JOURNAL OF MEDICINAL FOOD

J Med Food 6 (4) 2003, 359–364

© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

Identification of Nitric Oxide Metabolites in Various Honeys: Effects of Intravenous

Honey on Plasma and Urinary Nitric Oxide Metabolites Concentrations
Noori S. Al-Waili
Dubai Specialized Medical Center and Medical Research Laboratories,
Islamic Establishment for Education, Dubai, United Arab Emirates

ABSTRACT Honey has antibacterial activity, promotes healing, and enhances immunity. Its acidity, osmotic effects of its
high content of sugar, and hydrogen peroxide are assumed to be responsible for its effects. In this study, various honeys were
investigated for the presence of nitrite/nitrate, the stable nitric oxide (NO) metabolites, and the effects of intravenous infusion
of honey on urinary and plasma NO end products were studied in healthy sheep. Seven kinds of honey, different in their ori-
gin (three from Yemen, two from the United Arab Emirates, one from Germany, and one from India), color, and duration of
storage, were investigated for the presence of NO metabolites. The assessment of NO metabolites was performed before and
after exposure of the honey samples to heating (80°C for 1 hour) or ultraviolet light (for 24 hours). Seven healthy male sheep
were used for the study. Fresh unprocessed yellow honey (2 g/kg of body weight) was infused over a period of 45 minutes
to each fasting sheep. Plasma and urinary NO metabolites were measured before and after the infusion. All the honey sam-
ples examined had various concentrations of NO metabolites; the highest concentration was in the fresh dark honey collected
from Yemen, and the lowest in 1-year-stored dark honey collected from India. Darker or fresh honeys contained more NO
metabolites than light or stored honey. After heating, NO metabolites decreased in all the kinds of honey. After ultraviolet
exposure, NO metabolites were decreased in four kinds of honey, increased in one kind, and unchanged in two kinds. The
darker stored honey had more resistance to heating and ultraviolet exposure. Intravenous infusion of honey elevated urinary
NO metabolites from 8.4 6 7.4 mmol/L to 14.9 6 10 mmol/L during the first 60–90 min after infusion and to 35.2 6 34
mmol/L during the next 150–180 min. Plasma NO metabolites were increased during 1, 2, and 3 hours after infusion by 3%,
3.6%, and 17%, respectively. No side effects were reported with the use of intravenous honey. It might be concluded that
honey contains various concentrations of NO metabolites. Its intravenous infusion increased plasma and urinary NO metabo-
lites. It is assumed that NO might be responsible, in part, for the biological and therapeutic effects of honey.

KEY WORDS: • honey • intravenous • nitric oxide • plasma • urine

INTRODUCTION
H ONEY IS the regurgitated flower nectar gathered, trans-
formed, and stored in the honeycombs of bees. Hon-
eybees use nectar to make honey. Nectar is almost 80% wa-
ter with some complex sugars. Honeybees suck the nectar
out of the flower, and they store it in their honey stomach.
Indeed, honeybees have two stomachs: the honey stomach
and the regular stomach. On returning to the hives, honey-
bees pass the nectar onto other worker bees. These home
bees chew the nectar into simple sugar. The nectar is left in
the cell, allowing water to evaporate. Once the nectar is thick
the worker bees chew the nectar once more to make honey.
The enzymes that bees produce are invertase, which con-
verts most of the sucrose into fructose and glucose, and glu-
Received 25 May 2003. Accepted 1 July 2003.
Address reprint requests to: Dr. Noori S. Al-Waili, Director, Dubai Specialized Medical
Center and Medical Research Laboratories, P.O. Box 19099, Dubai, United Arab Emi-
rates, E-mail: noori786@yahoo.com
cose oxidase, which converts glucose into gluconic acid and
hydrogen peroxide.
Many reports showed that honey dressing is effective for
treating wound infections. 1–3 Honey can inhibit fungal
growth. 4 We have found that honey in various concentra-
tions inhibits growth of pathogenic bacteria in vitro and in
vivo.5–7 Honey accelerates eradication of bacterial infec-
tions, hastens the wound healing process, prevents wound
disruption in severe postoperative wound infections, and re-
duces inflammation.8,9 It could eradicate bacterial conjunc-
tivitis due to pathogenic bacteria, including Pseudomonas.5
Recently, we have found that honey increases antibody titer
against T-dependent and T-independent antigens during pri-
mary and secondary immune responses. 10 Honey stimulates
proliferation of B and T lymphocytes in cell cultures and
stimulates monocytes to release cytokines, which activate
immune responses. 11,12 In addition, honey shows antitumor
and antimetastasis effects and potentiates the antitumor ef-
fects of cytotoxic drug. 13
It is assumed that the antimicrobial activity of honey is
due to osmotic effects of its high sugar content. 14 Although

359
360 AL-WAILI
the level of hydrogen peroxide in honey is very low, it is
still effective as an antimicrobial agent.15 In nectar from var-
ious honeys there are undefined unique compounds, which
are also responsible for the biological effects of honeys.
These compounds are found in various concentrations in var-
ious nectars. Thus, various honeys have various antibacter-
ial effects.14 In general, the mechanism of wound healing
properties of honey is attributed to hydrogen peroxide and
osmotic effects, which cause absorption of water and body
fluid leading to inhibition of bacterial and fungal growth. 14
Nitric oxide (NO) is important for healing, bacterial
killing, viral inhibition, host immunity, and renal, cardio-
vascular, and nervous systems functions. It has been shown
that NO plays a role in host defense against various infec-
tions. 16 Killing of intracellular pathogens is mediated by
NO.17 Replication of many viruses can be inhibited by NO.18
NO is a very important mediator of immune responses. 17 It
inhibits tumor growth and metastasis.17 In the urinary sys-
tem, NO plays a role in the physiology and pathophysiol-
ogy of the penis, bladder, prostate, and nervous system con-
trolling bladder function. 19 NO causes natriuresis and
diuresis, increases renal blood flow and glomerular filtra-
tion rate, and inhibits sodium reabsorption. 19 The NO-de-
pendent vasodilator tone was maintained through the phys-
ical activation of endothelial cells, and NO may also
contribute to the regulation of blood flow and pressure.20
Wound healing involves platelet, inflammatory cell, fibro-
blast, and epithelial cells; all of them are capable of produc-
ing NO.21 It has been found that NO can reverse impaired
healing associated with diabetes mellitus and can enhance
bone healing. 22,23 Investigators have implicated NO in the
inflammatory and proliferative phases of wound healing. 24
NO is a free radical that is degraded to nitrite and nitrate.
Measurement of nitrite or nitrate, a stable end product of
NO, is an indirect measure of NO production. NO could be
detected as nitrite. It has been found that NO is an essential
mediator for memory in honeybees. 18 Almost all of the NO
synthase activity is located in the insect brain. A large
amount of NO synthase activity was detected in neurophils,
which are involved in processing olfactory function. Inhibi-
tion of NO synthase affects long-term memory.25 Antennal
lobes show staining patterns for the enzyme NO synthase,
which catalayzes the release of NO.26
We decided to study, first, whether raw unprocessed hon-
eys contain NO metabolites and, second, possible effects of
honey on plasma and urinary NO concentration after intra-
venous honey infusion to healthy sheep. This was planned
because we have found that intravenous infusion of raw nat-
ural honey into healthy sheep was safe and provoked bene-
ficial changes on biochemical investigations. 27
MATERIALS AND METHODS
Experiment 1: measurement of NO metabolites in
unprocessed honey
Seven types of honey were subjected to study. Three sam-
ples were collected from Yemen (fresh dark, 1-year-stored
dark, and 5-years-stored dark honeys), and two honey sam-
ples from Lootah Farm, United Arab Emirates (fresh dark
yellow honey and fresh light yellow honey). The remaining
samples were one from Germany (1-year-stored dark yel-
low honey) and one from India (1-year-stored dark honey).
Fresh honey was harvested within 3 months prior to testing.
Total nitrite/nitrate (NO metabolites) was measured in each
sample using Griess reagent (Assay Design, Ann Arbor,
MI). One milliliter of honey was diluted in 2 mL of reac-
tion buffer, mixed well, ultrafiltered through a 10,000 mol-
ecular weight cutoff (MWCO) filter, and used directly in the
assay. Nitrate concentration was determined by conversion
of the nitrate into nitrite and measured in the nitrite assay.
Fifty microliters of the diluted honey was pipetted into du-
plicate wells. Fifty microliters of the Griess reagents 1 and
2 was added into each well. The wells were mixed by shak-
ing the plate and then incubated at room temperature for 10
minutes, and the optical density of each well was read at
540 nm. For nitrate assay, 50 mL of honey samples was
pipetted into duplicate wells. Twenty-five microliters of
NADH and 25 mL of nitrate reductase were added to each
well. The wells were mixed and incubated for 30 minutes
at 37°C. Fifty microliters of Griess reagents 1 and 2 was
added to each well. After mixing the plate was incubated at
room temperature for 10 minutes. Optical density at each
well was read at 540 nm. This would measure total nitrate
and nitrite concentration in the honey samples. Therefore,
the nitrate concentration in the honey samples was equal to
total nitrite and nitrate minus nitrite concentration.
Experiment 2: effects of heating on honey content of
NO metabolites
All kinds of honey were subjected for heating to 80°C for
1 hour. NO metabolites were measured as described in ex-
periment 1.
Experiment 3: effect of ultraviolet exposure on honey
content of NO metabolites
Seven kinds of honey were subjected for ultraviolet light.
Each honey sample was put in a glass container and exposed
to ultraviolet B (UVB) light (TUV 15 W) for 24 hours. NO
metabolites were measured in each honey sample after UVB
exposure.
Experiment 4: effects of intravenous honey on plasma
and urinary NO metabolites in healthy sheep
Seven healthy male sheep, 25–30 kg, 6–8 months old,
were used for experimentation. They were bred on our farm
and fed regular diet. Physical examination and laboratory
hematological and biochemical tests revealed they were nor-
mal and healthy. After a 16-hour fast, blood samples were
withdrawn through a cannula, and urine was collected for
nitrite and nitrate assay. The cannula was fixed through a
posterior leg vein, and urine was collected though an exter-
nal condom fixed around the penis.
NO metabolites were estimated using a colorimeteric NO
assay kit to measure total NO after enzymatic conversion
 NITRIC OXIDE METABOLITES IN HONEYS 361

(Assay Design). The kit uses the NADH-dependent enzyme RESULTS

nitrate reductase for quantitative conversion of nitrate to ni-
trite before estimating nitrite using Griess reagents. Plasma
and urine samples were ultrafiltered through a 10,000
MWCO filter and used directly in the assay.
Pure fresh unprocessed honey, multifloral in origin, dark
yellow in color, collected from our local farm in the United
Arab Emirates was examined microbiologically to exclude
bacterial contamination. Honey (2 g/kg of body weight) dis-
solved in 100 mL of saline was given to each 16-hour-fasted
sheep through intravenous infusion, over a period of 45 min-
utes. Blood samples were withdrawn at 1, 2, and 3 hours
post-infusion. Urine samples were collected between 60–90
min and between 150–180 min post-infusion. The samples
were subjected to NO assay.
Statistical analysis
The analysis of variance test was used for statistical analy-
sis. P , .05 was considered significant.
All the seven types of honey examined were found to con-
tain NO metabolites (nitrite/nitrate). Total NO metabolites
ranged from 52.4 mmol/L in 1-year-stored dark honey col-
lected from India to 441 mmol/L in fresh dark honey col-
lected from Yemen (Table 1). Darker honey contained more
NO metabolites. Fresh honey, collected within 3 months
from hives, contained more NO metabolites than stored
honey. After heating for 1 hour, the total NO metabolites
and nitrite concentrations were reduced in all the various
honey samples. Regarding nitrite concentration, the reduc-
tion ranged from 2.5% in dark 5-years-stored honey to 32%
in fresh dark honey. The lowest reduction in total NO
metabolites was 6% in 5-years-stored honey, and the high-
est reduction was 32% in the fresh dark honey. UVB re-
duced NO metabolites in four types of the honey samples,
ranging from 8% in fresh dark yellow honey to 28% in 1-
year-stored dark yellow honey. Therefore, the effects of
heating and ultraviolet exposure on NO metabolites de-

TABLE 1. NO METABOLITES IN VARIOUS HONEYS BEFORE AND AFTER H EATING OR ULTRAVIOLET EXPOSURE

Level (mmol/L)

Country, types of honey, NO Unprocessed After After UVB

metabolites honey heating exposure

United Arab Emirates

Fresh dark yellow
Nitrite 6.86 6.4 (7%) 3.6 (47%)
Nitrate 296.9 272 (8%) 218 (26%)
Total 303.8 278.4 (8%) 222 (27%)
Fresh light yellow
Nitrite 2.743 6.3 (127%) 5 (192%)
Nitrate 195.3 185.7 (5%) 211 (8%)
Total 198 192 (3%) 216 (11%)
Germany
1-year-stored dark yellow
Nitrite 8.67 6.8 (21%) 4.1 (53%)
Nitrate 136.3 101 (26%) 101 (26%)
Total 145 108 (25%) 105 (28%)
Yemen
Fresh dark
Nitrite 16.4 11.1 (32%) 7.77 (52%)
Nitrate 425.4 291 (31%) 397 (7%)
Total 441.8 302 (32%) 405 (8%)
1-year-stored dark
Nitrite 25.2 22.8 (9%) 10.7 (58%)
Nitrate 331 282.2 (15%) 299 (10%)
Total 356.2 305 (14%) 310 (13%)
5-years-stored dark
Nitrite 35.4 43.5 (2.5%) 21 (41%)
Nitrate 131 121.5 (7%) 144 (9.8%)
Total 166.5 156 (6%) 165 (0.9%)
India
1-year-stored dark
Nitrite 7.30 6.4 (12%) 6.4 (12%)
Nitrate 45 30.3 (33%) 45.7
Total 52.3 46.7 (12%) 52.1

Data are percent changes in NO metabolites in comparison with unprocessed honey in the same honey type.
362 AL-WAILI
pended on the type of the honey and its duration of storage.
UVB increased NO metabolites by 9% in fresh light yellow
honey collected from the United Arab Emirates. NO metabo-
lites showed unremarkable changes after UVB in 1-year-
stored dark honey from India and 5-years-stored dark honey
from Yemen.
The total NO metabolites value of the honey infused to
the sheep was 303 mmol/L. After infusion, no side effects,
including anaphylactic reaction, shivering, difficulty in
breathing, or skin rash, were encountered in any of the
treated sheep. Urinary total nitrite/nitrite estimation was
8.4 6 7.4 mmol/L before honey infusion, which increased
to 14.9 6 10 mmol/L (44%) during 60–90 min and to 35.2
mmol/L (319%) during 150–180 min after honey infusion.
The mean total plasma NO metabolites concentration was
16.5 6 4.2 mmol/L before infusion, which was increased to
17 6 2.8 mmol/L (3%), 17.1 6 3.7 mmol/L (3.6%), and
19.3 6 3.2 mmol/L (17%) at 1, 2, and 3 hours after honey
infusion, respectively.
DISCUSSION
Results showed that various kinds of honey, fresh or
stored, yellow or dark, contained various concentrations of
NO metabolites. Hence, the concentration varies according
to the origin and color of the honey, its duration of storage,
and whether the honey was fresh or old. Darker honey con-
tains more NO metabolites. Storage and heating reduce
honey contents of NO metabolites. UVB exposure reduces
NO metabolites in some honeys and increases them in oth-
ers. Darker honeys, even stored for 5 years, had higher re-
sistance to heating and ultraviolet exposure regarding its
content of NO metabolites as compared with other types of
honey examined. Color of the honey and country of origin
were used to identify various kinds of honey in this exper-
iment.
The NO content of honey might be synthesized during
the honey-making process in the honeybees. The glandular
system, including the salivary gland (hypopharangeal
gland), might be the site for synthesis of NO in the honey.
We and others have found that saliva contained large amount
of NO metabolites.28,29 Further study to detect NO synthase
enzyme activity in the glandular gland or stomach of the
bees or their products is important to explore the source of
NO production.
Exposure of honey to ultraviolet irradiation for 24 hours
decreased, increased, or had no effect on NO metabolites.
UVB irradiation did not affect NO metabolites in 5-years-
stored dark Yemeni honey and in 1-year-stored dark Indian
honey. Fresh light yellow honey collected in the United Arab
Emirates revealed increased NO metabolites after UVB ir-
radiation. The other four samples showed decreased NO
metabolites after UVB irradiation. Darker honeys or older
honeys showed more resistance to UVB irradiation. There-
fore the effect of UVB irradiation might depend on the type
of honey and the duration of storage. It has been found that
UVB irradiation could increase or decrease NO production
in cells. The expression of NO synthase is induced by UVB
irradiation, and NO generation is increased by UVB irradi-
ation.30 UVB light (365 nm) caused relaxation of cavernosal
tissue by release of NO.31 Another study showed that the
generation of NO was induced by UVB irradiation, and NO
production was significantly increased 72 hours after irra-
diation of UVB (100 mJ/cm22 ) in skin cells.32 In contrast,
UVB light (2.5–25 mJ/cm22 ) decreases NO production in
keratinocytes and macrophages and suppresses NO synthase
type 2 gene expression in macrophages. 33 NO could prevent
skin damage induced by UVB.34 It is important to study the
effect of UVB irradiation on NO production by various hon-
eys collected from various flowers and nectars in vivo.
There is evidence that honey in a layer 1–2 mm thick ex-
posed for 15 minutes completely lost its non-osmotic activ-
ity.35 Sunlight exposure for 18 days results in complete loss
of honey activity.35 No loss of antibacterial activity was ob-
served when a thin layer of honey was exposed for 1 hour
to an ultraviolet (254 nm) lamp.36 Glucose oxidase is sen-
sitive to light, which is minimal at pH 8 but increases
markedly from pH 5 downwards. 37 We have found that ul-
traviolet exposure of dark yellow honey for 24 hours showed
little effect on antibacterial activity against some pathogens,
while it increased its activity against others pathogens (au-
thor’s unpublished data). Ultraviolet has been used to dis-
infect gram-negative bacteria in recycled water and to inac-
tivate bacteria.38,39 Ultraviolet could be used for sterilization
of honey. Honey could be contaminated by Clostridium bot-
ulinum during preparation, and Clostridium perfringens and
some other microorganisms have been found in some com-
mercial honey. 40,41 It was found that sterilization of honey
by filtration through microporous membranes is not practi-
cable because of the high viscosity of honey. Gamma-irra-
diation was used for honey sterilization.42 Ultraviolet could
be used for sterilization of honey. 39
Heating of various honeys to 80°C for 1 hour reduced
their NO metabolites. Reduction varied with various kinds
of honey. Greater reduction was found in fresh darker honey
collected from Yemen. It was found that increased body tem-
perature could increase or decrease NO production. In-
creased body temperature enhances synthesis of NO in
skin. 43 Whole-body heating does not increase cutaneous NO
concentration. 44 Heating decreases sensitivity to NO in vas-
cular smooth muscles.45 In addition, heating causes denat-
uration of glucose oxidation and causes loss of its activity
against some species.14 The glucose oxidase activity is very
labile to heating.46 Thermostable substances that are polar
and have antibacterial activity47 are present in honey. How-
ever, heat-stable antibacterial activity has been reported in
some honeys. 36 Heating honey for 1 hour at 75–80°C did
not destroy its activity against Bacillus subtilus.48 On the
contrary, exposure of honey to 100°C for 5 minutes or 56°C
for 1 hour caused complete loss of inhibition by 17%
honey. 48 Almost complete loss was found on heating honey
for 100°C for 10 minutes.49 Honey contains both heat-sta-
ble and heat-sensitive compounds. 48,49 We found that heat-
ing to 80°C for 1 hour reduced but not abolished completely
 NITRIC OXIDE METABOLITES IN HONEYS
the activity of raw honey or stored honey against the iso-
lates tested (author’s unpublished data). Heating did not
completely eliminate NO metabolites, but it reduced them.
This might explain our previous findings that heating of
honey reduced its antibacterial activity (author’s unpub-
lished data).
After intravenous infusion, honey increased levels of
plasma and urinary concentrations of NO end products. The
increments were due to either the honey content of NO, the
possible existence of NO synthase in the honey, or the ex-
istence of substances that activate host NO synthase to pro-
duce more NO. Since NO possesses a vast range of biolog-
ical activities, the increment in NO production is highly
important from physiological and pathological aspects.
Honey has several biological and therapeutic effects on bac-
terial and viral infections, wound healing, the immune sys-
tem, and tumors.5–7,49 In addition to its osmosis, acidity, and
hydrogen peroxide production, honey might show its thera-
peutic effects through increased NO production in the body
or at site of wounds or microbial infections. This will widen
the therapeutic uses of honey in medicine. However, a high
output of NO production may represent a potential risk to
humans. 17 Nevertheless, honey did not provoke vast incre-
ments in NO end products, particularly in plasma (the high-
est increment was up to 17%).
The antimicrobial activity of honey varies markedly with
its origin. 48 We have found that antimicrobial activity of
honey was decreased by exposure to heating and prolonged
storage (author’s unpublished data). Heating and prolonged
storage decreased NO metabolites identified in various kinds
of honey. In addition, the concentration of NO metabolites
varies in various types of honey. Investigators have found
that the antibacterial activities of various honeys continued
despite the addition of catalase to remove hydrogen perox-
ide.50 Catalase is present in plasma and wound exudate,
which may attenuate the antibacterial activity of honey.
Non-peroxide antibacterial activity present in different kinds
of honey has been recognized. 14 Such activity could be ex-
plained by the capability of honey to increase NO produc-
tion.
We have used fresh yellow honey for intravenous infu-
sion. It is important to study other kinds of honey such as
dark honey or stored honey and investigate their effects on
plasma and urinary NO metabolites.
The ability of honey to elevate NO production in the plasma
and urine is clinically important. Assessment of NO synthase
in the honey samples, and in plasma and urine before and af-
ter honey administration, might help to explain the mecha-
nism of elevated NO end product following honey infusion.
ACKNOWLEDGMENTS
The author would like to express sincere thanks to Haj
Saeed Lootah, Chairman of the Board of Trustees, Islamic
Establishment for Education for his support. Mr. N.S. Boni
and Mr. Amjed Ali provided assistance.
363
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