Yasmeen et al., The Journal of Animal & Plant Sciences, 22(2): 2012, Page:J.

416-419
Anim. Plant Sci. 22(2):2012
ISSN: 1018-7081

ANTIBACTERIAL ACTIVITY OF INDIGENOUS HERBAL EXTRACTS AGAINST

UREASE PRODUCING BACTERIA
R. Yasmeen, A. S. Hashmi, A. A. Anjum*, S. Saeed and K. Muhammad*

Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan
*
Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan
Corresponding author E. mail: [email protected]

ABSTRACT
Aqueous and alcoholic extracts of 14 local herbs (Aloe Vera, Azadirachta indica, Allium sativum, Calotropis procera,
Cannabis sativa, Carum capticum, Eucalyptus camaldulensis, Lantana camara, Mangifera indica, Mentha piperita,
Nigella sativa, Opuntia ficus indica, Piper nigrum and Zingiber officinalis) and four commercial products (Mentofin,
Suduri, Safi, Yucca) were evaluated for their in-vitro antibacterial activity against Proteus mirabilis by serial dilution
method. Complete random design (CRD) was followed. It was observed that with reference to rise in pH, Ammonia
concentration and urease activity in aqueous and alcoholic extracts of Allium sativum (pH: 8.5560, 8.8480,
Ammonia:4.42, 3.52 µg/mL, Urease: 0.009, 0.007 IU/mL respectively) had shown best results as compared to control
positive (pH: 9.03, Ammonia: 6.7µg/mL, Urease: 0.013 IU/mL). Alcoholic extracts of Mangifera indica (8.8820,
5.42µg/mL, 0.010 IU/mL), Mentha piperita (8.8880, 4µg/mL, 0.008IU/mL) Carum capticum (8.9540, 4.84µg/mL,
0.009IU/mL) and aqueous extract of Opuntia ficus indica (8.8100, 5.22µg/mL, 0.010 IU/mL) had weak activity against
P. mirabilis. Both aquous and alcoholic extracts of Euclyptus camalduensis (pH: 8.91, 8.96, Ammonia: 5.16, 5.06
µg/mL, Urease: 0.01, 0.01 IU/mL) had weak inhibitory effect. All commercial products had shown a strong antibacterial
activity (pH: 4.8-6.8, Ammonia: 0µg/mL, Urease: 0 IU/mL). Results of remaining herbal extracts were not significantly
different (p< 0.05) from positive control. It was concluded that all products had strong antibacterial activity against P.
mirabilis. Mentofin at dilution rate of 1/1000 had shown the best results with optimum inhibitory concentration.
Alcoholic extracts of few herbs had shown weak bactericidal activity. These herbs might be effective in in-vivo studies.
Key words: Antibacterial activity, Herbal extracts, Urease producing bacteria.

INTRODUCTION microbes are responsible for conversion of uric acid to

ammonia by secreting urease (Rothrock et al. 2008).
Urease producing bacteria in poultry litter are Gram negative bacteria are mainly responsible for
mainly responsible for polluted environment of the sheds. production of urease in litter. Most of these bacteria
Poultry birds are provided high-protein feed to meet their convert uric acid to urea. A few genera of these bacteria
optimum requirements (Gay and Knowlton, 2005). convert uric acid to ammonia (Kidd, 2011).
Metabolism of the protein produces uric acid and urea as Yucca plant extract in poultry drinking water
waste. This waste is eliminated along with intestinal has ability to inactivate urease producing bacteria in
microorganisms in droppings on litter. In presence of intestine and inhibit ammonia emission from litter
high humidity and ambient temperature, urease producing (Nazeer et al. 2002). Both Aloe vera leaf and gel has
bacteria grow and convert the urea into ammonia. antibacterial activity (Agarry et al. 2005). More than 135
Ammonia is a health hazard for poultry and human compounds have been isolated from different parts of
(Nicholson et al. 2004). The ammonia often accumulates neem tree, which have antibacterial activity (Biswas et al.
inside poorly ventilated or poorly managed poultry sheds 2002). Allium sativum has antibacterial activity against
particularly during winter season. Broilers in an Gram positive and Gram negative bacteria (Iwalokun et
environment having more than 25-50 ppm ammonia al. 2004). Calotropis procera latex demonstrates strong
show reduced body weight, immunosuppression and inhibitory effect against microbes (Kareem et al. 2008).
enhanced susceptibility to respiratory pathogens. This Cannabis sativa exhibited activity both against Gram-
polluted environment is serious health hazard for poultry positive and Gram-negative bacteria and also against the
workers as depicted by Occupational Safety and Health fungi. The seeds of Cannabis sativa have remarkable
Administration (OSHA) (Gay and Knowlton, 2005). impact on growth of broiler chicks and can help in
Poultry litter is rich in protein, urea, uric acid alleviating feed expenditure incurred on raising broiler
and microbes. Poultry litter contains 38% crude protein, chicks (Khan et al. 2010). Eucalyptus extracts displayed
52% fiber and minerals (Lanyasunya et al. 2006). Uric broad antibacterial activity against gram positive and few
acid is 70% of total nitrogen in poultry litter. Litter gram negative bacteria (Cock, 2009). It was observed that
Lantana camara was active against M. tuberculosis

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(Kirimuhuzya et al. 2009), Gram positive Bacillus cereus analyzed using ANOVA and Duncan’s multiple range
and Gram negative Salmonella typhi (Pour et al. 2010). tests by SPSS statistical program version 13.0.
Both seed and leaves of Mango (Mangifera indica) have
antimicrobial activity (Doughari et al. 2008; Sairam et al. RESULTS AND DISCUSSION
2003). M. piperita leaves showed the highest zone of
inhibition (17.24 mm) while stem showed least (15.82 Urease producing bacteria such as Proteus
mm) against 56 Gram negative bacteria (Sabahat and marabilis grew well in urea broth and showed the
Parween, 2005). Black pepper showed activity against enhanced urease activity that resulted in enhanced pH and
75% bacteria from Oral isolates (Nazia and Tariq, 2006). ammonia concentration. Aqueous and alcoholic extract of
Opuntia leaves inhibited intracellular virus replication Allium sativuma had shown weak but the best
and inactivate extra cellular virus (Ahmed et al. 1996). In antibacterial activity as compared to all other extracts in
the present study Extracts (aqueous and alcoholic) of the broth. Aqueous extract of Opuntia ficus indicab and
indigenous herbs were prepared and their antibacterial alcoholic extracts of Carum capticumb, Mentha piperitab
activity against urease producing bacteria was Piper nigrumb, Mangifera indicac and both the extracts of
determined. Euclyptus camaldulensisc showed poor antibacterial
activity. Either of the commercial products did not
MATERIALS AND METHODS support the growth of Proteus mirabilis. The pH of
Yucca (4.8972a) is less than the pH of the control
Isolation and identification of bacteria: Urease negative (6.820). The pH of the medium could be low
producing bacteria was isolated and identified as Proteus because of low pH of commercial product (Yucca: pH 3).
mirabilis from fresh poultry droppings (Bergay and Holt, The pH of control positive is high (9.0280) due to
1994). Twenty four hours old culture at dose rate of 106 absence of any herbal product and urease activity of the
Colony Forming Unit (CFU)/mL was used as inoculum bacteria. Ammonia concentration and urease activity in
(Babayi et al, 2004). the broth containing herbal commercial products was
zero. All the remaining alcoholic and aqueous extracts
Extracts of herbs: Aqueous and Alcoholic extracts of
failed to show antibacterial activity against Proteus
local 14 herbs were prepared. These herbs were Aloe
mirabilis. Both extracts of Allium sativum showed weak
Vera, Azadirachta indica, Allium sativum, Calotropis
antibacterial activity against P. mirabilis. Aqueous
procera, Cannabis sativa, Carum capticum, Eucalyptus
extracts of garlic shows antibacterial activity against
camaldulensis, Lantana camara, Mangifera indica,
multi drug resistant human enteric pathogenic bacteria
Mentha piperita, Nigella sativa, Opuntia ficus indica,
and alcoholic extracts of Carum capticum, Euclyptus
Piper nigrum and Zingiber officinalis.
camaldulensis, Mentha piperita, Mangifera indica and
Fresh leaves, rhizome and dry seeds of herbs
Piper nigrum show weak effect against the bacteria
(100 gm each) were homogenized in 50 mL distilled
(Iwalokun et al., 2004, Joe et al., 2009 and Ross et al.
water, macerated and centrifuged. It was used as aqueous
2001). Aqueous extract of Euclyptus camaldulensis had
extract (Babayi et al. 2004). Dried and powdered leaves,
inhibited growth of Bacillus subtilis and Staphylococcus
rhizome and seeds of herbs (5gm each) were
aureous (Babayi et al. 2004). Moreover, leaves of Mint
homogenized in 20 mL Alcohol. They were macerated
has antibacterial activity against P. vulgaris and P.
for 2 days and then filtered. The filtrate was evaporated
mirabilis (Sabahat and Parween, 2005). Alcoholic extract
in water bath at 70 °C. It was reconstituted in 10 mL
of Opuntia ficus indica and Piper nigrum did not show
distilled water and used as Alcoholic extracts (Joe et al.
antibacterial activity and hence did not interfere the
2009). Mentofin (Ewabo), Yucca, Suduri and Safi
urease activity such as increased production of Ammonia
(Humderd) were procured from local market.
in the broth. Both the aqueous and alcoholic extracts of
Antibacterial activity of the herbs was determined by 10
Aloe vera, Azadirachta indica, Calotropis procera,
fold dilution of the herbal extracts/products in Urea broth
Cannabis sativa, Lantana camara, Nigella sativa and
containing 105 CFU/ml of the bacteria. After 72 hours of
Zingiber officinalis did not show any antibacterial
incubation at 37 °C, change in pH, Activity of bacteria
activity against P. mirabilis. However, extracts of Allium
(growth: Yes/No), Ammonia concentration (µg/mL) and
sativum, Mentha piperita, carum capticum, Eucalyptus
urease activity (I.U/mL) were observed. For estimation of
camaldulensis, Opuntia ficus indica Mangifera indica
Ammonia and urease activity, Colorimetric method and
and Piper nigrum might be effective against urease
Nessler, s reagent was used, respectively (Massmann,
positive bacteria in-vivo. Mentofin (Ewabo) showed
1962). Optimum inhibitory concentration of herbs and
strong antibacterial activity even at 1:1000 dilution. It
products were determined. Antibacterial activity of the
stopped the bacterial growth thus no production of
herbal extracts and 4 herbal products were estimated and
urease. This resulted in decrease pH and no production of
results were obtained. The data thus obtained was
ammonia. It has ethereal oils of Mint and Eucalyptus that
might have shown antibacterial activity against urease

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Yasmeen et al., J. Anim. Plant Sci. 22(2):2012

producing bacteria. Similarly, Suduri and Safi (Humderd) It is concluded that alcohol or water extracts of
had also given same effect at 1:10 dilution and Yucca the indigenous herbs have antibacterial activity against
showed antibacterial activity at 1:10 dilution. It shows urease producing bacteria. A commercial product can
better results in-vivo study (Saif-ur-Rehman and therefore be prepared from locally available herbs to
Muhammad, 2011) control environmental pollution in and around poultry
sheds.

Table #1: Effect of herbal extracts on pH, ammonia and urease activity of avian strain of Proteus mirabilis.

Name of Herbs pH 0f culture Ammonia Urease activity

Concentration I.U/mL
µg/mL
Aloe vera (w) 9.0160 e 5.24 e 0.010c
A. vera (a) 9.0500 e 6.90 e 0.013 c
Aza. indica (w) 8.9400 e 5.90 e 0.011 d
Aza. Indica (a) 9.0220 e 5.62 e 0.011 d
Allium sativum (w) 8.5560 a 4.42 c 0.009 b
Allium sativum (a) 8.8480 c 3.52 a 0.007 a
Calo. procera (w) 9.0140 e 6.00 e 0.011 d
Calo. procera (a) 9.0139 e 6.01 e 0.011 d
Cannabis satival (w) 8.9440 e 5.24 e 0.010 c
Cannabis satival (a) 9.00 e 5.00 d 0.010 c
Carum capticum (w) 8.9440 e 5.32 e 0.010 c
Carum capticum (a) 8.9540 e 4.84 d 0.009 b
E.Camaldulensis (w) 8.9100 e 5.16 e 0.010 c
E. Camaldulensis (a) 8.9580 e 5.06 e 0.010 c
Lantana camara (w) 8.9520 e 5.32 e 0.010 c
Lantana camara (a) 9.0520 e 5.94 e 0.011 d
Mangifera indica (w) 8.9600 e 5.18 e 0.010 c
Mangifera indica (a) 8.8820 d 5.42 e 0.010 c
Mentha piperita (w) 9.0120 e 5.16 e 0.010 c
Mentha piperita (a) 8.8880 d 4.00 b 0.008 a
Nigella sativa (w) 9.0060 e 6.32 e 0.0118 e
Nigella sativa (a) 8.9780 e 5.30 e 0.010 c
O. f. indica (w) 8.8100 b 5.22 e 0.010 c
O. f. indicab (a) 9.0360 e 6.30 e 0.012 e
Piper nigrum (w) 8.9900 e 5.08 e 0.0102 c
Piper nigrum (a) 8.9080 e 4.80 d 0.009 b
Zin. Officinalis (w) 9.0260 e 6.36 e 0.0118 e
Zin. Officinalis (a) 9.0263 e 6.33 e 0.011 e
Figures in the column having similar superscripts are not significantly different, (p<0.05).
W: Aqueous extracts, a: Alcoholic extracts.

Table #2: Effect of Commercial products on pH, Ammonia and Urease activity of avian strain of Proteus
mirabilis.

Name pH 0f culture Ammonia Concentration µg/mL Urease activity

I.U/mL
Mentofin 6.8280b 0 0
Suduri 6.80600b 0 0
Safi 6.81000b 0 0
Yucca 4.8972a 0 0
Positive control 9.0280 6.70 0.013
Negative control 6.8200 0 0
Figures in the column having different superscripts are significantly different, (p<0.05).

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