caryophyllene, chamazulene, chamomile, germacrene D, mosquito repellent, nerolidol, pain, pain relief, spathulenol, Uncategorized

Stevia serratia for pain?

This post started as a story of some benefits of an unnamed road side shrub growing on the hillsides of Western Guatemala. The work is a collaboration of some Guatemalan scientists at Universidad de San Carlos de Guatemala, Guatemala City, Guatemala and Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. According to the authors (Reis Simas 2017), the locals have not named this shrub. This shrub was being investigated because it contains a natural product, chamazulene, found in chamomile and yarrow essential oil.  A blue drop of chamazulene from chamomile is part of the feature image.  All three are members of the same phylogenetic family.  What are the properties of the other five main compounds found in this road side shrub? Could they contribute to pain relief?  Do they have any other beneficial properties?

Stevia serratia, that grows naturally in fields and road sides in many locations, is related to yarrow and chamomile used to make essential oils.

 

Methods

About 40 g of aerial parts were harvested and hydrodistilled. The residue was extracted with pentane, which was latter evaporated. About 80mg of material was obtained. Gas chromatography mass spectrometry was used to identify constituents.

The S serratia essential oil was prepared as a 100 mg/mL carrier oil stock. Mice in groups of six were given doses of 10, 30 or 100 mg/kg by oral gavage.  The formalin pain model was used to create a biphasic source of pain.  Briefly,  20 μL of formalin (2.5% v/v) was injected  into the dorsal surface of the left hind paw. The time that the animal spent licking the injected paw was immediately recorded.  

Animal results

Phase 1:  Influenced by peripheral and central C-fibers and their afferent noci-receptors

Phase 2:  Influenced by central sensitization of spinal cord dorsal horn neurons after input of C-fibers.

* significant at p<0.05
As shown below, it is local anesthetics and anti-inflammatory NSAIDS that act on phase 1 and the opioid that act on receptors in the spinal cord.  

 

Formalin induced pain: TrpA1 ion channels and spinal cord pathways

Why does injecting a dilute solution cause pain?  McNamara and coworkers (2007) demonstrated the role of TrpA1 cation channels.  Let us take a closer at the relationship between pain, opioids and TRPA1. A. Opioid receptors exist in the brain and the dorsal horn (DH) of the spinal spinal cord. B. TrpA1 channels are in the dorsal root ganglion (DRH) neurons coming into the spinal cord. C. TrpA1 channels are also present in peripheral neurons. TrpA1 has a cytoplasmic (inside) loop that detects noxious stimuli. The reactive cysteines (Cys, yellow stars) may detect reactive oxygen species. The reactive lysine (Lys, orange diamond) may be reacting with formalin to form a Schiff base.

Ss_mechanism

 

 Chemical constituents

 

Ss_compounds

Reis Simas and coworkers identified 14 compounds in Ss essential soil. Only the top six are shown above. Ss was stated to contain 10x the chamazulene as chamomile flowers. Was this the compound responsible for early phase pain relief? The following are six publications that point to possibilities. The communication between opioid receptors and TRPA1 ion channels are examined. Algomedix was recently awarded a grant to study opioid receptor sparing TRPA1 ion channel inhibitors as a means of alleviating chronic pain.  

Chamazulene is an anti-oxidant

Capuzzoa and coworkers compared the anti-oxidant and free radical scavenging activity of chamazulene formed from a hydro distillate of chamomile with ascorbic acid, butylated hydroxytoluene (BHT) ,and α-tocopherol. The results indicated that chamazulene to be a more powerful anti-oxidant than the three reference compounds. Chamazulene was concluded to be an important factor for the antioxidant power of chamomile oil.

Nerolidol, a small molecules of many functions

Chan and coworkers (2016) published a very extensive (40 page) review on nerolidol that differentiated, where possible,  between the trans and cis,  (E)- and (Z)-enantomers. It would be impossible to do this review justice in one paragraph. Here are a few properties that might be useful for the cosmetic industry.

  • floral scent, induced by herbivores, may attract predators of herbivores
  • anti-oxidant
  • anti-microbial
  • anti-nociceptive
  • insect repellent for head lice and mosquitoes
  • enhanced skin penetration, good for trans dermal drug delivery, maybe not for cosmetics
  • low oral and dermal toxicity

The Chan review covers other aspects of toxicity that are of interest to the US FDA.  

β-Carylophylene and its oxide, a more complicated story

β-Caryophyllene is a selective  agonist of the mostly peripheral CB2 receptors (Fidt 2016).  β-Caryophyllene oxide (BCPO) does not bind to CB1 receptors.  Both are FDA approved additives in food and cosmetics (Fidt 2016).  These authors summarized proposed mechanisms of    β-aryophyllene oxide being anti-carcinogenic that might also apply to pain and pain reduction

  • BCPO contains methylene and epoxide exocyclic functional groups allowing for 
    covalent binding to protein  sulfhydryl and amino groups.  Do reactive cysteines and lysine  of TRPA1 react with BCPO?
  • BCPO reduction of cyclooxygenase 2 activity leading to a decrease in PGE2 was frequently mentioned in several cancer cell line studies in this review.  This would be phase 1 in our formalin pain model.

Germacrene D

Straden and coworkers (2003) compared the ability of (-)-germacrene D enantiomer with the (+)-enantiomer  to attract moths.  The former was found to be ten fold greater.  Release of germacrene D may be tied to other signals, at least in the case of forest tent caterpillars feeding on poplar trees (Arimura 2004).   Caterpillar feeding was found to induce the expression of  (-)-germacrene D synthase.  Arimura stated in their introduction that release of these volatiles by one insect herbivore may attract predatory insects that will eat the herbivore insect.  The presence of  insect attracting small molecule in cosmetics may be undesirable as well as common.

Spathulenol

Cantrell and coworkers (2018) examined an extract of Matricaria discoidea  DC. used by  Blackfoot Indians to repel mosquitoes. Aedes aegypti mosquitoes were chosen for study because of their reputation of being disease vectors.  Note that “pineapple weed” is in the same Asteraceaefamily as S serratia, yarrow, and chamomile.  The authors fractionated components of “pineapple weed” into its constituents.   In vitro assays were performed to assess biting/feeding as well as simply being attracted to a warm surface, i.e. repellent.  “The most active compounds were determined to be α-terpineol, spathulenol, and neryl isovalerate.”  All three of these compounds at at 25 nmoles/cm2 in ethanol were statistically similar to DEET reducing the percentage of biting insects by about 55% to 80% (DEET).   

So what neuronal receptors sense the low level heat that attract the Aedes mosquito to our skin for a blood meal?  The TRPA1 receptor of course! (Corfas 2015).  As a side note, family TrpV1 senses hot temperatures and TrpM8 senses cold temperatures.  It is remarkable to think that some of the essential oil topical anesthetics that target TrpA1 might also make good mosquito repellents.

Concluding thoughts

This post started out asking which other major components of Stevia serratia essential oil could explain the anti-nociceptive activity observed by Reis Simas and coworkers.

  • Oral Stevia serratia essential oil decreased what was likely to be TrpA1 nocireceptor mediated pain from the hind paw injection of formalin. 
  • The first major constituent, chamulazene is an anti-oxidant.  It would be interesting to evaluate its anti-nociception activity in a cell culture model pain involving agents that target reactive thiols in TrpA1.
  • The second major constituent,  nerolidol, has been reported to do a little bit of everything.  Does this compound have any affect on membrane potential of mammalian neuron like cells?  It would seem to interact with insect olfactory neurons.
  • The sixth and third major constituents are caryophyllene and its oxide.  The former is a selective cannabinoid 2, CB2, receptor agonist.  CB2 activation may explain some of the anti-nociceptive activity observed by Reis Simas. 
  • The fourth major constituent is  germacrene D also interacts with olfactory neurons of some insect species.   Does this compound do anything to change membrane potential in a mammalian neuron like cell culture model?
  • Spathulenol is the fifth major constituent of Ss essential oil.  This compound interacts with neurons of A aegypti mosquitoes in a manner that decreases biting.  Does it interact with mammalian neuron like cells in a cell culture model?
  • Many of the components in Ss essential oil play some role in the plant in how they interact with insects.  Could use in cosmetics be effective in local pain relief and function as an insect repellent?
  • Stevia serratia grows in the US Southwest.

References

Arimura G, Huber DP, Bohlmann J.(2004) Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1. Plant J. 37(4):603-16.

Cantrell CL, Ali A, Jones AMP. (2018)Isolation and identification of mosquito biting deterrents from the North American mosquito repelling folk remedy plant, Matricaria discoidea DC.PLoS One. 2018 Oct 31;13(10):e0206594

Capuzzo A, Occhipinti A, Maffei ME.(2014) Antioxidant and radical scavenging activities of chamazulene. Nat Prod Res.28(24):2321-3

Chan WK, Tan LT, Chan KG, Lee LH, Goh BH. (2016) Nerolidol: A Sesquiterpene Alcohol with Multi-Faceted Pharmacological and Biological Activities. Molecules. 2016 Apr 28;21(5). pii: E529.

Clapham DE (2015) Structural biology: Pain-sensing TRPA1 channel resolved. Nature. 2015 Apr 23;520(7548):439-41.

Corfas RA, Vosshall LB. (2015) The cation channel TRPA1 tunes mosquito thermotaxis to host temperatures. Elife. 2015 Dec 15;4. pii: e11750.

De Carvalho RBF, De Almeida AAC, Campelo NB, Lellis DROD, Nunes LCC.(2018)Nerolidol and its Pharmacological Application in Treating Neurodegenerative Diseases: A Review. Recent Pat Biotechnol. 2018;12(3):158-168.

Fidyt K, Fiedorowicz A, Strządała L, Szumny A. (2016)β-caryophyllene and β-caryophyllene oxide-natural compounds of anticancer and analgesic properties. Cancer Med.5(10):3007-3017. McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM.(2007) TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A. 2007 Aug 14;104(33):13525-30.

McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM. (2007) TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A.104(33):13525-30.

Reis Simas DL, Mérida-Reyes MS, Muñoz-Wug MA, Cordeiro MS, Giorno TBS, Taracena EA, Oliva-Hernández BE, Martínez-Arévalo JV, Fernandes PD, Pérez-Sabino JF, Jorge Ribeiro da Silva A.(2017) Chemical composition and evaluation of antinociceptive activity of the essential oil of Stevia serrata Cav. from Guatemala. Nat Prod Res. 2017 Nov 13:1-3.


Stranden M, Liblikas I, König WA, Almaas TJ, Borg-Karlson AK, Mustaparta H.(2003)(-)-Germacrene D receptor neurones in three species of heliothine moths: structure-activity relationships. J Comp Physiol A Neuroethol Sens Neural Behav Physiol.189(7):563-77.

 

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