Cannabis extracts were produced from a single proprietary cultivar using previously improved and widely used commercial extraction methods, including alcoholic extraction with ethanol and isopropanol and supercritical fluid CO2 extraction. For the latter, two fractions, S1 and S2, were prepared for analysis, each corresponding to a distinct extraction pressure setting. To guarantee wide chemical coverage, each extract was examined using a variety of complimentary analytical techniques. In total, 41 compounds were identified and annotated using gas chromatography mass spectrometry (GC–MS) in a non-targeted profiling approach, 15 phytocannabinoids were evaluated using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in a qualitative targeted assay, and 24 elements were quantified using inductively coupled plasma mass spectrometry (ICP–MS) (ICP-MS).
Principal Component Analysis (PCA) was used to analyze the chemicals discovered by GC–MS, revealing significant variance in the overall chemical profiles between samples depending on the extraction method used (Fig. 1). Of the 41 identified chemicals found by GC–MS, 33 were substantially different between at least two of the extracts (Table S1, Fig. 2; p 0.05 after Tukey post-hoc multiple comparison testing). Numerous long chain fatty acids, polyols, and carbohydrates, as well as sesqui-, tri-, and diterpenoids, are included in these molecules (Table S2). Compound annotations were determined using spectral databases rather than authentic standards. Thus, when structural isomers cannot be identified, annotations are assigned a numerical value (Fig. 2, e.g. pinitol). The statistics demonstrate considerable variances that may result in a variation in the product’s therapeutic potential (Fig. 2). For instance, sitosterol was substantially more abundant in the S1 and S2 fractions than in the IPA and EtOH fractions. This molecule is recognized to have numerous health benefits and is currently being investigated as a potential cancer preventive and treatment strategy, as well as an anticholesteremic agent32. Bisabolol was substantially more abundant in the IPA and EtOH extracts than in the S1 and S2 extracts. This substance is well-known for its anti-inflammatory and antimicrobial properties33. Palmitoleic acid is an anti-inflammatory carboxylesterase inhibitor34 that was abundant in the IPA, EtOH, and S2 extracts. Campesterol was found to be more abundant in the S1 and S2 extracts than in IPA and EtOH. Campesterol and other plant sterols are cholesterol-lowering compounds35 that may help prevent cancer36.
Principal Component Analysis (PCA) of substances discovered by gas chromatography–mass spectrometry (GC–MS) demonstrates the overall diversity in chemical profiles generated from each extraction. The first main component accounted for the lion’s share of variation (76%) (PC1). Green represents the ethanol extract; blue represents the isopropanol extract; and purple and yellow represent the supercritical CO2 fractions S1 and S2, respectively. Using Hotelling’s T2, the ellipse implies 95 percent confidence.
The heatmap depicts the chemicals identified and annotated by GC–MS that were significantly different in abundance throughout the four extracts (p 0.05 after Tukey post-hoc testing for multiple comparisons). Green represents the ethanol extract; blue represents the isopropanol extract; and purple and yellow represent the supercritical CO2 fractions S1 and S2, respectively.
While cannabinoids were found using GC–MS (Table S1), the uncontrolled decarboxylation occurring in the ionization source hinders interpretation; hence, a supplementary analysis utilizing a targeted UPLC–MS/MS assay was undertaken. 14 of the 15 phytocannabinoids discovered in this analysis were shown to be substantially different between extracts (Fig. 3, Table S3; p 0.05 after Tukey post-hoc testing for multiple comparisons).
The quantity of phytocannabinoids differed considerably by extraction method (p 0.05 after Tukey post-hoc testing for multiple comparisons). Between extractions, box plots illustrate the relative quantity of each component. Green represents the ethanol extract; blue represents the isopropanol extract; and purple and yellow represent the supercritical CO2 fractions S1 and S2, respectively.
In general, the EtOH and IPA extracts contain a greater concentration of phytocannabinoids (including CBD and 9THC) than the supercritical CO2 fractions (S1 and S2). A significant exception from this pattern was detected for CBDA, which was found in the highest concentrations in the supercritical CO2 S1 extract. This finding could be due to insufficient decarboxylation of CBDA to CBD, which was accomplished by heating the dried plant material prior to supercritical CO2 extraction. This is in contrast to the procedure employed when extracting with EtOH and IPA, which involved post-extraction decarboxylation.
Notably, in addition to variances in major phytocannabinoids, significant changes in a variety of under-researched minor phytocannabinoids were discovered. For instance, CBC was substantially more prevalent in the IPA and EtOH fractions than S1. CBC functions as a CB2 receptor agonist, exerting analgesic and anti-inflammatory properties37,38. CBC has already been implicated as a possible antidepressant in in vivo studies46. Additionally, as revealed in an ex vivo investigation utilizing isolated neurons from rats47, CBC can function as an agonist for TRPA1 channels. Additionally, it has been suggested to act as an analgesic for pain originating from efferent neuronal pathways39.
The greatest changes in abundance (more in EtOH and IPA than in S1 and S2) were identified for CBT, a minor phytocannabinoid detected in trace amounts in cannabis cultivars. Surprisingly, this chemical is also identified in one species of rhododendron, the particular variety utilized in traditional Chinese medicine to treat bronchitis and other respiratory ailments48. CBT was observed to lower intraocular pressure in rabbits in one of the few in vivo investigations conducted to date, implying that CBT may be a possible therapy for glacuoma49.
Cannabis plants have a large root system, which enables efficient mineral intake from the soil. Cannabis products, particularly seed extracts, have been found to be an excellent source of micro- and macronutrients such as P, K, Mg, Ca, Fe, Zn, Cu, and Mn. Apart from absorbing beneficial nutrients, cannabis plants can also be used to intentionally phytoremediate soil contaminated with hazardous heavy metals40. We profiled 24 elements using ionomics, including nutrients, minerals, and hazardous heavy metals. Ten elements (B, K, Mg, Mn, Na, Ni, S, Sr, P, and Mo) were significantly different in abundance between the four extracts, and all of them were more abundant in EtOH and/or IPA than in supercritical CO2 fractions (Fig. 4, Table S4). Notably, none of the dangerous heavy metals (Cd, Pb, and As) were significantly affected by the extraction procedure, and none were identified above the state of California’s statutory levels.
Significant differences in concentrations across elements according to extraction method (p 0.05 following Tukey post-hoc testing for multiple comparisons). Green represents the ethanol extract; blue represents the isopropanol extract; and purple and yellow represent the supercritical CO2 fractions S1 and S2, respectively.
Taken together, the data demonstrate the critical role of the extraction process in determining the overall chemical profile of the final extract, which will define the product’s potential bioactivity. This is especially true in the production of so-called “full spectrum,” “whole plant,” and/or “wide spectrum” extracts, which are gaining popularity as consumer items but lack evidence of therapeutic efficacy. While detecting and characterizing all possible molecular components in a complex matrix remains a significant challenge41, the approaches used in this study provide a useful snapshot of the major chemical constituents of these extracts and lay the groundwork for future studies examining the effect of extraction method on the therapeutic potential of full spectrum products. Notably, these findings highlight the importance of increased transparency and regulation regarding the labeling of full spectrum cannabis products in order to safeguard consumers and facilitate proper interpretation and comparison of multi-component goods.