Rhododendron is one of the largest genera of vascular plants and covers most of the Northern hemisphere. The first written reference to the species dates as far as 401 B.C. recording the toxicity of rhododendron honey. Plants of the genus are still known to cause intoxications, mainly due to consumption of contaminated honey. Despite their toxicity, rhododendrons have been used in ancient medical systems such as traditional Chinese and Ayurvedic medicine and also in European and North American folk medicine. The genus provided a large number of chemical compounds of which some indicated pharmacological activity (antimicrobial, antibacterial and antifungal activity). Toxicological studies of Rhododendron species addressed clinical data as well as the characterization of its toxic constituent, the grayanotoxin.
The genus Rhododendron: an ethnopharmacological and toxicological review
Grayanotoxin
Grayanotoxins , also known as andromedotoxins, acetylandromedol and rhodotoxin, represent a family of toxins that can be derived from the flowers, leaves and twigs of plants belonging to the genera of the Ericaceae family (e.g. Rhododendron, Pieris, Agarista and Kalmia genera) and that have been demonstrated to be of particular relevance in some reported clinical cases.
More than 25 isoforms have been isolated from Rhododendron but three members of this large family seem to play a fundamental role in the poisoning processes that may derive from the exposure.
The chemical structure of the grayanotoxin has been fully elucidated as diterpene, which is a polyhydroxylated cyclic hydrocarbon with a 5/7/6/5 ring structure that does not contain nitrogen.
The differences between the three main isoforms of the toxin involve the functional groups of the molecules (R1, R2, R3), as seen in the table below.
| GRAYANOTOXIN | R 1 | R 2 | R 3 |
| Grayanotoxin I | -OH | -CH 3 | Acetyl |
| Grayanotoxin II | -CH 2 | -CH 2 | -H |
| Grayanotoxin III | -OH | -CH 3 | -H |
According to its molecular structure, each isoform shows different chemical and physical properties, although the mechanism of action and pharmacokinetics remain the same for all the different forms of the toxin.
Chemical and physical properties
| GRAYANOTOXIN I | GRAYANOTOXIN II | GRAYANOTOXIN III |
| Empirical formula | C 22 H 36 O 7 | C 20 H 32 O 5 | C 20 H 34 O 6 |
| Systematic name | (3β,6β,14R)-3,5,6,10,16-pentahydroxygrayanotax-14-yl acetate | (3β,6β,14R)-grayanotox-10(20)-ene-3,5,6,14,16-pentol | (3β,6β,14R)-grayanotoxane-3,5,6,10,14,16-hexol |
| Molecular weight | 412.58 | 352.47 | 370.48 |
| Color/form | Crystals from ethyl acetate | Columnar crystals | - |
| Melting point | 258-260 °C to 267-270 °C, depending on rate of heating | 199-200 °C | - |
| Boiling point (at 760 mmHg) | 549.3 °C | 522.9 °C | 552.6 °C |
| Flash point | 184.3 °C | 237.9 °C | 250.9 °C |
| Solubilities | Soluble in hot water, alcohol, acetic acid, hot chloroform; very slightly soluble in benzene, ether, petroleum ether | - | - |
| Toxicity data in mice (i.p. LD 50) | 1.31 mg/kg | 26.1 mg/kg | 0.84 mg/kg |
Grayanotoxin I
Grayanotoxin II
Grayanotoxin III
Mechanism of action
Grayanotoxin has the ability to bind to voltage-gated sodium channels (Na v) within the cell membranes, interfering with the transmission of the action potential by blocking them and causing a persistent activation. The binding unit is the group II receptor site, localized on a region of the channel which is involved in the voltage-dependent activation and inactivation gating mechanism.
In general, the alpha subunit protein consists of 24 interconnected membrane spanning alpha-helixes organized as four repeats of 6 alpha-helixes each (S1-S6) with the four S6 domains delimiting the pore region. In a mutation analysis of Na v 1.4, Maejima et al. demonstrated that all four S6 domains give an important contribution in grayanotoxin interaction.
Moreover, it has been verified that the binding site for the toxin most probably resides on the internal surface of the membrane, as the hydrophilic analog used in the tests (desacyl asebotoxin VII) was found to be active only when applied from the cytoplasmic side. The binding of the substance generally takes place in the channel’s open state and, since the target compounds normally prevent sodium channel inactivation, the grayanotoxin itself impede the process and the activation voltage of the modified Na v channels shifts to the direction of hyperpolarization. Excitable cells (nerve and muscle) are thus maintained in a depolarized and activated state during which entry of calcium into the cells may be facilitated.
The voltage-gated sodium channels of the neurons represent a prominent target of grayanotoxins. Injecting intracerebroventricularly a small dose equivalent to 50 mg of honey in anaesthetized albino rats causes marked bradycardia and respiratory depression, while a much larger amount of extract (5 g/kg) injected intraperitoneally is needed to obtain the same result; moreover, bradycardia did not occur after the administration of the toxin in bilaterally vagotomized rats. These observations might indicate the important role of the central nervous system in rhodotoxin pathophysiology compared to the peripheral nervous system and, regarding the latter, the central role played by vagal stimulation in the grayanotoxin-induced bradycardia. It was also verified that M2-subtype of the muscarinic receptors that mediate vagal stimulation in the myocardium are involved in the cardiotoxicity processes but not in the respiratory toxicity ones. Thus, it is possible to state that the sites of cardiac and respiratory actions of the toxin are within the CNS and that the bradycardia is mediated by vagal stimulation at the periphery.
Kimura et al. studied the effects of grayanotoxin I on domain 4 segment 6 (D4S6) of rat skeletal muscle sodium channel, revealing that rhodotoxin requires a favorable condition for hydrophobic and hydrogen bonding to the channel in order to exert its pharmacological action. The introduction of hydrophilic lysine into the sites which are critical for grayanotoxin binding is thought to disturb the hydrophobic microenvironment around these sites, leading to the loss of the toxicity effects.
A Rare Cause of Complete Atrioventricular Block and Accelerate Nodal Rhythm;
Mad Honey Poisoning
On site of action of grayanotoxin in domain 4 segment 6 of rat skeletal muscle sodium channel
Distinct Sites Regulating Grayanotoxin Binding and Unbinding to D4S6 of Nav1.4 Sodium Channel as Revealed by Improved Estimation of Toxin Sensitivity
Geographical distribution of grayanotoxin
Toxic diterpenoids, such as rhodotoxin, have been found in the leaves of various species of Rhododendron, Leucothoe and Ericaceae but also in a number of products originating from the members of these families, such as honey, Labrador tea, cigarettes and a variety of decoctions used in alternative medicine. Grayanotoxins I and II have been found in the honey, leaves and flowers of Rhododendron ponticum and Rhododendron flavum in the eastern Black Sea area, while the first isoform has been identified in Rhododendron simsii from a case in Hong Kong. The II and III isoform of the toxin were found in the honey from Grouse Mountain (British Columbia, Canada), causing a similar type of poisoning.
Secondary products derived from plants, such as honey, can contain chemical compounds that, depending on their concentration and application, can be considered medicinal or poisonous. The so-called “mad honey” is a grayanotoxin-containing honey that can cause dramatic effects when ingested: the best studied and most numerous cases of grayanotoxin toxicity known thus far are indeed those classified as mad honey disease. Most of the local beekeepers produce honey at a small scale, so that the final product can be obtained from a small area or even a single bee hive. This means that it can contain a considerable concentration of the toxin.
Most known cases of mad honey disease are restricted to the Black Sea region, but geographical spreading of the Turkish population and, above all, transport and export of locally produced honey from Turkey may result in cases in other regions. Some cases have been reported from other parts of the Turkey, but also from Germany, Austria, Switzerland, Nepal, Reunion Island and Korea. Grayanotoxin concentrations were reported also in samples of North-American honey.
In regions where production has been scaled up, the final product is often a mixture of honey produced at different locations and this limits the chance for severe grayanotoxin contamination by dilution. Some beekeepers produce mad honey purposely for its supposed therapeutic effects.
Poisoning
The toxic effects of mad honey poisoning are rarely fatal and generally last for no more than 24 hours, showing dose-related symptoms that occur after a latent period of a few minutes to two or more hours. Yilmaz et al. reported that the quantity of honey that causes poisoning is generally between 5 and 30 g and patients often regain consciousness and feel better within hours, as grayanotoxins are metabolized and excreted rapidly; the heart rate and the blood pressure usually return to normal within 2-9 hours.
At this point no readily available testing of blood or urine exists for this toxin and contaminated honey may possibly be submitted to health departments for analysis. Identifying the cause of these symptoms relies upon taking a through medical history from the patient and a conclusive diagnosis can be reached after analyzing the pollen in the honey.
Range of toxicity
In various cases, 50 to 75 mL of contaminated honey have caused toxicity because of a particular grayanotoxin isoform present in the nectar of selected plants of the Ericaceae family.
Honey poisoning occurred in 66 adults following ingestion of 5 to 20 g (mean 13.45 ± 5.39 g) of honey produced from Rhododendron ponticum .
Intoxication also occurred in eight adults who had ingested 20 to 150 g of “mad honey” and in a 76-years-old man who ingested 10 azalea ( Rhododendron mucronulatum ) blossoms.
Mad honey intoxication: A case series of 21 patients
Clinical effects
Major clinical effects are burning of the mouth, perioral numbness and tingling, nausea, vomiting, diaphoresis, diarrhea, bradydysrhythmias, coma, altered mental state and seizures. In over 90% of the reported cases, significant hypotension (systolic blood pressure 70 mmHg) and bradycardia (pulse rate of 48 beats/minute) are present, while in about 70% of exposures the poisoning leads to diaphoresis, dizziness and altered mental status. Syncope has occurred in about 30% of cases.
After the ingestion of contaminated honey, onset occur within 30 to 120 minutes and the worst signs and symptoms generally last no more than 24-48 hours. Deaths have been recorded in the past, but with supportive care and management of hypotension and dysrhythmias prognosis is excellent and no reports of deaths have occurred in modern medical literature.
Transient blindness and blurred vision have been reported by heent examination.
The cardiovascular symptoms of the poisoning include hypotension and bradycardia when grayanotoxin is ingested in low doses; highest doses have caused various A-V conduction disturbances (mainly second and third degree AV block), syncope and asystole due to vagal stimulation. Asystole in particular was reported in one adult, after ingesting a few spoonfuls of “mad honey” obtained from the Rhododendron species. A non-ST segment elevation myocardial infarction was reported in an adult with no risk factors for cardiac disease, following mad honey ingestion from Rhododendron ponticum.
Grayanotoxins may also cause generalized muscle weakness, circumoral and extremity paresthesias, confusion, ataxia and seizures, as seen from a neurologic examination, and nausea, vomiting and excessive salivation from the gastrointestinal one.
Treatment
Four treatment appear to be effective after oral or parenteral exposure:
- Activated charcoal: it has to be administer as a slurry (240 mL water/30 g charcoal). The usual dose is from 25 to 100 g in adults and adolescents, 25 to 50 g in children from 1 to 12 years and 1 g/kg in infants who are less than 1 year old.
- Intravenous fluids: they are used to treat the initial hypotension. Pressors are rarely indicated but should be considered for profound hypotension that is unresponsive to adequate fluid challenge.
- Atropine: it is used for bradycardia. The adult dose consists of 0.5 mg injected intravenously and the injection may have to be repeated every 3 to 5 min. The maximum dose is 3 mg. The pediatric dose is 0.02 mg/kg injected intravenously or intraosseously or from 0.04 to 0.06 mg/kg for endotracheal administration and, if needed, it can be repeated once. For children, the maximum single dose and total dose are respectively 0.5 mg and 1 mg; for adolescent, they are 1 mg and 2 mg.
- Pacemaker: it can be considered if bradycardia is unresponsive to atropine.
Early gastrointestinal decontamination may be required for significant ingestions of plants parts of the Ericaceae family.
Case report
An 87-year old woman was admitted to the emergency department of Zonguldak (Turkey) after an episode of syncope. Physical examination revealed a depressed mental status, sever hypotension (60/30 mmHg) and bradycardia with 48 beats per minute. Electrocardiography showed sinus bradycardia with left bundle branch bloc and an extremely long QTc interval (810 ms, fig. 3), while cardiac and respiratory examinations and laboratory tests (blood count, cardiac markers and biochemical analyses) were normal.
The patient was immediately given intravenous fluid and dopamine. It was learned that, while she had no previous history of cardiac disease and was not taking drugs, she had been consuming a tablespoon of mad honey every morning for the last 3 months.
After the intravenous administration of 1 mg of atropine, heart rate and blood pressure improved dramatically and the course of the QTc interval revealed a significant decline (510 ms) on the next ECG. During the 48-hour monitoring, vital signs were stable and there were no heart rhythm problems. The patient did not require temporary pacing or any further treatment.
Transthoracic echocardiography showed an injection fraction of 25% with mild to moderate aortic insufficiency and mild mitral insufficiency. Based on the echocardiographic findings, left bundle branch block was considered to be chronic.
The patient underwent follow-up in the outpatient clinic after discharge and at the follow-up examination 3 weeks later she reported that she had not consumed “mad honey” during this period. No complaints were noted and the recovery electrocardiogram at post hospital period revealed QTc interval declining significantly to a value of 470 ms.
Extreme QT interval prolongation caused by mad honey consumption
Conclusions
Cases of honey intoxication should be anticipated everywhere. Some may be ascribed to an increased consumption of imported honey. Others may result from the ingestion of unprocessed honey with the increased desire of natural foods in the diet of Western Countries populations. This research for natural unprocessed foods, may result in more cases of grayanotoxin poisoning. Individuals who obtain honey from farmers who may have only a few hives are at increased risk. The pooling of massive quantities of honey during commercial processing generally dilutes any toxic substance. This is an example of the fact that not always the industrialization of the manufacturing process of foods means lower quality or healthiness.
Bad Bug Book - Grayanotoxin
Grayanotoxin Poisoning: ‘Mad Honey Disease’ and Beyond
Human health effects of Grayanotoxin I
Poisoning by mad honey: A brief review
