Cannabinoid Extraction

Marijuana refers to varieties of Cannabis having a high content of A9-tetrahydrocannabinol (A9-THC), which is the psychoactive ingredient of marijuana whereas industrial hemp refers to varieties of the Cannabis plant that have a low content of A9-THC. 

It is of note that in some embodiments, the specific cannabinoids isolated are those cannabinoids with suspected health benefits or suspected medicinal uses. For example, the cannabinoids and cannflavins may be used as anti-emetics, antinauseants, appetite stimulants, antiinflammatories, antioxidants, neuroprotectives, analgesics, suppressants for primary immune response, glaucoma remedies, antineoplastics, migraine headache remedies, menstrual pain remedies, anticonvulsants, anti-epileptics, or movement disorder remedies. The essential oils may be used for aromatherapy or as flavoring/scenting adjuvants. 

Several investigations have been carried out over the years to isolate THC from the plant material, mostly to determine its chemical structure or to investigate the phytochemistry of the plant. In 1942, Wollner, et al., (11) reported the isolation of tetrahydrocannabinol from cannabis extract red oil . Red oil was prepared by extraction of the plant material with ether, followed by distillation of the concentrated extract at room pressure followed by redistillation under reduced pressure (15-50 mm Hg). 

The plant material is extracted with a non-polar organic solvent. Useful solvents include lower alkanes, such as, for example, hexane, heptane or iso-octane. The extract containing THC, after solvent removal, is subjected to fractional distillation under reduced pressure and a first distillate is collected. In one embodiment of the present invention, the first distillate is again subjected to fractional distillation at reduced pressure and a second distillate is collected. The second distillate has a THC content of greater than 90% by wt. 

One kg of the fine powdered marijuana plant material [average % of THC was about 5.21%] was macerated with 6 L. hexanes (Hexanes GR from EM Sciences) in a percolator (9 in diameter from the top and 20 long, cone shaped) for 24 hours at room temperature and filtered. The macerate was reextracted with 5 L. hexanes for another 24 hours. The hexane extracts were combined and evaporated under reduced pressure at low temperature to give 110.7 g residue (11.07% extractives). The % of THC in the hexane extract was 41.21%. 

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In the studies of acute effects (1-5), marijuana was administered by smoking under double-blind conditions. The research assistant guided the subject in paced smoking procedures. Cigarettes that contained either 10 mg or 19 mg of A -tetrahydrocannabinol (THC) were compared with placebo cigarettes that contained inactive, cannabinoid-extracted marijuana with only trace amounts of THC. In the studies of chronic effects (6,7), chronic marijuana users were compared with nonusers, i.e., control subjects. In the initial study of chronic marijuana users (6), these users were also grouped according to frequency of marijuana use. 

C8 or C18 reverse-phase LC chromatography columns and guard columns with similar packing material have been predominantly used for the analysis of cannabinoids extracted from biological matrices. 

Cannabinoids were used in medicine in the form of their crude extracts many centuries ago. Lately the use of cannabis for so-called recreational purposes has become a national vice of substantial proportions. Several attempts have been made to focus the potentially useful pharmacological properties of marijuana into drug molecules with no abuse potential. 

Based on the role of endocannabinoids and cannabinoid receptors in several pathological conditions, the pharmacological manipulation of their levels or action is being developed as a therapeutic strategy. Enhancement of endocannabinoid signalling when this plays uniquely a protective role can be effected in a safer way using (i) cannabis extracts in which the presence of non-psychotropic cannabinoids with therapeutic activity per... 

The aim of the analysis of cannabinoids in plants is to discriminate between the phenotypes (drug-type/fiber-type). Quantification of cannabinoids in plant material is needed if it will be used in medicinal appHcations, e.g., in C. sativa extracts. The ratio between A9-THC and CBN can be used for the determination of the age of stored marijuana samples [84]. 

Usually the first step is an extraction of the desired compounds from plant material. This extraction can be done by different solvents, e.g., methanol [85], n-hexane [86], petroleum ether or solvent mixtures such as methanol/chloroform [87]. The use of a second liquid-liquid extraction (LLE) with 0.1 M NaOH after extraction with a non-polar solvent like n-hexane makes a separate analysis of acidic cannabinoids possible, which can be found... 

The best studied of the endocarmabinoids are anandamide (A -arachidonyl-ethanolamine, AEA)(1) and 2-arachidonylglycerol (2-AG)(2). Anandamide was first identified from porcine brain extracts by Devane and co-workers in 1992 [13], while 2-AG was first reported in 1995 to have been isolated from canine gut [14] and rat brain [15]. More recently, noladin ether (2-arachidonyl-glyceryl ether, 2-AGE)(3) [16], virodhamine (D-arachidonyl-ethanolamine)(4) [17] and A-arachidonyl-dopamine (NADA)(5) [18] were proposed as endogenous ligands for the cannabinoid receptors. In a subsequent publication, the authors failed to detect noladin ether in mammalian brains and questioned the relevance of this compound as an endocarmabinoid [19]. Anandamide, noladin ether and NADA have functional selectivity for CBi receptors, virodhamine is CB2 selective and 2-AG is essentially non-selective. 

Other therapeutic uses of cannabinoid agonists have been reported. The potential of cannabinoids as a treatment for asthma is supported experimentally. A CBi agonist, (i )-methanandamide (21), inhibited nerve growth factor (NGF)-induced airway hyperresponsiveness in vivo [251]. The antipruritic effect of cannabinoids has been reported, the action being mediated by both CBi and CB2 pathways [252]. Treatment with cannabis extract improved urinary tract symptoms of multiple sclerosis patients significantly in an open-label pilot study [253]. 

Forensic analysis of street drugs include that of cocaine together with excipients frequently encountered (579), amphetamines 080), and dyes found in heroin samples 081). An on-line photochemical derivitization of cannabinoids has been described 082). Other pharmaceutical agents studied in formation include nortriptyline in tablets. 083), glycyrrhizic acid from licorice extract 084, 585), pirimiphos methyl 086), digitalis glycosides 0S7), pilocarpine 088), and its antagonist atropine 009). 

Huizing and T. M. Malingre. Use of amberlite XAD-2 columns for the separation of cannabinoids from cannabis extracts. J Chromatogr 1981 205 444—450. 

Luo, J., J. H. Yin, H. Z. Wu, and Q. Wei. Extract from Fructus cannabis activating calcineurin improved learning and memory in mice with chemical drug-induced dysmnesia. Acta Pharmacol Sin 2003 24(11) 1137-1142. Degenhardt, L., W. Hall, and M. Lynskey. Exploring the association between cannabis use and depression. Addiction 2003 98(11) 1493-1504. Zajicek, J., P. Pox, H. Sanders, et al. Cannabinoids for treatment of spasticity and other symptoms related to multiple sclerosis (CAMS study) multicentre randomised placebo-controlled trial. Lancet 2003 362(9395) 1517-1526. 

CS375 Dhawan, K., S. Kumar, and A. CS385 Sharma. Reversal of cannabinoids (delta9-THC) by the benzoflavone moiety from methanol extract of Passiflora incarnata Linneaus in mice a... 

Method. The derivatives are formed by shaking the sample (dissolved in acetone) for 1 h at 45 °C with a 3-5 molar excess of recrystallized DNS-C1. The reaction is buffered at pH 10.8.0.25 ml of 1N sodium hydroxide is then added in order to hydrolyze the unchanged DNS-C1. The derivatives are extracted with 3 ml of ethyl acetate after addition of 1 ml of a saturated aqueous solution of sodium chloride to the reaction mixture. The organic phase is used for TLC on activated layers of silica gel G. The cannabinoids yield mono-DNS derivatives with the exception of cannabidiol which forms a bis-DNS derivative. The following solvent systems are satisfactory for separation of cannabinoids on silica gel A, benzene-acetone (9 1) B, cyclohexane-ethyl acetate (5 1) C, cyclohexane-acetone-diethylamine (20 4 1) and D, cyclohexane-acetone-triethylamine (20 4 1). The R f values of nine cannabinoids in the above solvent systems are given in Table 4.25. 

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