Methylglyoxal, a Bioactive Component In Manuka Honey Exhibits Virucidal Properties Against SARS-CoV-2


A new study by researchers from Qatar University has demonstrated that methylglyoxal (MG), a bioactive component in manuka honey exhibits virucidal properties against the SARS-CoV-2 coronavirus in vitro.

The study findings were published in the University’s website and has not been peer reviewed by any third parties yet.
To date there is no approved antiviral treatment for the COVID-19 disease. Despite a massive global vaccination programme, it is gradually coming to light despite massive cover ups, censorships and control of the COVID-19 narratives, there is now a growing number of breakthrough infections among the fully vaccinated with some leading to hospitalizations due to disease severity and even deaths in some cases.

It is expected that as more Delta sub-variants and other immune evasive variants emerge, the pandemic will soon take a more catastrophic turn. At the moment more than 242.9 million people globally have been infected with the SARS-CoV-2 coronavirus and more than 4.94 million people have died with America alone registering more than 751,000 COVID-19 deaths so far.
Scientists are desperately seeking suitable antivirals that would not be toxic to human body unlike the two big pharma antiviral candidates that are likely to get approved by the no longer U.S. FDA.
Numerous past studies have shown that Manuka honey has virucidal/antiviral effect.
Methylglyoxal (MG), a bioactive component in Manuka honey, has antiviral activity in vitro. MG may modify arginine residues in the functional domains of viral spike and nucleocapsid proteins, resulting in loss of charge, protein misfolding and inactivation.
The study team aim was to characterize the antiviral activity of Manuka honey against SARS-CoV-2 in vitro.
For the Manuka Honey-COVID-19 study, wild-type SARS-CoV-2 with titers of multiplicities of infection (MOI) 0.1 and 0.05 were incubated with 2-fold serial dilutions of 250+ Manuka honey (equivalent to 250 to 31 µM) in infection medium (Dulbecco’s Modified Eagle Medium + 2% fetal bovine serum + 100 units/ml penicillin + 100 µg/ml streptomycin) for 3 h.
Manuka honey treated and untreated control SARS-CoV-2 was incubated with confluent cultures of Vero cells in vitro for 1 h, cultures washed with phosphate-buffered saline and incubated in fresh infection medium at 37°C for 4 – 5 days until 70% of virus control cells displayed cytopathic effect.
The study team also studied the effect of scavenging MG in Manuka Honey with aminoguanidine (AG; 500 µM) on virucidal activity.
The antiviral activity of MG was judged by median tissue culture infectious dose (TCID50) assays. Data analysis was by logistic regression. TCID50 (mean ± SD) was deduced by interpolation.
The study findings showed that diluted Manuka honey inhibited SARS-CoV-2 replication in Vero cells.
SARS-CoV-2 was incubated in diluted Manuka honey in medium at 37°C for 3 h before adding to Vero cells.
It was found that Manuka honey dilutions down to 125 µM MG equivalents completely inhibited cytopathic effect of SARS-CoV-2 whereas 31.25 µM and 62.5 µM MG equivalents had limited effect.
Logistic regression and interpolation of the cytopathic effect indicated that the TCID50 = 72 ± 2 µM MG equivalents for MOI of 0.1. Prior scavenging of MG by addition of AG resulted in virus replication levels equivalent to those seen in the virus control without AG.
The study findings showed that Manuka honey has antiviral activity against SARS-CoV-2 when incubated with the virus in cell-free media at no greater than ca. 40-fold dilutions of 250+ grade. Anti-viral activity was inhibited by AG, consistent with the anti-viral effect being mediated by MG. Manuka honey dilutions in MG equivalents had similar antiviral effect compared to authentic MG, also consistent with MG content of Manuka honey mediating the antiviral effect.
Although Manuka honey may inactivate SARS-CoV-2 in cell-free culture medium, its antiviral activity in vivo for other than topical application may be limited because of the rapid metabolism of MG by the glyoxalase system and limited bioavailability of oral MG.
The study team is conducting more research as to how to address these limitations and tap the potential usage of methylglyoxal (MG) in Manuka honey to help with COVID-19.

Potential Effect of Honey and Its Main Components on Vital Organs Complications of Coronavirus

Coronaviruses are able to stimulate several inflammatory mediators, which leads to various organ dysfunctions including ARDS. Honey and its main components have anti-fibrotic activity by reducing the expression of inflammatory mediators involved in lung infection. It has been found that honey lowers levels of prostaglandins (PG) E2,8 PG2a,83 thromboxane B284 and increases nitric oxide end products.

These properties could help explain some biological and therapeutic properties of honey, particularly as an anti-bacterial agent or wound healing.46 Moreover, it is suggested that honey could be effective against human respiratory syncytial virus (RSV) by inhibition viral replication.42 Pulmonary fibrosis is a serious consequence of COVID-19 infection associated with ARDS.

Furthermore, COVID-19 may affect the respiratory system and cause ARDS to secrete inflammatory mediators related to pulmonary fibrosis such as transforming growth factor beta (TGF-β) and IL-1β. Indeed, chrysin could inhibit the cellular inflammatory response by improving the NF-кB signaling pathway and fibrotic response in a rat model of viral-induced acute lung injury.85

In addition, chrysin reduced inflammation, collagen deposition, malondialdehyde (MDA) levels in the lung in an experimental model of bleomycin-induced pulmonary fibrosis.86 Furthermore, it has been shown that kaempferol reduced pulmonary inflammation and fibrosis in the experimental model of silicosis.87

In some patients with COVID-19, pulmonary edema was observed accompanied by a decrease in the activity of the epithelial sodium channels and the ion channel of the E-protein on the pulmonary epithelial cells.88 In addition, chrysin inhibited alpha-naphthylthiourea (ANTU)-induced pulmonary edema in the animal model through regulating inflammatory responses and oxidative/nitrosative stress.89

Moreover, cardiovascular disturbances may occur in patients with COVID-19 due to the systemic inflammation. It has been shown that honey reduced the degree of infiltration of inflammatory cells and to preserve the morphology of myocardial fiber in heart attack model.90 In a rat model, chrysin has been found to have a protective effect on myocardial fiber structure in isoproterenol-induced acute myocardial infarction.91

Chrysin modulated the hemodynamic and ventricular functions in isoproterenol-induced acute myocardial infarction in a rat model through decreasing oxidative stress and also by reversing arterial ligation and peroxisome proliferator-activated receptor gamma (PPAR-γ) inhibition. Treatment with chrysin also led to an improvement in receptor for advanced glycation end-products (RAGE), inhibitor of nuclear factor kappa B (IKK-β) and nuclear factor kappa B (NF-κB) expressions and TNF-α levels. More interestingly, chrysin exerts cardiovascular protection by reducing apoptosis indices.91-95

The connection between COVID-19 and kidney damage is not clear. However, it was found that some patients with COVID-19 infection showed signs of kidney damage without previous history. There is some evidence linking COVID-19 infection to kidney damage:

1) the ability of coronavirus to attach to kidney cells and enter to the cell,

2) the decrease in blood oxygen,

3) the induction of systemic inflammation,

4) the formation of clots in the bloodstream that can block the blood vessels in the kidney.

In addition, honey and its main components may be effective for kidney inflammation associated with COVID-19 treatment. It has been reported that rosmarinic acid improved blood pressure by inhibiting angiotensin-converting enzyme activity in 2-kidneys 1-clip model in rats.96

Further, chrysin showed an inhibited therapeutic effect against adenine-induced CKD in a mouse model of focal cerebral ischemia/reperfusion injury by suppressing the NF-κB signaling pathway.97 It was suggested that clot formation in small and large blood vessels may be major factor in organ failures and death from COVID-19 and that inhibition of clot formation may be effective against organ failures and death from COVID-19.98

In this context, in vitro and in vivo assays confirmed the inhibitory effects of honey and its main components on platelet aggregation and blood coagulation.99-106 For example, the in vitro, in vivo, and ex vivo models showed the anti-thrombotic and anti-coagulant effect of quercetin.103 Indeed, several studies have reported that quercetin decreased, similar to other natural polyphenols (resveratrol, curcumin, ginkgo biloba and bilberry) diastolic pressure by potentiating eNOS activation, nitric oxide production107,108 and the activity of thrombin, formation of fibrin clots and blood clotting through modulating the coagulation cascade.103

Quercetin and apigenin were found to decrease collagen- and Adenosine diphosphate (ADP)-induced aggregation in platelet-rich plasma for 2 weeks in healthy volunteers.104 Moreover, rosmarinic acid exerted a mild anti-thrombotic effect though inhibiting platelet aggregation and fibrinolytic activity in anesthetized rats with tight ligature in the inferior vena cava below the left renal vein.105

The anti-thrombotic effects of caffeic acid was investigated on cerebral arterioles and venules of mice by intravital microscopy and also in vitro. Furthermore, caffeic acid was able to inhibit platelet-mediated thrombosis by the activating of p38, extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) and led to an increase in cyclic adenosine monophosphate (cAMP) levels and a decrease in the expression of P-selectin and integrin αIIbβ3 activation.106

Potential Effect of Honey and Its Main Components on Interaction Coronavirus With Nrf2 Signaling Pathways

Nuclear factor erythroid 2-related factor 2 (Nrf2) dependent antioxidant genes expression is markedly reduced in COVID-19 patients. Nrf2 stimulators may inhibit the replication of SARS-CoV2 and also related inflammatory genes expression.109 Previous studies indicated the Nrf2-mediated antioxidative effect of honey and its main components in various diseases.

In this regard, it was found that honey stimulated the Keap1/Nrf2 signaling in the epidermis and induced epidermal barrier recovery.110 Honey significantly improved hypertension via stimulation of Nrf2 in the kidney of hypertensive rats.111 In murine macrophages exposed to the lipopolysaccharides (LPS), Carthamus tinctorius L. honey induced the expressions of Nrf-2/Heme Oxygenase-1 (HO-1), leading to inhibition of inflammation.112 Chrysin ameliorated the neutrophils infiltration and other lung pathological damages through modulation of oxidative stress dependent Nrf2 pathway in lungs of rats exposed to carrageenan.113 Chrysin, luteolin and apigenin protected rat primary hepatocytes against oxidative stress through modulating Nrf2 signaling pathway.114

Regarding to stimulatory effects of honey on the Nrf2 signaling, this natural agent can potential effect to combat against SARS-CoV2.

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In an attempt to use honey as one of the treatments against COVID-19, antiviral properties of honey need to be exploited. Since ancient times, it is believed that honey is a valuable cure against pathogenic respiratory agents, including viruses that cause cough [26]. Several studies showed antiviral activity of honey against a wide range of viruses such as herpes simplex virus (HSV), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), adenovirus, and influenza viruses [27,28,29,30,31].

Furthermore, honey also possesses anti-inflammatory capacities and is recognized as a potent immune booster, which compliments it as an effective remedy to reduce the severity of viral diseases [32,33,34]. However, the therapeutic potential of honey against COVID-19 has still not been studied. Although many people believe that the antiviral effect of honey may work against SARS-CoV-2 and/or play an immunomodulatory role in COVID-19 patients, the potential mechanism of action still unclear.

Therefore, it is worth clarifying these points scientifically, based on previous reports. In this review, we describe the potential effects of honey as a natural remedy to support our ongoing combat against COVID-19.

The Medicinal Properties of Honey

Although there are different types of honey from various producer bees, the chemical composition of 100 g of the commonly consumed honeys include approximately 64.9–73.1% carbohydrates, 35.6–41.8% fructose, 25.4–28.1% glucose, 16.9–18% water, 1.8–2.7% maltose, 0.23–1.21% sucrose, and 0.50–1% proteins, vitamins, amino acids, and minerals [35].

Honeys display variability in chemical composition associated with botanical and geographical origin, bee species, and climate [36]. The majority of the medicinal properties of honeys were associated with their antioxidant phenolic compounds that vary between honeys, typically based on the floral origin of the honey [35].

Phenolic compounds are plant secondary metabolites founded in honey with diverse chemical structures including phenolic acids and polyphenols (e.g., flavonoids). Despite the variability in the chemical composition of honeys, the most abundant flavonoids are apigenin, quercetin, luteolin, chrysin, kaempferol, galangin, genistein, pinocembrin, and pinobanksin, while the most abundant phenolic acids are gallic acid, chlorogenic acid, syringic acid, vanillic acid, p-coumaric acid, p-hydroxybenzoic acid, and caffeic acid [35].

Based on the floral source, there are monofloral (from a single floral source) and multifloral (from diverse floral sources) honeys. Most honeys are monofloral, produced commonly by bees from the genus Apis, and named according to their respective plant species (e.g., Manuka honey).

Honeys produced by stingless bees (genus Meliponinae) are commonly multifloral [37]. However, which type of these two honeys can express superior therapeutic potentials (mainly based on its antioxidant activity) is still under investigation. A study of 10 different monofloral and multifloral honeys showed that the antioxidant activities, based on their phenolic content, of some monofloral honeys (i.e., heather > phacelia> honeydew > buckwheat) were higher compared to multifloral honeys, whereas other monofloral honeys (i.e., nectar–honeydew> lime > rape> goldenrods > acacia) showed lower antioxidant activities [38].

So far, the full process of absorption, metabolism, and excretion, which might be valid for all phenolic compounds, still requires clarification. Although the mechanisms behind the bioavailability of phenolic compounds have been addressed in few studies, only a few of these studies have specifically focused on those compounds derived from honey [39,40].

The utilization of phenolic compounds from honey in the clinical practice is often hampered by their very low bioavailability and absorption [41]. Understanding of the pharmacokinetics of phenolic compounds starts with phenol metabolism, which depends on hydrolysis reaction.

This reaction can be performed by the lactase phlorizin hydrolase and the cytosolic β-glucosidase called β-endoglucosidase enzymes and are present in the small intestine [42]. These enzymes are responsible for catalyzing the β-hydrolysis of the sugar in the glycosylated phenolic compounds so they can be absorbed by the small intestine [43]. Some compounds contain sugars that prohibit the absorption but are deglycosylated by enzymes of microfloras presented in the colon. The final metabolites can either be absorbed or excreted through the feces or kidneys [43].

Honey as an Immune System Booster

It is well-known that honey is an immune booster that improves the proliferation of T and B lymphocytes, stimulates phagocytosis, and regulates the production of vital pro-inflammatory cytokines from monocytes, such as tumor necrosis factor (TNF), interleukin 1 beta (IL-1β), and IL-6 [32,33].

On the other hand, honey also showed anti-inflammatory activity that inhibits the expression of these pro-inflammatory cytokines [34]. This dual immunomodulatory role of honey has been attributed to its antioxidant properties [34,44], which prevent and manage oxidative stress (Figure 3). The antioxidant activity of honey is positively correlated to its phenolic compounds content [45].

Molecules 25 05017 g003
Figure 3. The principal of oxidative damage and the role of antioxidants in scavenging free radicals. (A) The free radicals generated from endogenous sources, at limited concentrations, are considered important for regulation of cell maturation, in addition to their role in immune defense. Excessive concentrations of these unstable molecules can result from illness conditions and exogenous sources, and thus lead to oxidative damage (imbalance between free radical and antioxidant concentrations). This status results in cell injury/death based on the extremely high reactivity of free radicals with vital cellular molecules including lipids, amino acids, proteins, and DNA. (B) Antioxidants are necessary to stop oxidative damage by neutralizing free radicals. They own this unique role due to their capability to give an electron to free radicals that have unpaired electrons to make them stable and unharmful. ROS, reactive oxygen species; RNS, reactive nitrogen species (adapted from Al-Hatamleh et al., 2020 [36]).

According to the current literature, the severity of COVID-19 infection correlates with lymphocytopenia, and patients who died from COVID-19 had lower lymphocyte counts compared to survivors [46,47]. These data suggest that lymphocyte-mediated antiviral activity is poorly effective against COVID-19.

Despite lymphocytopenia, evidence for an exaggerated release of pro-inflammatory cytokines (i.e., IL-1 and IL-6) has been reported in the course of acute respiratory syndrome in COVID-19 infected patients, aggravating the clinical course of the disease [48].

Therefore, honey is anticipated to play a vital role in boosting the immune system as a supportive treatment for patients infected with COVID-19, and also for preventive measures for healthy individuals (Figure 4).

Molecules 25 05017 g004 550
Figure 4. Potential mechanisms of action of honey as an immunomodulatory agent. These mechanisms relied on the antioxidant activity of honey. This activity inhibits oxidative stress and results in stopping harm to the vital cellular components (lipids, amino acids/proteins, and DNA), which promotes lymphocytes proliferation and activation. On the other hand, inhibition of the mitogen-activated protein kinases (MAPK) and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways results in complicated cellular mechanisms finished with suppression of pro-inflammatory genes, and thus blocks the expression of pro-inflammatory cytokines. In addition, the antioxidant activity also reduces the release of arachidonic acid, which results in the oxidation of membrane phospholipids, and thus reduces its metabolites (leukotrienes and prostaglandins) that are considered as important inflammatory mediators [34,49].

On the other hand, studies showed that antioxidants could modulate the signal transduction pathways crucial to cellular responses including inflammation, survival, cellular proliferation, and death, that are affected by oxidative stress [50,51]. For example, the nuclear factor-erythroid-2-related factor-2 (Nrf2) can be modulated by antioxidants, which results in the activation of some Nrf2 target gene candidates (e.g., Nrf2, SLC48A1, SLC7A11, p62, HO-1, and Bcl-2 genes) that control antioxidant defense and autophagy [52].

Furthermore, inhibition of phosphodiesterases (PDE), which can result from antioxidant activity, promotes the intracellular cyclic AMP (cAMP) second messenger system. Therefore, activation of cAMP response element-binding protein (CREB) targets genes and the AMP-activated protein kinase (AMPK) pathway, which is the key regulator of autophagy and is also involved in the regulation of Nrf2 pathway [52].

Altogether, the potential immune booster activities of antioxidants from honey are not only limited to inducing lymphocytes proliferation and activation and inhibiting the production of pro-inflammatory cytokines, it also can induce autophagy machinery. Thus, promoting these three immune responses could help to fight against COVID-19.

The Antiviral Activity of Honey

Although the antimicrobial activities of honey have been well studied against many bacteria and fungi [53,54], its antiviral activities still need an extensive exploration so that it can be used as prevention and treatment of viral infections.

In 1996, Zeina et al. suggested that honey has antiviral activity against the Rubella virus in infected monkey kidney cells (Vero cells) in vitro [55]. After four days of incubation, 1 mL of honey (at a range of concentrations from 1:1 to 1:1000) was enough to kill 1 mL of the virus in the culture in all concentrations (10 to 109 virus/mL) without causing any cytotoxicity against the cells themselves [55].

At a concentration of 500 µg/mL, honey showed highest antiviral activity against HSV in vitro with a decrease in viral load at a concentration of 100 µg/mL [27]. Furthermore, honey is shown to be effective upon the topical use to treat recurrent skin lesions caused by HSV [56]. In addition, the antiviral activity of ‘Manuka’ (from mānuka tree flowers) and clover honeys against VZV has been reported in vitro [28].

Another study has shown that honeys including Manuka, Soba, Kanro, Acacia, and Renge have antiviral effects, and Manuka honey is the most potent antiviral candidate against influenza virus A/WSN/33 (H1N1) in the cultured Madin–Darby canine kidney (MDCK) cell line [29]. Moreover, extracts of honey, garlic, and ginger (HGG) mixture showed antiviral activity against influenza A virus isolates and is comparable to the standard antiviral drug Amantadine [57].

This in vitro study showed that HGG inhibited H1N2 replication in human peripheral blood mononuclear cells (PBMCs) and promoted cellular proliferation [57].
Previous reports on patients infected with HIV showed that consumption of honey helps to boost their immunity through the increase of lymphocytes proliferation, and generally improves their haematological and biochemical status (e.g., erythrocytes, haemoglobin, platelets, neutrophils, copper, and proteins levels) [58,59,60].

Meanwhile, another study also showed that consumption of honey in HIV positive subjects not only increases CD4 T lymphocytes counts but also decreases the viral load [61]. Other in vitro research on Manuka honey was carried out by Zareie, who examined its antiviral activity against RSV [31]. The inhibition and neutralization experiments showed a significant inhibitory effect on the progression of infection by honey through the inhibition of viral replication and the mRNA copy numbers of two viral genes [31].

It is reported that methylglyoxal, a compound in honey, serves as an antiviral agent for HIV [62,63]. Methylglyoxal affects the late stage of infection of HIV, where it blocks virion assembly and maturation [63].
It is known that the advanced glycation end-products (AGEs) of DNA and protein give rise to a major cell-permeant precursor “methylglyoxal”.

Methylglyoxal reacts with free amino groups of lysine and arginine and with thiol groups of cysteine, forming AGEs [64]. Thus, higher levels of methylglyoxal were associated with dysfunctioning glyoxalase system, the most important pathway for the detoxification of methylglyoxal, which is related to various diseases including diabetes, cardiovascular disorders, cancer, and central nervous system problems [65].

Recently, studies have shown that the methylglyoxal content of honey is responsible for much of the honey’s antimicrobial properties. It was proven that methylglyoxal effectively inhibited the growth of gram-positive and gram-negative bacteria.

These inhibitory effects were well-discovered, and it started when methylglyoxal levels reached 0.3 mM in media, causing alterations in the structure of bacterial fimbriae and flagella, which would limit bacteria adherence and motility [66]. However, there is no information precisely describing the mechanisms of activity for methylglyoxal against viruses. In the next section, the potential mechanisms of antiviral properties of honey are further discussed.

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