Journal of Biosciences and Medicines, 2014, 2, 34-40
Published Online August 2014 in SciRes. http://www.scirp.org/journal/jbm
http://dx.doi.org/10.4236/jbm.2014.26006
How to cite this paper: Njume, C., Gqaza, B.M., George, G. and Goduka, N.I. (2014) Inhibitory and Bactericidal Potential of
Some Indigenous Functional Food-Plants Used in the O.R. Tambo District Municipality of South Africa. Journal of
Biosciences and Medicines, 2, 34-40. http://dx.doi.org/10.4236/jbm.2014.26006
Inhibitory and Bactericidal Potential of
Some Indigenous Functional Food-Plants
Used in the O.R. Tambo District Municipality
of South Africa
Collise Njume1,2*, Bomkazi M. Gqaza3, Grace George3, Nomalungelo I. Goduka1
1Centre for Rural Development, Enkululekweni, Walter Sisulu University, Mthatha 5117, South Africa
2Department of Medical Microbiology, Walter Sisulu University, Mthatha 5117, South Africa
3Department of Medical Biochemistry, Walter Sisulu University, Mthatha 5117, South Africa
Received June 2014
Abstract
Antimicrobial resistance is a major problem in the management of infectious diseases. African in-
digenous functional food-plants such as Chenopodium album and Solanum nigrum may constitute
important sources of phytochemical constituents for the synthesis of antimicrobial compounds
against infectious organisms. The objective of this study was to determine the antimicrobial pro-
perties of Ch en opodiu m albu m and Solanum nigrum-l eaves used as functional food-plants in the
O.R. Tambo district municipality of South Africa. Organic and aqueous solvent-extracts of C. albu m
and S. nigru m were tested against Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa
(ATCC127853), Bacil lus subtilis (ATCC 6051), Escherichia coli (25922) and Enterococcus faecalis
(51299) using standard microbiological techniques. Ciprofloxacin was included in all the experi-
mental runs as positive control antibiotic. The aqueous extracts of both plants were the most ac-
tive with zones of inhibition diameters ranging from 0 mm - 20 mm and minimum inhibitory con-
centration (MIC50) values ranging from 0.63 mg/mL - 10 mg/mL. The positive control antibiotic
was highly active with zones of inhibition diameters ranging from 17 mm - 31 mm and MIC50 val-
ues from 0.0003 mg/mL - 0.0005mg/mL for all the bacteria tested. Both extracts were bactericidal
with minimum bactericidal concentration (MBC) ranges from 2.5mg/mL - 20mg/mL. From the re-
sults, it can be concluded that both plants possess compounds with antimicrobial properties, thus
validating scientifically their use in traditional medicine. However, more studies to document the
respective plant-principles responsible for antimicrobial activity of these plants would shed more
light on their functional properties.
Keywords
Antimicrobial Resistance, Sensitivity Tests, Indigenous Leafy Vegetables, Eastern Cape Province,
South Africa
*
Corresponding author.

C. Njume et al.
35
1. Introduction
Bacterial antimicrobial resistance against commonly used antibiotics is distressingly on the rise [1]. Patients in-
fected with resistant organisms are more likely to have longer more expensive hospital stays [2]. The modifica-
tion of chemotherapeutic agents to limit this problem has been greatly successful. However, many reports also
indicate that many of the drugs are being rendered obsolete by microbial drug-resistance [3]. As a result, the
treatment of microbial infection is becoming increasingly complicated. Physicians have now resorted to the use
of combination therapy, increasing the cost of treatment even more. Reports on Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis infections and antibacterial re-
sistance reveal the need for a constant search of new drugs against these organisms [4] [5]. E. coli is a major
cause of travellers’ diarrhoea, one of the most common forms of diarrhoea worldwide [6] [7]. Both E. coli and P.
aeruginosa are also major causes of urinary tract infections while S. aureus and E. faecalis are common causes
of nosocomial infections [8] [9]. B. subtilis infections are not common but few cases have been reported in the
literature in patients with oesophageal perforations [10]. The use of medicinal plants in the treatment of human
infections is a common practice in many remote areas of Africa with inadequate health care facilities. Chen o-
podium album and Solanum nigrum are functional food-plants with wide nutritional and medicinal importance
among rural communities in the O.R. Tambo District Municipality of South Africa [11]. They are jointly re-
ferred to as imifino ezikhulelayo in isiXhosa, meaning indigenous vegetable. C. album is locally known as imbi-
kicane while S. nigrum is known as umsobo [12] [13]. Both plants grow wildly in bushes, barren land and road-
side paths from where they are harvested either for nutritional or medicinal purposes. In some parts of India, C.
album is also used in ayurveda for treating anorexia, cough, dysentery, diarrhoea, oedema, piles and worm in-
festations [14]. Despite their medicinal uses, very little information is available in the literature about their
pharmacological potential. This is surprising considering the ever-increasing rate of antimicrobial resistance of
human infectious organisms against currently used drugs. The aim of this study therefore was to investigate the
antimicrobial properties of these plants in an attempt to identify cheap sources of compounds for the synthesis of
new drugs against medically important bacteria.
2. Materials and Methods
2.1. Bacterial Strains
Standard bacterial strains including Pseudomonas aeruginosa ATCC 127853, Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 51299 and Bacillus subtilis ATCC 6051
(American Type Culture Collection, Rockville, MD) obtained from the stock culture of the National Health
Laboratory Services (NHLS), Nelson Mandela Academic Hospital, Mthatha were used in this study. Ethical
clearance was obtained from the Eastern Cape Department of Health and the Ethics Committee of the Faculty of
Health Sciences, Walter Sisulu University (WSU). The organisms were cultured on nutrient agar (Oxoid Llt.,
Basingstoke, UK).
2.2. Collection and Preparation of Plant Material
The leaves of C. album and S. nigrum were harvested from home gardens and along bush paths in the vicinity of
WSU main campus in Mthatha in October 2012. The plants were identified by Dr. Kathleen Immelman of the
Department of Botany at WSU and voucher specimens were prepared and deposited in the Kei herbarium
(CN01 and CN02). The plant leaves were washed with tap water to remove dirt and soil particles. The plant
leaves were placed on cardboards and dried at 50 ˚C for 24 hours in a hot air oven (Heraeus, Schutzart). The
plant material was powdered (ATO Mix, Cambridge) and stored in airtight containers at 5 ˚C for further analy-
sis.
2.3. Preparation of Plant Extracts
Approximately 400 g of dried powdered plant material was exhaustively extracted in different solvents. The
plant material was separately soaked in 700 mL of concentrated hexane, acetone, ethanol, methanol and water in
2L volumetric flasks (Schott, Durban). The flasks were placed in an orbital shaker incubator (labcon, Marais-
burg) for 48 h [15]. The plant material was centrifuged at 1006.2 x g for 5 minutes and filtered through a fritted

C. Njume et al.
36
filter funnel of pore size 60 Å. The procedure was repeated twice and the three extracts combined and concen-
trated to dryness under vacuum (Büchi, Switzerland). The dried crude extract was collected in porcelain evapo-
rating dish (Haldenwanger, Berlin) and left open in a biosafety class 2 cabinet (Durban, South Africa) for com-
plete evaporation of residual solvents. The aqueous extracts were lyophilized [16]. A 2-g sample of each extract
was used for the preliminary bioassay, and where possible, another 2 g or more was put in universal bottles and
kept in the extract bank. Stock solutions were prepared by dissolving the extracts in 80% acetone (a concentra-
tion we found to be non inhibitory to any of the bacterial strains tested).
2.4. Screening of Crude Extracts for Antibacterial Activity
The agar-well diffusion method was used for this analysis [17]. Briefly, each bacterial suspension prepared in
0.9% saline (McFarland turbidity standard 0.5) was inoculated by spreading on Mueller Hinton agar (Oxoid Llt.,
Basingstoke, UK) plates and allowed to dry for 15 minutes. Wells (6mm in diameter) were punched into the
agar using a sterile stainless steel borer and filled with 70 μL of the extract at 100 mg/mL. Seventy microliters of
0.005 mg/mL ciprofloxacin and 80% acetone were included in all experiments as positive and negative controls,
respectively. The plates were incubated at 37˚C for 24 hours, after which the diameters of zones of inhibition
were measured in millimetres. The experiment was repeated twice, and means for zones were recorded.
2.5. Determination of Minimum Inhibitory Concentration (50% Susceptibility)
Based on their good antimicrobial activity in the screening, the aqueous extracts were selected for determination
of minimum inhibitory concentration (MIC50) using the micro broth dilution technique performed in 96-well
plates [18]. Two-fold dilutions of the extract and control antibiotic (Ciprofloxacin) were prepared in the wells
containing Mueller Hinton broth. The final extract concentration ranged from 20 - 0.31 mg/mL while that of the
control antibiotic ranged from 0.005 - 0.00015 mg/mL. Exactly 20 μL of an 18-hour old broth culture
(McFarland turbidity standard 0.5) of the bacteria was inoculated into 180 μL of extract-containing culture me-
dium. Negative control wells were prepared with culture medium only and bacteria suspension and broth only
respectively. An automatic ELISA micro plate reader (Tokyo, Japan) adjusted to 590 nm was used to measure
the absorbance of the plates before and after 24-hour incubation. The absorbencies were compared to detect an
increase or decrease in bacterial growth and the values plotted against concentration. The lowest concentration
of the test extract resulting in inhibition of 50% of bacterial growth was recorded as the MIC.
2.6. Determination of Minimum Bactericidal Concentration (MBC)
The MBC was determined following well established procedures [19]. Briefly, the entire content of the MIC
well (≈200 μL) was serially tenfold diluted in 0.9% saline. A loop-full was taken from each tube and inoculated
into Mueller Hinton agar plates and incubated for 24 h at 37˚C. The MBC was recorded as the lowest concentra-
tion of the extract or antibiotic that gave complete inhibition of colony formation of the test bacteria at the later
cultivation.
2.7. Statistical Analysis
The statistical package used for analysis was SPSS v18.0 (SPSS Inc., Chicago, IL). One-way analysis of vari-
ance (ANOVA) was used to compare the mean difference in inhibitory activities of extracts and control antibi-
otic, followed by Turkey’s post-hoc test. Differences were considered significant at P < 0.05.
3. Results
The zones of inhibition diameters of active plant extracts ranged from 0 mm - 20 mm while those for the control
antibiotic ranged from 17 mm - 31 mm. Hexane extracts of C. album and methanol extracts of both plants were
inactive (Table 1).
3.1. Minimum Inhibitory Concentration of Active Crude Extracts and Control Antibiotic
Based on agar-well results, the most active extracts (aqueous) were selected for MIC and MBC determination
alongside the positive control antibiotic. The activity of the aqueous extracts was confirmed with MIC50 values

C. Njume et al.
37
Table 1. Antimicrobial activity of crude extracts of S. nigrum and C. album as revealed by the agar-well diffu-
sion technique.
S. nigrum C. album
H A E M W H A E M W CIP
Bact
Bs 15 0 10 0 17 0 13 0 0 14 22
11 10 10 0 20 0 21 0 0 17 29
10 0 12 0 15 0 11 0 0 17 31
Sa 0 0 10 0 10 0 0 0 0 11 19
7 0 10 0 12 0 0 0 0 10 19
9 0 9 0 14 0 10 9 0 10 17
Ef 0 0 11 0 0 0 0 0 0 17 21
0 0 15 0 0 0 0 0 0 14 19
0 0 11 0 0 0 11 0 0 14 21
Ec 9 0 9 0 11 0 10 9 0 9 21
0 0 10 0 10 0 0 0 0 10 18
0 11 13 0 10 0 10 0 0 9 18
Pa 0 0 10 0 13 0 0 0 0 15 19
0 0 13 0 12 0 0 10 0 17 27
0 0 9 0 10 0 0 0 0 16 23
Mean ± SD 4.1 ± 5.4 1.4 ± 3.6 10.8 ± 1.7 0 10.3 ± 6.0 0 5.7 ± 6.8 1.8 ± 3.8 0 13.3 ± 3.1 21.6 ± 4.2
Last row data are Mean ± SD of 15 determinations for each plant crude extract; H: hexane; A: acetone; E: ethanol; M: methanol; W: water; Bact:
bacteria; Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Ef: Enterococcus faecalis; Ec: Escherichia coli; Pa: Pseudomonas aeruginosa.
of 0.63 mg/mL - 10 mg/mL and 0.63 mg/mL - 7.5 mg/mL for S. nigrum and C. album respectively (Figure 1).
3.2. Minimum Bactericidal Concentration of Active Crude Extracts and Control Antibiotic
Aqueous crude extracts of both plants were also bactericidal against the tested bacteria with MBC values rang-
ing from 2.5 mg/mL and 5.0 mg/mL - 20 mg/mL for C. album and S. nigrum respectively (Figure 2). MIC and
MBC values of 0.0003 mg/mL and 0.001 mg/mL respectively were recorded for the control antibiotic and were
the least values in the entire study (F igur e 1 & Figure 2).
4. Discussion
Medicinal plants may constitute an important source of therapeutic compounds against human infectious organ-
isms. Many plants have been reported to contain flavonoids, alkaloids, tannins, phenols, saponins or other sec-
ondary metabolites which serve as defence mechanisms against micro organisms, insects and animals [20].
These compounds are known to act in different ways to exert antimicrobial action. The results of this study in-
dicate that crude extracts of C. album and S. nigrum have the potential for further evaluation in the search for
antibacterial compounds. Gram-positive organisms; S. aureus, E. faecalis and B. subtilis were the most suscept-
ible in the entire study while Gram-negatives; E. coli and P. aeruginosa were less susceptible (Table 1, Figur e
1 & Figure 2). The difference in susceptibility between Gram-negative and Gram-positive bacteria to antimi-
crobial agents has been reported by other researchers [20] [21] and may be attributed to structural differences in
the cell wall of both organisms. Gram-negative bacteria have a lipid protective sheath around their cell walls
which seems to shield them from the effects of antimicrobial agents [22]. All the bacteria tested were highly

C. Njume et al.
38
Figure 1. Minimum Inhibitory Concentration (mg/mL) values of plant crude
extracts and control antibiotic tested against some bacteria of medical impor-
tance.
Figure 2. Minimum Bactericidal Concentration (mg/mL) values of plant
crude extracts and control antibiotic tested against some bacteria of medical
importan ce.
susceptible to Ciprofloxacin, the control antibiotic (Table 1, Fig ure 1 & Figure 2). The plant crude extracts
were relatively less active when compared to Ciprofloxacin (P < 0.05). This was expected as the control antibi-
otic is a purified compound with excipients to facilitate activity. The crude extracts on the other hand are made
of numerous compounds; some of which may have antagonistic properties against each other. Equally important
is the fact that the quantity of the active ingredient in the crude extracts may be in minute quantities, not enough
to exhibit the type of activity demonstrated by the control antibiotic. Of all the bacteria tested, B. subtilis and S.
aureus were most susceptible to aqueous extracts of the plants, producing large zones of inhibition diameters
(Table 1), low MIC and MBC values (Figure 1 & Figur e 2). However, there were no significant differences in
antibacterial activity between the aqueous extracts of C. album and S. nigrum (P > 0.05).
5. Conclusion
The current study illustrates the antibacterial properties of crude extracts of C. album and S. nigrum against
some selected bacteria of medical importance. The study shows that aqueous extracts of both plants are inhibi-
tory and bactericidal to S. aureus, B. subtilis, P. aeruginosa, E. co li and E. fa ec alis. These findings are consis-
tent with their folkloric use in the treatment of stomach-related morbidities in the O.R. Tambo District Munici-
pality of South Africa. However, more studies to document the plants active ingredients will shed more light on
their pharmacological relevance as antibacterial agents.

C. Njume et al.
39
Acknowledgements
We are grateful to the Department of Science and Technology (DST) and the National Research Foundation
(NRF), South Africa for funding this study through a grant awarded to the Research Chair for Indigenous
Knowledge System at Walter Sisulu University, South Africa.
References
[1] Falagas, M.E., Karageorgopoulos, D.E., Leptidis, J. and Korbila, I.P. (2013) MRSA in Africa: Filing the Global Map
of Antimicrobial Resistance. PLoS One, 8, e68024. http://dx.doi.org/10.1371/journal.pone.0068024
[2] Matsuura, G.T., Pharm, D. and Barg, N. (2013) Update on the Antimicrobial Manage me nt of Foot Infections in Pa-
tients with Diabetes. Clinical Diabetes, 31, 59-65. http://dx.doi.org/10.2337/diaclin.31.2.59
[3] Balsalobre, L.C., Dropa, M. and Matte, M.H. (2014) An Overview of Antimicrobial Resistance and Its Public Health
Signi ficance. Brazilian Journal of Microbiology, 45, 1-5. http://dx.doi.org/10.1590/S1517-83822014005000033
[4] Gupta, K., Hooton, T.M. and Stamm, W.E. (2001) Increasing Antimicrobial Resistance and the Management of Un-
complicated Community-Acquired Urinary Tract Infections. Annals of Internal Medicine, 135, 45-50.
http://dx.doi.org/10.7326/0003-4819-135-1-200 1070 30-00 01 2
[5] Tamma, P.D., Cosgrove, S.E. and Maragakis, L.L. (2012) Combination Therapy for Treatment of Infections with Gram
Negative Bacteria. Clinical Microbiology Reviews, 25, 450-470. http://dx.doi.org/10.1128/CMR.05041-11
[6] Mittal, R., Aggarwal, S., Sharma, S., Chhibber, S. and Harjai, K. (2009) Urinary Tract Infections Caused by Pseudo-
monas aeruginosa: A Minireview. Journal of Infection and Public Health, 2, 101-111.
http://dx.doi.org/10.1016/j.jiph.2009.08.003
[7] Can izalez-Roman, A., Gonzalez-Nunez, E., Vidal. J.E., Flores-Villasenor, H. and Leo n -Sicairos, N. (2013) Prevalence
and Antibiotic Resistant Profiles of Diarrheagenic Escherichia coli Strains Isolated from Food Items in North Western
Mexico. International Journal of Food Microbiology, 164, 36-45. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.03.020
[8] Sood, S., Malhotra, M., Das, B.K. and Kapil, A. (2008) Enterococcal Infections and Antimicrobial Resistance. Indian
Journal of Medical Research, 128, 111-121.
[9] Oli, A.K., Sungar, R., Shivshetty, N., Hosamani, R. and Rivansiddappa, K.C. (2012) A Study of Scanning Electron
Microscopy of Vancomycin Resistant Enterococcus faecalis from Clinical Isolates. Advances in Microbiology, 2,
93-97. http://dx.doi.org/10.4236/aim.2012.22012
[10] Jeon, Y.L., Yang, J.J., Kim, M.J., Lim, G., Cho, S.Y., Park, T.S., Suh, J.T., Park, Y.H., Lee, M.S., Kim, S.C. and Lee,
H.J. (2012) Combined Bacillus licheniformis and Bacillus subtilis Infection in a Patient with oesophageal Perforation.
Journal of Medical Microbiology, 61, 1766-1769 . http://dx.doi.org/10.1099/jmm.0.042275-0
[11] Njume, C., Goduka, N.I. and George G. (2014) Indigenous Leafy Vegetables (imifino, morogo, muhuro) in South Af-
rica: A Rich an d Unexplored Source of Nutrients and Antioxidants. African Journal of Biotechnology, 13, 1933-19 42 .
[12] Gq aza , B.M., Njume, C., Goduka, N.I. and George, G. (2013a) Nutritional Assessment of Chenopodium album L. (Im-
bikican e) Young Shoots and Mature Plant-Leaves Consumed in the Eastern Cape Province of South Africa. Intern a-
tional Proceedings of Chemical, Biological and Environmental Engineering, 27-28 July, Moscow, Russia. 53, 97-102.
[13] Gqaza, M.B., Njume, C., Goduka, I.N. and Grace, G. (2013b) The Proximate Composition of S. nigrum Plant -Leaves
Consumed in the Eastern Cape Province of South Africa. International Proceedings of Chemical, Biological and En-
vironmental Engineering, 27-28 July, Moscow, Russia. 53, 103-106.
[14] Yadav, N., Vasudeva, N., Singh, S. and Sharma, S.K. (2007) Medicinal Properties of Genus Chenopodium Linn.
Natural Product Radiance, 6, 131-134.
[15] Njume, C., Afolayan, A.J. and Ndip, R.N. (2011) Diversity of Plants Used in the Treatment of Helicobacter py-
lori-Associated Morbidities in the Nkonkobe Municipality of the Eastern Cape Province of South Africa. Journal of
Medicinal Plants Research, 5, 3146-3151.
[16] Castillo-Juárez, I., González, V., Aime-Aguilar, H., Martínez, G., Linares, E., Bye, R. and Romero, I. (2009) Anti-
Helicobacter pylori Activity of Plants Used in Mexican Traditional Medicine for Gastrointestinal Disorders. Journal of
Ethnopharmacology, 122, 402-405 . http://dx.doi.org/10.1016/j.jep.2008.12.021
[17] Boyanova, L., Gergova, G., Nikolov, R., Derejian, S., Lazarova, E., Katsarov, N., Mitov, I. and Krastev, Z. (2005) Ac-
tivity of Bulgarian Propolis against 94 Helicobacter pylori Strains in Vitro by Agar-Well Diffusion, Agar Dilution and
Disc Diffusion Methods. Journal of Medical Microbiology, 54, 481-483. http://dx.doi.org/10.1099/jmm.0.45880-0
[18] Bonacorsi, C., Raddi, M.S.G., Iracilda, Z.C., Sannomiya, M. and Vilegas, W. (2009) Anti -Helicobacter pylori Activity
and Immunostimulatory Effect of Extracts from Byrsonima crassa Nied. (Malpighiaceae). Complementary and Alter-
native Medicine, 9, 1472-6882. http://dx.doi.org/10.1186/1472-6882-9-2

C. Njume et al.
40
[19] Nethathe, B.B. and Ndip, R.N. (2011) Bioactivity of Hydnora africana on Selected Bacteria Pathogens: Preliminary
Phytochemical Screening. African Journal of Microbiology Research, 5, 2820-2826 .
[20] Madduluri, S., Rao, K.B. and Sitaram, B. (2013) In Vitro Evaluation of Antibacterial Activity of Five Indigenous
P lan ts Extracts against Five Bacteria Pathogens of Humans. International Journal of Pharmacy and Pharmaceutical
Sciences , 5, 679-68 4.
[21] Cock, I. (2007) Antibacterial Activity of Selected Australian Native Plant Extracts. The Internet Journal of Microbiol-
ogy, 4, 76-81.
[22] Silhavy, T.J., Kahne, D. and Walker, S. (2010) The Bacterial Cell Envelope. Cold Spring Harbor Perspectives in Bi-
ology, 2, 1-17. http://dx.doi.org/10.1101/cshperspect.a000414