© Med Sci Monit, 2005; 11(12): BR433-438 WWW. M ED S CI M ONIT .

COM
PMID: 16319779 Basic Research

Received: 2005.01.25
Accepted: 2005.06.20 The antimicrobial potential of honey from United Arab BR
Published: 2005.12.01
Emirates on some microbial isolates
Authors’ Contribution: Noori S. AL-Waili1 ABDEG, Mohammod Akmal2 BF, Faiza S. AL-Waili2 BF,
A Study Design
B Data Collection
Khelod Y. Saloom1 BCEG, Amjed Ali2 EF
C Statistical Analysis
D Data Interpretation
1
Al-Waili Charitable Foundation for Science and Trading, New York, NY, U.S.A.
E Manuscript Preparation
2
Dubai Specialized Medical Center, Islamic Establishment for Education, Dubai, United Arab Emirates
F Literature Search
G Funds Collection
Source of support: Departmental sources

Summary
Background: The study investigated activity of honey towards pathogens when grown in media contained hon-
ey, or when honey was added to cultures after inoculation.
Material/Methods: 1 – Staphylococcus aureus (S. aureus), Streptococcus pyogenes (S. pyogenes), E.coli and Candida albicans
(C. albicans) were cultured into broth containing 10–100% (wt/v) honey concentrations. 2 – Honey
was added to broth inoculated with isolates after inoculation. 3 – Optimum growth of isolates, ther-
apeutic period of honey, and time after addition of honey that showed optimum effect was meas-
ured.
Results: The optimum growth of E. coli and C. albicans was 10 hrs and S. aureus was 12 hrs. Honey (30–70%)
prevents growth of all isolates. Honey (80%) inhibited growth of small (1 ul) and large size of in-
oculum (10 ul) of E. coli and S. aureus when added to their cultures during 24 hrs after inocula-
tion. Honey inhibited growth of C. albicans when added during 2 to 6 hrs after inoculation. Honey
delayed the appearance of microbial growth on the plates. Reculturing of specimens collected
from media that showed no growth after addition of honey yielded recovery growth for E.coli and
C. albicans, and therapeutic period of honey for E.coli and S. aureus was 2–24 hrs and for C. albicans
was 2–6 hrs.
Conclusions: Honey prevents growth of the isolates and inhibits their growth when honey was added to growing
culture. The therapeutic period of honey and recovery growth of inhibited isolates necessitates ad-
justment of honey doses according to type of isolate and grade of growth.

key words: honey • Candida albicans • Streptococcus pyogenes • Staphylococcus aureus • E. coli

Full-text PDF: http://www.medscimonit.com/abstract/index/idArt/438827

Word count: 2766
Tables: 5
Figures: —
References: 24

Author’s address: Noori S. Al-Waili, Al-Waili Charitable Foundation for Science and Trading, New York, U.S.A., email: noori786@yahoo.com

Current Contents/Clinical Medicine • SCI Expanded • ISI Alerting System • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts BR433
Basic Research
Background
Honey is a drug more than a nutrient. Honey was valued
highly in the Middle East. It was mentioned in the Holy
Quran 1400 years ago (And thy LORD taught the bee to
build its cells in hills, on tree and in men’s habitations, then
to eat of all the produce of the earth and find with skill the
spacious paths of its LORD, there issues from within their
bodies a drink of varying colors, wherein is healing for men,
verily in this is a sign for those who give thought). It was also
mentioned in Holy Talmud and Holy Bible. Hippocrates
and Celsus used honey for wounds and ulcers. Prophet
Mohammed (SAWW) had recommended honey for treat-
ment of diarrhea. The antimicrobial activity of honey has
been demonstrated in vitro and in vivo. Laboratory stud-
ies and clinical trials have shown that honey is an effective
broad-spectrum antimicrobial agent [1–8].
The antibacterial effect of honey was studied when a loop-
ful specimen of each pathogen was inoculated into various
concentration of honey prepared in solid media or using
disc impregnated with honey. These techniques explored
effect of honey on growth of small number of isolates usu-
ally with use of 1 ul standard loop for inoculation. In ad-
dition, they planned to answer whether honey could pre-
vent multiplication of a single pathogen inoculated into
media prepared with various concentration of v/v honey.
Therefore, the purpose of the present study was 1 – to inves-
tigate antimicrobial activity of honey collected from United
Arab Emirates against large size of human pathogenic inoc-
ulums cultured in liquid broth, 2 – to investigate effect of
honey on microbial growth when it was added to their cul-
tures during 24 hrs after their inoculation into the liquid
broth, 3 – to measure the optimum growth of each isolate
after culturing into broth media, and 4 – to calculate ther-
apeutic period of honey.
Material and Method
Pathogenic isolates
Cultures of various human pathogenic strains were obtained
from Microbiology Department, Dubai Specialized Medical
Center and Medical Research Laboratories, Dubai. Species
included E.coli, S. aureus, S. pyogenes and C. albicans. These
strains were isolated from human specimens. The isolates
were identified by the standard bacteriological techniques.
Using a 10 uL standard loop, a colony of each isolate was
picked from a plate, grow in 10 ml nutrient broth, and used
after 24 h incubation in 37ºC. Bacterial growth was assessed
visually on solid media as: 0; no growth, 1; little growth, 2;
mild growth, 3; moderate growth, 4; heavy growth and 5;
very heavy growth. The test was duplicated for each culture
to verify the results.
Honey
Honey was collected from United Arab Emirates. It was dark
yellow in color and multifloral origin. Different concentra-
tions of honey were prepared. The concentrations were giv-
en as percent (w/v). Honey had a density of 1.40 g/ml. The
amount of honey necessary to achieve the required concen-
trations (10–100%, w/v) was aseptically weighted into ster-
ile test tubes. 28.57 ml of broth was added to 71.42 ml of
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Med Sci Monit, 2005; 11(12): BR433-438
honey (100 g of honey) to obtain 100% honey concentra-
tion. Nutrient broth was added to the tubes to make up to-
tal volume of the concentration required. Honey broth so-
lutions were mixed by stirring with sterile sticks.
Effects of honey on human pathogens
Cultures of the specimens of the isolates in broth contain-
ing different concentrations of honey were performed by
using a standard loop (10 ul). The specimen of each mi-
croorganism was taken from pure culture grown in the 10
ml nutrient broth as described above. These cultures were
incubated at 37ºC for 24 h. Then after a loopful (10 ul) of
the cultures of each of the specimens of microorganisms
was streaked onto agar plates to assess the viability of the
isolates. The streaked plates were incubated aerobically at
37ºC and inspected after 24 h. The same test was duplicat-
ed for each isolate to verify the obtained results
Effects of honey on microbial growth during various
times after inoculation
Using a standard loop (1 ul), specimens of gram positive
S. aureus, gram negative E.coli and C. albicans taken from
pure cultures were inoculated onto fresh 10 ml broth me-
dia. Twelve milliliters of the selected honey was added to the
broth media inoculated with the isolates to obtain 80% wt/v
honey concentration in the broth. The honey was added at
2, 4, 6, 8, 10, 12 and 24 hrs after inoculation of the microor-
ganisms. At each two hours intervals (2–24 hrs) after inoc-
ulation, a loopful (1 ul) of the cultures of each isolate was
streaked onto appropriate plates to assess the viability. The
streaked plates were incubated aerobically at 37ºC and in-
spected after 24h. In addition after each time interval, and
at 24 and 48 hrs after addition of honey to each microor-
ganism, specimens were taken with use of standard loop (1
ul) for subsequent culturing into fresh 10 ml nutrient broth
free of honey. After 24 h incubation at 37ºC, loopful speci-
mens (1 ul) were streaked onto solid media prepared in Petri
dishes to assess viability and growth of the isolates. This pro-
cedure was performed to identify antibacterial activity of a
single dose of 80% honey concentration. Same experiment
was repeated except for using 10 ul of loopful specimens in-
stead of 1 ul. If there was no inhibitory effect with use of 80%
honey, the dose of honey would be increased to 90 or 100%.
The optimum growth of each isolate was measured after cul-
turing into broth media. The time after addition of honey to
the growing isolates that showed optimum effect of honey
was calculated. Therapeutic period of honey was measured,
which means the time after inoculation of isolates during
which the addition of honey to the media could completely
inhibit growth of the growing isolates. The tests were repeat-
ed twice for verification of the results obtained.
Results
The minimum concentration of honey that prevents growth
of large size of inoculum (10 ul) of S. aureus or S. pyogenes
was 50%, of E. coli was 30% and C. albicans was 70%. The
most sensitive microorganism was E. coli (Table 1).
Table 2 showed that 80% of honey completely inhibited
growth of (I ul) E. coli and S. aureus when honey was added
to their cultures during 2–24 hrs after their inoculation. The
Med Sci Monit, 2005; 11(12): BR433-438 AL-Waili NS et al – The antimicrobial potential of honey from United Arab Emirates…

Table 1. Grade of microbial growth after 24 hrs culture into nutrient broth (control) and various concentrations of honey in broth.

Concentrations of honey (wt/v) percent

Isolates Control
10 20 30 40 50 60 70 80 90 100

E. coli 4+ 3+ 2+ 0 9 0 0 0 0 0 0 BR
S. aureus 4+ 3+ 1+ 1+ 1+ 0 0 0 0 0 0
Streptococcus
5+ 3+ 3+ 2+ 1+ 0 0 0 0 0 0
pyogens
C.albicans 5+ 3+ 3+ 3+ 1+ 1+ 1+ 0 0 0 0

Table 2. Inhibitory effect of honey (80 % wt/v) added at various times (2–24 hrs) following microbial culture (I ul of specimens of microbe
inoculated into 10 ml broth).

E. coli Staphylococcus aureus Candida albicans

No. Time Growth Growth after Growth Growth after Growth Growth after
of tubes (hrs)* when addition of honey when addition of honey when addition of honey
honey was honey was honey was
added Grade Times (hr)** added Grade Times (hr)** added Grade Times (hr)**
1 2 3+ 0 2 1+ 0 2 4+ 0 4
2 4 3+ 0 2 1+ 0 2 4+ 0 4
3 6 3+ 0 2 1+ 0 2 4+ 0 4
4 8 4+ 0 2 3+ 0 2 4+ 1+ 6
5 10 4+ 0 10 3+ 0 2 5+ 1+ 16
6 12 4+ 0 8 5+ 0 2 5+ 1+ 22
7 24 4+ 0 2 5+ 0 10 5+ 1+ 24
* Time after inoculation of the isolates at which honey was added to the culture media;
** Time taken to reach optimum effects of added honey.

time needed to achieve complete inhibition ranged between
2 and 10 hrs after the addition of honey. Furthermore, 80%
of honey completely inhibited growth of C. albicans when
honey was added 2 to 6 hrs after inoculation of the isolate
into the broth. Honey reduced growth of C. albicans from 5
+ to 1 + when added between 8 and 24 hrs after inoculation.
When complete inhibition of microbial growth with addition
of honey was obtained, reculturing of specimens from the
media into fresh broth free of honey yielded positive growth
for E. coli and C. albicans and negative growth for S. aureus.
After 24 hrs of incubation of plates, we observed that all the
plates streaked by specimens from the isolates taken 2–24 hrs
after addition of honey to their cultures yielded no growth
while control plates (without addition of honey) showed pos-
itive growth. Then after, the growth started to appear on the
plates streaked by specimens from honey broth media during
subsequent hours of incubation. This means that although
honey did not completely inhibit growth of some isolates at
certain time it clearly delayed appearance of their growth
on the solid media compared with the control.
When higher size of inoculum (10 ul) of microbial specimens
was used (Table 3), the addition of 80% of honey to the cul-
ture of E.coli (between 2 and 12 hrs) completely inhibited the
growth of the isolate, and reduced grade of the growth from
5 + to 1 + when honey added after 24 hrs. In this case when
higher concentration of honey was used (90 and 100%), no
growth was observed after 12 hrs. Honey completely inhib-
ited growth of S. aureus when it was added during 24 hrs af-
ter inoculation of the isolate. The time required to obtained
complete inhibition ranged from 6 to 18 hrs. Reculturing of
specimens from media that showed negative growth with ad-
dition of honey yielded positive growth of S. aureus and E.coli.
The number of growing isolates may be too high after inoc-
ulation to be completely inhibited or killed by such honey
concentration. Regarding C. albicans, the addition of honey
after 2, 4 and 6 hrs completely inhibited the growth while
the addition of honey after 10 to 24 hrs reduced the growth
from 5 + to 1 + (Table 4). Increasing concentration of hon-
ey to 100% completely inhibited the growth when added af-
ter 2 to 8 hrs and reduced the grade of growth to 1 + when
added after 10 to 24 hrs. The higher concentration of hon-
ey was not able to completely inhibit growth of growing C.
albicans. Multiple doses might be sufficient to inhibit this iso-
late. Reculturing in fresh broth media showed microbial re-
covery growth. This means that single dose of such concentra-

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Basic Research Med Sci Monit, 2005; 11(12): BR433-438

Table 3. Inhibitory effect of honey (80 % wt/v) added at various times (2–24 hrs) following microbial culture (I ul of specimens of microbe
inoculated into 10 ml broth).

E. coli Staphylococcus aureus

No. Time Growth after Growth after
Growth when Growth when
of tubes (hrs)* addition of honey addition of honey
honey was honey was
added Grade Times (hr)** added Grade Times (hr)**
1 2 1+ 0 2 2+ 0 6
2 4 2+ 0 2 3+ 0 6
3 6 3+ 0 6 3+ 0 8
4 8 5+ 0 6 4+ 0 14
5 10 5+ 0 16 5+ 0 16
6 12 5+ 0 20 5+ 0 18
7 24 5+ 1+ 24*** 5+ 0 10
* Time after inoculation of the isolates at which honey was added to the culture media;
** Time taken to reach optimum effects of added honey;
*** Became (0) at 12 hrs with use of 90% and 100% honey.

Table 4. Inhibitory effect of honey (80 % and 100% wt/v) added at various times (2–24 hrs) following C. albicans culture (I0 ul of specimens
of microbe inoculated into 10 ml broth).

Candida albicans (80% honey added) Candida albicans (100% honey added)
No. Time Growth after Growth after
Growth when Growth when
of tubes (hrs)* addition of honey addition of honey
honey was honey was
added Grade Times (hr)** added Grade Times (hr)**
1 2 5+ 0 6 5+ 0 4
2 4 5+ 0 8 5+ 0 6
3 6 5+ 0 8 5+ 0 8
4 8 5+ 1+ 24 5+ 0 40
5 10 5+ 1+ 62 5+ 1+ 40
6 12 5+ 1+ 60 5+ 1+ 36
7 24 5+ 1+ 48 5+ 1+ 24
* Time after inoculation of the isolates at which honey was added to the culture media;
** Time taken to reach optimum effects of added honey.

tions of honey was not enough to kill all the isolate growing
in broth media when honey added during 2–24 hrs after mi-
crobial inoculation. Table 5 shows the optimum growth for
E.coli, S. pyogenes and C. albicans, and optimum effect of add-
ed honey to growing isolates. Therapeutic period of honey
for C. albicans was shorter than E. coli or S. pyogenes. Except
for small size of inoculum of S. aureus, all isolates showed re-
covery growth when re cultured in media free of honey.
Discussion
The study reports the main following findings; 1 – Honey
prevents growth of large size of inoculums of microbes
inoculated separately in broth contained honey, 2 – The
most sensitive microorganism was E.coli when the isolates
were cultured separately in broth media, 3 – Honey inhib-
its microbial growth when added into media containing
growing isolate, 4 – The optimum growth of 1 ul speci-
mens of E. coli and C. albicans was 10 hrs and the optimum
growth of S. aureus was 12 hrs. When size of inoculum was
10 ul, the optimum growth for E. coli and S. aureus was
10 hrs and for C. albicans was 2 hrs, 5- Reculturing of spec-
imens collected from media that showed no growth after
addition of honey yielded recovery growth for E.coli and
C. albicans, 6 – The therapeutic period of honey for E.coli
and S. aureus was 2–24 hrs and for C. albicans was 2–6 hrs,

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Med Sci Monit, 2005; 11(12): BR433-438
Table 5. Times for optimum growth of various isolates and optimum effect of honey on their growth.
1 ul
Isolates Time for
pptimum
Optimum effect of added honey
growth Time (hrs)* Grade
(Grade 5 +)
E. coli 10 hrs 2–24 – 1–2+
S. aureus 12 hrs 2–24 – 0
C. albicans 10 hrs 2–4 – 2–4+
* Time at which honey added after inoculation to obtain optimum inhibitory effects;
** Reculture in broth media free of honey.
and 7 – Multiple doses of honey might be necessary to erad-
icate tested pathogens.
In all our experimentations, the test for each microorgan-
ism was duplicated to be sure that the effect is reproducible.
Approximately similar concentrations of honey were able to
prevent growth of lower size of inoculums (1 ul) of same iso-
lates [3]. It seems that honey has same potency to prevent
growth of low as well as high amount (10-fold) of isolates.
Honey inhibits growth of C. albicans, which was substantiated
by earlier studies showing that honey alone or honey mixed
with olive oil and beeswax was useful to treat cutaneous fun-
gal infection and seborrhoic dermatitis [9,10].
Honey prevents growth of gram positive, gram negative or
C. albicans. This property makes honey a good drug to be
applied at time of closing surgical wounds or wounds due
to various traumas. It is not necessary to wait until devel-
opment of sings and symptoms of established infection to
apply honey. This is simply because honey is a safe natural
product. Such practice is important because wounds are li-
able for contaminations even at intensive care units.
The present study has tested whether addition of various con-
centrations of honey to cultures of isolates during 2–24 hrs
after inoculation could inhibit its growth and multiplica-
tion. This means that honey would be exposed to a larger
number of microbes, which was expected to increase rapid-
ly during 24 hrs after inoculation. The single dose of honey
added to the growing microbes was tested further to identi-
fy whether it killed the microbes or just stop their growing.
It was found that bacteria could overcome the antibacterial
activity of honey after a period of inhibition [11]. However,
the appearance of microbial growth after initial inhibition
by a single dose of honey might be a result of inability of
such dose to kill all the growing isolates. This is supported
by the finding that reculturing of specimens of media that
showed no growth with honey yielded mild growth of iso-
lates. Therefore, multiple doses of honey might be neces-
sary to kill all the growing isolate.
After 24 hrs of incubation of plates, it was observed that all
the plates streaked by specimens from the isolates taken
2–24 hrs after addition of honey to their cultures yielded
no growth while control plates (without addition of honey)
AL-Waili NS et al – The antimicrobial potential of honey from United Arab Emirates…
Size of inoculums
10 ul
Optimum
Optimum effect of added honey BR
growth
Reculture** (Grade 5 +) Time (hrs)* Grade Reculture**
10 hrs 2–12 – 2–4+
10 hrs 2–24 – 1–2+
2 hrs 2–6 – 3–5+
showed positive growth. Then after, the growth stared to ap-
pear on the plates during subsequent hours. This means that
the addition of honey to the cultures of each microorganism
does not only inhibit completely or partially the growth of
the isolate but also delays appearance of microbial growth
on the plates, including plates that showed positive cultures
during 2–24 hrs after the addition of honey.
Studies have diluted honey by distilled water to obtain var-
ious volume/volume concentrations of honey [12–18]. We
used broth for dilution, which is closely matching wounds
that was a suitable media for microbial growth. Furthermore,
the percentage of honey concentration was made as weight
of honey in volume of nutrient broth. This is more accept-
able since various honeys have different densities. In addi-
tion, In vitro studies mostly involved honey incorporated to
the agar plate as nutrient or brain heart infusion then in-
oculate microorganism or using impregnated honey disc.
As honey diffuse it will be diluted and the inhibition ob-
tained did not match real honey concentration used. In ad-
dition, we found that disc impregnated with various con-
centrations of honey added to agar plate became dry due
to vaporization of fluid from the disc when media incubat-
ed into 73ºC for 24 h.
The variations in antibacterial activity of honey can be in the
amount of hydrogen peroxide and in the presence of ad-
ditional antibacterial components derived from the nectar
source [19]. However, we have found that honey increased
nitric oxide end products in various animals and humans’
biological fluids, decreased prostaglandin concentration and
enhanced antibody production [20–24]. We have proposed
that killing of microbes by nitric oxide production might
explain, in part, its antibacterial activity [3].
The main limitation of the study is that the number of iso-
lates was not known. The use of 1 µL or 10 µL of culture
broth of the organisms did not indicate the number of or-
ganisms surviving or to be killed by honey. In order to deter-
mine the sensitivity tests of organisms to a particular chemo-
therapeutic agent the number of organism to be eliminated
by the chemotherapeutic agent must be in proportion with
the agent. The weight of honey used in the experiment is
known but the number of bacteria to be killed is not meas-
ured. Therefore, measuring the number of bacteria by dilu-
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Basic Research
tion or optic density will be the next works for the authors.
Most of the previous works on the effects of honey on bac-
teria did not include number of viable organisms. The use
of 1+, 2+, 3+ etc to assess growth of subcultures from liquid
to solid media was not explained in terms of numbers of mi-
crobes represented by such numbers. However, the use of
such scoring for evaluation of microbial growth was used by
other investigators. In the next works when the number of
microorganism is known in addition to the weight of hon-
ey used, it will be easy to identify the potency of certain di-
lution of honey.
Conclusion
S. aureus, S. pyoyogenes, E.coli and C. albicans are common hu-
man pathogens. The results demonstrated that natural hon-
ey prevented growth of large size inoculum of single path-
ogenic isolate S. aureus, S. pyogenes, E.coli or C. albicans when
these isolates were cultured separately into media containing
honey. Furthermore, honey inhibited growth of each isolate
when honey was added to its growing culture. The optimum
growth of isolates and the therapeutic period of honey varied
according to the type of isolates and grade of their growth.
The therapeutic period of honey determined for each isolate
and the recovery growth of inhibited isolates after culturing
in media free of honey showed that the doses of honey used
to manage microbial infections must be adjusted according
to the type of isolates and grade of their growth.
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