5HMF Effects on Human Health
5HMF is considered beneficial to human health by providing antioxidative, anti-allergic, anti-inflammatory, anti-sickling, and anti-hyperuricemic effects.
5HMF draws great attention from our scientists at Ypera Inc. and other renowned scientists around the globe. The organic compound 5HMF known as 5-hydroxymethylfurfural (or HMF) is formed from reducing sugars in honey and various foods in acidic environments when they are heated through the Maillard reaction. 5HMF is easily absorbed from food through the gastrointestinal tract and, upon being metabolized into different derivatives, is excreted via urine.
5HMF is not only present in honey; it is nearly ubiquitous in our daily heat-processed, sugar-containing foodstuffs, from our breakfast cereals, bread, dairy products, and fruit juices to liquors at different concentrations. In most previous studies, 5HMF has been reported to have negative effects on human health, however, in more recent extensive studies, 5HMF has been proved to have a wide range of positive effects, such as antioxidative, anti-allergic, anti-inflammatory, anti-hypoxic, anti-sickling, anti-hyperuricemic effects.
5HMF is a cyclic aldehyde produced by sugar degradation through the Maillard reaction (a non-enzymatic browning reaction) during food processing or long storage of honey. The presence of simple sugars (glucose and fructose) and many acids, as well as minerals, in honey, can further enhance the production of this substance.
5HMF 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 5HMF formation in any food is multifactorial. Factors affecting the rate of 5HMF formation include temperature, pH, type of sugar, the concentration of divalent cations and water activity in the medium.
5HMF is a six-carbon heterocyclic organic compound containing both aldehyde and alcohol (hydroxymethyl) functional groups. The ring of the structure is centered on furan moieties, whereas the two functional groups, i.e., formyl and hydroxy-methyl groups, are linked at the second and fifth positions, respectively (Fig.1.). 5HMF is a solid, yellow substance that has a low melting point but is highly soluble in water.
5HMF 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.). 5HMF formation is correlated with chemical characteristics such as pH, free acid content, total acidity, lactone content and mineral content, which in turn are related to the floral source of collected honey samples.
Similarly to honey (Fig.3.), which is rich in glucose and fructose, most sugar-containing foods also contain 5HMF, although 5HMF occurs at very low concentrations and can even be absent in honey and food products.
5HMF in foods correlates with urine metabolites
Following oral or intravenous administration, 5HMF is mainly metabolized into three major metabolites: 5-hydroxymethylfuroic acid (HMFA), 2,5-furandicarboxylic acid (FDCA) and 5-(hydroxymethyl)-2-furoyl glycine (HMFG). It is estimated that 80–100% of the total amount is released within the first 24 h. Following its administration 5HMF is first oxidized to carboxylic acid and is conjugated with glycine, leading to the formation of HMFG. The concentration of free glycine is the rate-limiting step in this pathway. Subsequently, 5HMF undergoes further oxidation to yield FDCA.
An in vivo study reported the presence of three metabolites in urine following oral administration of labeled 5HMF (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 the species and the doses). In humans, 5HMF is completely cleared following its oral administration (using the juice of dried plum). Similarly, in another clinical study, 5HMF was shown to be completely excreted through urine within 48 h following oral administration at 240 mg/day.
5HMF in various food products
In addition to being found in honey, 5HMF 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, and tomato products, and 5HMF is released from sugar and carbohydrates after home cooking, indicating that 5HMF is ubiquitous in the human diet.
We consume several different food products, including bakery goods, milk, fruit juice, cereals, coffee, chocolate, soft drinks, vinegar, wine, nuts and grilled meat in our daily lives. The majority of these products undergo thermal treatments prior to consumption, such as boiling, baking, extrusion cooking, roasting, pasteurization, and other processing. These processes are performed not only to make the products more edible but also 1) for preservation (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. During thermal processing and preservation, the Maillard reaction or non-enzymatic browning may occur, where 5HMF is a common product whose extent of formation depends on the processing conditions.
5HMF in Cereals
According to Norwegian and German researchers, cereals and cereal products, including bread, are some of the most prominent sources of human consumption of 5HMF. The extent of 5HMF formation in cereal products is heavily dependent on many factors, including temperature, dough fermentation process, water activity and the presence of fruits, grains and other flavorings or additives (such as cocoa, malt, sucrose, glucose, salt, and honey).
5HMF in Coffee
Coffee is one of the most common drinks reported to contain 5HMF. The 5HMF 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. An investigation of 5HMF 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 and traditional Turkish coffee samples contained 5HMF over ranges of 336.03–362.05 and 213.02–238.99 mg/kg, respectively.
5HMF in Dairy products
5HMF is formed via side reactions during heat sterilization and browning processes. An investigation of the formation of 5HMF as a result of exposure to different storage temperatures (20, 30, 37 °C) and storage duration (up to 9 months) in liquid as well as in powdered infant milk, concluded that 5HMF formation follows a zero-order kinetics profile independent of storage temperature and milk type. In the case of traditional dairy products, there was a strong positive correlation between 5HMF concentration and the products’ flavors, colors and textures.
5HMF in Fruits and Vegetables
5HMF concentrations in dried apricot, peach, pear, fig, date, apple and pineapple products. HMF concentrations were highest in dates (1000 mg/kg) and plums (1100–2200 mg/kg). The mean range of 5HMF concentrations in other dried fruits was 1–780 mg/kg.
Due to their rich content of sugars and amino acids, fruits and vegetables contain high 5HMF levels. In a study involving jam products (prepared commercially and under laboratory conditions) stored at 20 and 35 °C for 12 months, a temperature-dependent relationship was established between 5HMF formation and storage duration. Additionally, a positive correlation between storage time and temperature with 5HMF formation has been observed for a variety of apple juice.
5HMF in other food products
It is concluded that there are likely no heat-processed food products that are free of 5HMF. The 5HMF concentrations in several food products are listed in (Table 1.).
5HMF is formed upon thermal treatment and in combination with other factors. As it is a product of the non-enzymatic Maillard reaction, there is no fixed concentration of 5HMF in different food items. The baking temperature, rate of saccharose degradation, the concentration of reducing sugars, type of sugar (glucose, fructose or others), water activity, the addition of other food additives, coloring agents, caramelization, storage time and temperature, type of storage and processing vary widely among different food items.
Therefore, 5HMF content varies among food items, even among those of the same type. The formation of 5HMF is inevitable, and comparing or ranking food items with respect to 5HMF concentration has yet to be performed precisely.
Positive effects of 5HMF on human health
5HMF as an antioxidant
ROS (Reactive Oxygen Species) 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 from stress to metabolic defects, neurodegenerative diseases or even neoplastic transformations. In a 5study, 5HMF showed a dose-dependent (0.8–6.4 mM) free-radical scavenging capacity. 5HMF also has significant protective effects on erythrocytes against ROS-induced damage. To investigate the protective effect and oxidative stress induced by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), the levels of ROS (Reactive Oxygen Species) and malondialdehyde (MDA: an indirect determinant of lipid peroxidation) production, the activity of the antioxidant enzymes glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) in erythrocytes pre-treated with 5HMF were determined. It was revealed that the contents of ROS and MDA were reduced in 5HMF-treated cells, while the activities of these enzymes were elevated relative to those of negative control cells. The ROS scavenging activity of 5HMF is imparted by its structure, which features functional reactive groups such as aldehyde oxygen, double bonds and another oxygen in its furan ring. These features can attract electrons easily and quench ROS (Reactive Oxygen Species).
5HMF also has a protective effect at the morphological and biochemical levels on hepatocytes damaged by hydrogen peroxide (H2O2)-induced oxidative stress, under which cells undergo morphological changes such as wrinkling, condensation of chromatin and splitting of nuclei—hallmarks of apoptotic and necrotic cells. However, in an H2O2-stressed human liver cell line (L02), cells treated with HMF (0.79 µM) could preserve their morphology better than non-treated cells could. 5HMF also reduces the levels of caspases 3 and 9 (executioner of apoptosis) in these cells. It was hypothesized that 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 and caspase 3.
5HMF against hypoxic injury
Oxygen is required for cell survival. Deficiency of oxygen (hypoxic condition) has numerous detrimental and even life-changing effects on health. Hypoxia may be induced by many factors, including altitude and self-related conditions such as ischemia, atherosclerosis, and cancer. Several cellular mechanisms are triggered and can ameliorate hypoxic conditions, among which extracellular signal-regulated kinase(ERK)-mediated transactivation of the transcription factor and hypoxia-inducible factors (HIF) are believed to participate. The mitochondrial membrane potential is also reduced and negatively affects hypoxic cells. In their in vitro study of the cell line ECV304 (human umbilical cord vein endothelial cell), Li et al.  showed that cells pre-treated with 5HMF (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 and necrotic cells also significantly declined. In their further study with a Kunming mice model, the authors showed that pre-exposure to 5HMF (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 5HMF increases survivability under hypobaric hypoxic conditions, it may be a potent therapeutic agent against acute mountain sickness (AMS), high-altitude cerebral edema (HACE) and high-altitude pulmonary edema (HAPE).
5HMF as an anti-allergen
Basophils and mast cells participate in the pathogenesis and manifestations of allergic reactions such as asthma, atopic dermatitis, and allergic rhinitis. RBL-2H3 cells are mast cells located in the mucosal layer. These cells express immunoglobulin Fc epsilon receptor type I (FcεRI) on their surface. The crosslinking of IgE with specific protein antigens and the binding of crosslinked IgE to FcεRI trigger intracellular signal transduction cascades. These events lead to Ca2+ influx and the release of mediators by degranulation, MAPK phosphorylation, upregulation of cytokine gene expression and increased ROS generation [23, 83, 84, 154]. A study showed that 5HMF acts at different stages to inhibit degranulation at doses of 0.01–0.30 µg/ml. In addition, 5HMF interferes with antigen-antibody crosslinking and antibody-receptor binding. 5HMF also blocks calcium (Ca2+) influx into IgE-sensitized bovine serum albumin-stimulated RBL-2H3 cells. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a major role in the production of ROS in IgE-mediated RBL-2H3 cells. H2O2 and NO, two major ROS, are known to regulate degranulation and Ca2+ signaling in mast cells [83, 84, 186]. A significant inverse association exists between the release of histamine and Ca2+ from intracellular stores and the superoxide anion or DPPH scavenging activities. The anti-allergen effect of 5HMF on cells is due to the blocking of histamine release and Ca2+ signaling through the compound’s free-radical scavenging activity [98, 167].
In another study using ovalbumin (OVA)-immunized BALB/c mice, HMF decreased the levels of total IgE and OVA-specific IgE. The study also showed that HMF-treated immunized mice exhibited lower levels of IFNγ (Interferon-gamma) and IL-4 (Interleukin 4) than those of untreated mice. Therefore, 5HMF may be a potent anti-allergic compound.
The use of 5HMF 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 acid in the blood lead to the development of hyperuricemia, which is the main cause of gout. In addition, many other pathological states are associated with hyperuricemia, including metabolic syndrome, heart failure, pulmonary disorder, and type 2 diabetes mellitus. Increased XO activity downregulates the anti-inflammatory transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) and accelerates inflammatory action. XO is also an endogenous producer of superoxide, a potent activator of nuclear factor kappa B (NFκB). NFκB acts as a transcription factor and upregulates the expression of nitric oxide synthase 2 (NOS2) and interleukin 8 (IL-8). 5HMF exerts an anti-inflammatory effect by downregulating NFκB and inhibits the activity of XO.
5HMF as an anti-sickling agent
Hemoglobinopathies such as sickle cell disease are life-threatening. 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 to morbidity. Although there are many anti-sickling 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, 5HMF can act as an effective anti-sickling agent. Studies showed that 5HMF orally administered in low concentrations is absorbed into the bloodstream from the gastrointestinal tract in transgenic (Tg) sickle mice. Subsequently, 5HMF 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, 5HMF allosterically shifts 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 5HMF survived longer under hypoxic conditions than did untreated mice, indicating the potential of 5HMF as an anti-sickling agent.
Tolerable daily intake (TDI) of 5HMF
The susceptibility of cells to 5HMF depends on the presence and expression levels of receptors, metabolism, structure and the enzyme activity of 5HMF. At the preclinical level, no toxic effects have been observed at daily doses ranging from 80 to 100 mg/kg body weight. The established TDI for HMF is 132 mg/day using a 40-fold safety margin. 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 is worth considering and appreciating.
5HMF is considered beneficial to human health by providing antioxidative, anti-allergic, anti-inflammatory, anti-hypoxic, anti-sickling, and anti-hyperuricemic effects, with many studies currently being conducted at preclinical levels.
HMFA (5-hydroxymethylfuroic acid)
FDCA (furandicarboxylic acid)
HMFG (5-(hydroxymethyl)-2-furoyl glycine)
ROS (reactive oxygen species)
Abdulmalik O, et al. 5-hydroxymethyl-2-furfural modifies intracellular sickle hemoglobin and inhibits sickling of red blood cells† Br J Haematol. 2005;128:552–561. DOI: 10.1111/j.1365-2141.2004.05332.x.[PubMed] [CrossRef]
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