Journal of Babylon University/Pure and Applied Sciences/ No.(5)/ Vol.(22): 2014
Yazi Abdullah Jassim Maha Jadoa Nushia
University of Babylon/ College of science/ Department of Biology
Crude extracts from dried garlic ( Allium sativum ) and cloves ( Eugenia caryophyllus ) were studied for their antimicrobial activity against Staphylococcus aureus , Staphylococcus epidermidis and
Pseudomonas aeruginosa.
. A total 60 clinical specimens were collected from patients clinically diagnosed as cases of bacterial infections in Marghan Hospital in Babylon. Gram positive bacteria were isolated from 43 specimens and 17 specimens were gram negative bacteria. Gram positive bacteria identified by cultural and biochemical tests , among those isolates , 24 isolates were Staphylococcus epidermidis and 19 isolates were Staphylococcus aureus . Gram negative bacteria identified by cultural and biochemical tests and all isolates were Pseudomonas aeruginosa . The effect of ethanolic and water extract were tested against isolating bacteria by using agar well diffusion method. Forty microliters of the dilution of Crude extracts was transferred to each well and sterile water served as a control.
Extract from cloves showed the highest activity with minimum inhibitory concentration (MIC) ranging from 100 to 400 mg/ml, .Garlic extract showed no inhibitory effect against these bacteria. The inhibitory activity of the herbs extracts decreased over the storage period 4 weeks at 4c◦.
Staphylococcus aureus , ةايب قا ةالدضر ةقا قارضايمقا تالريتير تاب دضامقال والنررقا تتابن صلختسم ريثأت ةسارد تمت
تاةمج .
Pseudomonas aeruginosa
تانتا ةا يع 17 ل ةا يع 43 نام د ا
ةالرتانزقا ائالزقا ل Staphylococcus epidermidis ةاللربتقا ةالدضر ةقا قارضايمقال
را ةغباصق ةابجضمقا تالريتيبقا تقزع . ورتر ف نتجرم ىلشتسم ف تهصيخشت مت ىبرم نم ة يع 60
اا ةااقزع 24 تاانتال ةاالضي ضمييقال ةاايعرزمقا قتاايضرلقا داتختااستر داراا ةغبااصق ةاابجضمقا تاالريتيبقا تااصخ . داراا ةغبااصق ةبقتااس
دارا ةغباصق ةبقتاسقا تالريتيبقا تصخ ل Staphylococcus aureus ةقزع 19
رتاشتن ا ةارلرا داتختاستر تالريتيبقا تاب قضاريقال ئتامقا صلختاسمقا
ةايقتةف والنررقا قتابن ص لختسم رهظا .
ريثأات سرد .
ةرطيسا ءتمقا دتختسال ةرل وا ف صلختسمق
ل Staphylococcus epidermidis
ةلضي ضمييقال ةيعرزمقا قتيضرلقا داتختستر
ا لضلرم نم ريتيقلريلتم
40
عبل ثي
تلريتير
تضلأ
ر تا تر
ةاايقتةلقا نأ . تاالريتيبقا ىاالع ريثأاات أ دضاامقا صلختااسم رااهمل مااق تاام ير
.
.عي رتسأ عررأ
(mg/ml)
ةتمق ْد 4
400 – 100 ناام ح لارااتل ىااندأ باامم زاايارتر ةاايقتع
ةجرتر ظلرقا ةرتف للاخ ورت تنتا صلختسملق ةيطيبمتقا
The increasing demand for high quality foods with less synthetic chemicals added means continuing search for alternative sources of antimicrobial compounds.
Spices and herbs used as seasoning agents in foods and beverages have been extensively studied for their antimicrobial activities. Garlic, onion, leek, cinnamon, allspice, cloves, oregano, thyme, savory, celery, parsley and angelica have been investigated (Beuchat& Golden, 198; Conner,1993;Elgayyar et al, 2001). The latest trend in food packaging includes the incorporation of antimicrobial substances in packaging materials (Hotchkiss,1995; Appendini and Hotchkiss, 2002 ; Brody et al ., 2001). For a long period of time, plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies. According to World Health Organization source to obtain a variety of drugs(Santos et al.,1995) medicinal plants would be the because not only does it decrease the use of synthetic , but it also provides added functions, such as provision of nutraceuticals and flavors (Han,2002). Therefore, such plants should be investigated to better understand their properties, safety and efficiency (Ellof,1998) Cloves ( Syzygium aromaticum , syn. Eugenia aromaticum or
Eugenia caryophyllata ) are the aromatic dried flower buds of a tree in the family
Myrtaceae (Banerjee & Das,2005) Cloves are used in Ayurveda, Chinese medicine and Western herbalism. Cloves are used as a carminative, to increase hydrochloric acid in the stomach and to improve peristalis (Saeed & Tariq, 2008). It is also used in
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dentistry where the essential oil of clove is used as anodyne for dental emergencies
(Gislene et al .,2000). cloves is a plant which is believed by herbalists to have antimicrobial effect. This plant has been tested in the present study to investigate their in vitro potential effect against Gram positive and Gram negative bacterial species(Saeed & Tariq,2008)…
The bacterial strains used in this study were Staphylococcus aureus ,
Staphylococcus epidermidis and Pseudomonas aeruginosa , obtained from Marghan
Hospital in Babylon. They were kept in 40% (v/v) glycerol and nutrient broth, and stored at -18

C. A working culture was prepared by inoculating a loopful of each isolates into 10 ml of nutrient broth, which was subjected to successive 24 hrs transfers before use .
:
Tow selected herbs in powder form namely ( Eugenia caryophyllus ) cloves and
( Allium sativum Linn.) garlic were collected from Hilla . Fifty grams of each sample was extracted in 500 ml of both ethyl alcohol 95% and distilled water at 50

C for 24 hrs, then filtered and concentrated by vacuum evaporation at 50

C, to obtain 50 ml of crude extract (Jatuapornchai, 2003). The extracts were kept in amber glass bottles and stored at 4

C.
Antimicrobial activities of crude extracts were assayed using the agar well diffusion method (Rauha et al., 2000) . Four wells (diameter 5 mm ) were made on each nutrient agar plate using a sterile cork borer and inoculated with test bacteria
Crude extracts were serially diluted to yield dilutions of 100, 125, 200, 250, 500, 750 and 1000 mg/ml. Forty microliters of the dilution was transferred to each well and sterile water served as a control.
Then, the plates were incubated at 37

C, for 24 hrs.
MICs (the lowest concentration of extract that resulted in the zones of inhibition with a diameter greater than 7 mm( Nascimento et al.
,2000 ) were determined. The tests were repeated at week 1, week 2, and week 4 to investigate the stability of the extracts during storage at 4

C.
The results in Table 1 were very useful for selecting herb extracts for further study.Garlic was screened out, as no inhibitory effect against any test bacteria was observed. This result was in accordance with a previous report (Onyeagba et al., 2004;
Poovarodom etal.,2009). It was also evident that the preparation of crude extracts using ethyl alcohol was preferable to water. Therefore, only extracts with ethyl alcohol were considered for further studies.
Cloves had the most pronounced antimicrobial effect, with MICs of 100-150 mg/ml It is important to note that extract of cloves could inhibit the growth of all test bacteria as shown in table 2 and 3.
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Journal of Babylon University/Pure and Applied Sciences/ No.(5)/ Vol.(22): 2014
Table 1:
Herb
Allium sativum
( garlic)
Extracts
Water
Ethanol
Diameters of the inhibition zone (mm)
MIC (mg/ml)
S. aureus
_
S. epidermidis
_
P. aeruginosa
_
_ _ _
Eugenia
caryophyllus
(cloves)
Water
Ethanol
400
100
400
150
150
150
aeruginosa):
The results in( Table 2 & 3) were important to note that extracts of
Eugenia caryophyllus could inhibit the growth of all test bacteria. The diameters of the inhibition zone varied in the range 0f 11 to 25mm for the gram-positive bacteria,(
S . aureus and S. epidermidis ), and from 9 to 16.6 mm for the gram-negative bacterium, ( P. aeruginosa) . This finding corresponded well to previous reports
(Kanika,2001; Jaturapornchai,2003; Petinaki et al.,2006) . Eugenia caryophyllus contain eugenol compounds, triterpenes and many organic acids, which can disturb cell activities in many ways, such as food absorbability and cell membrane disruption. Thus, cloves were capable of inhibiting both gram-positive and gram negative bacteria (Kanika,2001; Jaturapornchai,2003).
Table 2: Effect of alcohol cloves extract on Gram positive bacteria S. epidermidis and S. aureus
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Number of isolation S. epidermidis(mm) S. aureus
1 24 11.7
3
4
5
6
7
8
Storage 22
17.5
MIC (mg/ml)
11
21
19.3
17
25
15.5
22
12.8
17
19.6
23.3
11.2
Table 3: Effect of alcohol cloves extract on Gram negative bacteria P. aeruginosa .
Number of isolation
1
Diameters of the inhibition zone for on P. aeruginosa
9.8
5
6
7
8
2
3
4
11.7
10.6
17
9
12.8
16.6
9.9

The inhibitory effect of crude extracts marginally decreased over the storage period of 4 weeks at 4

C as shown in Table 4.
Therefore, MICs slightly increased, as can be seen in Table (4). However, cloves extract still have inhibitory activity against the test bacteria. Therefore, it is essential to take into account the stability of antimicrobial agents during storage when studying their MICs and their suitability in various applications.
Table 4:

.
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Journal of Babylon University/Pure and Applied Sciences/ No.(5)/ Vol.(22): 2014
Eugenia
caryophyllus
(cloves) period
1 day
1 week
2 weeks
4weeks
S. aureus
100
100
250
250
S. epidermidis P. aeruginosa
150 150
200
200
200
150
200
400
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