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Research Journal of Applied Sciences, Engineering and Technology 2(2): 133-137, 2010
ISSN: 2040-7467
© Maxwell Scientific Organization, 2010
Submitted Date: September 04, 2009 Accepted Date: September 24, 2009 Published Date: March 10, 2010
Corresponding Author: A.R. Oyi, Department of Pharmaceutics and Pharmaceutical Microbiology, Faculty of Pharmaceutical
Sciences, Ahmadu Bello University, Zaria -Nigeria
133
Formulation and Antimicrobial Studies of Coconut (Cocos nucifera Linne) Oil
A.R. Oyi, J.A. Onaolapo and R.C. Obi
Departm ent of Pharmaceutics and Pharmaceutical M icrobiology,
Faculty of Pharmaceutical Sciences, Ahm adu Bello University , Zaria -Nigeria
Abstract: Coco nut oil obtained from the nuts of Cocos nucifera was formulated into creams in orde r to
standardize its use an d present it in an elegant form. Using the fusion method, oil in water (o/w ) creams were
formulated in concentrations of 5 to 40% w/w of oil.The release of active ingredients from creams was
investigated using cream challenge and skin inoculation tests, whereby creams were exposed to various spots
on skin inoculated with Ps. aeruginosa ATCC 7853, E. coli ATCC 9637, P. vulgaris (clinical isolate),
B. subtilis ATCC 607 and C. albicans ATCC 10231. In addition A. niger (clinical isolate) and S. aureus ATCC
13709 were used for antimicrobial screening.The stability of creams was also evaluated using a standard
method. The results showed that active ingredients of the coconut oil were released from the creams; this was
shown from the good antimicrobia l activity of the cream confirming that all formulation ingredients were
compatible and did not interfere with activity of the oil. The creams were also found to b e stable, as a result
of their ability to withstand shock and maintain their physical characteristics.
Key words: Antimicrobial, fusion, skin penetration
INTRODUCTION
The non-precise standardization of dosage and
unhygien ic practice environments in traditional medicine
setups are factors militating against traditional medical
practitioners. Such unethical practices could lead to
preventab le hazards such as therapeutic failure, toxicity,
emergence of resistance and other adverse effects on the
consumers. Formulation into designed dosage forms is a
gateway towards scientific evaluation and standardization
of crude drugs.
Skin infection s are widely encountered in the tropics
with lots of orthodox remedies involving the use of
systemic antibiotics, the problems of drug resistance and
reported allergies are also abound. Coconut oil has been
confirmed to possess antimicrobial, antiviral and
antiprotozoal activities (Isaacs and Thormar, 1991;
Thormar, 1996; Enig, 2003). Phytochemical studies
indicated that lauric acid which is its major fatty acid
component was highly responsible for the activities of the
oil (Peat, 2003). Lauric acid has been documented to be
converted to monolaurin in the human body and it is the
antimicrobial agent found in hum an milk (Peat, 2003).
This study is aimed at carrying out formulation studies of
the oil, with the view o f utilizing comp atible excipients
and in compliance with cGMP. Standardization of oil
quantity according to activity will also be carried out.
Topical drug delivery has gained a lot of attention in the
last 30 years. The most cosmetically acceptable emollient
preparation is the cream due to its cosmetic appeal and the
ease with which it is washed off the skin. The
antimicrobial activities of coconut oil has been reported
earlier by Obi et al. (2005), prompting the present attempt
to explore its formulation into creams.
MATERIALS AND METHODS
This study was conducted in the Department of
Pharmaceutics and Pharmaceutical Microbiology,
Ahmadu Bello University, Zaria-Nigeria; November 2004
- February 2005.
Materials:
Coconut oil: This was obtained by extraction from
coconut fruits sourced locally.
Micro-organisms: Ps. aeruginosa ATCC 7853, E. coli
ATCC 9637, A. niger (clinical isolate), S. aureus ATCC
13709, P. vulgaris (clinical isolate), B. subtilis ATCC 607
and C. albicans ATCC 10231 which were all obtained
from the Department of Ph armaceutics and
Pharmaceutical Microbiology, Ahmadu Bello University,
Zaria-Nigeria.
Media: Nutrient agar (Biotec), Nutrient broth (Lab.M),
Sabouraud’s dextrose broth (SDB) and Sabouraud’s
dextrose agar (SDA) were from Oxoid.
Methods:
Extraction of coconut o il: The fresh endosperm or
coconut meat was separated manually from the shells and
then washed to rem ove dirt. The product was cu t into
pieces (about 1.27 cm or 12.7 mm in diameter) using a
Res. J. Appl. Sci. Eng. Technol., 2(2): 133-137, 2010
134
knife. It was weighed and milled using a local
crusher mill.
The resulting mass was mixed with lukewarm water and
chaff was filtered out using a cotton cloth. (The ratio of
milled meat to water was I:1). The residue (marc) was
firmly pressed and process repeated to ensure thorough
extraction of th e milky liquid oil. This liquid was heated
to boiling; at this po int the source o f heat was rem oved to
avoid boiling over and wastage of milk.
Heating effected the floating of oil as the top layer.
This was skimmed off and gradually ev aporated to
dryness over gentle heat in a shallow aluminum pan.
Constant stirring to avoid burning and discoloration was
carried out at this stage of drying. The oil was filtered off
and the deposit or “brownie” was firmly pressed to release
remaining oil. The oil was therea fter decanted. Finally,
the product was gently heated to dry all traces of moisture
and filtered through a cotton cloth to obtain the oil for
further use.
Formulation of coconut oil creams: Oil in water creams
(o/w) were selected for formulation due to the advantage
of easy removal from skin. The concentration of coconut
oil used for the formulation ranged from 5 – 40% w/w
using the formula on Table 1. Caution was exercised to
avoid contamination of cream during formulation by
working in a clean environment.
The following procedure was generally adopted:
CThe oil was weighed in a dry ceramic evaporating
dish (200 ml capacity). Oil so luble ingredients such
as cetostearyl alchohol, d "-tocopheryl acetate and
the parabens were weighted separately and
transferred to the oil. The mixture was melted over
the water bath at a temperature of 70ºC. The
temperature was not allowed to rise above 75ºC as
parabens are decomposed around that temperature
(Idson and Lazarus , 1986).
CPropylene glycol was weighted into a dry 100 ml
beaker; water was measured out and added leaving
behind about 10 ml. The sodium laurly sulphate and
cetrimide were added, after which the mixture was
heated to dissolve the ingredients and further raised
to 75ºC.
Note: Aqueous phase is generally required to be
heated 5ºC above the oily phase, since it will be
transferred to the later, otherwise, it will lower the
temperature of the oily mixture and cause local
congealing of the waxy components.
CThe temperatures of the oily phase and the aqueous
phase were taken using two different thermometers.
The aqueous phase was then added gently and
steadily to the oily phase without splashing and with
continuous stirring using a big glass rod. At this
temperature (70-72ºC), intimate mixing of the two
phases occurred and the continuous phase was
gradually added to the disperse phase. Initially w/o
emulsion will be formed but as more w ater is added,
phases will invert to a fine o/w emulsion (Idson and
Lazarous, 1986 ).
C The hot mixture was then transfe rred to an
insulating pad and stirring continued to ensure that
the waxy components did no t separate out. Cooling
was slowed down to allow adequate mixing and
prevent aeration.
CPerfume was added when mixture cooled to 43 –
45ºC to avoid chilling the cream and fac ilitate
mixing of perfume oil in the incompletely congealed
oil phase.
CThe citric acid was weight and dissolved in the
remaining water and solution gradually incorporated
into the cooled cream.
CThe pH of the cream was checked and adjusted to
4.5-6.5 where necessary using citric acid.
CFinally, the cream was cooled to 30-40ºC and
passed through a h and homogenizer.
CFor the batch containing bentonite and sodium
lauryl sulpate, the preservatives were added to the
oily phase together with d "-tocopheryl acetate.
After melting, the bento nite was inco rporated into
the oily phase.
All cream s were packaged into wid e mouthed glass
jars with plastic screw caps fitted with impermeable liners
and properly labeled.
Tests on coconut oil creams:
Emulsion type determination;
Dye test: A drop o f 1% amaranth solution wa s added to
a small quantity of cream on slide, covered with a cover
slip and examined microscopically under x40
magnification.
Sudan Red dye test: A few drop of 2% suda n red oily
solution was incorporated into the cream on a slide,
covered with a slip and examined microscopically.
Electrical condu ctivity test: The electrical conductivity
of each of the creams using a conductivity Meter (Jerway,
model 4010) was carried out as a co nfirmatory test for
emulsion type.
pH determination: pH paper strips with colour indicators
were used. Three readings were taken at once and
recorded. An interval of 20-25 min was given for the
paper to soak thoroughly after insertion into the creams.
Stability test: The acce lerated stability test method by
Garti et al. (1982) was used. According to this method
subjection of cream to conductivity test before and after
stress will indicate its stability. The magnitude of the
conductivity differential between the two readin gs will
indicate the degree of instability. The following procedure
was adopted at room temperature:
CCream was subjected to heat on water ba th (60-62ºC)
for ten minutes. It was removed and allowed to cool
to room temperature. The process of heating and
cooling was repeated.
Res. J. Appl. Sci. Eng. Technol., 2(2): 133-137, 2010
135
CCondu ctivity measurements were repeated on all
creams and results recorded.
CSteps (a) and (b) were repeated for two more days on
a daily basis and results recorded.
Preservative efficacy test: The British Pharmacopoeia
(1988) test for preservative efficacy of creams was
adopted to test the efficacies of lemon grass oil, parabens
and cetrimide.
Preparation of inoculum: The following organisms were
used as test organisms
C. albicans ATCC 1023
A. niger (laboratory isolate)
S. aureus ATCC 13709
Ps. aeruginosa ATCC 7853
The bacteria were incubated for 18 to 24 h at 37ºC on
nutrient agar slants while Candida and Aspergillus were
incubated at 25ºC for 48 and 96 h, respectively on SDA
slants. The cells were harvested using 10 mls sterile 0.1%
peptone water containing 1% polysorbate 80. Serial
dilutions were made from this stock to produce germ
counts of approximately 108 sfu/ml. The bacteria cells
were grown in nutrient broth and incubated at 37ºC for
18 h. The o vernight cultures were diluted 1 in 103 for
S. aureus and 1 in 104 for Ps. aeruginosa (Garrold et al.,
1963).
Antimicrobial activity testing on formulated creams:
The following categories of creams were used for the test
CCream preserved with methyl and propyl paraben
CCream preserved with methyl, propyl paraben and
cetrimide
CCream p reserved with lemon grass oil
CUnpreserved cream
CEach of the four tes ted creams was inoculated with
0.2 ml of the standardized cu lture
CThe incorporation was done using sterile spatula to
ensure even distribution. The cream was covered and
stored at room temperature
CAt the same time 0.2 ml of the incorporated
suspension was added to 20 ml sterile 1% peptone
water containing 1% polysorbate 80 (peptone water
control)
COne gram quantity of the creams were removed at 0,
6, 24, 48 h; 7, 14 and 28 days. Each withdrawn
sample was introduced aseptically into 9ml sterile
0.1% peptone water containing 1% polysorbate 80;
two further step wise dilutions were made to produce
1:100 and 1:1000 dilutions using same broth.
C1 ml aliquots of the last dilutions were each used to
prepare pour plates with 20mls of nutrient agar or
Sabouraud’s dextrose agar as appropriate. These
media were prepared with 0.5%w/v polysorbate 80
and 0.07% lecithin for the cetrimide containing
cream. These served as neutralizers for the
preservatives (Orth, 1979).
CControls using one milliliter of diluted cultures on
plain agar was done to confirm that the preservatives
were not antagonistic to the micro-organisms.
CThe peptone water controls were diluted and
examined at the same time as the zero hour samples
to obtain the initial number of micro-organisms per
gram. This was used as the baseline for reduction in
population of organisms with time.
CThe population (cfu) of organisms recovered after 3
days incubation for bacteria and 5 days for
fungi/yeast were determined. The difference in
population at zero hour and after the incubation
periods were recorded.
Test of activity of cream on skin:
CA 16 h culture of the organisms in above (preparation
of inoculum) w as standardized to contain 106 cfu/ml
with sterile normal saline.
CThe back portion of the hand, joints between the
fingers was inoculated with the standard organisms
above and dried
CCoconut oil cream was applied on the inoculated
spots.
CFive minutes later the area was swa bbed using sterile
swabs
CThe swabs were incubated overnight in 5 ml of sterile
nutrient broth at 37ºC
CTests we re replicated and co ntrols using normal
saline in place of cream were carried out at the same
time.
RESULTS
Emulsion type determination:
Dye test results: Microscopic examinations revealed that
all creams were o/w. With amaranth; the emulsion
background was pinkish with c olorless globules. However
with Sudan red which is oil soluble; globules were reddish
against a colorless background.
Confirmation of emulsion type was done by the
electrical conductivity test using the Jerway condu ctivity
meter, where all creams conducted electricity and thus
confirmed to be o/w.
Determination of pH: The pH values of all creams
varied between 6 and 6.5 Batches (Table 1) A, B and E
had pH of 6 while C and D were of pH 6.5; the
recommended by BPC (1994) range is 4.5–6.5.
Conductivity tests: The results revealed that batches A
and D had negligible variations while batches B and C
showed higher variations (Table 2). Batch C separated
into two layers after stress, while batch E was not tested
because it w as a colored p roduct.
Res. J. Appl. Sci. Eng. Technol., 2(2): 133-137, 2010
136
Table 1: Quantities of ingredients used for various batches
Quantities (g) and batches
-----------------------------------------------------------------------------------------------------------------
Ingredie nts ABCDE
Cocon ut oil 40 45 40 50 40
Propylene glycol 44444
Cetostearyl alcohol 752.7 2.7 -
Sodium lauryl sulphate 11--0.5
Methyl paraben 0.5 -0.2 0.2 -
Propyl paraben 0.1 -0.1 0.1 0.2
Citric acid 0.05 0.05 0.05 0.05 0.05
d- "-tocophe ryl acetate 0.1 0.1 0.1 0.10.1
Perfum e oil 0.4ml -0.4ml 0.4ml -
Distilled water 46.85 44.34 42.15 42.15 49.65
Lem on grass oil -0.5ml ---
Cetrimide --0.3 0.3 -
Benton ite ----5
Phenoxyethanol ----0.5
Batches A and B are o/w creams containing anionic emulsifying agent, Batches C and D are o/w creams containing cationic emulsifying agent. Batch
E contained anionic emulsifying agent and a finely divided solid.
Table 2: Conductivity measurem ent of creams before and after stress
Conductivity (microsiemens)
-------------------------------------------------------------------------------------------------------------------------------
S. No. Batch Before stress After; 1 st day 2nd day 3rd day
1A138 127 139 145
2B141 156 193 210
3C100 87 59 Not taken, Separated
4D117 112 114 107
Batche s A and D had negligible variatio n while B and C s howed h igher varia tion (Garti et al 1982)
Table 3: Effects of preserved and un preserved creams on the survival of A. niger on exposure for different times
Cream type 0 h 6 h 24 h 48 h 7days
----------------- ----------------- ------------------- -------------------- ----------------------
ababababab
UNP 1NG NG 1NG NG NG 1From
LGO 12NG 5NG NG NG NG 7 days to
PARA 1NG NG NG NG NG 2NG 28 days
CET 411NG NG 1NG NG ALL NG
PWC 11
UNP: Unpreserved cream, LGO: Cream preserved with lemon grass oil (batch B), PARA: Cream preserved w ith the Parabens (batch A ), CET: Cream
preserved with cetrimide (batch C), PWC : Peptone water control, a: 102cfu/ml, b: 103 cfu/ml, NG : No Gro wth
Table 4: Number o f surviving colonies of S.aureus after exposure to creams
0 h 6 h 24 h 48 h 7 days 14 days 28 days
Cream -------------- ---------------- ------------- ------------------ ----------------- ------------------ ------------------
type ababababababab
UNP NG 1NG NG NG NG NG NG NG NG NG NG NG NG
LGO NG 1NG NG NG NG NG NG NG NG NG NG NG NG
PARA NG NG NG NG NG NG NG NG NG NG NG NG NG NG
CET 11NG NG NG NG NG NG NG NG NG NG NG NG
PWC TNTC 65
a: 102cfu/ml, b: 103cfu/ml, TNTC: Too Numerous To Count
Table 5: Number o f surviving organisms of Can dida albicans after exposure to cream for different times
0 h 6 h 24 h 48 h 7 days 14 days 28 days
Cream -------------- ---------------- ------------- ------------------ ----------------- ------------------ ------------------
type ababababababab
UNP NG NG NG NG NG NG NG NG 1NG NG NG NG NG
LGO NG NG NG NG NG NG NG NG NG NG NG NG NG NG
PARA NG NG NG NG 5NG NG NG NG NG NG NGNG NG
CET NG NG NG 1NG NG NG NG NG NG NG NG NG NG
PWC TNTC 400
a: 102cfu/ml, b: 103cfu/ml, TNTC: Too Numerous To Count
Preservative efficacy test: The results indicated that all
creams including the unpreserved one were protected and
possessed antimicrobial activities (Table 3-6)
Skin activity test:All organisms did not survive the
cream treatment as no viable organism was observed.
DISCUSSION
The formulated co conut oil creams exhibited both
antibacterial and antifungal properties. This confirms that
formulating the oil into cream does not affect its activity
and that all excipients used did not affect the
Res. J. Appl. Sci. Eng. Technol., 2(2): 133-137, 2010
137
Table 6: Survival of Ps. aeruginosa to cream on exposure for varying periods
0 h 6 h 24 h 48 h 7 days 14 days 28 days
Cream -------------- ---------------- ------------- ------------------ ----------------- ------------------ ------------------
type ababababababab
UNP NG 3NG 1NG 1NG NG NG NG NG NG NG NG
LGO 1 NG 11NG NG NG NG NG NG NG NG NG NG
PARA 121122NG NG NG NG NG NG NG NG
CET NG 31NG NG 1NG NG NG NG NG NG NG NG
PWC TNTC 300
a: 102cfu/ml, b: 103cfu/ml, TNTC: Too Numerous To Count
antimicrobial activity of oil. In contrast to the pure oil
(Obi et al., 2005), the cream formulations displayed a
cidal activity. The active co mpound in the oil previously
identified to be monolaurin could have had an enhanced
penetration due to the presence of surface active
emulsifying agents used in formulating the cream since
emulsification of oils generally increases their
absorptivity (Aulton, 1988). The choice of an anionic
(sodium lauryl sulphate) and cationic (cetrimide)
emulsifying agents were to avoid incompatibility with the
selected preservatives notably phenolics and carboxylic
acids (Pharmaceutical Codex, 1994). The antimicrobial
activity of coconut oil had been attributed to the
carboxylic acid – monolaurin m etabolized to la uric acid
in the body .
The preservative efficacy test indicated a
synergistic action between the coconut oil and
preservatives, which showed 100% kill at 0 h. This is in
conformity with the finding of Thormar (1996) which
reputed that monocaprin a component of coconut oil
destroyed HIV, herpes virus and gonococci within a
minute. Since the unpreserved cream showed similar
results with the preserved, this indicates that preservation
may not be necessary. The conductivity test carried out
indicated that batches a and d w ere stable while b and c
were unstable. The two stable batches had oil to water
ratios close to 50:50 which has been generally observed
with stable creams (Martins et al., 1970).
CONCLUSION
This study has established that coconut oil can be
formulated into an elegant cream which is active on both
fungal and bacterial organisms. It also demonstrates the
possibility of stan dardizing the quality and quan tity of oil
to be used therapeutically in extemporaneous
preparations.
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