AASCIT Journal of Health
2017; 4(2): 17-21
http://www.aascit.org/journal/health
ISSN: 2381-1277 (Print); ISSN: 2381-1285 (Online)
Keywords
Neem Extracts,
Personal Hygiene,
Antibacterial Activity,
Agar-well Diffusion Method
Phytochemical Analysis
Received: July 16, 2017
Accepted: October 25, 2017
Published: November 25, 2017
Phytochemical Properties and the
in-vitro Antibacterial Activity of
Aqueous Extract of Neem
(Azadirachta indica) Seed oil on
Human Skin Microflora
Awache Ibrahim1, *, Yakubu Michael Nosano1, Longhull Benard1,
Yusuf Abdullahi1, Acha Maryfavour1, Gero Mohammed2
1
Department of Microbiology, Faculty of Pure and Applied Sciences, Federal University, Wukari,
Nigeria
2
Department of Consultancy and Production, National Research Institute for Chemical Technology,
Zaria, Nigeria
Email address
i.awache@fuwukari.edu.ng (A. Ibrahim)
*
Corresponding author
Citation
Awache Ibrahim, Yakubu Michael Nosano, Longhull Benard, Yusuf Abdullahi, Acha Maryfavour,
Gero Mohammed. Phytochemical Properties and the in-vitro Antibacterial Activity of Aqueous
Extract of Neem (Azadirachta indica) Seed oil on Human Skin Microflora. AASCIT Journal of
Health. Vol. 4, No. 2, 2017, pp. 17-21.
Abstract
The phytochemical components and antibacterial activity of aqueous extract of neem
seed oil (Azadirachta indica) on the bacterial species isolated from human skin was
investigated. The neem tree was observed to be devoid of any infection prior to cutting
the fruits (seeds) for laboratory analysis. Strategic body parts of fifteen (15) students
were swabbed using a sterile swab stick in the morning before each of them washes their
body at Federal University Wukari. Bacterial isolation was done using nutrient agar.
Identification of the bacteria isolates were done using standard techniques. Agar-well
diffusion method was used to determine the antibacterial effect of the neem oil extract on
a prepared nutrient agar. The neem seed oil extracts showed antibacterial activity against
all the test isolates. The zone of inhibition increased with increased volume. Therefore,
these results, is a further confirmation of claims by other studies on the importance of the
traditional use of neem oil against various skin ailments. Phytochemical extraction was
done using GC-MS QP2010 PLUS machine. Miliacin, Gedunin and Mahmoodin were
extracted at a very large amount; Tignic acid and Nimbolide were found to be in a large
quantity while Nimbin, Nimbidic acid, Nimbidinin and Nimbidin were found to be low.
Most of these phytochemical chemical components extracted from neem seed oil have
antibacterial activity. Others are antifungal, antimalarial and spermicidal. Therefore,
these results, further confirm the belief and other scientific studies on the importance of
the traditional use of neem seed oil in managing various human ailments. Some of the
isolates from the human skin are potential pathogens and may pose a public health
hazard to the community. Therefore, personal hygiene are strongly advocated to prevent
skin diseases.
1. Introduction
The use of different herbal mixtures and other active ingredients of medicinal plants
 AASCIT Journal of Health 2017; 4(2): 17-21
even in the health care systems, is in the increase worldwide.
In the rural regions of Africa and Asia, people have resorted
to using herbal mixtures to treat various kinds of human
ailments. Neem tree (Azadirachta indica), has gained wide
acceptance due to its effectiveness in treating various
systemic and skin ailments. The aqueous extracts of the
neem’s leaves, barks and seeds has proven to be effective [7].
The neem tree originated from the Asian parts of the world.
It is a tall perennial tree common with clear vegetation that
grows between 18m to 24m tall with a fast-growing and
robust twigs [6, 8]. The taxonomic grouping of neem tree is
as thus: Order = Rutales; Sub order = Rutinae; Family =
Meliaceae (mahogany family); Sub family = Melioideae;
Tribe = Melieae; Genus = Azadirachta and Species = indica
[6, 8].
In Nigeria, neem is prevalent; and has adapted to various
environments, even in the Sahara. This feature of neem
(strong adaptive ability to various environments), has given it
an edge especially in the aspects of erosion prevention,
therapeutic agent and afforestation programmes [6, 8, 17].
Separated into parts, its leaves are akin to a typical leaf
foliage with small white flowers. The leaves supports it’s
bunches of fruits with length of between 1.5cm to 2cm long.
These fruits each with a seed, are green when unripe and
yellow when ripe [17]. See figures 1, 2, 3 and 4.
The bark and leaf extracts has be proven to be effective in
the treatment and control of intestinal helminthiasis, leprosy,
respiratory disorders, constipation, rheumatism, chronic
syphilitic sores, blood morbidity, biliary afflictions, itching,
skin ulcers, burning sensations and pthysis. and apathetic
ulcer that have not manisfested [7]. They also possesses
interferon inducing and anti-tumour activities [9]. The twigs
are used as oral deodorant, toothache reliever and for
cleaning of teeth [14]. The fruits (seeds) of neem have one or
more kernels from which the oil is extracted. The oil is
extracted by crushing and pressing the kernels and then by
purifying the extract. Neem oil extracted from its seeds have
anti-fertility ability and also have the ability to stimulate cell
mediated immune response [14, 18]. It contains numerous
components. Miliacin forms the bitter principles of the oil,
tignic acid (5-methyl- 2-butanic acid) responsible for the
distinctive odour of the oil [16]. Other compounds include
nimbin, nimbolide, gedunin (antifungal, antimarial),
mahmoodin [antibacterial], which have antifungal,
antimalarial, antibacterial, spermicidal and anti-inflammatory
properties [8, 13, 15, 16, 19].
Numerous microbes colonizes human skin. These includes
but not limited to Staphylococcus spp, Corynebacterium spp,
Propionibacterium spp, Streptococcus spp and Pseudomonas
spp. Some of these microbes are pathogenic, others are
potential pathogens. The molecular mechanisms of microbial
virulence employed by these microbes depends on the
specific events involved in the host–microbe interaction.
However, some of these organisms may protect the host. [2].
Several studies on neem seed oil reported antimicrobial
activity against Escherichia coli and Kleibsiella pneumonia,
18
Candida albicans and Polio virus, oral pathogens and
dermatophytes [14]. Again, [4, 10 and 11] reported in their
studies reported bactericidal effect of neem seed oil on
Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa. This research investigated the phytochemical
components of the aqueous extract of neem seed oil and
tested the antibacterial activity (in vitro) of the neem seed oil
on the bacteria isolated from human skin in Wukari, Taraba
State, Nigeria.
2. Materials and Methods
2.1. Sample Collection and Preparation
The seed samples of Azadirachta indica were collected
from the Federal University Wukari, Taraba State, Nigeria.
The samples were immediately washed using tap water and
sun-dried for 5 days. The dried seeds were then crushed to
remove the kernels. The kernels were then further pulverized
using a pestle and mortar. Exactly twenty (50g) of the
powder was weighed using an electronic weighing balance
and dissolved into 500mL of boiled water. Using a spatula,
the mixture was stirred and allow to settle. After about 15 to
20 minutes, the oils in the kernels settled at the top. And the
oil was carefully decanted to get the neem seed oil extract.
2.2. Bacterial Analysis
Using a sterile swab stick, swab samples were collected
from strategic body parts of students (palms, armpits, necks
and faces) of Federal University Wukari, Taraba State,
Nigeria at about 8:00hr before taking their baths.
Immediately, the swab sticks were immediately streaked on
to a prepared nutrient agar and incubated at 37°C for 24h.
The distinct colonies that emerged after incubation were
identified using microscopy and then sub-cultured on to a
prepared nutrient agar to obtain a pure culture. Then, the pure
bacterial isolates obtained were Gram-stained and identified
by comparing their characteristics with those of known taxa
using the schemes of [3] and by the conventional
bacteriological test methods and by reference to the keys
outlined by [1]. See Table 1.
2.3. Antibacterial Analysis of the Sample
To determine the antibacterial activity of the aqueous
extracts of the neem seed oil, an aliquot (0.1mL) of the neem
seed oil, was obtained using sterile syringe and inoculated
onto prepared nutrient agar plates by agar well diffusion
method, using a sterile cork borer of diameter size 6.00mm,
to cut the solid Agar surface; (3 wells per 90 mm diameter
plate) as carried out by [26]. Incubation was done at 37°C for
24 hours. Zone of clearance (inhibition) was determined and
measured round the well. The minimum inhibitory
concentration (MIC) was interpreted as the lowest
concentration of the extract that did not show any visible
growth when compared to control plates. See table 2
19 Awache Ibrahim et al.: Phytochemical Properties and the in-vitro Antibacterial Activity of Aqueous Extract of
Neem (Azadirachta indica) Seed oil on Human Skin Microflora

2.4. Phytochemical Analysis of Neem Seed to increase to 280°C at a rate of 20°C ending with at 5
Oil (GC-MS Analysis) minutes. The total run time was 35 minutes. At a temperature
of 200°C, the MS transfer line was maintained with the
The samples of the twigs of Azadirachta indica collected source temperature maintained at 180°C. The GC-MS was
from Wapan-Nghaku around the Federal University Wukari, analyzed using an acceleration electron impact ionization at
Taraba State, Nigeria were immediately washed up using tap 70eV and data were evaluated using Total Ion Count (TIC)
water, crushed into a pieces using mortar and pestle. The for compound identification and quantification. The
pastes were sun-dried for 3 to 5 days. Using an electronic spectrums of the components were compared with the
weighing balance, exactly 1000g was weighed and sent to the database of spectrum of known components stored in the
Department of Consultancy and Production, National GC-MS library. See Table 3
Research Institute for Chemical Technology Zaria, Nigeria,
for phytochemical analysis using a Gas Chromatography –
Mass Spectrometry Analysis (GC –MS). GC-MS analysis 3. Results
was carried out using the GCMS-QP2010 PLUS Table 1. Showing bacterial species isolated from human skin.
SHIMADZU. The column used was Perkin Elmer Elite - 5
capillary column measuring 30m × 0.25mm with a film S/N Bacteria Isolated
1. Streptococcus spp
thickness of 0.25mm composed of 95% Dimethyl
2. Staphylococcus aureus
polysiloxane. Helium was the carrier gas used at a flow rate 3. S. epidermidis
of 0.5mL/min. And the sample injection volume that was 4. E. coli
utilized was 1µL, maintaining 250°C inlet temperature. 5. Pseudomonas aeruginosa
Initially, the oven temperature was programmed at 110°C for
4 min, then an increased to 240°C. And further programmed
Table 2. Showing the zone of inhibition of A. indica seed oil on the bacterial isolates from human skin.

Diameter of zone of clearance (mm) on Bacterial Isolates

Aqueous extract of neem seed oil (mL)
Streptococcus spp S. aureus S. epidermidis P. aeruginosa E. coli
0.1 03 05 04 06 2.5
0.2 3.8 6.3 5.8 06 3.8
0.3 5.2 08 7.2 09 06
0.4 07 09 09 10 09
0.5 9.3 11.3 11 13 10
Average 5.66 7.92 7.40 8.80 6.26

The results captured by this table 2 (above) showed that all

the microcorganisms above are sensitive to the Aqueous
extract of neem seed oil at various concentration. P.
aeruginosa and S. aureus were the most sensitive in this
study at all concentrations used. While Streptococcus spp and
E. coli were the least sensitive.

Figure 2. Fruits of Neem tree (Muthulinggam, N and Partiban, S., 2014).

Figure 3. Seed of A. indica with endocarp (Muthulinggam, N and Partiban,

Figure 1. Shows Azadirachta indica plant (Sultana et al., 2011). S., 2014).
 AASCIT Journal of Health 2017; 4(2): 17-21
Figure 4. Seed of A. indica without endocarp (Muthulinggam, N and
Partiban, S., 2014).
Table 3. Phytochemical properties of neem seed oil aqueous extract.
Phytochemical Composition of
Quantity Biological activity
neem seed oil
Miliacin +++
Tignic acid ++
Nimbin + Spermicidal
Nimbolide ++ Antibacterial
Gedunin +++ Antifungal
Mahmoodin +++ Antibacterial
Nimbidic acid + Anti-inflammatory
Nimbidinin + Anti-inflammatory
Nimbidin + Anti-inflammatory
Keys: (+ = Low amount, ++ = Large amount, +++ = Very large amount, - =
Absent) [8, 13, 15, 16]
4. Discussion
Table 1 shows the bacterial species isolated from human
skin. Table 2 presented the zone of inhibitions (mm) of
neem seed oil against the test bacteria. Table 3 presented
the phytochemical compounds extracted from the neem
seed oil.
The antibacterial activity of the neem seed oil extracts
showed growth inhibition on all tested bacteria. However, the
results showed that increased volume of the extract have
higher zone of clearance (in millimeter). On one hand, lesser
volume (mL) of the neem seed oil extract showed lesser
effect on E. coli and Streptococcus spp but the effect was
slightly high on S. epidermidis. However, P. aeruginosa and
S. aureus showed a high sensitivity on the same volume. On
the other hand, higher volume of the neem seed oil, indicated
considerably higher zone of inhibition on all the isolates.
These effect are more pronounced on P. aeruginosa, S.
aureus. S. epidermidis and E. coli accordingly. These results
agrees with several other similar studies [4, 10, 11, 14].
Differences in this study compared to other relevant studies
at different locations and time is most likely due to the
methods of processing, time and location of study.
The phytochemical components of need seed oil showed
that Miliacin, Gedunin and Mahmoodin were extracted at a
very large amount; Tignic acid and Nimbolide were found to
be in a large quantity while Nimbin, Nimbidic acid,
Nimbidinin and Nimbidin were found to be low. Most of
these phytochemical chemical components extracted from
20
neem seed oil have antibacterial activity. Others are
antifungal, antimalarial and spermicidal. Therefore, these
results, further confirm the belief and other scientific studies
on the importance of the traditional use of neem seed oil in
managing various human ailments [8, 13, 15, 16].
5. Conclusion
To this, it is safe to say that these results, further
revalidated other related studies on the importance of the use
of neem seed oil extract in managing various human ailments.
Therefore, routine personal hygiene is strongly advocated for
preventive measures against some of the diseases
aforementioned, as some of the isolates from human skin are
potential pathogens and may pose a public health hazard to
the community.
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Neem (Azadirachta indica) Seed oil on Human Skin Microflora

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