separations

Article Biological Activity of sativa L. Extracts Critically Depends on Solvent Polarity and Decarboxylation

Guillermo Moreno-Sanz 1, Carlos Ferreiro Vera 2 , Carolina Sánchez-Carnerero 2 , Xavier Nadal Roura 3 and Verónica Sánchez de Medina Baena 2,*

1 Abagune Research, Vitoria-Gasteiz, 01006 Álava, Spain; [email protected] 2 Phytoplant Research S.L., 14014 Córdoba, Spain; [email protected] (C.F.V.); [email protected] (C.S.-C.) 3 Ethnophytotech Research & Consulting S.L.U, 14002 Córdoba, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-95-71-01-629

 Received: 24 September 2020; Accepted: 15 October 2020; Published: 20 October 2020 

Abstract: Minor and non-cannabinoid molecules have been proposed to significantly contribute to the pharmacological profile of cannabis extracts. Phytoplant Research has developed highly productive cannabis cultivars with defined chemotypes, as well as proprietary methods for the extraction and purification of . Here, we investigate the effect of solvent selection and decarboxylation on the composition and pharmacological activity of cannabis extracts. A library of forty cannabis extracts was generated from ten different cannabis cultivars registered by Phytoplant Research at the EU Community Plant Variety Office. Plant material was extracted using two different solvents, ethanol and hexane, and crude extracts were subsequently decarboxylated or not. Cannabinoid content in the resulting extracts was quantified, and biological activity was screened in vitro at three molecular targets involved in hypoxia and inflammation (NF-κB, HIF-1α and STAT3). Changes in transcriptional activation were strongly associated to solvent selection and decarboxylation. Two decarboxylated extracts prepared with hexane were the most potent at inhibiting NF-κB transcription, while HIF-1α activation was preferentially inhibited by ethanolic extracts, and decarboxylated extracts were generally more potent at inhibiting STAT3 induction. Our results indicate that solvent selection and proper decarboxylation represent key aspects of the standardized production of cannabis extracts with reproducible pharmacological activity.

Keywords: cannabis; botanical extracts; decarboxylation; cannabinoids; THC; CBD

1. Introduction L. is an herbal medicine which contains at least 554 identified compounds including 150 phytocannabinoids, terpenophenolic active molecules which are nearly exclusive to this plant, and other phyto-molecular families such as terpenoids, flavonoids and oxylipins [1]. The main two cannabinoids responsible for the pharmacological properties of cannabis extracts are ∆9- (THC), the principal active component in drug-type cannabis, and (CBD), the major cannabinoid molecule found in fiber-type cannabis or hemp [2]. These compounds are formed in the cannabis plant as their 2-carboxylic precursors, ∆9-tetrahydrocannabinolic acid (THCA) and (CBDA), whom rapidly decarboxylate to render the neutral forms upon heating [3]. Pharmacological interest has been attributed to THCA and CBDA, as well as to other less-abundant, non-psychoactive cannabinoids such as (CBG), the molecular precursor of THC and CBD, and ∆9- (THCV) and (CBDV), their propylic

Separations 2020, 7, 56; doi:10.3390/separations7040056 www.mdpi.com/journal/separations Separations 2020, 7, 56 2 of 16 analogs [4]. THC was originally characterized as a partial agonist of G protein-coupled receptors, namely cannabinoid receptors type-1 and type-2 (CB1 and CB2 receptors, respectively). CBD is a rather “promiscuous” molecule, which interacts with a variety of physiological targets such as CB1 and CB2, as a negative allosteric modulator [5–7] producing biased agonism at both receptors and CB1-CB2 heteromers [8], G protein-coupled 55 (GPR55), and serotonin receptor 5-HT1A among others [9]. Although the mechanisms of their actions are not completely understood, THC and CBD remain the two cannabinoids available in the clinical practice, either in their synthetic form () or as a standardized cannabis extracts (Sativex, Epidiolex), for the management of nausea and vomiting associated to chemotherapy, spasticity secondary to multiple sclerosis, and epileptic seizures in pediatric syndromes such as Dravet or Lennox-Gastaut [10]. Although adverse effects of cannabis are primarily due to THC, many patients report cannabis extracts having better therapeutic activity and less side effects compared to dronabinol, such as dysphoria, depersonalization, anxiety, panic reactions, and paranoia [11]. Some authors contend that whole-plant cannabis extracts provide better pharmaceutical and mood-altering outcomes compared to isolated cannabinoids owing to (i) an increased therapeutic index due to synergistic actions and (ii) mitigated side effects of the primary active cannabinoid by other active or silent compounds within the phytocomplex [12,13]. Although the molecular underpinnings of this theoretical “entourage effect” remain to be elucidated, recent data seem to indicate that such synergistic interaction does not occur at cannabinoid receptors [14]. At present, many countries have passed legislation permitting the use of cannabis for medical purposes, generally bypassing the strict regulatory procedures that apply to pharmaceutical development [15]. Therefore, much research is required to guide regulation and to fully understand the implications of current industrial practices in the manufacturing of cannabis and hemp extracts for human consumption. Since 2012, Phytoplant Research SL has developed Cannabis sativa L. cultivars with distinct cannabinoid profiles, including varieties rich in minor cannabinoids, such as CBG, THCV and CBDV [16]. To date, 13 of these cannabis varieties have been registered at the Community Plant Variety Office (CVPO), the European Union agency that manages plant variety rights covering the 28 Member States (https://cpvo.europa.eu). Phytoplant Research SL has also patented proprietary methods for the purification of CBD, CBG and CBGA by crystallization [17] and for the purification of THC, THCA, THCV, CBDA, CBDV and CBGV by liquid-liquid chromatography [18]. Due to the relative high amounts of these cannabinoids in certain registered varieties (Table1), it is possible to purify them by direct crystallization from a primary cannabis extract obtained by percolation of plant material with an organic solvent followed by a step of crystallization and recrystallization in the same solvent. This method of purification is cost-effective and easily scalable, as it reduces the use of solvents and eliminates the need for chromatographic steps, while yielding isolated cannabinoids with a purity over 98%, compatible with the Good Manufacturing Practices (GMP) certification required for pharmaceutical production. Ethanol and supercritical carbon dioxide (CO2) are usually the preferred options, as polar and non-polar solvents, in the preparation of cannabis extracts for direct human consumption without further purification steps at pilot-industrial scale [19]. We hypothesized that cannabis extracts from the same plant variety obtained with two very different solvents, a polar alcohol and a non-polar alkane, and subject or not to complete decarboxylation might not only differ in their chemical composition but, more importantly, in their overall pharmacological properties. Cannabinoids are soluble in both polar and non-polar solvents, but the rest of cannabis components, such as terpenes, terpenoids, flavonoids and polyphenols, will have different extraction ratios depending on which solvent is used. It seems plausible to speculate that a polar solvent will favor the extraction of more polar molecules, like flavonoids and polyphenols, and disfavors the extraction of non-polar terpenes, and vice versa. The process of decarboxylation further modifies the chemical composition of extracts, not only by transforming acid cannabinoids into neutral, but also potentially affecting other organic acids and thermally-labile substances that may be present in the extract. In support of this hypothesis, a very recent publication describes the differential pharmacological activity of cannabis extracts varying in their decarboxylation degree at cannabinoid receptors [20]. In the present study we broadened our Separations 2020, 7, x FOR PEER REVIEW 3 of 15

(PHYRESCEL) was prepared by systematically processing 10 distinct cultivars developed by Phytoplant Research SL and characterizing their cannabinoid profile (Figure 1).

Table 1. Summary of Cannabis sativa L. varieties used in the present study. ND: Not determined.

Yield Yield Biomass Δ9- Main Cannabinoids Terpenes ethanol hexane VARIETY Phenotype yield THC Terpene profile Cannabinoid total (%) total (%) extraction extraction (g/m2) (%) (%) (%) Limonene::β- 1.000 :Guaiol 15.5– MONIEK Δ9-THC I (flower 19.0–26.0 0.75–0.90 ~ 1 > Myrcene > α- 28 28.4 22.0 only) Terpineol:Fenchol:β Pinene̴1 Myrcene:β- 500 11.0– Caryophyllene ~ 1 > α- MAGDA Δ9-THC I (flower 13.0–16.5 0.50–0.60 20 20 14.0 Pinene > β-Pinene > only) Limonene:Linalool ~ 1 1.000 Δ9- 5.00– MATI II (flower 10.5–14.5 ND ND 20 19.6 THC+CBD 6.00 only) 500 Δ9- 4.50– BEATRIZ II (flower 11.0–15.0 ND ND 18.8 17.8 THC+CBD 6.50 only) 515 (leaf 0.40– Myrcene > Phytol > β- SARA CBD III 7.0–10.5 0.30–0.45 20.1 18.7 and 0.60 Caryophyollene flower) 605 α-Pinene > Myrcene:β- (leaf 0.30– Caryophyllene ~ 1 > PILAR CBD III 4.5–8.5 0.25–0.50 12 11.7 and 0.65 Guaiol:β-Pinene:Phytol flower) ~ 1 780 Myrcene > β- (leaf 0.30 Caryophyllene > Phytol THERESA CBD+CBDV III 6.0–11.0 0.25–0.80 8.8 8 and –0.70 > Limonene:Linalool ~ flower) 1 555 Myrcene > (leaf 0.05 Guaiol:Phytol ~ 1 > β- AIDA CBG IV 3.5–8.5 0.30–0.40 9.5 7.6 and –0.11 Caryophyllene:α- flower) Pinene ~ 1 1.000 Separations 2020, 7, 56 Phytol > Myrcene > β- 3 of 16 (leaf 0.02 OCTAVIA CBG IV 5.0–11.0 0.15–0.20 caryophyllene:Ocimene 6.8 4.9 and –0.15 ~ 1 flower) scope to focus on non-classical cannabinoid880 targets that could mediatePhytol cellular > Myrcene responses > β- elicited by (leaf 0.03– caryophyllene:guaiol ~ JUANIcannabis CBG+CBGV extracts. A plantIV extract library (PHYRESCEL)3.0–6.0 was0.15 prepared–0.20 by systematically processing8.2 5.7 and 0.05 1 > α-Pinene : Ocimene 10 distinct cultivars developed byflower) Phytoplant Research SL and characterizing~ their 1 cannabinoid profile (Figure1).

Figure 1. Schematic workflow for the preparation and characterization of Phytoplant Research Cannabis Extract Library (PHYRESCEL). Composition of all 40 extracts was analyzed by GCMS. Ten cannabinoids (∆9-THC, ∆9-THCA, ∆9-THCV, CBD, CBDA, CBDV, CBG, CBGA, CBC and CBN) were identified and Figure 1. Schematic workflow for the preparation and characterization of Phytoplant Research quantified. PHYRESCEL entries were screened in vitro for their ability to inhibit the transcriptional Cannabisactivation Extract of NF-kB, Library HIF1a (PHYRESCEL). and STAT3, three Composition molecular targets of all involved 40 extracts in inflammation was analyzed and hypoxia.by GCMS. Ten cannabinoids (Δ9-THC, Δ9-THCA, Δ9-THCV, CBD, CBDA, CBDV, CBG, CBGA, CBC and CBN) were PHYRESCEL contained 40 different entries that were further screened for their pharmacological activity towards NF-κB, HIF-1α and STAT3, three molecular targets known to mediate cellular responses to inflammation and hypoxia. Separations 2020, 7, 56 4 of 16

Table 1. Summary of Cannabis sativa L. varieties used in the present study. ND: Not determined.

Main Cannabinoids Yield Ethanol Yield Hexane VARIETY Phenotype Biomass Yield (g/m2) ∆9-THC (%) Terpenes Total (%) Terpene Profile Cannabinoid Total (%) Extraction (%) Extraction (%) Limonene:Linalool: β-Caryophyllene: 1.000 MONIEK ∆9-THC I 15.5–22.0 19.0–26.0 0.75–0.90 Guaiol ~1 > Myrcene > 28 28.4 (flower only) α-Terpineol:Fenchol:β Pinene ~1 Myrcene:β- Caryophyllene ~1 > 500 MAGDA ∆9-THC I 11.0–14.0 13.0–16.5 0.50–0.60 α-Pinene > β-Pinene > Limonene:Linalool 20 20 (flower only) ~1 1.000 MATI ∆9-THC+CBD II 5.00–6.00 10.5–14.5 ND ND 20 19.6 (flower only) 500 BEATRIZ ∆9-THC+CBD II 4.50–6.50 11.0–15.0 ND ND 18.8 17.8 (flower only) 515 SARA CBD III 0.40–0.60 7.0–10.5 0.30–0.45 Myrcene > Phytol > β- Caryophyollene 20.1 18.7 (leaf and flower) 605 α-Pinene > Myrcene:β- Caryophyllene ~1 PILAR CBD III 0.30–0.65 4.5–8.5 0.25–0.50 12 11.7 (leaf and flower) > Guaiol:β-Pinene:Phytol ~1 780 Myrcene > β-Caryophyllene > Phytol > THERESA CBD+CBDV III 0.30 –0.70 6.0–11.0 0.25–0.80 8.8 8 (leaf and flower) Limonene:Linalool ~1 555 Myrcene > Guaiol:Phytol ~1 > AIDA CBG IV 0.05 –0.11 3.5–8.5 0.30–0.40 9.5 7.6 (leaf and flower) β-Caryophyllene: α-Pinene ~1 1.000 Phytol > Myrcene > β-caryophyllene: OCTAVIA CBG IV 0.02 –0.15 5.0–11.0 0.15–0.20 6.8 4.9 (leaf and flower) Ocimene ~1 Phytol > Myrcene > 880 JUANI CBG+CBGV IV 0.03–0.05 3.0–6.0 0.15–0.20 β-caryophyllene:guaiol ~1 > α-Pinene: 8.2 5.7 (leaf and flower) Ocimene ~1 Separations 2020, 7, 56 5 of 16

2. Materials and Methods

2.1. Plant Material Plant material consisted on female inflorescences with or without the attached leaves from ten different, asexually-propagated, medicinal cannabis varieties registered by Phytoplant Research at the CPVO (https://cpvo.europa.eu), namely: Aida, 2016/0167; Beatriz, 2017/0146; Juani, 2016/0117; Magda, 2017/0145; Mati, 2017/0147; Moniek, 2016/0114; Octavia, 2017/0148; Pilar, 2016/0115; Sara, 2015/0098 and Theresa, 2016/0116 (see Table1).

2.2. Reagents for Plant Material Extraction, Purification of Cannabinoids and Analysis of Cannabinoid Content n-hexane and absolute ethanol (Scharlab, Barcelona, Spain) were used as solvents for the extraction of plant material. Petroleum ether bp 40–60 ◦C (Scharlab, Barcelona, Spain) or n-hexane were used for the purification of cannabinoids by crystallization (CBD, CBG and CBGA), and purified water were used in addition to the n-hexane and absolute ethanol in the purification of cannabinoids by liquid-liquid chromatography (THC, THCA, CBDA, CBDV and CBGV) used in this study. LC–MS-grade ammonium formate from Sigma–Aldrich (San Luis, MO, USA) and acetonitrile, formic acid and methanol from Scharlab (Barcelona, Spain) were used for preparation of chromatographic mobile phases and working solutions. Standards (purity > 98%) of cannabinoids THC, THCA, THCV, CBG, CBGA, CBDV, CBD, CBDA, cannabidivarinic acid (CBDVA), (CBC), ∆8-tetrahydrocannabinol (∆8-THC), (CBN) and (CBL) were used to make the corresponding calibration curves. All standards were from Cerilliant (Round Rock, TX, USA), commercialized as a solution in methanol (1 mg mL 1). · − 2.3. Extraction Procedure 100 g of plant material were extracted, at room temperature, three times in organic solvent (n-hexane or ethanol) under constant stirring at 190 rpm in a mini OVAN OS10E S orbital shaker (Ovan, Barcelona, Spain). Extracts were then filtered under vacuum (Laboxact KNF, Trenton, NJ, USA) through a glass plate porous filter (Pobel, Madrid, Spain) with a pore size of 16–40 µm. Extracts were then evaporated to dryness in a RS-3000V rotaevaporator from Selecta (Barcelona, Spain) at 40 and 50 ◦C for hexane and ethanol, respectively. This process yielded a total of 20 extracts from Phytoplant varieties, 10 extracted with hexane, and 10 with ethanol. Dry extracts were then divided into two glass vials and one was placed into a “Conterm” oven (Selecta, Barcelona, Spain) and incubated at 150 ◦C for 60 min to induce decarboxylation of acid cannabinoids to their neutral forms. In this way, a total of 40 extracts were obtained: 20 decarboxylated (DEC) and 20 non-decarboxylated (NDC) (see Figure1).

2.4. Cannabinoid Purification CBD, CBG and CBGA cannabinoids were purified (>95%) by direct crystallization of the n-hexane extracts of petroleum ether extracts, and 3 recrystallizations in the same solvent in a 1L reactor and 1L jacketed filter (Vidrafoc, Barcelona, Spain) chilled to 18 C or 4 C with Digiterm 30 (Selecta) − ◦ ◦ and filtered under vacuum (Laboxact KNF, Trenton, NJ, USA) [17]. THC, THCA, CBDA and CBDV cannabinoids were purified (>95%) by liquid-liquid chromatography with CPC-1000-PRO and PLC 250 systems from Gilson (Middleton, WI, USA), as previously described [18].

2.5. Analysis of Cannabinoid Content For LC-DAD analysis, extracts (40 mg) were diluted with 5 mL of methanol, placed in an ultrasound bath for 25 min, and centrifuged for 5 min at 3000 rpm. Then, the supernatant containing cannabinoids was collected and further diluted 1:200 prior to analysis. An 1260 Agilent liquid chromatography (Santa Clara, CA, USA) set-up consisting of a binary pump, a vacuum degasser, a column oven, an autosampler and a diode array detector (DAD) equipped with a 150 mm length 2.1 mm internal × Separations 2020, 7, 56 6 of 16 diameter, 2.1 µm pore size Raptor ARC-18 column with a Raptor ARC-18 EXP guard column cartridge 2.7 µm, both from Restek (Bellefonte, PA, USA), was used for the quantification of cannabinoids. The device was controlled by OpenLAB CBS software (Agilent, Santa Clara, CA, USA). Full spectra were recorded in the range of 200–400 nm. The analysis was performed in isocratic mode using water with 5 mM ammonium formate and 0.1% formic acid and acetonitrile with 0.1% formic acid as mobile phases. Flow rate was set at 0.375 mL min 1, the injection volume at 3 µL and the injector needle · − was washed between injections with methanol. Chromatographic peaks were recorded at 225 nm. All determinations were carried out at 35 ◦C. All samples were analyzed in duplicate. The concentration of cannabinoids was calculated by peak area versus a calibration curve prepared with a commercial standard. The results for each cannabinoid were calculated as relative weight (%) in the extract.

2.6. Sample Preparation for Biological Activity Stock solutions of extracts and purified cannabinoids were prepared in DMSO at a concentration of 100 mg mL 1 and stored at 80 C until used. Experimental solutions were freshly prepared on · − − ◦ the day of the experiment by diluting stock solutions in DMSO. Final concentrations tested were 10, 25 and 50 µg mL 1 in the case of plant extracts and 5, 12.5 and 25 µg mL 1 for purified cannabinoids · − · − (THC, THCA, CBD, CBDA, CBDV, CBG, CBGA). Dosage range was determined to allow for direct comparison between the biological activity of purified cannabinoids and plant extracts, anticipating a 50% w/w concentration of the main cannabinoid in the extract.

2.7. Cell Lines Anti-NF-kB activity was determined using the Raw-KBF-Luc cell line, stably transfected with the plasmid KBF-Luc, which contains three copies of the NF-kB binding site from the major histocompatibility complex promoter, fused to a fragment of the simian virus 40 (SV40) promoter driving the firefly luciferase reporter gene. The increase in NF-κB levels activates the pCRE-Luc system, inducing the expression of the luciferase reporter gene. The determination of STAT-3 activity was performed using the Raw-STAT3-Luc cell line, which is stably transfected with the plasmid 4xM67 pTATA TK-Luc that contains STAT3 binding sites and whose activation induces the expression of the firefly luciferase reporter gene. The determination of HIF-1α activity was performed using the cell line NIH-3T3-Epo-Luc. This cell line is stably transfected with the plasmid Epo-Luc, which contains three tandem copies of the hypoxia response element (HRE) of the erythropoietin (EPO) promoter and whose activation induces the expression of the firefly reporter gene. The determination of cytotoxicity was performed using the mouse brain neuroblastoma cell line N2a obtained from ATCC (Manassas, VA, USA).

2.8. Determination of Biological Activity 100 µL of a cell suspension were seeded in 96-well plates (500,000 cells mL 1) and incubated for · − 24 h. Cells were then treated for 30 min either with cannabis extracts or purified cannabinoids and then stimulated with either LPS (1 µg mL 1, in the case of NF-κB and STAT-3) [21] or deferoxamine · − (150 µM, in the case of HIF-1α) for 6 h [22]. Deferoxamine, a potent hypoximimetic compound acting as an iron chelating agent, or LPS were used alone as positive control and cells without stimulus were used as negative control. Cells were then washed twice with PBS 1X and lysed in 100 µL of lysis buffer (25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 7% glycerol) for 15 min at room temperature under orbital shaking. Luciferase activity was determined with a TriStar LB 942 plate multi-reader (Berthold Technologies, GmbH & Co. KG, Bad Wildbad, Germany) following the instructions of the Luciferase Assay system from Promega. Resulting RLU (Relative Light Units) were Separations 2020, 7, 56 7 of 16 normalized to positive control (deferoxamine or LPS) in order to calculate the percentage of activity left. Percentage of inhibition was calculated as:

T B %inhibition = 100 − (1) ∗ C B − where T are the RLU of cells treated with cannabis extracts, C are the RLU of cells challenged with LPS of deferoxamine, and B are the RLU of un-stimulated control cells.

2.9. Determination of Cytotoxicity 100 µL of a cell suspension were seeded in 96-well plates (40,000 cells mL 1) and incubated for · − 24 h. Cells were then treated for 24 h either with cannabis extracts or purified cannabinoids and then the media removed and 50 µL of MTT was added (1.25 mg mL 1) and incubated for 4 h. Then the · − supernatant was removed and 100 µL of DMSO were added and the absorbance recorded at 550 nm with a TriStar LB 941 plate multi-reader (Berthold Technologies, GmbH & Co. KG, Bad Wildbad, Germany). Resulting absorbances were normalized to control (untreated cells) and blank (DMSO) to calculate the percentage of cell viability according to the following formula:

T B % viability = 100 − (2) ∗ C B − where T is the absorbance of the cannabis extract treated cells, C is the absorbance of the untreated cells and B is the absorbance of the blank.

2.10. Statistical Analysis Cannabinoid concentration is reported as percentage (w/w). Biological activity is expressed as the mean standard deviation of two or three independent assays. Dose-response inhibition of NF-κB ± activity was analyzed by a one-way ANOVA followed by a Dunnett’s multiple comparisons test with significance set at p < 0.05, using GraphPad Prism (Version 8.1.1) (San Diego, CA, USA).

3. Results Extraction yield was found to be cultivar-dependent, ranging from the less-producing variety, Octavia (main cannabinoid, CBG), to the largest producer, Moniek (main cannabinoid, THC). Although not significant differences were found, hexane extraction yields were slightly lower compared to ethanol: 4.9% vs. 6.8% for Octavia and 28.4% vs. 28% for Moniek (Table1). This may be indicative of a richer phytocomplex with more polar molecules like flavonoids and polyphenols in the case of ethanol whom, contrary to hexane, is nor cancerous nor toxic for the environment. Accordingly, average weight loss during decarboxylation ranged from 0.37% and 6.37% for hexane extracts and between 8.65% and 16.67% in the case of ethanol, likely due to the amount of water extracted by ethanol from the plant material. All extracts pass the residual solvents analysis accordance of European Pharmacopoeia 9.0. (2.4.24, Identification and control of Residual Solvents using Methodology system A) for Class I solvents the HEX cannabis extracts and for Class III solvents the ETH cannabis extracts. Of note, the salient carbon dioxide molecule generated during decarboxylation represents approximately 12% of the molecular weight of THCA, CBDA and CBGA [3]. Decarboxylation did not produce significant changes in the color or physical appearance of extracts. No significant differences were appreciated between solvents in the average total content of cannabinoids among extracts, which was 71.1 15.7 % for hexane and 68.0 10.0% for ethanol. In the ± ± case of non-decarboxylated extracts the average of total content in hexane and ethanol extracts were 74.30 12.15 % and 68.1 7.9% respectively (Supplementary Table S1). Figures2 and3 depict the ± ± relative concentrations of major and minor cannabinoids, a total of 13 is reported. Because of their low quantity and similar relative abundance across extracts, CBL, ∆8-THC, CBN, CBC and THCV were not tested in the in vitro assays. Separations 2020, 7, x FOR PEER REVIEW 7 of 15 Separations 2020, 7, x FOR PEER REVIEW 7 of 15 case of non-decarboxylated extracts the average of total content in hexane and ethanol extracts were 74.30case of ± non12.15-decarboxylated % and 68.1 ± 7.9% extracts respectively the average (Supplementary of total content Table in hexane S1). Figure and ethanols 2 and extracts 3 depict were the relative74.30 ± 12.15concentrations % and 68.1 of ±major 7.9% andrespectively minor cannabinoids, (Supplementary a total Table of 13S1). is Figurereported.s 2 andBecause 3 depict of their the lowrelative quantity concentrations and similar of relativemajor and abundance minor cannabinoids, across extrac ats, total CBL, of Δ8 13- THC,is reported. CBN, CBCBecause and of THCV their Separations 2020, 7, 56 8 of 16 werelow quantity not tested and in similarthe in vitro relative assays. abundance across extracts, CBL, Δ8-THC, CBN, CBC and THCV were not tested in the in vitro assays.

Figure 2. Cannabinoid composition of ethanolic extracts determined by HPLC-DAD. Concentrations Figure 2. Cannabinoid composition of ethanolic extracts determined by HPLC-DAD. Concentrations (%Figure w/w )2. are Cannabinoid expressed ascomposition mean ± standard of ethanolic deviation. extracts determined by HPLC-DAD. Concentrations (%(%w w//ww)) areare expressed expressed as as mean mean ± standardstandard deviation.deviation. ±

FigureFigure 3.3. Cannabinoid composition of hexane extracts determined by HPLC-DAD.HPLC-DAD. Concentrations (% wFigure/w) are 3. expressed Cannabinoid as mean compositionstandard of hexane deviation. extracts determined by HPLC-DAD. Concentrations (% w/w) are expressed as mean± ± standard deviation. w/w) are expressed as mean ± standard deviation. Figure4 displays the degree of transcriptional inhibition (as % of activation) at the most e ffective, Figure 4 displays the degree of transcriptional inhibition (as % of activation) at the most effective, non-toxic dose for each cannabis extract in PHYRESCEL at the three targets investigated, NF-κB non-toxicFigure dose 4 displays for each the cannabis degree extractof transcriptional in PHYRESCEL inhibition at the (as three % of targets activation) investigated, at the most NF effective,-κB (left (left panel), HIF-1α (middle panel) and STAT3 (right panel), following incubation with LPS or panel),non-toxic HIF dose-1α for (middle each cannabis panel) extract and STAT3 in PHYRESCEL (right panel), at the three following targets incubation investigated, with NF - LPSκB (left or deferoxamine. First, our results indicate that decarboxylated (DEC) extracts, rich in neutral cannabinoids deferoxamine.panel), HIF-1α First, (middle our panel) results and indicate STAT3 that (right decarboxylated panel), following (DEC) incubationextracts, rich with in LPSneutral or such as THC, CBD and CBG, were consistently more toxic for the cell lines used in the assay, cannabinoidsdeferoxamine. such First, as THC, our resultsCBD and indicate CBG, were that consistently decarboxylated more (DEC)toxic for extracts, the cell lines rich used in neutral in the which share some proliferative features of cancerous cells, than their non-decarboxylated (NDC) assay,cannabinoids which such share as some THC, proliferative CBD and CBG, features were consistently of cancerous more cells, toxic than for their the cell non lines-decarboxylated used in the counterpartsassay, which irrespectively share some proliferative of the solvent features used. Accordingly, of cancerous most cells, DEC than extracts their non (>95%)-decarboxylated were toxic (NDC) counterparts irrespectively1 of the solvent used. Accordingly, most DEC extracts (>95%) were at(NDC) a dose counterparts of 50 µg mL irrespectively− , with less of than the solvent 20% of use NDCd. Accordingly extracts exhibiting, most DEC cytotoxicity extracts at(>95%) the samewere toxic at a dose of 50· μg·mL−1, with less than 20% of NDC extracts exhibiting cytotoxicity at the same dose (Figure4). This observation−1 agrees with the literature, where the cytotoxic e ffect of neutral dosetoxic at (Figure a dose 4). of This50 μg·mL observation, with less agrees than with 20% the of NDC literature, extracts where exhibiting the cytotoxic cytotoxicity effect at of the neutral same cannabinoidsdose (Figure 4). on This immortalized observation cell agrees lines has with been the literature,thoroughly where noted the as cytotoxica limitation effect of cell-based of neutral assays in the pharmacological characterization of both cannabinoids [23,24] and cannabis extracts [25].

Therefore, we were not able to obtain complete dose-response information regarding the potency of most entries in PHYRESCEL, particularly in the case of either DEC_HEX or DEC_ETH groups (Figure4). However, despite this toxicity issue, we were able to draw relevant information regarding Separations 2020, 7, x FOR PEER REVIEW 8 of 15 cannabinoids on immortalized cell lines has been thoroughly noted as a limitation of cell-based assays in the pharmacological characterization of both cannabinoids [23,24] and cannabis extracts [25]. SeparationsTherefore,2020 we, 7 ,were 56 not able to obtain complete dose-response information regarding the potency9 of of 16 most entries in PHYRESCEL, particularly in the case of either DEC_HEX or DEC_ETH groups (Figure the4). However, differential despite effects ofthis solvent toxicity selection issue, we and were cannabinoid able to draw decarboxylation relevant information on the biological regarding activity the ofdifferential cannabis extracts.effects of Althoughsolvent selection this not alwaysand cannabinoid correlated decarboxylation with cannabinoid on content, the biological the overall activity results of suggestcannabis that extracts. DEC extractsAlthough displayed this not always higher potency,correlated with with a majoritycannabinoid of non-toxic content, ID50the overall values results in the rangesuggest of that 10–25 DECµg extractsmL 1, while displayed NDC extractshigher potency, were generally with a majority most effective of non at-toxi 50 µc gID50mL values1 with in a fewthe · − · − exceptionsrange of 10 (Figure-25 μg·mL4). −1, while NDC extracts were generally most effective at 50 μg·mL−1 with a few exceptions (Figure 4).

Figure 4. Screening of the biological activity of PHYRESCEL. The ability of cannabis extracts to inhibitFigure 4. the Screening activation of the of biological transcription activity factors of PHYRESCEL. NF-κB, HIF-1 Theα and ability STAT-3 of cannabis was measured extracts toin inhibit vitro. Degreethe activation of inhibition of transcription is expressed factors as reduction NF-ҡB, HIF of transcription-1α and STAT activation-3 was measured in percentage. in vitro. Color Degree legend of indicatesinhibition solventis expressed used as and reduction decarboxylation of transcription treatment. activation Blue: in Hexanepercentage./non-decarboxylated; Color legend indicates Red: Hexanesolvent /decarboxylated; used and decarboxylation Green: Ethanol /non-decarboxylated; treatment. Blue: Purple: Hexane/no Ethanoln-decarboxylated;/decarboxylated. Red: NA: notHexane/decarboxylated; active. Green: Ethanol/non-decarboxylated; Purple: Ethanol/decarboxylated. NA: not active. The molecular mechanism by which cannabinoids modulate NF-κB signaling remains controversial. For example,The molecular CBD but mechanism not THC has by been which shown cannabinoids to reduce the modulate activity of the NF- NF-κB κ signalingB pathway remains in BV-2 microglialcontroversial. cells For [26 example,]. However, CBD synthetic but not THC cannabinoid has been , shown to reduce non-antioxidant the activity CB1of the receptor NF-κB agonistpathway WIN in BV 55,212-2-2 microglial and phytocannabinoid cells [26]. However, CBN, synthetic which cannabinoid weakly binds dexanabinol, to CB1 receptors, non-antioxidant inhibited NF-CB1κ B receptor transcriptional agonist activity WIN 55,212 and apoptosis-2 and phytocannabinoid in a neuron-like cell CBN, line, which which weakly suggests binds that toneither CB1 thereceptors, antioxidant inhibited properties NF-κB of transcriptional cannabinoids noractivity binding and to apoptosis cannabinoid in a receptors neuron-like are cell responsible line, which for thissuggests effect that [27 ].neither Our results the antioxidant show how properties most extracts of cannabinoids did not block nor more binding than to 50% cannabinoid the transcriptional receptors activationare responsible of NF- forκB this in macrophages effect [27]. Our induced results by show LPS (Figurehow most4). Accordingly,extracts did THCnot block and CBDmore displayed than 50% cellularthe transcriptional toxicity at higheractivation doses of andNF-κB were in macrophages inactive towards induced NF-κ Bby at LPS the (Figure dose of 4). 10 Accordingly,µg mL 1 (Table THC2). · − Discrepanciesand CBD displayed with citedcellular literature toxicity may at higher be due doses to di andfferences were inactive in the cell towards lines used NF-κB as wellat the as dose in the of experimental10 μg·mL−1 (Table design 2). [Discrepancies25]. Nevertheless, with wecited were literature able to may identify be due four to cannabis differences extracts in the capable cell lines of inhibitingused as well NF- asκ Bin activationthe experimental more than design 40% [25]. at non-toxic Nevertheless, doses we (Table were2). able to identify four cannabis extracts capable of inhibiting NF-κB activation more than 40% at non-toxic doses (Table 2).

Separations 2020, 7, x FOR PEER REVIEW 9 of 15 Separations 2020, 7, 56 10 of 16 Table 2. Inhibition of LPS-induced NF-ҡB activation in Raw-KBF-Luc macrophages by cannabis extracts. Activity values for the most effective extracts (transcription activation < 60%) identified in Table 2. Inhibition of LPS-induced NF-κB activation in Raw-KBF-Luc macrophages by cannabis extracts. the screening assay, paired with their cannabinoid composition. Activity values of major Activity values for the most effective extracts (transcription activation < 60%) identified in the screening cannabinoids present in those extracts (THC, THCA, CBD and CBDA) are also presented. assay, paired with their cannabinoid composition. Activity values of major cannabinoids present in those extracts (THC, THCA,% CBD of Transcriptional and CBDA) Activation are also presented. Δ-9- Group Variety CBDV CBD CBDA THCV THCA CBG CBGA 10 μg·mL−1 25 μg·mL−1 50 μg·mL−1 THC % of Transcriptional Activation Group Variety CBDV CBD CBDA THCV ∆-9-THC THCA CBG CBGA ETH_NDC MAGDA10 µg mL107.91 ± 4.025 µg mL90.11 ± 17.050 µg mL 53.51 ± 1.98 0 0 0.67 0 9.71 45.34 0.1 0.67 · − · − · − ETH_NDC MAGDA 107.9 4.0 90.1 17.0 53.5 1.98 0 0 0.67 0 9.71 45.34 0.1 0.67 ETH_DEC BEATRIZ ± 131.01 ± 54.21 ± 0.19 41.36 0 0.42 25.5 0 1.34 0 ETH_DEC BEATRIZPILAR 131.0156.8 54.21- -0.19 41.360.4 68.00 0.420 025.5 1.25 00 1.341.88 0 0 HEX_DEC PILAR 56.8 - - 0.4 68.0 0 0 1.25 0 1.88 0 HEX_DEC SARA 57.9 ± 12.3 - - 0.99 83.79 0 0.8 3.0 0 2.48 0 SARA 57.9 12.3 - - 0.99 83.79 0 0.8 3.0 0 2.48 0 ± 1 1 12.5 1 5 µg mL5− μg·mL12.5−1 µg mL− 25 µg mL25− μg·mL−1 · · −1 · CBD 102.7 3.6 -μg·mL - ± ISOLATED THCCBD 111.3 12.4102.7 ± 3.6 -- - - STANDARDS ± ISOLATED CBDATHC 105.3 111.37.5 ±12.4 102 13.8- 109.1 3.1 - ± ± ± STANDARDS THCACBDA 107.9 4.7105.3 ± 7.5 109.3 11.7102 ± 13.8 112.4 8.9109.1 ± 3.1 ± ± ± THCA 107.9 ± 4.7 109.3 ± 11.7 112.4 ± 8.9 Contrary to their toxic potential, the inhibitory activity of these extracts did not seem to correlateContrary with to their their cannabinoid toxic potential, content. the inhibitory To further activity characterize of these extracts this finding, did not seem we conducted to correlate a refinedwith their dose-response cannabinoid experiment content. To with further extracts characterize of Magda this (ETH_NDC, finding, Figurewe conducted5A), Beatriz a refined (ETH_DEC, dose- Figureresponse5B), experiment Pilar (HEX_DEC, with extracts Figure of5 C)Magda and Sara(ETH_NDC, (HEX_DEC, Figure Figure 5A),5 BeatrizD) and (ETH_DEC, were able to Figure confirm 5B), theirPilar (HEX_DEC, potency and Figure toxic 5C) range, and with Sara all(HEX_DEC, of them inhibitingFigure 5D) NF- andκ Bwere activation able to confirm in a dose-dependent their potency mannerand toxic before range, reaching with all toxic of them concentrations. inhibiting NF Such-κB eactivationffect that wasin a notdose found-dependent when themanner same before plant varietiesreaching weretoxic subjectedconcentra totions. different Such processingeffect that conditionswas not found or with when pure the cannabinoids. same plant Therefore,varieties were it is plausiblesubjected toto speculatedifferent thatprocessing these extracts conditions may or contain with additionalpure cannabinoids. molecules Therefore, capable of it blocking is plausible NF- κtoB transcriptionalspeculate that activation, these extracts either may acting contain alone or additional as a part of molecules a phytocomplex. capable For of example, blockin flavonoidg NF-κB Cannflavintranscriptional A and activation, a phenanthrenequinone either acting alone derivative, or as a part Denbinobin, of a phytocomplex. present For in the example, cannabis flavonoid variety “Carma”,Cannflavin elicit A and synergistic a phenanthrenequinone effects in combination derivative, with CBD Denbinobin, and CBG present to inhibit in NF-theκ cannabisB activation variety [28]. Denbinobin“Carma”, elicit alone synergistic also inhibits effects NF- inκ combinationB[29]. with CBD and CBG to inhibit NF-κB activation [28]. Denbinobin alone also inhibits NF-κB [29].

Figure 5. Inhibition of LPS-induced NF-κB activation in Raw-KBF-Luc macrophages by cannabis Figure 5. Inhibition of LPS-induced NF-ҡB activation in Raw-KBF-Luc macrophages by cannabis extracts. Plant extracts from (A) Magda (ETH_NDC), (B) Beatriz (ETH_DEC), (C) Pilar (HEX_DEC) extracts. Plant extracts from (A) Magda (ETH_NDC), (B) Beatriz (ETH_DEC), (C) Pilar (HEX_DEC) and (D) Sara (HEX_DEC) were tested at different concentrations to further investigate their inhibitory and (D) Sara (HEX_DEC) were tested at different concentrations to further investigate their inhibitory effect and toxicity. Values are expressed as the mean standard deviation of 2–3 independent assays. effect and toxicity. Values are expressed as the mean ± standard deviation of 2–3 independent assays. ** p < 0.01; *** p < 0.001 vs. LPS alone. ** p < 0.01; *** p < 0.001 vs LPS alone. Hypoxia-inducible factor-1α (HIF-1α) has been identified as a key regulator of the hypoxic responseHypoxia with-inducible pleiotropic factor effects,-1α such (HIF- as1α) promotion has been of identified invasion, as angiogenesis, a key regulator switch of to the glycolytic hypoxic metabolism,response with and pleiotropic up-regulation effects, of cellsuch survival-related as promotion of molecules invasion, [30 angiogenesis,]. The HIF-1 αswitchsignaling to glycolytic pathway hasmetabolism, been proposed and up to-regula mediatetion theof cell neuroprotective survival-related role molecules for CB2 receptors [30]. The HIF in microglia-1α signaling [31]. pathway A single studyhas been has proposed reported theto mediate ability of the CBD neuroprotective to dose-dependently role for reduce CB2 receptors the expression in microglia of HIF-1 [31].α in A gliomasingle cellsstudy [ 32has]. reported Results the from ability our of biological CBD to dose screening-dependently suggest reduce that among the expression phytocannabinoids, of HIF-1α in glioma CBDV, CBDcells [32]. and Results CBG were from able our to biological reduce the screening activation suggest of HIF-1 thatα amonginduced phytocannabinoids, by deferoxamine, CBDV, with IC50s CBD and CBG were able1 to reduce the activation of HIF-1α induced1 by deferoxamine, with IC50s under under 12.5 µg mL− for CBD and CBDV, and 25 µg mL− for CBG (Table3). 12.5 μg·mL−1 for· CBD and CBDV, and 25 μg·mL−1 for· CBG (Table 3).

Separations 2020, 7, 56 11 of 16

Table 3. Inhibition of deferoxamine-induced HIF-1α activation in NIH-3T3- Epo-Luc cells by cannabis extracts. Table shows the activity values for the most effective extracts identified in the screening assay, paired with their cannabinoid composition. Activity values of major cannabinoids present in those extracts (THCA, CBD, CBDA, CBDV, CBG and CBGA) are also presented.

% of Transcriptional Activation Group Variety CBDV CBD CBDA THCV ∆-9-THC THCA CBG CBGA 10 µg mL 1 25 µg mL 1 50 µg mL 1 · − · − · − MONIEK 89.1 6.3 58.7 10.5 30.5 1.8 0 0 0 0 10.45 49.52 0.72 2.11 ± ± ± MAGDA 85.8 9.1 42.2 1.9 12.6 3.1 0 0 0 0 9.71 45.34 0.10 0.67 ETH_NDC ± ± ± MATI 104.8 4.8 70.2 3.6 38.5 17.3 0 3.16 44.26 0 6.76 26.95 1.14 2.70 ± ± ± PILAR 100.6 18 67.6 8.6 30.3 26.5 0 8.71 61.60 0 0.79 1.26 0.14 2.12 ± ± ± THERESA 93.3 27.1 61.1 34 26.1 16.2 2.59 9.48 34.90 0.08 0.65 0.42 0.08 0.53 ± ± ± AÍDA 103.2 9.8 82.2 7.7 29.3 2.7 0.35 11.54 0 0 0.35 0 57.86 0 ± ± ± ETH_DEC JUANI 80.2 7.7 40.1 14.3 - 3.27 5.10 0 0 0 0 56.14 0 ± ± MATI 99.6 35.9 29.2 14.7 - 0.13 37.91 0 0.62 29.53 0 2.71 0 ± ± THERESA 101.4 22.7 28.7 9.9 - 15.05 57.47 0 0.98 1.97 0 0.79 0 ± ± 5 µg mL 1 12.5 µg mL 1 25 µg mL 1 · − · − · − CBD 55.6 19.5 0.59 1.83 - ± ± CBDV 66.1 28.3 26 31.7 - ± ± ISOLATED CBDA 80.8 21.9 86.6 11.1 81.3 10.9 STANDARDS ± ± ± CBG 89.2 10.5 59.7 22 41.5 1.2 ± ± ± CBGA 75.8 23.8 86.4 41.6 61.4 18.1 ± ± ± THCA 106.4 9.1 103.1 3.9 85.2 25.7 ± ± ± THC 107.9 4.7 109.3 11.7 112.4 8.9 ± ± ± Separations 2020, 7, 56 12 of 16

Additional studies would be required to confirm and better characterize this finding. As for the extracts, ETH extracts were clearly more potent than HEX, with a total of nine ETH extracts found active at inhibiting HIF-1α (Figure4, Table3). Among these, five NDC extracts presented similar potencies, with IC50s in the 25–50 µg mL 1 range, irrespective of their major cannabinoid component · − (either CBDA, THCA or both). However, this observation did not correlate with the reported potency for acid cannabinoids in this assay, which were not active at the highest concentration tested (Table3). This may be suggestive of the presence of non-cannabinoid active molecules, selectively extracted by the polar solvent ethanol and not by the non-polar solvent hexane, occurring in five different cannabis varieties (Moniek, Magda, Mati, Pilar and Theresa). Several flavonoids have been shown to actively inhibit HIF-1a transcription [33]. Flavonoids are more polar than cannabinoids and, therefore, more likely to be extracted by ethanol than hexane, thus providing a plausible explanation as to why ETH extracts from the same cannabis cultivar are active, but not their HEX counterparts. On the other hand, the degree of inhibition displayed by the remaining four ETH_DEC extracts seems to correlate with their cannabinoid content, dominated by the presence of CBD, CBDV and CBG. However, the reason why their HEX_DEC counterparts were either not active or toxic remains unclear. Theresa and Mati varieties produced ETH_NDC and ETH_DEC extracts active at inhibiting HIF-1a with the ETH_DEC IC50 of <25 µg mL 1, more active than the ETH_NDC with a IC50 between 25–50 µg mL 1 · − · − Further studies are currently undergoing to help clarifying this finding by employing more powerful analytical and statistical techniques, such as quantitative time-of-flight (QTOF) detection and principal component analysis (PCA), similarly to what recently reported by other authors [25]. Signal transducer and activator of transcription 3 (STAT3), a member of the STAT families, is involved in regulating essential cellular functions in cancer, such as proliferation, differentiation, survival, invasion, angiogenesis, and metastasis [34]. STAT3 activation can be effectively modulated by several key receptors of the such as CB1 [35,36], CB2 [37] and G protein-coupled receptor 55 (GPR55), which has been postulated as a novel [38]. Further, THC induces trophoblast dysfunction in pregnant mice by suppressing STAT3 signaling [39]. Indeed, our results show how THC nearly abolished STAT3 activation at the lower dose tested (5 µg mL 1). · − All other cannabinoids tested were also able to suppress the activation of STAT3 induced by LPS except for CBGA (Figure6). Accordingly, all PHYRESCEL entries resulted active at, at least, one non-toxic dose, with DEC extracts being about five-times more potent than NDC extracts (Figure4). However, major differences between solvents were not observed, probably due to the efficacy of the cannabinoid components present in the extracts. While remarkable, more directed studies will be required to confirm and better characterize this inhibitory effect displayed by cannabinoids over LPS-induced STAT3 activation. Separations 2020, 7, 56 13 of 16 Separations 2020, 7, x FOR PEER REVIEW 12 of 15

Figure 6. Inhibition of LPS-induced STAT-3 activation in Raw-STAT3-Luc macrophages by pure isolated Figure 6. Inhibition of LPS-induced STAT-3 activation in Raw-STAT3-Luc macrophages by pure cannabinoids (A) THC, (B) CBD, (C) CBDV, (D) CBG, (E) THCA, (F) CBGV, (G) CBDA, (H) CBGA, isolated cannabinoids (A) THC, (B) CBD, (C) CBDV, (D) CBG, (E) THCA, (F) CBGV, (G) CBDA, (H) were tested at different concentrations of 5, 12.5 and 25 µg mL 1. Values are expressed as the mean CBGA, were tested at different concentrations of 5, 12.5 and· 25− g·mL−1. Values are expressed as the± standard deviation of 2–independent assays. mean ± standard deviation of 2–independent assays. 4. Conclusions 4. Conclusions Here we disclose the preparation and preliminary characterization of PHYRESCEL, a botanical drugHere library we containingdisclose the 40 preparation cannabis extracts and preliminary with varying characterization cannabinoid of content. PHYRESCEL, By screening a botanical the drugpharmacological library containing activity 40of PHYRESCEL cannabis extractsin vitro with, we varying were able cannabinoid to pin-point content. several By cannabis screening extracts the pharmacologicalthat differentially activity inhibited of thePHYRESCEL transcriptional in vitro, activation we were of NF-ableκ B,to HIF-1pin-pointα and several STAT3, cannabis three molecular extracts thattargets differentially known to mediate inhibited cellular the transcriptional responses to inflammation activation of and NF hypoxia.-κB, HIF- The1α andefficacy STAT3, of isolated three molecularminor cannabinoids targets known at inhibiting to mediate these cellular transcription responses factors to inflammation is also reported. and hypoxia. Our results The support efficacy the of isolatednotion that minor cannabis cannabinoids extracts atpossess inhibiting differential these transcription pharmacological facto propertiesrs is also reported. compared Our to isolated results supportcannabinoid the notion molecules. that cannabis The composition extracts possess and pharmacological differential pharmacological activity of different properties cannabis compared extracts toobtain isolated from a cannabinoid single cultivar molecules. differed largely The composition depending on and the organic pharmacological solvent used activity and the of subsequent different cannabisdecarboxylation extracts process.obtain from Although a single the cultivar translation differed value largely of these depending findings on is the unclear, organic we solvent expect theseused andfunctional the subsequent coordinates decarboxylation to aid in the identification process. Although of novel, thenatural translation modulators value of cellularof these responses findings tois unclear,pro-inflammatory we expect and these hypoxic functional stimuli present coordinates in certain to aid varieties in the of identification Cannabis sativa of L. novel, developed natural by modulatorsPhytoplant Research.of cellular responses to pro-inflammatory and hypoxic stimuli present in certain varieties of Cannabis sativa L. developed by Phytoplant Research. Supplementary Materials: The following are available online at http://www.mdpi.com/2297-8739/7/4/56/s1, SupplementaryTable S1: Cannabinoid Materials: composition The following of all are extracts available determined online at by www.mdpi.com/xxx/s1, HPLC-DAD. Concentrations Table S are1: Cannabinoid expressed as mean standard deviation. composition± of all extracts determined by HPLC-DAD. Concentrations are expressed as mean ± standard deviation.Author Contributions: Conceptualization, X.N.R. and V.S.d.M.B.; methodology, X.N.R., V.S.d.M.B., C.F.V., C.S.-C.; validation, G.M.-S., X.N.R., C.F.V. and V.S.d.M.B.; formal analysis, G.M.-S., C.F.V. and C.S.-C.; investigation, X.N.R., AuthorC.F.V. and Contributions: V.S.d.M.B.; data Conceptualization, curation, V.S.d.M.B.; X.N. writing—originalR. and V.S.d.M.B. draft; methodology, preparation, X.N. G.M.-S.;R., V.S. writing—reviewd.M.B., C.F.V., C.S. and- C.;editing, validation, X.N.R., G.M C.F.V.,.-S., G.M.-S.X.N.R., C.F. andV. V.S.d.M.B.; and V.S.d.M.B. supervision,; formal V.S.d.M.B.analysis, G.M All. authors-S., C.F.V. have and read C.S.- andC.; investigation, agreed to the X.N.publishedR., C.F. versionV. and ofV.S. thed.M.B. manuscript.; data curation, V.S.d.M.B.; writing—original draft preparation, G.M.-S.; writing— reviewFunding: andThis editing, research X.N. receivedR., C.F.V no., G.M external.-S. and funding. V.S.d.M.B.; supervision, V.S.d.M.B. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

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Acknowledgments: Technical assistance and scientific support from Juan Diego Unciti and Eduardo Muñoz from Emerald Health Biosciences España is gratefully acknowledged. Conflicts of Interest: C.F.V. and V.S.d.M.B. are employees of Phytoplant Research. X.N.R. and C.S.-C. are former employees of Phytoplant Research. G.M.-S. is a paid consultant to Phytoplant Research.

Abbreviations CBC: cannabichromene; CBD: cannabidiol; CBDA: cannabidiolic acid; CBDV: cannabidivarin; CBDVA: cannabidivarinic acid; CBG: cannabigerol; CBGA: cannabigerolic acid; CBN: cannabinol; CPVO: Community Plant Variety Office; HIF-1α: Hypoxia-inducible factor-1α; HPLC-DAD: high-performance liquid chromatography-diode array detection; IC50: inhibitory concentration 50; LPS: lipopolysaccharide; NF-κB: nuclear factor kappa-light- chain-enhancer of activated B cells; PCA: principal component analysis; PHYRESCEL: Phytoplant Research Cannabis Extract Library; QTOF: quantitative time-of-flight; STAT3: signal transducer and activator of transcription 3; THC: ∆9-tetrahydrocannabinol; THCA: ∆9-tetrahydrocannabinolic acid; THCV: ∆9-tetrahydrocannabivarin.

References

1. Hanuš, L.O.; Meyer, S.M.; Muñoz, E.; Taglialatela-Scafati, O.; Appendino, G. Phytocannabinoids: A unified critical inventory. Nat. Prod. Rep. 2016, 33, 1357–1392. [CrossRef][PubMed] 2. Russo, E.; Guy, G.W. A tale of two cannabinoids: The therapeutic rationale for combining tetrahydrocannabinol and cannabidiol. Med. Hypotheses 2006, 66, 234–246. [CrossRef][PubMed] 3. Moreno-Sanz, G. Can you pass the acid test? Critical review and novel therapeutic perspectives of δ9-tetrahydrocannabinolic acid A. Cannabis Cannabinoid Res. 2016, 1, 124–130. [PubMed] 4. ElSohly, M.A.; Radwan, M.M.; Gul, W.; Chandra, S.; Galal, A. Phytochemistry of cannabis sativa L. In Progress in the Chemistry of Organic Natural Products; Springer: Berlin/Heidelberg, Germany, 2017; pp. 1–36. 5. LaPrairie, R.B.; Bagher, A.M.; Kelly, M.E.M.; Denovan-Wright, E.M. Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor. Br. J. Pharmacol. 2015, 172, 4790–4805. [CrossRef] 6. Martínez-Pinilla, E.; Varani, K.; Reyes-Resina, I.; Angelats, E.; Vincenzi, F.; Ferreiro-Vera, C.; Oyarzabal, J.; Canela, E.I.; Lanciego, J.L.; Nadal, X.; et al. Binding and signaling studies disclose a potential allosteric site for cannabidiol in cannabinoid CB2 Receptors. Front. Pharmacol. 2017, 8, 744. [CrossRef] 7. Tham, M.; Yilmaz, O.; Alaverdashvili, M.; Kelly, M.E.M.; Denovan-Wright, E.M.; LaPrairie, R.B. Allosteric and orthosteric pharmacology of cannabidiol and cannabidiol-dimethylheptyl at the type 1 and type 2 cannabinoid receptors. Br. J. Pharmacol. 2019, 176, 1455–1469. [CrossRef] 8. Navarro, G.; Reyes-Resina, I.; Rivas-Santisteban, R.; De Medina, V.S.; Morales, P.; Casano, S.; Ferreiro-Vera, C.; Lillo, A.; Aguinaga, D.; Jagerovic, N.; et al. Cannabidiol skews biased agonism at cannabinoid CB1 and CB2 receptors with smaller effect in CB1-CB2 heteroreceptor complexes. Biochem. Pharmacol. 2018, 157, 148–158. [CrossRef] 9. Russo, E.B.; Marcu, J. Cannabis pharmacology: The usual suspects and a few promising leads. In Advances in Pharmacology; Academic Press: Cambridge, MA, USA, 2017; pp. 67–134. 10. MacCallum, C.A.; Russo, E.B. Practical considerations in administration and dosing. Eur. J. Intern. Med. 2018, 49, 12–19. [CrossRef] 11. Grinspoon, L.; Bakalar, J.B.; Zimmer, L.; Morgan, J.P.; McGregor, I.S.; Di Chiaro, G.; De Fonseca, F.R.; Navarro, M.; Carrera, M.R.A.; Koob, G.F.; et al. Marijuana addiction. Science 1997, 277, 749–752. [CrossRef] 12. Russo, E.B.; McPartland, J.M. Cannabis is more than simply ∆9-tetrahydrocannabinol. Psychopharmacology 2003, 165, 431–432. [CrossRef] 13. Russo, E.B. Taming THC: Potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br. J. Pharmacol. 2011, 163, 1344–1364. [CrossRef][PubMed] 14. Santiago, M.; Sachdev, S.; Arnold, J.C.; McGregor, I.S.; Connor, M. Absence of entourage: Terpenoids commonly found in Cannabis sativa do not modulate the functional activity of ∆9-THC at human CB1and CB2 receptors. Cannabis Cannabinoid Res. 2019, 4, 165–176. [CrossRef][PubMed] 15. Fitzcharles, M.A.; Eisenberg, E. Medical cannabis: A forward vision for the clinician. Eur. J. Pain 2018, 22, 485–491. [CrossRef] 16. Callado, C.S.-C.; Núñez-Sánchez, N.; Casano, S.; Ferreiro-Vera, C. The potential of near infrared spectroscopy to estimate the content of cannabinoids in Cannabis sativa L.: A comparative study. Talanta 2018, 190, 147–157. [CrossRef][PubMed] Separations 2020, 7, 56 15 of 16

17. Roura, X.N. Methods of Purifying Cannabinoids, Compositions, and Kits Thereof. WO Patent 2016116628, 28 July 2016. 18. Roura, X.N. Methods of Purifying Cannabinoids Using Liquid: Liquid Chromatography. WO Patent 2019145552A1, 1 August 2019. 19. Baldino, L.; Scognamiglio, M.; Reverchon, E. Supercritical fluid technologies applied to the extraction of compounds of industrial interest from Cannabis sativa L. and to their pharmaceutical formulations: A review. J. Supercrit. Fluids 2020, 165, 104960. [CrossRef] 20. Lewis-Bakker, M.M.; Yang, Y.; Vyawahare, R.; Kotra, L.P. Extractions of medical cannabis cultivars and the role of decarboxylation in optimal receptor responses. Cannabis Cannabinoid Res. 2019, 4, 183–194. [CrossRef] 21. Del Prete, D.; Taglialatela-Scafati, O.; Minassi, A.; Sirignano, C.; Cruz, C.; Bellido, M.L.; Muñoz, E.; Appendino, G. Electrophilic triterpenoid enones: A comparative thiol-trapping and bioactivity study. J. Nat. Prod. 2017, 80, 2276–2283. [CrossRef] 22. Minassi, A.; Rogati, F.; Cruz, C.; Prados, M.E.; Galera, N.; Jiménez, C.; Appendino, G.; Bellido, M.L.; Calzado, M.A.; Caprioglio, D.; et al. Triterpenoid hydroxamates as HIF prolyl hydrolase inhibitors. J. Nat. Prod. 2018, 81, 2235–2243. [CrossRef][PubMed] 23. Ruhaak, L.R.; Felth, J.; Karlsson, P.C.; Rafter, J.J.; Verpoorte, R.; Bohlin, L. Evaluation of the cyclooxygenase inhibiting effects of six major cannabinoids isolated from Cannabis sativa. Biol. Pharm. Bull. 2011, 34, 774–778. [CrossRef] 24. Brown, I.; Cascio, M.G.; Rotondo, D.; Pertwee, R.G.; Heys, S.D.; Wahle, K.W.J. Cannabinoids and omega-3/6 endocannabinoids as cell death and anticancer modulators. Prog. Lipid Res. 2013, 52, 80–109. [CrossRef] [PubMed] 25. Baram, L.; Peled, E.; Berman, P.; Yellin, B.; Besser, E.; Benami, M.; Louria-Hayon, I.; Lewitus, G.M.; Meiri, D. The heterogeneity and complexity of Cannabis extracts as antitumor agents. Oncotarget 2019, 10, 4091–4106. [CrossRef][PubMed] 26. Kozela, E.; Pietr, M.; Juknat, A.; Rimmerman, N.; Levy, R.; Vogel, Z. Cannabinoids ∆ 9-tetrahydrocannabinol and cannabidiol differentially inhibit the lipopolysaccharide-activated NF-κB and Interferon-β/STAT proinflammatory pathways in BV-2 microglial cells. J. Biol. Chem. 2010, 285, 1616–1626. [CrossRef] [PubMed] 27. Jüttler, E.; Potrovita, I.; Tarabin, V.; Prinz, S.; Dong-Si, T.; Fink, G.; Schwaninger, M. The cannabinoid dexanabinol is an inhibitor of the nuclear factor-kappa B (NF-κB). Neuropharmacology 2004, 47, 580–592. [CrossRef][PubMed] 28. Blanco, E.M.; Appendino, G.; Fiebich, B.L.; Grassi, G. A Composition Containing Non-Psychotropic Cannabinoids for the Treatment of Inflammatory Diseases. WO Patent 2009043836A1, 9 April 2007. 29. Sánchez-Duffhues, G.; Calzado, M.A.; de Vinuesa, A.G.; Appendino, G.; Fiebich, B.L.; Loock, U.; Lefarth-Risse, A.; Krohn, K.; Muñoz, E. Denbinobin inhibits nuclear factor-kappaB and induces apoptosis via reactive oxygen species generation in human leukemic cells. Biochem. Pharmacol. 2009, 77, 1401–1409. [CrossRef] 30. Kaur, B.; Khwaja, F.W.; Severson, E.A.; Matheny, S.L.; Brat, D.J.; van Meir, E.G. Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis. NeuroOncology 2005, 7, 134–153. [CrossRef] 31. Kossatz, E.; Maldonado, R.; Robledo, P. CB2 cannabinoid receptors modulate HIF-1α and TIM-3 expression in a hypoxia-ischemia mouse model. Eur. Neuropsychopharmacol. 2016, 26, 1972–1988. [CrossRef] 32. Solinas, M.; Massi, P.; Cinquina, V.; Valenti, M.; Bolognini, D.; Gariboldi, M.; Monti, E.; Rubino, T.; Parolaro, D. Cannabidiol, a non-psychoactive cannabinoid compound, inhibits proliferation and invasion in U87-MG and T98G glioma cells through a multitarget effect. PLoS ONE 2013, 8, e76918. [CrossRef][PubMed] 33. Deng, X.; Peng, Y.; Zhao, J.; Lei, X.; Zheng, X.; Xie, Z.-Z.; Tang, G. Anticancer activity of natural flavonoids: Inhibition of HIF-1α signaling pathway. Curr. Org. Chem. 2019, 23, 2945–2959. [CrossRef] 34. Yu, H.; Lee, H.; Herrmann, A.; Buettner, R.; Jove, R. Revisiting STAT3 signalling in cancer: New and unexpected biological functions. Nat. Rev. Cancer 2014, 14, 736–746. [CrossRef] 35. Zhou, H.; Zhang, Z.; Wei, H.; Wang, F.; Guo, F.; Gao, Z.; Marsicano, G.; Wang, Q.; Xiong, L. Activation of STAT3 is involved in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptors in rats. Brain Res. 2013, 1529, 154–164. [CrossRef] Separations 2020, 7, 56 16 of 16

36. Ciaglia, E.; Torelli, G.; Pisanti, S.; Picardi, P.; D’Alessandro, A.; Laezza, C.; Malfitano, A.M.; Fiore, D.; Zottola, A.C.P.; Proto, M.C.; et al. Cannabinoid receptor CB1 regulates STAT3 activity and its expression dictates the responsiveness to SR141716 treatment in human glioma patients’ cells. Oncotarget 2015, 6, 15464–15481. [CrossRef][PubMed] 37. Montecucco, F.; Lenglet, S.; Braunersreuther, V.; Burger, F.; Pelli, G.; Bertolotto, M.B.; Mach, F.; Steffens, S. CB (2) cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion. J. Mol. Cell. Cardiol. 2009, 46, 612–620. [CrossRef][PubMed] 38. Stanˇci´c,A.; Jandl, K.; Hasenöhrl, C.; Reichmann, F.; Marsche, G.; Schuligoi, R.; Heinemann, A.; Storr, M.A.; Schicho, R. The GPR55 antagonist CID16020046 protects against intestinal inflammation. Neurogastroenterol. Motil. 2015, 27, 1432–1445. [CrossRef][PubMed] 39. Chang, X.; Bian, Y.; He, Q.; Yao, J.; Zhu, J.; Wu, J.; Wang, K.; Duan, T. Suppression of STAT3 signaling by ∆9-Tetrahydrocannabinol (THC) induces trophoblast dysfunction. Cell. Physiol. Biochem. 2017, 42, 537–550. [CrossRef][PubMed]

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