Phytochemical Screening And Antioxidant Activity Of .

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GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-118Available online at GSC Online Press DirectoryGSC Biological and Pharmaceutical Sciencese-ISSN: 2581-3250, CODEN (USA): GBPSC2Journal homepage: https://www.gsconlinepress.com/journals/gscbps(R E S E A R C H A R T I C L E )Phytochemical screening and antioxidant activity of methanolic extracts of 53antimalarial plants from Bagira in Eastern DR CongoValentin Bashige Chiribagula 1, 2, 3, *, Amuri Salvius Bakari 1, Philippe Okusa Ndjolo 2, Byanga Joh Kahumba 1and Jean-Baptiste Lumbu Simbi 31 Laboratoirede Pharmacognosie - Faculté des Sciences Pharmaceutiques - Université de Lubumbashi- 27, av Kato,Commune Kampemba, Lubumbashi/ RD Congo.2 Service de Chimie thérapeutique- Faculté des Sciences Pharmaceutiques - Université de Lubumbashi- 27, av Kato,Commune Kampemba, Lubumbashi/ RD Congo.3 Laboratoire de chimie Organique - Faculté des Sciences Université de Lubumbashi- 2 av de la Maternité, Commune deLubumbashi /RD Congo.Publication history: Received on 02 August 2020; revised on 10 August 2020; accepted on 11 August 2020Article DOI: ractA previous study inventoried 53 plants used in traditional medicine in Bagira in Eastern Democratic Republic of Congo(DRC) in the management of malaria. During malaria disease, oxidative stress is responsible for the worsening of thepatient's condition. This study aims to identify phytochemical groups and to evaluate antioxidant activity of 53 plantsused in traditional medicine in Bagira to treat malaria. The phytochemical screening was carried out by conventionalreactions in solution and antioxidant activity used in vitro method with 1,1-diphenyl-2- picrylhydrazyl radical (DPPH).Chemical screening has identified secondary metabolites with both antimalarial and antioxidant potential such ascoumarins, steroids, saponins, tannins and terpenoids in more than 70% of plants. Antioxidant screening revealed forthe first-time antioxidant activity of 18 plants, among which Dalbergia katangensis, Dialium angolense and Solaneciocydoniifolius with IC50 1.6 µg / mL having the highest activities. This study shows that among plants used asantimalarial in Bagira several possess antioxidant power and contain many of groups presumed to be both antioxidantand antimalarial. This suggests that further studies continue to isolate compounds responsible for the proven activity.Keywords: Anti-free radical activity; DPPH; Bagira; Antimalarial; Phytochemistry Corresponding author Valentin Bashige ChiribagulaCopyright 2020 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-1181. IntroductionOxidative stress results from a profound imbalance between oxidative systems and the body's antioxidant capacities infavor of the former [1]. Unbalanced, it leads to irreversible cell damage [2] responsible for aging and many conditionssuch as obesity [3], type 2 diabetes [4], atherosclerosis [5], cancer [6] or virus diseases [7] requiring the use ofantioxidants. Several synthetic antioxidants used in the past have been abandoned because of their increased risk oftoxicity in favor of natural antioxidants [8], which motivates the screening of plants with antioxidant potential.Studies have shown that during a malarial disease oxidative stress occurs which can progress to cerebral malaria oranemia [9,10]. Thus, studies have been carried out with a view to seeking both plants with antioxidant and antimalarialpotential. This is the case with the work of Saliq et al [11] or Sulistyaningsih et al [12] like so many others [13–16].Another advantage of this approach is that it allows the discovery of new antimalarial molecules with new mechanismsof action likely to overcome the resistance problems facing current antimalarials [17,18]. This approach to screeningplants with dual potential has seen some studies lead to the isolation of natural molecules that are both antimalarialand antioxidant. This is the case of mammea A / AA cyclo D, a coumarin isolated from the stem bark of Mesua borneensis(P. F. Stevens), a Calophyllaceae [19] or that of Lonchocarpol A, a flavonoid isolated from the stem bark of Erythrinacrista-galli L., a Fabaceae [20]. Furthermore, the isolation of a bioactive molecule is conventionally preceded by thesearch for secondary metabolites with the desired potential. Beyond this interest, this screening also makes it possibleto provide new knowledge on the chemical composition of the plant concerned on the major phytochemical groups ofsecondary metabolites of plants. In the case of malaria, bibliographical reviews [21–23] have highlighted alkaloids,flavonoids, coumarins, quinones, steroids, terpenoids as phytochemical groups with antimalarial potential. Amongthese phytochemical groups, flavonoids, coumarins, and terpenoids are particularly reported as groups with antioxidantpotential [24,25].This study focused on 53 plants used in traditional medicine in Bagira, in the treatment of malaria, to assess theirantioxidant potential in vitro and to search for phytochemical groups with antiplasmodial potential. These plants comefrom an ethnobotanical study carried out on antimalarial plants from Bagira, such as the city of Bukavu in the easternDRC.2. Material and methods2.1. Plant materialThe plant material consisted of the leaves, stems, roots, flowers, fruits, and aerial parts of 53 plant species taken from adatabase of a survey we conducted in Bagira in 2013-2014. These plants have been collected in Bukavu in the companyof traditional healers and the herbaria created for this occasion were deposited at the IRS Lwiro herbarium where theidentity of the plants was determined (Table 2). After drying at room temperature, the plant material was ground usinga stainless-steel electric mill (Plymix PX-MFC 90 D, Belgium) and then kept cool before handling. The choice of organsto screen was related to availability at harvest. Thus, for the herbs, we screened the aerial parts consisting mainly ofleaves and stems without discrimination.2.2. Obtaining extractsThe extracts were obtained by maceration of 350 g of powder in 1.5 L of methanol (Sigma Aldrich, USA) for 72 hours atroom temperature then filtered through paper (Whatman, USA) and concentrated on a rotary evaporator (Büchi R -210,Switzerland) at a pressure of 180 mbar and a temperature of 40 C.2.3. Substrate and positive controlDPPH (Sigma Aldrich, United Kingdom) was used as a substrate for the evaluation of antioxidant activity. It wasprepared at 0.002% (w / v) in methanol. L-ascorbic acid (Sigma Aldrich, China) used as a reference antioxidantsubstance made it possible to prepare a standard curve with 5 successive dilutions of order 2 carried out from a solutionof ascorbic acid at 40 µg/mL ( y 0.0298X 0.0071; r2 0.9997).2.4. Identification of secondary metabolitesThe phytochemical screening was carried out using conventional reactions in solution in tubes, based on staining,precipitation, or the formation of foams. It consisted in looking for alkaloids, anthocyanins, coumarins, flavonoids,quinones, saponins, steroids, tannins and terpenoids for their antiplasmodial or antioxidant potential and cyanogenicheterosides for their toxic potential, following the protocols previously described [26–28].100

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-1182.4.1. AlkaloidsThe detection of alkaloids consisted in precipitating them using six precipitation reagents. Briefly, 1 g of powder of dryplant material was macerated in 10 mL of methanol at room temperature for 24 hours and then in an oven at 50 C for4 hours. The solution obtained was filtered then the marc washed three times with portions of hot methanol. The filtratewas evaporated to dryness in an oven at 50 C and the residue was collected twice with 2 mL of hot 1% hydrochloricacid solution (Sigma-Aldrich, USA). The acid solution obtained was basified with 1 mL of concentrated ammonia (SigmaAldrich, UK), placed in a separating funnel (VWR, Belgium) and then mixed with 5 mL of chloroform (Sigma-Aldrich,USA). After stirring, the two phases were separated, and the operation was repeated three times. The organic phase wasevaporated to dryness in the open air, the residue obtained was taken up in 0.5 mL of chloroform and the solution,transferred to a test tube, was mixed with 0.5 mL of 1% HCl thus forming two phases. The aqueous phase, which isabove, was removed using a Pasteur pipette. Six drops were placed on a microscope slide. Each of these drops wastreated with one drop of one of six precipitation reagents namely Dragendorff, Mayer, Hager, Wagner, Bertrand, andSonnenschein reagent. The presence of alkaloids was only considered certain if each of the six reagents gave aprecipitate.2.4.2. CoumarinsCoumarins were identified by the alkaline reaction. Briefly 0.5 g of the moistened various extracts was taken in a testtube. The mouth of the tube was covered with filter paper treated with 1 N NaOH solution. Test tube was placed for 5minutes in boiling water and then the filter paper was removed and examined under the UV light for yellow fluorescenceindicated the presence of coumarins.2.4.3. Flavonoids and anthocyaninsThe flavonoids have been demonstrated by the Shinoda test. Briefly, 5 g of plant material placed in an Erlenmeyer flaskwas infused in 50 mL of distilled water for 30 minutes. 5 mL of filtrate were then treated successively with 5 mL ofconcentrated HCl, 5 drops of isoamyl alcohol and 1 mg of magnesium shavings. The red-orange (flavone), red or redviolet (flavonones), cherry red (flavonol) coloration appeared in the supernatant layer if the solution contained theflavonoids. Likewise, the reaction carried out for two minutes in a water bath in the absence of magnesium chips allowedthe characterization of anthocyanins with the appearance of a red color.2.4.4. Cyanogenic heterosidesCyanogenic heterosides were identified by the reaction with picric acid. Briefly, 5 g of vegetable powder was placed inan Erlenmeyer flask with 10 mL of distilled water. The container was closed with a stopper to which was attached astrip of picrosodium paper lightly moistened with water and the contents were slightly heated (to 60 C). The yellowpicrosodium paper turned orange or red if the plant extract had released hydrocyanic acid.2.4.5. QuinonesQuinones were identified by the Borntrager test. Briefly, 5g of powdered plant material was macerated for 24 hours in50mL of petroleum ether. After filtration, 10 mL of ethereal filtrate was treated with 5 mL of 10% NH3. The appearanceof a purplish red color in the aqueous phase indicated the presence of free quinones and that of yellow or orange colors,the bound quinones.2.4.6. SaponinsSaponins were identified by the foaming reaction. Briefly, 10 g of coarsely ground plant material was treated with 100mL of distilled water to make a decoction for 30 minutes and the mixture was filtered through filter paper after cooling.15 mL of the decocts were then introduced into a test tube 16 mm in diameter and 160 mm in height. The contents ofthe tube were shaken tightly for one minute and then allowed to stand for 10 minutes. The appearance of a persistentfoam greater than 10 mm in height indicates the presence of saponins.2.4.7. Steroids and terpenoidsBoth steroids and terpenoids have been defied by the reaction with sulfuric acid. Briefly, 5 g of plant material wasmacerated for 24 hours in 100mL of petroleum ether. After filtration, the solvent was evaporated to dryness. In theresidue obtained, were added successively and with stirring, 2 mL of chloroform and three drops of concentratedsulfuric acid. The appearance of purple or green colorings indicated the presence of steroids. The identification ofterpenoids followed the same pattern as that of steroids. In addition to the reagents used for steroid testing, a few drops101

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-118of Hirschson reagent (concentrated acetic anhydride) were added to 4 mL of the acidified solution. Yellow stainingturning red indicated the presence of terpenoids.2.4.8. TanninsThe tannins were identified according to the protocol below: 5 g of plant material were infused in 50 mL of watercontained in an Erlenmeyer flask for 30 minutes. 5 mL of the infused was taken and mixed with 1 mL of 1% ferricchloride. The test was considered positive when either a precipitate appeared or a blue-green, dark blue or green color.15 mL of Stiasny reagent (10 ml 40% formalin and 5 mL concentrated HCl) was mixed with 30 mL of the infused andthe mixture was brought to a water bath at 90 C. The appearance of a precipitate indicated the presence of catecheticaltannins. The solution was then filtered, and the filtrate was saturated with sodium acetate before adding a few drops offerric chloride thereto. The formation of a precipitate in this case revealed the presence of gallic tannins.2.5. Antioxidant activity test with DPPHAntioxidant activity was assessed using the DPPH assay [29]. Briefly, 50 µL of extract or positive control prepared atdifferent dilutions of order 2 in methanol from a 100 µg / mL solution were interacted with 1950 µL of 0.002% DPPHin test tubes. (Nunc WVR, Germany). After mixing and incubating in the dark for 30 minutes, the absorbance of thesolution was read at 492 nm (Thermo Fisher Scientific Inc. spectrophotometer, Waltham, USA). The tests were carriedout in triplicate and the percentage of antioxidant activity was calculated by the formula:%AAO (Ab Ae)x100Ab(equation 1)with Ab absorbance measured in the presence of the negative control, Ae absorbance measured in the presence ofthe extract and % AAO Percent inhibition and expresses antioxidant activity. This percentage of activity made itpossible to generate the IC50 or concentration at which the extract has 50%, to categorize the extracts.2.6. Statistical analysis of dataGraphPad Prisme version 6 software (GraphPad Software, La Jolla, USA) was used to perform statistical analysis of thedata and generate the IC50s. The analysis of the variables was carried out by one-way ANOVA with the significance levelset at 95%.3. Results and discussion3.1. Phytochemical screeningThe results of the chemical screening show that each of the 53 plants contains at least 5 phytochemical groups out ofthe 10 sought. No plant contains cyanogenic heterosides and 3 species, Carica papaya, Entada abyssinica and Flueggeavirosa, all contain the 9 groups with therapeutic potential. Each organ contains at least 4 phytochemical groups and theleaves of Azadirachta indica as well as the fruits of Lantana camara with 8 groups each, contain the greatest number ofthe desired phytochemical groups (Table 1).Table 1 shows that antimalarial molecules have already been isolated for 12 plants. These are the species Artemisiaannua (terpenoids), Azadirachta indica (terpenoids), Bidens pilosa (flavonoids), Cajanus cajan (flavonoids), Cymbopogoncitratus (terpenoids), Erythrina abyssinica (flavonoids), Euphorbia hirta (flavonoids), Lantana camara (terpenoids),Ochna schweinfurthiana (flavonoids), Phyllanthus niruri (steroids), Physalis angulata (steroids) and Tithonia diversifolia(terpenoids). It also shows that 11 plants were so far phytochemically unrecognized. These are, Aframomum laurentii,Clematis villosa, Crassocephalum montuosum, Crassocephalum picridifolium, Dalbergia katangensis, Dialium angolense,Isoberlinia angolensis, Isoberlinia tomentosa, Julbernardia paniculata, Rothmannia engleriana, and Solaneciocydoniifolius.102

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-118Table 1 Phytochemical screening of 53 plants used as antimalarial drugs in Bagira (DRC) -a - a a a b a--a a- a a a -FETRFrFETERFrFlrFTRFrFlrFTRFlr-a a a a a - b a a a a a a a a a ( ) a -3Ageratum conyzoïdes L.(Asteraceae)4Artemisia annua(Asteraceae)5Azadirachta indica A.Juss ascariensis(Desv.) J.H. Kirkbr.(Fabaceae)Cajanuscajan(L.)Millsp. (Fabaceae)8L.L.9CaricaL(Caricaceae)papaya10Cassia occidentalis L.(Fabaceae)11Catharanthus roseus (L.)G Don. (Apocynaceae) a ( ) a a a a a ( ) a a a a --[39,40]Previouschemicalscreening-Hcn a b b b-a-a Steroids a ( ) a a [30] a a a a a -SaponinsTerpenoids a-a-a a TanninsAframomum laurentii(De Wild & T, Durand)K.Schum(Zingiberaceae) a a a a a onoidsAcacia polyacantha DeWild [31][32,33][34,35][36,37][38][41][42][43]103

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), opodiaceae)Chenopodiumopulifolium Schrad, d) Bern. MoensEx Trimen (Rubiaceae)Clematis villosa DC(Ranunculaceae)Crassocephalummontuosum (S. olium (DC) SMore (Asteraceae)Cymbopogoncitratus(DC) Stapf. (Poaceae)19Dalbergia katangensisLechenaud (Fabaceae)20Dialiumangolense(Welw EX Beth) Harms(Fabaceae)21Dialopsis africana Radck(Sapindaceae)22Ekebergia benguellensisWelwEXCDC(Meliaceae)23Eleusine indica (L)Gaertn (Poaceae)Entada abyssinica Steud.ex A. Rich. (Fabaceae)2425ErythrinaabyssinicaLam. Ex DC a virosa flora(Forssk) Roem, & Schult(Acanthaceae)L.FR a-- a a a - a -- a -[44]PA- - a a-[45]FETERFlrPA a a a - a a a - a a a - -a-a- -[46]PA- -- - -PA-- - - -FRFETFETERFETERFETERPA-a a ( ) - a a - a a -a a a b a -[47,48]FETRFrFETRFPAFRFrPA a a a a a a a b a a b a -- a a a- -a a a a -a-a a - a --[30] -a a a a[49][50,51][52,53][54,55][56,57][58][59,60]104

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-118293031Isoberlinia angolensis(Welw. Ex Benth.) Hoyle& Brenan (Fabaceae)Isoberlinia tomentosa(Harms) Craib & lbernardia iaceae)Mangifera indica L.(Anacardiaceae)35L36Moringa oleifera Lam.(Moringaceae)37Ochna schweinfurthianaF Hoffm (Ochnaceae)38Ocimum gratissimum L.(Lamiaceae)39Phyllanthusmuellerianus (Kuntze)Exell (Phyllanthaceae)40Phyllanthus niruri(Phyllanthaceae)L.41Physalis angulata(Solanaceae)L.42Piliostigma thonningii(Schum.) Milne-Redh.(Fabaceae)PA - - - --PA- -- - -FTRFrFETERFTRFrFlrFR a a -a - - a a a a a a a-a -a -a - -a a a a a--a -a b --[61]FETERFrFTRFrFERETFRFlrFETRFrFTRFRFrFTR-a a a -a-a-a a a-a- - ( ) -a a a a b a a a a -a -a a-a a a a a a a -a a b a a a a a -a a -a-b -a- a a a a a a a 105

Bashige et al. / GSC Biological and Pharmaceutical Sciences, 2020, 12(02), 099-11843Psidium icaceae)45Rothmannia engleriana(K.Shum)Keay(Rhubiaceae)46Senecio cineraria (DC)(Asteraceae)Solanecio cydoniifolius(O Hoffm.) C. Jeffrey(Asteraceae)Spilanthes mauritiana(A. Rich. Ex Pers.) DC.(Asteraceae)SyzygiumcordatumHochst. in C. raceae)52Trema orientalis (L.)Blume (Ulmaceae)53VernoniaamygdalinaDelille (Asteraceae)FrFETERFrFERETFERETPA-a a

potential. This is the case with the work of Saliq et al [11] or Sulistyaningsih et al [12] like so many others [13–16]. Another advantage of this approach is that it allows the discovery of new antimalarial molecul

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