Journal of Ethnopharmacology 169 (2015) 275–279
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Antibacterial activities of the methanol extracts, fractions and compounds from Fagara tessmannii Simplice B. Tankeo a, Francois Damen b, Maurice D. Awouafack b, James Mpetga b, Pierre Tane b, Jacobus N. Eloff c, Victor Kuete a,n a
Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, Cameroon c Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 February 2015 Received in revised form 20 April 2015 Accepted 22 April 2015 Available online 30 April 2015
Ethnopharmacological relevance: Fagara tessmannii is a shrub of the African rainforests used to treat bacterial infections, cancers, swellings and inflammation. In the present study, the methanol extract from the leaves (FTL), bark (FTB), and roots (FTR) of this plant as well as fractions (FTR1-5) and compounds isolated from FTR namely β-sitosterol-3-O-β-D-glucopyranoside (1), nitidine chloride (2) and buesgenine (3), were tested for their antimicrobial activities against a panel of Gram-negative bacteria including multidrug resistant (MDR) phenotypes. Materials and methods: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples; Column chromatography was used for the fractionation and purification of the roots extract whilst the chemical structures of compounds were determined using spectroscopic techniques. Results: Results of the MIC determinations indicated that the crude extracts from the roots as well as fraction FTRa4 were active on all the 26 tested bacterial strains. MIC values below 100 mg/mL were obtained with roots, leaves and bark extract respectively against 30.8%, 15.4% and 11.5% tested bacteria. The lowest MIC value below of 8 mg/mL was obtained with extract from the roots against Escherichia coli MC100 strain. The lowest MIC value of 4 mg/mL was also obtained with compound 3 against E. coli AG102 and Klebsiella pneumoniae ATCC11296 Conclusions: The present study demonstrates that F. tessmannii is a potential source of antimicrobial drugs to fight against MDR bacteria. Benzophenanthrine alkaloids 2 and 3 are the main antibacterial consituents of the roots of the plant. & 2015 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Buesgenine Chloramphenicol Dimethyl sulfoxide p-Iodonitrotetrazolium chloride β-sitosterol-3-O-β-D-glucopyranoside Nitidine chloride Keywords: Benzophenanthrine Buesgenine Antibacterial Fagara tessmannii Nitidine Rutaceae
1. Introduction Infectious diseases including bacterial infections continue to be a serious health concern worldwide. The situation is complicated by the appearance of multidrug-resistant (MDR) pathogens. Clinically, the continuous emergence of Gram-negative MDR bacteria drastically reduces the efficacy of the antibiotic arsenal and, consequently, increases the frequency of therapeutic failure (Rice, 2006). Approximately 60% of world's population still relies on medicinal plants for their primary healthcare (WHO, 2010). Medicinal plants have been used as a source of remedies since ancient times in Africa. Fagara tessmannii Engl. (Rutaceae) (Syn. n Correspondence to: Department of Biochemistry, Faculty of Science, #University of Dschang, P.O. Box 67 Dschang Cameroon. #Tel.: þ 237 77 355 927; fax: þ237 22 226 018. E-mail address: [email protected] (V. Kuete).
http://dx.doi.org/10.1016/j.jep.2015.04.041 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Zanthoxylum tessmannii), commonly known as “Trade olon (Liberia)” is a shrub of the African rainforests found in South-West, Centre, South and East (Mbaze et al., 2007). In Cameroon, the plant is known as “Ewoungea” or “Bongo.” The aqueous decoction of the plant is traditionally used to treat bacterial infections, cancers, swellings and inflammation (Kerharo and Adam, 1971; Mbaze et al., 2007). The decoction of the roots of the plant is used in West Africa in traditional medicine as a toothbrush (Kerharo and Adam, 1971). Previous phytochemical study on this medicinal plant led to the isolation of alkaloids (chelerythrine and dictamine), coumarins (scoparone, xanthotoxin) (Adesina, 2005; Mabry and Ulubelen, 1980), 2,6-dimethoxy-1,4-benzoquinone; 3β-acetoxy-16β-hydroxybetulinic acid, 3β, 16β-hydroxybetulinic acid (Mbaze et al., 2007). The bark of F. tessmannii previously inhibited the proliferation of leukaemia CCRF-CEM cells (Kuete et al., 2014). The compounds 2,6dimethoxy-1,4-benzoquinone and 3β-acetoxy-16β-hydroxybetulinic acid also have antimicrobial activities against Bacillus subtilis,
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Staphylococcus aureus, Escherichia coli, Streptomyces viridochromogenes, Mucor miehei, Chlorella vulgaris, and Scenedesmus subspicatus (Mbaze et al., 2007). In the present study, the bioguided fractionation was undertaken for in depth investigation of the antimicrobial activity of F. tessmannii.
2. Materials and methods 2.1. Plant material and extraction The leaves, bark and roots of F. tessmannii Engl. were collected in Lebialem, South-West Region of Cameroon (41100 N 91140 E / 4.1671N 9.2331E) in February 2012. The plant was identified at the National Herbarium (Yaoundé, Cameroon) where a voucher specimen was deposited under the reference number 25124/SRF/CAM [roots, leaves, bark]. The air-dried leaves (50 g), bark (50 g), and roots (2 kg) of F. tessmannii were extracted in 200 mL (leaves and bark) and 5 L (roots) of methanol (MeOH). The methanol extract was concentrated under reduced pressure to yield a dark residue that constituted the leaves (9.10%), bark (10.65%) and root extracts (8.70%).
and FTRb (20 g) respectively. The aqueous fraction (FTRb) did not have activity any substantial antibacterial activity and was not further investigated. The active fraction (FTRa; 96 g) was submitted to silica gel column chromatography (CC) eluting with dichloromethane (CH2Cl2), CH2Cl2/ethyl acetate (EtOAc), EtOAc/MeOH, and MeOH in increasing polarity to afford 105 fractions of 300 mL each. After comparative TLC five combined sub-fractions were finally obtained as follow: FTRa1 [CH2Cl2/EtOAc (100:0, and 90:10), 1.5 g], FTRa2 [CH2Cl2/EtOAc (80:20, 70:30, and 60:40), 8.5 g], FTRa3 [CH2Cl2/EtOAc (50:50, 40:60, 30:70, and 15:80), 3.2 g], FTRa4 [EtOAc/MeOH (100:0, and 90:10), 20.5 g], and FTRa5 [EtOAc/MeOH (80:20, 60:40, 70:30, and 0:100), 30 g]. Sub-fractions FTRa1 and FTRa5 did not have any substantial antibacterial activity whilst FTRa4 was the most active. Part of fraction FTRa4 (19.5 g) was therefore subjected to a purification on silica gel CC using a gradient mixture of CH2Cl2/MeOH to afford 30 new sub-fractions of 100 mL each. Sub-fraction 6 gave a white powder (1; 10 mg) while sub-fractions 8–9 were crystallized in a mixture of CH2Cl2/MeOH (9:1) to give a yellow powder compound (2; 20 mg). Sub-fractions 13–16 were further subjected separately to Sephadex LH-20 to give a red powder (3; 15 mg). 2.3. General procedure
2.2. Bioguided purification of the roots of F. tessmannii Part of the roots extract (135 g) was subjected to ethyl acetate (EtOAc)-water partition to afford two fractions named FTRa (103 g)
Mass spectral data [Electrospray ionization mass spectrometry (ESI-MS)] were measured on a Waters Synapt HDMS spectrometer. NMR spectra were recorded with a Varian spectrometer at 400 MHz.
Table 1 Bacterial strains used and their features Strains
Features and References
Escherichia coli ATCC10536 AG100 AG100A
Reference strain Wild-type E. coli K-12 AG100 ΔacrAB: KANR
AG100ATET AG102 MC4100 W3110 Enterobacter aerogenes ATCC13048 CM64 EA3 EA27 EA289 EA294 EA298 Enterobacter cloacae ECCI69 BM67 Klebsiella pneumoniae ATCC12296 KP55 KP63 K24 K2 Providencia stuartii NEA16 ATCC29916 PS2636 PS299645 Pseudemonas aeruginosa PA01 PA124
ΔacrAB mutant AG100, with over-expressing acrF gene; TETR ΔacrAB mutant AG100, owing acrF gene markedly over-expressed; TETR Wild type E. coli Wild type E. coli
Reference strains CHLR resistant variant obtained from ATCC13048 over-expressing the AcrAB pump Clinical MDR isolate; CHLR, NORR, OFXR, SPXR, MOXR, CFTR, ATMR, FEPR Clinical MDR isolate exhibiting energy-dependent norfloxacin and chloramphenicol efflux with KANR AMPR NALR STRR TETR KAN sensitive derivative of EA27 EA289 acrA: KANR EA 289 tolC: KANR
Viveiros et al. (2005) Kuete et al. (2010), Okusu et al. (1996), Viveiros et al. (2005) Viveiros et al. (2005) Elkins and Mullis (2007), Kuete et al. (2011) Baglioni et al. (2003) Baglioni et al. (2003), Sar et al. (2005)
Ghisalberti et al. (2005) Mallea et al. (1998), Mallea et al. (2003) Mallea et al. (1998), Mallea et al. (2003) Pradel and Pages (2002) Pradel and Pages (2002) Pradel and Pages (2002)
Clinical MDR isolates, CHLR Clinical MDR isolates, CHLR
Fankam et al. (2011) Fankam et al. (2011)
Reference strains Clinical MDR isolate, TETR, AMPR, ATMR, CEFR Clinical MDR isolate, TETR, CHLR, AMPR, ATMR AcrAB-TolC, Laboratory collection of UNR-MD1, University of Marseille, France AcrAB-TolC, Laboratory collection of UNR-MD1, University of Marseille, France
Chevalier et al. (2000) Chevalier et al. (2000) Fankam et al. (2011) Fankam et al. (2011)
Clinical Clinical Clinical Clinical
MDR MDR MDR MDR
isolate, isolate, isolate, isolate,
AcrAB-TolC AcrAB-TolC AcrAB-TolC AcrAB-TolC
Reference strains MDR clinical isolate
Tran et al. (2010)
Lorenzi et al. (2009)
AMP, ATMR, CEFR, CFTR, CHLR, FEPR, KANR, MOXR, STRR, TETR: Resistance to ampicillin, aztreonam, cephalothin, cefadroxil, chloramphenicol, cefepime, kanamycin, moxalactam, streptomycin, and tetracycline, respectively; MDR: Multidrug resistant.
S.B. Tankeo et al. / Journal of Ethnopharmacology 169 (2015) 275–279
277 12
11
H
OH HO HO
H
O OH
H3CO
H H
H3CO
O
10 9
7
1
2
O
3
O
10b
4b
10a 8
12a
6a
N5 6
Cl
4a
4
CH3
2
1
O O N
HO
CH3
OMe OH 3
Fig. 1. Chemical structures of compounds isolated from the roots of Fagara tessmannii. 1: β-Sitosterol-3-O-β-D-glucopyranoside; 2: nitidine chloride; 3: buesgenine.
Chemical shifts (δ) were quoted in parts per million (ppm) from the internal standard tetramethylsilane (TMS). Deuterated dimethyl sulfoxide (DMSO-d6), was used as solvent for the NMR experiments. Column chromatography was performed on silica gel Merck 60 F254 [(0.2–0.5 mm) and (0.2–0.063 mm)] 70–230 and 230–400 mesh (Darmstadt, Germany). Pre-coated silica gel 60 F254 thin layer chromatography (TLC) plates (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 50% sulphuric acid (H2SO4) followed by heating to 100 1C. 2.4. Antimicrobial assays 2.4.1. Chemicals for antimicrobial assay Ciprofloxacin Z98% (Sigma-Aldrich, St. Quentin Fallavier, France) was used as reference antibiotics (RA) against Gramnegative bacteria. p-Iodonitrotetrazolium chloride Z97% (INT, Sigma-Aldrich) was used as microbial growth indicator. 2.4.2. Microbial strains and culture media The studied microorganisms included sensitive and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, E. coli obtained from the American Type Culture Collection. Their bacterial feature are summarized in Table 1. Nutrient agar was used for the activation of the tested Gramnegative bacteria (Kuete et al., 2011). 2.4.3. INT colorimetric assay for MIC and MBC determinations The MIC and MBC determinations on the tested bacteria were conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods (Eloff, 1998) with some modifications (Kuete et al., 2009).
3. Results and discussion The structures of compounds from the roots of F. tessmannii was elucidated using physical and NMR data and comparison with literature. They were identified as a steroid known as β-sitosterol-3O-β-D-glucopyranoside C35H66O6 (1; m.p. 159 1C; m/z 583)(Al-Oqail et al., 2012) and two benzophenanthridine alkaloids namely nitidine chloride C21H18O4N (2; m.p. 181 1C; m/z 349)(Marek et al., 1999) and buesgenine C20H17NO5 (3; m.p. 177 1C; m/z 352)(Sandjo et al., 2014) (Fig. 1). Alkaloids (chelerythrine and dictamine), coumarins (scoparone, xanthotoxin) (Adesina, 2005; Mabry and Ulubelen, 1980), 2,6dimethoxy-1,4-benzoquinone; 3β-acetoxy-16β-hydroxybetulinic acid, 3β, 16β-hydroxybetulinic acid (Mbaze et al., 2007) were previously isolated from the bark of this plant. However, these compounds were
not detected in this study, either due to the fact that we used the roots as plant material or due to the purification method employed. These crude extract and fractions from F. tessmannii as well as compounds 1–3 were tested for their antimicrobial activities on a panel of 26 bacterial strains and the results are reported in Tables 2–4. The results of the MIC determinations of extract and fractions as shown in Table 2 indicated that the crude extracts from the roots as well as fraction FTRa4 were active on all the 26 tested bacterial strains. Extracts from the leaves (FTL), bark (FTB), as well as fractions FTRa, FTRa2 and FTRa3 displayed a selective activities, the MIC value below 1024 mg/mL being recorded respectively on 25/26 (96.2%), 22/26 (84.6%), 16/26 (61.5%), 4/26 (15.4%) and 3/26 (11.5%) tested bacteria. Sub-fraction FTRa1 and FTRa5 did not have any activity on the studied bacteria at the highest concentration tested. The antimicrobial activity of a phytochemical (crude extract) has been defined as significant when MIC is below 100 mg/mL, moderate when 100 mg/mL oMICo 625 mg/mL or low when MIC4100 mg/mL (Kuete, 2010). Therefore, extracts from the parts of F. tessmannii could be considered as promising herbal drug, as MIC values below 100 mg/mL were obtained with roots, leaves and bark extract respectively against 8/26 (30.8%), 4/26 (15.4%) and 3/26 (11.5%) tested bacteria. The lowest MIC value below of 8 mg/mL was obtained with extract from the roots against E. coli MC100 strain. Interestingly, the roots extract was more active (lower MIC value) than the reference compound ciprofloxacin against some MDR strains such as E. coli MC100, E, aerogenes EA294 highlighting its good antimicrobial potency. MBC values were also obtained with the crude extracts, fractions FTRa and sub-fraction FTR4 on many bacterial species (Table 3). However, the obtained value were generally high; A keen look of data from Tables 2 and 3 indicated that the ratio MBC/MIC were generally above 4, indicating that the studied extracts as well as the active fractions exerted bacteriostatic effects (Mbaveng et al., 2008). MIC and MBC of ciprofloxacin were also very high (4100 mg/ mL) on several pathogens, showing that most of the bacterial strains used were MDR phenotypes. Based on their inhibitory activities, extracts from roots and its best fractions FTRa were subjected to fractionation and purification using column chromatography. The resulting terpenoids (1), alkaloids (2 and 3) were further tested on 12 selected bacterial including reference ATCC strains and MDR species (Table 4). Compound 1 did not show any inhibitory activitiy when it was tested at up to 512 mg/mL. Alkaloid 2 displayed selective activities whilst alkaloid 3 was active on all the 12 tested bacteria. The activity of compounds is significant when MIC o10 mg/mL, moderate when 10 oMIC o100 mg/mL and low when MIC 4100 mg/mL (Kuete, 2010). Based on these, the inhibitory activity of compound 3 (MIC of 4 mg/mL) against E. coli AG102 and K. pneumoniae ATCC11296 could be considered as important. Benzophenanthrine
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Table 2 MICs (mg/mL) of the crude extract, fractions from Fagara tessmannii and ciprofloxacin on the panel of tested bacteria. Bacterial strains
Escherichia coli ATCC 10536 AG100 AG102 AG100A AG100Atet MC4100 W3110 Entrobacter aerogenes ATCC 13048 CM64 EA3 EA27 EA294 EA289 EA298 Pseudomanas aeruginosa PA01 PA124 Klebsiella pneumoniae ATCC11296 K2 KP55 KP63 Providentia stuartii ATCC29916 PS2636 PS299645 NAE 16 Enterobacter cloacae ECCI 69 BM67
Crude extracts
Fractions
FTR FTB
FTL
FTTa FTRa2 FTRa3 FTRa4
Reference drug CIP
128 16 128 256 128 32 o8
512 1024 1024 256 512 256 16
128 128 512 256 128 16 32
512 1024 – 512 – 1024 512
1024 – – – – – –
1024 – – – – 1024 –
128 128 1024 128 128 512 32
o1 8 32 4 32 64 o1
64 128 128 128 64 256 128
256 1024 1024 128 1024 512 –
128 512 512 256 128 256 128
1024 – 512 256 256 1024 –
– – – – 1024 – –
– – – – 1024 – –
64 512 256 128 128 256 512
64 16 4 2 128 16
64 512 512 –
– 256
256 –
– –
– –
128 1024
32 2
16 256 256 256
– 1024 1024 1024
1024 32 256 256
32 – 256 –
1024 – – –
1024 – – –
32 128 256 256
16 32 16 32
256 32 128 256
32 1024 64 1024
32 256 128 128
1024 512 1024 –
– – – –
– – – –
128 256 128 1024
64 32 128 256
256 1024 256 512 – 512
– –
– –
– –
1024 1024
128 32
Tested samples [methanol extract from the roots (FTR), bark (FTB) and leaves (FTL) of Fagara tessmannii; Fractions from the roots (FTRa-e) of Fagara tessmannii; CIP: ciprofloxacin]; (–): MIC 41024 mg/mL; Fractions FTRa1 and FTRa5 were not active at up to 1024 mg/mL.
alkaloids 3 and nitidine (2) are therefore the main active consituents of the roots of F. tessmannii. Interrestingly, the best compound, buesgenin (3) was found not toxic towards the normal AML12 hepatocytes (Sandjo et al., 2014). To the best of our knowledge, the antibacterial activity of the crude extract from the roots of F. tessmannii as well as compounds 2–3 against MDR bacteria is being reported for the first time. Nonetheless, compounds from the bark of this plant such as 2,6dimethoxy-1,4-benzoquinone and 3β-acetoxy-16β-hydroxybetulinic previously showed moderate antibacterial activities against the sensitive strains of Bacillus subtilis, S. aureus, E. coli, S. viridochromogenes, M. miehei, C. vulgaris, and S. subspicatus (Mbaze et al., 2007). Enterobacteriaceae, including K. pneumoniae, E. aerogenes and E. coli as well as P. aeruginosa have been classified as antimicrobial-resistant organisms of concern in healthcare facilities (Kuete et al., 2011; Savafi et al., 2005; Zager and McNerney, 2008). The good activities of the crude extract, fractions as well as compound 3 on many the tested microorganisms belonging to MDR phenotypes as observed herein confirmed that F. tessmannii is a potential source of antimicrobial drugs. The good antibacterial activities of various parts of this plant also justify its traditional uses (Mbaze et al., 2007) in the control of microbial infections.
Table 3 MBCs (mg/mL) of the crude extract, fractions from Fagara tessmannii and ciprofloxacin on the panel of tested bacteria. Bacterial strains
Escherichia coli ATCC 10536 AG100 AG102 AG100A AG100Atet MC4100 W3110 Enterobacter aerogenes ATCC 13048 CM64 EA3 EA27 EA294 EA289 EA298 Pseudomonas aeruginosa PA01 PA124 Klebsiella pneumoniae ATCC11296 K2 KP55 KP63 Providencia stuartii ATCC29916 PS2636 PS299645 NAE 16 Enterobacter cloacae ECCI 69 BM67
Crude extract
Fractions
Reference drug
FTR
FTB
FTR
FTRa4
CIP
1024 512 1024 1024 – 1024 128
– – – – – – 256
1024 1024 1024 – 1028 512 1024
1024 – – 1024 – 512 1024
4 16 256 256 128 16 64
512 1024 1024 1024 256 1024 512
1024 – – – – 1024 nt
512 – – 1024 1024 512 1024
512 – – 1024 1024 – –
256 256 256 256 256 128 16
1024 –
– nt
– –
– –
256 256
512 1024 – 1024
– nt – 128
1024 – – 256
512 1024 – –
64 16 128 128
1024 512 1024 –
– 1024 – –
1024 1024 1024 1024
1024 1024 1024 –
256 256 128 4256
– –
– nt
– –
– –
4256 256
Tested samples [methanol extract from the roots (FTR), bark (FTB) and leaves (FTL) of Fagara tessmannii; Fractions from the roots (FTRa-e) of Fagara tessmannii; CIP: ciprofloxacin]; (–): MBC 41024 mg/mL; nt: not tested as MIC was 41024 mg/mL; Fractions FTRa1-3 and FTRa5 were not tested or were not active at up to 1024 mg/ mL if MIC was observed; fraction FTTa showed MBC value of 512 mg/mL only against Klebsiella pneumoniae ATCC11296: tested concentration range: 8–1042 mg/mL for extracts and fractions and 2–256 mg/mL for CHL.
Table 4 MICs and MBC (mg/mL) of compounds from the roots of Fagara tessmannii on selected bacteria. Bacterial strains
Compounds, MICs and MBC (in parenthesis) 2
Escherichia coli ATTC10536 AG102 AG 100Atet Enterobacter aerogenes ATCC 13048 EA294 Kblebsiella pneumoniae ATCC11296 K2 KP55 Providencia stuartii ATCC29916 PS2636 Pseudomonas aeruginosa PA01 PA124
3
128 ( ) 256 ( ) 256 ( )
32 (512) 4 (512) 64 ( )
512 ( ) –
16 (128) 32 (512)
128 ( ) – 256 ( )
4 (256) 32(512) 64 (512)
512 ( ) 256 ( )
128 ( ) 32 (512)
256 ( ) –
32 (512) 512 ( )
Compound 1 (β-Sitosterol-3-O-β-D-glucopyranoside) was not active; 2: nitidine chloride; 3: buesgenine (–): MIC 4512 mg/mL; nt: not tested as MIC was 4512 mg/ mL: tested concentration range: 4–512 mg/mL.
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4. Conclusion The results of the present study are very interesting, taking in account the medical importance of the studied microorganisms. These data provided evidence that the crude extract from F. tessmannii as well as some of the compounds, and mostly buesgenine (3) antibacterial natural products to fight MDR phenotypes. Acknowledgements Authors are thankful to the Cameroon National Herbarium (Yaounde) for the plant identification. Authors are also thankful to UMR-MD1 (Mediterranean University, Marseille, France) for providing some clinical bacteria. References Adesina, S., 2005. The Nigerian zanthoxylum; chemical and biological values. Afr. J. Tradit. Complement.Altern. Med. 2, 282–301. Al-Oqail, M., Hassan, W., Ahmad, M., Al-Rehaily, A., 2012. Phytochemical and biological studies of Solanum schimperianum Hochst. Saudi Pharm. J. 20, 371–378. Baglioni, P., Bini, L., Liberatori, S., Pallini, V., Marri, L., 2003. Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor. Proteomics 3, 1060–1065. Chevalier, J., Pages, J.M., Eyraud, A., Mallea, M., 2000. Membrane permeability modifications are involved in antibiotic resistance in Klebsiella pneumoniae. Biochem Biophys. Res. Commun. 274, 496–499. Elkins, C.A., Mullis, L.B., 2007. Substrate competition studies using whole-cell accumulation assays with the major tripartite multidrug efflux pumps of Escherichia coli. Antimicrob. Agents Chemother. 51, 923–929. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med. 64, 711–713. Fankam, A.G., Kuete, V., Voukeng, I.K., Kuiate, J.R., Pages, J.M., 2011. Antibacterial activities of selected Cameroonian spices and their synergistic effects with antibiotics against multidrug-resistant phenotypes. BMC Complement. Altern. Med. 11, 104. Ghisalberti, D., Masi, M., Pages, J.M., Chevalier, J., 2005. Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes. Biochem. Biophys. Res. Commun. 328, 1113–1118. Kerharo, J., Adam, J., 1971. La pharmacopée Sénégalaise traditionnelle. Vigot Frères, Paris. Kuete, V., Nana, F., Ngameni, B., Mbaveng, A.T., Keumedjio, F., Ngadjui, B.T., 2009. Antimicrobial activity of the crude extract, fractions and compounds from stem bark of Ficus ovata (Moraceae). J. Ethnopharm. 124, 556–561. Kuete, V., Ngameni, B., Tangmouo, J.G., Bolla, J.M., Alibert-Franco, S., Ngadjui, B.T., Pages, J.M., 2010. Efflux pumps are involved in the defense of Gram-negative bacteria against the natural products isobavachalcone and diospyrone. Antimicrob. Agents Chemother. 54, 1749–1752. Kuete, V., 2010. Potential of Cameroonian plants and derived products against microbial infections: a review. Planta Med. 76, 1479–1491. Kuete, V., Alibert-Franco, S., Eyong, K.O., Ngameni, B., Folefoc, G.N., Nguemeving, J. R., Tangmouo, J.G., Fotso, G.W., Komguem, J., Ouahouo, B.M., Bolla, J.M.,
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Chevalier, J.M., Ngadjui, B.T., Nkengfack, A.E., Pages, J.M., 2011. Antibacterial activity of some natural products against bacteria expressing a multidrugresistant phenotype. Int. J. Antimicrob. Agents 37, 156–161. Kuete, V., Tankeo, S.B., Saeed, M.E., Wiench, B., Tane, P., Efferth, T., 2014. Cytotoxicity and modes of action of five Cameroonian medicinal plants against multifactorial drug resistance of tumor cells. J. Ethnopharmacol. 153, 207–219. Lorenzi, V., Muselli, A., Bernardini, A.F., Berti, L., Pages, J.M., Amaral, L., Bolla, J.M., 2009. Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species. Antimicrob. Agents Chemother. 53, 2209–2211. Mabry, T.J., Ulubelen, A., 1980. Chemistry and utilization of phenylpropanoids including flavonoids, coumarins, and lignans. J. Agric. Food Chem. 28, 188–195. Mallea, M., Chevalier, J., Bornet, C., Eyraud, A., Davin-Regli, A., Bollet, C., Pages, J.M., 1998. Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes. Microbiology 144, 3003–3009, Pt 11. Mallea, M., Mahamoud, A., Chevalier, J., Alibert-Franco, S., Brouant, P., Barbe, J., Pages, J.M., 2003. Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates. Biochem. J. 376, 801–805. Marek, R., Toušek, J., Dostál, J., Slavík, J., Dommisse, R., Sklenář, V., 1999. 1H and 13C NMR study of quaternary benzo[c]phenanthridine alkaloids. Magn. Reson. Chem. 37, 781–787. Mbaveng, A.T., Ngameni, B., Kuete, V., Simo, I.K., Ambassa, P., Roy, R., Bezabih, M., Etoa, F.X., Ngadjui, B.T., Abegaz, B.M., Meyer, J.J., Lall, N., Beng, V.P., 2008. Antimicrobial activity of the crude extracts and five flavonoids from the twigs of Dorstenia barteri (Moraceae). J. Ethnopharmacol. 116, 483–489. Mbaze, L.M., Poumale, H.M., Wansi, J.D., Lado, J.A., Khan, S.N., Iqbal, M.C., Ngadjui, B.T., Laatsch, H., 2007. Alpha-glucosidase inhibitory pentacyclic triterpenes from the stem bark of Fagara tessmannii (Rutaceae). Phytochemistry 68, 591–595. Okusu, H., Ma, D., Nikaido, H., 1996. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibioticresistance (Mar) mutants. J. Bacteriol. 178, 306–308. Pradel, E., Pages, J.M., 2002. The AcrAB-TolC efflux pump contributes to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes. Antimicrob. Agents Chemother. 46, 2640–2643. Rice, L.B., 2006. Unmet medical needs in antibacterial therapy. Biochem. Pharmacol. 71, 991–995. Sandjo, L.P., Kuete, V., Tchangna, R.S., Efferth, T., Ngadjui, B.T., 2014. Cytotoxic benzophenanthridine and furoquinoline alkaloids from Zanthoxylum buesgenii (Rutaceae). Chem. Cent. J. 8, 61. Sar, C., Mwenya, B., Santoso, B., Takaura, K., Morikawa, R., Isogai, N., Asakura, Y., Toride, Y., Takahashi, J., 2005. Effect of Escherichia coli wild type or its derivative with high nitrite reductase activity on in vitro ruminal methanogenesis and nitrate/nitrite reduction. J. Anim. Sci. 83, 644–652. Savafi, L, Savafi, D.N., Onlen, N, Ocak, S., Y, 2005. The prevalence and resistance patterns of Pseudomonas aeruginosa in intensive care units in a university hospital. Turk. J. Med. Sci. 35, 317–322. Tran, Q.T., Mahendran, K.R., Hajjar, E., Ceccarelli, M., Davin-Regli, A., Winterhalter, M., Weingart, H., Pages, J.M., 2010. Implication of porins in beta-lactam resistance of Providencia stuartii. J. Biol. Chem. 285, 32273–32281. Viveiros, M., Jesus, A., Brito, M., Leandro, C., Martins, M., Ordway, D., Molnar, A.M., Molnar, J., Amaral, L., 2005. Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes. Antimicrob. Agents Chemother. 49, 3578–3582. WHO, 2010. Tuberculosis, Fact sheet No. 104. World Health Organization, Geneva, Switzerland. 〈http://www.kff.org/globalhealth/upload/7883-02.pdf〉 (accessed 02.02.12). Zager, E.M., McNerney, R., 2008. Multidrug-resistant tuberculosis. BMC Infect. Dis. 8, 10.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antibacterial activities of the methanol extracts, fractions and compounds from Fagara tessmannii Simplice B. Tankeo a, Francois Damen b, Maurice D. Awouafack b, James Mpetga b, Pierre Tane b, Jacobus N. Eloff c, Victor Kuete a,n a
Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, Cameroon c Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 February 2015 Received in revised form 20 April 2015 Accepted 22 April 2015 Available online 30 April 2015
Ethnopharmacological relevance: Fagara tessmannii is a shrub of the African rainforests used to treat bacterial infections, cancers, swellings and inflammation. In the present study, the methanol extract from the leaves (FTL), bark (FTB), and roots (FTR) of this plant as well as fractions (FTR1-5) and compounds isolated from FTR namely β-sitosterol-3-O-β-D-glucopyranoside (1), nitidine chloride (2) and buesgenine (3), were tested for their antimicrobial activities against a panel of Gram-negative bacteria including multidrug resistant (MDR) phenotypes. Materials and methods: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples; Column chromatography was used for the fractionation and purification of the roots extract whilst the chemical structures of compounds were determined using spectroscopic techniques. Results: Results of the MIC determinations indicated that the crude extracts from the roots as well as fraction FTRa4 were active on all the 26 tested bacterial strains. MIC values below 100 mg/mL were obtained with roots, leaves and bark extract respectively against 30.8%, 15.4% and 11.5% tested bacteria. The lowest MIC value below of 8 mg/mL was obtained with extract from the roots against Escherichia coli MC100 strain. The lowest MIC value of 4 mg/mL was also obtained with compound 3 against E. coli AG102 and Klebsiella pneumoniae ATCC11296 Conclusions: The present study demonstrates that F. tessmannii is a potential source of antimicrobial drugs to fight against MDR bacteria. Benzophenanthrine alkaloids 2 and 3 are the main antibacterial consituents of the roots of the plant. & 2015 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Buesgenine Chloramphenicol Dimethyl sulfoxide p-Iodonitrotetrazolium chloride β-sitosterol-3-O-β-D-glucopyranoside Nitidine chloride Keywords: Benzophenanthrine Buesgenine Antibacterial Fagara tessmannii Nitidine Rutaceae
1. Introduction Infectious diseases including bacterial infections continue to be a serious health concern worldwide. The situation is complicated by the appearance of multidrug-resistant (MDR) pathogens. Clinically, the continuous emergence of Gram-negative MDR bacteria drastically reduces the efficacy of the antibiotic arsenal and, consequently, increases the frequency of therapeutic failure (Rice, 2006). Approximately 60% of world's population still relies on medicinal plants for their primary healthcare (WHO, 2010). Medicinal plants have been used as a source of remedies since ancient times in Africa. Fagara tessmannii Engl. (Rutaceae) (Syn. n Correspondence to: Department of Biochemistry, Faculty of Science, #University of Dschang, P.O. Box 67 Dschang Cameroon. #Tel.: þ 237 77 355 927; fax: þ237 22 226 018. E-mail address: [email protected] (V. Kuete).
http://dx.doi.org/10.1016/j.jep.2015.04.041 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Zanthoxylum tessmannii), commonly known as “Trade olon (Liberia)” is a shrub of the African rainforests found in South-West, Centre, South and East (Mbaze et al., 2007). In Cameroon, the plant is known as “Ewoungea” or “Bongo.” The aqueous decoction of the plant is traditionally used to treat bacterial infections, cancers, swellings and inflammation (Kerharo and Adam, 1971; Mbaze et al., 2007). The decoction of the roots of the plant is used in West Africa in traditional medicine as a toothbrush (Kerharo and Adam, 1971). Previous phytochemical study on this medicinal plant led to the isolation of alkaloids (chelerythrine and dictamine), coumarins (scoparone, xanthotoxin) (Adesina, 2005; Mabry and Ulubelen, 1980), 2,6-dimethoxy-1,4-benzoquinone; 3β-acetoxy-16β-hydroxybetulinic acid, 3β, 16β-hydroxybetulinic acid (Mbaze et al., 2007). The bark of F. tessmannii previously inhibited the proliferation of leukaemia CCRF-CEM cells (Kuete et al., 2014). The compounds 2,6dimethoxy-1,4-benzoquinone and 3β-acetoxy-16β-hydroxybetulinic acid also have antimicrobial activities against Bacillus subtilis,
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Staphylococcus aureus, Escherichia coli, Streptomyces viridochromogenes, Mucor miehei, Chlorella vulgaris, and Scenedesmus subspicatus (Mbaze et al., 2007). In the present study, the bioguided fractionation was undertaken for in depth investigation of the antimicrobial activity of F. tessmannii.
2. Materials and methods 2.1. Plant material and extraction The leaves, bark and roots of F. tessmannii Engl. were collected in Lebialem, South-West Region of Cameroon (41100 N 91140 E / 4.1671N 9.2331E) in February 2012. The plant was identified at the National Herbarium (Yaoundé, Cameroon) where a voucher specimen was deposited under the reference number 25124/SRF/CAM [roots, leaves, bark]. The air-dried leaves (50 g), bark (50 g), and roots (2 kg) of F. tessmannii were extracted in 200 mL (leaves and bark) and 5 L (roots) of methanol (MeOH). The methanol extract was concentrated under reduced pressure to yield a dark residue that constituted the leaves (9.10%), bark (10.65%) and root extracts (8.70%).
and FTRb (20 g) respectively. The aqueous fraction (FTRb) did not have activity any substantial antibacterial activity and was not further investigated. The active fraction (FTRa; 96 g) was submitted to silica gel column chromatography (CC) eluting with dichloromethane (CH2Cl2), CH2Cl2/ethyl acetate (EtOAc), EtOAc/MeOH, and MeOH in increasing polarity to afford 105 fractions of 300 mL each. After comparative TLC five combined sub-fractions were finally obtained as follow: FTRa1 [CH2Cl2/EtOAc (100:0, and 90:10), 1.5 g], FTRa2 [CH2Cl2/EtOAc (80:20, 70:30, and 60:40), 8.5 g], FTRa3 [CH2Cl2/EtOAc (50:50, 40:60, 30:70, and 15:80), 3.2 g], FTRa4 [EtOAc/MeOH (100:0, and 90:10), 20.5 g], and FTRa5 [EtOAc/MeOH (80:20, 60:40, 70:30, and 0:100), 30 g]. Sub-fractions FTRa1 and FTRa5 did not have any substantial antibacterial activity whilst FTRa4 was the most active. Part of fraction FTRa4 (19.5 g) was therefore subjected to a purification on silica gel CC using a gradient mixture of CH2Cl2/MeOH to afford 30 new sub-fractions of 100 mL each. Sub-fraction 6 gave a white powder (1; 10 mg) while sub-fractions 8–9 were crystallized in a mixture of CH2Cl2/MeOH (9:1) to give a yellow powder compound (2; 20 mg). Sub-fractions 13–16 were further subjected separately to Sephadex LH-20 to give a red powder (3; 15 mg). 2.3. General procedure
2.2. Bioguided purification of the roots of F. tessmannii Part of the roots extract (135 g) was subjected to ethyl acetate (EtOAc)-water partition to afford two fractions named FTRa (103 g)
Mass spectral data [Electrospray ionization mass spectrometry (ESI-MS)] were measured on a Waters Synapt HDMS spectrometer. NMR spectra were recorded with a Varian spectrometer at 400 MHz.
Table 1 Bacterial strains used and their features Strains
Features and References
Escherichia coli ATCC10536 AG100 AG100A
Reference strain Wild-type E. coli K-12 AG100 ΔacrAB: KANR
AG100ATET AG102 MC4100 W3110 Enterobacter aerogenes ATCC13048 CM64 EA3 EA27 EA289 EA294 EA298 Enterobacter cloacae ECCI69 BM67 Klebsiella pneumoniae ATCC12296 KP55 KP63 K24 K2 Providencia stuartii NEA16 ATCC29916 PS2636 PS299645 Pseudemonas aeruginosa PA01 PA124
ΔacrAB mutant AG100, with over-expressing acrF gene; TETR ΔacrAB mutant AG100, owing acrF gene markedly over-expressed; TETR Wild type E. coli Wild type E. coli
Reference strains CHLR resistant variant obtained from ATCC13048 over-expressing the AcrAB pump Clinical MDR isolate; CHLR, NORR, OFXR, SPXR, MOXR, CFTR, ATMR, FEPR Clinical MDR isolate exhibiting energy-dependent norfloxacin and chloramphenicol efflux with KANR AMPR NALR STRR TETR KAN sensitive derivative of EA27 EA289 acrA: KANR EA 289 tolC: KANR
Viveiros et al. (2005) Kuete et al. (2010), Okusu et al. (1996), Viveiros et al. (2005) Viveiros et al. (2005) Elkins and Mullis (2007), Kuete et al. (2011) Baglioni et al. (2003) Baglioni et al. (2003), Sar et al. (2005)
Ghisalberti et al. (2005) Mallea et al. (1998), Mallea et al. (2003) Mallea et al. (1998), Mallea et al. (2003) Pradel and Pages (2002) Pradel and Pages (2002) Pradel and Pages (2002)
Clinical MDR isolates, CHLR Clinical MDR isolates, CHLR
Fankam et al. (2011) Fankam et al. (2011)
Reference strains Clinical MDR isolate, TETR, AMPR, ATMR, CEFR Clinical MDR isolate, TETR, CHLR, AMPR, ATMR AcrAB-TolC, Laboratory collection of UNR-MD1, University of Marseille, France AcrAB-TolC, Laboratory collection of UNR-MD1, University of Marseille, France
Chevalier et al. (2000) Chevalier et al. (2000) Fankam et al. (2011) Fankam et al. (2011)
Clinical Clinical Clinical Clinical
MDR MDR MDR MDR
isolate, isolate, isolate, isolate,
AcrAB-TolC AcrAB-TolC AcrAB-TolC AcrAB-TolC
Reference strains MDR clinical isolate
Tran et al. (2010)
Lorenzi et al. (2009)
AMP, ATMR, CEFR, CFTR, CHLR, FEPR, KANR, MOXR, STRR, TETR: Resistance to ampicillin, aztreonam, cephalothin, cefadroxil, chloramphenicol, cefepime, kanamycin, moxalactam, streptomycin, and tetracycline, respectively; MDR: Multidrug resistant.
S.B. Tankeo et al. / Journal of Ethnopharmacology 169 (2015) 275–279
277 12
11
H
OH HO HO
H
O OH
H3CO
H H
H3CO
O
10 9
7
1
2
O
3
O
10b
4b
10a 8
12a
6a
N5 6
Cl
4a
4
CH3
2
1
O O N
HO
CH3
OMe OH 3
Fig. 1. Chemical structures of compounds isolated from the roots of Fagara tessmannii. 1: β-Sitosterol-3-O-β-D-glucopyranoside; 2: nitidine chloride; 3: buesgenine.
Chemical shifts (δ) were quoted in parts per million (ppm) from the internal standard tetramethylsilane (TMS). Deuterated dimethyl sulfoxide (DMSO-d6), was used as solvent for the NMR experiments. Column chromatography was performed on silica gel Merck 60 F254 [(0.2–0.5 mm) and (0.2–0.063 mm)] 70–230 and 230–400 mesh (Darmstadt, Germany). Pre-coated silica gel 60 F254 thin layer chromatography (TLC) plates (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 50% sulphuric acid (H2SO4) followed by heating to 100 1C. 2.4. Antimicrobial assays 2.4.1. Chemicals for antimicrobial assay Ciprofloxacin Z98% (Sigma-Aldrich, St. Quentin Fallavier, France) was used as reference antibiotics (RA) against Gramnegative bacteria. p-Iodonitrotetrazolium chloride Z97% (INT, Sigma-Aldrich) was used as microbial growth indicator. 2.4.2. Microbial strains and culture media The studied microorganisms included sensitive and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, E. coli obtained from the American Type Culture Collection. Their bacterial feature are summarized in Table 1. Nutrient agar was used for the activation of the tested Gramnegative bacteria (Kuete et al., 2011). 2.4.3. INT colorimetric assay for MIC and MBC determinations The MIC and MBC determinations on the tested bacteria were conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods (Eloff, 1998) with some modifications (Kuete et al., 2009).
3. Results and discussion The structures of compounds from the roots of F. tessmannii was elucidated using physical and NMR data and comparison with literature. They were identified as a steroid known as β-sitosterol-3O-β-D-glucopyranoside C35H66O6 (1; m.p. 159 1C; m/z 583)(Al-Oqail et al., 2012) and two benzophenanthridine alkaloids namely nitidine chloride C21H18O4N (2; m.p. 181 1C; m/z 349)(Marek et al., 1999) and buesgenine C20H17NO5 (3; m.p. 177 1C; m/z 352)(Sandjo et al., 2014) (Fig. 1). Alkaloids (chelerythrine and dictamine), coumarins (scoparone, xanthotoxin) (Adesina, 2005; Mabry and Ulubelen, 1980), 2,6dimethoxy-1,4-benzoquinone; 3β-acetoxy-16β-hydroxybetulinic acid, 3β, 16β-hydroxybetulinic acid (Mbaze et al., 2007) were previously isolated from the bark of this plant. However, these compounds were
not detected in this study, either due to the fact that we used the roots as plant material or due to the purification method employed. These crude extract and fractions from F. tessmannii as well as compounds 1–3 were tested for their antimicrobial activities on a panel of 26 bacterial strains and the results are reported in Tables 2–4. The results of the MIC determinations of extract and fractions as shown in Table 2 indicated that the crude extracts from the roots as well as fraction FTRa4 were active on all the 26 tested bacterial strains. Extracts from the leaves (FTL), bark (FTB), as well as fractions FTRa, FTRa2 and FTRa3 displayed a selective activities, the MIC value below 1024 mg/mL being recorded respectively on 25/26 (96.2%), 22/26 (84.6%), 16/26 (61.5%), 4/26 (15.4%) and 3/26 (11.5%) tested bacteria. Sub-fraction FTRa1 and FTRa5 did not have any activity on the studied bacteria at the highest concentration tested. The antimicrobial activity of a phytochemical (crude extract) has been defined as significant when MIC is below 100 mg/mL, moderate when 100 mg/mL oMICo 625 mg/mL or low when MIC4100 mg/mL (Kuete, 2010). Therefore, extracts from the parts of F. tessmannii could be considered as promising herbal drug, as MIC values below 100 mg/mL were obtained with roots, leaves and bark extract respectively against 8/26 (30.8%), 4/26 (15.4%) and 3/26 (11.5%) tested bacteria. The lowest MIC value below of 8 mg/mL was obtained with extract from the roots against E. coli MC100 strain. Interestingly, the roots extract was more active (lower MIC value) than the reference compound ciprofloxacin against some MDR strains such as E. coli MC100, E, aerogenes EA294 highlighting its good antimicrobial potency. MBC values were also obtained with the crude extracts, fractions FTRa and sub-fraction FTR4 on many bacterial species (Table 3). However, the obtained value were generally high; A keen look of data from Tables 2 and 3 indicated that the ratio MBC/MIC were generally above 4, indicating that the studied extracts as well as the active fractions exerted bacteriostatic effects (Mbaveng et al., 2008). MIC and MBC of ciprofloxacin were also very high (4100 mg/ mL) on several pathogens, showing that most of the bacterial strains used were MDR phenotypes. Based on their inhibitory activities, extracts from roots and its best fractions FTRa were subjected to fractionation and purification using column chromatography. The resulting terpenoids (1), alkaloids (2 and 3) were further tested on 12 selected bacterial including reference ATCC strains and MDR species (Table 4). Compound 1 did not show any inhibitory activitiy when it was tested at up to 512 mg/mL. Alkaloid 2 displayed selective activities whilst alkaloid 3 was active on all the 12 tested bacteria. The activity of compounds is significant when MIC o10 mg/mL, moderate when 10 oMIC o100 mg/mL and low when MIC 4100 mg/mL (Kuete, 2010). Based on these, the inhibitory activity of compound 3 (MIC of 4 mg/mL) against E. coli AG102 and K. pneumoniae ATCC11296 could be considered as important. Benzophenanthrine
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Table 2 MICs (mg/mL) of the crude extract, fractions from Fagara tessmannii and ciprofloxacin on the panel of tested bacteria. Bacterial strains
Escherichia coli ATCC 10536 AG100 AG102 AG100A AG100Atet MC4100 W3110 Entrobacter aerogenes ATCC 13048 CM64 EA3 EA27 EA294 EA289 EA298 Pseudomanas aeruginosa PA01 PA124 Klebsiella pneumoniae ATCC11296 K2 KP55 KP63 Providentia stuartii ATCC29916 PS2636 PS299645 NAE 16 Enterobacter cloacae ECCI 69 BM67
Crude extracts
Fractions
FTR FTB
FTL
FTTa FTRa2 FTRa3 FTRa4
Reference drug CIP
128 16 128 256 128 32 o8
512 1024 1024 256 512 256 16
128 128 512 256 128 16 32
512 1024 – 512 – 1024 512
1024 – – – – – –
1024 – – – – 1024 –
128 128 1024 128 128 512 32
o1 8 32 4 32 64 o1
64 128 128 128 64 256 128
256 1024 1024 128 1024 512 –
128 512 512 256 128 256 128
1024 – 512 256 256 1024 –
– – – – 1024 – –
– – – – 1024 – –
64 512 256 128 128 256 512
64 16 4 2 128 16
64 512 512 –
– 256
256 –
– –
– –
128 1024
32 2
16 256 256 256
– 1024 1024 1024
1024 32 256 256
32 – 256 –
1024 – – –
1024 – – –
32 128 256 256
16 32 16 32
256 32 128 256
32 1024 64 1024
32 256 128 128
1024 512 1024 –
– – – –
– – – –
128 256 128 1024
64 32 128 256
256 1024 256 512 – 512
– –
– –
– –
1024 1024
128 32
Tested samples [methanol extract from the roots (FTR), bark (FTB) and leaves (FTL) of Fagara tessmannii; Fractions from the roots (FTRa-e) of Fagara tessmannii; CIP: ciprofloxacin]; (–): MIC 41024 mg/mL; Fractions FTRa1 and FTRa5 were not active at up to 1024 mg/mL.
alkaloids 3 and nitidine (2) are therefore the main active consituents of the roots of F. tessmannii. Interrestingly, the best compound, buesgenin (3) was found not toxic towards the normal AML12 hepatocytes (Sandjo et al., 2014). To the best of our knowledge, the antibacterial activity of the crude extract from the roots of F. tessmannii as well as compounds 2–3 against MDR bacteria is being reported for the first time. Nonetheless, compounds from the bark of this plant such as 2,6dimethoxy-1,4-benzoquinone and 3β-acetoxy-16β-hydroxybetulinic previously showed moderate antibacterial activities against the sensitive strains of Bacillus subtilis, S. aureus, E. coli, S. viridochromogenes, M. miehei, C. vulgaris, and S. subspicatus (Mbaze et al., 2007). Enterobacteriaceae, including K. pneumoniae, E. aerogenes and E. coli as well as P. aeruginosa have been classified as antimicrobial-resistant organisms of concern in healthcare facilities (Kuete et al., 2011; Savafi et al., 2005; Zager and McNerney, 2008). The good activities of the crude extract, fractions as well as compound 3 on many the tested microorganisms belonging to MDR phenotypes as observed herein confirmed that F. tessmannii is a potential source of antimicrobial drugs. The good antibacterial activities of various parts of this plant also justify its traditional uses (Mbaze et al., 2007) in the control of microbial infections.
Table 3 MBCs (mg/mL) of the crude extract, fractions from Fagara tessmannii and ciprofloxacin on the panel of tested bacteria. Bacterial strains
Escherichia coli ATCC 10536 AG100 AG102 AG100A AG100Atet MC4100 W3110 Enterobacter aerogenes ATCC 13048 CM64 EA3 EA27 EA294 EA289 EA298 Pseudomonas aeruginosa PA01 PA124 Klebsiella pneumoniae ATCC11296 K2 KP55 KP63 Providencia stuartii ATCC29916 PS2636 PS299645 NAE 16 Enterobacter cloacae ECCI 69 BM67
Crude extract
Fractions
Reference drug
FTR
FTB
FTR
FTRa4
CIP
1024 512 1024 1024 – 1024 128
– – – – – – 256
1024 1024 1024 – 1028 512 1024
1024 – – 1024 – 512 1024
4 16 256 256 128 16 64
512 1024 1024 1024 256 1024 512
1024 – – – – 1024 nt
512 – – 1024 1024 512 1024
512 – – 1024 1024 – –
256 256 256 256 256 128 16
1024 –
– nt
– –
– –
256 256
512 1024 – 1024
– nt – 128
1024 – – 256
512 1024 – –
64 16 128 128
1024 512 1024 –
– 1024 – –
1024 1024 1024 1024
1024 1024 1024 –
256 256 128 4256
– –
– nt
– –
– –
4256 256
Tested samples [methanol extract from the roots (FTR), bark (FTB) and leaves (FTL) of Fagara tessmannii; Fractions from the roots (FTRa-e) of Fagara tessmannii; CIP: ciprofloxacin]; (–): MBC 41024 mg/mL; nt: not tested as MIC was 41024 mg/mL; Fractions FTRa1-3 and FTRa5 were not tested or were not active at up to 1024 mg/ mL if MIC was observed; fraction FTTa showed MBC value of 512 mg/mL only against Klebsiella pneumoniae ATCC11296: tested concentration range: 8–1042 mg/mL for extracts and fractions and 2–256 mg/mL for CHL.
Table 4 MICs and MBC (mg/mL) of compounds from the roots of Fagara tessmannii on selected bacteria. Bacterial strains
Compounds, MICs and MBC (in parenthesis) 2
Escherichia coli ATTC10536 AG102 AG 100Atet Enterobacter aerogenes ATCC 13048 EA294 Kblebsiella pneumoniae ATCC11296 K2 KP55 Providencia stuartii ATCC29916 PS2636 Pseudomonas aeruginosa PA01 PA124
3
128 ( ) 256 ( ) 256 ( )
32 (512) 4 (512) 64 ( )
512 ( ) –
16 (128) 32 (512)
128 ( ) – 256 ( )
4 (256) 32(512) 64 (512)
512 ( ) 256 ( )
128 ( ) 32 (512)
256 ( ) –
32 (512) 512 ( )
Compound 1 (β-Sitosterol-3-O-β-D-glucopyranoside) was not active; 2: nitidine chloride; 3: buesgenine (–): MIC 4512 mg/mL; nt: not tested as MIC was 4512 mg/ mL: tested concentration range: 4–512 mg/mL.
S.B. Tankeo et al. / Journal of Ethnopharmacology 169 (2015) 275–279
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