Total cannabidiol (CBD) concentrations and yields from traditional extraction methods: Percolation vs. maceration

Abstract

Introduction

Herbal medicines have been commonplace in Asia for many generations but have come back into fashion in Europe and North America with a 109.3% growth in sales in the US since 2000 (New Hope Network, 2019). Due to its pharmacological activity, Cannabis has become one of the botanicals in the public spotlight due to the change in regulatory structures in a growing number of countries, as well as the trail of politics following closely on its roots. Consumption of cannabidiol (CBD) chemotypes of Cannabis sativa L. [Cannabaceae], known as hemp by consumers, has grown exponentially and now are at or above 34% of the population as of a 2022 survey (ICSID, 2022).

Hemp extracts containing CBD are used most commonly for chronic pain, arthritis, joint pain, and anxiety (Corroon and Phillips, 2018). This current use mimics the traditional use of hemp for pain and anxiety (Balant et al., 2021; Russo, 2007). The method of administration most commonly used are liquids administered as sprays, drops, and tinctures (Corroon and Phillips, 2018) and this reflects the broader choice of consumers for non-Cannabis herbal medicines as well. Extractions using ethanol, are common to Cannabis products and throughout the medicinal plant field in general. These types of extractions, called tinctures, are increasingly being made in home kitchens due to the relaxation of laws that previously prohibited growing Cannabis in residential gardens.

Investigations of traditional preparations of medicinal plants, such as hemp, offers insight into the phytochemical diversity and the yield of actives. Two dominant techniques are used throughout the medicinal plant community to make tinctures (WHO, 2017). The most common is maceration which mixes the properly comminuted herbal materials with the menstruum (most commonly a hydroethanolic solvent with varying ratios of water and ethanol) and allowing the mixture to stand at a certain temperature, usually room temperature, with agitation for a defined period of time. Preparation of macerations is documented by Paracelsus in the 1500s, who used extended maceration times in low percentage of ethanol in hydroethanolic solutions (Raubenheimer, 1910). The lesser used technique, but notably quicker, is percolation. This procedure provides a continuous flow of the menstruum through the herbal material in a percolation cone (WHO, 2017).

The manner in which natural medicines are prepared influences the final composition of the herbal remedy (Politi et al., 2005b; Politi et al., 2005a). This in turn provides insights into what consumers are ingesting, and of the potential pharmacological activity, as well as synergic pharmacological effects that may result from a complex extract.

There has been a long standing debate on the superiority of extraction methods for medicinal plants (Raubenheimer, 1910). Unfortunately, the scientific focus on the traditional methods of extraction have faded from pharmaceutical sciences due to the focus on new-to-nature molecules. There are very few pharmacognosy university study programs remaining throughout the world. This movement away from natural products for drug discovery is correlated with the overall reduction in new leads in the drug development pipeline and a substantial decline in new drug approval (Li and Vederas, 2009). Regardless, traditional preparations continue to be utilized on every corner of the planet by shamans, herbalists, naturopaths, modern phytotherapists and integrative physicians, yet with little substantiation of optimal extraction conditions.

In the following experiments we used the long-standing traditional method documented in the Eclectic dispensatory which suggests the use of “rectified” ethanol (95% EtOH) (Felter and LLoyd, 1898). Moreover, due to the highly lipophilic nature (log P > 6) 95% EtOH is commonly used in Cannabis production facilities around the globe to pull these lipophiles out of flower. To this end, we replicated different home extraction techniques that are traditional, as well as low tech, and that have been used by many generations of herbalists—using Cannabis sativa L. [Cannabaceae] as our botanical, and the phytocannabinoid cannabidiol (CBD) as our target active compound. We investigated the traditional extraction technique of maceration and percolation and compared this for total CBD yield. To our knowledge this is the first comparison of the extraction of actives by percolation and maceration of a medicinal plant and/or C. sativa L.

Results

Discussion

These investigations include several limitations and opportunities for refinement. Resources were limited during the course of this project, which led to limits on the number of replicates for the extraction treatments to two replicates, when it would have been preferable to perform at least three replicates for adequate precision and accuracy. The amount of biomass used in percolation treatments also had to be limited to approximately 50 g due to the size of the percolation cone that was available, to mimic home extractions. Future follow up studies should match marc mass in the percolation and maceration experiments.

Further limitations of these investigations include the potential heterogeneity in CBD concentration of the biomass, potentially resulting in variable CBD concentrations in the starting biomass. Consistent distribution of biomass CBD content and particle size was managed by mixing the hemp biomass repeatedly and sifting the biomass through a #5 sieve for an effective medium approximation of CBD. Our analysis shows an RSD of [6.19%] for CBD concentration after triplicate samples of the hemp biomass and triplicate analysis of each sample.

In these experiments we compared the solvent recovery, yield of CBD, additional input of pressing marc in two disparate techniques of extractions; maceration and percolation. We also mimicked home extractions that a person not versed in laboratory extractions of medicinal plants could perform in a typical home kitchen. All items used; screen, oven, glassware and press, are either easily afforded in Western countries or are readily available in a typical household. In addition, the particle size of the flower, which had been baked at 250°F for decarboxylation, was consistent throughout both macerations and percolations.

These data show that in the maceration method, pressing the marc significantly increases the content of CBD in the menstruum. Our results show that an additional 8.19% (∼458 mg) of CBD was added to the menstruum from the pressing procedure. The step of pressing the marc to express the retained menstruum is often problematic for people making tinctures at home. We showed with an inexpensive and readily avialable wine press, that was not designed for pressing the biomass of tinctures, that there is a significant value in pressing the marc for an increase yield in active compound(s).

As opposed to menstruum recovery in the 2-week maceration, the total menstruum recovery in the percolation was improved by 4.931% as compared to the fully pressed macerated tincture and 13.398% of the unpressed macerated tincture. However, what most production facilities of medicinal plant tinctures employing maceration technique use is a press supported by a hydraulics. The above figures on menstruum recovery come from an inexpensive wine press. Thus, it is conceivable that a hydraulic press, generally not available in the herbalist’s kitchen would yield a higher menstruum recovery.

Interestingly, we attempted to press the marc from the percolation cone and did not recover any viable amount of menstruum (Table 4). Menstruum recovery for percolation was more efficient over maceration, even when pressing the macerated marc. These investigations show that percolation as an extraction technique, has a significantly increased extraction efficiency of CBD and menstruum from Cannabis tinctures as compared to a maceration.

Although the efficiency of percolation vs. maceration has been long debated, percolation is another extraction technique, traced back to ancient Greece, that has long been used for botanical medicines (Raubenheimer, 1910). Percolations are preferred by some medicinal plant extraction facilities because they are more time and space efficient. Instead of macerating solutions sitting for extended time periods in large drums, as traditionally done in the production of medicinal plant liquid extracts, percolations are finished in hours, rather than the traditional maceration time of 2–4 weeks. After two independent extractions the percolations extraction efficiency of CBD was 80.10% on average while the 2 week maceration extraction efficiency was 63.52% on average. This investigation provides data to supplement a long-held debate on the efficiency of percolations over macerations.

Percolation extraction requires a percolator, a cone shaped vessel narrowing at one end and open at both ends (Majeodunmi, 2015). Pharmaceutical grade percolation cones are not widely available for the home enthusiast. However, certain wine shaped glass bottles or sparkling water bottles can be adapted. In the attempts to keep this technique replicable to a consumer in a home kitchen we used a Perrier bottle, which is commonly used by herbalists, with the bottom of the bottle carefully scored and then removed. The plant material is scantly pre-moistened with the liquor, sits for 24 h and is then packed in a percolation chamber with the proper packing pressure. The liquor is then run through the packed herb in the percolator which has a stop cock at the end to control rate of flow, which depending on the herb, is from one to six drops a second (Raaman, 2006). A screw on cap on a Perrier bottle is often substituted in home extractions instead of a stop cock.

Another advantage of percolation may be preservation of phytonutrients. Select phytochemicals degrade more rapidly in a liquid medium than in a solid medium (Fairbairn et al., 1976; Rogers et al., 1998; Livesey et al., 1999; Perry et al., 2000; Bilia et al., 2002; Yang, 2008; Peschel, 2016). Phytocompounds in a macerating hydroethanolic solution, or sitting in tincture form on a shelf waiting for a consumer purchase, degrade on varying timescales. For example, caffeic acid derivatives, important herb constituents due to immunological influences (Kilani-Jaziri et al., 2017), have shown a remarkable ability to degrade in just a few months in hydroethanolic solutions (Livesey et al., 1999). Degradation in hydroethanolic solutions has also been shown for cannabinoids (Fairbairn et al., 1976; Peschel, 2016) and the lipophilic CB₂ ligands known as the alkylamides (Livesey et al., 1999; Perry et al., 2000).

These results are particularly pertinent to other medicinal plant extractions in that the identical techniques used for Cannabis in these experiments are used for a wide variety of medicinal plants. Thus, these results may be pertinent to other lipophilic compounds that are extracted from medicinal plants. For example, in the case of Echinacea spp., one of the groups of active constituents are the alkylamides. Specific alkylamides bind with similar affinities to CB₂ as the endocannabinoids (50–60 nM) (Matovic et al., 2007; Raduner et al., 2006) and have also shown PPARγ activity (Spelman, 2009). Previous research demonstrates that there is no difference in alkylamide concentrations in Echinacea purpurea roots extracted for 24 h versus 2 weeks in 70% ethanol (Spelman et al., 2009), however these results did not compare a percolated extraction.

The log P, a measure of lipophilicity, is less than ideal for optimal pharmacokinetics for both CBD and the alkylamides previously mentioned. Thus, these examples may be relevant to other lipophilic compounds in medicinal plants. Moreover, the topological polar surface area (PSA) for both compounds is also less than ideal for optimal pharmacokinetics suggesting that at the least, these two lipophiles may act similarly in crude extractions.

Finally, it should be noted that these extractions were carried out with CBD, not CBDA. Given that the PSA differs considerably between CBD (PSA = 40.5 Å²) and CBDA (PSA = 77.8 Å²) further extraction efficiency of CBDA in ethanol is expected due to the higher PSA. Under aqueous extraction conditions, superior extraction of tetrahydrocannabinolic acid (THCA) over tetrahydrocannabinol (THC) has previously been shown (Hazekamp, 2007). Thus, extraction from dried hemp that is not decarboxylated will likely be advantageous for extraction in ethanol.

To summarize, using common tools available in a home kitchen, an extraction performed by percolation has a statistically significant higher extraction yield on average of 16.59% CBD than a 2-week extraction (p = 4.419 × 10⁻⁹). In addition, the percolation method of extraction exhibited a superior performance of precision between replicates and menstruum recovery by 4.93% as compared to the combined liquor from the drain and pressed marc.

Methods and materials

Acknowledgments

References