β-Sitosterol Isolated from Cocoa Powder Functions to Increase Expression of Anti-Inflammatory Proteins in Trigeminal Neurons: Implications for Treatment of Migraine and TMJ Disorders

Previously published findings from our laboratory have shown that cocoa extract inhibits activation of trigeminal neurons in culture and in animals fed a cocoa enriched diet. Activation of trigeminal neurons is implicated in the underlying pathology of migraine, rhinosinusitis, and TMJ or jaw joint disorders. Within trigeminal neurons, proteins known as phosphatases restore cellular homeostasis and thus, suppress inflammatory and nociceptive responses. The goal of this study was to purify and identify the compound in cocoa powder responsible for increased phosphatase activity in trigeminal neurons. Crude extracts from cocoa powder were prepared using silica gel column chromatography and biological activity tested on primary cultures of trigeminal ganglia. Using immunocytochemistry, cultures were stained for the phosphatase MKP-1, which is a protein upregulated in response to treatment with the anti-inflammatory drug dexamethasone. Extracts displaying increased MKP-1 staining were then further purified and retested for MKP-1 activity. The greatest increase in MKP-1 activity was seen in a cocoa fraction containing β-Sitosterol that has a similar chemical structure to dexamethasone. This fraction was isolated with flash chromatography and quantified with LC/MS. β-Sitosterol was found to be 0.0234% (0.936mg/4g) the mass of cocoa powder. In conclusion, data from our study provide evidence for a mechanism by which cocoa exerts its anti-inflammatory and anti-nociceptive effects by inducing MKP-1 expression in trigeminal neurons. Furthermore, we speculate that the inclusion of β-Sitosterol as a dietary supplement would suppress activation of trigeminal neurons, and thus may be beneficial in the treatment of migraine and TMJ disorders.