Pulegol and Curcumol

Curcumol is isolated from roots of plants of the family Zingiberaceaegenus {1] Within that family is the G genus Curcumus. North Americans are familiar with this as the common turmeric. Wei and coworkers have published an excellent review on the potential of curcumol to treat various aspects of the cancer process. [1] Isopulegol is the alcohol form of the ketone (+)pulegone, a precursor in the synthesis of (-) menthol.. Isopulegol is a fully tested food and fragrance additive. [2] The only reason why these two apparently unrelated compounds are sharing space on the same post is a report that curcumol and menthol share a common binding site and method of modulating the α1β2γ2 version of GABAA. [3]

This TrpM8 agonist is also a GABAA agonist in cultured neurons [4] The Xu group of Shanghai China published a report that menthol and related compounds are also agonists of the GABAA receptor in cultured neurons from the CA1 region of the rat hippocampus. [4] Shown is a colorized version of of Figure 6 (Xu 2008) comparing 300 µM menthol related compounds on Cl currents evoked by 1µM GABA. [4] The structure images from this publication have been replaced by those from PubChem. Data for JE207 and “menthyl chloride” have been edited out because PubMed lists a different structure for “menthyl chloride” and does not list JE207 at all. These two compounds also failed to potentiate GABAA currents.

Modified from Figure 6 ref [4] ** p<0.01 compared to the control.

As a control, TrpM8 was found not to be expressed in these cultured neurons. [4] Menthol does not act on glutamate receptors. [4] these authors also investigated menthol as a treatment in a mouse model of epilepsy for the interested reader. [4] In another study published in 2008 Menthol and related compounds were investigated for modulation of recombinant human GABAA α1β2γ2S receptor currents expressed in Xenopus oocytes. [5] Sub-maximal EC20 GABA currents were enhanced by 3-300 μM (+)-menthol. The authors found a 2x increase in current by 50 μM menthol. menthol > isopulegol > isomenthol> alpha-terpineol >> cyclohexanol. [5] Flumazenil (a benzodiazepine binding site on GABAA antagonist) did not inhibit menthol enhancements. [5]

The Villmann Laboratory has a long history of studying the affects of terpenes from essential oils on GABAA receptors. In a 2014 publication they examined many terpenes in a medicinal plant,Sideritis, that is popular in Mediterranean countries. [6] Function of various isoforms of GABAA was measured by whole cell patch clamping of Xenopus oocytes. [6]

From reerence [6]

Note that carvacrol is better known as a ligand for the TrpA1 and TrpV3 Ca2+ selective cation channels according to PubChem. The these authors used enough GABA to obtain 5-10% of the maximal response. This was considered the best way to detect a positive allosteric modulator. [6] All experiments were carried out at room temperature (∼22oC). Details may be found in the publication.

More realistic concentrations and a human cell line from the Villman laboratory

A fluorescent membrane potential sensing blue dye (FLIPR Membrane Potential Blue) detected changes in membrane potential. The dye and the modulatory substances were added directly to the plates containing the HEK293 cells transfected with GABAA receptors. Recall that Cl currents through GABAA hyperpolarize the neuron and the HEK293 cell expressing functional GABAA.

The data are convincing that isopulegol is doing something, Changes in membrane fluidity at room temperature versus 37oC seems to be a seldom addressed parameter..


Why curcuminol? When viewed in PubChem 2D form, it’s structure is nothing like menthol’s look alike isopugelol. Like many pharmaceutical scientists, Liu et al were looking for alternatives to the benzodiazepine class of GABAA positive allosteric modulators. [3] Curcumol is a natural compound isolated from Rhizoma Curcumae oil. Extracts from this plant have been used a food condiment and in traditional Chinese medicine for thousands of years. [3] Of the three main ingredients in Rhizoma Curcumae oil contains curcumol, curcumin, and curdione. Curcumol is lipophilic and readily crosses the blood–brain barrier according to the authors’ review of the literature. [3] The authors also cited a study demonstrated oral bioavailability and ability to cross the blood brain barrier. [7] Patch clamp current traces in the publication will not be presented in this post. Additional structural information from rcsb.org and uniprot.org will be presented instead.

Methods in brief:

  • Primary cultures of mouse hippocampal were isolated from the brains of 15-day-old embryonic C57BL/6 J mice [3]
  • Rat subunits of GABAA α1β2γ2 were expressed in HEK293T cells. Site directed mutagenesis was used to modify the rat α5 sub unit. [3]
  • green fluorescent protein, part of the γ subunit was used to establish successful transfection for whole cell patch clamping studies that were performed at room temperature (23 ± 2 °C).

These are some highlights of figures 1 and 2. In panel 1c we see that 50 μM shifts the GABA dose response to the left. Curcumol increases the current from only 1 μM GABA by about 2x. Only moderate effects were seen with 100μM. This post will not consider an unrealistic concentration of 3 mM menthol. Figure 4 explored the use of flumazenil, an antagonist that binds to the diazepam site on the γ2 sub unit. [3]

Moving to HEK293 cells and site directed mutagenesis

Site directed mutagenesis of the β sub unit and α sub unit family member swapping were used to establish a site of interaction.

Mutagenesis of the β subunit

Two important sites on the β subunit for mutagenesis. Methionine 2886 resides in the second transmembrane domain (M2) that forms the wall of the channel. Tyrosine 444 is part of the cytoplasmic domain.

Data from reference [3] The GABAA channel image is from Research Gate **p< 0.01,* p<0.05 compared to GABA alone.

Both the Methione to tryptophan mutation at position 286 of the M2 region and the Tyrosine to trypophan in the cytoplasmic domain abolished menthol’s and curcumol’s ability to act as a GABA positive allosteric modulator. While the M3 to M4 loop is connects transmembrane domains that interact with the channel cavity M2 helix, this loop may have other functions.

α sub unit swapping

The protein structure database rcsb.org was visited to obtain a closeup of propofol binding to the interface between the β2 sub unit and α1 sub units. A similar structure could not be found for propofol and tonic α5 sub unit exoressubg GABAA.

The graphs were obtained from reference [3] The molecular images were obtained from rcsb.org as noted. The two images were screen captures with and without the backbone of the molecule. A “3d” image of propofol was obtained from PubChem and rotated in about the same orientation as the propofol in the structure.

Note that mutation of the the cytoplasmic domain of the β sub unit has no influence on how the ability of menthol to enhance GABA currents when α1 is swapped for α5. M286 of the β sub unit remains critical. While parts of the 3D structure of curcumol resemble those of isopulegol and menthol, the former is much larger. Perhaps he cytoplasmic loop is necessary to hold the molecule in place.

Ruling out the γ sub unit

It is starting to seem like any terpene can bind to any any allosteric modulatory site on GABAA. One last experiment on the infamous diazepam site to shows that this is not the case. [3]

The dashed lines indicate hydrogen bonds in the diazepam binding site.

Realistic concentrations in whole animals?

  • The toxicology study testing isopulegol as a food additive used a rodent dose of 0.03 mg/kg/day in one arm. [2]
  • While brain slices are hardly whole animals, menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, [7] This study used TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol potentiated gaboxadol (10 μM) currents. [7]
  • An epilepsy study used an i.p. isopulegol dose of 200 mg/kg (129.64 mM/kg) [8]
  • A mouse behavioral study used pulegone (100 or 200 mg/kg, i.p.) and menthol (100 or 200 mg/kg, i.p.) [9]

Tying things together … or not

the The two β sub unit mutations corresponding to mutations that abolish the structurally similar propofol binding also abolish curcumol modulation of GABAA. Propofol is an analgesic. Perhaps curcumol can also act as an analgesic? But wait! PubMed lists 137 entries for curcumol. Most of these publications focus on enzymes involved in carcinogenesis. PubMed has only 86 entries for pulegol. Most of these publications concern isopulegol’s use as a food and cosmetic additive. Eating a diet rich in turmeric root or smoking a cannabis strain/chemovar rich in isopulegol or menthol may be only a small part of a bigger part of exogenous agents that modulate GABAA.


  1. Wei, W., Rasul, A., Sadiqa, A., Sarfraz, I., Hussain, G., Nageen, B., Liu, X., Watanabe, N., Selamoglu, Z., Ali, M., Li, X., & Li, J. (2019). Curcumol: From Plant Roots to Cancer Roots. International journal of biological sciences, 15(8), 1600–1609. PMC free article
  2. Api AM, Belsito D, Bhatia S, Bruze M, Calow P, Dagli ML, Dekant W, Fryer AD, Kromidas L, La Cava S, Lalko JF, Lapczynski A, Liebler DC, Penning TM, Politano VT, Ritacco G, Salvito D, Schultz TW, Shen J, Sipes IG, Wall B, Wilcox DK.(2016) RIFM fragrance ingredient safety assessment, Isopulegol, CAS Registry Number 89-79-2. Food Chem Toxicol. 2016 Nov;97S:S129-S135.
  3. Liu, Y. M., Fan, H. R., Ding, J., Huang, C., Deng, S., Zhu, T., Xu, T. L., Ge, W. H., Li, W. G., & Li, F. (2017). Curcumol allosterically modulates GABA(A) receptors in a manner distinct from benzodiazepines. Scientific reports, 7, 46654. PMC free article
  4. Zhang, X. B., Jiang, P., Gong, N., Hu, X. L., Fei, D., Xiong, Z. Q., Xu, L., & Xu, T. L. (2008). A-type GABA receptor as a central target of TRPM8 agonist menthol. PloS one, 3(10), e3386. PMC free article
  5. Watt EE, Betts BA, Kotey FO, Humbert DJ, Griffith TN, Kelly EW, Veneskey KC, Gill N, Rowan KC, Jenkins A, Hall AC. Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol. Eur J Pharmacol. 2008
  6. Kessler, A., Sahin-Nadeem, H., Lummis, S. C., Weigel, I., Pischetsrieder, M., Buettner, A., & Villmann, C. (2014). GABA(A) receptor modulation by terpenoids from Sideritis extracts. Molecular nutrition & food research, 58(4), 851–862. PMC free article
  7. Lau, B. K., Karim, S., Goodchild, A. K., Vaughan, C. W., & Drew, G. M. (2014). Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons. British journal of pharmacology, 171(11), 2803–2813. PMC free article
  8. Silva MI, Silva MA, de Aquino Neto MR, Moura BA, de Sousa HL, de Lavor EP, de Vasconcelos PF, Macêdo DS, de Sousa DP, Vasconcelos SM, de Sousa FC. Effects of isopulegol on pentylenetetrazol-induced convulsions in mice: possible involvement of GABAergic system and antioxidant activity. Fitoterapia. 2009 Dec;80(8):506-13. free article
  9. da Silveira NS, de Oliveira-Silva GL, Lamanes Bde F, Prado LC, Bispo-da-Silva LB. The aversive, anxiolytic-like, and verapamil-sensitive psychostimulant effects of pulegone. Biol Pharm Bull. 2014;37(5):771-8. free article

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