like 3-(5-oxohexyl) plumbagin and plumbagin dimmers does not show any vero cell cytotoxicity (Suchaichit et al. 2021; Uc-Cachón et al. 2014). Structural activity relationship of various hit derivatives in plumbagin and thymol can be assayed further for assessing the compounds with increasing efficacy along with decreasing toxicity. Thus, exhaustive in vitro and in vivo studies need to be performed to identify the molecular mechanism of inhibition and host-drug interaction for effective lead optimization.

Conclusion

In vitro anti-theilerial activity of purified plumbagin and thymol compounds was confirmed. Plumbagin and thymol were found to be not cytotoxic to the bovine PBMC cells even at concentration which is 500 to 1000 times higher than the IC50 values. Plumbagin and thymol were found to have inhibitory effect on vero cell proliferation. Plumbagin with primary anti-theilerial activity could serve as a potential therapeutic phytochemical compound for the treatment of T. annulata. However, the efficacy of plumbagin against buparvaquone resistant strains of T. annulata is yet to be determined. Moreover, these compounds should undergo various other in vitro or in vivo tests for the effective lead selection and optimization.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 141 kb) (141.5KB, docx)

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection, writing first draft of manuscript were performed by ET and AP. Data analysis, manuscript review and editing were performed by AP, PP, SMR. Material preparation, manuscript review and editing SSD, NKS and GJ: Methodology. Funding acquisition and Supervision were done by AP and RM. All authors read and approved the final manuscript.

Funding

This work was supported by Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India, for their financial assistance (BT/PR10526/ADV/90/161/2014).

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

No experimental animals were used in this study and no invasive and painful methods were carried out on animals in this study.

Consent to participate

Not Applicable.

Consent for publication

All the authors have approved this manuscript and given consent to publish this data.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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