Summary
Intro:
· Spagyria is a Paracelsian alchemical medicinal plant modality often suggested to have novel phytochemical and pharmacological properties due to the inclusion of inorganic mineral salts derived from the spagyric preparation of medicinal plants
· A Spagyric and a control tincture were created from the leaves of Thujo occidentalis, sample treated with spagyric salificaiton and the control untreated without salification
· Occidentalis produces a highly bioactive, medicinal, and enigmatic specialized metabolite, thujone, a monoterpenoid ketone, which was quantified here
· Thujo occidentalis, like all plants, serve as a potential repository of inorganic macro and trace essential minerals, such as Potassium which was quantified here
· Potassium is a macronutrient that >95% of Americans are deficient in, leading to a reduced quality of life and greater susceptibility to disease and age-related illness
Hypothesis:
· We hypothesized that spagyric plant tincturing process extracts an entourage of macro and trace essential inorganic minerals in significantly higher quantities than non-spagyric tinctures
· As a result of salificaiton with an entourage of endogenous plant mineral salts, we further hypothesize there will be an effect on the quantity and potentially the quality and structure of the specialized metabolic profile, namely thujone.
Methods:
· Leaves of Thujo occidentalis were wild harvested from Lily Pond lake in the summer of 2022
· The leaves were Soxhlet extracted until solvent ran clear to create a control tincture
· Half of this control tincture was separated and turned into a spagyric. This process involved calcination of the remaining plant material, filtering out of water insoluble ash material, and then crystallization and salificaiton of the separated fraction with the crystallized salts to form the spagyric
· The samples were then prepared for GC-MS and ICP-MS analysis for the quantification of thujone and Potassium, and the qualitative assessment of further elemental complexity
Results:
· Potassium and Thujone standards were created achieving R^2 of 0.9998 and 0.9993 respectively
· No statistically significant difference was found in the quality of quantity of thujone between the control and the spagyric sample
· Potassium content in the spagyric tincture was 135% greater than that found in the control
· Elemental diversity was equal between the two, however quantity of qualitatively assessed elements can be assumed to be 10x higher in the spagyric sample than the control resultant from preparative dilution schemes
Discussion:
· Spagyric tincturing of Thujo occidentalis has no effect on the specialized constituent thujone, but does yield significant nutritional content
· The nutritional hypothesis was confirmed while the specialized metabolite hypothesis was rejected.
· Spagyric plant medicine represents a form of nutritional supplementation of essential inorganic macro and trace minerals
· Further research needs to be done on the effect of spagyric preparations with other plants species and their associated unique secondary metabolite profiles
· This work provides the first standardized quality assessment methodology for analysis of spagyric plant essences that can be employed in the future to assure the safety of spagyric medicines
Intro
Spagyric plant medicine represents a modern revival in Europe and the United States of an alternative natural medicine form that originates from a Paracelsian alchemical medicine modality. Plant spagyrics are often touted to have superior medicinal attributes compared to the conventional tincturing methods due to the inclusion of a plants endogenous nutritional mineral salts into the spagyric tincture which are said by Paracelsus to “activate” a plants specialized properties.
Thujo occidentalis, dubbed the ‘Tree of Life’ by Linnaeus is a hardwood cedar tree in the cupressaceae family. Occidentalis grows in US hardiness zones 2 through 7 and has become commonly cultivated as an ornamental tree and for the medicinal properties of its volatile oils, saps and bark.1 Occidentalis can be identified by its fan-like branches and flat scale like leaves and yellow-green to brownish slender seed cones. The bark is reddish-brown and peels off the tree in narrow longitudinal strips. It can grow anywhere from 10-20m tall, occasionally reaching 30m and has a trunk with about 0.4-1.6m in diameter at breast height. Oftentimes one can find mobs of deer resting under its branches and eating its leaves. A fresh leaf contains roughly 0.6% essential oils which contains predominantly thujone, isothujone, fenchone, sabines and alpha-pinen as the primarily expressed terpenoids.
The compound of interest for this experiment is the volatile medicinal and toxic compound thujone, a bicyclic monoterpenoid ketone that has enigmatic character in the literature.
Some studies suggest at certain concentrations thujone acts as a GABA-minergic receptor antagonist leading to excitability in the brain, while others suggest it is an agonist leading to neuro-inhibition. It also has activity at the 5HT3 receptor as a serotenergic antagonist with widely variable bioactivity (3). In addition, thujone exhibits low affinity for cannabinoid receptors. (4). Other studies have shown its Pro-apoptoptic and anti-angiogenic effects. It can be anticancer, antitumor, antiviral and mood elevating at lower doses, to neurotoxic, deliriating, and possibly psychoactive at higher doses.
Occidentalis is a cedar tree of which the leaves were used dominantly by the Ojibwe in medicinal and shamanic settings. They unknowingly used the multifaceted nature of this compound by employing the psychoactive affects through aroma-based applications as well as the medicinal affects through teas and tinctures. Today, it is mainly used in homeopathy as mother tincture or dilution with other synergetic herbs such as Echinacea purpurea, Echinacea pallida and Baptisia tinctorial (1).
Thujone has been used allopathically in clinical evidence-based phytotherapy for respiratory infections, as an adjuvant to antibiotics in severe bacterial infections, to treat the common cold, as antitumor, antirheumatic, anticancer, as a mood enhancer and other medicines (1). It was likewise chosen for this experiment due to the physical properties of the plant chemistry satisfying the core tenet of plant Paracelsian spagyria being the presence of essential oils within a plant. The traditional preparation of plant spagyrics is constituted of a tripartite essence containing essential oils, alcohols, and essential mineral salts from a singular plant species.
Thujone is regulated in commercial products, like the mythical absinthe, by the FDA due to its concentration dependent neurotoxic affects. Its use in traditional medicinal preparations, such as spagyria, open the door to novel alterations and configurations to the quantity and core structures of specialized phytochemicals which could potentially lead to alternative forms of bioactivity. Thujone containing plants have long been used in medicinal and shamanic practices across many cultures and can be found across a number of medicinal plants. Likewise, mineral salts originating from the earths crust are present as integral functional components of the primary metabolism and are essential inorganic nutrients to all carbon-based life.
Paracelsus suggested these mineral salts were vital to the structure and function of the plant (5). Studies show the majority of Americans are deficient in macro and trace minerals which leads to an array of diseases and significantly reduced quality of life. One study indicates 98% of Americans have a deficiency in the macronutrient Potassium alone. This is odd because Potassium should be a readily abundant mineral in our diet, being it is one of three of the most ubiquitous terrestrial plant minerals. Further, spagyrics often imply intra tincture reactions. The primary metabolic alkali plant salts potentially represent essential nutritional content which would not only contribute significantly to the medical efficacy of spagyric medicine, but the chemical constituency is often suggested to be altered (typically seen as an enhancement) in the process of combining purified essential oils, salts, and alcohols derived from singular plant species.
Through the inclusion of alkali plant salts, it is possible that the raw acidic phytocompounds present in essential oils, i.e. thujone, undergo acid-base reactions and pH changes, often leading to different physical characteristics (color changes, viscosity changes, miscibility changes) suggesting an alteration to the secondary metabolite profile, and thus further specialized bioactive properties as a result.
We investigated this hypothesis by collecting wild type occidentalis and creating an in-house spagyric by extracting the leaves according to the spagyric methodology put forth by Paracelsus (10) involving decoction, calcination, and salificaiton. We analyzed the treatment against a control without the salificaiton through two analyses. A chromatographic analysis quantized thujone via GC-MSMS and we further analyzed the proposed nutritional components via ICP-MS by quantizing the macro mineral Potassium (K) and qualitatively analyzing all other trace minerals of an occidentalis spagyric and the effectiveness of spagyric tincturing methodology in yielding nutritional content.
Methods
Fresh leaves of Thujo occidentalis were wild harvested from a cluster of trees around the east side of Lily Pond lake in Marquette, MI in early June. Leaves were immediately stored in 25F freezer for preservation until experiment was ready to be conducted. A Soxhlet extraction was employed to create a control tincture and a spagyric tincture was created via salificaiton on a fraction of the control tincture according to the spagyric SOP below. All samples were created in triplicate. All samples were prepared and analytical equipment was configured for analysis according to the GC-MS/MS SOP following the work of Alshishani et al and ICP-MS following the work of Ahmad, Ilyas, et al. Blanks were generated and standards were generated according to the schemes in the SOPs. Power of Hydrogen readings were recorded according to the pH SOP below for the control and the spagyric tincture to infer further understanding of the chemical changes. Figures of merit calculations will include: Limit of Detection, limit of quantification, accuracy and percent recovery generated and the total content of thujone, as well as Potassium in the control and spagyric tincture are declared in the results section.
Results
Power of Hydrogen
Control
5.611
Spagyric
6.154
Table A. Indicates the difference in pH between the control and spagyric tinctures of 0.543
Figure 1. Standard Curve displaying 0.5, 1, 5, 10, and 50 mg/L of Thujone standards. R-squared of 0.9993 and y = 3401.9x - 2659.7
Figure 2. Chromatogram of 100mg/mL Standard concentration of Thujone with peak appearing at 6.030 minutes
Figure 3. SIM mass spectra of the indicated standard peak displaying base peak parent ion of 152.25
Figure 4. Scan mass spectra of indicated peak showing target ion at 81 m/z with respective fragment ions, 95, 110, and 152 m/z.
Figure 5. Upper: Control tincture chromatogram displaying TIC and MIC with thujone peak indicated at 6.060 minutes. Lower: Zoomed in on TIC only of Control tincture chromatogram with thujone peak indicated at 6.060 minutes.
Figure 6. Mass spectral SIM of the above indicated peak with a parent ion displaying base peak at 152.25 m/z
Figure 7. Mass spectral SCAN of indicated peak displaying target ion 81 m/z and respective fragment ions of 95, 110, and 152 m/z
Figure 8. Upper: TIC and MIC chromatogram of spagyric tincture sample with thujone peak indicated at 6.062 minutes. Lower: Zoomed in TIC only chromatogram of spagyric tincture sample with thujone peak indicated at 6.062 minutes.
Figure 9. SIM mass specra of the indicated thujone peak with base peak parent ion at 152.25 m/z
Figure 10. SCAN mass spectra of the indicated peak displaying base peak target parent ion at 82 m/z and respective fragment ions 95, 110, 152 m/z peaks.
Statistical Analysis
ICP-MS Nutritional Analysis Report
Figure 11. DBN Calibration curve of Potassium standards 20, 100, 250 500, 1000 ppb with r^2 of 0.99986 and linear regression equation of C. = 0.0114371 * I – 61.22955
Figure 12. DBG Calibration curve of Potassium standards 20, 100, 250 500, 1000 ppb K with r^2 of 0.99986 and linear regression equation of C. = 2.042475 * X – 40.02927
Figure 13. Tables depicting qualitative assessment of trace metal analysis on controls tinctures, treated spagyric tincture and Yttrium blanks.
Figure 14. Tables depicting quantitative determination of Potassium (K) on control tinctures, spagyric tincture (treatment) and blanks.
Table 3. Displays the elemental complexity from a spagyric sample determined by qualitatively detected elements present in spagyric tincture with greater concentration in ug/L than in control tinctures (10x spagyric dilution accounted for). Green highlight represents a macro mineral, yellow represents trace minerals. Blue means not an essential nutrient but carries some form of bioactivity. Uncolored elements are non-essential. All elements were likewise present in the control, yet at 10x less estimated quantity. Mineral nutritional information taken from Olree et al (11).
Table 2. Figures of merit
Example equation for mg of K in whole treatment extract:
(Reported average value of K in spagyric sample from table * dilution factor of 10) = Average K PPM in sample
239.33 * 10 = 2393.33 PPM of K
Average K PPM in sample / 1000 = Average K mg/mL
2393.33 / 1000 = 2.393 mg of K/mL of extract
Mg/mL of K in extract * total number of mL in extract = total mg in whole extract
2.393 * 47 = 112.33 mg in whole spagyric sample extract
The control was not diluted 10x thus there is no correction for its reported values. All other sample preparations were the same between the two.
Discussion
Our two-part hypothesis was both rejected and failed to be rejected. The famous swiss physician Paracelsus suggested that spagyrically extracted medicinal plant salts were integral to the medicinal functioning of a plant. His alchemical botanical world view posited this in a supernatural manner, suggesting somehow the salts, derived from calcination, (or purified plant ‘body’ in his view) potentiated and actuated the inherent medicinal properties of the plant thus exalting its ‘spirit and soul’, or specialized metabolic profile, also referred to as ‘Sulphur and Mercury’ within alchemical allegory, into a superior refined quintessence. This suggests to the modern chemist, an alteration to the chemical constituency of the final tincture (spagyric). This is in contrast to plant essences of his contemporaries which consisted primarily of only the spirit and soul, or the entourage of specialized metabolites of plants, acquired through tincturing and decoction alone. What Paracelsus did not know was that the salts, which represent a major aspect of plants primary metabolite profile, likewise signified essential nutritional minerals which are necessary for proper functioning of all carbon-based life. As the infamous Dr. Linus Pauling stated, “All disease can be linked to a mineral [nutritional] deficiency”. One study suggested this is “specially so in a world population that is tending to divide itself into the underfed in the developing countries and the badly fed in the industrialized countries" Falquet Jacques et al. Nutritional related disease are one of the greatest threats facing the wellbeing of our world, studies published over the years have suggested upwards of 97% of Americans are deficient in the macro nutrient Potassium alone (9) thus contributing to a range of disorders and homeostatic imbalances. Congress has known since 1930’s of depleted mineral content in farmland and food stuffs from the grocery industrial complex. These shortages are leading to an incrementally slow starvation of those dependent on the western food supply chains or to those without centralized nutritional food sources. This study rejected the Paracelsian hypothesis that the addition of plant salts to their respective tincture have any significant effects on the specialized properties of the tincture, solely for our singular example of Thujo occidentalis. This experiment determined there was no significant effect on the presence or concentrations of the main specialized metabolite, thujone, in a spagyric preparation from Thujo occidentalis. However, we failed to reject the hypothesis that the spagyric tincturing methodology described by Paracelsus (10) hypothesized as an effective means of supplementing nutritional content into a plant medicine tincture. We found a 135.2% difference in the quantifiable amount of Potassium between the spagyric and the control tinctures.
A considerable amount of qualifiable data from the ICP analysis suggests the presence of a diverse profile of essential macro and micro minerals further supporting our hypothesis that spagyric plant tincturing is an effective method for extracting essential mineral nutrition from plants. The addition of this mineral salt entourage into the Thujo occidentalisspagyric tincture showed there was no significant alteration in quantity or chemical structure to the core specialized metabolite, thujone. This suggests Thujo occidentalis spagyric plant medicine may be used as a novel phytotherapy which employs both the specialized pharmacological action of thujone and simultaneously serving as a macro and micro mineral supplement which can have significant contributions to the long-term health, recovery from illness, active prophylaxis and metabolic homeostasis. Only when used in appropriate dose dependent manner, as thujone is readily a deliriant and potentially neurotoxic. We can infer that the methods described in the SOP adapted from Paracelsus can be applied to land-based plants to generate plant medicine supplements with significant nutritional value. Spagyric plant medicines lack a standardized approach to quality and/or nutritional assessment. With their popularity exploding in modern herbalist circles around the US and especially Europe, there should be a call for further analysis and a standardized method for quality assessment. Due to the radical chemical diversity and complexity of different plant primary and secondary metabolic profiles and the growing proponents of spagyric medicine, we suggest that further research needs to be conducted on other species and accessions of spagyric plant medicines to more accurately determine the effects on the secondary metabolite profile resulting from the spagyric tincturing of medicinal plants.
SOPs
*Warning*
Thujone is neurotoxic in high concentrations. Use care when handling.
*Warning*
Potassium is flammable and corrosive. Wear PPE when handling.
Harvesting of Thuja occidentalis
1. Find a healthy and balanced ecology that has Thujo occidnetalis specimens within it.
2. Harvest healthy leaves in the early morning before the sunrise from multiple different species.
3. Immediately place freshly harvested leaves into storage vessel (vaccum sealed bag) and place into freezer until ready for further preparation of samples.
Control and Spagyric Tincture Preparation
1. Wearing gloves to avoid contamination, homogenize the Thuja occidentalis sample into a fine powder.
2. Mass out 5-10 grams of Thuja occidentalis sample into a beaker and record and label accordingly.
3. Using a 20:1 volume mL/weight g solvent to herb material ratio, pour solvent over Thuja occidentalis samples and seal the top with parafilm.
*For example, if 5 grams of occidnetalis sample was measured in beaker, add 100mL of 95% EtOH solvent, if 6 grams, at 120 mL, etc.*
4. Assemble the Soxhlet apparatus.
5. Pour solvent with plant material into the thimble, allowing for the siphon to prime. *Add filter at the bottom of the thimble.*
6. Turn on heat so that solvent begins distilling at 78.2C. Run Soxhlet until the solvent runs clear through the siphon.
7. When solvent runs clear, remove the plant matter, disassemble the apparatus, place a funnel on top of the boiling flask containing the extract and press the plant matter through a coffee filter to reclaim any left over solvent absorbed in the plant matrix.
8. Filter the extract and separate it into two equal fractions, label one control and the other spagyric.
9. Take the solid plant matter and place it into a crucible and begin calcination.
10. Calcine the material between 260-550C until ash turns pure white. Usually 7-24 hours depending on temperature.
11. Remove the plant material/ash from the oven and crucible after about 6 hours and grind the plant matter with a mortar and pestle to encourage calcination.
12. Place material back into crucible then calcine again until plant material turns pure white, indicating total calcination and absence of carbon.
13. Place white calcined ash into small glass dish and pour water ~3 times its volume over the ash in order to dissolve the salts.
14. Filter the insoluble minerals out and then slowly dissolve the filtered water in order to crystallize the mineral salts.
15. When crystalline salts appear, collect them and pulverize them to a fine powder in a mortar and pestle, being careful not to spill any.
16. Add the purified and crushed mineral salts into the tinctures labeled “spagyric”.
17. Add the “spagyric” to a reflux apparatus and reflux the spagyric solution for ~4 hours on low heat (~65C).
18. Remove the spagyric tincture from the apparatus and put in a secure storage vessel away from light and label and date until they are ready for sample preparation and analysis.
GCMS Sample Preparation and Analysis Conditions
1. Remove the control and spagyric tinctures from storage and follow the below steps, repeating twice for processing both the control and the spagyric.
2. Ascertain a rotovap flask and annotate its weight.
3. Remove 1 mL of the tincture and place into the rotovap flask in order to reclaim the ethanol solvent. Remove the solvent from the tincture.
4. Weigh and record the tincture residue.
5. Add 10 times its weight in mL of GC grade MeOH and stir until fully dissolved., then transfer one mL of the sample into a GC vial.
6. Prepare the GCMS instrument conditions and gradient scheme according to these settings:
Employing a 30m long capillary column with 0.25mm inner diameter and 0.25um film thickness. A 10:1 split ratio and 1uL injection volume, Nitrogen carrier gas, with 1.5mL/mon flow rate, injector temperature set to 300C and initial oven temperature of 70C, held for 3 min, increased at 20C.min to 190C held for two minutes. MS will be set on EI (electron ionization mode) with ion source temperature at 250C. Mass spectra scanning range from 35 – 360 m/z.
7. Prepare a blank, collect the standards and the control and spagyric tincture samples and then inject the samples into the GC in triplicate.
ICPMS Sample Preparation and Analysis Parameters
(Repeat this process for both control and spagyric tincture sample prep.)
Microwave Digestion
1. Take 3 mL of the tincture and add it directly to a digestion vessel.
2. Add 5 mL of 65% HNO3 and 2 mL of 30%H2O2.
3. Cover samples and place into microwave digestor. Set digestion program at 1 min at 250 W, 1 min at 0 W, 5 min at 250 W, 5 min at 400 W, and 5 min at 600 W.
4. After cooling, transfer digested samples to volumetric flask and dilute to 50 mL using de ionized water and finally transfer to a 50 mL polyethylene flask.
ICPMS
5. Set the ICPMS to the following conditions:
(ICP-MS, 7900, Agilent) instrument with the following condition/parameters; Nebulizer gas-flow: ∼1 L min−1, Auxiliary gas-flow: ∼1 L min−1, Plasma gas-flow: ∼15 L min−1, Helium (He) gas-flow in Reaction Cell: ∼0.2 mL min−1, Reflected power: ∼45 W, Forward power: ∼1500 W, Analyzer vacuum: ∼6 × 10-5, Detector: EM, Replicates: 3, Sweeps/replicate: 100.
6. Inject the control and experimental spagyric tincture, blanks, and standards into the ICPMS.
Thujone Standards
1. Vortex each sample after it is prepared and store the samples in the 4C refrigerator after they are made.
2. Prepare the following thujone standards at concentrations of 100 mg/L, 50 mg/L, 10 mg/L, 5 mg/L, 1 mg/L, and 0.5 mg/L.
Potassium (K) Standards
1. Prepare the following Potassium standard using the concentration volume equation C1 * V1 = C2 * V2 at the following concentrations:
(10, 20, 50, 100 and 200 μg L−1).
These standard concentrations were based on the work of Serife, T. et al. and Ahmad et al.
(1) Naser, B., Bodinet, C., Tegtmeier, M., & Lindequist, U. (2005). Thuja occidentalis(Arbor vitae): A Review of its Pharmaceutical, Pharmacological and Clinical Properties. Evidence-Based Complementary and Alternative Medicine, 2(1), 69–78. doi:10.1093/ecam/neh065 10.1093/ecam/neh065
(2) Thuja occidentalis / eastern arborvitae | Conifer Species. American Conifer Society. https://conifersociety.org/conifers/thuja-occidentalis/ (accessed 2023-04-28).
(3) T. Deiml, R. Haseneder, W. Zieglgänsberger, G. Rammes, B. Eisensamer, R. Rupprecht, G. Hapfelmeier, α-Thujone reduces 5-HT3 receptor activity by an effect on the agonist-induced desensitization, Neuropharmacology, Volume 46, Issue 2, 2004, Pages 192-201, ISSN 0028-3908, 9. (https://www.sciencedirect.com/science/article/pii/S0028390803003940)
(4) Meschler, Justin P., and Allyn C. Howlett. "Thujone exhibits low affinity for cannabinoid receptors but fails to evoke cannabimimetic responses." Pharmacology Biochemistry and Behavior 62.3 (1999): 473-480.
(6) Alshishani, Anas Adib, et al. "Salting-out assisted liquid-liquid extraction method coupled to gas chromatography for the simultaneous determination of thujones and pulegone in beverages." International journal of food properties 20.sup3 (2017): S2776- S2785.
(7) Ahmad, Ilyas, et al. "ICP-MS based analysis of mineral elements composition during fruit development in Capsicum germplasm." Journal of Food Composition and Analysis 101 (2021): 103977.
(8) Falquet, Jacques, and J. P. Hurni. "The nutritional aspects of Spirulina." Antenna Foundation (1997)
(9) Fulgoni VL, III, Keast DR, Bailey RL, Dwyer J. Food fortificants, and supplements: where do Americans get their nutrients? J Nutr. 2011;141:1847–54.
(10) A. E. Waite - The Hermetic Writings Paracelsus Vol I. Concerning the Nature of Things, The Separation of Vegetables. pg. 167-168
(11) Olree, R. “Minerals for the Genetic Code”. 3rd ed.;Mark R. Andersun Pub.: Falcon
