Method for selective extraction of cannabinoids from a plant source

Abstract

Provided are methods for selective extraction of cannabinoids, for example cannabidiol (CBD), from a plant source, by using tailored extraction media.

Claims

1. A process for the extraction of cannabidiol from cannabis to yield a medium in microemulsion form which is devoid of water and enriched in cannabidiol, the process consisting essentially of: (a) heating cannabis to a temperature of between about 90° C. and 180° C. for about 5 minutes to 240 minutes, to convert cannabidiolic acid in the cannabis into cannabidiol thereby obtaining cannabis rich in cannabidiol; (b) mixing a first quantity of the cannabis rich in cannabidiol and a first quantity of an extraction medium consisting essentially of at least one oil, at least one hydrophilic surfactant and at least one co-surfactant to form a first microemulsion mixture which is devoid of water; (c) homogenizing the first microemulsion mixture under conditions to maintain the first microemulsion mixture devoid of water, wherein the homogenization is carried out at a pressure of between about 500 psi and 6,000 psi and at a temperature of between about 5° C. and about 70° C., for a period of time of between about 1 minute and about 60 minutes; (d) separating a cannabis biomass slurry from the first microemulsion mixture to obtain a first cannabidiol-loaded medium in a microemulsion form devoid of water; (e) mixing the cannabidiol-loaded medium in a microemulsion form that is devoid of water with a second quantity of cannabis to obtain a second mixture; (f) homogenizing the second mixture; and (g) separating a cannabis biomass slurry from the second mixture to obtain a medium in a microemulsion form which is devoid of water and enriched in cannabidiol, wherein the cannabidiol-loaded medium is in a microemulsion form which is devoid of water, wherein the at least one oil is selected from the group consisting of medium chain triglycerides, olive oil, soybean oil, canola oil, cotton oil, palmolein, sunflower oil, corn oil, isopropyl myristate, oleyl lactate, coco caprylocaprate, hexyl laurate, oleyl amine, oleic acid, oleyl alcohol, linoleic acid, linoleyl alcohol, ethyl oleate, hexane, heptanes, nonane, decane, dodecane, D-limonene, triacetin, neem oil, lavender oil, peppermint oil, anise oil, menthol, capsaicin, grape seed oil, pomegranate oil, avocado oil, sesame oil, fish oil, omega oils and omega fatty acids; wherein the at least one hydrophilic surfactant is selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, polyoxyethylene esters of saturated and unsaturated castor oil, ethoxylated monoglycerol esters, glycerol, ethoxylated fatty acids, ethoxylated fatty acids of short, medium and long chain fatty acids; and wherein the at least one co-surfactant is at least one polyol.

2. The process of claim 1, wherein the cannabidiol-loaded medium has a tetrahydrocannabinol content of at most 3 wt %.

3. The process of claim 1, wherein the cannabinoid-loaded medium has a cannabidiol content between about 0.1 and 12 wt %.

4. The process of claim 1, wherein the separating step comprises centrifuging the mixture.

5. The process of claim 4, wherein the centrifuging the mixture is followed by filtering.

6. The process of claim 1, wherein the weight ratio (wt./wt.) of cannabis to said first quantity of extraction medium is between 1:5 and 1:100.

7. The process of claim 1, wherein the step sequence (e)-(g) is repeated between 3 and 7 times.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIG. 1 shows CBD peak areas against heating temperature for plant samples heated at various temperatures for transforming CBDA to CBD as obtained by HPLC analysis.

(3) FIG. 2 shows CBD/CBDA peak areas ratio as obtained by HPLC analysis plotted against heating time at various heating temperatures.

(4) FIG. 3 shows CBD concentration in the extraction medium and in 40% water-diluted extraction media.

(5) FIG. 4 shows CBD concentration in the AX-1 extraction medium as a function of extraction duration.

(6) FIG. 5 shows the yield of CBD extraction from the plant using AX-1 extraction medium as a function of extraction duration.

(7) FIG. 6 shows the ratio between the CBD peak area and the peak areas of other cannabinoids in the plant sample (CBDA, THC and CBN) for various extraction durations.

(8) FIG. 7 shows CBD concentration in the AX-1 extraction medium as a function of plant-to-medium ratio.

(9) FIG. 8 shows the yield of CBD extraction from the plant using AX-1 extraction medium as a function of plant-to-microemulsion ratio.

(10) FIG. 9 shows the ratio between the CBD peak area and the peak areas of other cannabinoids in the plant sample (CBDA, THC and CBN) for various plant-to-medium ratios.

(11) FIG. 10 shows CBD concentration in the AX-1 extraction medium as a function of the number of extraction cycles.

(12) FIG. 11 shows the yield of CBD extraction from the plant using AX-1 extraction medium as a function of the number of extraction cycles.

(13) FIG. 12 shows the peak areas for CBD, CBDA, THC and CBN in the extraction medium samples as function of the number of extraction cycles.

(14) FIG. 13 shows the extraction concentration and yield of CBD and other cannabinoids using ethanol.

(15) FIG. 14 shows the extraction concentration and yield of CBD and other cannabinoids using petroleum ether.

(16) FIGS. 15A-15B show stability test results for 70 days carried out on CBD-loaded AX-1 and 5CS concentrates, respectively.

(17) FIG. 16 shows CBD levels in plasma at 0.5 and 2 hours from administration of CBD-loaded AX-1, CBD-loaded 5CS and crystalline CBD solubilized in olive oil (reference) formulations, in samples taken from rats.

(18) FIG. 17 shows TNF-α plasma levels form mice with induced acute paw inflammation as measured in blood samples taken after 24 hours of oral treatment with ‘5CS extracted media’ or ‘ethanol extraction’ at a dosage of 5, 25 and 50 mg plant source to kg body weight.

(19) FIG. 18 shows the paw-thickness of inflammated paw in mice treated with ‘5CS extraction media’ medium compared to extraction with ethanol dissolved in olive oil as measured 6 hours after treatment.

(20) FIG. 19 shows the measured ear thickness of DHT-induced rats.

(21) FIGS. 20A-D are pictures of rats' ears in DHT test: 24 mg/kg BW of 5CS formulation (FIG. 20A), 48 mg/kg BW of 5CS formulation (FIG. 20B), non-treated DHT-induced (FIG. 20C), and naïve rats (FIG. 20D).

DETAILED DESCRIPTION OF EMBODIMENTS

(22) Effect of Plant Pre-Heating

(23) As explained hereinabove, at least a portion of the CBD is found in the plant in the form of CBDA. Decarboxylation of CBDA can be carried out by heating the plant at controlled conditions to obtain the desired CBD. The plants used in the following examples were various hybrids of Cannabis sativa and Cannabis indica.

(24) For evaluating the effect of heating the plant source prior to the extraction process on the extraction yield, the content of cannabinoid species in various strains of dried cannabis plants were profiled prior to heating by HPLC.

(25) Plant samples (a mixture of flowers, leaves and stems was used) were roughly chopped and heated in air atmosphere at a temperature of between 90 and 170° C. for between 10 and 120 minutes. The samples were then extracted with ethanol (10 ml per 100 mg of plant) for 30 minutes under stirring at 30-35° C. Ethanol was used as a solvent for a reference to the conversion of CBDA to CBD, as determined by HPLC. The sample was then filtered through cotton wool to obtain an extract, which was analyzed by HPLC.

(26) HPLC analysis was carried out by using the following conditions: C18 column, mobile phase-gradient of methanol/water (69/31 v/v %) to 100% methanol, flow rate 0.3 ml/min.

(27) CBD peak areas were plotted against heating temperature (FIG. 1). FIG. 2 presents the ratio between CBD and CBDA peak areas against heating time at various heating temperatures.

(28) As evident from the results, heating increases significantly the concentration of CBD in the plant sample, thereby increasing the content of the desired extractable cannabinoid specie in the samples. When the samples were heated at a lower temperature range (140° C.), maximum CBDA-to-CBD transformation was observed after 60-90 minutes of heating. For higher temperatures (160° C.), 10-25 minutes of heating were sufficient to obtain desired levels of CBD in the samples.

(29) Long heating at 140° C. (for 60-90 min) or short heating at 160° C. (for 10-25 min) are suitable for reaching the highest amount of CBD in the plant as well as high conversion of CBDA to CBD. Above 170° C. no CBDA was identified after already 10 min, however degradation products appear at early stages.

(30) Thus, all the following extraction were done, based on these results, with plants that were heated at 160° C. for 15 min.

(31) Extraction Medium and Preparations

(32) As noted above, the extraction media used for the extraction process are self-assembled systems which are formed in a spontaneous manner. Therefore, several compositions of the extraction media were prepared by simple mixing of ingredients at 25-70° C. An exemplary process for preparing the extraction medium involves mixing together the oil, the surfactant and the co-surfactant (and where applicable also a solvent, a co-solvent and/or a phospholipid) until a homogenous, clear (transparent) mixture is obtained. In case the surfactants or oil are solid at room temperature, heating can be applied while mixing to allow full dissolution and formation of the empty extraction medium.

(33) The extraction medium is then slowly added to the pre-heated and chopped plant to allow appropriate wetting and then mixed and homogenized. Another variation of the process includes adding solid plant parts (leaves or buds for examples) stepwise to the empty (un-loaded) extraction medium until a homogeneous slurry is obtained.

(34) Extraction was carried out under heating with our without inert atmosphere, thereby solubilizing CBD into the extraction medium. The mixture was allowed to settle to the bottom of the mixing vessel before filtration and/or centrifugation.

(35) Table 1 provides details of exemplary formulations used in the process of the present disclosure.

(36) TABLE-US-00001 TABLE 1 Formulations of extraction medium Formulation 5CS Formulation AX1 Component wt % Component wt % Oil MCT 3.6 R-(+)-Limonene 5 Hydrophilic Polysorbate 80 35.37 Polysorbate 80 45 surfactant (Tween 80) (Tween 80) Cremophor EL 42.57 castor oil* Co-surfactant Propylene glycol 8.46 Propylene glycol 45 (PG) (PG) Solvent — — Ethanol 5 Phospholipid Phosal 50 PG** 10 — — *Polyoxyl 35 castor oil **Phosal 50 PG composed of 1.5-2.5% wt ethanol, >500 ppm ethylenemethylketone, 0.5 wt % water, 33.8-41.2 wt % propylene glycol, <50.0 wt % phosphatidylcholine, >6 wt % lyso-phosphatidylcholine

(37) The formation of commonly known emulsions, which are typically a dispersion of two immiscible liquids formed in the presence of emulsifiers, are based on the reduction of the interfacial tension between the two phases such that the dispersed droplets are covered by an emulsifier's layer to retard aggregation, flocculation, coalescence and phase separation. Since emulsifiers do not reduce the interfacial tension to zero and the coverage is not complete, emulsions require application of relatively high shear forces of multistage homogenizer to reduce the droplets size upon preparation of the emulsion. The resulting non-uniform droplets have a strong tendency to coalesce and/or result in phase-separate, thereby stabilizing the system energetically. Thus, emulsions show a relatively non-uniform and large droplet size, which are unstable over prolonged periods of time (i.e. the droplet size increases due to coalescence or can even result in phase separation). Moreover, in a typical emulsion the droplet size is far from being homogenous, resulting in milky, white-opaque appearance. Extraction with an emulsion media leads to very fast phase separation and very limited amount of extraction load.

(38) Contrary to known emulsions, the extraction media used in the process of disclosed herein have zero interfacial tension, and therefore are spontaneously formed as energetically balanced systems, which are characterized by a small and uniform droplet size, resulting in transparent systems. Due to their energetic balance, the extraction media used in the process (and as a result also the cannabinoid-loaded medium) are stable for prolonged periods of time, maintain their droplet size and size uniformity also upon dilution with aqueous liquids, making them suitable for formulation into various pharmaceutical compositions and enabling their administration in a variety of administrations routes and forms.

(39) Additional exemplary formulations are detailed in Table 2.

(40) TABLE-US-00002 TABLE 2 Formulations of extraction medium Formulation OR103(2) slow release Formulation OR210SE Component wt % Component wt % Oil Triacetin 5 MCT 5 Hydrophilic Labrasol 25 L-1695- sucrose surfactant Cremophor EL 35 mono/dilaurate 60 castor oil* Co-surfactant Propylene glycol 20 Propylene glycol 20 (PG) (PG) Solvent Isopropyl alcohol 5 Isopropyl alcohol 5 (IPA) (IPA) Phospholipid Phosal 50 PG* 10 Phosal 50 PG* 10 *Phosal 50 PG composed of 1.5-2.5% wt ethanol, >500 ppm ethylenemethylketone, 0.5 wt % water, 33.8-41.2 wt % propylene glycol, <50.0 wt % phosphatidylcholine, >6 wt % lyso-phosphatidylcholine

(41) The ability of formulations comprising olive oil instead of MCT (as the oil component in the extraction medium of formulation 5CS), as well as the ability of diluted media (40 wt % water) to extract CBD from a plant source was also evaluated. As seen in FIG. 3, olive oil is also suitable as an oil component in the extraction media. Further, diluted media also show the capability of extracting CBD form the plant material, and even show CBD concentrations slightly higher compared to the concentrated formulation.

(42) Extraction of CBD from Plant Samples by Extraction Media

(43) Cannabinoid Profile of Plant Samples

(44) Various strains of cannabis were tested in the extraction process of the present disclosure. Samples of the plants were evaluated for cannabinoids profiles prior to extraction with the extraction medium by ethanol extraction (as described above) and HPLC analysis. The cannabinoids profiles of the various strains are provided in Table 3.

(45) TABLE-US-00003 TABLE 3 Cannabinoids profile of some strains Strain CBVD (%) CBG (%) CBD (%) Δ.sup.9-THC (%) CBN (%) M1 — — — 1.1 Trace M1-L 0.5 0.1 6.7 0.7 Trace M(1)-1 0.2 1.7 12.3 0.6 Trace M(1)-3 0.5 0.2 10.6 0.5 Trace M(3)-1 0.8 0.1 11.0 0.5 Trace M(3)-2 0.6 0.1 9.4 0.4 Trace

(46) All extraction medium processes described herein were carried out on plant samples heated at 160° C. for 15 minutes. All of the following experiments were carried out on M(1)-1 strain.

(47) Effect of Extraction Duration

(48) Plant samples (after heating) were mixed with AX-1 extraction medium at a weight ratio of 1:40. The mixtures were then homogenized at room temperature using lab Silverson homogenizer L5M-A for 30 minutes. After homogenization, each sample was centrifuged at 4000 rpm for 20 minutes or filtered through cotton wool. Samples were prepared in triplicates.

(49) Analysis of cannabinoids content in the extracts was carried out by HPLC vis-à-vis calibration curves. FIGS. 4 and 5 show the concentration of CBD in the extracts and the extraction yield, respectively, as a function of the extraction time. The ratio between the CBD peak area and those of other cannabinoids in the plant sample (CBDA, THC and CBN) are shown in FIG. 6.

(50) As can clearly be seen from the results, the extraction medium is highly selective towards CBD. After one round of extraction, the medium contains at least 35-folds CBD compared to THC and selectivity is reduced as the extraction time increases. The ratio of CBD to CBDA starts from 27:1 and drops to at least 14-folds CBD compared to CBDA. This suggest that extraction of the plant source with extraction media as described herein may be used to obtain CBD-rich products with significantly lower concentrations of THC compared to other commercially available products.

(51) As observed from FIG. 4, a plateau is reached after 20 min of extraction. Beyond the 20 min no significant increasing in CBD concentration or extraction yield is achieved. Further, as seen from FIG. 5, a single extraction process yielded extraction of between 55 and 75% of CBD from the plant source. For those reasons a duration of 30 min was chosen for the following extractions.

(52) As seen in FIG. 6, Examining the ratio of CBD to other cannabinoids shows that the longer the extraction the lower the ratio, which means that extra time allows another cannabinoid to be extracted more rapidly compared to the CBD.

(53) Effect of Plant-to-Microemulsion Ratio

(54) The effect of the plant-to-medium ratio on the extraction efficiency was assessed by analyzing mixtures of varying plant source to extraction medium ratios.

(55) Plant samples (after heating) were mixed with AX-1 extraction medium at a weight ratio of between 1:15 and 1:60 (plant:medium). The mixtures were then homogenized at room temperature using Silverson homogenizer for 30 minutes. After homogenization, each sample was centrifuged at 4000 rpm for 20 minutes or filtered through cotton wool. Samples were prepared in triplicates.

(56) Analysis of CBD content in the extracts was carried out by HPLC vis-à-vis a calibration curve. FIGS. 7 and 8 show the concentration of CBD in the extracts and the extraction yield, respectively, as a function of the plant-to-medium ratio. The ratio between the CBD peak area and those of other cannabinoids in the plant sample (CBDA, THC and CBN) are shown in FIG. 9.

(57) Although the extraction yields decreases upon increasing the plant:medium ratio, in all weight ratios the selectivity of extraction is evident. The selectivity is controlled by the preference of the CBD molecule to interact with the surfactants tails and system core in comparison to the THC molecule, with a predominant factors being the polarity and structure of the molecule. This suggests that selective extraction of various cannabinoids may be tailored by varying the polarity of the extraction medium.

(58) Multiple-Extractions Process

(59) Increasing the CBD concentration in the extraction medium was carried out by a multi-extraction process. For the multi-extraction process a number of extraction cycles are carried out by using the same quota of extraction medium for several extraction cycles, in each cycle a fresh sample of plant is extracted according to the following procedure.

(60) A heated plant sample was mixed with AX-1 extraction medium at a weight ratio of 1:15. The mixture was then homogenized at room temperature using Silverson homogenizer for 30 minutes. After homogenization, the sample was centrifuged at 4000 rpm for 20 minutes or/and filtered through cotton wool. After separating the CBD-loaded medium from the spent biomass, the CBD-loaded medium was weighed and a new sample of plant was added at a weight ratio of 1:15 (plant:medium). Homogenization and separation were carried out for the new mixture. Two additional such cycles of extraction were carried out, amounting to a total of 4 extraction cycles. A total of 3 multi-extraction processes were carried out.

(61) Samples of the medium were taken in between cycles to assess the effect of the number of cycles on the cannabinoids profile and CBD loading of the medium.

(62) Analysis of CBD content was done according to the description hereinabove. FIGS. 10 and 11 show the concentration of CBD in the extracts and the extraction yield, respectively, as a function of the number of extraction cycles. The content of the various cannabinoids in the samples is shown in FIG. 12.

(63) As evident from the results, the CBD content in the extraction medium increases by at least 2-folds as a result of the multi-extractions process. However, as the extraction medium becomes loaded with CBD, the extraction efficiency of the extraction medium decreases compared to the extraction efficiency at the first cycle of extraction due to the proximity of the CBD content to the maximum loading capacity of the extraction medium. Regardless of this decrease, the selectivity of extraction is maintained throughout the process cycles.

(64) Reference Extraction Media

(65) To demonstrate the selectivity of the extraction media described herein towards specific cannabinoids, and especially towards the extraction of CBD, plant samples were also extracted with either ethanol or petroleum ether. Both solvents are known and used to extract cannabinoids. The process of extraction was identical to that carried out with the extraction medium of the present disclosure, as described above.

(66) The results are provided in FIGS. 13-14, which show the quantities ratio between CBD and other extracted cannabinoids, as determined by HPLC.

(67) As can clearly be seen, extraction carried out in both ethanol and petroleum ether showed a CBD:THC ratio of at most 22:1, while extraction with the extraction medium showed CBD:THC ratios of ˜35:1. Namely, the extraction media described herein provides high selectivity to the extraction of CBD over other cannabinoids, enabling obtaining an extraction product with extremely low levels of THC.

(68) Further, the lading capacity of CBD in the extraction medium is significantly higher than that obtained for either ethanol or petroleum ether, as can be seen in Table 4, attesting to the ability of the extraction medium to quantitatively extract CBD from the plant source.

(69) TABLE-US-00004 TABLE 4 comparative CBD loading of extraction medium vs. ethanol and petroleum ether Extraction process CBD loading (mg/ml) AX-1 >6*  Ethanol 1.2 Petroleum ether 0.9 *after 1 extraction cycle. Up to 22 mg/ml were obtained after 4 extraction cycles

(70) Stability of Formulations

(71) 5CS and AX2 extraction media (see Table 5-1) were loaded with 5 wt % CBD and incubated at three different temperature (4, 25 and 40° C.) under different conditions (without protection, with the addition of 600 ppm α-tocopherol acetate and under nitrogen atmosphere). Both the concentrate and a diluted microemulsion (80% water) were tested.

(72) TABLE-US-00005 TABLE 5-1 Formulations of microemulsions for stability tests Formulation 5CS Formulation AX2 Component wt % Component wt % Oil MCT 3.6 MCT 5 Oleic acid 2 Hydrophilic Polysorbate 80 35.37 Polysorbate 80 35 surfactant (Tween 80) (Tween 80) Cremophor EL 42.57 Cremophor EL 32 castor oil* castor oil* Glycerol 6.5 Co-surfactant Propylene glycol 8.46 Propylene glycol 9 (PG) (PG) Solvent — — Ethanol 5.5 Phospholipid Phosal 50 PG** 10 Phosphatidylcholine 5 *Polyoxyl 35 castor oil **Phosal 50 PG composed of 1.5-2.5% wt ethanol, >500 ppm ethylenemethylketone, 0.5 wt % water, 33.8-41.2 wt % propylene glycol, <50.0 wt % phosphatidylcholine, >6 wt % lyso-phosphatidylcholine

(73) The visual appearance of the samples were recorded after 30 days of incubation. The results are detailed in Table 5-2.

(74) TABLE-US-00006 TABLE 5-2 Stability of CBD-loaded media Incubation 5CS AX2 Extraction temper- Concen- 80% Concen- 80% Conditions ature trate dilution trate dilution No protec-  4° C. Stable Stable Stable Stable tion 25° C. Stable Stable Stable Stable 40° C. Yellowish N/A Yellowish N/A 600 ppm  4° C. Stable Stable Stable Stable α- 25° C. Stable Stable Stable Stable tocopherol acetate 40° C. Yellowish Stable Yellowish Stable Nitrogen  4° C. Stable Stable Stable Stable atmosphere 25° C. Stable Stable Stable Stable 40° C. Stable Yellowish Stable Yellowish

(75) As clearly seen, the CBD-loaded media are stable over a wide variety of conditions, namely most of the tested samples remained transparent, without any indication of phase separation or precipitation.

(76) Long-Term Stability

(77) 5CS and AX1 extraction media (see Table 1 above) were used to extract CBD from a plant source according to the following procedure: 1:15 w/w ratio of plant:ME, extraction time of 30 minutes under homogenization at 200° C. Two extraction cycles were carried out, and samples were collected for each cycles. The samples were incubated at three different temperature (4, 25 and 40° C.) for 70 days. The samples were not diluted (i.e. the test was carried out on concentrate samples).

(78) As clearly seen in FIGS. 15A and 15B, the CBD-loaded media in concentrate form are stable over a long period of time, with no change in color or observance of phase separation or precipitation.

(79) PK Study

(80) The pharmacokinetic (PK) profile was assessed by measuring the CBD concentration in plasma after oral administration of AX-1 and 5CS CBD-loaded formulations, as compared to CBD solubilized in olive oil.

(81) Male rats (250 g on average) were used for this PK study, which was carried out in two stages: in the first stage, crystalline CBD solubilized in olive oil was administered orally to the rats, and at a second stage either AX-1 or 5CS CBD-loaded formulations were orally administered via gavage. Blood samples were collected at different time points into heparinized EDTA-K3 tubes and stored on ice. The plasma was separated from each sample by pre-cold centrifugation at 3,000 rpm and stored in clean sterilized tubes at −80±10° C. The rats were sacrificed after 24 from administration.

(82) FIG. 16 shows the CBD levels in plasma at 0.5 and 2 hours after administration of different doses of the formulations. For both AX-1 and 5CS formulations, the CBD concentration measured in the plasma was higher after 0.5 hr, compared to CBD solubilized in olive oil. After 2 hr from administration, the CBD plasma level was similar for AX-1 and olive oil, while 5CS system showed significant increase in CBD concentration.

(83) Thus, formulations of this disclosure show rapid bioavailability and increase levels of CBD in the plasma compared to olive oil solutions, with Tmax of 0.5 vs. app. 4 hrs.

(84) In-Vivo Studies

(85) Response to Pain

(86) Response to pain and anti-inflammatory activity in mice of the extracted cannabis plant source (CBD-loaded) media of this disclosure were assessed by oral administration of extraction from a cannabis plant using 5CS extraction media compared to traditional ethanol extraction.

(87) 5CS extraction medium and ethanol extraction were prepared separately at four different concentrations which were equivalent to 5, 10, 25 and 50 mg plant material/kg body weight rat according to the following protocol.

(88) Three female Sabra mice at the age of 8 weeks old were maintained for 7 days in the SPF unit prior to study initiation. 40 μl of 1.5% (w/v) Zymosan A (sigma) suspended in 0.9% saline was injected into the sub-planter surface of the right hind paw of each mouse. Immediately after induction, extraction from a cannabis plant source was given orally to the inflammation induced mice. Extraction was performed by traditional ethanol extraction or by ‘5CS Extraction Medium’. As a positive control three induced mice were left untreated. The mice treated with the ‘extracted medium’ were administered orally directly with loaded 5CS, while the ‘ethanol extraction’ the ethanol was first evaporated and the precipitated material was re-suspended in olive oil.

(89) The therapeutic effect was evaluated in various administration dosage of extracted material including 10, 25 and 50 mg extracted plant source to each kg body weight. After 6 hr from treatment the swelling of the inflammated paw was measured using a caliper. In addition, after 24 hour form treatment, TNF-α (tumor necrosis factor) levels were measured using an ELISA kit (R&D system) according to the manufacturer's instruction.

(90) TNF-α was assessed by ELISA kit of plasma samples taken 24 hours after oral treatment. FIG. 17 shows the TNF-α levels for 5CS extracted media compared to extraction by ethanol and dispersion in olive oil. FIG. 18 shows the paw-thickness of inflammated paw in mice treated with extracted medium 5CS compared to extraction by ethanol dispersed in olive oil.

(91) Administrating ‘5CS extracted medium’ significantly reduced the paw's swelling (compared to the control untreated mice) within all given dosage (5, 25 and 50 mg/kg) in comparison to the ethanol extraction treated mice which showed a much lesser reduction. These results indicate that the inflammation is reduced in greater efficiency using the ‘extraction medium’ compared to extraction with ethanol. TNF-α plasma levels were considerably lower in the mice treated with the ‘5CS extraction medium’ compared to that extracted with ethanol at all dosage tested.

(92) The reduction in TNF-α levels (250 compared to 350 pg of control untreated mice) using 5CS extraction medium was seen even when using relatively low dosage (5 mg/kg), while using ethanol extraction at the same dosage did not affect the TNF-α levels, which were almost similar to those measured in the untreated mice (320 vs 350 pg, respectively).

(93) As seen from FIG. 18, in all dosages tested, mice administered with the CBD-loaded medium of the present disclosure showed lower TNF-α levels after 24 hours from administration resulting in reduced inflammation compared to that of the CBD extracted by ethanol. This attests to the improved extraction, release and permeation (performance) of the extraction media.

(94) Further, as seen in FIG. 17, mice administered with the extraction medium of the present disclosure showed a more significant reduction in paw thickness in all dosages tested as compared to identical dosages of CBD extracted in ethanol and dissolved in olive oil. Namely, the formulations of the present disclosure have an improved anti-inflammatory activity as compared to standard ethanol CBD extractions.

(95) Delayed-Type Hypersensitivity (DTH)

(96) CBD was shown to reduce inflammation response and pain-effected by inflammatory reaction. Without wishing to be bound by theory, inflammation reduction is achieved by various mechanisms, including agonist and antagonist binding to CB1 receptors, adenosine receptors and other GPCRs, involving the reduction of inflammatory cytokines and chemokines levels, such as IL-2, IL-6, TNF-α, MCP-1, etc.

(97) The therapeutic effect of oral administration of CBD-loaded formulations of this disclosure as anti-inflammatory agents. The CBD effect was evaluated using rat model of inflammation—Delayed Type Hypersensitivity (DHT) model. In this test, the reduction in ear swelling after inflammation-induction following treatment was measured.

(98) The belly of male rats (average weight 250 g) was shaved and challenged 10 times with 500 μl of 2% oxazolone (400 mg oxazolone dissolved in 16 ml acetone and 4 ml mineral oil). The next day (referred to herein as day 1), 500 μl of CBD formulation oral treatment was given via gavage. On day 6, the ear thickness of the rats was measured using a caliper.

(99) Rats were challenged with another dose of 50 μl of 0.5% oxazolne, and a second oral treatment of 500 μl CBD formulation was administered 2-hours after challenge. The ear thickness was measured again 12 and 24 hours after challenge, and blood samples were taken for serum preparation.

(100) Samples composition: two doses were administered of extracted CBD in 5CS with a dose of 24 mg/kg BW and 48 mg/Kg BW (BW=Body Weight), compared to control of Naïve rats and rats with DTH-induction that were not given any treatment.

(101) As seen in FIGS. 19 and 20A-D, a significant reduction in ear thickness and inflammatory appearance (redness and edema) as a result of the treatment with CBD extracted with 5CS was obtained compared to DTH-induced rats that were not treated. The anti-inflammatory effect of CBD extracted with 5CS is more significant than that seen for Ethanol extractions with both dose regiments.