- Research article
- Open Access
Multidrug resistant bacteria are sensitive to Euphorbia prostrata and six others Cameroonian medicinal plants extracts
BMC Research Notes volume 10, Article number: 321 (2017)
Multidrug resistant (MDR) bacteria are responsible for therapeutic failure and there is an urgent need for novels compounds efficient on them.
Eleven methanol extracts from seven Cameroonian medicinal plants were tested for their antibacterial activity using broth micro-dilution method against 36 MDR bacterial strains including Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa and Staphylococcus aureus.
Euphorbia prostrata extract was found active against all the 36 tested bacteria including Gram-negative phenotypes over-expressing efflux pumps such as P. aeruginosa PA124, E. aerogenes CM64 and E. coli AG102. E. prostrata had minimal inhibitory concentrations values between 128 and 256 µg/mL on 55.55% of the studied microorganisms. Other plants extract displayed selective antibacterial activity.
Results obtained in this study highlight the antibacterial potential of the tested plants and the possible use of E. prostrata to combat bacterial infections including MDR phenotypes.
Bacterial multidrug-resistance is the ability of bacteria to grow in the presence of antibiotics at concentrations that were previously inhibitory. Treating infections caused by multidrug-resistant (MDR) bacteria is a challenge more and more difficult to solve within hospital units . Although the resistance of bacteria to antibiotics is a natural adaptation phenomenon, the rapid emergence of MDR phenotypes is mainly due to misuse of antibiotics, which increases the selection pressure and favors the appearance MDR microorganisms . In hospitals, patients infected by these bacteria stay for long time, which impacts on the cost of treatment. Faced with this crisis, it is important to develop new antibacterial molecules effective vis-à-vis of MDR bacteria and medicinal plants offer a suitable alternative. According to WHO, 80% of world population uses medicinal plants for their health needs; antibacterial potential against the multidrug resistant phenotypes of many of them like Afromomum citratum, Afromomum melegueta, Imperata cylindricum, Cinnamomum zeylanicum, Dioscorea bulbifera, Dorstenia psilurus has already been demonstrated [3, 4]. In order to contribute to the discovery of active substances from medicinal plants, this study was designed to assess the antibacterial potential of different parts of Aloe buettneri A. Berger (Asphodelaceae), Alchornea floribunda Müll. Arg. (Euphorbiaceae), Crinum purpurascens Herb. (Amaryllidaceae), Euphorbia prostrata Ait. (Euphorbiaceae), Markhamia tomentosa K. Schum. (Bignoniaceae), Viscum album L. (Loranthaceae) and Rauwolfia macrophylla Ruiz & Pav. (Apocynaceae) against MDR Gram-positive and Gram-negative phenotypes.
Plant material and sample preparation
Different part of the investigated plants including leaves, stem, stem bark, bark or whole plant (Table 1) were harvested in different regions of Cameroon. The plants were then identified at Cameroon National Herbarium where the voucher specimens are available (Table 1). The dry powders (200 g) of each part of plants were soaked in methanol for 48 h; the filtrates obtained after filtration paper through Whatman No. 1 were concentrated under reduced pressure and the obtained extracts were kept at 4 °C for further biological tests.
The presence of compounds belonging to different classes of secondary metabolites was determined according to described methods .
Chemicals for antimicrobial assay
The reference antibiotic (RA) used against bacteria were chloramphenicol and ciprofloxacin (Sigma-Aldrich, St. Quentin Fallavier, France) meanwhile the bacterial growth indicator was p-iodonitrotetrazolium chloride ≥ 97% (INT, Sigma-Aldrich).
Microbial strains and culture media
Microorganisms used in this study included 36 multidrug-resistant strains of Gram-negative belonging to Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa species and Gram-positive bacteria belonging to and Staphylococcus aureus species (Table 3). Their bacterial features were previous reported . Mueller–Hinton Agar (Sigma) was used to activate the microorganisms whilst Mueller Hinton broth (MHB; Sigma) was used for antibacterial assays.
INT colorimetric assay for MIC and MBC determinations
The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of different plant extracts were determined by a by the rapid INT colorimetric assay according to described methods . Chloramphenicol and ciprofloxacin were the reference drugs for Gram-negative and Gram-positive respectively. Each plant extract was dissolved in a 5% DMSO solution; an aliquot of 100 μL was added to the wells of a microplate containing 100 μL of MHB; then serial dilution was performed. A bacterial suspension corresponding to 0.5 McFarland scale was prepared and diluted 100 times in the sterile MHB. Afterwards, 100 μL of the bacterial suspension was added to all wells and the plate was incubated at 37 °C for 18 h. Bacterial growth was detected by adding 40 μL of INT (0.2 mg/mL) and the appearance of pink color indicated bacterial growth; the lowest concentration of the extract where no color change was observed was recorded as MIC.
For the determination of MBCs, we used new 96-well plates containing 150 μL of MHB in which we added an aliquot of 50 μL from the wells corresponding to MIC as well as upper concentrations. Those microplates were incubated at 37 °C for 48 h and revelation was done as mentioned above and the lowest concentration indicating the absence of bacterial growth was considered as MBC. Each of the experiments was carried out in triplicate.
The classes of secondary metabolites present in extracts of different parts of plants were detected and results are summarized in Table 2. Alkaloids, triterpenes, sterols, flavonoids, polyphenols and saponins were screened in all plant extracts. Other classes of compounds were selectively distributed.
The results of antibacterial tests are summarized in Table 3. It appears that the plant extract from E. prostrata was active against all tested bacterial strains (36/36) with MIC between 128 and 256 µg/mL on 55.55% (20/36) of the studied microorganisms. The methanol extracts of leaves and stems of V. album as well as other plant extracts displayed selective activities on MDR Gram-negative as well as on methicillin-resistant S. aureus (MRSA) strains. Leaves extract of R. macrophylla were active against 83.33% (30/36) of the tested bacteria while only 3/36 (8.33%) of the studied bacteria were sensitive to the bark extract. Leaves extract of M. tomentosa was active on 75% (27/36) of bacterial strains while the extracts of bark were active only on 30.55% (11/36). The methanol extract from the leaves of C. purpurascens showed activity against all MRSA strains and 22/29 Gram-negative bacteria tested; except bacteria belonging to P. aeruginosa species, all other bacterial species studied herein were susceptible to C. purpurascens leaves extract. E. coli, P. aeruginosa, E. aerogenes and P. stuartii strains were mostly resistant to the action of the extract of leaves of A. floribunda. The extract of A. buettneri leaves had weak activity, its inhibitory effects being observed against 3/36 (8.33%) bacterial strains.
The emergence of diseases due to MDR bacterial strains is a phenomenon of growing concern worldwide, being qualified by WHO as a “slow-moving tsunami” . Medicinal plants are a promising alternative for the discovery of new anti-infective agents capable to fight against MDR phenotypes; hence, several phytochemicals have been tested against multi-resistant phenotypes [3, 8].
According to Simões et al. , a plant extract or phytochemicals can be considered as antimicrobials if the MIC obtain during in vitro tests is in the range 100–1000 μg/mL. The antibacterial activity obtained with E. prostrata extract is important as the extract was active on all MDR bacterial strains tested. MIC value of 256 μg/mL was recorded on strains over-expressing efflux pumps AcrAB-TolC (E. coli AG102 and E. aerogenes CM64) and MexAB-OprM (P. aeruginosa PA124) as well as against all MRSA strains. This remarkable activity on Gram-negative as well as Gram-positive bacteria may be due to the presence of phytochemicals that exhibit antimicrobial potential such as quercetin and kaempferol; in fact, their antibacterial properties’ vis-à-vis MRSA and multi-resistant Propionibacterium acnes were reported [10, 11]. Moreover, Tala et al.  have highlighted the in vivo anti-salmonella potential of this plant. The extracts from leaves of R. macrophylla and M. tomentosa exhibited better antibacterial activity than those obtained from their barks; this selective activity may be due the qualitative and/or quantitative difference in phytochemical contents of parts of the plants. The antibacterial activity obtained with R. macrophylla extracts may be due to the alkaloids present in this plant; in effect, Erasto et al.  have demonstrated the anti-mycobacterial extracts properties of alkaloids extracts from R. macrophylla. Viscum album extracts from various parts of plant were active on all bacterial species used in this study. This reflects the broad spectrum of activity phytochemical compounds available in these extracts like the triterpenes, flavonoids, alkaloids . Pseudomonas aeruginosa strains (PA01 and PA124) were not susceptible to the leaves extract of C. purpurascens although the fact that this was active on 29 strains out of the 36 tested (including all the MRSA strains); Voukeng et al.  have highlighted the involvement of efflux pumps-type RND as the major phenomenon of resistance of Gram-negative bacteria herein studied vis-à-vis of some plant extracts. The susceptibility of the studied MDR bacteria vis-à-vis of A. floribunda extracts varied depending on the part of the plant used; these results corroborate those obtained by Siwe et al. . Who got MIC value of 130 μg/mL and 2000 μg/mL with the methanol extracts of the leaves and bark respectively against S. aureus ATCC 25923. Okoye and Ebi  showed that fractions from leaves extract of A. floribunda contained mostly terpenoids, and possessed antibacterial activity against P. aeruginosa, Salmonella keitambii and Bacillus subtilis. Likewise, some flavonoids isolated from this plant such as taxifolin had MIC value of 225 μg/mL on the S. sobrinus .
This study highlights the efficacy of some Cameroonian medicinal plants against MDR phenotypes and the results obtained can serve as preliminary test for further experiments to isolate phytochemicals constituents with wide range antibacterial activity.
American type culture collection
- E. prostrata :
National Herbarium of Cameroon
p-iodonitrotetrazolium chloride ≥ 97% (INT, Sigma-Aldrich)
minimal bactericidal concentration
minimal inhibitory concentration
Cornejo-Juárez P, Vilar-Compte D, Pérez-Jiménez C, Ñamendys-Silva SA, Sandoval-Hernández S, Volkow-Fernández P. The impact of hospital-acquired infections with multidrug-resistant bacteria in an oncology intensive care unit. Int J Infect Dis. 2015;31:31–4.
Aly NY, Al-Mousa HH, Al Asar el SM. Nosocomial infections in a medical–surgical intensive care unit. Med Princ Pract. 2008;17:373–7.
Voukeng KI, Kuete V, Dzoyem JP, Fankam AG, Noumedem KJA, Kuiate JR, Pages JM. Antibacterial and antibiotic-potentiation activities of the methanol extract of some Cameroonian spices against Gram-negative multi-drug resistant phenotypes. BMC Res Notes. 2012;5:299.
Kuete V, Teponno RB, Mbaveng AT, Tapondjou LA, Meyer JJM, Barboni L, Lall N. Antibacterial activities of the extracts, fractions and compounds from Dioscorea bulbifera L. BMC Complement Altern Med. 2012;12:228.
Harborne JB. Phytochemical methods. New York: Chapman and Hall; 1973.
Voukeng KI, Beng PV, Kuete V. Antibacterial activity of six medicinal Cameroonian plants against Gram-positive and Gram-negative multidrug resistant phenotypes. BMC Complement Altern Med. 2016;16:388.
Reinl J. UN declaration on antimicrobial resistance lacks targets. Lancet. 2016;388(10052):1365.
Kuete V. Medicinal plant research in Africa: pharmacology and chemistry. Oxford: Elsevier; 2013.
Simões M, Bennett RN, Rosa EA. Understanding antimicrobial activities of phytochemicals against multidrug resistant bacteria and biofilms. Nat Prod Rep. 2009;26(6):746–57.
Lim YH, Kim IH, Seo JJ. In vitro activity of kaempferol isolated from the Impatiens balsamina alone and in combination with erythromycin or clindamycin against Propionibacterium acnes. J Microbiol. 2007;45(5):473–7.
Hirai I, Okuno M, Katsuma R, Arita N, Tachibana M, Yamamoto Y. Characterisation of anti-Staphylococcus aureus activity of quercetin. Int J Food Sci Technol. 2010;45(6):1250–4.
Tala DS, Gatsing D, Fodouop CPS, Fokunang C, Kengni F, Namekong DM. In vivo anti-salmonella activity of aqueous extract of Euphorbia prostrata Aiton (Euphorbiaceae) and its toxicological evaluation. Asian Pac J Trop Biomed. 2015;5(4):310–8.
Erasto P, Mbwambo HZ, Nondo OSR, Lall N, Lubschagne A. Antimycobacterial, antioxidant activity and toxicity of extracts from the roots of Rauvolfia vomitoria and R. caffra. Spatula DD. 2011;1(2):73–80.
Singh BN, Saha C, Galun D, Upreti DK, Bayry J, Kaveri SV. European Viscum album: a potent phytotherapeutic agent with multifarious phytochemicals, pharmacological properties and clinical evidence. RSC Adv. 2016;6:23837–57.
Siwe NX, Krause RWM, van Vuuren SF, Tantoh ND, Olivier DK. Antibacterial activity of the roots, stems and leaves of Alchornea floribunda. J Ethnopharmacol. 2014;151:1023–7.
Okoye FBC, Ebi GC. Preliminary anti-microbial and phytochemical investigation of the extracts and column fractions of Alchornea floribunda leaves. J Pharm Allied Sci. 2007;4:1.
Kuspradini H, Mitsunaga T, Ohashi H. Antimicrobial activity against Streptococcus sobrinus and glucosyltransferase inhibitory activity of taxifolin and some flavanonol rhamnosides from kempas (Koompassia malaccensis) extracts. J Wood Sci. 2009;55:308–13.
Telefo PB, Moundipa PF, Tchouanguep FM. Inductive effect of the leaf mixture extract of Aloe buettneri, Justicia insularis, Dicliptera verticillata and Hibiscus macranthus on in vitro production of estradiol. J Ethnopharmacol. 2004;91:225–30.
Tan PV, Enow-Orock EG, Dimo T, Nyasse B, Kimbu FS. Evaluation of the anti-ulcer and toxicity profile of Aloe buettneri in laboratory animals. Afr J Tradit CAM. 2006;3(2):8–20.
Metowogo K, Agbonon A, Eklu-Gadegbeku K, Aklikokou AK, Gbeassor M. Anti-ulcer and anti-inflammatory effects of hydroalcohol extract of Aloe buettneri A. Berger (Lilliaceae). Trop J Pharm Res. 2008;7(1):907–12.
Ajaghaku DL, Obasi O, Umeokoli BO, Ogbuatu P, Nworu CS, Ilodigwe EE, Okoye FBC. In vitro and in vivo antioxidant potentials of Alchornea floribunda leaf extract, fractions and isolated bioactive compounds. Avicenna J Phytomed. 2017;7:80.
Mesia GK, Tona GL, Nanga TH, Cimanga RK, Apers S, Cos P, Maes L, Pieters L, Vlietinck AJ. Antiprotozoal and cytotoxic screening of 45 plant extracts from Democratic Republic of Congo. J Ethnopharmacol. 2008;115(3):409–15.
Okoye CBF, Osadebe OP, Proksch P, Edrada-Ebel RA, Nworu SC, Esimone OC. Anti-inflammatory and membrane-stabilizing stigmastane steroids from Alchornea floribunda leaves. Planta Med. 2010;76(2):172–7.
Fomogne-Fodjo MCY, Van Vuuren S, Ndinteh DT, Krause RWM, Olivier DK. Antibacterial activities of plants from Central Africa used traditionally by the Bakola pygmies for treating respiratory and tuberculosis-related symptoms. J Ethnopharmacol. 2014;155(1):123–31.
Nkanwen ERS, Gatsing D, Ngamga D, Fodouop SPC, Tane P. V. Afr Health Sci. 2009;9(4):264–9.
Yoshida T, Namba O, Chen L, Liu Y, Okuda T. Ellagitannin monomers and oligomers from Euphorbia prostrata Ait. and oligomers from Loropetalum chinense OLIV. Chem Pharm Bull. 1990;38(12):3296–302.
Mosango DM. Euphorbia prostrata aiton. 2008. https://www.prota4u.org/protav8.asp?h=M1,M10,M11,M12,M14,M15,M16,M18,M19,M22,M23,M25,M26,M27,M34,M36,M4,M6,M7,M9&t=Euphorbia,prostrata&p=Euphorbia+prostrata#Protologue. Accessed 18 Sep 2016.
Alanis-Garza BA, González-González GM, Salazar-Aranda R, Waksman de Torres N, Rivas-Galindo VM. Screening of antifungal activity of plants from the northeast of Mexico. J Ethnopharmacol. 2007;114(3):468–71.
Bakhshi DG, Langade GD, Desai SV. Prospective, open label study of Euphorbia prostrata extract 100 mg in the treatment of bleeding haemorrhoids. Bombay Hosp J. 2008;50(4):577–83.
Ibrahim B, Sowemimo A, Spies L, Koekomoer T, van de Venter M, Odukoya AO. Antiproliferative and apoptosis inducing activity of Markhamia tomentosa leaf extract on HeLa cells. J Ethnopharmacol. 2013;149:745–9.
Tantangmo F, Lenta BN, Boyom FF, Ngouela S, Kaiser M, Tsamo E, Weniger B, Rosenthal PJ, Vonthron-Senecheau C. Antiprotozoal activities of some constituents of Markhamia tomentosa (Bignoniaceae). Ann Trop Med Parasitol. 2010;104(5):391–8.
Ali S, El-Ahmady S, Ayoub N, Singab NA. Phytochemicals of Markhamia Species (Bignoniaceae) and their therapeutic value: a review. Eur J Med Plants. 2015;6(3):124–42.
Aladesanmi A, Iwalewa E, Adebajo A, Akinkunmi E, Taiwo B, Olorunmola F, Lamikanra A. Antimicrobial and antioxidant activities of some Nigerian medicinal plants. Afr J Trad Complement Altern Med. 2007;4(2):173–84.
Deliorman D, Calis I, Ergun F. Studies on the vascular effects of the fractions and phenolic compounds isolated from Viscum album ssp. album. J Ethnopharmacol. 2000;72:323–9.
Ofem OE, Eno AE, Imoru J, Nkanu E, Unoh F, Ibu JO. Effect of crude aqueous leaf extract of Viscum album (mistletoe) in hypertensive rats. Indian J Pharmacol. 2007;39(1):15–9.
Nazaruk J, Orlikowski P. Phytochemical profile and therapeutic potential of Viscum album L. Nat Prod Res. 2016;30(4):373–85.
Yusuf L, Oladunmoye MK, Ogundare AO, Akinyosoye FA, Daudu OAY, Hassan GA. Antimicrobial and antioxidant properties of mistletoe (Viscum album) growing on cola (Cola nitida) tree in Akure North, Nigeria. J Microbiol Res Rev. 2013;1(3):35–41.
Cebović T, Spasić S, Popović M. Cytotoxic effects of the Viscum album L. extract on Ehrlich tumour cells in vivo. Phytother Res. 2008;22(8):1097–103.
Mollel NP. Rauvolfia caffra Sond. 2007. http://www.prota4u.org/protav8.asp?p=Rauvolfia+caffra. Accessed 22 Sep 2016.
Amer MMA, Court WE. Root wood alkaloids of Rauwolfia macrophylla. Planta Med. 1981;43(1):94–5.
Nasser AMAG, Court WE. Stem bark alkaloids of Rauvolfia caffra. J Ethnopharmacol. 1984;11(1):99–117.
Nasser AMAG, Court WE. Leaf alkaloids of Rauwolfia caffra. Phytochemistry. 1983;22(10):2297–300.
Nasser AMAG, Court WE. Alkaloids of Rauwolfia caffra seeds. Planta Med. 1983;47(4):242–3.
Milugo KT, Omosa KL, Ochanda OJ, Owuor OB, Wamunyokoli AF, Oyugi OJ, Ochieng WJ. Antagonistic effect of alkaloids and saponins on bioactivity in the quinine tree (Rauvolfia caffra sond.): further evidence to support biotechnology in traditional medicinal plants. BMC Complement Altern Med. 2013;13:285.
Njau EFA, Alcorn J, Ndakidemi P, Chirino-Trejo M, Buza J. Antimicrobial and antioxidant activity of crude extracts of Rauvolfia caffra var. caffra (Apocynaceae) from Tanzania. Int J Biol. 2014;6(4):156–67.
IKV carried out the study; IKV and VK designed the experiments and wrote the manuscript; VK and VPB supervised the work; IKV and VK provided culture media, bacterial strains and other facilities. All authors read and approved the final manuscript.
Authors are grateful to the Cameroon National Herbarium (Yaounde) for plants identification, Mr. Nganou Blaise and Tala Donald Sedric for their technical support.
The authors declare that they have no competing interests.
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The datasets supporting the conclusions of this article are presented in this main paper. Plant materials used in this study have been identified at the Cameroon National Herbarium where voucher specimens are deposited.
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Voukeng, I.K., Beng, V.P. & Kuete, V. Multidrug resistant bacteria are sensitive to Euphorbia prostrata and six others Cameroonian medicinal plants extracts. BMC Res Notes 10, 321 (2017). https://doi.org/10.1186/s13104-017-2665-y
- Euphorbia prostrata
- Medicinal plant
- Multidrug resistance