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Ethnobotanical Leaflets 13: 1186-96. 2009. Studies
on the Antimicrobial Properties and Phytochemical
Screening of Garlic (Allium
sativum)
Extracts
M. J. Olusanmi and J. E. Amadi Department of Plant Biology University of Ilorin, PMB 1515, Ilorin, Nigeria Issued 01 September 2009 Abstract This study was carried
out to evaluate the antimicrobial properties of garlic (Allium sativum) extracts on three fungi namely Aspergillus
flavus, Curvularia
lunata and Fusarium
moniliforme using the pour plate method. A phytochemical
screening of the extracts was also carried out to determine the constituents
in garlic. Water, ethanol and acetone were the extractants
used. Results showed that radial growth in all the three test organisms was
impaired by the addition of the extracts in the culture medium used. The test
organisms differed in their reaction to the different extracts but on the
whole, growth inhibition increased with the concentration of each extract. Phytochemical screening of the different extracts showed
that garlic contains important compounds such as carbohydrates, reducing
sugars, lipids, flavonoids, ketones,
alkaloids, steroids and triterpenes. Tannins were,
however, not detected in any of the extracts under the conditions of this
study. The significance of these results is discussed. Key words: Garlic, extracts, constituents, antimicrobial,
inhibition. Introduction Garlic (Allium sativum)
qualifies as an important vegetable because not only is it an indispensable
cookery ingredient, it can well and delightfully be eaten as and for itself
(Anon, 2009). The Allium genus belongs to the Liliaceae family comprising onions, leeks, shallots,
asparagus etc. Modern garlics are of only two
species; Allium
sativum,
the soft necks and A. ophioscorodon,
the hard necks. Taste, storage ability and suitability in growing are the
critical factors in selecting garlic classes of interest. The hard necks have
more intense flavours but less storage capabilities
while the soft necks are excellent keepers but often milder. Hard necks are
generally grown in cooler climates while the soft necks grow closer to the
equator (Al-Zahim et al., 1997). Garlic
requires a sunny spot and the soil should be rich but not too rich or the
tops will overdevelop. The medicinal value of
plants has assumed a more important dimension in the past few decades. This
is due largely to the discovery that extracts from plants contain not only
minerals and primary metabolites but also a diverse array of secondary
metabolites with antioxidant potentials (Sofowora,
1993, Okigbo et
al. 2009a). The use of plant extracts in traditional medicine is a
worldwide practice. Medicinal plants form the basis of primary health care
for majority of the people living in the rural and remote areas in According to Sofowora (1993), African medicinal plants rank highest
among plants used in the investigations of antimicrobial properties. This
could be due to their high traditional medicinal use and also the ease of
carrying out such tests. Among medicinal plants of African origin are Psidium guajava
(guava), Azadirachta indica
(neem), Vernonia
amygdalina (bitter leaf), Anacardium
occidentales (cashew), Allium
cepa (onion) and A. sativum
(garlic). The organs or parts used in these plants vary from one plant to the
other and these include the leaves, bark, roots, stem, flowers, fruits and
even the seeds (Farombi, 2003, Nguelefack
et al., 2005). While garlic is
primarily used as a herb to enhance many food dishes in various cultures
(Table 1), it contains many substances which studies have shown act together
to prevent disease and age-related conditions (Anon, 2009). According to
Johnson et al. (2008), biodiversity
provides mankind enormous direct benefits and indirect essential services
through natural ecosystem function and stability. Initial reports of
antimicrobial activity of garlic showed that allicin
(allyl 2-propene thiosulfinate),
a notable flavonoid in garlic is formed when garlic
cloves are crushed (Cavallito et al., 1945; Ross et al.,
2000). Allicin formation follows the action of an
enzyme, allinase of the bundle sheet cells upon the
alliin of the mesophyl
cells. When crushed, A. sativum yields allicin, a powerful antibiotic and antifungal compound (phytoncide).
However, due to poor bioavailability, it is of limited use for oral
consumption. Garlic also contains some sulphur-containing
compounds such as alliin, ajoene,
diallylsulphide, dithin,
S-allylcysteine and enzymes as well as some non sulphur-containing compounds including vitamin B,
proteins, minerals, saponins and flavonoids.
Yukihiro et al., 2002 have reported a phytoalexin
called allixin in garlic. The
objective of this study is to evaluate the potential of garlic (A. sativum L.) extracts in inhibiting the growth of
three phytopathogenic fungi, A. flavus, C. lunata and F.
moniliforme in vitro. This study will
also determine the phytochemical constituents of
garlic and the efficiency of their extraction using different solvents. Materials and Methods Materials
used and Extraction Procedure Cured
bulbs of A. sativum were obtained from Idi-Ape Market in The
aqueous, ethanol and acetone extracts of garlic were prepared following one
of the various acceptable procedures. After removing the papery skin of A.
sativum cloves, 25g were weighed into a beaker,
surface-sterilized with sodium hypochlorite for 2 minutes, rinsed twice with
sterile distilled water and then crushed using sterile mortar and pestle. The
resultant paste was soaked in 100ml of any of the chosen extractants
for 24 hours. The extract was then filtered through two layers of sterile Whatman No. 1 filter papers into conical flasks and used
as stock. The extract was stored in the refrigerator at 3oC for
subsequent use.
Concentration of extract = Weight of garlic cloves (g) /
Volume of extractant (ml) = 25g / 100ml = 0.25 g/ml
Effect of Extracts on Growth of Test Organisms Four concentrations of garlic extracts were tested
against three pathogenic fungi namely A.
flavus, C. lunata and
F. moniliforme. One hundred and twenty (120) millilitres
of sterile molten agar was amended separately with 5, 10, 20 and 40ml of the
stock extract. The amended agar medium was dispensed into sterile Petri
dishes and allowed to solidify. Each concentration was replicated three
times. The control experiment comprised three agar plates amended with
sterile distilled water. Two diagonal lines were drawn with a marker to cross
each other at the centre on the reverse side of each Petri dish. Each plate
was inoculated at the centre with a 2mm-diameter mycelial
plug cut from the edge of a growing culture using a sterile cork borer.
Growth was measured daily along the diagonal lines at the back of the plates. Phytochemical Screening The aqueous, ethanol and acetone
extracts of A. sativum were screened for the
presence of secondary metabolites using the procedure of Sofowora
(1993). Two (2) milliliters of each extract was measured into a test tube for
each of the tests and concentrated by evaporating the extractant
in a water bath. Tests were carried out for carbohydrates, reducing sugars,
tannins, polyphenols, lipids, flavonoids,
ketones, alkaloids, steroids and triterpenes. This aspect of the research was carried out
in the Department of Chemistry of the University of Ilorin, Ilorin, Nigeria. Results and Discussion Growth Inhibition Radial growth in all the organisms
tested in this study was inhibited by the three extracts investigated. The
effectiveness of the extracts in hindering growth of the test fungi differed
with the extracts, the organisms and also between concentrations. At the
highest concentration (40ml extract: 120ml molten agar) tested in this study,
all the extracts inhibited growth completely in all the test fungi. At 20ml
extract: 120ml molten agar concentration, the acetone extract was most
effective against A. flavus inhibiting growth completely while permitting
slight growth in both C. lunata and F. moniliforme. At the same 20:120 concentration of the
aqueous and ethanol extracts, both A. flavus and C. lunata recorded slight radial growth. Only F. moniliforme was
completely inhibited at this concentration. At
both the 5:120 and 10:120 concentrations, growth was recorded in all the test
fungi with more growth occurring at the 5:120 concentration than at 10:120 of
all the extracts. At the lowest concentration (5ml extract: 120ml molten
agar), the aqueous extract was more inhibitory to the growth of F. moniliforme
than any of the other two extracts. However, there was more growth in
all the test fungi in the control plates than in any of the extract
concentrations tested under the conditions of this study. Observations from the control plates
also showed that C. lunata was the fastest
growing organism among the three organisms used in this study while F. moniliforme
was the slowest. Phytochemical Screening Screening
tests showed that the constituents of A.
sativum extracts differed with respect to the extractant employed. The aqueous and ethanol extracts of A.
sativum showed similar reactions in all the
tests carried out while the acetone extract showed slightly different
reactions with the same reagents (Table 2). Both the aqueous and ethanol
extracts were found to contain carbohydrates, reducing sugars, lipids, flavonoids, ketones, alkaloids,
steroids and triterpenes. In the acetone extract
alkaloids, steroids and triterpenes were absent
((Table 3). Tannins and polyphenols were not
detected in any of the A. sativum extracts under the conditions of this study. Growth inhibition was observed in
the present study when A. sativum extracts were incorporated into potato
dextrose agar (PDA) medium. All the extracts irrespective of the extractant used were effective against the three fungi
tested though the level of antimicrobial activity differed slightly. At
higher concentrations the extracts completely inhibited the growth of the
fungi tested in this study. The acetone extract was inhibitorier
to the radial growth of all the test fungi. This may be due to the additional
effect of the extractant. Antifungal activities
have been detected in crude medicinal plant extracts of 20 plant species
against Fusarium oxysporum
causing wilt disease in Solanum melongena L. It has been reported that the potency of
any plant extract depends on its concentration and the method of extraction.
The systemic screening of antimicrobial plant extracts represents a
continuous effort to find new compounds with potential to act against
multi-resistant pathogenic bacteria and fungi (Shariff
et al., 2006). Investigations into
the chemical and biological activities of plants during the past two
centuries have yielded compounds for the development of modern synthetic
organic chemistry and the emergence of medicinal chemistry as a major route
for the discovery of novel and more effective therapeutic agents (Roja and Rao, 2000). The result of phytochemical
screening showed that water and ethanol extracted more components from crude
garlic extract than acetone under the conditions of this study. While
carbohydrates, reducing sugars, lipids, flavonoids,
ketones, alkaloids, steroids and triterpenes were extracted both in water and ethanol,
acetone could not extract alkaloids, steroids and triterpenes. Farombi (2003)
and Okigbo et
al. (2009a) had reported some of these components in many other plants.
In most traditions, decoctions or infusions of herbs are usually made with
either alcohol or water as the solvent. This may be related to their
efficiency in extracting most of the active principles in plants. At times,
marked differences exist between the phytochemical
profile of alcoholic and aqueous extracts of plants. For A. sativum, the aqueous extract is
recommended because no vital phytochemical
constituent seemed to be left out and also because of probable unwanted
effects that alcohol which is another drug on its own may produce. The inhibitory activity of A. sativum and
its healing power has been reported (Cavallito,
1945). This may be due to the presence of active components like flavonoids, alkaloids, steroids and triterpenes.
Flavonoids are potent water-soluble antioxidants
and free radical scavengers, which prevent oxidation, cell damage and have
strong anticancer activity. They also lower the risk of heart diseases (Sofowora, 1993). Alkaloids have been documented to
possess analgesic, antispasmodic and bactericidal effects (Okigbo et al., 2009b). Garlic powder preparations have
been reported to be of some clinical use in subjects with mild hypertension. The fungi used for this study are
economically important for different reasons. A. flavus produces a violent toxin
called aflatoxin. It is a highly toxic substance
which has some carcinogenic effects and may cause cancer of liver in humans
and animals (Sharma, 2005). Curvularia lunata is a member of the family Dematiaceae
of the order Moniliales. It occurs on rice and many
other crops, causing leaf spots, blights, grain deformation, grain discolouration and even root rot. Fusarium moniliforme belongs to the family Tuberculariaceae and occurs on many crop plants such as
rice (Oryza sativa L.), maize (Zea mays L.)
and sorghum (Sorghum vulgare L.). It causes serious wilting in the host
plants as well as diseases like foot rot, seedling blight, stalk rot and top
rot (Pandey, 2008). More research activities may be
required to elucidate the exact structures of the active principles in garlic
responsible for the observed antimicrobial properties. References Al-Zahim, N., Newberry, J. H. and Ford-Lloyd,
B. V. (1997. Classification of genetic variation in garlic (Allium sativum L.).
Hortscience,
36:1102-1104. Anon, 2009. www.growingtaste.com/vegetables/garlic.shtml Awosika, F. 1993. Traditional medicine
as the solution to Nigeria Health problems. Clinical pharmacology and Herbal Medicine, 9 (3): 26-31. Cavallito, C. J., Bailey, J. H. and Buck,
J. 1945. Allicin, the antibacterial principle of Allium sativum. III. Its
precursor and “essential oil” of garlic. J.
Am. Chem. Soc.67:1032. Farombi, E. O. 2003. African indigenous
plants with chemotherapeutic potentials and biotechnological approach to the
production of bioactive prophylactic agents. African Journal of Biotechnology 2(12): 662-671. Johnson, M., Maridass, M. and Irudayaraj,V. 2008). Preliminary Phytochemical
and Anti-Bacterial Studies on Passiflora edulis. Ethnobotanical Leaflets
12: 425-432. Nguelefack,T. B,Watcho,
P., Wansi, S. L. and Kamany,
A. 2005. Effects of the methanolic leaf extract of Alchornea cordifolia (Schum. & Thonn.) Muell. Okigbo, R. N., Anuagasi, C. L. and Amadi, J.
E. 2009a. Advances in selected medicinal and aromatic plants indigenous to Research 3(2): 086-095. Okigbo, R. N., Anuagasi,
C. L., Amadi, J. E. and Ukpabi,
U. J. 2009. Potential Inhibitory Effects of Some African
Tuberous Plant Extracts on Escherichia
coli, Staphylococcus aureus and Candida albicans. International Journal of Integrative Biology, 6(2): 91-98. Pandey, P. B. 2008. Plant Pathology. S. Chand and Company Ltd., Roja, G. and Rao,
P. S. 2000. Anticancer compounds from tissue cultures of medicinal plant. J. Herbs, Spices Med. Plants 7:71-102. Ross, Z. M., Maslin, D. J. and Hill, D. J. 2000. The effect of steam
distilled garlic oil on lactic acid and other enteric bacteria. 4th
Symposium on European Microbiological Societies. FEMS Microbiol. Rev. 12(G43): 137. Shariff, N., Sudharshana,
M. S., Umesha, S., and Hariprasad,
P. 2006. Antimicrobial activity of Rauvolfia tetraphylla and Physalis minima leaf and callus extracts. African Journal of Biotechnology 5:946-950. Sharma, O. P. 2005. Textbook of
Fungi. 14th Reprint. Tata Mc Graw-Hill
Publishing Company Ltd., Sofowora, A. 1993. Medicinal Plants and Medicine in Books, Yukihiro, K., Makoto, I., Jiro, Y., Naoki,
K., Naoto, U., Isao, S., Nagatoshi, I. and Kazuhisa, O. 2002. Pharmacokinetic study of allixin,
a phytoalexin produced by garlic. Chem. Pharm. Bull., 50: 352-363. Table 1. Nutritional
value per 100g (3.5oz) of raw garlic.
Table 2. Phytochemical
Observation of A. sativum Extracts.
Table 3. Phytochemical
Constituents of A. sativum Extracts.
+
Present - Absent |
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