Improvement of Secondary Metabolites from Phyllanthus odontadenius against Malaria by Mutagenesis

Aims: Majority of deaths in children aged under 5 years are due to Plasmodium falciparum malaria. Malaria deaths in children decreased but malaria remains a major killer of children, taking the life of a child every 2 minutes. This study aims to investigate the increasing of the in vitro antiplasmodial activities by mutagenesis techniques using gamma-rays (Cs-137) or sodium azide (NaN 3 ) as mutagens. It will allow the importance of mutagenesis use as tools for improvement of secondary metabolites against malaria parasites using chemical or physical mutagens. Study Design: Laboratory experiment tests : identification of plant material, immersion of seeds in SA (sodium azide) solutions or irradiation by Gamma-rays (Cs-137) of P. odontadenius seeds for improvement of secondary metabolites against malaria parasites, in vitro culture of seeds followed by the in situ culturing of plantlets for obtaining material of study, phytochemical screening of Phyllanthus odontadenius aerial parts to determine the change of compounds in comparison to controls, in vitro antiplasmodial tests for the determination of SA concentrations or those of gamma-rays doses which killing 50% of malaria parasite populations (IC 50 ). Place and Duration of Study


INTRODUCTION
Malaria is an erythrosis or erythropathy transmitted from one human to another by infected female Anopheles mosquito bites, called "malaria vectors", which bite mainly between dusk and early morning [1,2].Malaria is responsible for major socioeconomic disturbances in all subtropical and tropical countries where it is endemic [3,4,5].
Malaria is the most important parasitic disease in tropical areas.In 2016, an estimated 216 million cases of malaria occurred worldwide thus 90% for the WHO African Region, 7% for the WHO South-East Asia Region and 2% for the WHO Eastern Mediterranean Region [6].
Most deaths caused by malaria in 2015 are estimated to have occurred in the WHO African Region at 92%, at 6% for the WHO South-East Asia Region and with 2% the WHO Eastern Mediterranean Region [7].The vast majority of deaths (99%) are due to Plasmodium falciparum malaria with 70% of the global total to have occurred in children aged under 5 years.Number of malaria deaths in children is estimated to have decreased by 29% since 2010, but malaria remains a major killer of children, taking the life of a child every 2 minutes [7].
There are currently no registered vaccines against malaria or any other human parasite.An experimental vaccine against P. falciparum, RTS, S/AS01, is the most advanced and currently evaluated in a large clinical trial in some African countries [2].The DRC is among the 34 of the 41 high-burden countries with the highest prevalence of malaria, which had obtained, in the past 3 years (2014 -2016), the increases in external funding for their malaria control program [6].The spread of muti-drug resistant P. falciparum such as those of ARTEMISININ [6] in addition of that of chloroquine has highlighted the urgent need to develop to find new medicinal principles with different mechanisms of action, preferably inexpensive drugs that are affordable for developing countries where malaria is prevalent.
Vegetable resources have already proved their worth, as they are source of discovery of two major antimalarial drugs: quinine from Cinchona sp (Cinchona sp) and artemisinin from Artemisia annua.These two natural products have served as the basis for the hemisynthesis of many major antimalarial drugs [3].Secondary metabolites produced by plants through the secondary metabolism pathways are often the keystone in interactions between plants and their environment.Traditional medicine using plant extracts continues to provide health coverage for over 80% of world's population, especially in developing world, but also in modern allopathic medicine through use of purified or derived components obtained from chemical hemisynthesis [5,7,8,9].
African tropical forests are rich in plant genetic resources [10,11].However, deforestation of many forest ecosystems for crop field needs, firewood, construction, works of art, etc. and domestication problems of most plant species lead to the reduction in biodiversity and consequently to plant protection potential of these ecosystems which are felt to be inestimable losses [12].Martinov (2009) are used in DRC and in several regions of the world for treatment of several diseases [13,14,15,16].The in vitro antiplasmodial activity of Phyllanthus specimens varies according to the regions where the plant was harvested and according to the Harvest period, and does not allow users to use it appropriately for various virtues [16,17,18,19].

Plants of Phyllanthaceae family
Plant biotechnology offers opportunities in plant breeding [20].The search for new crop varieties have such as base on obtaining plant varieties with agronomic or sometimes pharmaceutical characteristics through use of mutagens [21,22].These mutagens are likely to modify the biochemical or physiological mechanism of plant in a random or targeted way.These mutagens could also allow the accumulation of adaptation of genes under different conditions [23,24].
This study aimed to investigate improvement of secondary metabolites of P. odontadenius against malaria through in vitro antiplasmodial activity of some Plasmodium falciparum strains using some mutagens such as gamma rays and sodium azide (SA).

Plant Materials
Plant extracts of Phyllanthus odontadenius from irradiated seeds by gamma rays (Cs-137) or from seeds dipped in Sodium Azide (SA) solutions and cultured in fields were used as biological materials.Physical and chemical Mutagenic techniques were used for gamma irradiation of P. odontadenius seeds or those of soaked seeds in SA resumption respectively in [25,26,27] and [28].
For the physical mutagenesis, seeds of P. odontadenius obtained from drying fruits harvested on the Kinshasa university site were irradiated with gamma rays from Cesium-137 (Cs-137) source in the Conservatome Lisa I Irradiator at the Department of Biochemistry, General Atomic Energy Commission (GAEC).The dose rate was 1.21 Gy/min [29,30].
For the chemical treatment, 100 seeds were placed in the Eppendorf microtubes (1.5 mL) and then imbibed in sterilized water for 1 h with agitation on shaker.The stock solution of sodium azide (Merck) was prepared in phosphate buffer (1 M), pH 3, filtered and stored frozen it at -20°C.Stock solution was diluted successively in water as well as in phosphate buffer of pH 3 to obtain various concentrations for the treatment of seeds.Distilled water removed and seeds were kept under various concentrations of sodium azide for 2 h 30 of time with continuous agitation on shaker at room temperature (25°C±2).Immediately after treatment of sodium azide, seeds were washed thoroughly in distilled water to reduce residual effect of sodium azide on the seed coat during 4-5 times.A portion of seeds were submerged in deionized water for the same period of time served as control.
The first generation of irradiated seeds or dipped seeds thus as plants obtained were designed as M1.
The in vitro susceptibility to extracts was determined by measurement of [ 3 H]hypoxanthine incorporation into parasite nucleic acids using the method of [36,37].
Antimalarial activity of the CRPf strain K1 was determined as concentration of drugs inducing 50% of growth inhibition (IC 50 ) by nonlinear regression analysis from the dose-response relationship as fitted by software -ICEstimator 1.2 (http://www.antimalarialicestimator.net) [38].

Determination of the Increasing of In vitro Antiplasmodial Activity
The comparison of In vitro antiplasmodial activities from plant extracts (report) was released between control plant extracts (CPE) against treated plant extracts (TPE) by the follow relationship: This relationship could be translate in percentage by the last formula from [39]:

Control In vitro antiplasmodial activity
The averages of in vitro antiplasmodial activities from control plant extracts realized with three trials were presented in Tables 1 and 2.  With regard to Fig. 1, it turns out that gamma radiation increased in vitro antiplasmodial activity up to 7.6 (Fig. 1a) at the dose of 150 Gy in M1.This in vitro antiplasmodial activity is enhanced up to 660% (Fig. 1b) than of the control.In M2, it increased up to 5.3 (Fig. 1c) times compared to the control; this in vitro antiplasmodial activity was improved by 430% (Fig. 1d) compared to the control.125 and 150 Gy are the two doses of gamma irradiation that showed very high values compared to the control.

2° In vitro antiplasmodial activity of extracts from treated plants on CRPf strain K1.
Fig. 2 illustrates the increasing In vitro antiplasmodial activity between the aqueous extracts from M1 and M2 plants obtained by gamma-irradiated seeds against control plant extracts on the CRPf strain K1.
In contrast to the effect of Phyllanthus odontadenius extracts from treated plants on P. falciparum isolates in both M1 and M2, these extracts did not show an increase in antiplasmodial activity in vitro on CRPf strain K1 in M1 (Fig. 2).It is in M2 where there is an increase of in vitro antiplasmodial activity from doses from150 to 225 Gy compared to the control.Plant extracts at 225 Gy increased the in vitro antiplasmodial activity up to 2.48 (Fig. 2c) compared with the control and improves this activity by 147.57% (Fig. 2d).With regard to Fig. 3, it turns out that SA increased in vitro antiplasmodial activity up to 10.15 (Fig. 3a) at the concentration of 10 mM in M1.This in vitro antiplasmodial activity is enhanced up to 915% (Fig. 3b) than of the control.In M2.1, it increased up to 6.39 times at the concentration of SA of 17.5 mM (Fig. 3c); this in vitro antiplasmodial activity was improved by 538.92% (Fig. 3d) compared to the control.Regarding M2.2, the in vitro antiplasmodial activity increased up to 9.11 (Fig. 3e) and SA enhanced up to 810.87% (Fig. 3f) the in vitro antiplasmodial activity than of the control.The concentration of 10 mM of SA showed in general the best in vitro antiplasmodial activities compared to all excepted 17.5 mM of SA in M2.1 and M2.2.

2° In vitro antiplasmodial activity of extracts from treated plants on
Chloroquine-Resistant Plasmodium falciparum strain K1.With regard to Fig. 4, it turns out that SA increased in vitro antiplasmodial activity up to 1.7 at the concentration of 15 mM in M1 on CRPf strain K1 (Fig. 4a).This in vitro antiplasmodial activity is enhanced up to 70.37% (Fig. 4b) than of the control.In M2.1, it increased up to 1.24 times at the concentration of SA of 17.5 mM (Fig. 4c); this in vitro antiplasmodial activity was improved by 24.43% (Fig. 4d) compared to the control.Regarding M2.2, the in vitro antiplasmodial activity increased up to 4.39 times at the concentration of 10 mM of SA and thus the SA enhanced up to 338.83% the in vitro antiplasmodial activity than of the control (Fig. 4e  and 4f).The concentration of 10 mM (M2.2) of SA showed the best in vitro antiplasmodial activity against all concentrations of SA.

Control in vitro antiplasmodial activities
Regarding values of the control in vitro antiplasmodial activities, they differ greatly from one to another.In vitro antiplasmodial activity of M1 presented high value than those of M2, except for the M1 value on the isolate P. falciparum.This difference could be explained by difference of plant material due by composition of plant because secondary metabolites in plants differ from one species to another and take an essential part in the plant metabolism and development [40,41,42].Phyllanthus species contain alkaloids, polyphenols, flavonoids, tannins, tepenes and steroids which could be explain the in vitro antiplasmodial activities showed in the control plant extracts [15,19,42] and [43].Phyllanthus species also contain sapogenin steroids which could explain their in vitro antimalarial activity [44,45].

Increasing of in vitro antiplasmodial activities
The in vitro antiplasmodial activities were increased greatly with the isolates of P. falciparum than that of CRPf strain K1.These results showed the capacity of resistance for the strain K1.  [47,48].For P. odontadenius, the gamma-rays values of DL30 and LD50 from gamma-rays obtained are respectively 79,05 Gy and 155, 10 Gy [49].
On CRPf strain K1, the M1 treated plant extracts showed low effect against the parasite, only in M2 where the in vitro antiplasmodial activity increased up to 2.48 (225 Gy) times in comparison to control plant extracts; those value of the in vitro antiplasmodial activity exceeded up to 147.57% than of control.In comparison to clinical P. falciparum, CRPf strain K1 not followed the letal doses (79.05 Gy and 155.10 Gy).This in vitro antiplasmodial activity on CRPf strain K1 at 225 Gy could be explained by metabolism of others compounds differed to those produced at 125 and 150 Gy.
For chemical mutagenesis by SA, the in vitro antiplasmodial activities against parasites in M1 increased up to 10.15 (10 mM) and 1.7 (15 mM) respectively for the isolates P. falciparum and CRPf strain K1.SA improved thus respectively the in vitro antiplasomodial activities to 915% and 70.37% in comparison to control plant extracts In M2.1, in vitro antiplasmodial activities increased up to 6.39 (17.5 mM) and 1.24 (17.5These different values could be explained the use of mutagens in plant breeding for improvement of antimalarial drugs or the improvement of secondary metabolites against malaria parasite by mutagenesis using physical or chemical mutagenesis.Concrete aims of using plant genetic resources (PGR) in crop improvement are : to develop cultivars that are specifically adapted to abiotic or biotic stresses, to assure sustainable production in high-yielding environments through reduced application of agrochemicals and increased nutrient and water efficiency ; and to open production alternatives for farmers through development of industrial, energy or pharmaceutical crops [50].

CONCLUSION
In this study, we proved that it's possible to improve the in vitro antimalarial activities using physical and chemical mutagenesis techniques.Our work illustrated an increasing in vitro antiplasmodial activity plant extracts treated by irradiation of P. odontadenius seeds or immersion of P. odontadenius seeds in SA solutions.
Plant extracts from treated seeds by gamma rays have significant effects on in the in vitro antiplasmodial activities than control plant extracts respectively with 660% in M1 and 430% in M2.For SA, the in vitro antiplasmodial activities of plant extracts from immersed seeds in sodium azide exceeded those of control plant extracts to 915% (M1), 538.92% (M2.1) and 810.87% (M2.2).
125 and 150 Gy are the doses of Gamma-rays used for improving the in vitro antiplasmodial activities against isolates of P. falciparum and 225 Gy could be used as dose for improvement of in vitro antiplasmodial activity against the strain K1 of P. falciparum resistant to chloroquine.10, 15 and 17.5 mM were the various concentrations of SA used for improving in vitro antiplasmodial activities against isolates of P. falciparum or chloroquine-resistant P. falciparum strain K1.

CONSENT
It is not applicable.We do not use patient or laboratory animals during our work.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.

Fig. 1
Fig. 1 illustrates the increasing In vitro antiplasmodial activity between the aqueous extracts from M1 and M2 plants obtained by gamma-irradiated seeds against control plant extracts on isolate of Plasmodium falciparum.

Fig. 3
Fig. 3 illustrates the increasing in vitro antiplasmodial activity between the aqueous extracts from M1 and M2 plants obtained by immersion of P. odontadenius seeds in SA solutions against control plant extracts on isolate of Plasmodium falciparum.

Fig. 1 .Fig. 2 .
Fig. 1.In vitro antimalarial activity of P. odontadenius plant extracts obtained from gamma irradiated seeds (Cs137) on clinical isolates of P. falciparum.a: ratio between IC 50 values (μg/ml) obtained from irradiatedplant extracts with that of control plant extracts in M1; b: The increasing of in vitro antimalarial activity in % compared to control in M1; c: ratio between IC 50 values (μg/ml) obtained from irradiatedplant extracts with that of control plant extracts in M2; d: The increasing of in vitro antimalarial activity in % compared to control in M2

Fig. 4
Fig. 4 illustrates the increasing of in vitro antiplasmodial activity between the aqueous extracts from M1 and M2 plants obtained by immersion of P. odontadenius seeds in SA solutions against control plant extracts on the CRPf strain K1.

Fig. 3 . 2 Fig. 4 .
Fig. 3.In vitro antimalarial activity from plant extracts of P. odontadenius obtained by immersion of seeds in SA solutions on isolates of P. plasmodium.a: Comparison of IC 50 (μg/ml) between treated plant extracts and control plant extracts in M1; b: Improvement of in vitro antimalarial activity in % compared to control in M1; c: Comparison of IC 50 (μg/ml) between treated plant extracts and control plant extracts in M2.1; d: Improvement of in vitro antimalarial activity in % compared to control in M2.1; e: Comparison of IC 50 (μg/ml) between treated plant extracts and control plant extracts in M2.2; 5.3f: : Improvement of in vitro antimalarial activity in % compared to control in M2.2

Strains and Methods 2.2.1 Clinical isolates of P. falciparum
[31]Sister32,33,34]ele, Mont-Ngafula, Kinshasa.Venous blood samples were collected in tubes containing 1% heparin and transported in carboglass until INRB laboratory.4ml of venous blood were centrifuged for 5 min at 3000 rpm to separate the plasma and the erythrocytes.1ml of erythrocytes were mixed with 9 ml of RPMI 1640 containing 25mM HEPES, 25mM sodium.In addition, antimalarial activity assays were performed by microscopic techniques adapting[31]method at the National Institute of Biomedical Research (NIBR) in Kinshasa/ Gombe, DR.Congo [27,32,33,34].

Table 1 . Averages of in vitro antiplasmodial activity from control plant extracts using gammarays as physical mutagen
50(1.12±0.55μg/ml)buthigh in vitro antiplasmodial activity in comparison to M2 plant extracts which were showed high IC 50 (9.68±2.21μg/ml)butlow in vitro antiplasmodial activity.3.1.2Increasing of in vitro antiplasmodial activities of plant extracts by irradiation (Cs137) of Phyllanthus odontadenius seeds1° In vitro antiplasmodial activity of Extracts from treated plants on Isolate of Plasmodium falciparum from human blood.

Table 2 . Averages of in vitro antiplasmodial activity from control plant extracts using Sodium Azide as chemical mutagen Strains IC 50 (μg/ml)
plants from immersed seeds in Sodium Azide solutions field I; M2.2:The second generation of plants from immersed seeds in Sodium Azide solutions field II
mM) and improved respectively to 538.92% and 24.43% in comparison to control plant extracts.In M2.2, the in vitro antiplasmodial activities of treated plant extracts on parasite, isolates of P. falciparum and CRPf strain K1, increased respectively up to 9.11 (15 mM) and 4.39 (10 mM) in comparison of the control plant extracts.These values showed an improvement of in vitro antiplasmodial activity respectively up 810.87% and 338.83% in comparison to the control plant extracts.For the breeding program of P. odontadenius species by SA, values of LD30 and LD50 were respectively 4.76 mM and 10.99 mM [27].Regarding results, plant extracts from 10 mM in M1 on the clinical isolate of P. falciparum and in M2.2 on CRPf followed values of LD30 and LD50.Others values obtained for example at 15 and 17.5 mM could be explained in the effects of mutagens on change of secondary metabolites [21,22].