Inhibitory and Bactericidal Potential of Some Indigenous Functional Food-Plants Used in the O.R. Tambo District Municipality of South Africa

doi:10.4236/jbm.2014.26006

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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

Email: *[email protected]; *[email protected]

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.

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.

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.

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.

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.

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.

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