Selenium Content, Anthelmintic, Antioxidant and Antibacterial Activities of Artocarpus Heterophyllus Lam. From Ubangi Ecoregion in Democratic Republic of the Congo

Over 80% of the African population relies on medicinal plants for the management of several diseases and to meet their primary healthcare [1]. In the therapy of several ailments, traditional medicine is showing a bright future and as it is performed as a good clinical practice. Plants are known to be the source of several medicines such as anthelmintic drugs. However, helminths are recognized as one of the major problems in farms for the animal’s production and constitute a public health concern as well in the tropics. Most of diseases caused by helminths are of a chronic and devastating in nature; they probably cause morbidity and greater economic and social deprivation [2]. Currently, gastrointestinal nematodes become more resistant to available anthelmintic Abstract


Introduction
Over 80% of the African population relies on medicinal plants for the management of several diseases and to meet their primary healthcare [1]. In the therapy of several ailments, traditional medicine is showing a bright future and as it is performed as a good clinical practice. Plants are known to be the source of several medicines such as anthelmintic drugs. However, helminths are recognized as one of the major problems in farms for the animal's production and constitute a public health concern as well in the tropics. Most of diseases caused by helminths are of a chronic and devastating in nature; they probably cause morbidity and greater economic and social deprivation [2]. Currently, gastrointestinal nematodes become more resistant to available anthelmintic drugs; this situation causes a serious problem in the treatment of helminthiases [3]. However, the chemotherapy is and remains the only treatment and effective tool to cure and control helminth infections, as effective vaccines are not yet developed until then. The random use i.e. the overuse or the misuse of synthetic anthelmintic drugs can be the cause of resistance to these drugs by the parasites.
Moreover, these drugs are unaffordable due their high cost [4].
With respect to this alarming situation of resistance, a way can be paved for the use of herbal medicines as alternative anthelmintic and could help in the prevention of resistance development. In low-income countries, plants are used in ethnoveterinary and are a source of many potent and powerful drugs [5]. Artocarpus heterophyllus Lam. called jackfruit tree, is a species of tree in the Artocarpus genus (Moraceae) [6]. It has been reported that Artocarpus heterophyllus is a good diuretic and laxative remedy. In the Democratic Republic of the Congo (DRC), the leaves of jackfruit tree are used for their anthelminthic properties mainly on animals [6].
To our knowledge, few investigations reported on the chemical constituents and the biological properties of this species. The main aim of the current work was to investigate on the anthelmintic, antioxidant and antibacterial activities of ethanolic extracts and organic acid fraction.

Plant Material Collection
The selection of plant species used in this study was based on previous ethnobotanical surveys [6].

Preparation of Extracts
The dried and powdered plant material (50g) was repeatedly extracted with 400 ml of ethanol (95%) by cold percolation for 48 hours [7]. The filtrate obtained was concentrated to dryness under reduced pressure using a rotary evaporator. Organic acid fraction (triterpenic acids) was extracted as follow: the powdered stem bark of Artocarpus heterophyllus (40g) were macerated with 400 mL of dichloromethane-methanol-NH4OH (100:1:1; v/v/v) and then percolated with 300 mL of the same solvent mixture at room temperature. The extract was concentrated under reduced pressure until 100mL (pH 10). The resulting solution was then mixed with citric acid 5% (1:1; v/v) to precipitate organic/triterpenic acids. The resulting fractions were evaporated to dryness on an evaporator apparatus. All extracts were weighed and kept in dark inside hermetic flasks at 4°C.

Phytochemical Analysis
Thin Layer Chromatographic Analysis: The phytochemical screening was performed on the aqueous and organic extracts for the identification of alkaloids, saponins, total polyphenols, flavonoids, tannins, anthocyanins, leuco-anthocyanins, quinones, terpenes and steroids following the standard protocol as described by Bruneton [8] and Tiwari et al. [9].

Phenolics Contents
Total Phenolics Content: Total phenolic content of methanolic extracts (Methanol 80%) was determined according to the Folin-Ciocalteu method as described previously [7]. A calibration curve of gallic acid (0.025-0.4 mg mL1) was prepared, and phenolic contents were determined in triplicate from the linear regression equation The results are expressed in mg equivalent of quercetin per g (mg QE/g) of dry vegetal material [10].

Total Anthocyanins Content:
The determination of anthocyanin content of the extracts was performed in accordance with the procedure reported by Bahati et al. [10].

Determination of Mineral Content
The determination of mineral content was carried out by the

Evaluation of Radical Scavenging Activity
The evaluation of the antioxidant activity was performed using the ABTS and DPPH assays in accordance with Kapepula et al.

In Vitro Anthelminthic Activity
The in vitro anthelmintic activity of Artocarpus heterophyllus was evaluated as per the method reported by Yashaswini [11].
The tested samples prepared from the ethanol and organic acid

Antibacterial Activity
The antibacterial activity of Artocarpus heterophyllus leaf extracts was performed following the micro-well dilution method on two bacteria strains namely Escherichia coli and Staphylococcus aureus. And the minimum inhibitory concentration (MIC) was obtained [7].

Data Analysis
Each concentration was tested in triplicate for each assay, and at

Phytochemical Analysis
Phytochemical analysis performed by TLC showed the presence of phenolic compounds as major compounds. These phenolic compounds are responsible of antiradical activity of plants, since this potential is strongly attributed to them [12]. By comparing with the used standards, phenolic acids such as cafeic acid and chlorogenic acid are identified in the plant [13,14]. These results were like those previously described that indicate the presence of flavonoids in the leaves of A. heterophyllus [15]. Figure 2 shows the presence of coumarins (blue fluorescent spots) in almost all the extracts of the tested samples. Concerning anthraquinones, Figure 2 shows the presence of anthrones (red spots) [16,17]. The

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Karunanayake [14][15][16][17] in A. heterophyllus [18] that indicated the presence of these chemical compounds in A. heterophyllus. The presence of these compounds in a plant species could confer some biological properties, such as antioxidant activity and anthelmintic activity [4]. The alkaloid test gave a negative response using Draggendorff reagent. The results of secondary metabolite assays are presented in Table 1 below.  [19,20]. Studies have shown that not only extrinsic factors (such as geographic and climatic factors), genetic factors, but also the degree of maturation of the plant and the duration of storage has a strong influence on the content of polyphenols [21,22].

Mineral Compounds
The mineral composition of the leaf extract of Artocarpus heterophyllus is given in the figures. The analysis of both figures above showed that calcium is the microelement which has a remarkable predominance in terms of concentration in the leaves of this plant followed by potassium compared to other detected microelements (magnesium, sodium and phosphorus) (Figure 1). While Selenium is the microelement that has a high content followed by iron, nickel and manganese while other microelements such as Barium, Cobalt, Zinc and Copper present low concentration (Figure 2). Soetan et al. [23] reported that minerals are inorganic nutrients, usually required in small amounts from less than 1-2500 mg per day, depending on the mineral. As with vitamins and other essential food nutrients, mineral requirements vary with animal species. For example, humans and other vertebrates (like ruminants) need large amounts of calcium for construction and maintenance of bones and normal function of nerves as well as muscles. Phosphorus is an important constituent of adenosine triphosphate (ATP) and nucleic acid and is also essential for acid-base balance, bone and tooth formation. acid metabolism [24]. Deficiencies of cobalt in ruminants cause anorexia, wasting of skeletal muscle, fatty livers, hemosiderosis of the spleen and anaemia [23]. The interactions between nutrition and diseases, nutrition and drug metabolism have been reported. Excessive intake of some minerals can disrupt the homeostatic balance and causes toxic side effects.

Antioxidant Activity
The antioxidant activity of tested extracts determined by ABTS and DPPH assays is presented in Table 2

In Vitro Anthelmintic Activity
The in vitro anthelmintic activity is depicted in  [6]. It was reported earlier that the tannins and phenolic were known to interfere with the energy generation in helminth parasites by uncoupling oxidative phosphorylation or they bind to the free protein of the gastrointestinal tract of the worms and led to the death while the presence of flavonoids and polyphenolic compounds were also responsible for anthelmintic activity [25]. The presence of these compounds in a plant species could confer some biological properties, such as antioxidant activity, antibacterial activity as well as the anthelmintic activity [4]. Albendazole have the property to be fixed on the nicotinic receivers at acetylcholine of the parasitic worms as thus mimics the action of acetylcholine. This fixing induces the permeability change of the post-synaptic membrane causing a muscular contraction, followed by the spastic paralysis and finally, the death of the worms [27]. On the contrary, the polyphenols like tannins would have the capacity to bind to proteins and, consequently would modify the physical properties and biochemical parasitic worms. Indeed, they would interact with the worms while being fixed at the macromolecules of the cuticule or of the sheath of the L3s larvae (containing proteins rich in proline and hydroxyproline) or would fix them to the secrete enzymes over the worms and would block their activity [28]. It was considered that the interactions between tannins and proteins of the exoskeleton (the cuticule), the alimentary canal even the genital structures of the worms would affect some essential functions like the nutrition or reproduction [29].

Antibacterial Activity
The antibacterial avtivty of the leaves of A. heterophyllus is presented in the Table 4. The results of antibacterial activity showed E. coli and S. aureus are not sensitive to A. heterophyllus with the CMIs>1000 μ g/mL) This antibacterial activity is however weak (MIC>1000 μ g/mL) [30] and would be due to the presence of phenolic and triterpenic compounds present in the tested extracts.
The bioactivity would be due to the nature of their wall [31]. Indeed, S. aureus is a positive gram bacterium. Its wall is thick (several layers) and would be the pharmacological target of the biologically active compounds present in A. heterophyllus leaves. These results corroborate with studies on the antimicrobial properties of the secondary metabolites of plant origin [32,33].