Nutritional and Antimicrobial Properties of Ocimum gratissimum Leaves
Fred O.J. Oboh,
Honeybell I. Masodje
Stephen A. Enabulele
Fresh green Ocimum gratissimum leaves were analysed
for protein content, moisture, ash, minerals and antimicrobial activity. The
fresh leaves had a moisture content of 81.35%, a protein content of 1.21% and
an ash content of 0.57%. On a fresh weight basis mineral content was as follows:
phosphorus 52.4, selenium 0.007, iron 7.9 and zinc 1.5 μg g-1.
An aqueous extract of the leaves inhibited the growth of the gram positive bacterium
Staphylococcus aureus and the gram negative bacterium Escherichia
coli. The nutritional implications of the results are discussed.
Leafy vegetables are a good source of dietary fibre, carotenoids, vitamin C,
folate, phytochemicals and certain minerals, but have low concentrations of
protein, digestible carbohydrates and lipids (Wills et al.,
1998). They are easy to cultivate and provide an inexpensive means of combating
vitamin and mineral deficiency in less developed regions of the world. Ocimum
gratissimum is found throughout the tropics and subtropics, both wild and
cultivated. Its greatest variability occurs in tropical Africa, where it probably
has its origin and India (Osuji et al., 1995).
The leaves of the plant, which are highly appreciated for their pleasant aroma
are used as seasoning and are eaten as a vegetable. The plant has interesting
medicinal properties (Gill, 1992). While, its essential
oil have been extensively investigated (Simon et al.,
1990), little is known about its nutritional composition and antimicrobial
activity. In the present study, the moisture, protein, ash and mineral (phosphorus,
selenium, iron and zinc) content of O. gratissimum leaves and the antimicrobial
effect of their aqueous extract on the bacteria Escherichia coli and
Staphylococcus aureus were determined. The nutritional and food processing
implications of the results are discussed.
MATERIALS AND METHODS
This study was conducted in Benin City, Nigeria in the months of May and June 2007.
Vegetables: Leaves of O. gratissimum were purchased from markets
in Benin City. They were dark green in colour and freshly cut.
Microorganisms: Escherichia coli (ATCC 35218) and Staphylococcus aureus (N 315 methicillin resistant/gram + ve) were obtained respectively from the Veterinary Research Institute of Nigeria, Vom, Plateau State and the Nigerian Institute of Oceanography and Marine Research, Victoria Island, Lagos.
Reagents: All reagents were analytical grade.
Moisture: Shredded fresh vegetable (10 g) was dried in a thermostatically
controlled ventilated oven at 105°C until constant weight was obtained. The
loss in weight was recorded as moisture content (AOAC, 1984).
Sample preparation for protein, ash and mineral analysis: Leaves were cut into tiny pieces and dried in a ventilated oven at 60°C for 5 days to constant weight. The dried vegetable was ground into powder and stored in airtight bottles for analysis.
Crude protein: Dried and pulverised leaf (0.2 g) was digested in 2 mL
concentrated H2SO4 in the presence of selenium catalyst,
until a clear digest was obtained (AOAC, 1984). The nitrogen
content of diluted digest was determined colourimetrically at 630 nm according
to Charlot (1964). Protein was calculated as: nitrogen
content x 6.25.
Ash: For the determination of ash content, dried pulverised vegetable was ashed at 550°C in a muffle furnace.
Minerals: Minerals were obtained by ashing 2.0 g dried and ground sample
in a muffle furnace at 550°C. The ash was dissolved in 10 mL, 20% nitric acid
and filtered through a dry 9 cm acid washed Whatman No. 541 filter paper into
a 100 mL volumetric flask. The filtrate was made up to the mark with deionised
water and the resulting solution was used for the analysis of phosphorus, zinc
and iron. Phosphorus was determined colourimetrically according to Charlot
(1964). To mineral solution (10 mL) in a 50 mL volumetric flask was added
0.2 mL of 0.5% para-nitrophenol indicator solution. To this was added an ammonia
solution (6 N) dropwise, with gentle shaking until the appearance of a yellow
colour. Nitric acid (1 N) was then added in the same manner until the solution
turned colourless. Finally, ammonium molybdate/ammonium vanadate mixed reagent
(5 mL) was added. The solution was made up to 50 mL with distilled water; the
flask was stoppered and its contents well mixed. The flask was allowed to stand
for 30 min and the absorbance of the solution measured at 400 nm. Phosphorus
content of the solution was read off a calibration graph prepared using potassium
dihydrogen phosphate as standard. Zinc and iron were determined using an atomic
absorption spectrophotometer (Buck Scientific VGP 210) at 630 nm (Okalebo,
1985). Selenium was determined titrimetrically (Charlot
and Benzier, 1957). To an aqueous, extract of the leaves were added 5 mL
starch solution and 1 g potassium iodide. The mixture was stirred, allowed to
stand for 15 sec and titrated with 0.05 N sodium thiosulphate solution. The
end point was marked by a change from a dirty or turbid solution to a transparent
red colour. Analysis were done in duplicate.
Preparation of crude water extract of leaves: Aqueous extract of
O. gratissimum leaves was prepared by blending 10 g fresh leaves in 100
mL peptone water (15 g peptone in 1 L water). The water extract was filtered
through Whatman No. 1 filter paper and tested for antimicrobial activity.
Preparation of crude antibiotic discs: Sterile Whatman No. 1 paper was
punched into 5 mm diameter disc sizes. The Whatman discs were placed in Mac
Cartney bottles and sterilised in an autoclave at 120°C for 15 min. The bottle
was transferred into a hot air oven at 60°C to dry for 30 min. An aqueous extract
of the leaves (1.0 mL) was transferred into a sterile Bijou bottle containing
sterile discs. The sterile crude discs were allowed to soak in extract for 6
h for proper absorption, after which they were removed and allowed to dry (Cheesebrough,
Antimicrobial assay of extract: Aqueous extract of O. gratissimum
leaves was screened in vitro for antimicrobial activity against Staphylococcus
aureus and Escherichia coli. Nutrient agar (7 g) was added to 250
mL distilled water in a flask. This was stirred and autoclaved at 115°C and
then cooled to 50°C. A portion of the medium (20 mL) was poured into a sterile
petri dish and allowed to solidify. The sterility of the medium was confirmed
by allowing it to stay for 8 h and observing no contamination. An isolate colony
of each test organism was subcultured on nutrient broth and incubated at 37°C
for 8 h. This was then spread on the entire plate medium to ensure uniform growth.
The crude extract discs were incubated immediately for 24 h at 37°C (Ogbulie
et al., 1998). Antimicrobial assays were carried out in quadruplicate.
Zones of inhibition were observed using a hand lens for proper magnification
and the zones measured.
RESULTS AND DISCUSSION
Ocimum gratissimum leaves were purchased freshly cut and were dark green in colour. Their moisture, protein and ash content are shown in Table 1. Included for comparisons are published data for some locally available vegetables, cassava leaves, cabbage, cowpea leaves and sweet potato leaves.
Water was the dominant component, constituting 81.35% of O. gratissimum leaves and 79-85% of the others. Protein constituted 1.21% of fresh O. gratissimum leaves, lower than the published values for fresh cassava leaves (6.0%), cabbage (1.4%), fresh cowpea leaves (4.1%) and sweet potato (4.6%). Ash constituted 0.57% of fresh O. gratissimum leaves. Overall, foods with a high water and dietary fibre content provide a low energy density contribution to the meal and create a feeling of satiety. The leaves had a low protein content.
Generally, fresh leafy vegetables have low protein content. This protein is
mostly in the form of enzymes, rather than acting as a storage pool, as in grains
and nuts (Wills et al., 1998). The leaves of cassava,
cowpea and sweet potato and cabbage appear to be better sources of dietary protein
than O. gratissimum leaves. Thus fresh O. gratissimum leaves may
not be an important source of dietary protein. Ash content was low, indicating
a low mineral content. The concentration in O. gratissimum leaves, of
the macromineral phosphorus and the trace minerals, selenium, iron and zinc
were determined. Table 2 shows the mineral content of the
leaves. Included for comparison are the published values for fresh cassava
and cowpea leaves and cabbage and the recommended daily allowances for the minerals.
Phosphorus constituted 52.4 μg g-1 (i.e., 0.4% of RDA was present
in 100 g of fresh leaves), zinc 1.5 μg g-1 (i.e., 1.0% of RDA
in 100 g fresh leaves) and iron 7.9 μg g-1 of fresh O. gratissimum
leaves. These were lower than published values for cabbage and cowpea leaves
except for the iron content of cabbage, which was lower. The concentration of
selenium in leaves was 0.007 μg g-1 (1.3% of RDA in 100 g of
fresh leaves). The iron contained in 100 g fresh O. gratissimum leaves
represents about 5-8% of its RDA. The contributions of the other minerals studied
to their total dietary requirements appear to be small. Thus O. gratissimum
leaves may be a minor dietary source of these minerals. It is important to note
however, that the nutritional value of vegetables depends not only on the concentration
of nutrients in the produce, but also on the amount consumed in the diet. Also,
since vegetables are usually eaten in combination with other dietary components,
some of which may be better sources of the minerals under consideration, they
could be of value in supplementing the minerals available from these better
||Zones of inhibition (cm) of bacteria by aqueous extract of
Ocimum gratissimum leaves
The effect of an aqueous extract of Ocimum gratissimum on the gram positive bacterium Staphylococcus aureus and the gram negative bacterium Escherichia coli was shown in Table 3.
Both showed sensitivity to the extract, giving zones of inhibition of 1.0 and
1.2 cm, respectively. Thus the aqueous extract of Ocimum gratissimum
exhibited broad spectrum antimicrobial activity by inhibiting the growth of
gram + ve and gram-ve organisms. Leafy vegetables are high moisture, low acid
produce, which support the growth of a wide range of microorganisms. Thus great
care is needed when processing them, in order to minimize poisoning, especially
those, for example S. aureus and E. coli, which produce heat stable
toxins that may not be destroyed by heat treatment such as cooking (James
and Kuipers, 2003; Fellows and Axtell, 2001; Schmidt,
1983). Staphylococcus aureus and Escherichia coli are common
food poisoning bacteria. The antimicrobial activity of O. gratissimum leaves
could inhibit the growth of these bacteria on the vegetable itself and (by extraction
during cooking, if the antimicrobial principle is heat stable) in food, e.g.,
sauces and stews in which it is an ingredient, thus protecting the consumer
from their harmful effect.
Ocimum gratissimum leaves contained mainly water. Protein and ash were minor components. The elements studied in this study occur in appreciable concentrations in Ocimum gratissimum leaves. However, except for its iron content, which could be a major contributor to its RDA, the potential contribution of the other elements studied-phosphorus, selenium and zinc, to their RDA appears to be minor. The cold aqueous extract of the leaves inhibited the growth of S. aureus and E. coli.
1: AOAC., 1984. Official Methods of Analysis. 14th Edn., Association of Official Analytical Chemists, Washington, DC., USA., pp: 522-533.
2: Charlot, G., 1964. Colorimetric Determination of Elements Principles and Methods. 1st English Edn., Elsevier Publishing Company, Amsterdam, London, New York, pp: 320-322.
3: Charlot, G. and D. Bezier, 1957. Quantitative Inorganic Analysis. 1st Edn., Methuen and Co. Ltd., London, pp: 691.
4: Cheesbrough, M., 2000. District Laboratory Practice in Tropical Countries. 1st Edn., Press Syndicate of the University of Cambridge, Cambridge UK., pp: 132-143.
5: Fellows, P. and B. Axtell, 2001. Opportunities in Food Processing. A Handbook for Setting up and Running a Small Food Business. 1st Edn., Technical Centre for Agricultural and Rural Cooperation, Wageningen, Netherlands, ISBN: 929081246X, pp: 298.
6: Gill, L.S., 1992. Ethnomedical Uses of Plants in Nigeria. 1st Edn., University of Benin Press, Benin City, ISBN: 978-2027-20-0, pp: 180-181.
7: James, I.F. and B. Kuipers, 2003. Preservation of Fruits and Vegetables. 4th Edn., Agromisa Foundation, Waganingen, ISBN: 90 77073 302, pp: 86.
8: Madamba, R., G.J.H. Grubben, I.K. Asante and R. Akromah, 2006. Vigna nguiculata (L.) Walp. In: Plant Resources of Tropical Africa 1. Cereals and Pulses, Brink, M. and G. Belay (Eds.). PROTA Foundation/Backhuys Publishers/CTA Wageningen, Netherlands, ISBN: 90-5782-170-2, pp: 221-229.
9: Ogbulie, J.N., J.C. Uwaezuike and S.I. Ogiehor, 1998. Introductory Microbiology Practical. 1st Edn., Springfield Publishers, London, pp: 162.
10: Okalebo, J.R., 1985. A simple wet ashing technique for phosphorus, potassium, calcium and magnesium analysis in plant tissue in a single digest. Kenyan J. Sci. Technol., 6: 129-133.
11: Osuji, P.O., S. Fernandez-Riviera and A. Odenyo, 1995. Improving Fibre Utilisation and Protein Supply in Animals Fed Poor Quality Roughages. ILRI Nutrition Research and Plans. In: Rumen Ecology Research and Planning, Wallace, R.J. and A. Lahlou-Kassi (Eds.)., Vol. 1. International Livestock Research Institute, Addis Ababa, pp: 1-22.
12: Schmidt, T.R., 1983. The use of citric acid in the canned fruit and vegetable industry. Biotech. Products Division, Miles Laboratories Inc., Technical Bulletin, pp: 24.
13: Simon, J.E., J. Quinn and R.G. Murray, 1990. Basil: A Source of Essential Oils. In: Advances in New Crops, Janick, J. and J.E. Simon (Eds.). Timber Press, Portland, pp: 484-489.
14: Van Gastel, S. and A. van den Wijngaart, 1997. Small Scale Production of Weaning Foods. 1st Edn., Agromisa and CTA, Netherlands, ISBN: 90-72746-76-7, pp: 68.
15: Wardlaw, G.M. and M. Kessel, 2002. Perspectives in Nutrition. 5th Edn., McGraw Hill, New York, USA., ISBN: 0-07-228784-5.
16: Wills, R., B. McGlasson, D. Graham and D. Joyce, 1998. Postharvest an Introduction to the Physiology and Handling of Fruit Vegetables and Ornamentals. 4th Edn., CAB International, ISBN: 0 85199 264 1, pp: 15-32.