ABSTRACT

The increase of Plasmodial parasite resistance to available antimalarial drugs underscores the emergency to research alternatives to new drugs development. Medicinal plants traditionally used against malaria are potential source of new molecules; however, their efficiency must be scientifically validated. The present study is therefore aimed at evaluating the antiplasmodial potential and the cytotoxicity effect of alkaloids from Coffea Arabica and Coffea canephora. Alkaloids were extracted in acidic medium from powder of the dry leaves of each plant. Characterization of alkaloids was carried out using Mayer and Wagner reagents. Antiplasmodial tests based on the fluorescence of SYBR green were carried out on two isolates of Plasmodium falciparum. The revelation was made by flow cytometry. Toxicological evaluation of the extracts had been carried out through hemolysis test, erythrocyte sensitivity as well as cytotoxicity on Vero cells and Raw cells using spectrophotometry. Evaluation of antiplasmodial activity showed that C. arabica was active on isolates Pf3D7 and PfDd2 with resistance index of 0.78 and IC₅₀ values of 9.53 ± 1.51 µg/mL and 7.48 ± 0.93 µg/mL respectively; On the other hand, C. canephora showed a weak activity on the strain Pf3D7 (IC ₅₀ >100µg/mL) and a moderate activity on the strain PfDd2 (IC₅₀ = 85.55 ± 1.17 µg/ mL). The toxicological profile was favorable for both extracts concerning hemolysis test (HC ₅₀ >1000 µg/mL) and for cytotoxicity test (CC ₅₀ >30 µg/mL). The present study revealed that these extracts could constitute glimmers of hope by offering an alternative to the management of malaria.

Key Words: Coffea arabica, Coffea canephora, Alkaloids, Antiplasmodial activity, Cytotoxicity

INTRODUCTION

Malaria is one of the deadliest parasitic diseases in the world [1]. It is one of the most important health problems in tropical and subtropical regions [2]. In sub-Saharan Africa, malaria infected more than 200 million people in 2020 indicating its exponential increase attributed to the interruption of malaria-control services during the wake of Covid-19 pandemics [3, 4]. In Cameroon, malaria is still an important public health threat; it remains the first cause of morbidity and mortality among the most vulnerable groups [5, 6].

Plasmodium falciparum is the main parasite responsible for over 95% of malaria cases [7, 8]. The control strategy of early and effective management of malaria has not been satisfactory. Unfortunately, the increasing spread of drug-resistant P. falciparum strains has worsened the situation [9, 10]. The rise in resistance of Plasmodium falciparum to conventional antimalarial drugs in humans [9] has therefore led to research of new drugs with new modes of action.

Research of new cures from medicinal plant species used in the treatment of malaria depends on precise and specific ethnopharmacological information obtained from local healers [2]. In Cameroon, research about the evaluation of antiplasmodial activity [11] of medicinal plants such as Coffea used for the treatment of malaria in traditional medicine is not new [12, 13]. The present study is aimed at comparing the antiplasmodial activity of Coffea arabica and Coffee canephora.

MATERIALS AND METHODS

Plant collection

Plants were harvested in the Western region of Cameroon more specifically in Bantoum for Coffea robusta and Bangoua for Coffea arabica. Botanical identification numbers are given at the National Herbarium of Cameroon based on comparison with documented samples by botanists for Coffea arabica and Coffea canephora 58228/HNC and 67456/ HNC, respectively. Extractions were made in the Laboratory of Pharmacognosy and Pharmaceutical Chemistry of the Faculty of Medicine and Biomedical Sciences (FMSB) of the University of Yaoundé I. Various tests were carried out in the Laboratory of Phytobiochemistry and Study of medicinal Plants of the Faculty of Sciences, University of Yaoundé I.

Extraction of total alkaloids

Extraction was performed on the samples according to a slightly modified method described by Kemajou et al. (2012) [14]. Five hundred grams (500 g) of leave powder from each plant species weighed on an ARCHMED brand electronic scale (sensitivity of 0.01g and a range of 510 g) were macerated with 1800 mL of methanol for 48 hours with constant stirring using a PRO HPS stirrer -7 Lab Plus Series. This operation was repeated twice. The crude extracts that had been obtained after filtration through Whatman paper N°3 and concentration under reduced pressure using a BUCHI R-201 rotary evaporator were acidified with 0.5N hydrochloric acid and then placed in a separatory funnel with 100 mL of chloroform. The collected aqueous phase was alkalinized by 6 mL of a 33% ammonia solution to reach a pH between 9 and 10, then placed again in a separatory funnel with 100 mL of chloroform. The chloroformic phase was then collected, dried using anhydrous magnesium sulfate, and then filtered to obtain total alkaloids.

In vitro antiplasmodial activity test

Preparation of stock solutions of total alkaloids, chloroquine, and artemisinin

A stock solution of the different total alkaloids (100 mg/mL) was prepared by dissolving 100 mg of total alkaloids in 1mL of dimethylsulfoxide (DMSO). Chloroquine (98 %. Sigma-Aldrich) and Artemisinin (98%, Sigma-Aldrich) were prepared at a concentration of 1 mM. Preparation of intermediate concentrations was carried out by adding either 10 µL of total alkaloids (100 mg/mL) or 20 µL of chloroquine or artemisinin (1mM) in 190 µL or 180 µL of incomplete RPMI1640 medium respectively contained in a 96-well microplate followed by a geometric dilution of order 5 leading to concentrations ranging from 8 to 5000 µg/ mL for total alkaloids and from 0.016 to 10 µM for Artemisinin and Chloroquine.

Cultivation of the malaria parasite

Two strains of plasmodium falciparum, one chloroquine-sensitive (NF54-E) and another multi-resistant (Dd2) were cultured according to the method described by Trager and Jensen in 1976 [15]. The strains were cultured in human red blood cells of group O Rhesus positive at 4% hematocrit in RPMI 1640 medium (500 mL) supplemented with 25 mM HEPES, 0.50% Albumax I, 45 µg/ mL of hypoxanthine, and 20 µg/ mL gentamicin (0.5mL) and incubated at 37ºC in a humidified incubator containing 92% N₂, 5% CO₂ and 3% O₂. The medium was replaced daily by a complete RPMI medium to allow parasite growth in culture. Subsequently, blood smears were made, stained with Giemsa 10%, and then observed under optical microscope objective 100 with immersion oil to follow all the stages of the cell cycle and evaluate the parasitemia.

Antiplasmodial activity test

The antiplasmodial activity test was evaluated according to the method described by Smilkstein et al. (2004) [16], based on the fluorescence of SYBR Green I. Ninety microliter s (90 µL) of parasite suspension synchronized in stage at rings of 2% parasitemia and 1% hematocrit were incubated with 10 µL of different concentrations of each pre-diluted total alkaloid, Artemisinin or Chloroquine solution. Plates were incubated 72 hours at 37° C. in a humidified incubator consisting of 92% N₂, 5% CO_2, and 3% O₂. Final concentrations in the test plates ranged from 0.8 to 500 µg/ mL (DMSO < 1%) for each extract and from 0.0016 to 1µM (DMSO 0.1%) for Artemisinin and Chloroquine in a final volume of 100 µL. The experiments were carried out in duplicates.

After 72 hours of incubation, 100 µL of SYBR Green I buffer [6 µL of 10,000 × SYBR Green I (Invitrogen) + 600 µL of red blood cell lysis buffer {Tris (25 mM; pH 7.5)} + 360 µL of EDTA (7.5 mM) + 19.2 µL of parasite lysis solution {saponin and 28.8 µL of Triton X-100 (0.08%; v/v)}] were added to each well and incubated 1 hour in the dark at 37 °C. Fluorescence was measured using an Infinite M200 (Tecan) Microplate reader with excitation and emission wavelength of 485 and 538 nm, respectively.

Resistance index was determined through IC₅₀ ratio expressed as IR = CI₅₀ extracted on PfDd2 / CI₅₀ of the same extract on 3D7. A resistance index below 1 indicates that the inhibitor (plant extract) acts preferentially on the resistance strain.

Kinetics of growth inhibition of multi-resistant strain (PfDd2) in vitro

Evaluation of the kinetics of the growth inhibition of the resistant strain in vitro was carried out using the method described by Le Manach et al. (2013) [17]. On an asynchronous culture (containing all stages of development) of P. falciparum, different concentrations of the extracts were added and the mixture was incubated. The antiplasmodial activity was evaluated using a method based on nuclear staining with SYBR Green and expressed as an IC₅₀ value_. For each extract of the total alkaloids, three incubation times were used, namely 24 h, 48 h, and 72 h. Subsequently, the ratio of normalized IC₅₀ compared to 72 hours IC₅₀ was used to classify the action time effect (fast or slow) of the different extracts.

Cytotoxicity assay

Cytotoxicity assessment of the total alkaloid extracts was evaluated according to the method described by Bowling et al. (2012) and Al-Daghistani et al. (2021) [18-20]. Vero cell line (ATCC CRL 1586) from normal African green monkey kidney and Raw (ATCC #TIB-71) from murine macrophages were maintained in modified complete Dulbecco Eagle medium supplemented with 10% fetal bovine serum, bicarbonate of sodium 0.2% (w/v) with a penicillin-streptomycin combination 1% (v/v). A cell with a density of 10,000 cells per well in a 100 μL suspension was seeded on 96-well plates and incubated at 37°C for 24 hours at 5% CO₂ to reach 90% confluence. Ninety microliters (10 μL) of total alkaloid extracts or control were added after 24 hours with an initial 500 μg/ml concentration. Podophyllotoxin as Positive control (20 µM) was added. Thereafter, 10 µL of Resazurin solution (0.15 mg/mL in sterile PBS), was added to all wells and incubated for an additional 4 hours at the same condition. Fluorescence was subsequently read via a Magelan multi-well plate fluorescence reader, Infinite M200 (Tecan) at the excitation and emission wavelengths of 530 and 590 nm respectively [18]. Selectivity Index (SI) was used as a parameter of the clinical significance of test samples by comparing general toxins and selective inhibitory effect of P. falciparum using the following equation [21]:

In addition, the cytotoxicity activity of our extracts was evaluated according to the cytotoxicity criteria for crude extracts as established by the American National Cancer Institute (NCI) stipulating that IC₅₀ of the extract < 30 µg/ mL [22].

Data processing

Data collected were transferred to an Excel table for interpretation and calculation. Fluorescence values obtained were used to calculate the inhibition percentages using Microsoft Excel 2016 software. Inhibitory concentrations 50 (IC₅₀) was then determined using the concentration-response curves plotted to the logarithm of the concentration as a function of the percentage inhibition. The software used was Graph-pad software Prism 9.

RESULTS AND DISCUSSION

Determination of yields led to obtaining values for each extract as is shown in Table 1.

Table 1. Extraction yield

This result shows that from the same initial mass of plant powder, Coffea canephora is richer in alkaloids with a yield of 0.08% than Coffea arabica contents 0.05%.

The difference between these two yields could be explained by each plant's contents in alkaloids. Indeed, caffeine is the major alkaloid of these two species [23], and caffeine content is higher in C. canephora [24]. In addition, some parameters like climate and culture conditions can modify the chemical composition [25] and influence extraction yield.

In vitro antiplasmodial test of total alkaloids of the two species and the resistance, the index is presented in Table 2.

Table 2. Antiplasmodial activity of total alkaloids and resistance index

Pf: Plasmodium falciparum; IC_50: Inhibitory Concentration 50; AT: Total Alkaloids; ND: Not determined

The results were interpreted as follows: high activity (IC ₅₀ < 5 µg/ mL), active (5 ≤ IC ₅₀ < 50 µg/ mL), moderate activity (50 ≤ IC₅₀ < 100 µg/ mL), inactive (IC₅₀ > 100 µg/ mL) [26].

Total alkaloids extract from dried leaves of Coffea arabica is active both on the chloroquine-sensitive Pf3D7 strain and on multi-resistant PfDd2 strain with IC _{50 values} of 9.53 ± 1.51 µg/ mL and 7.48 ± 0.93 µg/ mL respectively, while total alkaloid extract from dried leaves of Coffea canephora showed weak activity (IC₅₀ >100 µg/ mL) on the Pf3D7 strain and moderate activity (IC₅₀ = 85.55 ± 1.17 µg/ mL) on the PfDd2 strain (Table 2). These results are different from those of Lacroix et al. (2011) [27] who worked on ethyl acetate extract of leaves of Coffea arabica. This difference may be due to the nature of the extract and the solvent used. Indeed, ethyl acetate will weakly extract alkaloids and more other compounds such as mono and diglycosidic flavonoids [28]. These results show that alkaloids could therefore be responsible for the antiplasmodial activity of the Coffea genus. Moreover, the antiplasmodial activity of total alkaloids of C. arabica being greater than that of C. canephora while alkaloid extraction yield being higher in C. canephora (Table 1) would suggest some differences in the alkaloid composition of the leaves of these two species. Furthermore, antiplasmodial activity (Table 2) may show that extracts could act preferentially on the multi-resistant PfDd2 strain with a resistance index of 0.78 (IR<1) probably because it acted on the resistance genes carried on the genomes of multi-resistant isolates (PfDd2) but absent in chloroquine-sensitive isolates (3D7).

Concerning kinetics of in vitro growth inhibition of multi-resistant strains (PfDd2), IC₅₀ values were determined for the two standards (chloroquine and artemisinin) as well as for the two total alkaloid extracts after 24, 48, and 72 hours of totals culture time incubation (Figure 1 and Table 3).

Table 3. Data on inhibition kinetics and speed of action

R: Ratio CI ₅₀ 24h / CI ₅₀ 72h; ND: not determined

The 24-hour and 72-hour dosage of AT1, Artemisinin and Chloroquine gave IC ₅₀ values of 78.33 ± 0.00; 63.28 ± 0.00 and 970.9 ± 0.01 (24 hours) and 27.56 ± 0.00; 22.16 ± 0.00 and 491.4 ± 0.00 (72 hours) respectively. Calculated ratios show that IC₅₀s were 2.89; 2.86 and 1.97 times higher at the 24-hour time point compared to those generated at the 72 hour time point (Table 3). These three days assessment data are a good indicator that the AT1 extract and the standards state with rapid action. AT2 extract values on the other hand were high at the three points (24, 48, and 72 hours) describing slow action. These results show that unlike total alkaloid extract of C. canephora (AT2), total alkaloid extract of C. arabica (AT1) could act at all evolutionary stages of the parasites (rings, trophozoites, schizonts) explaining the alkaloid difference composition hypothesis of the two species.

Biological efficiency is generally not due to in vitro cytotoxicity when the selectivity index (SI) is higher or equal to 10 [29]. In this study, in vitro cytotoxicity is said to be low when the Selectivity index is less than 10 and high when the Selectivity index is higher than 10 according to Waiganjo et al. (2020) [21]. Results are summarized and presented in Table 4 in which total alkaloid extracts of C. arabica and C. canephora have high selectivity index (SI ≥ 10) compared to Vero and Raw cells, and their IC_50s were > 30 µg/ mL (Table 4). This result indicates that the total alkaloids of the two species of the Coffea are non-cytotoxic [29].

Table 4. Selectivity index (SI) of total alkaloid extracts assayed against Vero, Raw cells, and the Dd2 resistant strain of P. falciparum

AT1: Total alkaloids of C. arabica; AT2: Total alkaloids of C. canephora; ND: not determined

SI: Selectivity Index

CONCLUSION

For the first time, a promising antiplasmodial and antimalarial activity presenting an interesting selectivity index has been demonstrated about total alkaloids of two species of Coffea (C. arabica and C. canephora). Results revealed that C. arabica showed a better activity with very low cytotoxicity. This plant is particularly interesting for further investigation as its phytochemical composition is poorly known, precisely concerning alkaloids. All these results justify their uses in traditional medicine for the treatment of malaria and can be a substrate for Improved Traditional Medicines (MTA).

Acknowledgments: None

Conflict of interest: None

Financial support: None

Ethics statement: None

[1]    Pradines B, Dormoi J, Briolant S, Bogreau H, Rogier C. La résistance aux antipaludiques. Rev Francoph Lab. 2010;2010(422):51-62.

[2]    Tona L, Cimanga RK, Mesia K, Musuamba CT, De Bruyne T, Apers S, et al. In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the Democratic Republic of Congo. J Ethnopharmacol. 2004;93(1):27-32. doi:10.1016/j.jep.2004.02.022.

[3]    Carlson CJ, Colwell R, Hossain MS, Rahman MM, Robock A, Ryan SJ, et al. Solar geoengineering could redistribute malaria risk in developing countries. Nat Commun. 2022;13(1):1-9. doi:10.1038/s41467-022-2963-w.

[4]    WHO. WHO report on Malaria. Available from: https://www.who.int/news-room/fact sheets/detail/malaria. 01 February 2022

[5]    Aborode AT, David KB, Uwishema O, Nathaniel AL, Imisioluwa JO, Onigbinde SB, et al. Fighting COVID-19 at the expense of malaria in Africa: the consequences and policy options. Am J Trop Med Hyg. 2021;104(1):26-9.

[6]    Alkandahri MY, Patala R, Berbudi A, Subarnas A. Antimalarial activity of curcumin and kaempferol using structure-based drug design method. J Adv Pharm Educ Res. 2021;11(4):87-90. doi:10.51847/q7yYE310JY.

[7]    Odono AE, Obeta UM, Etukudoh NS, Ali DO. Effet of Artesunate on serum bilirubin and albumin in Swiss Wistar Rats. Int J Pharm Phytopharmacol Res. 2021;11(6):8-14. doi:10.51847/8XI7ujhDVb.

[8]    Titanji VPK, Zofou D, Ngemenya MN. the antimalarial potential of medicinal plants used for the treatment of malaria in Cameroonian folk medicine. Afr J Trad Cam. 2008;5(3):302-21.

[9]    PNLP. Plan Stratégique nationale de lutte contre le paludisme 2011-2015. Rapport Minsante Cameroun. 2016:1-82.

[10] Kopa KT, Tane P, Wabo KH, Tala MF, Tchinda TA, Zofou D, et al. Activité antiplasmodiale in vitro des composés isolés des écorces du tronc de Vitex thyrsiflora. CR Chem. 2016;19(7):807-11. doi:10.1016/j.crci.2015.10.007.

[11] Miotto O, Almagro-Garcia J, Manske M, MacInnis B, Campino S, Rockett KA, et al. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nat Genet. 2013;45(6):648-55. doi:10.1038/ng.2624.

[12] Bernadin D, Siendou C, Marius KK, Moreto S. Usages ethnobotaniques des plantes par les populations de la Sous-Préfecture de Gonaté (Centre-Ouest de la Côte d'Ivoire). Int J Innov Appl Stud. 2021;34(2):369-79.

[13] Frédérich M, Jacquier MJ, Thépenier P, De Mol P, Tits M, Philippe G, et al. Antiplasmodial activity of alkaloids from various Strychnos species. J Nat Prod. 2002;65(10):1381-6. doi:10.1021/np020070e.

[14] Kémajou A, Mba L, Bagda AA. Effect of drying on the active principles of medicinal plants: case of total alkaloids from the bark of Alstonia boonei Wild, an antimalarial plant. Nat Technol. 2012;7:62-6.

[15] Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193(4254):673-5. doi:10.1126/science.781840.

[16] Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother. 2004;48(5):1803-6. doi:10.1128/AAC.48.5.1803-1806.2004.

[17] Le Manach C, Scheurer C, Sax S, Schleiferböck S, Cabrera DG, Younis Y, et al. Fast in vitro methods to determine the speed of action and the stage-specificity of antimalarials in Plasmodium falciparum. Malar J. 2013;12(1):424-30. doi:10.1186/1475-2875-12-424.

[18] Kurdi L, Alhusayni. Cytotoxicity effet of 5-fluorouracil and bee products on the MCF-7 Human Breast Cancer Cell Line in vitro. Int J Pharm Phytopharmacol Res. 2020;10(2):19-26.

[19] Bowling T, Mercer L, Don R, Jacobs R, Nare B. Application of a resazurin-based high throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis. Int J Parasitol Drugs Drug Resist. 2012;2:262-70. doi:10.1016/j.ijpddr.2012.02.002.

[20] Al-Daghistani HI, Abu-Niaaj LF, Bustanji Y, Al-Hamaideh KD, Al-Salamat MN, Nassar HM, et al. Antibacterial and cytotoxicity evaluation of Arum hygrophilum Bioss. Eur Rev Med Pharmacol Sci 2021;25(23):7306-16. doi:10.26355/eurrev20211227424.

[21] Itharat A, Houghton JP, Eno-Amooquaye E, Burke PJ, Sampson JH, Raman A. In vitro cytotoxic traditionally to treat cancer. J Ethnopharmacol. 2004;90(1):33-8. doi:10.1016/j.jep.2003.09.014.

[22] Waiganjo B, Moriasi G, Onyancha J, Elias N. Antiplasmodial and Cytotoxic Activities of Extracts of Selected Medicinal Plants Used to Treat Malaria in Embu County, Kenya. J Parasitol Res. 2020;17:1-12. doi:10.1155/2020/8871375.

[23] Ashihara H, Suzuki T. Distribution and biosynthesis of caffeine in plants. Front Biosci. 2004;9(2):1864-76. doi:10.2741/1367.

[24] Magalhães STV, Guedes RNC, Demuner AJ, Lima ER. Effect of coffee alkaloids and phenolics on egg-laying by the coffee leaf miner Leucoptera coffeella. Bull Entomol Res. 2008;98(5):483-9. doi:10.1017/S0007485308005804.

[25] Belitz HD, Grosch W, Schieberle P. Coffee, tea, cocoa. Food Chem. 2009:938-70.

[26] Kamaraj C, Kaushik NK, Rahuman AA, Mohanakrishnan D, Bagavan A, Elango G, et al. Antimalarial activities of medicinal plants traditionally used in the villages of Dharmapuri regions of South India. J Ethnopharmacol. 2012;141(3):796-802. doi:10.1016/j.jep.2012.03.003.

[27] Lacroix D, Prado S, Kamoga D, Kasenene J, Namukobe J, Krief S, et al. Antiplasmodial and cytotoxic activities of medicinal plants traditionally used in the village of Kiohima, Uganda. J Ethnopharmacol. 2011;133(2):850-5. doi:10.1016/j.jep.2010.11.013.

[28] Ferhat M. Research of bioactive substances of Centaurea microcarpa coarse and hard. [Online]. Master memory. Algeria: University of M'sila; 2009. Available from: https://www.memoireonline.com/04/10/3329/m_Recherche-de-substances-bio-actives-de-centaurea-microcarpa-coss-et-dur11.html. Consulted on May 27, 2020

[29] Vonthron-Sénécheau C, Weniger B, Ouattara M, Bi FT, Kamenan A, Lobstein A, et al. In vitro antiplasmodial activity and cytotoxicity of ethnobotanical selected Ivorian plants. J Ethnopharmacol. 2003;87(2-3):221-5. doi:10.1016/s0378-8741(03)00144-2.