Frenchol

Frenchol is a primary terpene of basil and closely related to borneol. This is a journey that took some strange twists. Two groups of scientists were looking for neurological disease treatments. The paradoxical thing is that neither of the frenchol/fenchone targets are expressed in the brain in large amounts. They are both expressed in the gastrointestinal tract according to material from Proteinatlas.org.

Frenchol & frenchone: the enigma

A toxicology review did not have much to say about frenchol toxicology except that the information was very limited. Frenchol does have some structural homology with l-borneol and borneol acetate. [1] . This brief study gave a list of foods that contain frenchol. Ocimum (basil) species are a well recognized source of frenchol. Ginger, nutmeg, thyme, black pepper, and pistachio oil are also on the list. [1] It took a lot of digging but two extremely different targets were uncovered in the peer reviewed literature.

• Frenchol and many other terpenes were tested as activators of the transient receptor vanilloids (Trp) channel TRPA1. These channels respond to noxious compounds and pain.
• The second potential target is the free fatty acid receptor 2. This G protein coupled receptor is thought to be a player in the gut-brain access that is activated by short chain “fatty acids” like butyrate and propionate. FFAR2 is thought to be responsible for appetite suppression by short chain fatty acids. Alternate agonists of this receptor might bypass the need for the perfect intestinal microbiota.

TrpA1, a classical terpene target

A group out of Osaka Japan tested the hypothesis that frenchol (frenchyl alcohol) and frenchone would behave similarly to borneol in terms of activating/inactivating the nociceptor TrpA1 Ca²⁺ channel. [2] They were interested in this receptor’s role in inflammatory pain associated with cancer more so than its role in response to noxious environmental stimuli. [2] Human TRPA1 (the gene) was transfected into human embryonic kidney cells 293 (HEK293). Increases in intracellular Ca²⁺ were measured with the Ca²⁺ sensitive dye Fura2. [2] Whole cell patch clamping was also used to detect the flow of positive charges (Ca²⁺ and small amounts of Na⁺) though the over expressed TRPA1 (the protein) channels. Camphor is considered a blocker of TrpA1 while menthol and allyl isothocyanate (AITC) of Wasabe mustard are considered activators. [2].

Takaishi Fig  1-2 Borneol and frenchol inhibit AITC

Fura-2 is a small molecule that may become trapped inside a cell,  When excited with 340 nm light Frua-2’s  fluorescence at 510 nm increases when it binds Ca²⁺ The Ca²⁺ binding 510 nm fluorescence change is less at 380 nm.   Takaishi et al used the highly sensitive  510 nm Em _340ex /  510 nm Em _380ex to calculate the intracellular Ca²⁺ concentration [2]    Camphor is considered a blocker of TRPA1 while menthol and allyl isothocyanate (AITC) of Wasabe mustard are considered activators. [2].  Some of Figure 2 of the Takaishi paper requires the assumption that basal intracellular Ca²⁺ was subtracted from that of the addition of the agonist.  It would appear that camphor and borneol act on basal TRPA1 and/or other receptors that control intracellular Ca²⁺.  Note:  much of the other compounds tested have been edited out of Figure 1 to keep this post focused on borneol, fenchol, and fenchone. 

Whole cell patch clamping was also used to detect the flow of positive charges (Ca²⁺ and small amounts of Na⁺) though the over expressed TRPA1 (the protein) channels.    The membrane potential was clamped at -60 mV for all conditions.  The lines going up from the baseline are voltage ramp-pulses from -100 to +100 mV (500 ms) that were applied every 5 s.  The bigger the spike the greater the current.  Upward spikes are anions (-) flowing into the cell or cations (+) flowing out of the cell.  The huge dips upon adding AITC are Ca²⁺ ions flowing into the cell.  Smaller upward spikes upon adding the terpene could mean less opened TRPA1 channels that are over expressed in these HEK293T cells. [2]

Takaishi Fig 4: Borneol and frenchol inhibit two TRPA1 agonists

Excerpts from Figure 4.  Add 1 mM menthol and a downward trajectory is seen in the trace indicating an inward movement of positive charges.  Addition of 1mM borneol shuts off this inward movement.  Wash out the borneol while keeping the menthol constant, the inward movement of positive charges continues.  Wash out the menthol as well and the current ceases.  Flufenamic acid (FFA) is yet another noxious compound that opens TrpA1 channels.[2]

Takaishi Fig 5: dose response curves for fenchol and borneol

In figure 5 the influence of frenchol and borneol on AITC activation of TrpA1 was presented.  Since 1mM terpene is not reasonably achievable in a whole animal, dose response curves were prepared

RPA1 mutants  T874, an amino acid that appears to hydrogen bond with menthol. [2] , S873V and T874L in transmembrane helilx 5 and Y812A in transmembrane helix 3 abolished the inhibitory action of borneol.  Fenchol was not tested in this system of mutants.

Searching 144,156 compounds for a FFAR 2 agonist

Free Fatty Acid Receptor 2 (FFAR2) is another potential target of frenchol and perhaps frenchone and borneol.  FFAR2 is a G protein coupled receptor that signals through Gαi and G_αq leading to shut down of adenylyl cyclase activity and IP3. The second featured paper is a US/India collaboration between neuroscientists and experts in the gut microbiome. [3]  These investigators started with the observation that those with Alzheimer’s Disease were more likely to have disruptions in short chain fatty acid producing bacteria in their colons.  [3] 

• Amino acid sequences of human and mouse FFAR2 from UniProt.org
• Used three online programs to predict the tertiary structure of these receptors
• Used AutoDock to screen databases for potential ligands. Indofine Natural products and IBScreen NP are two examples. [3] 
• Then tested top scoring ligands on the neuroblastoma cell line SK-N-SH
• Looked at two G protein coupled receptor responses: decreased cAMP and increased ca²⁺

Counter to what might be presumed from the ProteinAtlas.org website,  FFAR2 was expressed in the nuroblastoma cell line.  Carbacol is an agonist of the acetylcholine Ca²⁺ channel.  Its use serves as a positive control for the Fura4 Ca²⁺ indicator.  An additional objective was to demonstrate increased proteosomal degradation of Aβ.

The FFAR2 receptor should be coupled to Gα_i and therefore tend to turn off production of cAMP.  Acetate turns down cAMP to a little more than half that of forskolin alone.   Frenchol, requiring DMSO as a solvent, was the only really hydrophobic terpene tested.  Though dissimilar in structure, 10 µM also inhibited forskolin stimulated cAMP production.  Fura-4 was used to detect increases in intracellular Ca²⁺.    

A FFAR2 antagonist inhibits Aβ protection

.  Further addition of Aβ25–35 (25 µM) for 2 hours decreased cell viability, an outcome that was increased by the introduction of 10 µM FFAR2 inhibitor CATPB.  CATBP is an inverse agonist of FFAR2 that inhibits MAPK signaling and augments forskolin induced increases in cAMP.   The Western blot for TRPA1 from Devicia (2019) [4] has been added just to show that TRPA1 has been shown to be produced in a different neuroblastoma cell line. .  Even mRNA data such as those on ProteinAtlas.org indicate that neither TrpA1 nor FFAR2 is significantly expressed in the normal brain.Why FFAR2? Would THC binding to CB1 coupled to Gα_i mitigate the toxicity of Aβ peptides? The greater expression of CB1 in the brain begs the question of why not use THC to induce the proteolysis of Aβ peptide aggregates? How much of this proteolysis is dependent on Gα_i and/or Gαq dependent pathways? How much is the link between Alzheimer’s Disease and dysregulated intestinal microbiota more to do with FFAR on immune cells?

Looking to the gut

As interesting as the FFAR2 study is, it is hard to reconcile it with the sparse expression of FFAR2 mRNA in the normal brain.  FFAR2 and CB1 are both expressed in satiety peptide hormone CCK secreting neuroendocrine I cell in the duodenum. [5]  An unknown in this study is whether FFAR2 is expressed on the apical/luminal or the basolateral/blood surface.  The virtual sterility of the duodenum means that the luminal short chain fatty acid concentration is negligible.  [5]  The The authors speculated that FFAR2 on the basolateral side senses when short chain fatty acids from the gut get to a certain threshold in the blood. [5] Yes, TRPA1 mRNA can be found in I cells and they assist in CCK secretion. [6]

Natural ligands and receptor binding

Fenchol/fenchone/borneol might activate TRPA1 and FFAR2. Let’s think about this alternative GI mode some more. According to a Talavera review [7] TRPA1 has some endocannabinoid ligands as well as some familiar terpenes.

compound	EC50 µM	IC50 µM	technique
4-Hydroxy-2-nonenal	1.9±  0.7		mTRPA1, CHO, Ca2+ imaging
annandamide	10 ±2		rTRPA1, HEK293, Ca2+ imaging
Arachidonic acid	13 ±4		hTRPA1, HEK293, Ca2+ imaging
camphor		660	rTRPA1, HEK293, electrophys
menthol	7 to 278	56 to >000	5 different systems

It is interesting to note that note much is known about TRPA1 inhibitors. Inhibiting TRPA1 would decrease intracellular Ca²⁺ whereas the Razazan study proposes that frenchol binding to FFAR2 increases intracellular Ca²⁺ via Gαq. [3]

Razazan (2021) supplemental data

The FFAR2 study had a interesting table of Vino binding energies in the supplemental data.  Binding energies are in units of kcal/mol.  In general, the more negative the binding energy, the more stable the interaction.  A positive binding energy indicates that energy is required to get the ligand to interact with the receptor. 

Some terpenes close to those featured on this site that are predicted to have favorable binding energy to FFAR2 are shown.

Note that structurally related compounds fenchol, frenchone, and borneol are predicted to H-bond to Tyr238. According to annotation on UniProt, The Y238A mutation results in partial loss of propionate G-protein coupling. The nearby N238A mutation results in “complete loss of acetate-induced G protein-coupled receptor activity.” His 242 and Arg 255 are also crucial for G protein receptor activity coupling.

Concluding comments

So many ligands bind to different receptors. TRPA1 and FFAR2 binding would have opposite affects on intracellular Ca²⁺ and CCK secretion. I cells might encounter frenchol in the diet before the liver has a chance to process it. It would be interesting to do the experiment. Many ligands have multiple potential receptors, not always in the same cell.

References

1. Api AM et al. (2015) RIFM fragrance ingredient safety assessment, Fenchyl alcohol, CAS registry number 1632-73-1  Food and Chemical Toxicology 84 (2015) S25eS32 free article
2. Takaishi, M., Uchida, K., Fujita, F., & Tominaga, M. (2014). Inhibitory effects of monoterpenes on human TRPA1 and the structural basis of their activity. The journal of physiological sciences : JPS, 64(1), 47–57. PMC free article
3. Razazan A, Karunakar P, Mishra SP, Sharma S, Miller B, Jain S and Yadav H (2021) Activation of Microbiota Sensing – Free Fatty Acid Receptor 2 Signaling Ameliorates Amyloid-β Induced Neurotoxicity by Modulating Proteolysis-Senescence Axis. Front. Aging Neurosci. 13:735933 PMC free article
4. Devecia HA , Akyuvab Y, Nura G, Nazıroğluc M (2019)Alpha lipoic acid attenuates hypoxia-induced apoptosis, inflammation and mitochondrial oxidative stress via inhibition of TRPA1 channel in human glioblastoma cell line. Biomedicine & Pharmacotherapy 111 (2019) 292–304
5. Sykaras, A. G., Demenis, C., Case, R. M., McLaughlin, J. T., & Smith, C. P. (2012). Duodenal enteroendocrine I-cells contain mRNA transcripts encoding key endocannabinoid and fatty acid receptors. PloS one, 7(8), e42373. PMC free article
6. Purhonen AK, Louhivuori LM, Kiehne K, Kerman KE, Herzig KH. TRPA1 channel activation induces cholecystokinin release via extracellular calcium. FEBS Lett. 2008 Jan 23;582(2):229-32. free article
7. Talavera et al(2020) Mammalian transient receptor potential TRPA1  channel:  from structure to disease.  Physiol Rev 100: 725–803