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Deleterious effects of Artemisia infusions on Paramecium, Vibrio and Plasmodium

September 27, 2018 - 19:44 -- Pierre Lutgen

 

DELETERIOUS EFFECTS OF ARTEMISIA INFUSIONS ON PARAMECIA, VIBRIO AND PLASMODIUM

                                                    For the figures see attached pdf

Jerôme Munyangi,1,University of Kolwezi, Democratic Republic of Congo 

Lucile Cornet-Vernet,2, Maisons de l’Artemisia, Paris

Constant Tchandema,3, CAE, Lubumbashi

Pierre Lutgen,4 IFBV-BELHERB, Luxembourg 

 

Abstract

Paramecium like Plasmodium is an apicomplexan parasite. As in vivo trials with antimalarial drugs are difficult to perform, mostly for ethical reasons and as in vitro trials may be meaningless because the protozon Plasmodium, which needs a specific culture medium which interfers itself with the antimalarial drug, may react in a completely different way in vivo, where antimalarial drugs are dismantled by metabolism. Cultures of Paramecium tetraurelia are easy to cultivate and to handle. In vitro trials were run in 2014-2015 at the Laboratoire de Biologie cellulaire et moléculaire de l’Université Paris 11 with infusions of Artemisia annua and Artemisia afra and showed several detrimental effects on the behaviour of this parasite. This confirms similar effects on Vibrio fischeri and Vibrio cholerae which had been identified earlier.

Introduction

1. Artemisia annua and Artemisia afra

The in vivo effects of Artemisia annua and Artemisia afra on Plasmodium falciparum have been documented in scientific publications since 2005 in Kenya, Cameroon, Mozambique, Uganda, Togo, Senegal, Ethiopia, Mali, Benin and RDCongo

   Mueller et al,. The potential of Artemisia annua L. as a locally produced remedy for malaria in the tropics : agricultural, chemical and clinical aspects. Journal of Ethnopharmacology ; 73 (2000). p. 487–493.

   Whole-Leaf Artemisia annua based antimalarial Drug . Report on Proof of concept, 2005 International Centre of Insect Physiology and Ecology (ICIPE), Kenya.

  Chougouo-Kengne R. et al. Etude comparative de l’efficacité thérapeutique de l'artésunate seule ou en association avec l'amodiquine et de la tisane d'Artemisia annua cultivée à l'ouest du Cameroun. s.l.: Annales de Pharmacie, Université de Kinshasa, avril 2012. pp. 147-158. Vol. volume 4, numéro1.

   Gebeyaw Tiruneh et al,. Use of the plant Artemisia annua as a natural anti-malarial herb in Arbaminch town. Ethiopian Journal of Health Sciences, 2010. Vol.2, No.2

   Ogwang P. et al. Use of Artemisia annua L. Infusion for malaria prevention: Mode of action and benefits in a Ugandan community. British Journal of Pharmaceutical Research. 2011. pp. 124-132. Vol. 1(4).

   Ogwang, P. et et al. Artemisia Annua L. Infusion Consumed Once a WeekReduces Risk of Multiple Episodes of Malaria: A Randomised Trial in a Ugandan Community. s.l. : Tropical Journal of Pharmaceutical Research, June 2012. pp. 445-453 doi.org/10.4314/tjpr.v11i3.14. Vol. 11 (3).

   Onimus, M., Carteron, S. et Lutgen, P. The surprising efficiency of Artemisia annua powder capsules. s.l. : Medicinal and Aromatic Plants, 2013. Vol. 2-3.

   Weathers PJ, Lutgen P et al,. Whole plant approaches to therapeutic use of Artemisia annua, L. (Asteraceae). /IN: Artemisia annua. - Pharmacology and Biotechnology. Heidelberg : Eds., T Aftab, JFS Ferreira, MMA Khan, M Naeem Springer, 2013. 

   Tchandema, C. et Lutgen, P. In vivo trial on the therapeutic effects of encapsulated Artemisia annua and Artemisia afra. s.l. : Global Journal for Research Analysis, June 2016. pp. p 228-235. Vol. Vol 5.

   Resistance mitigating effect of Artemisia annua phytochemical extracts in cultures of Plasmodium falciparum and Olasmodium berghei and Plasmodium yoelii, Lucy N Kanghete, A Hassanalai, PhD Thesis, Jomo Kenyatta University, 2014, Nairobi

   Zime-Diawara H. et al. Etude de l’efficacité et de la tolérance d’une tisane à base d’Artemisia annua L. (Asteraceae) cultivée au Bénin pour la prise en charge du paludisme simple. International Journal of Biochemical Sciences. 9(2): 692-702, 2015

   N Bati Daddy, P Weathers. Artemisia annua dried leaf tablets treated malaria resistant to ACT and i.v. artesunate: case reports. Phytomedicine. 2017, 15; 32: 37–40.

   J. Munyangi, P. Lutgen, L Cornet-Vernet. Artemisia Plants: A Deadly Weapon Against Tropical Diseases Int J Clin Res Trials 2016, 1: 109. http://dx.doi.org/10.15344/ijcrt/2016/109

In this most recent clinical trial a team of medical doctors in RDCongo, J. Munyangi and M. Idumbo, have run randomized clinical trials on a large scale in the Maniema province with the participation of some 1000 malaria infected patients. The trials were run in conformity with the WHO procedures and compared Artemisia annua and Artemisia afra with ACTs (Coartem and ASAQ). For all the parameters tested herbal treatment was significantly better than ACTs: faster clearance for fever and parasitemia, absence of parasites on day 28 for 99.5% of the Artemisia treatments and 79.5% only for the ACT treatments. A total absence of side effects was evident for the treatments with the plants, but for the 498 patients treated with ACTs, 210 suffered from diarrhea, and/or nausea, pruritus, hypoglycemia etc. The efficiency was equivalent for Artemisia annua and Artemisia afra. More important even is the observation for the total absence of gametocytes after 7 and 28 days treatment with the herb.

The effects of Artemisia annua and Artemisia afra on other parasites, helminths and micobacteria has also been studied in vivo by our research group. Against all these diseases it shows a surprising activity and efficacy.

 

2. Paramecia tetraurelia

Paramecia is an unicellular eukariot. It is easily cultivated in the laboratory, its observation under the microscope is evident (120 μm large) and permits to follow the cellular cycle, the behaviour, the morphogenesis, its secretions and a large set of other biological functions. Its autogamy produces 100% homozygotic clones where toxicological impacts are easy to follow. It has the elongated form of a slipper (Pantoffeltierchen in German with a dorsal-ventral polarity. The dorsal side carries two pulsatile vacuoles which also control osmolarity. The oral aperture is on the bottom side and allows to swallow bacteria. Metabolic waste is expulsed by the anal cytoprocte. A very specific feature of Paramecium are thousands of cilia on its surface which contribute to great motility and allow forward and backward swimming. At the encounter of an obstacle Paramecium will retreat brusquely. Cilia also serve as antennae for chemical signals (attraction and repulsion)

   Itsuki Kunita, Shigeru Kuroda,Attempts to retreat from a dead-ended long capillary by backward swimming in Paramecium. Front Microbiol. 2014; 5: 270.

   Valentine MS, Van Houten JL Methods for Studying Ciliary-Mediated Chemoresponse in Paramecium. Methods Mol Biol. 2016;1454:149-68.

Paramecium needs large quantities of oxygen which it imports exlusively through its large skin surface.

   Rüdiger Wehner, Walter Gehring: Zoologie. 23. neu bearbeitete Aufl. Stuttgart 1995, ISBN 3133674234 This ciliated protozoan can alter its swimming behaviour in response to a change in the ionic concentration of the environment. Calcium serves as a messenger in all eukaryotes, from man to protozoa, including ciliates. Ca²⁺ may govern widely different processes, including cell movements, cytokinesis, morphogenesis. Cells dispose not only of Ca²⁺ influx channels, but also of intracellular release channels. It is likely that the Artemisia infusions act on the SERCA channels of Paramecium, but we do not know by which constituent. In this ciliated protozoan alveolar sacs underlie the somatic cell membrane. These sacs are are targets of Ca⁺² stimulation.

   EM Ladenburger, H Plattner, Calcium-release channels in Paramecium. PLos ONE 6(11) e27111

   Hinrichsen RD, Fraga D, Russell C (1995) The regulation of calcium in Paramecium. Adv Sec Mess Phosphoprot Res 30:311–338

Inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺ dependent ATPase) calcium pumps have an impact on internal Ca²⁺ stores in Paramecium. External application of inhibitors may dramatically alter the typical behavioral and electrophysiological responses of Paramecium to extracellular chemical stimulation.

   J Wassenberg, KD Clark, Effect of SERCA Pump Inhibitors on Chemoresponses in Paramecium. Journal of Eukaryotic Microbiology. 2007 https://doi.org/10.1111/j.1550-7408.

The influence of potassium on calcium has been studied more extensively in the case of Toxoplasma gondii, another apicomplexan. The reduction of extraparasitic K⁺ and calcium fluxes within the parasite are knwn to activate to activate the parasite’s motility machinery. For instance, buffers containing K⁺ levels that mimic the high concentration normally found within host cells block the motility of extracellular parasites. Similarly, intraparasitic calcium fluxes activate and regulate motility related events.

   M D. Lavine, G Arrizabalaga. Exit from Host Cells by the Pathogenic Parasite Toxoplasma gondii Does Not Require Motility, Eukaryotic Cells 2008, 7, 131-140

   T Endo, K Yagita.Effect of Extracellular Ions on Motility and Cell Entry in Toxoplasma gondii Journal of Eukaryotic MicrobiologyJ Propozool 1990, 37 :133-138

When the K⁺ concentration of the surrounding medium is lowered, the cells initiatially accelerate forward swimming. However, when they are transferred to a solution of higher K+ concentration, they show transient backward swimming and then recover.

   T Oka, Y Nakaoka, F Oosawa. Changes in membrane potential during adaptation to external potassium ions in Paramecium caudatum. J. exp. Biol. 126, 111-117 (1986)

   J Larsen, P Satir. Analysis of Ni²⁺-induced arrest of Paramecium axonemes : Journal of cell science, 1991, 99. 33-40

Artemisia plants are very rich in potassium and basically don’t contain any sodium

   E Brisibe, J Ferreira, Nutritional characterisation and antioxidant capacity of different tissuesof Artemisia annua L. Food Chemistry 2009, 125, 1240-6.

Paramecium has a mitochondrion. It has been shown that silica nanoparticles induce an oxidative stress which could play an important role of the mitochondrial membrane damage and the cell apoptosis. Artemisia plants are rich in silica nanoparticles.

   Sun L, Li Y, Liu X, Jin M, Zhang L, Du Z, Guo C, Huang P, Sun Z. Cytotoxicity and mitochondrial damage caused by silica nanoparticles. Toxicol In Vitro. 2011 Dec;25(8):1619-29

   Asweto CO, Wu J, Alzain MA, Cellular pathways involved in silica nanoparticles induced apoptosis: A systematic review of in vitro studies. Environ Toxicol Pharmacol. 2017 Dec;56:191-197.

   Pierre Lutgen. Silica particles inhibit sporozoite invasion, promote IgE, inhibit CYP3A4 and provoke bursting of infected erythrocytes Pharmacy & Pharmacology International Journal Volume 6 Issue 2 - 2018

Paramecium has been used to evaluate sludge toxicity, pyrethroid toxicity, Paramat toxicity. Many bioassays for herbicides, fungicides, insecticides, antimicrobials, heavy metals are based on Paramecium

   Norikazu MiyoshiTomonori KawanoTomonori Kawano. Use of Paramecium Species in Bioassays for Environmental Risk Management: Determination of IC50 Values for Water Pollutants. Journal of Health Science 2003, 46(6):429-435

Nickel is known as one of the most effective immobilization agents for protozoa. I was found to have a high toxicity on Paramecium bursaria. At concentrations of 5x10¯² g/dmᵌ it completely immobilizes the cells, stops rotary movement and leads to deformation and death. In humans, it accumulates to a large extent in the liver.

   Patrycja Zagata Leśnicka, Katarzyna Możdżeń. Acute toxicity of metals : Nickel and Zinc on Paramecia Bursaria. J Microbiol Biotechnol and Food Sciences 2015, 128-131

   Bovee E. Nickel sulfate as an anesthetic for protozoa. 1958 Turtox News, 36, p.78 Madoni P. The acute toxicity of nickel to freshwater ciliates. 1999. Environmental pollution, 109, 2000, p. 53-59

   T A. Glassman, J Suchy. Spectrophotometric evidence for the formation of a 2-nickel-adenosine triphosphate complex. Biochemistry, 1973, 12 (13), pp 2430–2437

The solubility of nickel from medicinal plants in aqueous infusions is higher than that of other metals and it has been estimated that the daily intake of Ni⁺⁺ from infusions of medicinal herbs is 4-5 times higher than from tap drinking water.

    J Mirslawski, A Paukszto, Determination of the Cadmium, Chromium, Nickel and Lead in selected Polish medicinal plants Biol Trace Elem Res 2018, 182, 147

Many Asteraceae plants are known as hyperaccumulators of heavy metals and Artemisia plants are part of this familiy. They are even used for bioremediation of contaminated soils. E Alirzayeva,

   T Shirvani, Heavy metal accumulation in Artemisia species from the Azerbaijan flora. For Snow Lands Res 2006, 80.3 XX-XY

   S Siebert, N Rajakaruna, N Schutte. A new nickel accumulator in the Asteraceae in South Africa. http://opus.sanbi.org/bitstream/20.500.12143/5435/1

A detailed study on the influence of tin oxide SnO₂ nanoparticles on Paramecia tetraurelia was made in Algeria. An increase in the number of Paramecium at low concentrations of SnO₂ and its inhibition at high concentrations was noticed. An obvious effect of hormesis : low-dose stimulation and high-dose inhibition.

    T Bouarroudj, M Benloucif, Cytotoxic effect of SnO₂ on Paraecium tetraurelia. Studia Universitate Vasile Goldis 2016, 26, 323-330

The antimalarial drugs, quinacrine, quinine and mefloquine induce calcium-dependent backward swimming in Paramecium calkinsi. These drugs are also toxic to Paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. This is definitely the case for pyrethroids.

   Nori VS, Barry SR. Toxic effects of antimalarial drugs in Paramecium: role of calcium channels. J Comp Physiol A. 1997;180(5):473-80.

   Steven B. Symington, Aiguo Zhang, Characterization of Pyrethroid Action on Ciliary Calcium Channels in Paramecium tetraurelia. Pesticide Biochemistry and Physiology, 1999. 65, 181–193 The Alveolates (Paramecium, Tetrahymena) are close relatives of the Apicomplexa (Plasmodium, Toxoplasma).

The toxic effects of artesunate and dihydroartemisinin on the growth metabolism of Tetrahymena thermophila were studied by microcalorimetry. The results showed that: : low concentrations of artesunate (<or=1 mg L(-1)) and dihydroartemisinin (<or= 2 mg L(-1)) promoted the growth metabolism of T. thermophila , whereas high concentrations of artesunate (1-60 mg L(-1)) and dihydroartemisinin (2-60 mg L(-1)) inhibited its growth.

 

   Shen XS, Su Q, Qiu ZP, et al. (2010). Effects of artemisinin derivative on the growth metabolism of Tetrahymena thermophila BF5 based on expression of thermokinetics. Biol Trace Elem Res 136:117–25

 

 

MATERIALS AND METHODS

 

  1. Preparation of Artemisia infusions

 

A mixture of Artemisia leaves and twigs (Artemisia annua or Artemisa afra) was used in powder form. 2 g are weighted on a precision electronic scale, placed in a tube and a litre of boiling demineralized  water at 100°C is added. pH of the cold infusion was measured routinely and  is between 7.0 – 7.8 for the different media used in the Paramecium test.

The original infusion was used at different dilutions.

For the trials we used progressive dilutions : 100, 50, 20, 10, 0 mg/L

 

2.  Preparation of the Paramecia tetraurelia culture

The techniques used for the culture of Paramecium are those described by Sonneborn revised by Janine Beisson

   T.M. Sonneborn, Chapter 12 Methods in Paramecium Research, , 10.1016/S0091-679X(08)61758-6, (241-339), (1970).

   Janine Beisson, Mireille Bétermier,Mass Culture of Paramecium tetraurelia. 2010, Cold Harbor Spring Protocols

Paramecium tetraurelia feeds on bacteria and develops best at 27°C with 4 to 5 divisions per day. The culture medium was BHB: an infusion of wheat grass, containing 50 μM of calcium and 0.4 µM of Na⁺ and the day before its use Klebsiella pneumoniae were added. A fresh culture is established every 3 days in order to use Paramecium during its exponential growth phase.

We prepared infusions with 2, 4, 6, 8 g/L of Artemisia powdered leaves and added them to the culture, before adding the Paramecia

 

  1. Measurement of growth kinetics

The growth kinetics of Paramecia vs time were established by counting them under the microscope (Leica DL 1000) following the procedure of Bouaricha at a 10x enlargment. The cell number is counted immediately after addition of the Artemisia infusion, and than after one hour and after 72 hours. 

For some measures we also used a binocular magnifier Nikon with continuous zoom. A camera (CCD 2048*2048px) is mounted on this magnifier allowing video recording.

    Bouaricha, H., Berrebbah, H., Grara, N., Djebar, M.R. 2012. Response of paramecium sp. with respect to an insecticide (Proclaim): growth, content of MDA, Ache activity and respiratory metabolism. Journal of Applied Sciences Research. 8(8): 4172-4180

 

  1. Percentage of response

Percentage of response is a parameter used to evaluate the xenobiotic effect of Artemisia via the inhibition of cell growth of protists

Percentage of response is calculated by the formula

Response (%) = (CN-EN) / CNx100

where CN is the number of control cells and EN the number of treated cells. Positive values of response percentage indicate an inhibition of growth, while negative values indicate a stimulation of growth

 

  1. Mortality rate

Mortality rate is established by counting the cells up to 72 hours

 

  1. Malformation rate

Malformations are detected by observations under the microscope at an enlargment of 100 following the procedure of Azouz. Malformations include changes in scape, loss of cilia, budding and sprouting on the membrane.

    Azouz Z., Berrebah H., Djebar R. (2011). Optimization of Paramecium tetraurelia growth kinetics and its sensitivity to combined effects of azoxystrobine and cyproconazole. Afri. J.Microbiol. Res. 5(20): 3243-3250.

 

  1. Modification in the endocytose and exocytose functions

These modifications are evaluated after the addition of picric acid and are based on the number of digestive vacuoles. The observations are made by placing the Paramecia between slides and plates under the microscope (Leica DL 1000) at an enlargment of 10.

 

  1. Study of the calcium receptors

Was based on the SERCA inhibition.

 

RESULTS

  1. Previous results  showing the effect of Artemisia infusions on Vibrio fischeri and Vibrio cholerae

In 2008 IFBV-BELHERB had run some experiments with Artemisia annua infusions on Vibrio fischeri. (P Lutgen unpublished). Infusions were  prepared by adding a liter of boiling tap water to 5 g of the dry Artemisia annua herb and leaving to infuse for 10 minutes before filtering.

Fig 1. Vibrio fischeri inhibition by Artemisia annua      

This effect on Vibrio was later confirmed by the University des Montagnes in Cameroun. Among 8 bacterial strains tested Vibrio cholerae showed the greatest antibacterial sensitivity to Artemisia annua essentiel oil

Rosine D.Chougouo K., Pierre R. Fotsing K., Jonas Kouamouo ,Bibiane Domum T., Pierre Lutgen, Lazare Kaptué .Antibacterial , Antifungal and larvicidal Activity of the Essential Oil Extracted by Hydro-Distillation from Artemisia annua Grown in West-Cameroon. 6th MIM Pan-African Malaria Conference. 6-11 October 2013, Durban , South Africa

 

In 2009 IFBV-BELHERB had used Paramecium to study the influence of UV radiation and Artemisia annua infusions on the sterilization of waste water (report Pollutec Paris dec 2009).

  1. Dose-effect relationship on swimming behaviour.

 

This parameter is studied by comparing controls with Paramecia treated with different doses of Artemisia infusion

Fig.2 describes the swimming behaviour at different doses. For the control Paramecia we notice an increase starting after 10 minutes and reaching a plateau after 50 to 60 minutes. At the beginning Paramecia treated with Artemisia infusion behave in the same way, but rapidly a dose dependant inhibition is noticeable. At a concentration of 100mg/L the movement is completely inhibited. At low concentrations of 20 mg/L the swimming speed was reduced and sometimes backward swimming was noticed

Fig.2. Effect of increasing concentrations of Artemisia infusions on the swimming behaviour of Paramecia

 

Observation of immobilized Paramecia

This observation was done under the microscope at an enlargement of 40 on plates having small cavities. These immobilized Paramecia are still alive. Some have lost their cilia. Pictures with further enlargements were obtained with the binocular lens, a Basler camera coupled with an USB 3.0 computer

  1. Study of exocytosis

This was done by comparing Paramecia treated and immobilized by Artemisia infusions with those immobilized by picric acid

In Paramecia tetraurelia, a crucial step in the secretory process is the transport of secretory organelles  to the cell surface, before exocytosis can occur. It has been postulated that micro- tubules might represent long-range signals or guiding rails for secreta transport. An enhanced Ca²⁺ ot the attack of xenobiotics may trigger this excretion process.

We could not observe this feature in Artemisia treated parasites although their cellular membrane was inflated. In the control Paramecia some excreta could be observed around the  buccal cavity.

 

  1. Comparing the effect of the two Artemisia species used :  annua and afra.

For growth rate the comparisons are always made during the exponential growth phase of the parasites.

For Artemisia annua we were not able to notice a difference in the behaviour of the cells treated by the infusion and the control cells at the early stages of the treatment. Only after one hour do we notice a difference in the growth rate and the inhibition steadily increases, and is dose dependent. After 48 hours growth inhibition is complete.

For Artemisia afra we notice an immediate decrease in the Paramecia count which progressively becomes more sever for higher doses.

Percentage of response was established acording to the procedure of  Wong

   Wong C.K., CheungY., 1999: Toxicological assessment of coastal sediments in HongKong using a flagellate Dunalliella tertiolecta. Environ. Poll., 105:175-183.

For both plants we notice a dose dependence. For Artemisia annua we find 38.26% at 2g/L for the reponse percentage  and 88.26% at 8g/L. For Artemisia afra it is 11.0% at 2 g/L and 90.3% at 8/gL.

Mortality rate

For Artemisia annua the mortality rate is of 4.2% after 1 one hour treatment at 6g/L. After 72 hours of the 6g/L treatment it reaches 16.35%.

For Artemisia afra we notice a mortality rate of 4.15% after 72 hours of exposure to 6g/L. At the dose of 8g/L we notice 16.35%.

Morphological changes and malformations.

The control Paramecia keep their elongated shape and a regular trajectory. Those treated by both Artemisia infusions see a significant and dose dependant increase of invaginations

 

Evolution of number of digestive vacuoles.

The Artemisia infusion treatments lead to a significant diminution in the number of digestive vacuoles when  compared to controls.

Controls carry between 7 to 10 vacuoles. For Artemisia annua at a dose of of 4g/L we only find 4 vacuoles an at 4 g/L and this number goes down to 2 at 8 g/L. For Artemisia afra we also notice a 80% diminution at the dose of 8g/L.

 

SERCA  receptors.

A dose dependent decrease of SERCA receptors was noticed for Paramecia treated by Artemisia infusions

 

DISCUSSION

 

All the parameters we have studied show a deleterious effect of Artemisia infusions on Paramecia tetraurelia. Similar effects were found for other apicomplexa. The ease of Paramecia culture and handling justifies further studies on the effect of Artemisia plants. The eukaryote Paramecium carries a nucleus, a mitochondria and a cytoskeleton. The impact of toxic substances on Paramecia could thus be extrapolated to pluricellular organisms. It is also worthwile to consider the hypothesis that the effect Artemisia has on the motility of Paramecium may also play a role in the gliding behaviour of Plasmodium sporozoites, and consequently the invasion of hepatocytes.

 

The work of Jerome Munyangi shows that the effects of Artemisia annua and Artemisia afra are similar. Artemisia afra does not contain artemisinin. Artemisia afra is an indigenous plant of Africa. WHO/EDM/TRM/2000.1 stipulates that if  documentary evidence shows that a plant has been used over three or more generations  for a specific health related purpose, there is no requirement for pre-clinical toxicity testing.

 

It remains to be assessed if either the prooxidant organic constituants of Artemisia or the minerals it contains are responsible for the detrimental effects on Paramecium.

 

 

 

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