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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article                                                                                    Research Article

Potentiation of antimalarial activity in Artemisia annua cultivated in the presence of fodder peanuts and beneficial microorganisms

Ferdinand Kouoh Elombo *¹, JJ Alladoum Kadounia ^{1, 2}, M Mbiandjeu Tchoumke ¹, J O Kuamou Nzoutap ¹, D Nwaga ²

1. University of Yaoundé I, Faculty of Sciences – Department of Biochemistry, Laboratory of Pharmacology and Toxicology, 812 Yaoundé, CAMEROON. 

2. Laboratory of Soil Microbiology/ Biotechnology Centre & Department of Microbiology, Faculty of Science, University of Yaoundé 1, PO Box 812 Yaoundé, Cameroon.   

Introduction 

In Cameroun, malaria is strongly endemic. Every year, we register six million cases of malaria and our health establishments report 4000 deaths around. The WHO estimates that around 11,000 people die of malaria in Cameroon each year. Artemisia annua, the source of new drugs Artemisinin and its derivatives have not, to date, induced resistance in Plasmodium. They are therefore widely used to treat malaria, mainly in South-East Asia and Africa¹. However, the chemical production of Artemisia annua biomolecules remains too expensive and unprofitable. Also the antimalarial active biomolecules in Artemisia annua plant are low concentrated. Research into optimizing their extraction and concentration is a current challenge². Promoting an inexpensive and more profitable cultivation route is a challenge to take up. A more environmentally sound and productive approach is the use of beneficial microorganisms such as biofertilizers and legume nodulating bacteria (LNB) ³. Although several studies have investigated the production of secondary metabolites of interest in Artemisia annua plants in different cultivation systems, only a few of them have studied the effect of microbial biofertilizers on these plants. The objective of this work was to evaluate the efficiency of microbial biofertilizers associated with a forage legume to improve the biomass and quality of A. annua. More specifically (i) evaluate the impact of fodder peanuts and microbial biofertilizers on the parameters of growth, development and biomass yield in A. annua, (ii) analyze the major phytochemical compounds of A. annua, (iii) determinate anti-oxidant as well as antiplasmodial activities of Artemisia annua extracts biofertilized or not.

Materials and methods  

Plant Material

Seeds of A. annua were grown on a non-sterilized soil substrate using non-randomized bloc design. Two standard treatments were established: control (C) and forage peanut (Arachis pintoi: Ara) alone⁴.  With the latter one we made some combinations:  Ara + special organic matter (MOS), Ara + Biochar (Bio) and finally a combination of Ara + mycorrhizal fungi (CMA) + endophyte bacteria (End) as previously described^{5, 6, 7}. Infusion was made by steeping 5g of dried leaves and stems in 1L of boiled water for 15 minutes. Whatman paper no. 3 was used to filter. The resulting filtrate was evaporated at 45°C in an oven prior for next experiences. 

Biochemical analysis

Chlorophyll a and chlorophyll b were measured at wavelengths 645 and 655 nm. Carotenoids were measured at 480. Each sample measurement was performed in three replicates and the pigment content was calculated as previously described by Bulda et al.⁸; Total polyphenols were determined as described by Kouoh et al⁹. Briefly, Gallic acid was used as a standard. The polyphenols content were expressed in mg gallic acid equivalent/g of Dry Matter (mgEqAG/ g of DM). Total flavonoid determination was made as described by Kouoh et al⁹. Quercetin was used as the standard. The flavonoid content was deduced from the calibration line and expressed in mgEqQ/g of DM as described ⁹. The total antioxidant capacity (TAS) of the extracts was determined based on the reduction of molybdenum, in the form of molybdate ion M₀O4^2-, to molybdenum M₀O²⁺ as previously described¹⁰. Sulfhydryl groups, or thiols, were determined using spectrophotometric assays, with Ellman’s reagent (DTNB - 5,5′-dithio-bis-(2-nitrobenzoic acid)), producing a yellow product measured at 412 nm. The reduction of 5,5'-dithio-bis-2-nitrobenzoic acid (Ellman's reagent, DTNB) by the (-SH) groups of glutathione produces 2-nitro-5-mercapturic acid. This is a yellow colored complex that absorbs at 412 nm¹¹. 

In Vitro Antiplasmodial Activity

The Trager technique¹² was used with slight modification as previously described¹³. Briefly, in a humidified incubator consisting of N (92%), CO₂ (5%), and O₂ (3%), fresh human group O⁺ red blood cells at 4% hematocrit in complete RPMI medium ((Gibco, UK) cells were used to culture the chloroquine-sensitive Plasmodium falciparum strain 3D7 and the multiresistant Plasmodium falciparum strain Dd2. The medium was supplemented with 25 mM HEPES (Gibco, UK), 0.50% Albumax I (Gibco, USA), 0.1 mM hypoxanthine (Gibco, USA),, and 20 μg/mL gentamicin (Gibco, China)).   The temperature of incubation was   37°C. The parasite were synchronized at the ring stage before testing antiplasmodial activity. This was done by treating them with 5% (w/v) sorbitol for 10 minutes as previously described¹⁴. The in vitro antiplasmodial activity was evaluated according to the method described by Smilkstein et al.¹⁵. Briefly, a 96-well microplate titer with 90 μL of the parasite suspension at the ring stage of 2% parasitemia and 1% hematocrit was added 10 μL of the various concentrations of extracts, artemisinin, and chloroquine. Then, plates were incubated for 72 hours at 37°C in a CO₂ incubator. This experiment was done in triplicates and the final plant extract concentration ranged from 0.01258 to 200 μg/ml. Prior to 1 hour of incubation in the darkness, 100 μL of SYBR Green was added into each well. At an excitation and emission wavelength of 485 and 538 nm respectively, the result of the antiplasmodial activity was read using an ELISA fluorescence microplate reader (Tecan Infinite M200). Duncan's test allowed the means to be compared with each other at the 5% threshold (risk of error). 

RESULTS AND DISCUSSION 

Forage peanut and biofertilizers increased biomass yield in A. annua. Arachis pintoï (Ara) combined to special organic matter (MOS) is the most efficient treatment (Table 1). However beneficial microorganisms associated with forage peanut may produce bioactive metabolites by acting as signals that activate the expression of genes involved in their biosynthesis¹⁶, increasing the activity of antioxidant enzymes such as superoxide dismutase, catalase and peroxidase (Table 2). It’s also known that microorganisms associated with forage peanut can improve the absorption of nutrients favorable to the good growth of the Artemisia plant³.

Table 1: Influence of peanut and biofertilizers on biomass yield in A. annua. Values in the same column followed by the same letter are not significantly different at the 5% threshold.

Fresh aerial biomass (g/plant)

Fresh root biomass (g/plant)

Dry aerial biomass (g/plant)

We also noticed an impact of fodder peanut and microbial biofertilizers on photosynthetic pigment, sulfur compound and tripeptide contents (Table 2).

Table 2: Effect of fodder peanut and microbial biofertilizers on photosynthetic pigment, sulfur compound and tripeptide content. Values in the same column followed by the same letter are not significantly different at the 5% threshold. (Ara): fodder peanut; (MOS): Special Organic Matter; (Bio): Biochar; (CMA):  Mycorrhizal Fungi; (End): Endophyte bacteria.

Chlorophyll content (mg/l)

Carotenoid content (mg/l)

Glutathione content (µmol/gE)

Fodder peanut alone or combined with microbial biofertilizers increases biomass yield in Artemisia annua and stimulates more antioxidant power, flavonoids, total polyphenols, total thiols and glutathione while organic matter has weaker effects on these parameters. For all these raisons, we have chosen extracts of A annua cultivated without biofertilizer and with “Ara+CMA+End” to see what could be the effect of microbial biofertilizers associated with fodder peanut on Plasmodium falciparum. Hence, in Table 3, cultivating A. annua in the presence of “Ara+CMA+End” inhibited Plasmodium falciparum Dd2 (PfDd2) and 3D7 (Pf3D7) strains. Moreover, we had 50% inhibitory effect of PfDd2. Microbial biofertilizers, including “Ara+CMA+End” associated with fodder peanut enhance plant health, nutrient uptake, and soil quality and strengthen agricultural sustainability¹⁷. This study provide an evidence that biofertilizers applied to fodder peanut could kill or inhibit the malaria-causing Plasmodium falciparum parasite.

Table 3: IC50 determination in strainds Pf3D7 and PfDd2. AnY = biofertilized Artemisia annua; AnB= none biofertilized Artemisia annua; M±ET= Mean ± standard deviation.

                                    Parameters

Samples

CI50 M±ET (µg/mL)

PfDd2

Pf3D7

Artemisia Annua 

aqueous extracts 

AnY

31±2,05

34,14 ± 2,71

AnB

60,68± 13,07 

47,69 ± 6,34 

Positive Controls 

Artemisinin

0,025 ± 0,005 

0,035 ± 1E-05 

Chloroquine

0,733 ± 0,09 

0,045 ± 0,003

CONCLUSION 

Integrated soil fertility management (ISFM) involving fodder peanut and beneficial microorganisms (mycorrhizal fungi and   endophyte bacteria) may be the best option to provide the highest bioactive molecules content with the best anti-oxidant and antiplasmodial capacity in A. annua. At least, ISFM improve Artemisia annua biomass production for better active therapeutic ingredients. 

Acknowledgements : We would like to thank the staff of the National Herbarium of Cameroon for helping us locate and identify the Artemisia annua plants.

Funding : This work received funding from the Cameroonian Ministry of Higher Education throughout the special allowance for the modernization of research

Disclosure : The study was independently designed by the authors and the funding body had no role in Lab experiments, analysis and interpretation of the data. 

Competing interests : The authors declare that they have no personal relationships or known competing financial interests that could have influenced the work reported in this paper.

Author’s contributions

JJ Alladoum Kadounia performed the analyses, processed the data and drafted the manuscript.

J O Kuamou  Nzoutap collected the samples and performed the analyses

Ferdinand Kouoh Elombo conceived the study, contributed to data processing, analyses and manuscript writing. 

M Mbiandjeu Tchoumke performed the analyses

D Nwaga contributed to manuscript writing. 

All authors read and approved the final manuscript.

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