Patients often source cannabis inflorescence for medical purposes in one-month doses. Additional flexibility exists for consumers in adult use markets, but storage of inflorescence over one-month periods is not uncommon. Since the entire inflorescence sample is not consumed in one dose, patients must open and close the storage container numerous times to obtain a portion of the inflorescence. Continually opening the container allows terpenes which have evaporated from the inflorescence to escape the storage container and contributes to reduction of inflorescence terpene content over time. Other sources which can affect the terpene content of inflorescence include cultivation, harvest, storage, and curing (drying) methods, chemotype genetics, as well as storage conditions and age of the inflorescence. These factors present challenges for patients and consumers seeking inflorescence of reliable terpene content.
A novel method to preserve, replenish, and establish a desired terpene content of cannabis inflorescence was investigated. A total of 38 inflorescence samples were investigated in this study (Table 1). Nine samples were stored without an external volatile source (controls) while 27 samples were stored in the presence of an external volatile. Eight of the nine controls samples experienced a loss in terpene content over the duration of storage. The aged one-year DjG control unexpectedly experienced an increase in terpene content after 2 weeks of storage as compared to initial, which may have been caused by sample heterogeneity. It should be noted that the control samples from the two harvests of DjG experienced different rates of terpene loss. The aged one-year DjG sample experienced a 9.41% loss while the aged one-month DjG sample experienced a 51.6% loss of terpene content after 4 weeks of storage. It is likely the aged one-month DjG had a higher percentage of more volatile terpenes as compared to the aged one-year DjG. Thus, the aged one-month DjG was more susceptible to terpene loss in the experiments described here. The differing rates of terpene loss from inflorescence of the same chemotype, but different harvests, is further illustrative of the inherent variability of cannabis inflorescence terpene content.
The repeatability and robustness of the approach was investigated by testing the system using different chemotypes, storage times, percentages of external volatiles, and types of external volatiles. The ‘Chemotype’ group also included differences in inflorescence cultivation locations, different harvests and age, ground versus intact inflorescence, initial terpene content (typical versus depleted), and neat volatiles stored in a glass container versus volatiles impregnated into an inert matrix. Furthermore, quantitative terpene analysis for the different sites was performed by different testing labs, utilizing different analysts. Experiments at Site Two mimicked patient use by opening the storage jars twice per week. Under all conditions, all preservation samples (n = 27) successfully maintained a higher terpene content as compared to controls after 2 and 4 weeks (Site One) and 2, 4, and 6 weeks (Site Two) of storage, illustrating the robustness of the method. Variables that were statistically meaningful to the effectiveness of the novel system were identified by main effects analysis and ANOVA modeling. The amount and type of external terpene utilized were found to be statistically meaningful parameters towards the effectiveness of the system for terpene preservation and augmentation. Conversely, chemotype and storage time did not impact the novel’s system ability to preserve terpenes. Explicitly, all factors that were varied in the chemotype group, most notably the initial terpene content of the inflorescence, did not impact the method’s ability to preserve or augment the terpene content of the inflorescence, further illustrating the robustness of the novel system. All durations of storage were found to be effective towards terpene preservation, illustrating that the maximum storage duration has not been identified, however as results indicate it is greater than 6 weeks.
As described previously, samples from the DjG chemotype were almost entirely depleted of terpene content (0.153 and 0.170%) due to the processing and grinding methods applied to the inflorescence post-harvest. Conversely, samples from the Cre chemotype had an initial terpene content of 1.49%. This terpene content is within the expected range for inflorescence available for medicinal or recreational use (Jin et al. 2020). For both sample groups the novel system was able to increase the terpene content of the inflorescence, illustrating the system is effective independent of initial terpene content of the material. Results illustrate the novel system was able to replenish the terpene content of the DjG inflorescence samples. The largest augmentation was achieved by using α-pinene resulting in an increase from 0.153 to 0.910% terpene content for the inflorescence samples. Although 0.910% inflorescence terpene content would be regarded as lower than what is typical for medicinal or recreational use, the + 495% change illustrates a proof of concept for the system’s ability to replenish terpenes from terpene depleted inflorescence. Figure 3 illustrates that the degree of inflorescence terpene content augmentation is directly related to the amount of external terpene is used. In this study terpene content augmentation was maximized by storing inflorescence samples in the presence of external volatiles representing ~ 42–58% the weight of the inflorescence. However, larger percentages could be utilized to further augment the terpene content of both terpene containing or terpene depleted inflorescence. Thus, providing patients and manufacturers the ability to establish a desired terpene content for their inflorescence. The rate of terpene augmentation, relative to the percent external volatile utilized, was similar for all inflorescence groups (Table 3). When adjusting for the weight of the individual inflorescence samples, the terpene content augmentation was within 3.4% for all three inflorescence groups, illustrating the reproducibility of the method.
The repeatability of the approach to selectively modify the inflorescence terpene profile was investigated. Inflorescence samples from different harvests were stored in the presence of a formulated 8-part terpene mixture. The percent difference of the terpene profiles was compared before and after storage. Preliminary results indicate a 39.5% reduction in terpene profile variance for the different inflorescence samples of the DjG chemotype. It should be noted that the approach was effective even in the extreme scenario investigated in this study, in which the inflorescence samples differed in age by approximately 1 year. Similar terpene profiles obtained in cannabis inflorescence samples from two independent harvests, separated by 1 year, illustrates a proof of concept for improving batch to batch consistency of terpene content cannabis inflorescence. Site Two experiments showed that variability in the terpene profile caused by aging, and different rates of terpene loss, of inflorescence is reduced when using the novel system. This development may address a common real-world scenario, in which patients and consumers source inflorescence of the same chemotype at different periods of time. Patients and consumers may expect reliable terpene profiles (which is often correlated to efficacy and experience) from inflorescence of the same chemotype. However, as illustrated in this work and previous research, the terpene profiles of the inflorescence may vary due to age, batch (harvest), and other factors. Results reported here illustrate the variance between inflorescence samples can be significantly reduced when the novel packaging approach is utilized.
In this proof of concept study, botanically derived terpenes were utilized as the external volatile source. However, methods for extracting terpenes from cannabis are well established and terpenes extracted from cannabis could be reintroduced to the inflorescence using the novel system described here. Similar to preliminary results, utilizing the novel system with chemotype specific terpenes as the ‘external’ volatiles, is expected reduce terpene loss over time, and batch to batch variability of the terpene profile. It is worth noting that any terpenes or other volatiles utilized as an external volatile source, requires control of potential manufacturing impurities which could be introduced into the inflorescence. The reintroduction of chemotype specific terpene profiles may provide a path for pharmaceutical development via the FDA’s Botanical Drug pathway, as all ingredients would originate from the same botanical (cannabis) source, with the improved batch to batch consistency offered by the novel system.
The accuracy of adjusting the terpene profile to match a formulated blend was assessed by comparing the recoveries of each terpene in the inflorescence after storage, to the terpene profile of the formulated blend. The recoveries were found to be dependent upon terpene class, monoterpenes which contain 2 isoprene units (C10H15) were observed to have the highest rate of terpene infusion as compared to sesquiterpenes, which contain 3 isoprene units (C15H24). Both isomers of the monoterpene pinene had the highest rate of terpene infusion for the 8 terpenes investigated (Table 2) and produced inflorescence with a higher terpene content as compared to other external volatile sources (Table 1 and Fig. 2a). These factors may indicate that terpene volatility, in which monoterpenes are more volatile as compared to sesquiterpenes, determines the rate of infusion. Characterizing the infusion rates of various terpenes is a future direction for this research. Conversely, the ability to selectively adjust the terpene profiles of inflorescence samples to be dominated by individual terpenes was achieved with an accuracy of 95.4% for α-pinene and 89.0% for β-myrcene.
Although further clinical research would be required to satisfy the medical and scientific community, there is widespread popular belief that, different chemotypes of cannabis are associated with specific pharmacological effects. The characteristic terpene profiles and total terpene content of various cannabis chemotypes may be responsible for the differences in perceived pharmacological and medicinal benefits (Hazekamp et al. 2016; Mudge et al. 2019; Lewis et al. 2018). For example, inflorescence with a terpene profile dominated by β-myrcene is often associated with calming or sedative effects (Sarma et al. 2020). Similarly, an α-pinene dominant terpene profile may have efficacy towards treating anxiety, as the inhalation of α-pinene has been linked to anxiolytic-like activity in mice and rats (Satou et al. 2014; Zhang and Yao 2019). Due to cost and availability, the pulmonary delivery of cannabis inflorescence (either through vaporization or combustion) is the main delivery method for both medicinal and recreational cannabis users. The so-called ‘entourage effect’, or the concept that whole plant products have improved efficacy as compared to their individual chemical constituents, is the proposed benefit of this delivery form (Russo 2011). Further research on the entourage effect is required, but recent research has been shown that pulmonary delivery of whole plant cannabis products may be more advantageous for treating specific pain types (neuropathic), as compared to other routes of cannabinoid absorption, such as oromucosal (Rabgay et al. 2020). However, variability and/or loss of inflorescence terpene content may reduce the enhanced cannabinoid pharmacology associated with the entourage effect. Variability in cannabinoid-terpene synergism may lead to unreliable experience and efficacy of medicinal and recreational cannabis inflorescence. Achieving batch to batch consistency and stability of all phytochemical components, including terpenes, is an important step towards the pharmaceuticalization of cannabis inflorescence and an unmet need in the cannabis industry (Koltai et al. 2019). The novel system described here addresses the drawbacks associated with terpene loss from inflorescence over time and has the potential to improve experiences for both medical and recreational cannabis users.
Experiments reported in this manuscript assessed the practicality and cost of the novel packaging system. It was experimentally determined that approximately 41.3 mg of external terpenes are required to maintain the native terpene content of 3.5 g of inflorescence (a common amount available at dispensaries). Since terpene extraction methods from both cannabis and botanical sources are well established, and can be achieved at relatively large scales, 41.3 mg of terpenes adds minimal cost to a final product. Several inert matrices of sizes ≤1 cm3 are available which are capable of housing this volume of terpenes. Finally, the compartment which houses the terpene impregnated matrix can be incorporated directly into current inflorescence packaging. Thus, the authors expect the components of the novel system to add minimal cost to individual inflorescence sale units. The practicality of the novel system is improved by the fact that manufacturers and consumers are not required to handle neat terpenes, as positive results were obtained from inflorescence samples stored in the presence of terpenes impregnated into an inert matrix.