Bisabolol

North Americans will recognize sage and chamomile [1] as a source of α-bisabolol. According to Floraplex Terpenes, Sour Diesel is a strain that contains α-bisabolol among many other terpenes. Camomile tea has been used as a sleep aid due to its calming effects. A clinical trial has been conducted . Participants received six tablet a day that were standardized to contain up to 2.5 mg of (-)-α-bisabolol and ≥ 2.5 mg of apigenin per tablet. Three tablets were taken at a time, twice a day. The total daily dose of (-)-α-bisabolol is assumed to be 15 mg. Dramatic changes in sleep parameters were not observed, perhaps as a result of using the wrong dose.The method of administration may have had issues too, judging from the featured studies.

(-)-α-bisabolol against Rotenone induced Parkinson’s Disease

The featured study came out of the United Arab Emerates (the UAE group). These authors discussed plants many North Americans may not have heard about as being sources of bisabolol. Studies on this site have a unifying hypothesis: A compound in a folk medicine of my country will mitigate the symptoms of a disease that is costing my country a lot of money. These authors chose the pesticide Rotenone and the Parkinson’s Disease like symptoms it causes in rats. [1]

1. Introduction to Rotenone and what was concluded

Section 1 is for readers who want to know the conclusion before getting immersed into the details. A bit of introductory material is provided.

The target is oxygen radicals from the Mitochondria

Glucose and other six carbon sugars in foods that we eat is broken down into carbon dioxide and acetyl-co-enzyme A (acetyl-CoA). AcetylCoA is transported into the mitochondria where the tricarboxylic acid (TCA) cycle further renders the acetyl of acetyl-CoA into CO2 and electrons in the form of NADH and FADH2. These molecules are simply of electrons and H+. The H+ are pumped out to establish an electrochemical gradient that will be used to the work of making ATP. The electrons have to go somewhere. If Complex I is blocked, that somewhere is O2 to produce super oxide. Under optimal conditions O2 is reduced to H2O in complex IV.

What is Rotenone?

The pesticide Rotenone inhibits electron transport thorough Complex such that the electron reduces O2 to super oxide. Complex I inhibitors like Rotenone freely penetrate the blood brain barrier and have the potential to accumulate in the lipid rich brain. The lipids tend to be polyunsaturated and targets of lipid peroxidation by superoxide. Inhibition of complex I results in a downstream decrease in the production of ATP. Rotenone induces anatomical, behavioral, and biochemical changes similar to those seen in Parkinson’s Disease:, like nigrostriatal dopaminergic neuro-degeneration. [2]

Last figures 1^st

Panels from figures 9 and 10 from reference [1]  The structure of (-)α-bisabolol is shown in the upper right hand corner.  The UAE group included some structural information.  T he number of rotatable bonds tells us that this molecule is fairly flexible.  The single -OH group is the only hydrogen bond donor and acceptor.    is fairly hydrophobic.  The two double bonds make is a  good scavenger of ABTS radicals  

• TBARS is a measurement of MDA
• MDA is a degradation of product of
• Lipid peroxides LOOH that result when rotenone block complex I leading to reactive oxygen species.  Double bonds in unsaturated lipids are a good target for reactive oxygen species.
• ATP is the consequence of a functional electron transport chain without blockage.   ATP is the energy currency of the cell and used in large amounts by the brain.
• ABTS is used to measure the antioxidant capacity of a mixture in the food industry.

A snapshot of the experimental protocol

In this particular study both Rotenone and bisabolol were injected directly into the abdominal cavity of the rats.  Experimental Animals

• 5-6 months old male adult albino Wistar rats
•  280–300 g
• ROT  50X stock solution in dimethyl sulfoxide and diluted in sunflower oil, of 2.5 mg/mL.
• BSB diluted in oil to prepare the dose of 50 mg/kg body weight. injected intraperitoneally 30 min before ROT

Rats were divided into four groups:

1. sunflower oil injected  (vehicle control)
2. Rotenone injected group (ROT),
3. BSB injected 30 min before Rotenone (BR)
4.  BSB injected 30 min before  vehicle  (BSB).

The brain was removed as rapidly as possible, and the brain hemispheres were separated.   One hemisphere  was used for biochemical assays and the other for histology.  Mitochondria were separated from cytosol and lipid membranes.  Commercial kits were used to measure enzyme activities and protein amounts.

2. Parkinson’s Disease and dopaminergic neuron sparing

The enzyme tyrosine lhydroxylase is involved in the synthesis of dopamine. These neurons are compromised in Parkinson ’s disease. The UAE group used antibodies against this enzyme to detect the cell bodies of these important neurons. The projections into the striatum were also counted by a pathology who did not know which experimental the brains came from.

3 Protecting antioxidant enzymes

The figure examines the activities of two enzymes that scavenge reactive oxygen species and two small molecule indicators of their performance.  GSH will oxidize in the absence of super oxide dismutase (SOD) and catalase (CAT).  Malondialdehyde is again a marker of lipid peroxidation.  SOD activity is measured in units of “Units” per mL whereas ctalase activity is measured in units of nmoles H₂O₂ per minute per mL that are converted to H₂O. 

Rotenone destroys at least two key anti-oxidant enzymes. Bisabolol mitigates some of the damage.

4-5.  Reactive glia, caretakers of neurons gone bad

Astrocytes are the second most abundant cells in the brain that take care of neurons by releasing GSH and SOD and take up the neurotransmitter glutamate, [1]  Microglia are the resident macrophage of the brain that serve an immune function. This caretaker role of glia was obtained from  an Internet image with a dead link.  The UAE group used GFAP as a marker of reactive astrocytes.  Iba was used as a marker of reactive microglia.  The immunohistochemistry of figure 5 are not presented in this post.  When these caretaker cells of neurons are set off they produce the induced nitric oxide synthase (iNOS) and matrix metalloprotease 9 (mmp9).  In the reduced state this cysteine binds a catalytic zinc keeping the enzyme inactive. Reaction with something like superoxide will release the inhibitory peptide. iNOS produces the vasodialator nitric oxide (NO).    Super oxide from the Rotenone blocked complex I (MC-I) reacts with NO to form peroxynitrite ONOO-.  Nitrite is marker for this lipid and a protein damaging reactive nitrogen species that is notorious for damaging neurons.    NO also reacts with the cysteine switch of the inactive mmp9 so as to turn it into an active protease. 

Reactive microglia are the ones that release cytokines yet their amounts are only doubled with Rotenone. Reactive astrocyes are tripled with Rotenone. Bisbolol seems to help. Now it is time to see the UAE group’s data for the cytokines being produced by activated microglia.

Figures 6-7 Rotenone brain damage is because of cytokines

For this post Figures 6 and 7 from reference [1] are being combined. The cartoon is from reference [3] , a review on ways to protect the brain from ischemic stroke damage. Many proteins of “cellular pathways” have been replaced by arrows for clarity. The point is that cytokines can promote the transcription of each other’s genes in a positive feedback cytokine storm manner. The UAE group favored the hypothesis that damage is caused by COX-2 activation. [1] The review that this image was adapted from states that small preconditioning insults like bacterial LPS and exercise released cytokines can lead to changes in gene expression that lead to cerebral ischemia tolerance. [3] These small insults result in an ipi-genetic change or induction translation inhibiting micro RNAs. [3]The length of times needed for IT documented in this review is longer than the 20 min preconditioning with bisabolol. The UAE group cited a report that bisabolol inhibits Cox2. Things really appear to be more complicated than Cox-2 being the lynch pin.

Note that bisabolol (BSB) increases the amounts of Cox2 and iNOS compared to the control. There appears to be a misprint in the figure legend of the online text of this paper. p < 0.05 CON vs. ROT The * is clearly over the bisabolol only bar. Perhaps this is what the Garcia-Bonilla review meant by mild inflammatory events leading to cerebral ischemic tolerance. [3]

The inflammasome is a macromolecular assembly which like mmp9, has a cysteine switch with a reactive thiol that senses reactive oxygen species.  The inflammasome is a protease assembly that cleaves the inactive pro-cytokines IL-1β and and more. [4]  This review did not get into specific molecular mechanisms of reactive oxygen species activation of the NRLP3 component of the inflammasome.  [4]  The review did cover different sources of ROS that include the mitochondria. [4]

Figures 8-9 Apoptotic pathways

Images of Western blots used to quantitate protein levels from the UAE study have been omitted from this post. They have been replaced with an image of apoptotic pathways. Apoptosis is commonly referred to as “programmed cell death.” The mitocondria control the intrinsic pathway.

Other studies.

• Bisabolol Mice were pretreated orally with BISA (50, 100 and 200 mg/kg) or vehicle, The antinociceptive effects on mustard oil, formalin, acetic acid, and capsaicin pain were assessed. [5] Bisabolol improved all four pain model. The various antagonists were tested to determine which of many pathways were involved with no resolution.[5]
• A study evaluated behavioral effects of bisabolol on anxiety. The elevated plus maze (EPM) and  open field tests were the stressors. Bisabolol was given as  intraperitoneal treatments of 0.5, 1, 2, 5, and 10 mg/kg. GABAA and HT receptor antagonists led the investigators to conclude that bisabolol acts on the GABAA receptor ion channel. [6]
• A Brazilian group [7] removed sciatic nerves from mice to compare the compound action potential (CAP) characteristics of bisabolol with the voltage gated Na+ channel blocker lidocaine at a concentration of 1mM and 1mM of the voltage gated K+ channel blocker 4-AP. . CAP recordings in the absence and presence of (−)-α-bisabolol (0.5, 1, 5 and 10 mM). Briefly, the mice sciatic nerves were carefully removed and desheathed. (−)-α-Bisabolol was diluted in the vehicle (physiological solution added to 0.1% Tween 80), at concentrations of 0.5, 1, 5 and 10 mM, washing procedure was 1 mL/min. [7]
• The same United Arab Imirates group published a followup study with male albino Wistar rats. were pre- and co-treated with intraperitoneal injection of α-bisabolol (25 mg kg⁻¹) daily for 10 days along with the subcutaneous injection of the β-adrenergic agonist isoproterenol (85 mg kg⁻¹) at an interval of 24 h for two days (9th and 10th day). Note that bisabolol was used in a more refined preconditioning scheme in this study. Iso increases the heart rate and in so doing so, creates a challenge for the mitochondria of the heart to obtain enough oxygen. ISO injections induced myocardial infarction as determined by cardiac proteins in the blood. [8] MI as evidenced by the elevated cardiac marker enzyme in serum and altered oxidative stress markers in the total heart and lysosomal fractions. ISO also caused activation of NLRP3 inflammasomes that lead to the production of proinflammatory cytokines. α-Bisabolol mitigated damage caused by isoproterenol. α-bisabolol attenuated oxidative stress and inflammation by inhibiting NLRP3 inflammasome.

Conclusion

See section 1 ! The target of (-) α-bisabolol is super oxide from stressed out mitochondria [1,8]. The inflammasome is secondary. This does not preclude other targets.

References

1. Javed, H., Meeran, M., Azimullah, S., Bader Eddin, L., Dwivedi, V. D., Jha, N. K., & Ojha, S. (2020). α-Bisabolol, a Dietary Bioactive Phytochemical Attenuates Dopaminergic Neurodegeneration through Modulation of Oxidative Stress, Neuroinflammation and Apoptosis in Rotenone-Induced Rat Model of Parkinson’s disease. Biomolecules, 10(10), 1421. PMC free article
2. Cabezas R, El-Bachá RS, González J, Barreto GE. Mitochondrial functions in astrocytes: neuroprotective implications from oxidative damage by rotenone. Neurosci Res. 2012 Oct;74(2):80-90
3. Garcia-Bonilla, L., Benakis, C., Moore, J., Iadecola, C., & Anrather, J. (2014). Immune mechanisms in cerebral ischemic tolerance. Frontiers in neuroscience, 8, 44.   PMC free article
4. Shao, B. Z., Xu, Z. Q., Han, B. Z., Su, D. F., & Liu, C. (2015). NLRP3 inflammasome and its inhibitors: a review. Frontiers in pharmacology, 6, 262. PMC free article
5. Leite, G., Fernandes, C. N., de Menezes, I. R., da Costa, J. G., & Campos, A. R. (2012). Attenuation of visceral nociception by α-bisabolol in mice: investigation of mechanisms. Organic and medicinal chemistry letters, 2(1), 18. PMC free article
6. Tabari MA, Tehrani MAB. Evidence for the involvement of the GABAergic, but not serotonergic transmission in the anxiolytic-like effect of bisabolol in the mouse elevated plus maze. Naunyn Schmiedebergs Arch Pharmacol. 2017 Oct;390(10):1041-1046.
7. Alves Ade M., Gonçalves J.C., Cruz J.S., Araújo D.A. Evaluation of the sesquiterpene (−)-alpha-bisabolol as a novel peripheral nervous blocker. Neurosci Lett. 2010;472:11–15. free article
8. Nagoor Meeran MF , Azimullah S , Laham F , Tariq S , Goyal SN , Adeghate E , Ojha S .(2020) α-Bisabolol protects against β-adrenergic agonist-induced myocardial infarction in rats by attenuating inflammation, lysosomal dysfunction, NLRP3 inflammasome activation and modulating autophagic flux. Food Funct. 2020 Jan 29;11(1):965-976.