A crazy family of look alike compounds with different targets
Tea tree oil is the volatile oil obtained by distillation from the leaves and terminal branchlets of the narrow-leaf tea tree Melaleuca alternifolia, a native plant of the northern coast of Australia.  Different chemotypes were discussed in this review. True tea tree oil seems to be defined by the International Standards Organization Iso 4730 list of acceptable The featured image contains structures and data of terpenes in Iso 4730. Four of the top five on the list are derivatives of terpinene. #12 on this list, limonene, is structurally very similar to terpinolene. The data de Groot and coauthors led them to conclude that allergic reactions resulted from oxidation products of the oil.  The rest of this post will explore these closely related terpenes.
According to Poison Control, tea tree oil is very poisonous when swallowed. Tea tree oil may even cause neurological symptoms when absorbed through the skin. The post features research studies of components of tea tree oil given orally to rodents.
α-Terpinene, voltage gated Ca2+ channels or adenosine A2A?
A study out of Brazil examined the ability of Dysphania ambrosioides essential oil to relax the rat trachea. α-Terpinene was determined to be a major constituent of the essential oil .  α-Terpinene did not affect basal tone but did induce relaxation in response to high KCl and 5-HT, aka serotonin. The authors speculated that the target was voltage gated calcium channels.  Being similar to adenosine A2A ligand limonene, another explanation is presented. Limonene modulation of adenosine A2A is covered on the G-protein coupled receptor GPCR page. The core of this image was posted to this link on research gate with modifications from Cardiovascular Physiology Concepts.
The 5-HT/serotonin receptor fires via Gq subunits that serve to increase intracellular Ca2+. Ca2+ binds to calmodulin (CaM) that regulates myosin light chain kinase (MLCK). MLCK phosphorylates the myosin light chain. This phosphorylation activates ATPAse activity that causes movement of the myosin head upon actin: contraction. Agonist binding to the adenosine A2A receptor increases cAMP, that activates Protein kinase A that phosphorylates and inhibits MLCK. More work is needed to prove that α-terpinene is acting on the adenosine A2A receptor.
γ-Terpinene, multiple GPCR targets or just CB1/2?
This particular study also came out of Brazil. Mice were orally given γ-TPN orally at 6.25, 12.5, and 25 mg/kg.  This comes out to be 46 μM for the 6.25 mg/kg dose assuming the mouse has the density of water. This dosage was tested for analgesia in response to injecting the right hind paw with formaldehyde. Pain was measured by the time the mice spent paw licking. The γ-TPN doses were doubled for capsaicin injections and halved for glutamate.  The 3.25 mg/ kg oral dose was effective in reducing glutamate induced paw pain.  This dose was chosen for further study to evaluate the mechanism of action.
- Opioid receptor antagonist Naloxone inhibited morphine and γ-TPN analgesia.
- ATP gated K+ channel blocker Glibenclamide blocked γ-TPN analgesia. The authors pointed out that central opioid analgesia pathways impacting the dorsal horn of the spinal cord involves voltage gated KATP channel activity.
- Muscarinic acetylcholine receptor antagonist Atropine blocked muscarinic receptor agonist Philocarpine analgesia. 
- Nicotinic acetylcholine antagonist Mecamylamine blocked both nicotine and γ-TPN analgesia. 
Three of the four pharmaceuticals operate on G-protein coupled receptors. We have some evidence that γ-TPN is a ligand for CB1 as well.  A quick search of PubMed reveals that CB1 heterodimerizes with with the delta-opioid receptor, In the periaquiductal gray area, mucarinic receptor signalling modulates endocannabinoid pathway production. There appears to be CB2/nicotinic receptors cross talk presented in the literature.
α-Terpineol, dopamine d2 and CB1/2 receptors
This particular mouse study also came out of Brazil. The authors were interested in a natural treatment for depression in humans. They argued that since neuro inflammation might be a source of depression in humans, they could create depression in their mouse model by dosing with the Gram negative bacterial toxin lipopolysaccharide (LPS). They used two doses of oral α-terpineol, 100 and 200 mg/kg, before and after the LPS.  With an atomic mass of 154.25 gmol-1, this is about 65 and 130 mM. Two tests were used to measure depression and lack of motivation in mice. Mice were tapped to the wall by their tails. How much time did they spend immobile? Mice were sprayed with a sucrose solution. How much time did they spend grooming? For inhibitor tests, mice were treated with inhibitors and α-terpineol before treatment with LPS. In these tests α-terpineol decreased the time before movement when the mouse was hung upside down by its tail and increased the time it spent licking sucrose off its fur and brought these parameters back to those of mice not treated with LPS.  Down arrows indicate a statically significant reversal of the α-terpineol improvement of the LPS deficit.
|Haloperidol||nonselective dopaminergic receptor antagonist||↓ p<0.01||No effect|
|Sulpiride||selective dopamine D2 receptor antagonist||↓ p<0.01||No effect|
|Propranolol||β-adrenoceptor antagonist||No effect||No effect|
|Caffeine||nonselective adenosine receptor antagonist||No effect||No effect|
|AM281||selective CB1 receptor antagonist / inverse agonist||↓ p<0.01||No effect|
|AM630||selective inverse agonist for the CB2 receptor||↓ p<0.01||No effect|
The authors didn’t really discuss whether movement when hung upside down depends on reflexes or the emotional status of the mouse. The role of CB2 and the immune response to LPS was also not discussed. This group did offer some brilliant insight into structural similarities between α-terpineol and some CB1/2 ligands.
Let’s look to Pharma Education for some definitions. A partial agonist binds to and activates a receptor but to a lesser extent than a full agonist. An inverse agonist binds to the receptor and creates effects opposite that of the agonist. Note that these synthetic ligands also have a 3-5 carbon alkyl chain that resembles that of endocannabinoid AEA. Brilliant though this observation might be, it seems unreasonable that terpineol might be a full agonist of CB1 or CB2. Note that phyto and synthetic cannabinioids share the 3-5 carbon alkyl chain of AEA. Given the similarity in structures with synthetic cannabinoids distal from the alkyl group shared by endocannabinoids, could terpiniol be a positive allosteric modulator of endo cannabinoids? Recent work regarding α-terpiniol in a CB1-/- and a CB2-/- knock out mouse pain model also suggests that α-terpeineol may modulate CB1. 
Terpinen-4-ol and GABAA
This study comes out of João Pessoa, PB, Brazil. The motivation came from a a desire for a better treatment for epilepsy.  This study took two approaches to inducing epilepsy by inhibiting the action of gamma-amino butyric acid, or GABA. PTZ is a direct GABA antagonist. The authors chemically inhibited the production of GABA in the brains of the mice.  GABA works by opening chloride channels that hyperpolarize the post synaptic neuron. This hyperpolarization increases the treshhold for action potentials that result in seizures. See the GABAA page for details. Diazapam is a positive allosteric modulator of GABAA, it makes small amounts of GABA go further in opening the GABAA Cl– channel. This study addressed the hypothesis that a major comment in tea tree oil could also positively modulate GABAA Cl– channels.
- Mice were divided into 6 groups (n = 8 per group).
- The negative control received 5% Tween 80, aka the vehicle for dissolving drugs.
- The positive control received 4 mg/kg diazepam (DZP). DZP is a positive allosteric modulator of GABAA.
- Treatment groups received intraperitoneally (i.p.) injections with terpinen-4-ol 4TRP (25, 50, 75, or 150 mg/kg). Figure 2 of  Lower doses showed a lot of variability in increasing time to a seizure. The 200 mg/kg showed the same latency time as 4mg/kg diazepam, a positive allosteric modulator of GABAA.
- After 30 minutes the mice were injected with seizure inducing pentylenetetrazol PTZ (60 mg/kg, i.p.) and observed for at least 15 min and watched for forelimb clonus.
- Seizure activity was also induced with mercaptopropionic acid that inhibits the production oaf γ-aminobutyric acid.
- EEG recordings and intracerebral injections of drugs were performed
- The dorsal root ganglion (DRG) were collected and cultured for patch clamp recordings. Not shown in this post, the amplitude of depolarizing currents was decreased. 
That FLU inhibited DZP but not terpinen-4-ol should not be interpreted as evidence that 4TRP does not act on GABAA, just not at the site shared by FLU and DZP. The use of two different protocols to inhibit GABAA adds strength to this argument. See the  link for more details of this study.
TrpA1, study edited for terpinene family from tea tree oil
Monarda fistulosa, or the wild bergamot is a source of medicinal essential oil used by natives of North America.  Isolates of this essential oil were tested individually on TrpV1 expressed in HEK293 cells.  Intracellular Ca2+ was measured with a fluorescent indicator dye. TrpA1 is the Trp family member with reactive thiols and affinity for noxious compounds.
Carvacrol is very similar to the terpinene family presented on this post. Carvacrol increases the Ca2+ influx into the HEK cells almost 6x compared to the non-transfected cells (NT). It would appear that having a hydroxyl group is needed for the terpinene family member to have a 2-3x increase in [Ca2+]i change in this model.
Four closely related compounds, four different targets? The most exciting entry in this post may be the Vieira et al  insight that α-terpineol shares structural homology with phyto and synthetic cannabinoids.  Tea tree oil defining terpene terpinen-4-ol, with its unusually placed hydroxyl group, seems to be a GABAA agonist.  What about α- and γ-terpinenes? [2,3] Could they be interacting with adenosine A2A or a number of other G protein coupled receptors that include CB1/2? That many isolated tea tree oil compounds can be successfully given orally to rodents for a therapeutic action brings us to the Paracelsus notion that “Dose makes the poison.”
- de Groot AC, Schmidt E. Tea tree oil: contact allergy and chemical composition. Contact Dermatitis. 2016 Sep;75(3):129-43. free article
- Leal-Cardoso JH, Barbosa R. Myorelaxant action of the Dysphania ambrosioides (L.) Mosyakin & Clemants essential oil and its major constituent α-terpinene in isolated rat trachea. Food Chem. 2020 May 1;325:126923 PMC free article
- Passos, F. F., Lopes, E. M., de Araújo, J. M., de Sousa, D. P., Veras, L. M., Leite, J. R., & Almeida, F. R. (2015). Involvement of Cholinergic and Opioid System in γ-Terpinene-Mediated Antinociception. Evidence-based complementary and alternative medicine : eCAM, 2015, 829414. PMC free article
- Vieira, G., Cavalli, J., Gonçalves, E., Braga, S., Ferreira, R. S., Santos, A., Cola, M., Raposo, N., Capasso, R., & Dutra, R. C. (2020). Antidepressant-Like Effect of Terpineol in an Inflammatory Model of Depression: Involvement of the Cannabinoid System and D2 Dopamine Receptor. Biomolecules, 10(5), 792. PMC free article
- Nóbrega, F. F., Salvadori, M. G., Masson, C. J., Mello, C. F., Nascimento, T. S., Leal-Cardoso, J. H., de Sousa, D. P., & Almeida, R. N. (2014). Monoterpenoid terpinen-4-ol exhibits anticonvulsant activity in behavioural and electrophysiological studies. Oxidative medicine and cellular longevity, 2014, 703848. PMC free article
- Bilbrey JA, Ortiz YT, Felix JS, McMahon LR, Wilkerson JL. Evaluation of the terpenes β-caryophyllene, α-terpineol, and γ-terpinene in the mouse chronic constriction injury model of neuropathic pain: possible cannabinoid receptor involvement. Psychopharmacology (Berl). 2021 Nov 30.
- Ghosh, M., Schepetkin, I. A., Özek, G., Özek, T., Khlebnikov, A. I., Damron, D. S., & Quinn, M. T. (2020). Essential Oils from Monarda fistulosa: Chemical Composition and Activation of Transient Receptor Potential A1 (TRPA1) Channels. Molecules (Basel, Switzerland), 25(21), 4873. PMC free article
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