Journal:The impact of extraction protocol on the chemical profile of cannabis extracts from a single cultivar

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Full article title The impact of extraction protocol on the chemical profile of cannabis extracts from a single cultivar
Journal Scientific Reports
Author(s) Bowen, Janina K.; Chaparro, Jacqueline M.; McCorkle, Alexander M.; Palumbo, Edward; Prenni, Jessica E.
Author affiliation(s) Colorado State University, Charlotte’s Web Inc.
Primary contact jprenni at colostate dot edu
Year published 2021
Volume and issue 11
Article # 21801
DOI 10.1038/s41598-021-01378-0
ISSN 2045-2322
Distribution license Creative Commons Attribution 4.0 International
Website https://www.nature.com/articles/s41598-021-01378-0
Download https://www.nature.com/articles/s41598-021-01378-0.pdf (PDF)
Emblem-important-yellow.svg This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed.

Abstract

The last two decades have seen a dramatic shift in cannabis legislation around the world. Cannabis products are now widely available, and commercial production and use of phytocannabinoid products is rapidly growing. However, this growth is outpacing the research needed to elucidate the therapeutic efficacy of the myriad of chemical compounds found primarily in the flower of the female Cannabis plant. This lack of research and corresponding regulation has resulted in processing methods, products, and terminology that are variable and confusing for consumers. Importantly, the impact of processing methods on the resulting chemical profile of full spectrum cannabis extracts is not well understood. As a first step in addressing this knowledge gap, we have utilized a combination of analytical approaches to characterize the broad chemical composition of a single cannabis cultivar that was processed using previously optimized and commonly used commercial extraction protocols, including alcoholic solvents and supercritical carbon dioxide. Significant variation in the bioactive chemical profile was observed in the extracts resulting from the different protocols, demonstrating the need for further research regarding the influence of processing on therapeutic efficacy, as well as the importance of labeling in the marketing of multi-component cannabis products.

Keywords: Cannabis, processing methods, extract, cultivar, chemical analysis

Introduction

Cannabis sativa L. is a pharmacologically important annual plant that produces bioactive phytocannabinoids and other secondary metabolites that have demonstrated therapeutic potential for a wide variety of human health conditions.[1][2][3][4][5] Cannabis sativa L. can be broadly divided into three categories based on genomic diversity and chemical composition.[6] Specifically, based on the analysis of 340 cannabis varieties—including grain hemp, fiber hemp, CBD hemp, marijuana, and feral populations—the distinct groups were described as:

  • fiber/grain hemp with low cannabinoid content;
  • cannabis with narrow leaflets (colloquially described as sativa) and high cannabinoid content (i.e., CBD hemp and marijuana); and
  • cannabis with broad leaflets (colloquially described as indica) and high cannabinoid content (i.e., CBD hemp and marijuana).

Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most extensively studied Cannabis sativa L.-derived phytocannabinoids and are the only compounds currently available by prescription in the United States.[7] In addition to these two major neutral phytocannabinoids, acidic versions such as tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), and cannabichromenic acid (CBCA); minor versions such as cannabigerol (CBG), cannabinol (CBN), and cannabichromene (CBC) ; and varinic versions such as tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), and cannabigerovarin (CBGV) have also exhibited promising in vitro and in vivo results for treatment of various human health conditions.[4] For example, as reviewed by Franco et al.[4], there is preliminary evidence that these understudied bioactive compounds have anti-inflammatory, anti-microbial, anti-proliferative, anti-convulsive, and neuroprotective properties. Furthermore, these minor phytocannabinoids are emerging as potential treatment strategies for anxiety, nausea, diabetes, acne, metabolic syndrome, obesity, pain, colorectal cancer, breast cancer, and more. Finally, in addition to phytocannabinoid compounds, there are a multitude of other bioactive compounds found in cannabis, including terpenes and terpenoids[8][9][10][11][12], flavonoids[13][14], bibenzyl[15], stilbenoids[16][17], and hydroxycinnamic acids.[18][19]

There is a growing body of work exploring cannabis polypharmacy in terms of potential synergistic effects, commonly referred to as the entourage effect, that may contribute to or modulate the therapeutic properties of cannabis extracts. Synergistic effects have been proposed in research exploring combinations of phytocannabinoids[20][21], as well as other bioactive secondary metabolites such as terpenes and/or terpenoids.[22][23] This has also been shown with human endocannabinoids in vitro, though in vivo studies are notably lacking. For example, it has been demonstrated that the endocannabinoid 2-arachidonoylglycerol shows enhanced activity in the presence of 2-acylglycerol esters, which alone are inactive.[24] This effect has also been noted for organisms other than cannabis. Combining multiple terpenes from a tropical Amazonian plant was demonstrated to have a synergistic effect that was more toxic to a parasite than the terpenes alone[12], and combining multiple terpenes was more effective at inhibiting growth of a protozoa than the terpenes alone.[25] Conversely, there is also some evidence suggesting that cannabis polypharmacy could results in negative interactions or potential toxicity.[26]

Many distillate and isolate products are readily available to consumers, including those containing CBD, CBDV, CBC, CBG, CBGA, CBN, Δ8-THC, Δ9-THC, and THCV. All but Δ9-THC products can be purchased online and shipped anywhere in the United States. There has also been a surge in marketing of so called "full spectrum" products which capitalize on the idea of the synergistic entourage effect. However, because these products are not regulated by the U.S. Food and Drug Administration (FDA) as dietary supplements, there is not a clear definition of what denotes a high-quality cannabis product, and phrases such as "whole plant," "full spectrum," and "broad spectrum" further muddy the waters for consumers.

The composition of a commercial cannabis extract will in large part be determined by the genetics of the starting plant material.[27] While previous work has evaluated extraction parameters with the goal of optimizing recovery of the major phytocannabinoids[28][29][30] (28,29,30), the impact of processing methodology on the comprehensive composition of full spectrum extracts is not well understood. Commonly utilized commercial extraction approaches include the use of alcoholic solvents (e.g., ethanol and isopropanol) to more advanced technologies using supercritical carbon dioxide (CO2). Solvent extraction represents the lowest cost option; however, this method runs the risk of leaving behind trace organic solvent contamination. This is more of a concern with hydrocarbon solvents such as methanol, acetone, and butane, which are toxic for human consumption. Extraction with supercritical CO2 requires investment in specialized equipment but has multiple advantages, including “tunability” by modifying temperature and pressure conditions for more precise extraction, the potential reuse of CO2[31], and the lack of any residual solvents.

Given the lack of research into the therapeutic effects of phytocannabinoids and other bioactive secondary metabolites in humans, coupled with the potential for synergistic effects and variability in commercial processing methods, there is a critical need for additional research to characterize the numerous chemical compounds found in cannabis extracts and how this chemical profile is impacted by production choices. As a first step, we have conducted a comprehensive qualitative chemical analysis of cannabis extracts from a single high-CBD cultivar generated using three previously optimized commercial extraction protocols. The results of this study lay the groundwork for evaluation of the impact of processing method on chemical variation in full-spectrum consumer products and represent an important step towards enabling industry standardization.

Results and discussion

Cannabis extracts were generated from a single proprietary cultivar using previously optimized and commonly used commercial extraction procedures, including alcoholic extraction with ethanol and isopropanol and extraction with supercritical CO2. For the latter, two fractions were generated for analysis, S1 and S2, corresponding to different pressure settings during the extraction. Each extract was analyzed using a combination of complementary analytical approaches to ensure broad chemical coverage. Overall, 41 compounds were detected and annotated by gas chromatography–mass spectrometry (GC–MS) using a non-targeted profiling approach, 15 phytocannabinoids were evaluated by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) using a qualitative targeted assay, and 24 elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS).

Principal component analysis (PCA) was performed for the compounds detected by GC–MS, demonstrating that there is significant variation in the overall chemical profiles between samples based on extraction method (Figure 1). Of the 41 annotated compounds detected by GC–MS, 33 were significantly different between at least two of the extracts (Supplementary Information 1, Table S1; Figure 2) (p < 0.05 after Tukey post-hoc testing for multiple comparison). These compounds include multiple long chain fatty acids, polyols and carbohydrates, and sesqui-, tri-, and diterpenoids (Supplementary Information 1, Table S2). Compound annotations were determined based on searching against spectral databases rather than comparison to authentic standards. Thus, when structural isomers could not be distinguished, annotations are denoted with a number (e.g., pinitol in Figure 2). The data reveal significant differences that could result in variation of therapeutic potential of the product (Figure 2). For example, sitosterol was significantly enriched in the S1 and S2 fractions as compared to IPA and EtOH. This compound has multiple known health benefits and is used as a potential prevention and therapy for treatment of cancer and as an anticholesteremic drug.[32] Bisabolol was significantly enriched in the IPA and EtOH extracts as compared to S1 and S2. This compound has known anti-inflammatory and anti-microbial properties.[33] Palmitoleic acid is a carboxylesterase inhibitor that has anti-inflammatory effects[34] that was enriched in the IPA, EtOH, and S2 extracts. Campesterol was enriched in the S1 and S2 extracts compared to IPA and EtOH. Plant sterols such as campesterol are cholesterol-lowering compounds[35] that may act in cancer prevention.[36]


Fig1 Bowen ScientificReports21 11.png

Figure 1. Principal component analysis (PCA) for compounds detected by GC–MS, demonstrating the overall variation in chemical profiles generated from each extraction. The majority of variation (76%) was observed in the first principal component (PC1). Green = ethanol extract; Blue = isopropanol extract; Purple and Yellow = supercritical CO2 fractions S1 and S2, respectively. Ellipse indicates 95% confidence using Hotelling’s T2.

Fig2 Bowen ScientificReports21 11.png

Figure 2. Heatmap of the compounds detected and annotated by GC–MS, showing significantly different in abundance across the four extracts (p < 0.05 after Tukey post-hoc testing for multiple comparisons). Green = ethanol extract; Blue = isopropanol extract; Purple and Yellow = supercritical CO2 fractions S1 and S2, respectively.

While cannabinoids were detected by GC–MS (Supplementary Information 1, Table S1), the unregulated decarboxylation that occurs in the ionization source complicates interpretation; thus, a complementary analysis was performed using a targeted UPLC-MS/MS assay. From this analysis, 14 of the 15 detected phytocannabinoids were significantly different across the extracts (Supplementary Information 1, Table S3; Figure 3) (p < 0.05 after Tukey post-hoc testing for multiple comparison).


Fig3 Bowen ScientificReports21 11.png

Figure 3. Phytocannabinoids significantly different in abundance by extraction method (p < 0.05 after Tukey post-hoc testing for multiple comparisons). Box plots indicate relative abundance of each compound between extractions. Green = ethanol extract; Blue = isopropanol extract; Purple and Yellow = supercritical CO2 fractions S1 and S2, respectively.

In general, there is a trend of higher abundance of phytocannabinoids (including CBD and Δ9-THC) in the EtOH and IPA extracts compared to the supercritical CO2 fractions (S1 and S2). A notable break in this trend was observed for CBDA, which was observed to be in highest abundances in the supercritical CO2 S1 extract. This result could reflect incomplete decarboxylation of CBDA to CBD, which was performed by heating the dried plant material prior to extraction with supercritical CO2. This is in contrast to the process used when extracting by EtOH and IPA, where decarboxylation was performed in the liquid phase post extraction.

Interestingly, in addition to differences in the major phytocannabinoids, significant differences were observed for multiple under-researched minor phytocannabinoids. For example, CBC was observed to be significantly more abundant in the IPA and EtOH fractions as compared to S1. CBC acts as a CB2 receptor agonist that has anti-nociceptive and anti-inflammatory effects.[37][38] CBC has been implicated as a potential anti-depressant in previous in vivo studies.[39] Furthermore, CBC can act as an agonist for TRPA1 channels, as demonstrated in an ex vivo study using isolated nerves from rats.[40] It has also been implicated as an analgesic for pain localized on efferent neural pathways.[41]

The largest differences in abundance (higher in EtOH and IPA as compared to both S1 and S2) were observed for CBT, a minor phytocannabinoid found in cannabis varieties at trace levels. Intriguingly, this compound is also found in one species of rhododendron, the specific type used in traditional Chinese medicine to treat bronchitis and other respiratory ailments.[42] In one of the only in vivo studies to date, CBT was found to decrease the intraocular pressure in rabbits, suggesting CBT as a potential therapeutic for glacuoma.[43]

Cannabis plants have a wide root system, which can facilitate efficient uptake of elements from the soil. Cannabis products, in particular seed extracts, have been shown to be a good source of both micro- and macro-elements, including phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn). In addition to the absorption of beneficial nutrients, cannabis plants can also be exploited for the intentional phytoremediation of toxic heavy metals from soil.[44] Here, we performed ionomics profiling of 24 elements, including nutrients, minerals, and toxic heavy metals. Ten elements—boron (B), K, Mg, Mn, sodium (Na), nickel (Ni), sulfur (S), strontium (Sr), P, and molybdenum (Mo)—were significantly different across the four extracts, and all of these were higher in abundance in EtOH and/or IPA as compared to the supercritical CO2 fractions (Supplementary Information 1, Table S4; Figure 4). Importantly, none of the toxic heavy metals—cadmium (Cd), lead (Pb), and arsenic (As)—were significantly impacted by extraction method, and none were detected above regulatory levels set by the state of California.


Fig4 Bowen ScientificReports21 11.png

Figure 4. Elements significantly different in concentration by extraction method (p < 0.05 after Tukey post-hoc testing for multiple comparisons). Green = ethanol extract; Blue = isopropanol extract; Purple and Yellow = supercritical CO2 fractions S1 and S2, respectively.

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  32. ↑ Rudkowska, Iwona; AbuMweis, Suhad S.; Nicolle, Catherine; Jones, Peter J.H. (1 October 2008). "Cholesterol-Lowering Efficacy of Plant Sterols in Low-Fat Yogurt Consumed as a Snack or with a Meal" (in en). Journal of the American College of Nutrition 27 (5): 588–595. doi:10.1080/07315724.2008.10719742. ISSN 0731-5724. http://www.tandfonline.com/doi/abs/10.1080/07315724.2008.10719742. 
  33. ↑ Kamatou, Guy P. P.; Viljoen, Alvaro M. (1 January 2010). "A Review of the Application and Pharmacological Properties of α-Bisabolol and α-Bisabolol-Rich Oils" (in en). Journal of the American Oil Chemists' Society 87 (1): 1–7. doi:10.1007/s11746-009-1483-3. ISSN 0003-021X. http://doi.wiley.com/10.1007/s11746-009-1483-3. 
  34. ↑ de Souza, Camila Oliveira; Valenzuela, Carina A.; Baker, Ella J.; Miles, Elizabeth A.; Rosa Neto, José C.; Calder, Philip C. (1 October 2018). "Palmitoleic Acid has Stronger Anti-Inflammatory Potential in Human Endothelial Cells Compared to Oleic and Palmitic Acids" (in en). Molecular Nutrition & Food Research 62 (20): 1800322. doi:10.1002/mnfr.201800322. https://onlinelibrary.wiley.com/doi/10.1002/mnfr.201800322. 
  35. ↑ Heggen, E.; Granlund, L.; Pedersen, J.I.; Holme, I.; Ceglarek, U.; Thiery, J.; Kirkhus, B.; Tonstad, S. (1 May 2010). "Plant sterols from rapeseed and tall oils: Effects on lipids, fat-soluble vitamins and plant sterol concentrations" (in en). Nutrition, Metabolism and Cardiovascular Diseases 20 (4): 258–265. doi:10.1016/j.numecd.2009.04.001. https://linkinghub.elsevier.com/retrieve/pii/S0939475309000957. 
  36. ↑ Shahzad, Naiyer; Khan, Wajahatullah; Md, Shadab; Ali, Asgar; Saluja, Sundeep Singh; Sharma, Sadhana; Al-Allaf, Faisal A.; Abduljaleel, Zainularifeen et al. (1 April 2017). "Phytosterols as a natural anticancer agent: Current status and future perspective" (in en). Biomedicine & Pharmacotherapy 88: 786–794. doi:10.1016/j.biopha.2017.01.068. https://linkinghub.elsevier.com/retrieve/pii/S0753332216324052. 
  37. ↑ Udoh, Michael; Santiago, Marina; Devenish, Steven; McGregor, Iain S.; Connor, Mark (1 December 2019). "Cannabichromene is a cannabinoid CB 2 receptor agonist" (in en). British Journal of Pharmacology 176 (23): 4537–4547. doi:10.1111/bph.14815. ISSN 0007-1188. PMC PMC6932936. PMID 31368508. https://onlinelibrary.wiley.com/doi/10.1111/bph.14815. 
  38. ↑ Zurier, Robert B.; Burstein, Sumner H. (1 November 2016). "Cannabinoids, inflammation, and fibrosis" (in en). The FASEB Journal 30 (11): 3682–3689. doi:10.1096/fj.201600646R. ISSN 0892-6638. https://onlinelibrary.wiley.com/doi/abs/10.1096/fj.201600646R. 
  39. ↑ Broeckling, Corey D.; Ganna, Andrea; Layer, Mark; Brown, Kevin; Sutton, Ben; Ingelsson, Erik; Peers, Graham; Prenni, Jessica E. (20 September 2016). "Enabling Efficient and Confident Annotation of LC−MS Metabolomics Data through MS1 Spectrum and Time Prediction" (in en). Analytical Chemistry 88 (18): 9226–9234. doi:10.1021/acs.analchem.6b02479. ISSN 0003-2700. https://pubs.acs.org/doi/10.1021/acs.analchem.6b02479. 
  40. ↑ Chong, Jasmine; Wishart, David S.; Xia, Jianguo (1 December 2019). "Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis" (in en). Current Protocols in Bioinformatics 68 (1). doi:10.1002/cpbi.86. ISSN 1934-3396. https://onlinelibrary.wiley.com/doi/10.1002/cpbi.86. 
  41. ↑ Xiong, Wei; Cui, Tanxing; Cheng, Kejun; Yang, Fei; Chen, Shao-Rui; Willenbring, Dan; Guan, Yun; Pan, Hui-Lin et al. (4 June 2012). "Cannabinoids suppress inflammatory and neuropathic pain by targeting α3 glycine receptors" (in en). Journal of Experimental Medicine 209 (6): 1121–1134. doi:10.1084/jem.20120242. ISSN 1540-9538. PMC PMC3371734. PMID 22585736. https://rupress.org/jem/article/209/6/1121/41297/Cannabinoids-suppress-inflammatory-and-neuropathic. 
  42. ↑ Johnson, Sarah A; Prenni, Jessica E; Heuberger, Adam L; Isweiri, Hanan; Chaparro, Jacqueline M; Newman, Steven E; Uchanski, Mark E; Omerigic, Heather M et al. (20 February 2021). "Comprehensive Evaluation of Metabolites and Minerals in 6 Microgreen Species and the Influence of Maturity" (in en). Current Developments in Nutrition 5 (2): nzaa180. doi:10.1093/cdn/nzaa180. ISSN 2475-2991. PMC PMC7897203. PMID 33644632. https://academic.oup.com/cdn/article/doi/10.1093/cdn/nzaa180/6041711. 
  43. ↑ Haugen, John-Erik; Tomic, Oliver; Kvaal, Knut (1 February 2000). "A calibration method for handling the temporal drift of solid state gas-sensors" (in en). Analytica Chimica Acta 407 (1-2): 23–39. doi:10.1016/S0003-2670(99)00784-9. https://linkinghub.elsevier.com/retrieve/pii/S0003267099007849. 
  44. ↑ Adesina, Ifeoluwa; Bhowmik, Arnab; Sharma, Harmandeep; Shahbazi, Abolghasem (14 April 2020). "A Review on the Current State of Knowledge of Growing Conditions, Agronomic Soil Health Practices and Utilities of Hemp in the United States" (in en). Agriculture 10 (4): 129. doi:10.3390/agriculture10040129. ISSN 2077-0472. https://www.mdpi.com/2077-0472/10/4/129. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added.

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