Nerolidol

This post follows the Chan review [1] with some added comments and exploration of their more biology references of this hard core chemistry review. The trans isoform is the most common. An extract of the wood of Myrocarpus fastigiatus is the only source of cis-nerolidol mentioned in the feature review. [1] A study on the cytotoxic effects of cis-nerolidol is examined in depth to make the point that lipid membranes are the target of cis-neruolidal. The Chan review came out of Malaysia and is every thing one ever wanted to know about nerolidol to write an enormous grant application. The post will foucs on key points that hint that HMG CoA reductase [3] might be a target for trans nerolidal.

Trans, cis-nerolidol, the good, bad, and racemic

Nerolidol comes in two stereo isomers, or enantiomers, at carbon 3. The R(+) rotates polarized light to the right while the S(-) rotates polarized light to the left. S is for sinister. These forms are largely ignored in the literature. Cis-Trans isomers about the double bond between carbons 6 and 7 are a much bigger deal. One is toxic, one is not. “Cis” and “trans” are Latin for “this side of” and “the other side of,” Z is for the German word zusammen meaning together and entgegen meaning going in opposite directions.

Sources of trans and cis nerolidol [1]
  • Piper claussenianum (Miq.) C.DC.(81.4%) [1] review. A publication from Brazil described the extraction of the flowers and leaves in greater detail. They found (+)-(E)-nerolidol in the leaves, and (+)-linalool and (+)-(E)-nerolidol in the inflorescences (flower) essential oil. {2}North Americans are more familiar with Piper nigrum that gives us black peppercorns. Trans aka 6E
  • Myrocarpus fastigiatus Allemao (80.0%) [1] This is a Brazilian plant that most North Americans probably are not familiar with. Cis aka 6Z

Nerolidol may be synthesized from linalool. Note the similarity in structures. The authors have depicted the 6Z, aka cis, isoforms as scrunched up molecules. While the double bond between carbons 6 and 7 restricts rotation, there is rotational freedom about carbons 8 and 9. Nerolidol is a largely hydrophobic molecule. It was predicted to easily pass through the lipid bilayer of the cell membrane to reach intracellular targets. This post makes the argument that a target may be the lipid bilayer too.

Cis and Trans double bonds in fatty acids have different afects on lipid bilyers, hence the maligning of trans fatty acids. Based on the available studies, it can be deduced that the non-toxic dose of trans-nerolidol for animal is up to 2000 mg/kg. Cis Nerolidol is another matter. Some times we don’t want our membranes to become more fluid and leaky.Many of the publications that the Chan review cited were a racemic mixture of cis and trans. [1] The following is a quick overview of an extensive review cherry picking for references to membranes.

6.1 Antioxidant Activity [1]


This review mentions Fenton chemistry generation of hydroxyl radicals from H2O2 and super oxide. The GSH peroxidase system is also mentioned as are catalase and superoxide dismutase.They mentioned reduction of lipid peroxidation. Only lipids with double bonds are subject to peroxidation. Does nerolidol scavenge those reactive oxygen species? Must it also partition into the membrane to do so? What are the consequences of peroxidized nerolidol in the membrane?

The reaction reaction with super oxide has pretty much the same general lipid peroxy radical outcome. The lipid peroxy radical is reactive.

6.2. Antibacterial Activity [1]

The review covered studies regarding nerolidol antibacterial activity against pathogens. No mention was made of antibacterial activity against beneficial “probiotic” bacteria.

  • “ by disrupting the cell membranes as indicated by the increased leakage of K+ ions from the bacterial cells”
  • “was shown to cause damage to the cell membrane, leading to the leakage of macromolecules and eventually cell lysis”
  • “The high content of trans-nerolidol was suggested to be responsible for the antibacterial activity …”

At this point we do not know if the membrane leakage is due to cis-nerolidol, inhibition of the mevalonate pathway in bacteria (see below), or something else entirely.

6.3. Anti-Biofilm Activity [1]
  • The review discussed the importance of biofilms but didn’t really mention the chemical composition.
  • “The anti-biofilm activity was attributed to the presence of cis- and trans-nerolidol in the essential oil of C. odorata.“ The cis isomer was more effective than the trans.
  • Another study cited in this review “also compared the anti-biofilm activities of the cis,trans-nerolidol and cis-nerolidol on the pre-formed biofilm by C. albicans.” The racemic mix was more effective at biofilm reduction than cis-nerolidol.
  • “Thus, it was suggested that trans-nerolidol, which was the main constituent in EO of Piper claussenianum (Miq.) C. DC., Piperaceae leaf, may responsible for the observed anti-biofilm activity in reducing the viability of the pre-formed biofilms.
6.4. Anti-Fungal Activity [1]


Mention was given to use of essential oils to combat fungal pathogens. The plant and human studies cited mentions trans nerolidol. Inhibition of mycelium growth and spore formation was cited. This study suggested that trans nerolidal is an effective fungicide. There did not seem to be a direct comparison with cis-nerolidol. A compound that is selectively toxic to fungal pathogens and not their mammalian hosts would be exciting. One very speculative possibility is differences in the regulation of HMG-CoA reductase in fungi, that includes yeast, and mammals. [3] Yeast have a HMG-CoA reductase degradation complex (HRD complex) that tags geranylgeranyl pyrophosphate bound HMG CoA reductase for proteosome degradation.[3] This post is not digging deep enough as to whether this negative feed back loop exists in pathogenic molds as well as yeasts.


adapted from Figure 5 reference [3]. See the original paper for details regarding the HRD complex.
6.5. Anti-Parasitic Activity

Nerolidol could be an inhibitor at the early step in the mevalonate pathway, a pathway that is used to make terpenes. This pathway is Statins are inhibitors of HMG CoA reductase. This enzyme is present in the endoplasmic reticulum of humans and parasites.

The first parts of the mevalonate pathway. HMGS HMG CoA synthase, HMGR HMG CoA reductase
6.6. Insect Repellent Activity [1]

This section discussed larvae cidal and insecticidal activity. There was not really discussion of potential pathways that might overlap those in mammals and potential targets of this terpene. This post will not explore how much the vevalonate pathway is involved in phremone synthesis and whether cis-nerolidal is acting on HMG CoA reductase in this pathway.

6.7 anti-ulcers [1]

The Chan reviewed studies of chemically induced ulcers rather tn Helicobacter pylori. The membrane component in terms of Cox1 and PLA2 activity that produce PGE2 was not mentioned. The original paper suggested free radical scavenging.

6.8 skin penetration enhancer activity [1]

The structure of nerolidol was proposed to make it suitable to disrupt the alignment within lipid lamellae of the stratum corneum. Chan et al cited references ranking lipiphiliicty: nerolidol > farnesol > limonene > linalool > geraniol > carvone > fenchone > menthol in facilitating transdermal delivery of pharmaceuticals. Chan et al did not mention which isomer of nerolidol where used. The stratum corneum is a layer of dead cells that are embedded matrix of lipids, ceramides, and cholesterol. PubChem has some images of crystalline structures from cholesterol and a typical fatty acid palmitic acid. Nbd ceramide looks like it would be pretty good at forming crystals.

Some images from PubChem.
6.9. Anti-Nociceptive and Anti-Inflammatory Activity [1]

This section contained and excellent review of the literature. Ion channels, NSAIDS, and arachidonic acid pathways were discussed. Could perturbation of the lipid bilayer affect ion channel gating and/or phospholipase A2 activity?

6.10. Anti-Cancer and Anti-Tumor Activity [1]

The trans isoform seems to be the one with anti tumor activity. Trans-nerolidal was cited as being non cyto toxic. [1] The Chan review did not speculate as to whether trans-nerolidal could be acting on an enzyme lide HMG CoA reductase that would be needed for the synthesis for lipid building blocks required for cell growth.

7 Pharmacology Studies [1]

skipping 7.1 in vitro studies

7.2. In Vivo Studies
Two rodent studies were extensively discussed as starting points for human clinical trials.

7.3. Toxicological Studies

This section was also an extensive review of the literature, which often included a racemic mixture of nerolidol.

In the liver [2]

This study came out of the University of Londrina in Paraná, Brazil. The authors wished to address potential hepatooxicity of nerolidol, a common terpene in plant based medicines. HepG2/C3A cells were originally derived from a hepatocellular carcinoma.

Cis- not trans-nerolidol is toxic [2]

Cell viability was measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In the presence of antioxidant reducing equivalents MTT is reduced to to a purple colored compound. (Figure 1)c The cell’s ability to produce ATP is dependent on the mitochondrial membrane potential, which was measured with the fluorescent dye Rhodamine 123.

Figures 1 and 3 from reference [6] Figure1 & 3 *Statistically significant when compared to control (p ≤ 0.05) by anova and Dunnett’s test.

Note that around 200 μM cis-nerolidol the two different indexes of cell viability start to drop to substantially less than half of the baseline.

cis-Nerolidol slows HepG2 cell growth. [2]

cis neurolidol slows cell growth aka cell viability index. Cell survival index was measured by growing cells on a conductive plate by using the xCELLigence real-time cell analysis single-plate instrument. As cells multiply and cover the plate the electrical resistance increases. If cells start to detach the resistance decreases. It is not certain if Figures 2 and 5 are from the same experiment. They seem to belong together and are presented as such.

Figures 2 and 5 from reference [6] *Statistically significant when compared to control (p ≤ 0.05) by anova and Dunnett’s test.

Both assays indicate a tipping point between 150 and 250 μM cis-neurolidol.

Cis-nerolidol is not that genotoxic [2]

The comet assay detects DNA damage in single cells. Cells are lysed in a medium called agarose. An electric potential is applied causing negatively charged DNA to migrate towards the anode. Damaged DNA will look like a comet: A big blob of chormosomal DNA wrapped up in DNA followed by a tail of damaged DNA. DNA synthesis occurs in the S phase of the cell cycle. Gap1 is a checkpoint to make sure everything is ready for DNA synthesis. Gap2 is a check point for mitosis, cell division.

Fugues 4 and 6 from reference [6] *Statistically significant when compared to control (p ≤ 0.05) by anova and Dunnett’s test
Cis-nerolidol, ER stress, and global effects on gene transcription [2]

The authors presented data from real time (quantitative) PCR experiments that can be a bit overwhelming even for scientists. The interested reader can view the entire figure at the provided link. [2]

Figure 7 The transcripts that were increased over 5c by cis-nerolidol. Figure 8 microscopic images of exemplary cells. showing ER damage.
  • BBC3 is a mediator TP53 apoptosis in response to ER stress. TP53 shuts down the cell cycle and initiates apoptosis in response to DNA damage and other stresses.
  • ERN1 is a protein kinase and endonuclease that mediates the unfolded protein response in the endoplasmic reticulum that participates in protein folding. The endonuclease part is specific for the XBP1 mRNA that gets trimmed to code for a transcription factor for unfolded protein response genes.
  • Cyp2C19 is a cytochrome P450 involved in the metabolism of poly unsaturated fatty acids. This P450 is responsible for hydroxylation of a double bond in the terpene limonene.
  • MYC is a transcription factor that controls transcription of genes for other transcription factors. It doesn’t really fit into the other three transcripts that have been up regulated over 5x.

Conclusions

cis-Nerolidol seems to act as a membrane disruption. Those membranes could be the cell membrane, mitochondrial membranes, or endoplasmic eticulum membranes. trans-Nerolidol appears to be an inhibitor of HMG CoA reductase. Both may be scavengers of reactive oxygen species.

As hopeful as trans nerolidol may seem, there are only two clinical trials registered on http://www.clinicaltrials.gov. One is a German study for treatment of head lice. A study from Congo tested an essential oil containing carvone, eugenol, geraniol, and nerolidol as a cervical spray for treating human papilloma virus. The interesting thing is that the cis or trans isomer is not mentioned. One may purchase 90% pure trans-nerolidol at Terpene University.

References

  1. Chan, W. K., Tan, L. T., Chan, K. G., Lee, L. H., & Goh, B. H. (2016). Nerolidol: A Sesquiterpene Alcohol with Multi-Faceted Pharmacological and Biological Activities. Molecules (Basel, Switzerland), 21(5), 529. PMC free article
  2. Biazi BI, Zanetti TA, Baranoski A, Corveloni AC, Mantovani MS. Cis-Nerolidol Induces Endoplasmic Reticulum Stress and Cell Death in Human Hepatocellular Carcinoma Cells through Extensive CYP2C19 and CYP1A2 Oxidation. Basic Clin Pharmacol Toxicol. 2017 Oct;121(4):334-341. PMC free article
  3. Burg, J. S., & Espenshade, P. J. (2011). Regulation of HMG-CoA reductase in mammals and yeast. Progress in lipid research, 50(4), 403–410. PMC free article

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