3-carene

The featured study comes primarily from the Korean Institute of Science and Technology. Woo et al tell us that 3-carene is a component of pine essential oil. [1] The American College of Healthcare Science lists 3-carene as a component of many pine family extracts that also includes products such as juniper berry essential oil. Askgrowers.com lists strains high in 3-carene. This site even mentions 3-carene as a treatment for insomnia. Strawberry Cough is on of many strains mentioned as being a source of 3-carene.

These authors were looking for a more natural treatment of insomnia. The authors wished to improve upon pharmaceutical targets of the GABAA receptor such as ZPD diazepam and zolpidem [1]. In doing so they wished to avoid deleterious side effects such as cognitive impairment, tolerance, headache, nausea, and rebound insomnia [1]

The protocol

This time line is a combination of Yoo et al Figures 2A and 3A. Figure 2 worked out the 3-carene dose response. Figure 3 demonstrated the optimum dose plus or minus the GABAA antagonist Flu.

This figure is a combinationof panels 2A and 3A from reference [1]. The first protocol, figure 2, was to establish the dose. The second protocol, virtually the same as the first, was to establish the role of GABAA receptors by treatment with the antagonist Flu, Flumazenil.

Both figures examined Sleep Latency and sleep duration. This post will provide the reader extra explanation of these two parameters. At first blush Phentobarbital appears to be a misspelling of phenobarbital, a GABAA agonist that has sedative properties and may be used to treat insomnia and anxiety. Phentobarbital is also a GABAA agonist used to treat insomnia.

This particular image of the GABAA receptor has been modified from one on Basic Medical Key. Note that the sleep aid Xolpidem and the antagonist Flumazenil share similar sites.

The top view of theGABAA receptor and is from reference [2] It shows major and minor rigid body conformational changes upon ligand binding. Predicted interaction sites of 3-carene are from reference [1] The image of the GABAA receptor from medicalkeys.com has been recolored to match the interaction residues from ref [1]
Sleep latency

Woo and coauthors acknowledged that this was only a preliminary study that lacked high powered measurements in brain activity. Nonetheless, sleep latency can be regarded as a measurement of how fast 3-carene interacts with the GABAA receptor.

From Woo et al [1]
sleep duration

In panel 2C Woo et al were establishing a dose. In panel 3C they showed that the increase in “sleep duration” was inhibited by the GABAA antagonist Flu. We just have to remember that this may not be true REM sleep. The rats are just too “zonked out” to right themselves when placed on their backs.

4. Electrophysiological measurement

Adult mice (7~9 weeks) were anesthetized until they stopped breathing. They were decapitated and their brains removed. The brains were placed in an artificial cerebral spinal fluid. Transverse sections (300 μm) containing hippocampus were prepared. To block the spontaneous excitatory post synaptic currents (EPSC), APV (50 μM) and CNQX (20 μM) were added into artificial CSF. Slightly different techniques were used for small inhibitory post synaptic currents (sIPSC) Inhibitory post synaptic potentials arise from currents that increase the amount of depolarization needed to achieve an action potential. CNQV blocks a non-NMDA ion channel that response to glutamate. APV is an antagonist of NMDA receptors (ion channels) for glutamate.Flumazenil (FLU) is an antagonist of GABAA-BZD receptors,

In panel 4A we have some representative traces of from control mouse brains and those that had been treated with 3-carene. Note that the downward dips have three characteristics

  1. number per unit time
  2. amplitude
  3. how long they last.

Panel 4B is an average of sIPDC from the control, 3-carene, and 3-carene with the GABAA inhibitior Flu.

Figures 4A-B from Woo et al [1] Images of exponential decay have been added for clarity

This particular image of exponential decay is for fluorescence intensity. I0 is the initial intensity. I is the intensity at time t. Tau, τ, is the decay constant. Exponential decay is a common theme in nature that can also describe the decay in hyperpolarization that is the same function flipped vertically. Yoo et al. found that the increase in τ significantly increased with 3-carene. This increase was blocked by the GABAA antagonist. 3-carene did nothing to change the frequency or amplidute of sIPSC.

How does this apply to flower power for sleep?

Woo et al presented convincing evidence that 3-carene has sedative properties, at least at it pertains to sleep. Another study of cannabis chemotypes associated with anxiolytic properties found that 3-carene content of the four most and four least effective chemotypes in relieving anxiety. 3-carene was correlated with more anxiety R = −1.000, p=0.046. [3] A simple explanation that 3-carene is inducing GABAA desensitization. A recent review covers methods of turning GABAA off. [4] Could these accessory proteins and the mechanics of turning GABAA off differ from one person to the next? A repeat of the Chemotype for Anxiety [3] study for sleep might be useful. Naturally these studies would be even more useful if they came with genetic tests for the user for personalized plant based medicine.

References

  1. Woo, J., Yang, H., Yoon, M., Gadhe, C. G., Pae, A. N., Cho, S., & Lee, C. J. (2019). 3-Carene, a Phytoncide from Pine Tree Has a Sleep-enhancing Effect by Targeting the GABAA-benzodiazepine Receptors. Experimental neurobiology, 28(5), 593–601. PMC free article
  2. Kim, J. J., Gharpure, A., Teng, J., Zhuang, Y., Howard, R. J., Zhu, S., Noviello, C. M., Walsh, R. M., Jr, Lindahl, E., & Hibbs, R. E. (2020). Shared structural mechanisms of general anaesthetics and benzodiazepines. Nature, 585(7824), 303–308. https://doi.org/10.1038/s41586-020-2654-5. PMS free article
  3. Kamal, B. S., Kamal, F., & Lantela, D. E. (2018). Cannabis and the Anxiety of Fragmentation-A Systems Approach for Finding an Anxiolytic Cannabis Chemotype. Frontiers in neuroscience, 12, 730. PMC free article
  4. Castellano, D., Shepard, R. D., & Lu, W. (2021). Looking for Novelty in an “Old” Receptor: Recent Advances Toward Our Understanding of GABAARs and Their Implications in Receptor Pharmacology. Frontiers in neuroscience, 14, 616298. PMC free paper
  5. Api AM, Belmonte F, Belsito D, Botelho D, Bruze M, Burton GA Jr, Buschmann J, Dagli ML, Date M, Dekant W, Deodhar C, Fryer AD, Gadhia S, Jones L, Joshi K, La Cava S, Lapczynski A, Lavelle M, Liebler DC, Na M, O’Brien D, Penning TM, Ritacco G, Romine J, Sadekar N, Salvito D, Schultz TW, Sipes IG, Sullivan G, Thakkar Y, Tokura Y, Tsang S. RIFM fragrance ingredient safety assessment, δ-3-carene, CAS Registry Number 13466-78-9. Food Chem Toxicol. 2018 Dec;122 Suppl 1:S771-S779. doi: 10.1016/j.fct.2018.11.049. Epub 2018 Nov 24. PMID: 30481572.

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