Folk medicine inspiration from the Brazilian Rain Forest
Baccharis uncinella ,used by the Laklaño Indians, caught the attention of several groups in Brazil that included Laboratório de Etnofarmacologia. . In their introduction the authors had some interesting things about how leaves of Baccharis uncinella have been used by the Laklaño Indians in the state of Santa Catarina, Brazil. This link takes the reader to an interactive map to explore where this plant is found.
Laklaño Indians, more about
- This group has been living in the valleys and plateau borders since 1914,
- About 800 individuals in the contacted group were exposed to the dominant culture at State of Santa Catarina (Ibirama Indian Reserve).
- The exchange introduced the use of salt in cooking and alcoholic beverages
- The authors mentioned an Indian named Congo Patté who, according to the authors, spoke of symptoms of hypertension: “heaviness in the head”, nape pain, nausea, and discouragement. 
- B. uncinella tea was used as a sedative as well as to “regulate blood pressure.”
- Congo Patté was reported to have remembered telling men to avoid a Brazilian hard liquor and to drink the B. uncinella tea as a means of calming down during disputes with the loggers.
Ascari et al., did not discuss salt induced hypertension versus emotional stress induced hypertension in their introduction. They favored a sedative mechanism in their discussion of their results. 
The two collection sites
Both collections of harvested leaves were dried for seven days at room temperature, stored at −18°C, and extracted with oil. The coordinates of the locations were entered in GPS coordinates.net.
- State of Paraná, Brazil ( BU-PR), altitude of 1050 m, 25°06′23′′ South and 50°00′39′′West
- State of Santa Catarina, Brazil (BU-SC), altitude of 440 m, 27°16′19′′ South and 49°49′60′′ West. This is in the Laklaño Reserve, at the locality of Pouso Redondo,
The terpene profiles
This post is not reporting minor terpenes in reference  The EO samples were obtained by hydrodistillation of about 1.2 kg of milled leaves. The samples were partitioned into ethyl ether. The organic solutions were dried with anhydrous Na2SO4, to yield about 10 g of each.  Extracts were analyzed by gas chromatography. Samples were identified based on retention of standards.
|Essential oil||monoterpene/sesquitepene ratio||limonene||α-pinene||spathulenol||β-caryophyllene||caryophyllene oxide|
- This study used 35-45 g, two month old, male CF1 mice.
- The B. uncinella essential oils were diluted in 1% Tween-80. Diazepam and pentobarbital were dissolved in saline.
- B. uncinella essential oils (always 10 mL/kg body weight of mouse) and vehicles were administered intraperitoneally.
- saline or 1% Tween -80, negative controls
- diazepam (2 mg/kg, positive control)
- 50 or 100 mg/kg BU-PR
- 50 or 100 mg/kg BU-SC
Hypnotic (sleep) activity
Thirty minutes after receiving essential oils and the Tween-80 alone, mice received sodium pentobarbital (35 mg/kg, i.p.). The sleeping time (time elapsed between loss and recovery of righting reflex was recorded with a cutoff time of 180 minutes. 
Note that the Bu-SC is dominant for β-caryophyllene. Bu-PR has no α-pinene, a GABAA agonist. Bu-SC has a little α-pinene.
The standard treatment regimen was followed. This time rectal temperatures were measured for groups of six mice before treatments (t = 0) and 15, 30, 60 and 120 min after drug administration.
Pentobarbital (50 mg/kg, i.p.) was used as the positive control. According to the authors repeated measures ANOVA confirmed that the body temperature was significantly decreased (F6.51 = 6.73; p < 0.01) by all treatments in comparison to controls at 15 and 30 min. Only rectal temperatures of the pentobarbitol treatment mice were lower than the control mice at 60 min. There was no significant difference between the two doses of BU-PR and/or Bu-SC. The mechanism of phentobarbitol lowering of body temperature is too complicated for this post. GABAA receptors tend to inhibit stimuli that increase body temperature.
Mice were individually habituated to a 45 × 25 × 20 cm activity cage for 10 min prior to receiving one of the standard treatments. After receiving their treatment they were returned to the activity cage. Their movement were digitally recorded for 30 minutes. Eight to 10 mice were in each group for this experiment. Diazepam at 1 mg/kg was used as positive control.
Diazepam, a drug used to treat anxiety, is a positive allosteric modulator of GABAA. Recall that spathulenol and caryophyllene oxide are in larger amounts in Bu-Pr. 
Another Brazilian study  examined the effects of essential oil from the leaves of Croton conduplicatus on sleep and physical responses to painful stimuli. Caryophyllene oxide composed about 12% of this essential oil. The GABAA antagonist flumazenil blocked the rat behavior changes induced by the essential oil.  Flumzenil is a competitive inhibitor of positive allosteric GABAA modulators binding to the benzodiazepine site. Here is a direct quote from Wikipedia: “Flumazenil does not antagonize all of the central nervous system effects of drugs affecting GABA-ergic neurons by means other than the benzodiazepine receptor (including ethanol, barbiturates, and most anesthetics)Z) recognition site.” Wikipedia authors also put the ethanol binding site of GABAA on the α-subunit. The benzodiazepine site is between γ and α subunits.
Japanese study of a North American bush
Chromolaena odorata is a North American bush that some may know as the Christmas bush. This plant was introduced to Africa where it is considered an invasive species. ..and paradoxically a source of phytochemicals. A group out of the Graduate School of Pharmaceutical Sciences, Kyoto University investigated the ability of a major terpene from the essential oil of this plant to act as a sedative in mice.  Based on gas chromatography, mass spectrum analysis, caryophyllene oxide makes up 43.75% of Chromoloena essential oil. 
- Mice were placed in glass cages, 60 cm wide, 30 cm long, 34 cm high
- Four pieces of filter paper were hung in the corners of the cages. The test substances were added to the filter paper.
- After 1 hour, mice were placed in the center of the glass cage.
- Mice were monitored by video for 1 hr. Locomotor activity was how many times they crossed 10cm grid marks per 5 min.e
- The area under the curve (AUC) of locomotor activity was calculated using the trapezoid rule on a plot of counts per 5 min (y-axis) as a function of time (x-axis) .
As much as the dosing and possibility of rats moving around in search of food connected to the smells, this study more accurately captures the experience of humans engaging in “aroma therapy. ”
- Figure 2 of this publication demonstrated a significant decrease in wandering about the cage at concentrations of 0.000004 to 0.04 mg essential oil per cage. The decrease seen at 0.4 mg was not significant. 
- Somewhat ambiguous effects were seen in cage wandering for purified β-caryophyllene and caryophyllene oxide.
Caffeine-Induced Stimulation of Locomotor Activity in Mice
- Diazepam (5 mg/kg) used as a positive control was administered orally 30 min before caffeine administration. t=-60
- Mice were injected intraperitoneally with 25 mg/kg caffeine (n = 6/group) t=-30
- Mice were placed in the vapor phase cages for 30 minutes. t=-30
- Locomotor activity was measured for an hour. T = 0 to +60
Caffeine, not surprisingly, more than doubled cage wandering. (p<0.01) Diazepam, on top of caffeine, decreased cage wandering compared to caffeine alone. (p<0.05) The same decrease was seen for caryophyllene oxide on top of caffeine compared to caffeine alone. (P<0.01)
Did the mice roam around less because they had lost motor control? Mice were trained on a Rota-rod treadmill for two days. Mice are started at 5 rpm for 3 minutes. The velocity is increased to 40 rpm. Dougnon and Ito concluded t the decrease in locomotor activity was not due to lack of coordination. 
Pentobarbital-Induced Sleeping Test
- A negative control in some groups 3 mg/kg of flumazenil, a specific GABAA receptor antagonist, was administered intraperitoneally 15 min before diazepam administration. T=-45 min
- As a positive control in other groups the GABAA agonist Diazepam (3 mg/kg) was administered orally 30 min before sodium pentobarbital injection. t=-30
- Sodium pentobarbital (42 mg/kg) was injected intraperitoneally to mice (n = 6/group) t=0
- Mice were immediately placed in a cage filled with vapor of the sample. t=0
- Sleeping time was defined as the time difference between loss and recovery of the righting reflex.
Sleep duration was decreased by the GABAA inhibitor flumazenil in the Diazepam group but not the caryophyllene oxide group. 
The authors gravitated towards caryophyllene oxide binding to the CB2 receptor.  As we are about to see, there is some evidence that similar compounds bind to a different site than flumazenil.
Caryophyllenes and the neurosteroid site
The PubChem entry for caryophyllene epoxide has an interesting quote regrading the metabolism of (-)-caryophyllene, “the metabolism was shown to progress through (-)-caryophyllene oxide since the latter compound also afforded 14-hydroxy caryophyllene as a metabolite.”
The Janzen group performed some electrophysiology studies of caryolanol in HEK293 cells expressing various forms of GABAA.  While β-Caryolanol was shown to be a negative allosteric modulator in some systems, it had no significant effect on cultured hippocampus neurons. 
The premise of the Janzen study is taht sesquiterpenes become hdyroxylated in the process if “digestion” that could also mean metabolism by the liver based on references in PubChem.
Pregnanolone is a neurosteroid, see panel (a) in the above figure compiled from reference . Pregnanolone is making some hydrogen bonds with Threonine 309, arginine 399, and glutamine 245. While caryolanol is not the same thing as caryo[hyllene oxide, we can imagine latter fitting into this hydophobic pocket with a few amines to hydrogen bod with. This is the general theme of the Jansen publication. The only issue is these oxidation products of sequiterpenes tend to be negative allosteric modulators of GABAA under many of the experimental conditions that they used. The potential of the epoxide group of caryophyllene oxide reacting with something in one variety of GABAA receptor, or another, adds a different and highly speculative twist!
- Ascari J, Sens SL, Nunes DS, Wisniewski A Jr, Arbo MD, Linck VM, Lunardi P, Leal MB, Elisabetsky E. Sedative effects of essential oils obtained from Baccharis uncinella. Pharm Biol. 2012 Jan;50(1):113-9. PMC free article
- Oliveira Júnior RG, Ferraz CAA, Silva JC, de Andrade Teles RB, Silva MG, Diniz TC, Dos Santos US, de Souza AVV, Nunes CEP, Salvador MJ, Lorenzo VP, Quintans Júnior LJ, Almeida JRGDS. (2018) Neuropharmacological effects of essential oil from the leaves of Croton conduplicatus Kunth and possible mechanisms of action involved. J Ethnopharmacol. 2018 Jul 15;221:65-76. free article
- Dougnon, G., & Ito, M. (2021). Essential Oil from the Leaves of Chromolaena odorata, and Sesquiterpene Caryophyllene Oxide Induce Sedative Activity in Mice. Pharmaceuticals (Basel, Switzerland), 14(7), 651. PMC free article
- Janzen D, Slavik B, Zehe M, Sotriffer C, Loos HM, Buettner A, Villmann C. Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABAA receptor function in vitro. J Neurochem. 2021 Oct;159(1):101-115 free article