Shapla et al. Chemistry Central Journal (2018) 12:35
https://doi.org/10.1186/s13065-018-0408-3

Open Access

REVIEW

5‑Hydroxymethylfurfural (HMF) levels
in honey and other food products: effects
on bees and human healthUmmay Mahfuza Shapla1†, Md. Solayman1,2*†, Nadia Alam3, Md. Ibrahim Khalil1,3 and Siew Hua Gan4*

Abstract
An organic compound known as 5-hydroxymethylfurfural (HMF) is formed from reducing sugars in honey and various
processed foods in acidic environments when they are heated through the Maillard reaction. In addition to processing, storage conditions affect the formation HMF, and HMF has become a suitable indicator of honey quality. HMF iseasily absorbed from food through the gastrointestinal tract and, upon being metabolized into different derivatives, isexcreted via urine. In addition to exerting detrimental effects (mutagenic, genotoxic, organotoxic and enzyme inhibitory), HMF, which is converted to a non-excretable, genotoxic compound called 5-sulfoxymethylfurfural, is beneficial
to human health by providing antioxidative, anti-allergic, anti-inflammatory, anti-hypoxic, anti-sickling, and anti-hyperuricemic effects. Therefore, HMF is a neo-forming contaminant that draws great attention from scientists. This review
compiles updated information regarding HMF formation, detection procedures, mitigation strategies and effects of
HMF on honey bees and human health.
Keywords: 5-Hydroxymethylfurfural, Mutagen, Carcinogen, Antioxidant, Anti-allergen
Introduction
Honey is a sweet natural product produced by honeybees (Apis mellifera), which gather nectar from flowers
before converting it to nutritious food. Honey is mainlycomposed of water (15–20%) and two sugars (dextroseand levulose), with the presence of small amounts ofat least 22 other more complex sugars (80–85%, w/w)
[41, 179]. Honey has also been reported to contain an
intricate mixture of nitrogenous compounds, lactone,
proteins, antibiotic-rich inhibine, enzymes, phenol antioxidants, aroma compounds, amino and organic acids,
gluconic acid, phenolic acids, flavonoids, minerals, vitamins, 5-hydroxymethylfurfural (HMF) and other phytochemicals [20, 56, 181, 183]. Honey composition varies*Correspondence: naeem40thju@gmail.com; gan.siewhua@monash.edu†
Ummay Mahfuza Shapla and Md. Solayman contributed equally to thiswork
1
Laboratory of Preventive and Integrative Bio‑medicine, Departmentof Biochemistry and Molecular Biology, Jahangirnagar University, Savar,
Dhaka 1342, Bangladesh
4
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan,
47500 Bandar Sunway, Selangor, Malaysia
Full list of author information is available at the end of the article

depending on its floral, geographical and entomologicalsources [13, 56]. In addition, external features such as
seasonal and environmental factors, honey processing,and storage time and conditions have crucial effects onhoney’s composition [58, 76, 104].
Honey is considered both nutritional and medicinal,
although the presence of certain constituents, for example, heavy metals (even in trace amounts), some alkaloids,and HMF and its derivatives may contribute to honey’s
toxicity [77, 152]. HMF is a cyclic aldehyde produced bysugar degradation through the Maillard reaction (a nonenzymatic browning reaction) during food processing or
long storage of honey [102]. The presence of simple sugars (glucose and fructose) and many acids, as well as minerals, in honey can further enhance the production of thissubstance [92]. HMF concentration is widely recognized
as a parameter affecting honey freshness because it istypically absent (or is present in only very small amounts
in fresh honeys), while its concentration tends to riseduring processing and/or because of aging. Previous
studies have reported that honey stored at low temperatures and/or under fresh conditions has low or minimal© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Shapla et al. Chemistry Central Journal (2018) 12:35

Page 2 of 18

HMF concentrations, while aged and/or honey stored atcomparatively higher or medium temperature has high
HMF concentrations. In addition to storage conditions,the use of metallic containers and honey floral sources
are critical factors affecting HMF levels (Table 1). Hence,higher HMF concentration is indicative of poor storage conditions and/or excess heating of honey [48, 81].
Therefore, the Codex Alimentarius Standard commission

has set the maximum limit for HMF in honey at 40 mg/
kg (with a higher limit of 80 mg/kg for honeys originating from tropical regions) to ensure that the product has
not undergone extensive heating during processing and issafe for consumption [6].
HMF is not only present in honey; it is nearly ubiquitous in our daily heat-processed, sugar-containing foodstuffs, from our breakfast cereals, breads, dairy products,

Table 1 Variation in HMF concentration in honey samples based on their storage time and geographical sourcesCountry

Storage time

Storage temperature (°C)

HMF concentration (mg/kg)

References

Bangladesh> 1.5 years

20–25

3.18–703.10

[76]

India

Fresh

–

0.15–1.70[160]

Malaysia (year)

<1

4–5

0.26–68.99

[108]

>1

25–30

206.06–383.39

[81]

Asia

>2

25–30

986.57–1131.76

Nepal

–

25–29

30.36–56.10

Pakistan–

4–5

ND–6.00[3]––

23.18–27.37

[74]

Iran

–

–

0.04–17.20[104]

TurkeyFresh–0.00–11.50[188]< 6 months–

19.20–28.6[91]

8.60–39.00[188]

1 year20 ± 5

Europe

[131]

Italy

Fresh

–

1.23–5.95[47]

Croatia––

0.00–23.69[173]

Czech Republic

4 years

10.30–44.20[80]

Poland––20 ± 2

0.70–3.50[89]

Portugal–Room temperature

18.00–94.00

[66]

Spain

< 3 years–

0.00–21.39[116]

Northwest Spain< 2 years

0.00–1.60[120]

Switzerland< 7 years− 30
40.00–112.00[148]

AfricaAlgeria< 1 year

4–6

1.73–480.00[36, 82]

Burkina Faso

< 1 year

0–4

3.00–27.50[103]

Ethiopia< 6 months–

0.68–6.56[25, 85]Kenya< 1 year

3.70–389.36[112]

Morocco–4

3.20–52.60[169]

Nigeria< 6 months–

0.66–1.43[16]

Tanzania––

5.00–26.40[115]

25 ± 2South America
Argentina–

0.05–2.94[21]

1.48–34.08

[34]

–

0.00–14.70[8]–

1.50–115.20[5, 162]< 1 year4

3.30–15.90[9]–

Room temperature

9.01–21.96[106]

2 years− 18

1.30–12.40[71]

Within 2 months–

Argentinean Patagonia> 3 years

Brazil–Cuba
Mexico23 ± 2

North America

Australia
Australia

ND not detected, HMF 5-hydroxymethylfurfuralShapla et al. Chemistry Central Journal (2018) 12:35

and fruit juices to liquors at different concentrations [17,37, 114, 137, 155, 170]. Therefore, HMF is considered
one of the main quality indexes of different commercial
whey proteins, molasses and many other products [40].
In most previous studies, HMF has been reported tohave negative effects on human health, such as cytotoxicity toward mucous membranes, the skin and the upper
respiratory tract; mutagenicity; chromosomal aberrations; and carcinogenicity toward humans and animals[59, 94, 107]. However, in more recent extensive studies,
HMF has been proved to have a wide range of positiveeffects, such as antioxidative [193], anti-allergic [187],
anti-inflammatory [86], anti-hypoxic [96], anti-sickling[1], anti-hyperuricemic effects [97].
It has been reported that humans may ingest between
30 and 150 mg HMF daily via various food products;
however, safe levels of HMF consumption are not wellclarified. The reason is that HMF’s metabolism, biotransformation and excretion and thus clearance rate from thebody depend on the organ function of an individual [176],
which have not been considered. The aim of this review isto describe the effects of HMF present in honey, which,if broadly analyzed, can be used to promote more widespread application of honey with special medicinal implications. Upon highlighting the HMF content in honey,a general discussion of the HMF content in other foods
and HMF’s detection, optimized formation, mitigationand eradication from food is provided. For this purpose,
all related articles relevant to the topic of “honey andother foods, HMF: its toxicity and therapeutic effects”
were included, regardless of the time of publication. To
our knowledge, this is the first study to extensively reporton HMF in honey from all over the world and to provide
a general overview to explore its effects on both bee andhuman health.

Formation of HMF in honeyHMF is a six-carbon heterocyclic organic compoundcontaining both aldehyde and alcohol (hydroxymethyl)functional groups. The ring of the structure is centeredon furan moieties, whereas the two functional groups,
i.e., formyl and hydroxy-methyl groups, are linked at thesecond and fifth positions, respectively (Fig. 1). HMF is asolid, yellow substance that has a low melting point but ishighly soluble in water [142].
HMF is considered the most important intermediate
product formed during two reactions (i) acid-catalyzed
degradation of hexose and (ii) decomposition of 3-deoxyosone in the Maillard reaction (Fig. 2) [46]. HMF formation is correlated with chemical characteristics such aspH, free acid content, total acidity, lactone content andmineral content, which in turn are related to the floral source of collected honey samples. The presence ofPage 3 of 18

Fig. 1 Chemical structure of HMF

simple sugars such as glucose and fructose and of many
acids has been reported to be favorable for honey production [47].
Similarly to honey, which is rich in glucose and fructose, most sugar-containing foods also contain HMF [71].
Nevertheless, although HMF occurs at very low concentrations and can even be absent in both fresh honey andfood products, heat treatment and/or prolonged storage conditions can enhance further HMF production.
Moniruzzaman et al. [108] reported the mean HMF concentration in Malaysian honeys stored for 2 months at
4–5 °C to be 35.98 mg/kg. In contrast, Khalil et al. [81]found that HMF concentrations in Malaysian honeystored at 25–30 °C for more than a year could reach very
high levels (118.47–1139.95 mg/kg). [76] also observedhigh HMF levels (3.18–703.10 mg/kg) in honey samples
from Bangladesh stored for more than a year at room
temperature (20–25 °C). Therefore, HMF level is not only
indicative of honey freshness but also of storage durationand conditions (Table 1).

Factors affecting HMF formation in honeyIn addition to being directly produced when heatingsugar from the degradation of hexoses under acidic conditions at high temperatures and/or during the Maillard
reaction [15], HMF is produced from the oligo- and polysaccharides that can yield hexoses upon hydrolysis. However, HMF appears to be more selectively produced fromketo-hexoses such as fructose [141]. Interestingly, there
are two reasons why higher yields of HMF are obtained
from fructose (ketose) than from glucose (aldose). First,
the reactivity of glucose is lower than that of fructose,with a lower enolization rate [92]. Enolization is believed
to be the rate-determining step of HMF production. Second, fructose forms an equilibrium mixture of difructoses and dianhydrides and thus internally blocks mostreactive groups, leading to the formation of certain byproducts. In contrast, glucose forms true oligosaccharides that still contain reactive reducing groups, posing
a higher risk for cross-polymerization with the reactive
intermediates, including HMF [92].
Honey has been reported to contain many different
types of sugars. In fact, a recent review by Solayman
et al. [163] indicated that honey contains approximately

Shapla et al. Chemistry Central Journal (2018) 12:35

Fructopyranos

Page 4 of 18

Fructofuranose

5-Hydroxymethylfurfural (HMF)Intermediate product 1

Intermediate product 2

Fig. 2 Formation of HMF in honey

39.44% fructose, 28.15% glucose and 3.19% sucrose. The
cyclic aldehyde HMF is also produced in honey by thedegradation of related sugars [81]. Heating of honey during its processing reduces its viscosity, which can prevent
crystallization or fermentation [161]. In addition to heating, several other factors influence the formation of HMF
in honey, such as honey’s physicochemical properties(pH, free acid content, total acidity, lactone content andmineral content), water activity (­aw), the use of metallic
containers [182], and thermal and photochemical stress[164].
HMF is easily formed at low temperatures in the presence of low-pH or acidic conditions [93], while hightemperature and long storage duration increase its concentration to a large extent. Nevertheless, a different
pathway is proposed in dry and pyrolytic conditions
under which HMF is formed from fructose and sucrose.
In addition to temperature and pH, the rate of HMF
formation in honey is also dependent on honey’s moisture content [62, 63]. Therefore, many steps are taken to
maintain low moisture content in honey samples, including gamma irradiation and heat treatment to inhibit
HMF formation.
The rate of HMF formation is also dependent on the
fructose:glucose ratio and the type of sugars formed
because it has been reported that at pH 4.6, fructose
has five times more reactivity than glucose, and a highfructose:glucose ratio will accelerate the reaction [93].
Turhan [175] showed that temperature and duration ofheat treatment may both affect HMF formation in honeysamples. Moreover, it was shown that heating honeysamples collected from Anatolia in Turkey at 135 °C
for 100 s produced similar amounts of HMF as thatyielded by heating samples to 150 °C for 40 s. According to [150], there is a logarithmic relationship between
the storage time and HMF levels in honey. In their study
on Malaysian honey samples [81], showed that honeysamples stored for 3–6 months had HMF values below
the International Honey Commission (IHC) limit fortropical honey (< 80 g/kg); however, samples stored for
12–24 months had HMF concentrations above the recommended level.
Khalil et al. [81] investigated the correlation between
the physicochemical properties of honey and HMF formation and found that there was a strong correlation
between free acids and total acidity, while there was onlya moderate correlation between pH and lactones. In
another study conducted on four honey samples (eucalyptus, sulla orange and chestnut), Fellico et al. [47]reported a relationship between HMF concentration andheating time, acidity and pH. A similar relationship wasindicated in another study [30], which reported a proportional increase in HMF formation with an increase intemperature by one degree, moisture content and water
activity. In addition, the concentrations of metallic ionssuch as manganese, zinc, magnesium, and iron(II) present in honey have also been reported to have a positiveeffect on HMF formation [122].

Shapla et al. Chemistry Central Journal (2018) 12:35

HMF in foods correlates with urine metabolites
Following oral or intravenous administration, HMF
is mainly metabolized into three major metabolites:
5-hydroxymethylfuroic acid (HMFA), 2,5-furandicarboxylic acid (FDCA) and 5-(hydroxymethyl)-2-furoyl glycine(HMFG), with the possible formation of a fourth metabolic product termed 5-sulfoxymethylfurfural (SMF)[67]. However, studies designed to investigate the distribution of radio-labeled [U-14C]-HMF and its metabolites
by whole-body autoradiography have revealed that neither HMF nor its metabolites are accumulated. It is estimated that 80–100% of the total amount of radioactivity
is released within the first 24 h following its administration [55, 61]. HMF is first oxidized to carboxylic acid andis conjugated with glycine, leading to the formation of
HMFG. The concentration of free glycine is the rate-limiting step in this pathway. Subsequently, HMF undergoes
further oxidation to yield FDCA.
In an in vivo study, [61] reported the presence of
three metabolites in urine following oral administration of labeled HMF (10–500 mg/kg), where the relative
amounts of FDCA, HMFA, and HMFG excreted were
2–6, 78–85 and 5–8%, respectively (depending on thespecies and the doses). In humans, HMF is completely
cleared following its oral administration (using the juiceof dried plum) [130]. Similarly, in another clinical study
by Hardt-Stremayr et al. [67], HMF was shown to becompletely excreted through urine within 48 h following
oral administration at 240 mg/day, while SMF was notdetected in urine, as also reported in other studies.
HMF in various food products
In addition to being found in honey, HMF is also present
in dried fruits (> 1 g/kg), products containing caramel,
instant coffee (up to 6.2 g/kg), apple juice, citrus juices,beer, brandy, milk, breakfast cereal, baked foods, andtomato products, and HMF is released from sugar andcarbohydrates after home cooking, indicating that HMF
is ubiquitous in the diet.
We consume several different food products, including bakery goods, milk, fruit juice, cereals, coffee, chocolate, soft drinks, vinegar, wine, nuts and grilled meat inour daily lives. The majority of these produces undergo
thermal treatments prior to consumption, such as boiling, baking, extrusion cooking, roasting, pasteurizationand other processing. These processes are performed
not only to make the products more edible but also 1) forpreservation (by means of reducing microbial load and/or eliminating enzymatic activities) and 2) to generate
more desirable sensory (color, aroma and taste) and texture properties. Nevertheless, extensive processing produces adverse effects by introducing undesirable, harmful
and non-nutritive compounds or by reducing nutritive

Page 5 of 18

value, fresh appearance and taste. During thermal processing and preservation, the Maillard reaction or nonenzymatic browning may also occur, where HMF is acommon product whose extent of formation depends on
the processing and preservation conditions [87].
Sugar

Sugar is a disaccharide composed of fructose and glucose.
It is produced exclusively from sugar beet and sugar cane.
Although the extraction and purification processes arevery simple, HMF is formed as a result of heat. Polovková and Šimko [128] analyzed brown (n = 25) and whitesugars (n = 13) from local markets in the Bratislava territory, the capital city of the Slovak Republic. Upon preparing the sugar samples, HMF levels were determinedby high-performance liquid chromatography coupledwith a diode array detector (HPLC-DAD) at 284 nm. Surprisingly, white sugar was free of HMF, but brown sugar
was found to contain HMF (0.17–6.45 mg/kg). The presence of HMF in brown sugar may be due to the additionof treacle to the preparation preserved at 50 °C to maintain its liquidity. Similarly, using HPLC, Risner et al. [139]found similar ranges of HMF concentration (11.9–16.4
and 12.3–23.3 mg/kg) for light and dark-brown sugars,
respectively.
Cereals

According to Norwegian and German researchers, cereals and cereal products, including bread, are some of themost prominent sources of human exposure to HMF [2,73]. The extent of HMF formation in cereal products isheavily dependent on many factors, including temperature, dough fermentation process, water activity and thepresence of fruits, grains and other flavorings or additives(such as cocoa, malt, sucrose, glucose, salt, and honey).
Mańkowska et al. [101] investigated the HMF concentrations in 41 food products. Wheat bread with cranberries was reported to contain the highest amount of
HMF (210 mg/kg), followed by breakfast cereals, i.e.,
honey wheat loops (85.09 mg/kg). The lowest amount of
HMF was reported to occur in gluten-free sponge cakes
and whole-grain oatmeal. Sweetened breakfast cereals
contained HMF at 25.55 mg/kg, which was higher thanthe mean HMF concentration (18.40 mg/kg) in bakery
products. In another study, the mean HMF concentrations in cereals, i.e., mixed grains (240 mg/kg), cornflakes(7–114 mg/kg) and wheat-based cereals (6–132 mg/kg),
were also determined to be high [144, 145]. The products
for which the dough fermentation process and the typesof flour were specifically affected were again reported to
be bread and other bakery products, including cookies.
Additionally, HMF concentrations in different types offlours, including wheat, whole wheat, bread flour and ryeShapla et al. Chemistry Central Journal (2018) 12:35

flour, were investigated. The HMF concentration in breadmade with rye flour was determined to be the highest(mean 26.88 mg/kg), perhaps due to the high amino acidcontent.
Different types of dried fruits (raisins, cranberries,
palms, strawberries, red currants and apple) are added tocereal products, which normally contribute to high HMF
concentrations. In fitness and rice-wheat flakes with raisins added, the HMF concentrations were reported to be
6.78 and 11.70 mg/kg, respectively. The lowest (6.06 mg/kg) HMF concentration was reported to occur in crunchy
products containing raisins and plums. On the other
hand, red fruits such as apples, strawberries and red
currants confer some of the highest concentrations of
HMF to food products, including oatmeal (47.62 mg/kg) [101]. Similarly, bread containing dried fruits tend tohave higher HMF concentrations than does white bread[137, 156], indicating that the main contributor of HMF
is dried fruits.
CoffeeCoffee is one of the most common drinks reported tocontain HMF. The HMF concentration in coffee depends
on the brewing processes or types of coffee (mocha,
espresso, filtered coffee or plunger-brewed coffees) used,
as well as the amount of sugar added to it. Mortas et al.
[111] investigated HMF concentrations in Turkish coffees (either prepared traditionally or of the instant variety) using HPLC coupled with a diode array detector.
The authors reported that before brewing, instant andtraditional Turkish coffee samples contained HMF overranges of 336.03–362.05 and 213.02–238.99 mg/kg,
respectively. However, following brewing, the HMF concentration increased by 32.29–55.83% (in instant coffee)
and 74.12–224.75% (traditional coffee), respectively.
Arribas-Lorenzo and Morales [15] used reversed-phase
HPLC coupled with UV detection to detect HMF concentrations in three types of ground coffee consumed
by the Spanish and found significant differences in theconcentrations observed. HMF levels were 110 mg/kg(natural coffee: produced by traditional roasting of coffee
beans), 625 mg/kg (torrefecto coffee: obtained by adding
sucrose prior to the roasting process) and 1734 mg/kg(blended coffee: a combination of natural and torrefacto
ground coffee in various proportions). However, soluble coffee contained the highest level (2480 mg/kg). The
authors concluded that the daily HMF intake of a highcoffee consumer in Spain is approximately 122.42 μg/kg,
indicating that heat contributes to HMF formation.
Dairy products

HMF is formed via side reactions during heat sterilization and browning processes. Albalá-Hurtado et al. [4]Page 6 of 18

investigated the formation of HMF as a result of exposure to different storage temperatures (20, 30, 37 °C) andstorage duration (up to 9 months) in liquid as well as inpowdered infant milk. They found that HMF formation
follows a zero-order kinetics profile independent of storage temperature and milk type. A powdered infant milk
sample stored at 37 °C for 12 months contained higher
amounts of HMF and furfural compounds (31.5 µmol/L)than those present in liquid milk (2.5 µmol/L).
In another study on ultra-high temperature processed(UHT) milk, no significant variation in HMF levels wasobserved for samples stored at 4 and 8 °C. However,storage at room temperature led to a two-fold increased
formation of HMF [33]. In the case of traditional Indian
dairy products, there was a strong positive correlation
between HMF concentration and the products’ their flavors, colors and textures [70]. Morales and Jiménez-Pérez[110] used micellar electrokinetic capillary chromatography and reported a mean HMF concentration of 29.5 µg/
kg for many infant milk-based formulas.
Fruits and vegetables

Due to their rich content of sugars and amino acids,
fruits and vegetables contain high HMF levels. In a study
involving jam products (prepared commercially andunder laboratory conditions) stored at 20 and 35 °C for
12 months, a temperature-dependent relationship wasestablished between HMF formation and storage duration [133]. Additionally, a positive correlation between
storage time and temperature with HMF formation has
been observed for two varieties of apple juice (Golden
amasya and delicious) [32]. Ordóñez-Santos et al. [121],
who investigated changes in HMF levels in bottled
tomato puree stored at 20 °C for 180 days, reported anegative correlation between HMF formation and thecontent of organic acids such as ascorbic, citric and mallic acids.
Murkovic and Pichler [114] analyzed HMF concentrations in dried apricot, peach, pear, fig, date, apple andpineapple products. HMF concentrations were highest in
dates (1000 mg/kg) and plums (1100–2200 mg/kg). The
mean range of HMF concentrations in other dried fruits
was 1–780 mg/kg [114]. On the other hand, in a study
by Ruf ían-Henares et al. [146] on commercially dehydrated vegetables, HMF was not detected, except in cabbage, tomato and artichoke (58.60, 18.20 and 6.97 mg/kg
respectively).
Oil concentration in products may also affect HMF formation. To investigate this hypothesis, Fallico et al. [45]roasted defatted and ground hazelnuts containing different amounts of hazelnut oils or oil containing saccharose
and/or hexanol. The defatted hazelnuts roasted in highoil concentrations exhibited increased HMF formation.

Shapla et al. Chemistry Central Journal (2018) 12:35

Non-defatted hazelnuts with saccharose contained thehighest HMF concentration (372 mg/kg), while defatted
hazelnuts with saccharose contained the lowest (33.5 mg/kg). Because the HMF concentration increased from 66.5
to 144.0 mg/kg in non-defatted samples upon prolonged
roasting (from 30 to 60 min respectively), the authors
also concluded that HMF levels increased with the duration of the heat treatment applied to the products.
Other miscellaneous food products

It is concluded that there are no heat-processed foodproducts that are free of HMF. The HMF concentrations
in several food products are listed in Table 2.
Comparison of HMF levels in honey and different foodproducts

HMF is not present naturally in food products. The
compound is formed upon thermal treatment and incombination with other factors. As it is a product of
the non-enzymatic Maillard reaction, there is no fixed
concentration of HMF in different food items. The baking temperature, rate of saccharose degradation, concentration of reducing sugars, type of sugar (glucose,fructose or others), water activity, the addition of otherfood additives such as HMF-containing sweeteners, coloring agents, caramelization, storage time and temperature, type of metallic storage and processing container
vary widely among different food items. Therefore, HMF
content varies among food items, even among those of
the same type. Nevertheless, honey is a safer food item
than other processed foodstuffs with respect to its HMF
concentration. Turhan et al. [174] showed that the initial heating temperature and time are not directly correlated with HMF concentration in honey. Nectar honeyand honeydew honey processed at 95 °C for 90 min and
90 °C for 75 min showed HMF levels lower than 40 mg/
kg. Storage temperature and storage duration in particular directly influence the development of HMF in stored

Page 7 of 18

honey [44, 81]. Unlike for honey, in the processing ofother foodstuffs, comparatively higher temperatures(during baking, roasting), longer duration times, and different additives are required, which profoundly affect the
HMF content in the foods. For example, cookies baked
at high temperature contain 10–100 times more HMF(167.4–1100.1 mg/kg) than cookies baked at 200 °C (9.9–39.6 mg/kg) [10]. Fresh cookies baked at 300 °C and withsaccharose added during processing have been reported
to contain as much as 1100 mg/kg HMF. Even ammonium bicarbonate addition may dramatically increase
the HMF content (above 3500 mg/kg) of saccharose-containing cookies baked at 220 °C. In investigating 22 coffee
samples, Murkovic and Pichler [114] reported HMF concentrations ranging from 300 to 1900 mg/kg. Murkovic
and Bornik [113] found that HMF formation was dramatically increased in coffee roasted at 240 °C within the first
3 min, with concentrations reaching up to 900 mg/kg. In
the case of bread, HMF values vary widely, from 3.4 to
176.1 mg/kg, depending on the fermentation conditions,the leavening agents added, the thickness of the crust andcrumb, and the type of bread (oat bread, white bread or
wheat bread) [137]. According to Raimirez-Jimenez et al.
[137], the HMF content of biscuits (15.6 mg/kg) is higher
than that of confectionaries such as doughnuts and croissants (9.5 mg/kg). Thus, the formation of HMF is inevitable, and comparing or ranking food items with respect to
HMF concentration cannot be performed precisely.

Detection methods of HMF
Accurate quantitative analysis of HMF is of great importance because HMF is a marker of quality deterioration,
thermal processing and other adulteration practices.
HMF is also important in clinical research and therapeutics. The IHC has recommended three main methods for
HMF determination: two spectrophotometric methods
by White and Winkler and a reversed-phase high-performance liquid chromatography (HPLC) method [191].

Table 2 HMF concentrations in different food products
Food type

HMF concentrations (mg/kg)

Detection method

Country

References

Must syrup

3500–11,000

GC–MS

Spain

[147]

Molasses

100

Sugarcane syrup

100–300

Palm syrup<3

Prunes237

HPLC

Norway[73]

Dark bear

13.3

Canned peaches

5.8

Raisins

5.0

Balsamic vinegar

316–3250

HPLC

Italy

[171]

GC–MS gas chromatography–mass spectrometry, HPLC high performance liquid chromatography

Shapla et al. Chemistry Central Journal (2018) 12:35

Prior to the availability of the spectrophotometricmethods, both optical and chemical methods were used.
In his newer spectrophotometric method, White utilizeda clarified honey sample containing 0.1% sodium bisulfate as a reference and as honey solution without sodium
bisulfate as a sample [180]. On the other hand, the Winkler method utilizes honey solutions with p-toluidine andbarbituric acid [184]. Nevertheless, although these methods are fast, they lack good sensitivity and specificity.
Moreover, the method described by Winkler requires theuse of p-toluidine, which is carcinogenic. Although the
HPLC method is relatively more expensive, it is advantageous with respect to both labor and time. In addition,
the method is deemed as an automated and sensitive
method that can exclude many interferences from otherrelated compounds [185]. Nevertheless, although HPLC
is a sophisticated technique, the method is still not satisfactory to some who have recommended further development and modification of the method [13].
Reyes-Salas et al. [138] reported an electrochemical
approach for HMF detection. In this method, a single
and sharp reduction signal was created at − 1100 mVversus argentum or argentum chloride, while borate wasused as a supporting electrolyte. Another method is theion exchange liquid chromatography–photodiode array
detection technique described by Yuan and Chen [189],
which is consistent with Winkler’s method. Anothermethod involves automated flow injection, as reported
by Iglesia et al. [38], which is based on the operating
principle of the Winkler method and provides a detection range of 5–40 ppm. Micellar electrokinetic capillarychromatography (MEKC) is another rapid method thatuses caffeine as a standard. The technique is suitable for
rapid quantification of HMF, particularly in honey samples, without requiring sample pretreatment [140]. A
unique and efficient rapid screening technique is direct
analysis in real time (DART) coupled with time-of-flightmass spectrometry (TOF-MS), which has been reported
to yield a chromatogram with high resolution [134].
The method can quantitatively analyze HMF concentrations in a more precise manner than other methods andrequires no (or very little) sample pretreatment.

Optimization of HMF formation in different foods
HMF is formed unavoidably in most foods containing
monosaccharides (such as glucose, galactose, or fructose)during processing as a function of temperature and storage period. The process of HMF formation in any food ismultifactorial. Factors affecting the rate of HMF formation include temperature, pH [62], type of sugar [93], concentration of divalent cations [64] and water activity [63,90] in the medium. Hence, to control and optimize thelevel of HMF formation is a troublesome task. Therefore,

Page 8 of 18

food processors must know the effects of different factors
so that necessary precautions can be adopted previously
to limit and/or optimize the synthesis of HMF in food.
To understand the conditions under which HMF would
be formed only at desirable levels, a more fundamental
and systematic approach must be adopted. The possible
effects of these factors must be studied simultaneously
and in combination. Toker et al. [172] recommended central composite face-centered design (CCFD) of response
surface methodology (RSM) as a useful experimental
technique for this purpose. According to Hunter [72],this versatile and effective systematic tool can be used todetermine the optimal levels of the contributing factors
for the parameters concerned. The mathematical expression derived by this methodology can be employed todevelop predictive models upon setting the levels of thevarious influencing factors to reach the optimum HMF
concentration in food.

Hazard posed by HMF to honey bees
The productivity of apiculture is highly dependent on
the health, survival and quality of honey bees. Therefore,
improving the health quality of bee food is an important priority. Normally, sources of carbohydrates forhoney bees are the nectar and honeydew collected fromplants, as processed by foragers. However, in the case ofcommercial honey production, the food and nutrientsof cultured bees are depleted following the collection of
the autumn yield from hives. Furthermore, honey bees’
natural resources tend to wane during winter. Therefore,
bee keepers must use alternative sources to replenish thecarbohydrate demand of bees, such as sucrose, high fructose corn syrup (HFCS), various fruit sugars and invert
sugars [117]. Among these, however, compared withsucrose, HFCS is considered a dynamic source due to its
practical storage, transportation facility and liquidity as
opposed to the crystallization method. HFCS is produced
commercially from corn starch by using enzymes suchas alpha amylase, amylo-glucosidase and invertase [26,29], whereas sucrose is produced by acid hydrolysis alone[18]. Regardless, these sources are not safe because HMF
and its daughter products (formic and levulinic acids)
are formed, thus posing a potential threat to honey bees
(Fig. 3). Beet sugar, which is also used to feed bees, hasbeen reported to contain up to 475 mg/kg HMF, depending on the processing and storage conditions [177]. In
another study, a sugar solution containing 30–150 mg/
kg of HMF was used to feed honey bees and was foundto cause 15–58.7% of deaths of caged bees within 20 days
[78]. Krainer et al. [88] reported that the LD50 values of
HMF for bee larvae fed for more than 6 days were 4280
and 2424 mg/kg on days 7 and 22, respectively. Interestingly, they found that 7-day larvae emerging after being

Shapla et al. Chemistry Central Journal (2018) 12:35

Page 9 of 18

Fig. 3 HMF effects on honey bee and human healthfed HMF for 6 days were more sensitive than were adult
bees, but the emerging adult bees at day 22 were less sensitive. Moreover, the authors concluded that HMF used
as a supplement in food does compromise colony fitness
but may not cause great losses of brood. It is hypothesized that HMF causes bees to experience dysentery-like
symptoms and ulcers in the gastrointestinal intestinal
tract [18], leading to their death.

Effects of HMF on human health
HMF and other congeners exert both detrimental andpositive effects on human health (Fig. 3).
Adverse effects on human health

HMF and its derivatives have been confirmed to confer
genotoxic, mutagenic, carcinogenic, DNA-damaging,organotoxic and enzyme inhibitory effects.

Shapla et al. Chemistry Central Journal (2018) 12:35

HMF as an indirect mutagen

Florin et al. [51] investigated the mutagenic activity ofcertain compounds, including HMF, towards four mutant
strains of Salmonella typhimurium. Liver extracts frommethylcholanthrene-induced rats were used for metabolic activation of the investigated compounds, and it
was confirmed that HMF is not a mutagen. In contrast,
Lee et al. [94] demonstrated that HMF is an indirect
mutagen because it is converted to an active metabolite,
the sulfuric acid ester 5-sulfo-oxymethylfurfural (SMF),
with mutagenicity towards S. typhimurium TA104.
HMF is enzymatically activated to SMF by sulfotransferases (SULT) contained in rat liver extracts enrichedwith the sulfo group donor 3′-phosphoadenosine-5′phosphosulfate (PAPS). This finding is further supported
by another study in which HMF, 2,5-bishydroxymethylfuran (metabolite of HMF), furfuryl alcohol (FFA) and5-methyl-FFA showed mutagenicity towards S. typhimurium TA100 expressing human SULT1C2 but not
towards the parental strains [60].
Another study using FVB/N (FVB) mice expressing hSULT1A1/1A2 revealed that HMF has a genotoxic
effect. Mice orally administered single doses of 900 or
1300 mg/kg showed significant DNA damage of theirrenal cells, as detected by an alkaline single cell gel electrophoresis assay. Another furan derivative, 2,5 dimethylfuran (DMF), also causes DNA damage in the kidney andthe colon [69]. In an in vitro erythropoietic micronucleus
assay, rat bone marrow cells were exposed to DMF at aconcentration of 0.1 mM for 1 h. The study indicated that
DMF exhibits genotoxicity toward hematopoietic cells[53].
In another study conducted on five cell lines possessing different levels of SULT1A1 activity (mouse L5178Y,
no activity; Chinese hamster: V79-Hp-PST, high activity;
V79, negligible activity; human: HEK293, higher activity; and Caco-2, low activity), HMF exerted DNA damage when exposed at a concentration of 100 mM for 3 h[42]. However, it was also determined that HMF poses
DNA-damaging effects irrespective of the SULT1A1
activity of the cell lines. In addition, 5-HMF was determined to cause chromosomal aberrations in a Chinese
hamster V79-derived cell line constitutively expressing
human sulfotransferase SULT1A1 and CYP2E1. In fact,
5-HMF is a potent inducer of sister-chromatid exchange
at a concentration of 19.8–3808.0 µM in cells exposedfor 32 h [59]. Nishi et al. [118] confirmed that 5-HMF
causes chromosomal aberrations of V79 cells at 15.8 mM.
In both their pre-clinical and clinical studies, Pastoriza
et al. [125] indicated that orally administered HMF isconverted to reactive SMF after being absorbed throughthe gastrointestinal tract. SMF forms a DNA-SMF adductin mice’s kidney, leukocytes and liver cells, as well as inPage 10 of 18

leukocytes of pre-adolescent populations. In addition,
SMF is not properly excreted through urine due to renal
reabsorption, thus allowing SMF to accumulate in theplasma, making it available to react with cellular proteinsand DNA.
HMF as a dual player in carcinogenesis

HMF and its derivative SMF have been confirmed to bepotent carcinogens in several studies at the preclinicallevel. The compounds cause neoplastic transformations
in several organs, including the colon and the skin. Coloncancer involves the development of a multistep stage inwhich micro-adenomas and aberrant crypt foci (ACF)
are the morphological markers [14]. HMF acts as an initiator as well as a promoter of ACF [28]. A study was conducted on multiple intestinal neoplasia mice that were
subcutaneously administered with a single dose of HMF(500 mg/kg) or SMF (25 mg/kg) within 3–6 days after
birth. After 12 weeks, administration of both HMF and
SMF increased the number of small intestinal adenomas
and flat dysplastic lesions (flat ACF) in the large intestine[168]. The mice had a mutant copy of the tumor suppressor gene adenomatous polyposis coli (APC). Mutationin the APC gene leads to the development of adenomas
in the small and large intestines, analogously to humanfamilial adenomatous polyposis syndrome (FAP) [127,129].
In a study by Zhang et al. [192], 45% of F344 female
rats administered with HMF at 250 mg/kg twice per day
by oral gavaging were found to develop a large intestinal
ACF on day 30. HMF increases both the number and sizeof ACF. In contrast, an extensive study (with two mousemodels: wild-type FVB/N and genetically engineered
mice having several copies of human SULT1A1 andSULT1A2 at chromosome 9), Florian et al. [50] yielded
opposite findings (i.e., neither HMF nor its metabolite SMF induces ACF and colonic tumors). HMF also
induces skin papilloma. Upon topical application of sulfoxymethyl and chloromethyl derivatives of HMF, themice were found to develop papillomas on their skin.
Another derivative of HMF, 5-chloromethyfurfural,
has been found to induce hepatocarcinoma in B6C3F1
male rats at a very early age [166]. Schoental et al. [153]showed that rats subcutaneously administered with HMF(200 mg/kg), developed renal lipomatous tumors. In contrast to these studies, a study by Zhao et al. [194] using
the A375 cell line indicated that HMF can induce apoptosis and G0/G1 arrest in DNA-damaged cells via thereactive oxygen species (ROS)-mediated signal transduction pathway. Thus, it is concluded that HMF is a potent
anti-carcinogen.

Shapla et al. Chemistry Central Journal (2018) 12:35

HMF as an organotoxic agent

HMF has cytotoxic effects at high concentrations, as thecompound causes irritation in the mucous membranes,
skin, eyes and upper respiratory tract [109]. SMF was
revealed to be a strong nephrotoxic agent in an in vivo
study in which male FVB/N mice were intraperitoneallyadministered with SMF (250 mg/kg). After 5–11 days
post-treatment, the mice either died or became morbid, possibly due to liver damage or more severe renal
damage, particularly at the proximal tubules. A histopathological study revealed extensive proteinaceous
casts and necrosis in the affected area [22]. The organic
anion transporters type 1 and 2 (OAT1 and OAT2) arehighly conserved in different species, including rat andhuman, and are usually expressed on the cellular basolateral membrane of the proximal tubules. The transporters
mediate the uptake of different organic anions, including SMF, from the blood stream as their substrates andare concentrated into tubular cells, which mostly affectthe proximal tubules [31]. Another study using humanembryonic kidney cells (HEK293) that constitutively
express OAT1 and OAT2 transporters supports thismechanism [19]. Glutathione is an important endogenous antioxidant in the body. An ex vivo study involving two mammalian cell lines, V79 and Caco-2, indicatedthat HMF decreased cellular glutathione levels in a concentration-dependent manner at 50 mM and 120 mM,
respectively [79].
HMF as an enzyme inhibitor

The human genome encodes 16 DNA-dependent DNA
polymerases. Among them, three are involved in nuclear
DNA replication, while the remaining are involved in the
repair system [24, 52]. Human polymerase γ is a multifunctional enzyme with DNA polymerase, terminaltransferase, 5′-deoxyribose phosphate (dRP) lyase andpolynucleotide synthetase activities. The primary structure of the polymerase includes a nuclear localization
signal, a BRCA1 carboxy terminus domain, a proline-richregion, a pol β-like region and a pol X region [27, 135,136]. DNA pol γ shares a sequence homology with theterminal deoxynucleotidyl transferase (TdT) that catalyzes the addition of deoxyribonucleotide to the 3’ endof dsDNA or ssDNA in a template-independent manner[124, 143]. HMF competitively inhibits DNA pol γ and
TdT with respect to the deoxynucleotide substrate andDNA template primer with 50% minimum inhibitory
concentration ­(IC50) values of 26.1 and 5.5 µM, respectively [105].

Page 11 of 18

Positive effects of HMF on human health
HMF as an antioxidant

ROS are produced as toxic by-products of the body’s
aerobic metabolism. The species oxidize cellular macromolecules such as proteins, membrane lipids, and DNA
and cause cellular damage. The consequences range fromstress to metabolic defects, neurodegenerative diseases
or even neoplastic transformations [159]. In a study by
Zhao et al. [193], HMF showed a dose-dependent (0.8–
6.4 mM) free-radical scavenging capacity. HMF also hassignificant protective effects on erythrocytes against
ROS-induced damage. To investigate the protective effectand oxidative stress induced by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), the levels of ROS andmalondialdehyde (MDA: indirect determinant of lipid
peroxidation) production, the activity of the antioxidant
enzymes glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) in erythrocytes pretreated with HMF were determined. It was revealed thatthe contents of ROS and MDA were reduced in HMFtreated cells, while the activities of these enzymes were
elevated relative to those of negative control cells [193].
The ROS scavenging activity of HMF is imparted by its
structure, which features functional reactive groups suchas an aldehyde oxygen, double bonds and another oxygenin its furan ring. These features can attract electrons easily and quench ROS [176].
HMF also has a protective effect at the morphological and biochemical levels on hepatocytes damaged byhydrogen peroxide (­H2O2)-induced oxidative stress,
under which cells undergo morphological changes suchas wrinkling, condensation of chromatin and splitting of
nuclei—hallmarks of apoptotic and necrotic cells. However, in a H­ 2O2-stressed human liver cell line (L02), cells
treated with HMF (0.79 µM) could preserve their morphology better than non-treated cells could. HMF also
reduces the levels of caspases 3 and 9 (executioner of
apoptosis) in these cells [178]. Ding et al. [39] hypothesized the underlying protective biochemical mechanism,
which may be due to inhibition of apoptosis by accelerating the transition of cells in the S phase to the G2 or
M phase, as well as decreased levels of nitric oxide andcaspase 3.
HMF against hypoxic injury

Oxygen is required for cell survival. Deficiency of oxygen(hypoxic condition) has numerous detrimental and evenlife-changing effects on health. Hypoxia may be inducedby many factors, including altitude and self-related conditions such as ischemia, atherosclerosis and cancer [126].
Several cellular mechanisms are triggered and can ameliorate hypoxic conditions, among which extracellularsignal-regulated kinase(ERK)-mediated transactivation

Shapla et al. Chemistry Central Journal (2018) 12:35

of the transcription factor and hypoxia-inducible factors(HIF) are believed to participate [151]. The mitochondrial membrane potential is also reduced and negatively
affects hypoxic cells [75]. In their in vitro study of thecell line ECV304 (human umbilical cord vein endothelial cell), Li et al. [96] showed that cells pre-treated with
HMF (200 µg/ml for 1 h) before being exposed to hypoxic
conditions (0.3% oxygen for 24 h) exhibited increased
mitochondrial membrane potential and decreased phosphorylated ERK levels. The number of apoptotic andnecrotic cells also significantly declined. In their further
study with a Kunming mice model, the authors showedthat pre-exposure to HMF (100 µg/ml, 1 h) significantly
attenuates the extent of hypobaric hypoxia-induced permeability of the blood–brain barrier (BBB). Pre-exposure also decreases the extent of neuronal damage in the
CA1 region of the hippocampus. Thus, because HMF
increases survivability under hypobaric hypoxic conditions, it may be a potent therapeutic agent against acutemountain sickness (AMS), high-altitude cerebral edema(HACE) and high-altitude pulmonary edema (HAPE)[95].
HMF as an anti‑allergen

Basophils and mast cells participate in the pathogenesis and manifestations of allergic reactions such asasthma, atopic dermatitis and allergic rhinitis. RBL-2H3
cells are mast cells located in the mucosal layer. Thesecells express immunoglobulin Fc epsilon receptor typeI (FcεRI) on their surface. The crosslinking of IgE withspecific protein antigens and the binding of crosslinked
IgE to FcεRI trigger intracellular signal transduction cascades. These events lead to ­Ca2+ influx and the releaseof mediators by degranulation, MAPK phosphorylation,
upregulation of cytokine gene expression and increased
ROS generation [23, 83, 84, 154]. Yamada et al. [187]showed that HMF acts at different stages to inhibit
degranulation at doses of 0.01–0.30 µg/ml. In addition,
HMF interferes with antigen-antibody crosslinking andantibody-receptor binding. HMF also blocks calcium­(Ca2+) influx into IgE-sensitized bovine serum albuminstimulated RBL-2H3 cells. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a major rolein the production of ROS in IgE-mediated RBL-2H3 cells.
­H2O2 and NO, two major ROS, are known to regulatedegranulation and ­Ca2+ signaling in mast cells [83, 84,186]. A significant inverse association exists between therelease of histamine and C­ a2+ from intracellular storesand the superoxide anion or DPPH scavenging activities. The anti-allergen effect of HMF on cells is due to theblocking of histamine release and C­ a2+ signaling throughthe compound’s free-radical scavenging activity [98, 167].

Page 12 of 18

In another study using ovalbumin (OVA)-immunized
BALB/c mice, HMF decreased the levels of total IgE andOVA-specific IgE. The study also showed that HMFtreated immunized mice exhibited lower levels of IFNγ
(Interferon gamma) and IL-4 (Interleukin 4) than thoseof untreated mice. Therefore, HMF may be a potent antiallergic compound [7].
The use of HMF for other pathologic conditions

Uric acid is the end product of purine catabolism. The
final two steps of the purine catabolic pathway are catalyzed by a critical enzyme, xanthine oxidase (XO). Uric
acid is mainly excreted via urine. High levels of uric acidin the blood lead to the development of hyperuricemia[123], which is the main cause of gout. In addition, manyother pathological states are associated with hyperuricemia, including metabolic syndrome, heart failure, pulmonary disorder and type 2 diabetes mellitus [68]. Increased
XO activity downregulates the anti-inflammatory transcription factor peroxisome proliferator-activatedreceptor-γ (PPARγ) and accelerates inflammatory action[57]. XO is also an endogenous producer of superoxide,a potent activator of nuclear factor kappa B (NFκB) [99].
NFκB acts as a transcription factor and upregulates theexpression of nitric oxide synthase 2 (NOS2) and interleukin 8 (IL-8). HMF exerts an anti-inflammatory effectby downregulating NFκB [86] and inhibits the activity of
XO [97].
HMF as an anti‑sickling agent

Hemoglobinopathies such as sickle cell disease are lifethreatening. The underlying mechanism is polymerization of abnormal hemoglobin (sickle hemoglobin, HbS)
under hypoxic conditions. The pathophysiology is manifested by the deformation of red blood cells, loss of resilience and occlusion of tiny blood capillaries, leading tomorbidity [65, 100, 165]. Although there are many antisickling agents, these agents are not free of side effects.
Many agents also exhibit reduced bioavailability in minimal doses and react to non-target proteins. In addition,
HMF can act as an effective anti-sickling agent. Abdulmalik et al. [1] showed that HMF orally administered in
low concentrations is absorbed into the blood stream
from the gastrointestinal tract in transgenic (Tg) sickle
mice. Subsequently, HMF can penetrate the erythrocytes
and form stable a Schiff-base adduct with the N terminal
αV11 nitrogen of HbS in a symmetrical fashion. Following the formation of such an adduct, HMF allostericallyshifts the oxygen equilibrium curve to the left and prevents the sickling of erythrocytes. In fact, the authors
also reported that Tg sickle mice pre-treated with HMF
survived longer under hypoxic conditions than did

Shapla et al. Chemistry Central Journal (2018) 12:35

untreated mice, indicating the potential of HMF as ananti-sickling agent.

Tolerable daily intake (TDI) of HMF
Many studies have revealed that different cells respondto 5-HM-induced cytotoxicity differently. The susceptibility of cells to HMF depends on the presence andexpression levels of receptors [119], metabolism [157],
structure [149] and the enzyme activity of HMF [158]. At
the preclinical level, no toxic effects have been observed
at daily doses ranging from 80 to 100 mg/kg body weight[2]. Zaitzev et al. [190] established the TDI for HMF as
132 mg/day using a 40-fold safety margin. The European
Food Safety Authority (EFSA) [43] established a threshold of concern of 0.54 mg/day for the intake of furan
derivatives used as flavoring agents in Europe. However,in their study involving 268 Spanish school children, Pastoriza et al. [125] found that although the students’ daily
HMF intake, well-distributed throughout the day, was
10–70 mg, a statistically significant level of SMF, a potent
toxic metabolite of HMF, was found in their plasma. In
addition, most experiments concerning the health effectsof HMF have been carried out in vitro, ex vivo and at theexperimental animal level. Thus, based on the data available to date, it is not possible to ascertain a TDI. In addition, further research, particularly at the clinical level,paving the way for recommending a TDI for HMF isworth considering and appreciating.
Mitigation strategies
There is no particular strategy for mitigating the formation of HMF in honey due to the numerous precursors of
HMF and the types of reaction orders involved [35]. HMF
is formed following a zero-order kinetic process in an
exponential manner [54]. Various mechanistic strategies
may be adapted to mitigate the formation of HMF. Thereis a positive relation between a high temperature-time
profile and acidic pH for the formation of HMF, which
can be modified [62]. In fact, the reduction of thermal
input can significantly decrease the formation of HMF,
which can be achieved by various means, for example, by
adjusting the temperature of the processing oven whenthe moisture content is high or, conversely, by decreasing the temperature when the moisture content is low.
Reduced thermal input can also be achieved by heating
food to a low temperature for a prolonged period by vacuum frying or by dielectric heating (microwave and radiofrequency). In the case of dielectric heating, an external
alternating current is applied, causing the dielectric molecules in food products to undergo rotation and friction,
thus ensuring the rapid generation of uniform heat. In
addition, because water molecules are the main target of
this mechanism, heat is generated in the portion of foodPage 13 of 18

containing water [195]. Because long storage periods leadto high HMF formation in honey [81], shortening thestorage period can reduce the formation of HMF.

Strategy for removing HMF and other furans
To remove already formed HMF, a post-process vacuum
technology can be exploited. In this technology, depending on the physical and chemical properties of the compounds to be removed, the temperature, pressure andtime can be adjusted. The effectiveness of this technology
depends on food composition, moisture content and thenature of the compound to be removed [12, 132]. Viscosity minimizes the diffusion of molecules through thematrix and thus compromises the effect of the process
in dry foods [49]. Because honey is liquid or semi-liquid,
the process can effectively remove HMF from honey. The
technology can be applied to any finished product without any modification of any of the process parameters[11].
Conclusion and future directions
Multiple factors affect HMF production in honey andother food products. Therefore, determining how to prevent or reduce the formation of HMF remains a challenge. In addition, the determination of the compound’s
concentration and the low efficiency of removal methods
make it difficult to establish a complete database of HMF
concentrations in different monofloral and multifloral
honeys. As a constituent of processed foods, HMF has
both profoundly adverse and beneficial effects on humanand bee health. Some effects of HMF on human healthand its carcinogenic as well as anti-carcinogenic properties remain inconclusive, with many studies conducted
only at preclinical levels.
Abbreviation
AAPH: 2,2′-azobis (2-amidinopropane) dihydrochloride; ACF: aberrant crypt
foci; AMS: acute mountain sickness; APC: adenomatous polyposis coli; BBB:
blood–brain barrier; CAT: catalase; CCFD: central composite face-centered
design; CYP2E1: cytochrome P450 2E1; DART: direct analysis in real time;
DMF: 2,5-dimethylfuran; DPPH: 2,2-diphenyl-1-picrylhydrazyl; DRP: deoxyribose phosphate; EFSA: European Food Safety Authority; ERK: extracellularsignal-regulated kinase; FAP: familial adenomatous polyposis syndrome; FcεRI:
express immunoglobulin Fc epsilon receptor type I; FDCA: 2,5-furandicarboxylic acid; FFA: furfuryl alcohol; GPx: glutathione peroxidase; IFNγ: interferon
gamma; IgE: immunoglobulin E; IHC: International Honey Commission; IL-4:
interleukin 4; H2O2: hydrogen peroxide; HACE: high-altitude cerebral edema;
HAPE: high-altitude pulmonary edema; HbS: sickle hemoglobin; HFCS: highfructose corn syrup; HIF: hypoxia inducible factors; HMF: 5-hydroxymethylfurfural; HMFA: 5-hydroxymethylfuroic acid; HMFG: 5-(hydroxymethyl)-2-furoyl
glycine; HPLC-DAD: high-performance liquid chromatography coupled witha diode array detector; MAPK: mitogen activated protein kinase; MAD: malondialdehyde; MEKC: micellar electrokinetic capillary chromatography; NADPH:
nicotinamide adenine dinucleotide phosphate; NFκB: nuclear factor kappa
B; NOS2: nitric oxide synthase 2; OAT1 and OAT2: organic anion transporters
type 1 and 2; OVA: ovalbumin; PAPS: 3′-phosphoadenosine-5′-phosphosulfate;
PPARγ: peroxisome proliferator-activated receptor γ; ROS: reactive oxygen species; RSM: response surface methodology; SMF: 5-sulfoxymethylfurfural; SOD:

Shapla et al. Chemistry Central Journal (2018) 12:35

superoxide dismutase; TDI: tolerable daily intake; SULT: sulfotransferase; TdT:
terminal deoxynucleotidyl transferase; TOF-MS: time-of-flight mass spectrometry; UHT: ultra-high temperature processing; XO: xanthine oxidase.
Authors’ contributions
UMS, MS and MIK participated in designing the overall idea and discussionsof views represented in the paper. UMS and MS were involved in searching
the literature and preparing the draft manuscript. NA, MIK and SHG provided
the overall concept and critically edited and approved the final version of themanuscript. All authors read and approved the final manuscript.
Author details
1
Laboratory of Preventive and Integrative Bio‑medicine, Departmentof Biochemistry and Molecular Biology, Jahangirnagar University, Savar,
Dhaka 1342, Bangladesh. 2 Department of Biochemistry, Primeasia University,
Banani 1213, Bangladesh. 3 School of Medical Sciences, Universiti Sains Malaysia,
16150 Kubang Kerian, Kelantan, Malaysia. 4 School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia.
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Not applicable.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Received: 10 January 2018 Accepted: 27 March 2018

References
1. Abdulmalik O et al (2005) 5-hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibits sickling of red blood cells†. Br J
Haematol 128:552–561
2. Abraham K, Gürtler R, Berg K, Heinemeyer G, Lampen A, Appel KE(2011) Toxicology and risk assessment of 5-Hydroxymethylfurfural in
food. Mol Nutr Food Res 55:667–678
3. Akhtar S, Ali J, Javed B, Hassan S, Abbas S, Siddique M (2014) Comparative physiochemical analysis of imported and locally produced Khyber
Pakhtunkhwa honey. Global J Biotechnol Biochem 9:55–59
4. Albalá-Hurtado S, Veciana-Nogués MT, Mariné-Font A, Vidal-Carou MC(1998) Changes in furfural compounds during storage of infant milks. J
Agric Food Chem 46:2998–3003
5. Alfredo ASC, da Silva CG, Marchini SLC (2004) Características físicoquímicas de amostras de mel de Melipona asilvai (Hymenoptera:
Apidae). Ciência Rural 34:644–650
6. Alimentarius C (2001) Revised codex standard for honey. Codex Stan
12:1982
7. Alizadeh M, Khodaei H, Mesgari Abbasi M, Saleh-Ghadimi S (2017)
Assessing the effect of 5-hydroxymethylfurfural on selected components of immune responses in mice immunised with ovalbumin. J Sci
Food Agric 97:3979–3984
8. Aloisi PV (2010) Determination of quality chemical parameters of honeyfrom Chubut (Argentinean Patagonia) Chilean. J Agric Res 70:640–645
9. Alvarez-Suarez JM et al (2010) Antioxidant and antimicrobial capacity ofseveral monofloral Cuban honeys and their correlation with color, polyphenol content and other chemical compounds. Food Chem Toxicol
48:2490–2499
10. Ameur LA, Mathieu O, Lalanne V, Trystram G, Birlouez-Aragon I (2007)
Comparison of the effects of sucrose and hexose on furfural formation
and browning in cookies baked at different temperatures. Food Chem
101:1407–1416
11. Anese M, Manzocco L, Calligaris S, Nicoli MC (2013) Industrially applicable strategies for mitigating acrylamide, furan, and 5-hydroxymethylfurfural in food. J Agric Food Chem 61:10209–10214

Page 14 of 18

12. Anese M, Suman M, Nicoli MC (2010) Acrylamide removal from heatedfoods. Food Chem 119:791–794
13. Anklam E (1998) A review of the analytical methods to determine thegeographical and botanical origin of honey. Food Chem 63:549–562
14. Archer MC et al (1992) Aberrant crypt foci and microadenoma as markers for colon cancer. Environ Health Perspect 98:195
15. Arribas-Lorenzo G, Morales FJ (2010) Estimation of dietary intake of
5-hydroxymethylfurfural and related substances from coffee to Spanish
population. Food Chem Toxicol 48:644–649
16. Ayansola AA, Banjo AD (2011) Physico-chemical Evaluation of theAuthenticity of Honey Marketed in Southwestern Nigeria. J Basic Appl
Sci Res 1:3339–3344
17. Bachmann S, Meier M, Kanzig A (1997) 5-Hydroxymethyl-2-furfural(HMF) in lebensmitteln. Lebensmittelchemie 51:49–50
18. Bailey L (1966) The effect of acid-hydrolysed sucrose on honeybees. J
Apic Res 5:127–136
19. Bakhiya N, Monien B, Frank H, Seidel A, Glatt H (2009) Renal organic
anion transporters OAT1 and OAT3 mediate the cellular accumulation of 5-sulfooxymethylfurfural, a reactive, nephrotoxic metaboliteof the Maillard product 5-hydroxymethylfurfural. Biochem Pharmacol
78:414–419
20. Ball DW (2007) The chemical composition of honey. J Chem Educ
84:1643
21. Baroni MV, Arrua C, Nores ML, Fayé P, del Pilar Díaz M, Chiabrando GA,
Wunderlin DA (2009) Composition of honey from Córdoba (Argentina):
assessment of North/South provenance by chemometrics. Food Chem
114:727–733
22. Bauer-Marinovic M, Taugner F, Florian S, Glatt H (2012) Toxicity studies
with 5-hydroxymethylfurfural and its metabolite 5-sulphooxymethylfurfural in wild-type mice and transgenic mice expressing humansulphotransferases 1A1 and 1A2. Arch Toxicol 86:701–711
23. Beaven MA, Metzger H (1993) Signal transduction by Fc receptors: the
Fc&RI case. Immunol Today 14:222–226
24. Bebenek K, Kunkel T (2004) DNA repair and replication. Adv Protein
Chem 69:137–165
25. Belay A, Solomon W, Bultossa G, Adgaba N, Melaku S (2013) Physicochemical properties of the Harenna forest honey, Bale, Ethiopia. Food
Chem 141:3386–3392
26. Blanchard P, Geiger E (1984) Production of high fructose corn syrup in
the USA. A review article. Sugar Technol Rev 11:1–94
27. Bork P, Hofmann K, Bucher P, Neuwald A, Altschul S, Koonin E (1997)
A superfamily of conserved domains in DNA damage-responsive cell
cycle checkpoint proteins. FASEB J 11:68–76
28. Bruce WR et al (1993) Diet, aberrant crypt foci and colorectal cancer.
Mutat Res 290:111–118
29. Buck AW (2001) High fructose corn syrup food science and technology.
Marcel Dekker, New York, pp 391–412
30. Bulut L, Kilic M (2009) Kinetics of hydroxymethylfurfural accumulation and color change in honey during storage in relation to moisture
content. J Food Process Preserv 33:22–32
31. Burckhardt B, Burckhardt G (2003) Transport of organic anions acrossthe basolateral membrane of proximal tubule cells. In: Reviews of physiology, biochemistry and pharmacology. Springer, Berlin, pp 95–158
32. Burdurlu HS, Karadeniz F (2003) Effect of storage on nonenzymaticbrowning of apple juice concentrates. Food Chem 80:91–97
33. Cais-Sokolińska D, Pikul J, Danków R (2004) Measurement of colour
parameters as index of the hidroxymethylfurfural content in the UHT
sterilised milk during its storage. Electron J Pol Agric Univ 7:1–3
34. Cantarelli M, Pellerano R, Marchevsky E, Camiña J (2008) Quality ofhoney from Argentina: Study of chemical composittion and trace elements. J Argent Chem Soc 96:33–41
35. Capuano E, Ferrigno A, Acampa I, Ait-Ameur L, Fogliano V (2008)
Characterization of the Maillard reaction in bread crisps. Eur Food Res
Technol 228:311–319
36. Chefrour C, Draiaia R, Tahar A, Kaki YA, Bennadja S, Battesti M (2009)
Physicochemical characteristics and pollen spectrum of some northeast algerian honeys. Afr J Food Agric Nutr Dev 9:1276–1293
37. Claeys WL, Van Loey AM, Hendrickx ME (2003) Influence of seasonalvariation on kinetics of time temperature integrators for thermallyprocessed milk. J Dairy Res 70:217–225

Shapla et al. Chemistry Central Journal (2018) 12:35

38. De la Iglesia F, Lázaro F, Puchades R, Maquieira A (1997) Automatic
determination of 5-hydroxymethylfurfural (5-HMF) by a flow injection
method. Food Chem 60:245–250
39. Ding X, Wang M-Y, Yao Y-X, Li G-Y, Cai B-C (2010) Protective effect of
5-hydroxymethylfurfural derived from processed Fructus Corni on
human hepatocyte LO2 injured by hydrogen peroxide and its mechanism. J Ethnopharmacol 128:373–376
40. Dogan M, Sienkiewicz T, Oral RA (2005) Hydroxymethylfurfural content
of some commercial whey protein concentrates. Milchwissenschaft
60:309–311
41. Doner LW (1977) The sugars of honey—a review. J Sci Food Agric
28:443–456
42. Durling LJ, Busk L, Hellman BE (2009) Evaluation of the DNA damaging
effect of the heat-induced food toxicant 5-hydroxymethylfurfural (HMF)
in various cell lines with different activities of sulfotransferases. Food
Chem Toxicol 47:880–884
43. EFSA (2005) Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on arequest from the commission related to flavouring group evaluation
13: furfuryl and furan derivatives with and without additional sidechain substituents and heteroatoms from chemical group 14. EFSA J.
215:1–73
44. Escriche I, Visquert M, Carot JM, Domenech E, Fito P (2008) Effect ofhoney thermal conditions on hydroxymethylfurfural content prior topasteurization. Food Sci Technol Int 14:29
45. Fallico B, Arena E, Zappala M (2003) Roasting of hazelnuts. Role of oil incolour development and hydroxymethylfurfural formation. Food Chem
81:569–573
46. Fallico B, Arena E, Zappala M (2008) Degradation of 5-hydroxymethylfurfural in honey. J Food Sci 73:C625–C631
47. Fallico B, Zappala M, Arena E, Verzera A (2004) Effects of conditioning
on HMF content in unifloral honeys. Food Chem 85:305–313
48. Fallico B, Zappalà M, Arena E, Verzera A (2004) Effects of heating process
on chemical composition and HMF levels in Sicilian monofloral honeys.
Food Chem 85:305–313
49. Flink J, Karel M (1970) Retention of organic volatiles in freeze-dried solutions of carbohydrates. J Agric Food Chem 18:295–297
50. Florian S, Bauer-Marinovic M, Taugner F, Dobbernack G, Monien BH,
Meinl W, Glatt H (2012) Study of 5-hydroxymethylfurfural and its
metabolite 5-sulfooxymethylfurfural on induction of colonic aberrant
crypt foci in wild-type mice and transgenic mice expressing humansulfotransferases 1A1 and 1A2. Mol Nutr Food Res 56:593–600
51. Florin I, Rutberg L, Curvall M, Enzell CR (1980) Screening of tabaccosmoke constituents for mutagenicity using the Ames’ test. Toxicology
15:219–232
52. Friedberg EC, Feaver WJ, Gerlach VL (2000) The many faces of DNApolymerases: strategies for mutagenesis and for mutational avoidance.
Proc Natl Acad Sci 97:5681–5683
53. Fromowitz M et al (2012) Bone marrow genotoxicity of 2, 5-dimethylfuran, a green biofuel candidate. Environ Mol Mutagen 53:488–491
54. Gentry TS, Roberts JS (2004) Formation kinetics and application of
5-hydroxymethylfurfural as a time–temperature indicator of lethality forcontinuous pasteurization of apple cider. Innov Food Sci Emerg Technol
5:327–333
55. Germond JE, Philippossian G, Richli U, Bracco I, Arnaud MJ (1987) Rapidand complete urinary elimination of [14C]-5-hydroxymethyl-2-furaldehyde administered orally or intravenously to rats. J Toxicol Environ
Health 22:79–89
56. Gheldof N, Wang X-H, Engeseth NJ (2002) Identification and quantification of antioxidant components of honeys from various floral sources. J
Agric Food Chem 50:5870–5877
57. Gibbings S et al (2011) Xanthine oxidoreductase promotes the
inflammatory state of mononuclear phagocytes through effects on
chemokine expression, peroxisome proliferator-activated receptor-γ
sumoylation, and HIF-1α. J Biol Chem 286:961–975
58. Gidamis AB, Chove BE, Shayo NB, Nnko SA, Bangu NT (2004) Quality
evaluation of honey harvested from selected areas in Tanzania withspecial emphasis on hydroxymethyl furfural (HMF) levels. Plant Foods
Hum Nutr 59:129–132
59. Glatt H, Schneider H, Liu Y (2005) V79-hCYP2E1-hSULT1A1, a cell line forthe sensitive detection of genotoxic effects induced by carbohydrate

Page 15 of 18

60.

61.

62.
63.
64.
65.
66.
67.

68.
69.

70.
71.
72.
73.
74.
75.
76.

77.
78.
79.

80.
81.

pyrolysis products and other food-borne chemicals. Mutat Res Genet
Toxicol Environ Mutagen 580:41–52
Glatt H, Schneider H, Murkovic M, Monien BH, Meinl W (2011) Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium
strains engineered for expression of various human and rodent sulphotransferases. Mutagenesis 27:41–48
Godfrey VB, Chen L-J, Griffin RJ, Lebetkin EH, Burka LT (1999) Distribution and metabolism of (5-hydroxymethyl) furfural in male F344 rats
and B6C3F1 mice after oral administration. J Toxicol Environ Health PartA 57:199–210
Gökmen V, Açar ÖÇ, Köksel H, Acar J (2007) Effects of dough formulaand baking conditions on acrylamide and hydroxymethylfurfuralformation in cookies. Food Chem 104:1136–1142
Gökmen V, Açar ÖÇ, Serpen A, Morales FJ (2008) Effect of leavening
agents and sugars on the formation of hydroxymethylfurfural in cookies during baking. Eur Food Res Technol 226:1031–1037
Gökmen V, Şenyuva HZ (2006) Effects of some cations on the formationof acrylamide and furfurals in glucose–asparagine model system. Eur
Food Res Technol 225:815–820
Goldberg MA, Brugnara C, Dover GJ, Schapira L, Lacroix L, Bunn HF(1992) Hydroxyurea and erythropoietin therapy in sickle cell anemia.
Seminars in oncology. Elsevier, Amsterdam, pp 74–81
Gomes S, Dias LG, Moreira LL, Rodrigues P, Estevinho L (2010) Physicochemical, microbiological and antimicrobial properties of commercial
honeys from Portugal. Food Chem Toxicol 48:544–548
Hardt-Stremayr M, Mattioli S, Greilberger J, Stiegler P, Matzi V,
Schmid MG, Wintersteiger R (2013) Determination of metabolites of
5-hydroxymethylfurfural in human urine after oral application. J Sep Sci
36:670–676
Hayden MR, Tyagi SC (2004) Uric acid: a new look at an old risk marker
for cardiovascular disease, metabolic syndrome, and type 2 diabetes
mellitus: The urate redox shuttle. Nutr Metabol 1:10
Høie AH, Svendsen C, Brunborg G, Glatt H, Alexander J, Meinl W,
Husøy T (2015) Genotoxicity of three food processing contaminantsin transgenic mice expressing human sulfotransferases 1A1 and 1A2
as assessed by the in vivo alkaline single cell gel electrophoresis assay.
Environ Mol Mutagen 56:709–714
Hossain SA, Pal PK, Sarkar PK, Patil GR (1999) Sensory characteristicsof dudh churpi in relation to its chemical composition. Zeitschrift fürLebensmitteluntersuchung und-Forschung A 208:178–182
Huber GW, Iborra S, Corma A (2006) Synthesis of transportation
fuels from biomass: chemistry, catalysts, and engineering. Chem Rev
106:4044–4098
Hunter JS (1959) Determination of optimum operating conditions by
experimental methods. Ind Qual Control 15:6–11
Husøy T et al (2008) Dietary exposure to 5-hydroxymethylfurfural from
Norwegian food and correlations with urine metabolites of short-term
exposure. Food Chem Toxicol 46:3697–3702
Iftikhar F, Masood MA, Waghchoure ES (2011) Comparison of Apis
cerana, Apis dorsata, Apis florea and Apis mellifera honey from different
areas of Pakistan. Asian J Exp Biol Sci 2:399–403
Iijima T (2006) Mitochondrial membrane potential and ischemic neuronal death. Neurosci Res 55:234–243
Islam A, Khalil I, Islam N, Moniruzzaman M, Mottalib A, Sulaiman SA, Gan
SH (2012) Physicochemical and antioxidant properties of Bangladeshi
honeys stored for more than 1 year. BMC Complement Altern Med
12:177Islam M, Khalil M, Gan SH (2014) Toxic compounds in honey. J Appl
Toxicol 34:733–742
Jachimowicz T, El Sherbiny G (1975) Zur Problematik der Verwendung
von Invertzucker für die. Bienenfütterung Apidologie 6:121–143
Janzowski C, Glaab V, Samimi E, Schlatter J, Eisenbrand G (2000)
5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging
potential and reactivity towards cellular glutathione. Food Chem Toxicol 38:801–809
Kalabova K, Vorlova L, Borkovcova I, Smutna M, Vecerek V (2003)
Hydroxymethylfurfural in Czech honeys. Czech J Anim Sci 48:551–557
Khalil M, Sulaiman S, Gan S (2010) High 5-hydroxymethylfurfural concentrations are found in Malaysian honey samples stored for more than
1 year. Food Chem Toxicol 48:2388–2392

Shapla et al. Chemistry Central Journal (2018) 12:35

82. Khalil MI et al (2012) Physicochemical and antioxidant properties ofalgerian honey. Molecules 17:11199–11215
83. Kim K, Kim Y, Kim HY, Ro JY, Jeoung D (2008) Inhibitory mechanism ofanti-allergic peptides in RBL2H3 cells. Eur J Pharmacol 581:191–203
84. Kim Y, Lee YS, Hahn JH, Choe J, Kwon HJ, Ro JY, Jeoung D (2008)
Hyaluronic acid targets CD44 and inhibits FcɛRI signaling involving
PKCδ, Rac1, ROS, and MAPK to exert anti-allergic effect. Mol Immunol.
45:2537–2547
85. Kinati C, Tolemariam T, Debele K (2011) Quality evaluation of honeyproduced in Gomma Woreda of South Western Ethiopia Livestock. Res
Rural Dev 23:190
86. Kitts DD, Chen X-M, Jing H (2012) Demonstration of antioxidant andanti-inflammatory bioactivities from sugar–amino acid Maillard reaction
products. J Agric Food Chem 60:6718–6727
87. Kowalski S, Lukasiewicz M, Duda-Chodak A, Zięć G (2013) 5-Hydroxymethyl-2-furfural (HMF)—heat-induced formation, occurrence in foodand biotransformation–a review. Pol J Food Nutr Sci 63:207–225
88. Krainer S, Brodschneider R, Vollmann J, Crailsheim K, Riessberger-Gallé
U (2016) Effect of hydroxymethylfurfural (HMF) on mortality of artificially reared honey bee larvae (Apis mellifera carnica). Ecotoxicology
25:320–328
89. Krauze A, Zalewski RI (1991) Classification of honeys by principal
component analysis on the basis of chemical and physical parameters
Zeitschrift fuer Lebensmittel-Untersuchung und. Forschung 192:19–23
90. Kroh LW (1994) Caramelisation in food and beverages. Food Chem
51:373–379
91. Küçük M, Kolaylı S, Karaoğlu Ş, Ulusoy E, Baltacı C, Candan F (2007) Biological activities and chemical composition of three honeys of different
types from Anatolia. Food Chem 100:526–534
92. Kuster B (1990) 5-Hydroxymethylfurfural (HMF). A review focussing on
its manufacture. Starch Stärke 42:314–321
93. Lee HS, Nagy S (1990) Relative reactivities of sugars in the formationof 5-hydroxymethylfurfural in sugar-catalyst model systems1. J Food
Process Preserv 14:171–178
94. Lee Y-C, Shlyankevich M, Jeong H-K, Douglas JS, Surh Y-J (1995)
Bioactivation of 5-hydroxymethyl-2-furaldehyde to an electrophilic andmutagenic allylic sulfuric acid ester. Biochem Biophys Res Commun
209:996–1002
95. Li M-M, Wu L-Y, Zhao T, Wu K-W, Xiong L, Zhu L-L, Fan M (2011) The
protective role of 5-hydroxymethyl-2-furfural (5-HMF) against acutehypobaric hypoxia. Cell Stress Chaperones 16:529
96. Li MM et al (2011) The protective role of 5-HMF against hypoxic injury.
Cell Stress Chaperones 16:267–273
97. Lin S-M, Wu J-Y, Su C, Ferng S, Lo C-Y, Chiou RY-Y (2012) Identification and mode of action of 5-hydroxymethyl-2-furfural (5-HMF) and1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) as
potent xanthine oxidase inhibitors in vinegars. J Agric Food Chem
60:9856–9862
98. Li YX, Li Y, Qian ZJ, Kim MM, Kim SK (2009) In vitro antioxidant activity of
5-HMF isolated from marine red alga Laurencia undulata in free-radicalmediated oxidative systems. J Microbiol Biotechnol 19:1319–1327
99. Lorne E et al (2008) Role of extracellular superoxide in neutrophil
activation: interactions between xanthine oxidase and TLR4 induce
proinflammatory cytokine production. Am J Physiol Cell Physiol
294:C985–C993
100. Magdoff-Fairchild B, Swerdlow PH, Bertles JF (1972) Intermolecularorganization of deoxygenated sickle haemoglobin determined by X-ray
diffraction. Nature 239:217–219
101. Mańkowska D, Majak I, Bartos A, Słowianek M, Łącka A, Leszczyńska J(2017) 5-hydroxymethylfurfural content in selected gluten-and glutenfree cereal food products
102. Markowicz DB, Monaro E, Siguemoto E, Séfora M, Valdez B (2012) Maillard reaction products in processed foods: pros and cons. In: Valdez
B (ed) Food industrial processes-methods and equipment, 1st edn.
InTech, Rijeka, pp 281–300
103. Meda A, Lamien C, Millogo J, Romito M, Nacoulma O (2005) Physiochemical Analyses of Burkina Fasan Honey. Acta Vet Brno 74:147–152
104. Mehryar L, Esmaiili M, Hassanzadeh A (2013) Evaluation of somephysicochemical and rheological properties of iranian honeys and theeffect of temperature on its viscosity. Am Eurasian J Agric Environ Sci
13:807–819

Page 16 of 18105. Mizushina Y et al (2006) 5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase.
Arch Biochem Biophys 446:69–76
106. Mondragón-Cortez P, Ulloa J, Rosas-Ulloa P, Rodríguez-Rodríguez R,
Resendiz Vázquez J (2013) Physicochemical characterization of honeyfrom the West region of México. CyTA J Food 11:7–13
107. Monien BH, Engst W, Barknowitz G, Seidel A, Glatt H (2012) Mutagenicity of 5-hydroxymethylfurfural in V79 cells expressing human SULT1A1:
identification and mass spectrometric quantification of DNA adducts
formed. Chem Res Toxicol 25:1484–1492
108. Moniruzzaman M, Khalil MI, Sulaiman SA, Gan SH (2013) Physicochemical and antioxidant properties of Malaysian honeys produced by Apis
cerana, Apis dorsata and Apis mellifera. BMC Complement Altern Med
13:43
109. Morales FJ (2008) Hydroxymethylfurfural (HMF) and related compounds
process-induced food toxicants: occurrence, formation, mitigation, andhealth risks. Wiley, New Jersey, pp 135–174
110. Morales FJ, Jiménez-Pérez S (2001) Hydroxymethylfurfural determination in infant milk-based formulas by micellar electrokinetic capillarychromatography. Food Chem 72:525–531
111. Mortas M, Gul O, Yazici F, Dervisoğlu M (2017) Effect of brewing process
and sugar content on 5-hydroxymethylfurfural and related substancesfrom Turkish coffee. Int J Food Prop 20:1866–1875
112. Muli E, Munguti A, Raina S (2007) Quality of honey harvested and processed using traditional methods in rural areas of Kenya. Acta Vet Brno
76:315–320
113. Murkovic M, Bornik MA (2007) Formation of 5-hydroxymethyl-2-furfural(HMF) and 5-hydroxymethyl-2-furoic acid during roasting of coffee. Mol
Nutr Food Res 51:390–394
114. Murkovic M, Pichler N (2006) Analysis of 5-hydroxymethylfurfual in coffee, dried fruits and urine. Mol Nutr Food Res 50:842–846
115. Muruke MH (2014) Assessment of quality of Tanzanian honey based on
physicochemical properties. Food Sci Qual Manag 33:61–72
116. Nalda MN, Yagüe JB, Calva JD, Gómez MM (2005) Classifying honeys
from the Soria Province of Spain via multivariate analysis. Anal Bioanal
Chem 382:311–319
117. Neupane K, Thapa R (2005) Alternative to off-season sugar supplement
feeding of honeybees. J Inst Agric Anim Sci 26:77–81
118. Nishi Y, Miyakawa Y, Kato K (1989) Chromosome aberrations induced bypyrolysates of carbohydrates in Chinese hamster V79 cells. Mutat Res
Lett 227:117–123
119. Ohmi K, Kiyokawa N, Takeda T, Fujimoto J (1998) Human microvascularendothelial cells are strongly sensitive to Shiga toxins. Biochem Biophys
Res Commun 251:137–141
120. Olga E, María F-G, Carmen SM (2012) Differentiation of blossom honeyand honeydew honey from Northwest Spain. Agriculture 2:25–37
121. Ordóñez-Santos LE, Vázquez-Odériz L, Arbones-Maciñeira E, RomeroRodríguez MÁ (2009) The influence of storage time on micronutrientsin bottled tomato pulp. Food Chem 112:146–149
122. OumaáAnam O (1995) Influence of metal ions on hydroxymethylfurfural formation in honey. In: Analytical proceedings including analyticalcommunications. Royal Society of Chemistry, London, pp 515–517
123. Pacher P, Nivorozhkin A, Szabó C (2006) Therapeutic effects of xanthineoxidase inhibitors: renaissance half a century after the discovery ofallopurinol. Pharmacol Rev 58:87–114
124. Pandey V, Modak MJ (1987) Biochemistry of terminal deoxynucleotidyltransferase (TdT): characterization and mechanism of inhibitionof TdT by P1, P5-bis (5’-adenosyl) pentaphosphate. Biochemistry
26:2033–2038
125. Pastoriza de la Cueva S, Álvarez J, Végvári Á, Montilla-Gómez J, CruzLópez O, Delgado-Andrade C, Rufián-Henares JA (2017) Relationshipbetween HMF intake and SMF formation in vivo: an animal and humanstudy. Mol Nutr Food Res 61:1600773
126. Pattinson KT, Sutherland AI, Smith TG, Dorrington KL, Wright AD (2005)
Acute mountain sickness, vitamin C, free radicals, and HIF-1α. Wilderness Environ Med 16:172–175
127. Paulsen JE, Løberg EM, Ølstørn HB, Knutsen H, Steffensen I-L, Alexander
J (2005) Flat dysplastic aberrant crypt foci are related to tumorigenesis
in the colon of azoxymethane-treated rat. Can Res 65:121–129

Shapla et al. Chemistry Central Journal (2018) 12:35

128. Polovková M, Šimko P (2017) Determination and occurrence of
5-hydroxymethyl-2-furaldehyde in white and brown sugar by highperformance liquid chromatography. Food Control 78:183–186
129. Preston S et al (2008) The development of duodenal microadenomas in FAP patients: the human correlate of the Min mouse. J Pathol
214:294–301
130. Prior RL, Wu X, Gu L (2006) Identification and urinary excretion of
metabolites of 5-(hydroxymethyl)-2-furfural in human subjects following consumption of dried plums or dried plum juice. J Agric Food
Chem 54:3744–3749
131. Qamer S, Ahmad F, Latif F, Ali SS, Shakoori AR (2008) Physicochemical analysis of Apis dorsata honey from terai forests, Nepal. Pak J Zool
40:53–58
132. Quarta B, Anese M (2012) Furfurals removal from roasted coffee powder
by vacuum treatment. Food Chem 130:610–614
133. Rada-Mendoza M, Sanz M, Olano A, Villamiel M (2004) Formation ofhydroxymethylfurfural and furosine during the storage of jams andfruit-based infant foods. Food Chem 85:605–609
134. Rajchl A, Drgová L, Grégrová A, Čížková H, Ševčík R, Voldřich M (2013)
Rapid determination of 5-hydroxymethylfurfural by DART ionization with time-of-flight mass spectrometry. Anal Bioanal Chem
405:4737–4745
135. Ramadan K, Maga G, Shevelev IV, Villani G, Blanco L, Hübscher U (2003)
Human DNA polymerase λ possesses terminal deoxyribonucleotidyltransferase activity and can elongate RNA primers: implications fornovel functions. J Mol Biol 328:63–72
136. Ramadan K, Shevelev IV, Maga G, Hübscher U (2004) De novo DNA synthesis by human DNA polymerase λ, DNA polymerase μ and terminal
deoxyribonucleotidyl transferase. J Mol Biol 339:395–404
137. Ramirez-Jimenez A, Guerra-Hernández E, García-Villanova B (2000)
Browning indicators in bread. J Agric Food Chem 48:4176–4181
138. Reyes-Salas EO, Manzanilla-Cano JA, Barceló-Quintal MH, JuárezMendoza D, Reyes-Salas M (2006) Direct electrochemical determinationof hydroxymethylfurfural (HMF) and its application to honey samples.
Anal Lett 39:161–171
139. Risner CH, Kiser MJ, Dube MF (2006) An aqueous high-performanceliquid chromatographic procedure for the determination of 5-hydroxymethylfurfural in honey and other sugar-containing materials. J FoodSci 71:C179–C184
140. Rizelio VM, Gonzaga LV, Borges GdSC, Micke GA, Fett R, Costa ACO(2012) Development of a fast MECK method for determination of
5-HMF in honey samples. Food Chem 133:1640–1645
141. Román-Leshkov Y, Chheda JN, Dumesic JA (2006) Phase modifiers
promote efficient production of hydroxymethylfurfural from fructose.
Science 312:1933–1937
142. Rosatella AA, Simeonov SP, Frade RF, Afonso CA (2011) 5-Hydroxymethylfurfural (HMF) as a building block platform: biological properties,
synthesis and synthetic applications. Green Chem 13:754–793
143. Roychoudhury R, Jay E, Wu R (1976) Terminal labeling and addition of
homopolymer tracts to duplex DNA fragments by terminal deoxynudeotidyl transferase. Nucleic Acids Res 3:101–116
144. Rufian-Henares J, De la Cueva S (2008) Assessment of hydroxymethylfurfural intake in the Spanish diet. Food Addit Contam 25:1306–1312
145. Rufián-Henares J, Delgado-Andrade C, Morales F (2006) Analysis ofheat-damage indices in breakfast cereals: influence of composition. J
Cereal Sci 43:63–69
146. Rufián-Henares JA, García-Villanova B, Guerra-Hernández E (2008)
Occurrence of furosine and hydroxymethylfurfural as markers ofthermal damage in dehydrated vegetables. Eur Food Res Technol
228:249–256
147. Ruiz-Matute A, Soria A, Sanz M, Martínez-Castro I (2010) Characterization of traditional Spanish edible plant syrups based on carbohydrate
GC–MS analysis. J Food Compos Anal 23:260–263
148. Ruoff K et al (2007) Quantitative determination of physical and chemical measurands in honey by near-infrared spectrometry. Eur Food Res
Technol 225:415–423
149. Safo MK, Abdulmalik O, Danso-Danquah R, Burnett JC, Nokuri S, Joshi
GS, Musayev FN, Asakura T, Abraham DJ (2004) Structural basis for the
potent antisickling effect of a novel class of five-membered heterocyclic aldehydic compounds. J Med Chem 47:4665–4676

Page 17 of 18

150. Sancho MT, Muniategui S, Huidobro JF, Simal Lozano J (1992) Aging ofhoney. J Agric Food Chem 40:134–138
151. Sang N, Stiehl DP, Bohensky J, Leshchinsky I, Srinivas V, Caro J (2003)
MAPK signaling up-regulates the activity of hypoxia-inducible factors
by its effects on p300. J Biol Chem 278:14013–14019
152. Sanna G, Pilo MI, Piu PC, Tapparo A, Seeber R (2000) Determination ofheavy metals in honey by anodic stripping voltammetry at microelectrodes. Anal Chim Acta 415:165–173
153. Schoental R, Hard G, Gibbard S (1971) Histopathology of renal
lipomatous tumors in rats treated with the “natural” products, pyrrolizidine alkaloids and α, β-unsaturated aldehydes. J Natl Cancer Inst
47:1037–1044
154. Schroeder J, Kagey-Sobotka A, Lichtenstein L (1995) The role of thebasophil in allergic inflammation. Allergy 50:463–472
155. Schultheiss J, Jensen D, Galensa R (1999) Hydroxymethylfurfural undfurfural in kaffeeproben: HPLC-biosensor-kopplung mit supressionstechnik. Lebensmittelchemie 53:159
156. Serpen A, Gökmen V, Mogol BA (2012) Effects of different grain mixtures on Maillard reaction products and total antioxidant capacities ofbreads. J Food Compos Anal 26:160–168
157. Severin I, Dumont C, Jondeau-Cabaton A, Graillot V, Chagnon MC(2010) Genotoxic activities of the food contaminant 5-hydroxymethylfurfural using different in vitro bioassays. Toxicol Lett 192:189–194
158. Shinohara K, Kong ZL, Miwa M, Tsushida T, Kurogi M, Kitamura Y,
Murakami H (1990) Effect of mutagens on the viability and someenzymes of a serum-free cultured human histiocytic lymphoma cellline, U-937. Agric Biol Chem 54:373–380
159. Sies H (1985) Oxidative stress: introductory remarks. In: Sies H (ed)Oxidative stress. Academic Press, London, pp 1–8
160. Singh N, Bath PK (1997) Quality evaluation of different types of Indian
honey. Food Chem 58:129–133
161. Singh N, Singh S, Bawa A, Sekhon K (1988) Honey—its food uses. Indian
Food Packer 42:15–25
162. Sodré GdS, Marchini LC, Moreti ACdCC, Otsuk IP, Carvalho CALd (2011)
Physico–chemical characteristics of honey produced by Apis mellifera
in the Picos region, state of Piauí. Brazil Revista Brasileira de Zootecnia
40:1837–1843
163. Solayman M, Islam M, Paul S, Ali Y, Khalil M, Alam N, Gan SH (2015)
Physicochemical properties, minerals, trace elements, and heavy metals
in honey of different origins: a comprehensive review. Compr Rev FoodSci Food Saf. 5:219–233
164. Spano N et al (2006) An RP-HPLC determination of 5-hydroxymethylfurfural in honey: the case of strawberry tree honey. Talanta 68:1390–1395
165. Stetson CA Jr (1966) The state of hemoglobin in sickled erythrocytes. J
Exp Med 123:341
166. Surh Y-J, Liem A, Miller JA, Tannenbaum SR (1994) 5-Sulfooxymethylfurfural as a possible ultimate mutagenic and carcinogenic metabolite of
the Maillard reaction product, 5-hydroxymethylfurfural. Carcinogenesis
15:2375–2377
167. Suzuki M, Nakamura T, Iyoki S, Fujiwara A, Watanabe Y, Mohri K, Isobe K,
Ono K, Yano S (2005) Elucidation of anti-allergic activities of curcuminrelated compounds with a special reference to their anti-oxidative
activities. Biol Pharm Bull 28:1438–1443
168. Svendsen C, HUSØY T, Glatt H, Paulsen JE, Alexander J (2009) 5-Hydroxymethylfurfural and 5-sulfooxymethylfurfural increase adenoma andflat ACF number in the intestine of Min/+ mice. Anticancer Res
29:1921–1926
169. Terrab A, González AG, Díez MJ, Heredia FJ (2003) Characterisation of
moroccan unifloral honeys using multivariate analysis. Eur Food Res
Technol 218:88–95
170. Teixidó E, Santos F, Puignou L, Galceran MT (2006) Analysis of 5-hydroxymethylfurfural in foods by gas chromatography–mass spectrometry. J
Chromatogr A 1135:85–90
171. Theobald A, Müller A, Anklam E (1998) Determination of 5-hydroxymethylfurfural in vinegar samples by HPLC. J Agric Food Chem
46:1850–1854
172. Toker OS, Dogan M, Ersöz NB, Yilmaz MT (2013) Optimization of thecontent of 5-hydroxymethylfurfural (HMF) formed in some molasses
types: HPLC-DAD analysis to determine effect of different storage time
and temperature levels. Ind Crops Prod 50:137–144

Shapla et al. Chemistry Central Journal (2018) 12:35

173. Tucak Z, Periškić M, Škrivanko M, Konjarević A (2007) The influence ofthe botanic origin of honey plants on the quality of honey. Agriculture
13:234–236
174. Turhan I, Tetik N, Karhan M, Gurel F, Tavukcuoglu HR (2008) Qualityof honeys influenced by thermal treatment LWT-Food. Sci Technol
41:1396–1399
175. Turhan K (2009) Effects of thermal treatment and storage on hydroxymethylfurfural (HMF) content and diastase activity of honeys collectedfrom middle Anatolia in Turkey. In: Innovations in chemical biology.
Springer, Berlin, pp 233–239
176. Ulbricht RJ, Northup SJ, Thomas JA (1984) A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions. Toxicol Sci 4:843–853
177. Van der Zee R, Pisa L (2010) Bijensterfte 2009–10 en toxische invertsuikersiroop. NCB Rapp. 2:1–15
178. Wang M-Y et al (2010) Investigation on the morphological protective
effect of 5-hydroxymethylfurfural extracted from wine-processed Fructus corni on human L02 hepatocytes. J Ethnopharmacol 130:424–428
179. White J, Doner LW (1980) Honey composition and properties. Beekeep
US Agric Handb 335:82–91
180. White J Jr (1979) Spectrophotometric method for hydroxymethylfurfural in honey. J Assoc Off Anal Chem 62:509–514
181. White JW Jr, Kushnir I (1967) Composition of honey. VII. J Apic Res
6:163–178
182. White Júnior J (1978) Honey. Adv Food Res 22:287–374
183. White JW (1975) Composition of honey. Honey: a comprehensive
survey. Heinemann, London, pp 157–206
184. Winkler O (1955) Beitrag zum Nachweis und zur Bestimmung von
Oxymethylfurfurol in Honig und Kunsthonig. Zeitschrift für Lebensmitteluntersuchung und-Forschung A 102:161–167
185. Wootton M, Ryall L (1985) A comparison of Codex Alimentarius
Commission and HPLC methods for 5-hydroxymethyl-2-furaldehyde
determination in honey. J Apic Res 24:120–124

Page 18 of 18

186. Xiao PG, Xing ST, Wang LW (1993) Immunological aspects of Chinese
medicinal plants as antiageing drugs. J Ethnopharmacol 38:159–165
187. Yamada P, Nemoto M, Shigemori H, Yokota S, Isoda H (2011) Isolationof 5-(hydroxymethyl) furfural from lycium chinense and its inhibitory
effect on the chemical mediator release by basophilic cells. Planta Med
77:434–440
188. Yilmaz H, Küfrevioğlu Öİ (2001) Composition of honeys collected from
Eastern and South-Eastern Anatolia and effect of storage on hydroxymethylfurfural content and diastase activity. Turk J Agric For 25:347–349
189. Yuan J-P, Chen F (1998) Separation and identification of furaniccompounds in fruit juices and drinks by high-performance liquid
chromatography photodiode array detection. J Agric Food Chem
46:1286–1291
190. Zaitzev AN, Simonian TA, Pozdniakov AL (1975) Hygienic standards forhydroxymethylfurfural in food products. Vopr Pitan 1:52–55
191. Zappala M, Fallico B, Arena E, Verzera A (2005) Methods for the determination of HMF in honey: a comparison. Food Control 16:273–277
192. Zhang X-M, Chan CC, Stamp D, Minkin S, Archer MC, Bruce WR (1993)
Initiation and promotion of colonic aberrant crypt foci in rats by
5-hydroxymethy1-2-furaldehyde in thermolyzed sucrose. Carcinogenesis 14:773–775
193. Zhao L et al (2013) In vitro antioxidant and antiproliferative activities of
5-hydroxymethylfurfural. J Agric Food Chem 61:10604–10611
194. Zhao L, Su J, Li L, Chen J, Hu S, Zhang X, Chen T (2014) Mechanistic
elucidation of apoptosis and cell cycle arrest induced by 5-hydroxymethylfurfural, the important role of ROS-mediated signaling pathways.
Food Res Int 66:186–196
195. Zhao Y, Flugstad B, Kolbe E, Park JW, Wells JH (2000) Using capacitive(radio frequency) dielectric heating in food processing and preservation–a review. J Food Process Eng 23:25–55