Optimization of supercritical carbon dioxide fluid extraction of seized cannabis and self-emulsifying drug delivery system for enhancing the dissolution of cannabis extract
Graphical Abstract
Introduction
Extraction is critical to analyzing plant constituents and assaying activity to aid in formulation development. Selecting extraction conditions is important as optimal conditions provide the desired plant bioactive compounds with minimal decomposition [1]. Design of Experiment (DOE) is an advanced, versatile tool for systematically testing production steps during research and development [2]. DOE can be used across scientific fields for designing screens, comparing independent variables, identifying transfer functions, optimization, and robust design [3]. This requires less time, is cheaper, and uses fewer chemicals than the traditional One Factor at a Time trialing. Additionally, it has been used to identify interaction effects and characterize surface responses [4].
Eco-friendly, sustainable extraction techniques, such as pressurized liquid extraction, microwave-assisted extraction, ultrasound-assisted extraction, and supercritical fluid extraction (SFE), are gaining attention in research and development fields involving bioactive plant compounds. SFE is particularly beneficial owing to the ability of supercritical fluids to penetrate solid matrices deeper and faster than other phases [5]. This arises from the physical properties of a supercritical fluid, specifically, its density and viscosity, which are comparable to the liquid and gas phase, respectively, and the diffusivity, which is intermediate between gas and liquid. Importantly, SFE commonly employs carbon dioxide as the extraction solvent, which is suited for medical applications as it is inert, non-toxic, economical, easily accessible, and easily removed, and is already approved as a food-grade solvent [6]. Extracts can be obtained via SFE at low temperatures and selectively isolate solvent-free products without byproducts. Additionally, the physicochemical properties of carbon dioxide at supercritical conditions favor the extraction of non-polar compounds [7]. While the requirement for high-pressure makes this technique relatively expensive compared to conventional methods, its operational costs are economically acceptable, particularly for value-added products [8], [9].
Cannabis (also called hemp and marijuana; family: Cannabaceae) contains a number of medically valuable bioactive constituents, including cannabinoids (cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC)) and terpenes [10]. Cannabinoids are non-polar compounds and are thus well-suited for SFE. Previous work reported the extraction of bioactive compounds from cannabis plants using SFE, which produced purer extracts than other techniques, such as maceration [11], [12], [13], [14], [15], [16], [17]. Furthermore, SFE can separate other groups of compounds (e.g., terpenes) from cannabis for use in applications such as food additives, cosmetics, and aromatherapy. Cannabinoid-containing products are already used in medicine for a variety of conditions. For example, Sativex®, an oromucosal spray composed of approximately 2.5 mg CBD and 2.7 mg THC per 100 μL, has been developed for treating moderate to severe spasticity due to multiple sclerosis [18]. Epidiolex®, an oral solution composed of 100 mg/mL CBD, is used to treat seizures associated with Lennox-Gastaut syndrome or Dravet syndrome [19].
As cannabis extracts and cannabinoids are poorly water-soluble with high octanol/water partition coefficients, they are slowly absorbed via the gastrointestinal mucosa, resulting in low bioavailability when taken orally [20], [21]. Several techniques can enhance the solubility, dissolution, and bioavailability of poorly water-soluble drugs. Particle size reduction techniques, such as mechanical micronization or nanosization (e.g., jet milling, ball milling, high-pressure homogenization), and engineered particle size control (e.g., cryogenic spray process, crystal engineering) can enhance drug solubility and dissolution. Drug bioavailability can be improved using self-emulsifying drug delivery systems (SEDDS), complexation with cyclodextrins, polymeric micelles, freeze-dried liposomes, and solid lipid nanoparticles [22].
SEDDS, or self-emulsifying oil formulations, are lipid-based formulations that incorporate isotropic mixtures of natural or synthetic oils, solid or liquid surfactants, and co-surfactants. Upon exposure to aqueous media (such as gastrointestinal fluids), they undergo self-emulsification to form oil-in-water microemulsions or nanoemulsions with a droplet size ranging from 20 nm to 200 nm and are called self-microemulsifying drug delivery systems (SMEDDS) or self-nanoemulsifying drug delivery systems (SNEDDS), respectively [23]. The distinction between SMEDDS and SNEDDS varies in the literature, with SMEDDS reported to have droplet sizes ranging from less than 50 nm to less than 250 nm and SNEDDS defined as less than 100 nm [24], [25], [26]. In contrast, SEDDS typically produce an emulsion with droplet sizes between 100 nm and 300 nm, although they have also been described as greater than 300 nm [27]. SEDDS possess several advantages over conventional drug delivery systems: enhanced bioavailability, reduced local irritation of the gastrointestinal tract, physical and thermodynamic stability, and industrial scalability [28].
SEDDS formulations typically incorporate P-glycoprotein inhibitors, as P-glycoprotein is known to decrease the oral bioavailability of several drugs. P-glycoprotein reduces absorption and oral bioavailability by increasing drug excretion from hepatocytes and renal tubules [29]. Tween® 80, reported to be a P-glycoprotein inhibitor, is a primary surfactant in SEDDS formulations [30], [31], [32]. It permeabilizes the plasma membrane lipid bilayer by inserting into the lipid tails. It can also interact with the polar head of the plasma membrane and disrupt hydrogen and ionic bonds, which can help to inhibit P-glycoprotein activity [33], [34].
Seized cannabis (cannabis confiscated by law enforcement) is destined for destruction following adjudication. Rather than destroying seized cannabis, it could find use in research and development. This work took advantage of seized cannabis to optimize the SFE system using the Box-Behnken design. SEDDS were prepared from cannabis extract obtained under the optimal conditions to enhance dissolution. The formulation parameters were optimized using the 32 factorial design to minimize droplet size and emulsification time.
Section snippets
Materials
Carbon dioxide was purchased from Air Liquide Ltd., Thailand. Tween® 80, Span® 80, and ethanol (95%) were purchased from P. C. Drug Center Co., Ltd., Thailand. Organic coconut oil was obtained from Thai Pure Coconut Co., Ltd., Thailand. Methanol and isopropanol (HPLC grade) were purchased from Fisher Scientific, UK. Sodium hydroxide pellets and hydrochloric acid (37%) were purchased from Carlo Erba, France. Standard CBD and THC (> 99.0% purity) were isolated and purified from seized cannabis
Optimal SFE system
The Box-Behnken design of the SFE system provided extraction yields between 3.03% (Condition 5) and 7.23% (Condition 8) (Table 1). The CBD and THC contents in the cannabis extract were compared to those in the cannabis raw material, with the extract contents more important to formulation development (Table 1). The CBD content in the cannabis extract ranged from 4.89% (Condition 11) to 14.00% (Condition 1), while the raw material contained between 0.32% (Condition 11) and 0.63% (Condition 12)
Discussion
Pressure plays an important role in both the overall extraction yield and the quantity of bioactive compounds extracted. Increasing the pressure from 17 MPa to 34 MPa is reported to increase the yield of cannabis extract and its THC content [14]. However, this work found that increasing the pressure increased the extraction yield but decreased the CBD and THC content. This supports the general principle that higher pressure increases solvent strength and decreases extraction selectivity, making
Conclusions
This work used the Box-Behnken design to optimize SFE for seized cannabis. The highest CBD and THC content was obtained at 18 MPa and 40 °C without the use of ethanol. Winterized cannabis extract was prepared as SEDDS to enhance dissolution. The optimal formulation, a mixture of coconut oil and 2:1 Tween® 80:Span® 80, was prepared using the 32 factorial design. A 45:40 w/w surfactants mixture:coconut oil ratio provided the smallest droplet size and shortest emulsification time. The SEDDS
CRediT authorship contribution statement
Chaowalit Monton: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration. Natawat Chankana: Conceptualization, Methodology, Formal analysis, Investigation. Surang Leelawat: Conceptualization, Visualization. Jirapornchai Suksaeree: Conceptualization, Methodology, Formal analysis. Thanapat Songsak: Conceptualization, Visualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was funded by a grant from the Research Institute of Rangsit University (Grant no. 93/2562), which also provided support for proofreading and English language editing. The authors would like to thank Miss Kemjira Sriputorn, Miss Kultida Kongin, and Mr. Supadit Santasanasuwan for their research assistance.
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