Annals of Agricultural Science 61 (2016) 247–250

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Impact of Moringa aqueous extract on pathogenic bacteria and fungi

in vitro
Latifa A. Al_husnan ⇑, Muneera D.F. Alkahtani
Princess Nora Bint Abdul Rahman University, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Moringa peregrine have many benefits. In this study aqueous extract of Moringa plant inhibited the activ-
Received 7 April 2016 ity of these bacteria which include Bacillus cereus; Staphylococcus aureus; Pseudomonas aeruginosa,
Accepted 25 June 2016 Klebsiella pneumonia, Escherichia coli; Enterococcus cloacae; Salmonella typhi and; Proteus vulgaris.
Available online 27 December 2016
Moringa extracts has shown an impact on the growth of bacteria on the Blood with inhibition zone vari-
able (23.5 ± 0.45 to 12.5 ± 0.50 mm) according to the type of bacteria. The mean growth inhibition per-
Keywords: centages were 85.9 ± 0.42 to 65.3 ± 0.34 nm against all tested bacteria. As regards to fungi, high
Moringa
potency extract displayed zones of inhibition of P10 mm, moderate potent extracts gave zones of inhi-
Pathogenic bacteria
Fungi
bition between <10 and 9 mm. The results indicated that, Moringa aqueous extract played variable anti-
Minimum inhibition concentration (MIC) fungal activity ranged from high (18 ± 0.54 mm), moderate (13.2 ± 0.58 mm) and low (6.6 ± 0.47 mm).
The inhibition zones diameter in millimeters against A. niger, A. flavus, P. italicum, F. oxysporum, R. stolo-
nifer, Alternaria sp., C. albicans, C. parapsilosis were 15.2 ± 0.52, 12.4 ± 0.55, 10.5 ± 0.26, 9.4 ± 0.71,
13.2 ± 0.58, 6.6 ± 0.47, 12 ± 0.44 and 18 ± 0.54, respectively. On the other hand, the mean inhibition of
growth (as percentages) were 75.2 ± 0.55, 59.4 ± 0.75, 58.2 ± 0.63, 46.5 ± 0.63, 62.5 ± 0.77, 24.5 ± 0.65,
20.3 ± 0.75 and 80.00 ± 0.70% respectively. Thus, the aqueous extract of Moringa leaves showed antimicro-
bial activity against tested bacteria, fungi and yeasts at different concentrations.
Ó 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

Introduction calcium, phosphorus, iron, vitamin D, essential amino acids, as well

Moringa oleifera (Moringaceae), also known as ‘‘the tree of life”,
is mainly native to India and Africa. It is considered one of the most
useful trees in the world because every part of the Moringa tree can
be used for food, medication and industrial purposes (Moyo et al.,
2011). In particular, the leaves can be eaten fresh in salad, cooked,
or stored as dried powder for many months without loss of nutri-
tional value. Apart from treating malnutrition, in rural areas of
Uganda, its leaves are used to treat a wide range of medical condi-
tions such as HIV/AIDS-related symptoms, bronchitis, ulcers,
malaria and fever (Kasolo et al., 2010). In this regard, leaf extracts
of M. oleifera have been reported to exhibit antioxidant activity
both in vitro and in vivo due to their abundance of phenolic acids
and flavonoids (Vongsak et al., 2013; Al Khateeb et al., 2013). Pre-
vious phytochemical analysis of M. oleifera from different countries
have shown that the leaves are particularly rich in potassium,
Peer review under responsibility of Faculty of Agriculture, Ain-Shams University.
⇑ Corresponding author.
E-mail address: bio-tech-321@hotmail.com (L.A. Al_husnan).
as known antioxidants such as carotene, vitamin C, and flavonoids
(Mbikay, 2012). M. peregrina (Forssk Fiori) is also widely grown,
but to a much lesser extent than M. oleifera in Saudi Arabia, India
and south of Iran (Ghodsi et al., 2014). The specific components of
Moringa preparations that have been reported to have hypotensive,
anticancer, and antibacterial activity include 4-(40 -O-acetyl-a-L-
rhamnopyranosyloxy)benzylisothiocyanate, (-a-L-rhamnopyrano-
syloxy) benzyl isothiocyanate, niazimicin, pterygospermin, benzyl
isothiocyanate, and 4-(a-L rhamnopyranosyloxy) benzyl glucosi-
nolate. It is also rich in a number of vitamins and minerals as well
as other more commonly recognized phytochemicals such as the
carotenoids (including b-carotene or provitamin A) (Nepolean
et al., 2009). Bacteria and fungi are of human and veterinary
importance as outlined later. Bacillus cereus has been implicated
in food-borne intoxication (Granum and Lund, 1997). Escherichia
coli, Staphylococcus aureus and Pseudomonas aeruginosa cause dis-
eases like mastitis, abortions and upper respiratory complications
(Fraser, 1986). Streptococcus faecalis is a pathogenic bacteria
commonly found in the intestines of birds (Granum and Lund,
1997). Penicillium notatum induces hypersensitivity, pneumonitis

http://dx.doi.org/10.1016/j.aoas.2016.06.003
0570-1783/Ó 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
248 L.A. Al_husnan, M.D.F. Alkahtani / Annals of Agricultural Science 61 (2016) 247–250

in animals. Candida albicans is reported to cause vaginitis and yeast Table 2

mastitis. These necessitate searching for antibiotics that could be Screening of antifungal activity of (100 mg/ml) aqueous extract of Moringa leaves.

used against microbes. Moringa oleifera Lam is one of the best Tested fungi/yeasts Clear zones diametera (mm) Mean inhibition %b
known, widely distributed (Anwar et al., 2007). For this reason, Aspergillus niger 15.2 ± 0.52 75.2 ± 0.55
the purposes of this study was to treat pathogenic bacteria, fungi Aspergillus flavus 12.4 ± 0.55 59.4 ± 0.75
and yeasts using aqueous Moringa plant extract as antimicrobial Penicillium italicum 10.5 ± 0.26 58.2 ± 0.63
and impact on biofilm formation. As well as determination of Min- Fusarium oxysporum 9.4 ± 0.71 46.5 ± 0.63
Rhizopus stolonifer 13.2 ± 0.58 62.5 ± 0.77
imum inhibition concentration (MIC) for Moringa aqueous extract. Alternaria sp. 6.6 ± 0.47 24.5 ± 0.65
Candida albicans 12 ± 0.44 20.3 ± 0.75
Material and methods Candida parapsilosis 18 ± 0.54 80.00 ± 0.70
a
The test was done using the diffusion agar technique, well diameter: (6.0 mm).
b
Moringa oleifera (Moringaceae) leaves were collected from Viro- Broth micro-dilution method, the experiments were performed in triplicate and
logical greenhouse of the Microbiology Dept., Faculty of Agricul- the data are expressed in the form of mean ± SE.

ture, Ain Shams University, Egypt.

Preparation of Moringa leaves extract
Leaves extract were prepared according to Oludura. The fresh
leaves were air dried at room temperature to constant weight.
The dried leaves were ground into powder. A weight of 25 g of
the powdered leaves was extracted in 100 ml of demonized dis-
tilled water in conical flask (aqueous extract). The conical flask
was shaken at 120 rpm for 30 min. The extract was filtered by fil-
tration system using membrane filter (pore size 0.45 lm) before
use.
Microorganisms
For this study, eight isolates of bacteria and ten Fungi and yeast
isolates (Tables 1 and 2) were obtained from Microbiology Lab. of
the Microbiology Dept., Faculty of Agriculture, Ain-Shams Univer-
sity, Egypt.
Antibacterial activity
Disc diffusion assay
The susceptibility of the bacteria to Moringa aqueous extract
was estimated by measuring the diameter of zone inhibition and
values as average of three replicates according to Narms (2002).
Antifungal activity
Agar well diffusion assay
The susceptibility of the fungi to Moringa aqueous extract was
estimated on Sabouraud dextrose Agar (SDA) by measuring the
diameter of zone inhibition and values as average of three repli-
cates according to Albuquerque et al. (2006).
Mean growth inhibition percentage by ELISA reader
The bacterial suspension was diluted to (106 CFU/ml) using
Mueller Hinton Broth (MHB). As well as the fungi suspension
was diluted to (106 cfu/ml) in Sabouraud dextrose broth (SDB).
The adjusted microbial inoculums (100 ll) were added to each
well of sterilized 96 well flat – bottomed micro titer plate contain-
ing the tested concentration (100 mg/ml) of Moringa aqueous
extract. As a result, last inoculums concentration of (5  105 CFU/
ml) was obtained in each well. Two wells containing bacterial sus-
pension without Moringa aqueous extract (Growth control) and
two wells containing media only as background control were
included in this plate. Optical density was measured at 620 nm
after 24 h at 37 °C incubation using ELISA micro plate reader
(Sun Rise-TECAN, Inc., USA). The percentage of bacterial growth
reduction (GR) was calculated using as reference the control treat-
ment (without extract) as:
GR% ¼ C  T=C  100
where C = cell concentration (control) and T = cell concentration
(extract treatment). Three replicates were considered. The results
were recorded as means ± SE of the triplicate experiment (NCCLS/
CLSI, 2007).
Determination of minimum inhibition concentration (MIC)
MIC of Moringa aqueous extract was determined by the broth
micro dilution method as approved by the guidelines of Clinical
and Laboratory Standards Institute (NCCLS/CLSI, 2007).
Antivirulence activity
Biofilm formation assay of bacterial isolates
Tissue culture plate method as a quantitative test as described
by Christensen et al. were implemented in this study. The inocu-
lated trypticase soy broths with tested bacteria were incubated
at 37 °C for 24 h. The cultures were diluted 1:100 with broth med-
ium. Individual well of sterilized 96 wells of flat bottom polystyr-
ene Tissue culture plates (Sigma-Aldrich, USA) were filled with
200 ll of diluted cultures. Negative control wells were filled with
200 ll of diluted broth medium. After incubation, the wells were
washed with 0.2 ml of phosphate buffer (pH, 7.2) three times for
removing free floating bacteria by gentle tapping. Biofilm formed
by bacteria adherent to the wells were fixed by 2% sodium acetate
and stained by crystal violet (1%). Excess stain was removed by
deionized water and dried. Optical density of stained bacteria
adherent recorded by using ELISA micro-titer - plate reader (Sun
Rise – TECAN, Inc., USA).

Table 1
The growth inhibition of Moringa aqueous extract on bacterial isolates using micro-dilution method and measured by ELISA plate reader.

Bacterial isolates Growth inhibition (O.D.620) Bacterial isolates Growth inhibition

a a
ELISA reader Inhibition zone ELISA reader Inhibition zone (mm)
B. cereus 85.9 ± 0.42 21.8 ± 0.55 P. aeruginosa 69.9 ± 0.20 12.5 ± 0.50
K. pneumonia 75.5 ± 0.78 15.2 ± 0.48 S. aureus 75.6 ± 0.70 15.5 ± 0.60
E. cloacae 74.63 ± 1.2 23.4 ± 0.27 S. typhi 79.7 ± 0.36 23.5 ± 0.45
E. coli 85.0 ± 0.27 20.8 ± 0.55 P. vulgaris 65.3 ± 0.34 15.5 ± 0.56
a
Mean of 3 replicates ± standard error ((100 mg/ml)/well).
 L.A. Al_husnan, M.D.F. Alkahtani / Annals of Agricultural Science 61 (2016) 247–250
Results
Bioactivity of Moringa aqueous extract
The aqueous extract of Moringa leaves were assessed for antimi-
crobial activity to inhibit pathogenic microorganisms for repre-
senting a good alternative to the use of traditional antimicrobials
in therapy.
Antibacterial activity
The results of the clear zone inhibition diameter and mean
growth inhibition percentages in Table 1 showed the antibacterial
activity of aqueous Moringa leaves extract against bacterial iso-
lates. The results indicated that, the effect of the tested extract
showed variable inhibition zones ranging from 23.5 ± 0.45 to
12.5 ± 0.50 mm according to the type of bacteria. The mean growth
inhibition percentages were 85.9 ± 0.42 to 65.3 ± 0.34 against all
tested bacteria. Moringa extract, showed low inhibition effect on
growth of P. aeruginosa while had high antibacterial activity
against B. cereus compared with the other tested bacteria (Table 1).
Antifungal activity
The application of The Moringa leaves aqueous extract can be
used as inhibitor of Aspergillus niger; A. flavus, Alternaria spp., Fusar-
ium spp. R. stolonifer and Penicillium sp.; Candida. albicans; C. glab-
rata and C. Tropicalis. As regards to fungi, high potency extract
displayed zones of inhibition of P10 mm, moderate potent
extracts gave zones of inhibition between 10 and 9 mm. Antifungal
potential of aqueous extract of Moringa leaves (100 mg/ml.) was
tested against fungi and yeasts using inhibition zone diameter by
agar well diffusion and by mean growth inhibition percentage
using micro-dilution method. The results were exhibited that, Mor-
inga aqueous extract exhibited variable antifungal activity ranged
from high (18 ± 0.54 mm), moderate (13.2 ± 0.58 mm) and low
(6.6 ± 0.47 mm) as shown in Table 2.
In the presence of the total extract, the inhibition zones diame-
ter against A. niger, A. flavus, P. italicum, F. oxysporum, R. stolonifer
Alternaria sp., C. albicans, C. parapsilosis were 15.2 ± 0.52 mm,
12.4 ± 0.55 mm, 10.5 ± 0.26 mm, 9.4 ± 0.71 mm, 13.2 ± 0.58 mm,
6.6 ± 0.47 mm, 12 ± 0.44 mm and 18 ± 0.54 mm respectively. On
the other hand, the mean growth inhibition percentages with the
total extract against the same tested fungi and yeasts were
75.2 ± 0.55%, 59.4 ± 0.75%, 58.2 ± 0.63%, 46.5 ± 0.63%, 62.5 ± 0.77%,
24.5 ± 0.65%, 20.3 ± 0.75% and 80.00 ± 0.70%, respectively. The total
extract experienced low potency towards both Alternaria sp.; F.
oxysporum and C. albicans (Table 2).
Minimum inhibitory concentrations (MIC)
According to the results showed in Table 3, the minimum inhi-
bitory concentration, MIC, of Moringa leaves aqueous extract
Table 3
MICs (mg/ml) of aqueous extract of Moringa leaves against bacterial, fungi and yeasts
isolates.
Bacterial isolates MIC (mg/ml) Fungi and yeasts
B. cereus 0.652 A. niger
K. pneumonia 1.275 A. flavus
E. cloacae 1.246 P. italicum
E. coli 1.265 F. oxysporum
P. aeruginosa 5.265 Rh. stolonifer
S. aureus 1.234 Alternaria sp.
S. typhi 0.675 C. albicans
P vulgaris 2.125 C. tropicalis
249
against tested bacteria, fungi and yeasts showed different concen-
trations at which no fungal growth is detected (Alternaria sp. and C.
albicans). The aqueous extract against bacterial isolates indicated
that, MIC was variable according to the type of bacteria (range
from 0.652 to 5.265 mg/ml) against all tested bacteria. Moringa
extract, showed low concentration effect on growth of B. cereus
(0.652 mg/ml) while had high inhibitory effect on growth against
P. aeruginosa (5.265 mg/ml) compared with the other tested bacte-
ria (Table 3). Regarding fungi and yeasts, high MIC against F. oxys-
porum (18.75 mg/ml); P. italicum (17.95 mg/ml); moderate MIC
against A. flavus (9.75 mg/ml); low MIC against A. niger (3.52 mg/
ml) and R. stolonifer (3.20 mg/ml).
Discussion
Evaluating the antibacterial and antifungal activity of M. oleifera
extracts against bacteria; fungi and yeasts isolates was performed
in this work. For this, the ethanol extracts of pods, seeds, leaves,
stems and flowers of M. oleifera were tested against microbial
pathogens. The antibacterial and antifungal activities were deter-
mined by micro-dilution, based on the M27 and M38-A3-A2 CLSI
standards.
The leaf extracts of Moringa oleifera showed varying antimicro-
bial activity on wide range of microorganisms. The extract was
more effective than traditional antibiotics to combat the patho-
genic microorganisms studied. The chance to find antimicrobial
activity was more apparent in chloroform than petroleum ether
extracts of the same plants. The plant could be a source of new
antibiotic compounds. Further work is needed to isolate the sec-
ondary metabolites and study of metabolic interchanges in bacte-
rial metabolic pathways when applying this extract.
The difference in bacterial response to the Moringa extracts was
possibly due to the nature of the bacterial species. The M. oleifera
acetone extract, however, showed greater anti-bacterial activity
against Gram-negative bacteria than Gram-positive bacterial
strains. These results are in contrast to other researchers’ findings
who reported that most plant extracts have more activity against
Gram–positive bacteria (Ashafa and Afolayan, 2009). Moringa olei-
fera Lam is one of the best known, widely distributed plants
(Anwar et al., 2007). For this reason, the purposes of this study
was controlling pathogenic bacteria, fungi and yeasts using aque-
ous Moringa plant extract as antimicrobial and their impact on bio-
film formation. As well as determining Minimum inhibition
concentration (MIC) for Moringa aqueous extract against tested
microbial pathogen.
In conclusion, these findings have proven that genetic diver-
gence is very high in these Moringa species and it can therefore
be inferred from the data that the Moringa populations will be a
very good germplasm material for the future breeding and
improvement of this economically important tree crop. Genotypes
that are far apart identified based on their genetic similarity coef-
ficient (like T-01, T-06, M-01, and M-02) should be selected for
future breeding (Rufai et al., 2013).
References
Al Khateeb, W., Bahar, E., Lahham, J., Schroeder, D., Hussein, E., 2013. Regeneration
MIC (mg/ml)
and assessment of genetic fidelity of the endangered tree Moringa peregrina
3.52 (Forssk.) Fiori using inter simple sequence repeat (ISSR). Physiol. Mol. Biol.
9.75 Plants 19, 157–164.
17.95 Albuquerque, C.C., Camara, T.R., Marian, R.D.R., Willadino, L., Marcelino, C., Ulisses,
18.75 C., 2006. Antimicrobial action of the essential oil of Lippia gracilis Schauer. Braz.
3.20 Arch. Bio. Tech. 49 (4), 527–535.
ND Anwar, F., Latif, S., Ashraf, M., Gilan, A.H., 2007. Moringa oleifera: a food plant with
multiple medicinal uses. Phytother. Res. 21, 17–25.
ND
Ashafa, A.O.T., Afolayaan, A.J., 2009. Assessment of the antimicrobial activity of the
4.50
root extracts from Chrysocoma ciliata L. Afr. J. Microbiol. Res. 3 (11), 700–703.
250 L.A. Al_husnan, M.D.F. Alkahtani / Annals of Agricultural Science 61 (2016) 247–250
Fraser, C.M., 1986. The Merck Veterinary Manual. Merck, New Jersey.
Ghodsi, R., Sadeghi, H., Asghari, G., Torabi, S., 2014. Identification and cloning of
putative water clarification genes of Moringa peregrina (Forssk.) Fiori in E. coli
Xl1 blue cells. Adv. Biomed. Res. 27, 3–57.
Granum, P.E., Lund, T., 1997. Bacillus cereus and its food poisoning toxins. FEMS
Microbiol. Lett. 157, 223–228.
Kasolo, J.N., Bimenya, G.S., Ojok, L., Ochieng, J., Ogwal-Okeng, J.W., 2010.
Phytochemicals and uses of Moringa oleifera leaves in Ugandan rural
communities. J. Med. Plants Res. 4, 753–757.
Mbikay, M., 2012. Therapeutic potential of Moringa oleifera leaves in chronic
hyperglycemia and dyslipidemia: a review. Front. Pharmacol. 3, 24.
Moyo, B., Masika, P.J., Hugo, A., Muchenje, V., 2011. Nutritional characterization of
Moringa (Moringa oleifera Lam.) leaves. Afr. J. Biotechnol. 10, 12925–12933.
NARMS, 2002. National Antimicrobial Resistance Monitoring System, Enteric
Bacteria. CDC, USA.
‘‘NCCLS/CLSI” National Committee for Clinical Laboratory Standards/Clinical and
Laboratory Standards Institute (2007): Performance Standards for
Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement,
M2–A9 and M7–A7. Wayne, P.A., U.S.A.
Nepolean, P., Anitha, J., Emilin, R.R., 2009. Isolation, analysis and identification of
phytochemicals of antimicrobial activity of Moringa oleifera Lam. Curr. Biotica.
(3), 33–39
Rufai, Shamsuddeen, Hanafi, M.M., Rafii, M.Y., Ahmad, S., Arolu, I.W., Ferdous,
Jannatul, 2013. Genetic dissection of new genotypes of drumstick tree (Moringa
oleifera Lam.) using random amplified polymorphic DNA marker. BioMed Res.
Int., 6
Vongsak, B., Sithisarn, P., Mangmool, S., Thongpraditchote, S., Wongkrajang, Y.,
Gritsanapan, W., 2013. Maximizing total phenolics, total flavonoids contents
and antioxidant activity of Moringa oleifera leaf extract by the appropriate
extraction method. Ind. Crops Prod. 44, 566–571.