Phytochemical Study and Anthelmintic Activity of Nine Congolese Medicinal Plants

Background: Despite advances in hygiene and preventive medicine, parasitic diseases, particularly digestive parasitosis, remain a public health problem in tropical countries. Elaborate the ethnobotanical survey carried out in Brazzaville Congo, 21 plants (divided into 20 families, 20 genera) were identified, among them 9 were selected for this study. These were: Ageratum conyzoides (L.) L., Rauvolfia mannii Stapf, Aloe buettneri A. Berger, Garcinia kola Heckel, Piper guineense Schumach & Thonn., Aframomum alboviolaceum (Ridl.) K. Schum., Plagiostyles africana (Müll .Arg.) Prain, Morinda lucida Benth, Cogniauxia podoleana Baill. Materials and Methods: The in vitro evaluation of the anthelmintic activity of the aqueous extracts of these 9 plants was determined at concentrations of 10, 25 and 50 mg/mL against Lumbricus Original Research Article Merveille et al.; JPRI, 33(14): 63-76, 2021; Article no.JPRI.65697 64 terrestris used as animal support in comparison to the Mebendazole (20 mg/mL). The phytochemical screening realized by TLC to detect phytochemical families in plant, also, the antioxidant potential with DPPH radical was determined by UV-Visible spectrophometry in comparison to the Vitamin C. Results: Thus, P. guineense, A. conyzoides and A. alboviolaceum are those which presented significant vermicidal effect, after Mebendazole. Phytochemical screening into TLC revealed the copresence of alkaloids, coumarins, flavonoids, tannins, sterols and terpenes.The antioxidant potential of the aqueous extracts of these different plants in comparison to vitamin C, has shown that the species A. Buettineri and A. alboviolaceum presented the best potential compared to 7 other species studied, with the concentration needed to reduce 50% of the DPPH radical (CR50) respectively 0.096 mg/mL for A. Buettineri, 0.098 mg/mL for A. alboviolaceum Berger and 0.015 mg/mL for vitamin C. Conclusion: There is modern drugs against intestinal worms, it is necessary to identify the plants with anthelmintic activity, to determine phytochemical compounds that would be responsible for their use by traditional healers to treat the intestinal parasitosis.


INTRODUCTION
The parasites are very widespread in the world, but this presence is especially observed in the tropical zones. The number of subjects suffering from intestinal parasitosis is particularly high among African populations with low levels of economic and social organization, with poor hygienic conditions [1,2]. There are various modern drugs to fight against intestinal parasitosis. However, the difficult access to modern anthelmintics and, moreover, the phenomena of resistance developed by gastrointestinal parasites prompt African populations to have recourse to nature, to plants which are easily accessible and less expensive [3]. This is how ethnobotany and ethnopharmacology are working to identify reputedly biologically active plants whose modern research must specify the properties and seek new bioactive molecules [4,5]. The promotion of endogenous plant-based recipes and practices is now a scientific concern. Indeed, different techniques have been implemented to extract natural substances or active ingredients of plant origin [5][6][7]. Several plants which fight against intestinal parasites have been inventoried and studied in Congo [8,9]. With an aim of widening this repertory, the present study was identified 21  The richness of secondary metabolites of plants leads to their use in the manufacture of phytomedics, in agriculture to control pests through plant protection effects, in cosmetics as perfume or additives, in the agri-food industry as an ingredient, food preservative [10][11][12]. Also, these plants are used in several preparations to remedy various diseases such as malaria, diarrhea, microbial and bacterial infections, skin troubles [5,4].

Animal sample
Lumbricus terrestris Linn., commonly called earthworms, has been used as biological material to carry out vermicidal tests on [8,13]. They were collected in Brazzaville (Congo) near the river located at Diata (4 ° 16 '21,747' 'South ; 15 ° 14' 34,725 '' East) in the Makélékélé district. Their mass and their size varied from 0.4 to 3 g and 7-15 cm respectively.

Preparation of the aqueous extracts
According to the preparation methods collected from traditional hearlers, a mass of 30 g of powder from each organ was either macerated during 24 h for G. kola, P. africana, P. guineense or brought to a boil during 20 min for A. alboviolaceum A. conyzoides, A. buettneri, C. podoleana, M. lucida, R. manii in 300 ml of distilled water. After filtration and concentration in a rotary evaporator (BUCHI Water bath B-480), the dry extracts obtained are put in the bottles for analysis.

Preparation of drug solutions:
The extracts used to carry out the vermicidal tests were prepared by dissolving 0.2; 0.5 and 1 g of dry extract in 20 mL of distilled water.

Evaluation of the anthelminthic activity (Vermicide test)
The method of Ajayieoba [14] with slight modifications was used for carrying out the vermicidal tests of the aqueous extracts. A range of three concentrations (10, 25 and 50 mg/mL) has been prepared for each plant extract. Mebendazole (20 mg/mL) was used as the standard anthelmintic. Earthworms were placed in sets of five in petri dishes:  Lot 1: negative control lot, has been treated with distilled water only ;  Lot 2: test lot, was treated with plant extracts at different concentrations ;  Lot 3: positive control lot, was treated with Mebendazole (20 mg/mL), standar drug.
The behavior (hypermobility and mortality) of the worms in each dish was observed during 24 h. The paralysis and lethality times (100%) were noted. The experiment is repeated three times.

Phytochemical screening by TLC
The TLC was determined with the extract macerated in organic solvents (1g in 100 mL) in order of increasing polarity (Hexane, Chloroform, Ethylacetate, Ethanol). Specifics developers were used to identify the chemical families present in plants extract [6,7,15,16] (Table 1).

Total polyphenols
The determination of total polyphenols is made by UV-Visible spectrophotometry with the Folin-Ciocalteu reagent according to the method of Singleton [17]. Gallic acid, prepared under the same conditions as the plant samples, was used as the reference compound. The contents expressed in equivalent micrograms of gallic acid per gram of dry matter (µg EAG/g DM).

Total Flavonoids
The total flavonoids contents of the aqueous extracts are determined with aluminum trichloride (AlCl 3 ) according to the method of Swain [18,19].
Catechin prepared under the same conditions as the plant samples was used as the reference compound. The results obtained are expressed in micrograms of catechin equivalent per gram of dry matter (µg ECAT / g DM).

Condensed and hydrolyzable tannins
The condensed and hydrolyzable tannins are measured respectively by sulfuric vanillin and iron trichloride (FeCl 3 ) according to Swain [18,19].

Evaluation of antioxidant activity by spectrophotometry UV-Visible
For each extract, a range of concentrations (2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, and 0.125 mg/mL) is prepared in absolute ethanol. 1 mL of these samples was added to 1.5 mL of ethanolic solution of DPPH radical (0.03 mg/mL). The mixture obtained is well agitated and left to incubate for 30 min at room temperature. The absorptance of the mixture is read at 517 nm with a UV-Visible spectrophotometer against a blank consisting of 1 ml of absolute ethanol (without extract) and 1.5 mL of DPPH solution. The reference positive control was ascorbic acid (vitamin C) [20].
The percentage reduction of the DPPH radical is given using the following formula: R (%) = (Ab -Ae) / Ab × 100 (Eqn. 1) R: percentage reduction of the DPPH radical; Ab: Absorbance of white; Ae: absorptance of the sample.
The concentrations necessary to reduce 50% (CR 50 ) of radical DPPH were given with the Excel software.

Paralysis time of Lumbricus terrestris Linn
The worms were suspected to be paralyzed when they can no longer move completely in the petri dish [8,13].   (10,25 and 50 mg/mL) compared to the standar drug Mebendazole (20 mg/mL) which caused paralysis of the worms after 5 min. The constant contact of the worms with extracts has led to hyper-mobility and a tendency to want to get out of the petri dish. The permeability of the worms caused their paralysis and some resolved over time before dying. This phenomenon has been observed on the same type of worms during works carried out by Ongoka in Congo and Guissou in Burkina [8,13]. The increasing order of paralysis time of worms by extracts at all concentrations was the same and presented as follows: Table 2).

Mortality time of Lumbricus terrestris Linn
The death time of worms based on concentrations showed in Table 2 for each species and the standar drug Mebendazole. Earthworms are considered dead when they were stationary 5 min after having noted the death time. The shorter time of the total lethality time (100%) of worms proved that the extract exhibited a strong vermicidal activity [14]. The results obtained showed that the death time of worms depends on the concentration of plant extracts, so at 10 and 25 mg/mL this time looks longer than at 50 mg/mL. In comparison with the standar drug (Mebendazole), the aqueous extract of the seeds of P. guineense showed the best vermicidal activity against L. terrestris followed by the aqueous extracts of the leafy stems of A. conyzoides and A. alboviolaceum. Analysis of these results points out that P. guineense (40 <

Preliminary Phytochemical Com position of Plant Extracts
The chromatographic profile of the extracts from the 9 anthelmintic plants investigated revealed the presence of five chemical families, namely sterols / terpenes, coumarins, flavonoids, tannins and alkaloids (chromatograms 1-4).
Observation of the chromatograms 1 and 3 of the extracts of the plants macerated in chloroform, obtained with eluting mixture Hexane / Chloroform / Ethyl acetate, after revelation with the Lieberman-Büchard reagent, KOH at 5%, respectively showed the presence of several spots of different colors (blue green, yellow, brown), compounds of the triterpene / sterol type at 365 nm UV and blue, yellow and green molecular imprints characteristic of coumarins [7,15].

E.M = Mean values ± Standard error of means of three experiments
The extracts of the roots of R. mannii, C. podoleana, the seeds of G. kola, P. guineense, the leaves of A. buettneri, C. podoleana and the roots barks of M. lucida and the leafy stems of A. conyzoides, revealed the co-presence of sterols, terpenes and coumarins. Some authors also prouved the presence of terpenes, sterols, coumarins, flavonoids and tannins in the leaves of A. conyzoides, the seeds of P. guineense and the leaves of M. lucida noting the presence of alkaloids for A. conyzoides and their absence for M. lucida [27,28]. Also, the work of Munikishore et al., and Okunade, respectively, isolated two flavonoid-type molecules and determined the compounds present in the plant A. conyzoides [29,30].
The chromatographic profile 2 of the polar extracts (ethanol-water) after revelation, showed the presence of blue, green, yellow, orange spots, blue green and orange fluorescent spots, characteristics of flavonoids [7,15].These compounds are less abundant in the extracts of P. africana trunk bark and the roots of C. podoleana.  [31].The absence of alkaloids, terpens and sterols as well as the presence of tannins, flavonoids, coumarins in the leaves of P. africana was prouved by some authors [32].Streaks of alkaloids are observed only in the roots of R. mannii and the leaves of C. podoleana. Two alkaloids, vincajamine and reserpine, have been isolated from the leaves and roots of R. mannii, respectively [33,34].In addition, studies of Bosi et al. indicated the presence of alkaloids of the pyrrolizidine type in the whole plant of A. conyzoides [35]. Data from the literature have shown that the plant C. podoleana contains the purgative reserpine, the leaves and roots contain the alkaloids and terpenes-steroids [36].The phytochemical screening of the seeds the species G. kola showed the presence of alkaloids, showed the presence of tannins, flavonoids, steroids, flavonoids on the one hand the absence of alkaloids on the other [37,38].The absence of alkaloids in G. kola could be explained by the maturity stage of the harvested fruit, because studies of Morabandza et al. showed that the concentration of metabolites in the mesocarp of G. kola fruit increases with the stages of fruit [39]. Though, the absence of these secondary metabolites in some plants studied, could be justified by influences of certain environmental factors of the place of harvest such as climate, soil composition and season of sample collection [40].These compounds may be responsible for the vermicide effect of plants against earthworms Lumbricus terrestri. [41,42].

Total polyphenols
The total polyphenol contents were expressed in equivalent micrograms of gallic acid per gram of dry matter according to the calibration curve for gallic acid (y = 0.0152x + 0.0045; R² = 0.9997). As shown in Fig. 3A,

Antioxidant Activity by Spectrophotometry UV-Visible
The significant reduction of the DPPH radical by the plant extracts was shown in Figs. 4 and 5.
Thus, the extracts of A. alboviolaceum (92.35%) followed by P. africana (86.9%) and A. buettneri (80.5%) showed significant antioxidant activity at 0.5 mg / mL compared to the other six plants although it was moderately low compared to ascorbic acid (96.23%). In addition, the leafy stems of A. alboviolaceum exhibited similar efficacy (92.35%) to that of the standard compound ascorbic acid (Fig. 4). The CR 50 value is the concentration of extract reducing 50% of the DPPH radical, and values of CR 50 have been presented in Table 3

Correlation between the Contents (Total Phenols, Flavonoids, Tannins) and the CR 50
Correlations between the proportioned total compound (total polyphenols, flavonoids total, tannins condensed and hydrolyszables) and the concentration of reduction at 50% of radical DPPH have been established (Fig. 6). The coefficients were respectively R 2 = 0.046 and R 2 = 0.014 for total polyphenols and tannins hydrolyzables. The results proved, there is no correlation between the contents total polyphenols, tannins hydrolyzables with the concentration of reduction of DPPH radical for the majority of the species. In addition, this correlation remains still low between the total flavonoids, the tannins condensed with the concentrations of reduction which have coefficients about R 2 = 0. 0833 and R 2 = 0.0935. The presence of one of these families of compounds could in part confer antioxidant activity in plants, and the presence of all of these families in plants could amplify this antioxidant potential of plants. Bouyahya's study proved the implication of the content of phytocomponents, mainly flavonoids, in the antioxidant potential [43]. In addition, flavonoids and phenols are recognized for their antioxidant power, also, the antioxidant activity determined although low could derive from the presence and synergy of all these phytocompounds in the selected plants [44,45,46].

CONCLUSION
In order to contribute to the promotion of traditional medicine, precisely in the treatment of intestinal parasitosis, phytochemical screening, evaluation of antioxidant and anthelmintic activities of aqueous extracts from the organs of the 9 plants listed, have been carried out. Phytochemical screening made it possible to highlight 3 phytochemical families, namely alkaloids, phenolic compounds (coumarins, flavonoids, and tannins), steroids and terpenes. The contents of total polyphenols and condensed tannins are quite high compared to those of total flavonoids and hydrolysable tannins. The antiradical test by spectrophotometry with regard to DPPH radical, revealed the significant manifestation of the antioxidant activity of the 9 plants studied, and this, in comparison with vitamin C. With regard to the standar drug Mebendazole, the aqueous extract of seeds of P. guineense, A. conyzoides and A. alboviolaceum exhibited the best vermicidal activity against Lumbricus terrestris. would justify the use of said plants in the treatment against intestinal worms. The results of anthelmintic activity in vitro obtained cannot be the same to those obtained in vivo. Thus, the determination and identification of the chemical structures of these anthelmintic phytocompounds would be necessary to complete this work and be able to carry out in vivo tests in order to formulate anthelmintic plant products in our country.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.