J Basic Pharmacol Toxicol.

2019;3(1):14-18 14

JOURNAL OF BASIC
Original Research Article PHARMACOLOGY AND
TOXICOLOGY

Phytochemical screening and antibacterial potentials of the garlic (Allium

sativum) extracts against clinical isolates
Fadiji A E *
Department of Pure and Applied Biology, Ladoke Akintola University of Technology, P.M.B 4000, Ogbomoso, Oyo State, Nigeria

Abstract

This study investigated various phytochemicals and antibacterial activity of garlic (Allium sativum) bulb extract on five clinical
bacterial pathogens using the agar well diffusion method. These bacteria include; Pseudomonas aeruginosa, Staphylococcus
aureus, Klebsiella sp, Escherichia coli, and Salmonella typhi. Two different extracts were obtained from the bulbs of garlic
(ethanol-soluble and methanol-soluble extracts). Phytochemical screening of the garlic bulb extracts indicated the presence of
glycosides, steroids, phlobatanins, flavonoid, alkaloids, terpenoids and carbohydrate while saponin, phenolics and tannins were absent. There
were zones of inhibitions around the wells which indicate that the organisms were sensitive to both ethanol and methanol extract
of garlic having inhibitory strength on the test organisms ranging from 2-32 mm. The MIC and MBC revealed the isolates behaved
differently in their sensitivity ranging from ranged from 20 - 76 mg/mL for ethanolic extract, while methanolic extract has ranged
from 13 - 62 mg/mL. The methanol extract of the garlic bulb was absolutely more effective against five pathogenic bacteria than
the ethanolic extract. This study indicates that Allium sativum had great antibacterial effect thus confirming its usage in herbal
medicine.

Keywords: Allium sativum, antibacterial activity, garlic bulb, clinical isolates, phytochemical screening

effective agents that can be used to fight infectious diseases

1. Introduction [6].
Allium sativum, popularly known as garlic is one of the
Plants contain numerous biologically active compounds species of the onion family Alliaceae and belongs to the
which have the potential for development as medicinal plant order Liliales [7]. Among all the Allium species, garlic
agents. Traditional medicines already form the basis of is the most important [8]. Garlic is commonly called
therapeutic use in the developing countries, but of recent, ‘Tantanwa’ in Hausa. Medicinal applications of garlic have
there has been an increase in the use of herbal medicines in been known for many ages [9]. The plants are broadly used
the developed world too [1]. Plants provide an alternative as antibiotics and are effective against cancer,
strategy in the search for new drugs. There is a rich arthrosclerosis and diabetes [10, 11]. Garlic has been
abundance of plants reputed in traditional medicine to reported to be effective in reducing blood plasma
possess protective and therapeutic properties [2]. It is likely cholesterol and blood pressure. It also inhibits platelet mass
that plants will continue to be a valuable source of new formation [12]. The most active components present inside
molecules which may, after possible chemical fresh garlic are alliin and an enzyme called alliinase. Its
manipulation, provide new and improved drugs [3]. medicinal claims have included cures for cold, toothaches,
Phytochemicals found in plants include alkaloids, coughs and other viral infections, open wounds and evil
glycosides, essential oil, saponins, tannins, steroids, demons [4]. The aim of this study was to assess the
terpenoids, resins, flavonoids, proteins and others [4]. These phytochemical profile and antimicrobial activities of
chemicals are bioactive compounds present in medicinal ethanolic and methanolic extracts of A. sativum bulb.
plant parts and can be used for therapeutic purposes [5].
These inherent bioactive principles differ from plant to plant
as a result of their biodiversity and they produce significant 2. Materials and methods
physiological effects in human body. Bacterial resistance to
antibiotics is a serious challenge, clinicians and the 2.1 Test organisms
pharmaceutical industries are battling with and intensive
studies are ongoing globally to address this challenge. The bacterial species used in this study, Salmonella typhi,
Screening of medicinal plants is one of the most researched Escherichia coli, Klebsiella sp, Pseudomonas aureginosa,
area with the hope of getting safer, newer, and more and Staphylococcus aureus were obtained from the
Department of Microbiology, University of Ilorin Teaching

*
Corresponding author; E-mail: [email protected]
Fadiji AE. Antibacterial potentials of A. sativum in clinical isolates 15

Hospital (UITH), Ilorin, Kwara state. Purity of the cultures 2.7 Antimicrobial Assay of the Plant Extracts
was checked at regular intervals as described by
Acheampong et al. [13]. Prepared sterile nutrient agar plate was inoculated with
standardized organisms of 0.1ml of a day old culture. Glass
2.2 Plant collection, identification and processing spreader was used in spreading the inocula evenly on the
surface of the agar and excess are drained off. A sterile cork
Samples of the bulbs were bought at Ipata Market, Ilorin, borer of 5 mm diameters was used to make five (5) ditches
Kwara State. The plant samples were confirmed at the on the plates. The bacteria were inoculated into nutrient agar
herbarium unit of the Department of Pure and Applied with varying concentrations of the extracts 250 mg/ml, 100
Biology, Ladoke Akintola University of Technology, mg/ml, 50 mg/ml and 25 mg/ml were prepared from the
Ogbomoso. The fresh samples were sundried for a week, stock concentration of the extracts. 0.5 ml of each
ground into a fine powder and kept in plastic containers until concentration of the extracts was dispensed into each of the
further use at room temperature (28 ± 1 °C). The scales ditches on the plates that are appropriately labeled. The fifth
around the fresh bulbs of garlic were removed and the bulbs ditch in the plates was picked as control by adding 0.5 ml of
were washed and rinsed properly in tap and sterile distilled appropriate solvent use for the different extraction. The
water, respectively. The bulbs were macerated, dried in an plates were done in duplicates and left on the bench for few
electric oven and milled mechanically before being stored minutes for the extract to diffuse into the agar and later
in a plastic container. incubation at 37 0C for 24 hr. After incubation, the zone of
clearance around each ditch was measured using a metric
2.3 Preparation of ethanolic extracts ruler by taking measurement of the zone of clearance around
the ditch. The diameter of the cork borer was removed from
One hundred grams (10 g) of each of the plant parts the diameter of the zone of clearance and this made or
(seed, leaf and stem bark) were soaked into 100 ml of the represented the antibacterial activity measured or diameter
solvent (95% ethanol) in different air-tight sterile jars of the zone of inhibition.
respectively at room temperature and kept on a shaker (90
rpm) with uniform shaking for 24 hr. The solvents 2.8 Determination of Minimum Inhibitory Concentration (MIC)
containing the extracts were decanted filtered with a muslin of the Extracts
cloth and then with Whatman no. 1 filter paper respectively.
Further extraction of the ground samples was done with the Broth dilution method was used to determine MIC.
same volume of 95% ethanol, decanted and filtered two Varying concentrations of the extracts were used that ranged
more times. The filtrates from each round of extraction were from 10.0 mg/ml to 100 mg/ml. 0.5 ml of each concentration
combined and were evaporated to dryness in small, open - of the extracts were dispensed to 9 ml of nutrient broth
mouth jars and then packed in separate clean dry bottles and containing 0.5 ml of standardized test organisms (bacteria
stored at room temperature until required. and fungi) cells. The tubes were incubated aerobically at 37
0
C for 24 hr for bacterial controls were equally set up by
2.4 Methanolic Extract using solvents and test organisms without the extract. The
tube with the least concentration of extract that does not
The methanol extract of the garlic bulb was prepared show growth after incubation was picked as the minimum
using the procedure described by Ameh et al. [4]. Twenty- inhibitory concentration (MIC) [16].
five gram (25 g) of the powdered sample was soaked in a
mixture of methanol and distilled water in the ratio of 3:2 2.9 Determination of Minimum Bactericidal Concentration
for four days and later filtered to obtain the methanol (MBC)
extracts. The mixture was first concentrated by evaporation
using a water bath at 100°C for 1 hr. The culture tubes used in MIC (Minimum Inhibitory
Concentration) determination that did not show turbidity or
2.5 Sterility of extracts any visible growth after the period of incubation were drawn
with a syringe (0.5 ml) and dispense onto the surface of
Each of the extracts was tested for growth of Nutrient agar. The inocula were seeded on the surface of the
contaminants by using the procedure of Fadiji et al. [14]. media. The plates were incubated at 37 0C. The lowest
The plates were observed for growth. Absence of microbial concentration of the extract that inhibits the growth of the
growth in the extract indicated their sterility. Sterile extracts organisms on the plates after incubation was regarded as
were used to test for antimicrobial efficacy. Minimum Bactericidal Concentration (MBC) [14].

2.6 Standardization of inoculums 2.10 Phytochemical screening

Standardized inoculums of each tested organism were The phytochemical screening of the garlic bulb extract
obtained by making their respective suspension up to 0.5 was carried out according to the procedures described by
McFarland standard as observed in the spectrophotometer Trease and Evans [17] and Fadiji et al. [14].
and as described by Barry et al. [15].
J Basic Pharmacol Toxicol. 2019;3(1):14-18 16

2.11 Statistical analysis sp, S. aureus, and Salmonella typhi varied from one extract
to another. The methanolic extracts had the best MIC and
The data obtained in this study were expressed as mean MBC on all the clinical isolates tested compared to the
± SEM and subjected to analysis by one-way ANOVA. p aqueous extracts. Both extracts had considerable MIC and
values of 0.05 or less were considered significant using MBC values for all the pathogenic bacteria tested. The
SPSS software version 17.0. values ranged from 20 - 76 mg/mL for ethanolic extract,
while methanolic extract has ranged from 13 - 62 mg/mL.
These concentrations gave bactericidal and fungicidal
3. Results effects after 24 hr of incubation. They are therefore regarded
as the lowest concentrations of the extract sufficient to kill
3.1 Antimicrobial effects of aqueous and ethanolic extract of a defined proportion of viable organisms at a specified
plant samples period (Table 2). All values from the treatments were
significantly (p < 0.05) different from each other.
The screening for antimicrobial activity of the plant
samples used in this study revealed that the plant extracts 3.3 Phytochemical analysis of the plant samples

Table 1. Antimicrobial effects of ethanolic and methanolic extract of Alium sativum (garlic) on tested organisms
Name of Zone of inhibition (mm)
Organisms
Ethanolic extract Methanolic extract
250 100 50 25 Control 250 100 50 25 control
mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL

Pseudomonas 25 ± 0.01 13 ± 0.17 10 ± - - 29 ± 0.04 17 ± 0.03 13 ± - -

Aeruginosa 0.03 0.13
Escherichia coli 26 ± 0.03 17 ± 0.02 - - - 32 ± 0.03 22 ± 0.06 13 ± 9 ± 0.13 -
0.03
Salmonella 19 ± 0.08 12 ± 0.01 9 ± 0.01 3 ± 0.13 - 24 ± 0.05 19 ± 0.11 11 ± 5 ± 0.01 -
Typhi 0.19
Klebsiella sp 11 ± 0.01 7 ± 0.05 - - - 16 ± 0.01 11 ± 0.02 5 ± 0.12 - -
S. aureus 6 ± 0.03 2 ± 0.02 - - 13 ± 0.17 8 ± 0.04 3 ± 0.11 - -
P level (0.05) *** *** *** *** *** *** *** *** *** ***
KEY: - = No Zone of inhibition. Values expressed as mean ± SEM; *** = Means square significant at p < 0.001

had varying effects on the growth of the clinical isolates. All The phytochemical analysis of the plant extracts had
the plant extracts have inhibitory strength on the test shown that the tested plant parts contain some active
organisms ranging from 2-32 mm. The methanolic extract components. The ethanolic and methanolic extracts of

Table 2. Minimum inhibitory concentration and minimum bactericidal concentration of both ethanolic
and methanolic of extracts of Allium sativum
Microorganisms Ethanolic Extract Methanolic extract
MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL)
Pseudomonas 30 ± 0.02 59 ± 0.19 21 ± 0.13 46 ± 0.11
Aeruginosa
Escherichia coli 45 ± 0.014 68 ± 0.15 33 ± 0.08 52 ± 0.13
Salmonella 25 ± 0.08 52 ± 0.17 19 ± 0.09 37 ± 0.03
Typhi
Klebsiella sp 20 ± 0.02 34 ± 0.07 13 ± 0.05 26 ± 0.18
S. aureus 48 ± 0.14 76 ± 0.09 39 ± 0.01 62 ± 0.06
p level (0.05) *** *** *** ***
KEY: - = Not determined, MIC= Minimum Inhibitory concentration, MBC= Minimum bactericidal
concentration, MFC= Minimum fungicidal concentration.
Values expressed as mean ± SEM; *** = Means square significant at p < 0.001

of Alium sativum had the highest zone of inhibition Alium sativum showed that it contains glycosides, steroids,
approximately on all pathogens tested with extracts having phlobatanins, flavonoid, alkaloids, terpenoids and
29 mm and 32 mm against Pseudomonas aeruginosa and carbohydrate in a tangible amount with more presence in the
Escherichia coli at 250 mg/mL (Table 1). The lowest methanolic extract was observed when compared to the
inhibition was recorded in ethanolic extracts with 2 mm ethanolic extract, while saponin, phenolics and tannins were
against S. aureus at 100 mg/mL. absent (Table 3).
Table 3. Phytochemical analysis of aqueous and ethanolic extract of
3.2 Minimum inhibitory concentration and minimum bactericidal Alium sativum (Garlic)
/ fungicidal concentration of aqueous and ethanolic extracts of S. Allium sativum (Garlic)
plant samples on the tested organism No. Chemical Ethanolic Methanolic
constituents
1 Alkaloids ++ +++
The MIC and MBC values obtained for the plant extracts 2 Tannins - -
on Escherichia coli, Pseudomonas aeruginosa, Klebsiella 3 Saponins - -
Fadiji AE. Antibacterial potentials of A. sativum in clinical isolates 17

4 Phlobatanins + ++ Conclusion
5 Flavonoids + ++
6 Terpenoids + +++
7 Glycosides + +++ The present study, therefore, shows that A. sativum bulb
8 Steroids - + extracts have useful antimicrobial properties. The strong
9 Phenolics - - antibacterial activities showcased by both the ethanolic and
KEY +++ = More Strongly Present, ++ = Strongly Present, + = Present, -
= Absent
methanolic extracts of A. sativum bulb against the test
organisms brought about its likely recommendation for
frequent input of garlic in pharmaceutical and foods and
4. Discussion products. Structural elucidation of the bioactive compounds
is recommended in order to be able to ascertain the actual
In this study, the five clinical bacterial pathogens were antimicrobial activity of the various phytochemicals. Also,
studied to assess the antimicrobial properties of garlic. The efforts should be made by pharmaceutical companies
findings showed that they were sensitive to the plant towards the development of novel drugs which are of natural
extracts. The plant extracts showed varying degrees of origin.
antimicrobial activity on the microorganisms. This is in
agreement with the work of Rojas et al. [18] on ten
medicinal plants. References
Phytochemical screening of the garlic bulb extracts
1. El-Mahmood, A. M., Ogbonna, O. B., Raji, M. (2010). The
indicated the presence of glycosides, steroids, phlobatanins,
antibacterial activity of Azadarichta indica (neem) seeds
flavonoid, alkaloids, terpenoids and carbohydrate while
extracts against bacterial pathogens associated with eye
saponin, phenolics and tannins were absent. These bioactive and ear infections. J Med Plant Res. 4(14).
compounds found in most plants are believed to be 2. Kayode A.A.A., Kayode, O.T. (2011). Some Medicinal
responsible for the observed antibacterial activities of the Values of Telfairia occidentalis: A Review. American
plant extract. A previous study attributed the antimicrobial Journal of Biochemistry and Molecular Biology, 1: 30-38.
effect of plant extract to the presence of these secondary 3. Shah, J.S., Shah, M.B., Goswami, S.S., Santani, D.D.
plant metabolites [19]. Similar phytochemical analysis of (2006). Mechanism of action of antiulcer activity of bark
plant extracts of Ocimum gratissmum revealed the presence ex-tracts of Manikara hexandra against experimentally
of alkaloids, flavonoids, cardiac glycosides, steroidal induced gastric ulcers in rats. Phcog. Mag., 2: 40-45.
terpenes and tannins [14]. The presence of active 4. Ameh, G. I., Eze, S. C., Omeje, F. U. (2013).
compounds in the bulb extract of A. sativum could be used Phytochemical screening and antimicrobial studies on
to establish its usage in traditional medicine. the methanolic bulb extract of Allium sativum L. African
The inhibitory activities of the plant extract agree with Journal of Biotechnology Vol. 12(14), pp. 1665-1668.
5. Nwadiaro PO, Nwachukwu I (2007). Inhibition of
the report of Leven et al. [20]. Nwadiaro and Nwachukwu
Pathogenic Fungi by ethnobotanical extracts of
[5] linked the antimicrobial activities of plants to the
Cymbopogon citrates, Ceiba pentandra and Loranthus
presence of tannins, alkaloids, flavonoids and saponins. It bengwelensis. Nig. J. Biotechnol. 18(1-2):61-65.
has been reported that various plants that are rich in 6. Natarajan, V., Veugopal, P.V., Menon, T. (2003). Effect of
alkaloids, tannins and glycosides possess antimicrobial Azadirachta indica (Neem) on the growth pattern of
activity against a number of microorganisms [21]. This dermatophytes. Indian Journal of Medical Microbiology
suggests that the plant extract used in this study has a 21(2):98-101.
general antimicrobial activity. The antibacterial potential of 7. Rocio, M. C., Rion, J. L., (1982) A Review of Some
A. sativum bulb extract is more pronounced in the Antimicrobial Substances Isolated
methanolic extract than its ethanolic extracts. This is in from Medicinal Plants Reported in the Literature Review
agreement with an earlier report of Gomaa and Hashish [22] of Phytochemical Analysis on Garlic 1978 – 1972.
in which the inhibitory property of garlic extracts on the Phytother. Review. 3:117-125.
growth of some microorganisms including Salmonella typhi 8. Alli JA, Boboye BE, Okonko IO, Kolade AF, Nwanze JC.
was reported. 2011. In-Vitro assessments of the effects of garlic (Allium
sativum) extract on clinical isolates of Pseudomonas
The varying inhibition sizes recorded in this study is an
aeruginosa and Staphylococcus aureus. Advances in
indication of the bioactive strength of phytochemicals
Applied Science Research 2(4):25-36.
contained in the garlic bulb. The varying MICs and MBCs 9. Akintobi OA, Nwanze JC, Ogele JO, Idowu, A.A.,
of the plant extracts on the test organisms are in agreement Onianwa, O., Okonko, I.O. (2013). Antimicrobial activity
with the report indicating that microorganism exhibits a of Allium sativum (Garlic) extract against some selected
different level of susceptibility to plant extracts [23]. Lower pathogenic bacteria. Nature and Science 2013;11(1).
MIC and MBC values were recorded in methanolic extracts 10. Chiba, O., Hughes B. G., Lawson L. D., (1998)
as compared with ethanolic extract is an indication that Antimicrobial Effects of Allium sativum L. (garlic), Allium
methanolic extract has more antibacterial strength than the ampeloprassum (elephant garlic) and Allium cepa (onion),
ethanolic extracts. garlic compounds and commercial garlic supplement
products. Phytother. Vol 5:154- 158.
11. Ghazanfari, T., Hassan, Z. M., Brahimi, M. E. (2002)
Immunomodulatory activity of a protein isolated from
J Basic Pharmacol Toxicol. 2019;3(1):14-18 18

garlic extract on delayed type hypersensitivity. Int. 18. Rojas JJ, Ochoa VJ, Ocampo SA, Munoz JF (2006).
Immunopharmacology. Vol 2: Pp 151-154. Screening for Antimicrobial activity of ten Medicinal
12. Mayeux, P. R. and Babayi, H., Okogun, I. J., Ijah, J. J. Plants in Colombian Folkloric Medicines: A possible
(1997) The antimicrobial activities of methanolic extracts alternative in the treatment of non-nosocomial
of Eucalyptus camadulensis and Terminalia catapa against infections. Comp. Altern. Med. 6:2-12.
some pathogenic microorganisms. J. Nig. Soc. Ext. Biol. 19. Gandhiraja N, Sriram S M V, Srilakshini JK, Sasikumar
16(2): 106-111 (s). C, Rajeswari R (2009). Phytochemical screening and
13. Acheampong YB, El-Mahmood AM, Olurinola PF (1988). antimicrobial activity of the plant extracts of Mimosa
Antibacterial properties of the liquid antiseptic TCP. pudica L., against selected microbes. Ethnobot. Leaflets
Indian J Pharm Sci. 3: 183-186. 13:618-624.
14. Fadiji, A. E., Omomowo, I., Omomowo, O., Lanrewaju, 20. Leven M, Vanden-Berghe DA, Marten I, Vilientnick A,
A., Akande, D. (2018). Antimicrobial efficacy Lomweas EC (1979). Screening of higher plants for
andphytochemical screening of aqueous and ethanolic biological activity. Planta Medica 36:311-312.
extracts of Ocimum gratissimum (scent leaf) leaf against 21. Adebajo AO, Adewumi CO, Essien, EE (1983).
some clinical isolates. Journal of Basic Pharmacology Antiinfective agent of higher plants. International
Toxicology 2, 19-24. Symposium of Medium Plants. 5th ed. University of Ife,
15. Barry AL, Thornsberry C. (1980). Susceptibility testing: Nigeria. Pp. 152-158.
Diffusion test procedure. Manual of Clinical 22. Gomaa, N. F., Hashish, M. H., (2003) The Inhibitory
Microbiology. 3rd Edition, ASM Washington DC, USA. Effect of Garlic (Allium sativum) on the Growth of Some
16. Egharevba HO, Odigwe AC, Abdullahi MS, Okwute SK, Microorganisms. J. Egyt. Public Health Assoc. 78 (5-6):361-
Okogun JI (2010). Phytochemical Analysis and Broad 372. (s).
Spectrum Antimicrobial Activity of Cassia Occidentalis 23. Banso A, Adeyemo SO (2007). Phytochemical and
L. (whole plant). New York Sci. J., 2010; 3(10): 74-81. Antimicrobial Evaluation of Ethanolic extract of
17. Trease GE, Evans WC. Pharmacognosy. 15th Ed. Dracaena mannii Bark. Nig. J. Biotech. 18(1-2):27-32
London: Saunders Publishers; 2002. pp. 42–44. 221–229,
246–249, 304–306, 331–332, 391–393.