Cannabinoids, Phenolics, Terpenes and Alkaloids of Cannabis

Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.


Introduction
Cannabis sativa L. belongs to the plant family Cannabaceae, which only has one genus (Cannabis) with only one highly variable species, C. sativa. This is one of the oldest plants grown for food, fiber, and medicine. It grows in all habitats, ranging from sea level to temperate to alpine foothills. The plant originated in Western Asia and introduced to western medicine during the early 19th century. Cannabis has a long history of being used as a medicine to treat a variety of ailments, including asthma, epilepsy, fatigue, glaucoma, insomnia, nausea, pain, and rheumatism [1].
Cannabis is primarily a dioecious plant (male and female flowers occur on individual plants); it is only occasionally found as a hermaphrodite (male and female flowers on the same plant). It flowers under a short photoperiod (below 12 h of light) and continues growing vegetatively during the longer photoperiod days.
The plant is a chemically complex species, due to its numerous natural constituents [2]. Cannabinoids, a specific chemical class found in cannabis, are produced in the glandular trichomes of the plant. Among the cannabinoid constituents of cannabis, ∆ 9 -tetrahydrocannabinol (∆ 9 -THC), which is naturally present in the form of an acid (∆ 9 -tetrahydrocannabinolic acid, ∆ 9 -THCA), is the main psychoactive constituent of the plant. Decarboxylation of the acid with age or heat is required to form the pharmacologically active ∆ 9 -tetrahydrocannabinol [3]. Cannabidiol (CBD), another cannabinoid of current interest, is reported to be active as an antiepileptic agent, particularly for the treatment of intractable pediatric epilepsy [4,5].
Other than ∆ 9 -THC and CBD, tetrahydrocannabivarin (THCV), cannabinol (CBN), cannabigerol (CBG), and cannabichromene (CBC) are four other major cannabinoids also identified in C. sativa. Modern studies report that the pharmacological effects of phytocannabinoids result from their ability to interact with cannabinoid receptors and/or with other kinds of pharmacological targets, including non-cannabinoid receptors [6]. Thus far, 2 of 29 more than 500 constituents have been reported from Cannabis, out of which 125 are classified as cannabinoids. The non-cannabinoid constituents include non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others.
The present review discusses the chemistry of all identified major Cannabis constituents including cannabinoid and non-cannabinoid constituents (non-cannabinoid phenols, flavonoids, terpenes, and alkaloids), with special emphasis on the chemical structures, methods of isolation, and identification. This review also updated the chemistry of 125 cannabinoids, 42 phenolics, 34 flavonoids, 120 terpenes and 2 alkaloids. The last review on cannabis chemistry was published by our group in 2017 as a chapter book which focused on the new constituents reported between 2005 and 2017 only but, in this review, we provided a chemical account on the chemistry of major constituents (323 compounds) of cannabis from 1940 up to now along with their structures. For the reader, this review should provide almost all the information of cannabinoids and non-cannabinoids, their methods of isolation, identification and structures in one place. In addition, these 323 compounds include 12 new compounds (five cannabinoids, one cannabispiran, three flavonoids, and three terpenes). The inclusion of all the 323 chemical structures along with all other details makes this review unique.
Molecules 2021, 26, x FOR PEER REVIEW 6 of 29 using silica and alumina column chromatography, resulting in waxy and non-waxy fractions. The farnesyl prenylogue of cannabigerol (sesquicannabigerol, 46) was isolated from one of the waxy fractions. Its structure was established on the basis of NMR spectroscopic analysis ( 1 H-NMR, 13 C-NMR) and semisynthesis from cannabigerol (31) [33]. The CBG type cannabinoids are shown in Figure 3.
Shoyama et al. isolated cannabidivarinic acid (CBDVA, 52) from the benzene extract of Thai cannabis, which was chromatographed on a polyamide column eluted with
Recently, two new CBD homologues with n-hexyl and n-heptyl side chain were isolated from the hexane extract of C. sativa and their chemical structures were assigned as cannabidihexol (CBDH, 54) and cannabidiphorol (CBDP, 55). The two compounds were purified using a semi-preparative C18 HPLC using a mixture of ACN/0.1 aqueous formic acid as a mobile phase. The fractions containing 52 and 53 were analyzed by HRESMS. The chemical structures of CBDH and CBDP were determined by HNMR, C-NMR, UV, and HR-ESI-MS and confirmed by stereoselective synthesis [22,23].
In 2020, a CBD dimer was isolated from the hexane extract of hemp and named Cannabitwinol, (CBDD, 56). The hexane extract was chromatographed on a Sigel column, which was eluted with hexane/CH 2 Cl 2 followed by semi preparative C18-HPLC using a mixture of ACN/H 2 O/formic acid (7:3:0.1) as the mobile phase. The chemical structure of CBDD (56) was determined by applying a plethora of 1D and 2D NMR at −30 • C and was confirmed by HRESIMS and MS/MS spectrometry. The authors confirmed the chemical structure of 56 as two units of CBD connected by a methylene bridge and suggested that the dimerization of CBD occurred as a result of enzymatic reaction medicated by a one-carbon donor enzyme like methylene tetrahydrofolate [40]. All of the CBD type cannabinoids are shown in Figure 4. H2O:MeOH (1:1-1:6). Its structure was elucidated using UV, IR, and 1 H NMR spectroscopic analysis [16]. Cannabidiorcol (CBD-C1, 53) was identified in the hexane extract of Lebanese hashish by combined gas chromatography-mass spectrometry [39].
Recently, two new CBD homologues with n-hexyl and n-heptyl side chain were isolated from the hexane extract of C. sativa and their chemical structures were assigned as cannabidihexol (CBDH, 54) and cannabidiphorol (CBDP, 55). The two compounds were purified using a semi-preparative C18 HPLC using a mixture of ACN/0.1 aqueous formic acid as a mobile phase. The fractions containing 52 and 53 were analyzed by HRESMS. The chemical structures of CBDH and CBDP were determined by HNMR, C-NMR, UV, and HR-ESI-MS and confirmed by stereoselective synthesis [22,23] In 2020, a CBD dimer was isolated from the hexane extract of hemp and named Cannabitwinol, (CBDD, 56). The hexane extract was chromatographed on a Sigel column, which was eluted with hexane/CH2Cl2 followed by semi preparative C18-HPLC using a mixture of ACN/H2O/formic acid (7:3:0.1) as the mobile phase. The chemical structure of CBDD (56) was determined by applying a plethora of 1D and 2D NMR at -30 0 C and was confirmed by HRESIMS and MS/MS spectrometry. The authors confirmed the chemical structure of 56 as two units of CBD connected by a methylene bridge and suggested that the dimerization of CBD occurred as a result of enzymatic reaction medicated by a onecarbon donor enzyme like methylene tetrahydrofolate [40]. All of the CBD type cannabinoids are shown in Figure 4.

Cannabicyclol (CBL) Type Three3 Compounds)
Krote and Sieper isolated cannabicyclol (CBL, 64) from hashish by thin layer chromatography, but its structure was correctly elucidated by Mechoulam and Gaoni in 1967, based on spectral data [47,48]. The relative configuration of CBL (64) was determined by X-ray analysis in 1970 [49]. Cannabicyclolic acid (CBLA, 65) was obtained from the benzene extract of cannabis. The benzene extract was chromatographed on a polyamide column using methanol water as a mobile phase. CBLA was isolated as a methylated derivative and considered to be an artifact formed when CBCA is naturally irradiated during storage [50]. Cannabicyclovarin (CBLV, 66) was detected in the ether extract of Congo marihuana and was identified by GLC and GCMS [51]. CBLA (65) was determined to be produced during the natural irradiation of cannabichromenic acid (CBCA, 68), proving that CBLA is not a natural cannabinoid [50]. The chemical structures of CBL-type cannabinoids are shown in Figure 4.
Molecules 2021, 26, x FOR PEER REVIEW 10 of 29 et al. from the ethanolic extract of cannabis, which was chromatographed on a silica gel column [56] and identified by GCMS. The two ethoxy cannabitriols (92)(93) are most likely artifacts, since ethanol was used in their isolation from Cannabis [3,57].

Miscellaneous Types Cannabinoids (30 Compounds)
A total of thirty miscellaneous type cannabinoids (Figures 7 and 8 (99), and cannabichromanone-C5 (CBCN-C5, 105) after micropreparative GC and TLC. Their chemical structures were determined by mass and NMR spectroscopic analysis [58]. Cannabicitran (CBT, 104) was isolated from an ethanolic extract of Lebanese hashish. It was purified by counter-current distribution and silica gel chromatography. Its chemical structure was determined by GCMS, IR and 1 H NMR analyses [59]. The isolation of cis-Δ 9 -THC (101) was reported from a petroleum extract of marihuana by Smith and Kampfert in 1977. The extract was purified on a florsil column followed by preparative TLC [60].
Cannabiripsol (CBR, 103) was isolated from a South African Cannabis variant after hexane extraction and chromatography on silica and polyamide columns. Its chemical structure was determined by spectral means (IR, GCMS, UV, 1 H NMR) and by synthesis [61]. Cannabitetrol (CBTT, 104) was obtained and identified from the hexane extract of Mexican marijuana grown in Mississippi using silica gel column chromatography [62]. ElSohly and Slade reported the details of the isolation and chemical identification of compounds 106-109 [2].

Non-Cannabinoids
In addition to cannabinoids, more than 400 non-cannabinoid constituents have been isolated and/or identified from the cannabis plant. These non-cannabinoids belong to various chemical classes [2,3,66]. The major non-cannabinoid constituents are classified into four major categories: non-cannabinoid phenols, flavonoids, terpenes, alkaloids.

Spiro-Indans (16 Compounds)
Sixteen spiro-indan type compounds were isolated from cannabis (126-141, Figure  9). Compound 126 was isolated in 1976 from an Indian cannabis variety using silica gel column chromatography and given the name cannabispiran [67]. In the same year, the compound was also isolated by another research group from a South African variety and named cannabispirone; cannabispirenone (127) was also identified from the South African cannabis variety [68]. Bercht et al. used NMR and mass spectrometric analysis to prove the chemical structures of 126 and 127, while Ottersen et al. used X-ray crystallography to confirm the chemical structure of 126 [67]. The cannabispirenone isomer (128), with interchangeable methoxy and hydroxyl groups, was isolated from Mexican marihuana, and its chemical structure was established by 1 H NMR and EIMS analysis, [69]. Cannabispiradienone (129) was isolated from Thai cannabis, and its chemical structure was elucidated based on 1 H NMR spectroscopy and confirmed by hydrogenation to give cannabispiran (126) [70]. Two spiro-indans named cannabispirol (130) and acetyl cannabispirol (131) were detected by Yukihiro and Nishioka in the benzene extract of the dried leaves of Japanese cannabis. The benzene extract was chromatographed on a polyamide column followed by silica gel chromatography to yield compounds 130 and 131 [71]. Compound 130 was also isolated from a South African variety of cannabis grown in Mississippi by Turner's group and named β-cannabispirol. The orientation of the hydroxyl group was determined by 1 H NMR analysis of 130 and its acetate derivative (131) [61]. Three spiroindans were isolated from an ethanolic extract of a seized hashish sample from Saudi Arabia and were chemically identified as 5-hydroxy-7-methoxyindan-1-spiro-cyclohexane (132), 7-hydroxy-5-methoxyindan-1-spiro-cyclohexane (133), and 5,7-dihydroxyindan-1spiro-cyclohexane (134) through a combination of spectral and chemical analysis. The Cannabiripsol (CBR, 103) was isolated from a South African Cannabis variant after hexane extraction and chromatography on silica and polyamide columns. Its chemical structure was determined by spectral means (IR, GCMS, UV, 1 H NMR) and by synthesis [61]. Cannabitetrol (CBTT, 104) was obtained and identified from the hexane extract of Mexican marijuana grown in Mississippi using silica gel column chromatography [62]. ElSohly and Slade reported the details of the isolation and chemical identification of compounds 106-109 [2].

Non-Cannabinoids
In addition to cannabinoids, more than 400 non-cannabinoid constituents have been isolated and/or identified from the cannabis plant. These non-cannabinoids belong to various chemical classes [2,3,66]. The major non-cannabinoid constituents are classified into four major categories: non-cannabinoid phenols, flavonoids, terpenes, alkaloids.

Spiro-Indans (16 Compounds)
Sixteen spiro-indan type compounds were isolated from cannabis (126-141, Figure 9). Compound 126 was isolated in 1976 from an Indian cannabis variety using silica gel column chromatography and given the name cannabispiran [67]. In the same year, the compound was also isolated by another research group from a South African variety and named cannabispirone; cannabispirenone (127) was also identified from the South African cannabis variety [68]. Bercht et al. used NMR and mass spectrometric analysis to prove the chemical structures of 126 and 127, while Ottersen et al. used X-ray crystallography to confirm the chemical structure of 126 [67]. The cannabispirenone isomer (128), with interchangeable methoxy and hydroxyl groups, was isolated from Mexican marihuana, and its chemical structure was established by 1 H NMR and EIMS analysis, [69]. Cannabispiradienone (129) was isolated from Thai cannabis, and its chemical structure was elucidated based on 1 H NMR spectroscopy and confirmed by hydrogenation to give cannabispiran (126) [70]. Two spiro-indans named cannabispirol (130) and acetyl cannabispirol (131) were detected by Yukihiro and Nishioka in the benzene extract of the dried leaves of Japanese cannabis. The benzene extract was chromatographed on a polyamide column followed by silica gel chromatography to yield compounds 130 and 131 [71]. Compound 130 was also isolated from a South African variety of cannabis grown in Mississippi by Turner's group and named β-cannabispirol. The orientation of the hydroxyl group was determined by 1 H NMR analysis of 130 and its acetate derivative (131) [61]. Three spiro-indans were isolated from an ethanolic extract of a seized hashish sample from Saudi Arabia and were chemically identified as 5-hydroxy-7-methoxyindan-1-spiro-cyclohexane (132), 7-hydroxy-5-methoxyindan-1-spiro-cyclohexane (133), and 5,7-dihydroxyindan-1-spiro-cyclohexane (134) through a combination of spectral and chemical analysis. The methanolic fraction of hashish was subjected to flash chromatography and further purified through silica gel column chromatography to afford compounds 132-134 [72]. Isocannabispiran (135) has been isolated from a Panamanian variety of cannabis by repeated chromatography. The structure was chemically elucidated as 5 -hydroxy-7 -methoxy-spiro-(cyclohexane-1,1 -indan)-4-one by spectroscopic means as well as direct comparison with cannabispiran (126) [73]. Radwan et al. (2008) isolated 7-O-methyl-cannabispirone (136) from an extract of a high potency cannabis variety using normal phase chromatography followed by C 18 -HPLC [32], while isocannabispiradienone (137) and α-cannabispiranol (138) were obtained from the dichloromethane extract of decarboxylated C. sativa hemp that was subjected to C 18 flash chromatography, followed by silica gel gravity column chromatography and HPLC. The chemical structures were established using HR-ESIMS and NMR ( 1 H, 13 C, HSQC and HMBC) data [66]. Recently, three new cannabispirans (139-141) have been obtained from the leaves of C. sativa. Cannabispirketal (139) and the glycoside, α-cannabispiranol-4 -O-βglucopyranose (140) were isolated from an ethanolic extract, and their chemical structures were determined by 1D NMR ( 1 H NMR, 13 C NMR) and 2DNMR (COSY, HSQC, HMBC and ROESY) [74]. In 2018, Nalli et al. isolated prenylspirodienone (141) and proved its chemical structure by extensive NMR and ESI-MS analysis. Moreover, they proposed the biosynthetic pathway from this compound [75].

Flavonoids (34 Compounds)
Thirty-four flavonoids were isolated from C. sativa (168-201), which could be categorized into seven basic chemical skeletons that can be methylated, glycosylated (C or O glycosides), prenylated, or geranylated ( Figure 13). The seven chemical structures of the

Flavonoids (34 Compounds)
Thirty-four flavonoids were isolated from C. sativa (168-201), which could be categorized into seven basic chemical skeletons that can be methylated, glycosylated (C or O glycosides), prenylated, or geranylated ( Figure 13). The seven chemical structures of the

Flavonoids (34 Compounds)
Thirty-four flavonoids were isolated from C. sativa (168-201), which could be categorized into seven basic chemical skeletons that can be methylated, glycosylated (C or O glycosides), prenylated, or geranylated ( Figure 13). The seven chemical structures of the flavonoid aglycones are orientin, vitexin, isovitexin, apigenin, luteolin, kaempferol and quercetin. The details of the isolation and chemical structures of 19 flavonoids (168-171, 173-175, 178-184, 187-188, and 193-195) isolated from C. sativa were reviewed by turner et al. in 1980 [3]. The flavonoid glycosides, vitexin (172), cytisoside (176) and cytisoside glucoside (177), were identified from Canadian cannabis plants grown from seeds, where the authors used TLC, a hydrolytic test and UV spectroscopic analysis to determine their chemical structures [86]. Crombie et al. isolated two methylated isoprenoid flavones named Canniflavone 1 and Canniflavone 2 from a Thailand strain of cannabis [79]; four years later, Barrett et al. isolated the same two compounds and named them Canniflavin A (189, prenyl flavone) and Canniflavin B (190, geranyl flavone) from the ethanolic extract of C. sativa. The structures were elucidated by using UV, 1 H NMR and 13 C NMR spectroscopic techniques [87]. In 2008, Radwan el al isolated another methylated isoprenoid flavone, canniflavin C (191), along with 6-prenylapigenin (185) and chrysoeriol (192), from a high potency variety of C. sativa grown in Mississippi polar fractions by using combination of various chromatographic techniques, such as VLC, silica gel column chromatography, and RP-HPLC. The geranyl moiety in Canniflavin C (191) is attached to the C-8 instead of the C-6 as in Canniflavin B based on 1D and 2D NMR analysis [80]. The glycoside apigenin-6,8-di-C-β-D-glucopyranoside (186) was isolated from the methanolic extract of hemp [88], Two flavonoid glycosides (196)(197) were isolated from the pollen grains of the male plants of a Mexican variety of C. sativa that was cultivated at the University of Mississippi. Their chemical structures were identified as kaempferol-3-O-sophoroside (196) and quercetin-3-O-sophoroside (197) based on 1D and 2D NMR and UV experiments [89]. The flavonoid glycoside Rutin (198) was isolated for the first time from hemp pectin. The ethanolic extract was purified by macroreticular resin, silica gel column chromatography, and Sephadex-LH-20. Spectroscopic methods (ESI-MS, 1 H NMR, 13 C NMR) were used for identification of its chemical structure [84].
The flavonoids Quercetin (199), Naringenin (200), and Naringin (201) were identified and quantified in the hydroalcoholic extract of hemp inflorescence from monoecious cultivars grown in Central Italy. Four cultivars (Ferimon, Uso-31, Felina 32 and Fedora) were analyzed at four stages of growth from flowering to ripening using HPLC-PDA. Naringenin (200) and its glycoside (Naringin, 201) was detected only in Fedora 17 and Ferimon cultivars, respectively, while Quercetin (199) was present in the hydroalcohlic extract of the four cultivars [90,91]. Naringenin (200) was also detected and quantified in the industrial hemp of Futura 75 cultivar cultivated in Italy and the quantification was performed by HPLC-DAD-MS of the water extract [91].

Terpenes (120 Compounds)
Previous publications have reported 120 [2,92] or more [93,94] terpenes in cannabis. Our search for terpenes with the correct chemical structures yielded a total of 120 terpenes. Terpenes, or isoprenoids, consist of the second largest class of cannabis constituents. These compounds are responsible for the characteristic aroma of the plant. Terpenes can be classified into five main classes: monoterpenes, sesquiterpenes, diterpenes, triterpenes, and miscellaneous terpenes. Out of 120 terpenes, there are 61 monoterpenes (C 10 skeleton), 51 sesquiterpenes (C 15 skeleton), 2 diterpenes, (C 20 skeleton), 2 triterpenes (C 30 skeleton), and 4 miscellaneous compounds.
As the aroma of C. sativa L. is of high importance for the detection of illicit marijuana trafficking, the composition of the emitted aroma constituents was investigated, using a direct gas chromatographic analysis of the headspace components. The marijuana standard was grown and harvested at the University of Mississippi, and real samples were obtained from Customs' seizures. The samples were prepared by weighing 1 g of each sample, placing it in a microvial, and heating the sample at 65 • C for 1 h. From the microvial, 5 mL of the headspace air was withdrawn using a gas-tight syringe and directly injected into the gas chromatograph. A total of 18 terpenic compounds were detected, out of which three compounds had not been previously reported, namely 2-methyl-2-heptene-6-one (227), fenchyl alcohol (254), and borneol (256), [101].
The volatile oil of C. sativa of Mexican origin was prepared, and a total of 17 monoterpenes were identified in the oil through GC-MS analysis. Six oxygenated monoterpenes had not been identified previously, namely nerol (223), geraniol (224), carvacrol (230), 1,8-cineol (250), 1,4-cineol (251), and camphor (258) [60]. Later that year, piperitenone (231) was detected and identified in the volatile oil of Cannabis through GC-MS analysis and retention time matching [102]. Ross and ElSohly studied the composition of cannabis essential oil prepared by steam distillation of marijuana buds, by collecting the oil using the lighter-than-water volatile oil apparatus. The study included the preparation of volatile oil from fresh buds, as well as samples that were dried and stored at room temperature at three time points: 1 week, 1 month, and 3 months. The composition of the oils was then determined by GC-MS and GC-FID, where the percentage compositions of each component determined by the GC-FID method, and the presence of the terpenes was identified by GC-MS. There was a total of 68 components identified in the study, with 57 identified as monterpenes, sesquiterpenes, and other compounds, such as ketones and esters. The fresh buds' oil was determined to have the greatest composition of monoterpenes, at approximately 92.48%. Sesquiterpenes represented 6.84% of the total percent composition and other compounds represented only 0.68%. Analysis of the three-month samples showed a decrease in the percent composition of monoterpenes and an increase in the percent composition of sesquiterpenes and other compounds, where the monoterpenes represented 62.02% composition, the sesquiterpenes were 35.63%, and other compounds represented 2.35% of the volatile oil. Three new oxygenated monoterpenes were reported for the first time in this study, namely, ipsdienol (225), cis-carveol (240) and cis-sabinene hydrate (248) [104].

Sesquiterpenes (51 Compounds)
In 1942, a study was conducted for the analysis of the higher boiling point fraction of Egyptian hashish, resulting in the identification of one sesquiterpene, α-caryophyllene (α-
The volatile oil of C. sativa from Mexico was studied by Bercht and Paris in 1974, and a total of 17 monoterpenes were identified in the oil through GC-MS analysis. A sesquiterpene, nerolidol (278), was identified for the first time [60]. Using GC/MS and GC retention time, α-gurjunene (279) was detected for the first time in C. sativa resin [102].

Triterpenes
Two triterpenes have been identified in cannabis ( Figure 17). In 1971, analysis of the ethanolic extract of Cannabis roots resulted in the identification of two triterpenes, friedelin (friedelan-3-one, 316) and epifriedelanol (317), via spectral data and comparison with authentic samples [108].

Miscellaneous Terpenes
A total of four miscellaneous terpenes have been identified in cannabis ( Figure 17). Two of them, vomifoliol (318) and dihydrovomifoliol (319), are isophorone-type compounds and were isolated from Dutch hemp [60]. Both were identified from the stems and leaves of the plant through isolation, spectral data comparison, and synthesis from (+)-αionone. The other two miscellaneous-type compounds were identified from the volatile oil of C. sativa, namely β-ionone (320) and dihydroactinidiolide (321) [83].

Alkaloids
Only two spermidine alkaloids (322-323) have been identified in C. sativa ( Figure 18). In 1975, the first spermidine alkaloid was isolated from a methanolic extract of cannabis roots from a Mexican variant grown in Mississippi, and it was identified as cannabisativine (316) by X-ray crystallography [109]. Later the same year, the same compound was isolated from an ethanolic extract of the dry leaves and small stems of a Thailand variant [110]. The ethanolic extract was extracted and acid-base partitioned, as well as subjected to column and thin-layer chromatography followed by crystallization of the alkaloid from acetone.
A year after cannabisativine was reported, the second spermidine alkaloid, namely anhydrocannabisativine (323), was isolated from the dry leaves and small stems of cannabis of the Mexican variety grown in Mississippi [111]. The compound was isolated through a series of acid-base extractions and silica-gel chromatography. The identity of the compound was proven by spectral data analysis and by the conversion of cannabisativine (322) to anhydrocannabisativine (323). In 1978, the spermidine alkaloid was isolated from the roots and leaves of a Mexican variant [112]. In 1977, the Mississippi group also identified anhydrocannabisativine (322) in 15 different Cannabis variants using TLC eluted with chloroform: acetone: ammonia (1:1:1) [113].

Triterpenes
Two triterpenes have been identified in cannabis ( Figure 17). In 1971, analysis of the ethanolic extract of Cannabis roots resulted in the identification of two triterpenes, friedelin (friedelan-3-one, 316) and epifriedelanol (317), via spectral data and comparison with authentic samples [108].

Miscellaneous Terpenes
A total of four miscellaneous terpenes have been identified in cannabis ( Figure 17). Two of them, vomifoliol (318) and dihydrovomifoliol (319), are isophorone-type compounds and were isolated from Dutch hemp [60]. Both were identified from the stems and leaves of the plant through isolation, spectral data comparison, and synthesis from (+)-α-ionone. The other two miscellaneous-type compounds were identified from the volatile oil of C. sativa, namely β-ionone (320) and dihydroactinidiolide (321) [83].

Alkaloids
Only two spermidine alkaloids (322-323) have been identified in C. sativa ( Figure 18). In 1975, the first spermidine alkaloid was isolated from a methanolic extract of cannabis roots from a Mexican variant grown in Mississippi, and it was identified as cannabisativine (316) by X-ray crystallography [109]. Later the same year, the same compound was isolated from an ethanolic extract of the dry leaves and small stems of a Thailand variant [110]. The ethanolic extract was extracted and acid-base partitioned, as well as subjected to column and thin-layer chromatography followed by crystallization of the alkaloid from acetone.
A year after cannabisativine was reported, the second spermidine alkaloid, namely anhydrocannabisativine (323), was isolated from the dry leaves and small stems of cannabis of the Mexican variety grown in Mississippi [111]. The compound was isolated through a series of acid-base extractions and silica-gel chromatography. The identity of the compound was proven by spectral data analysis and by the conversion of cannabisativine (322) to anhydrocannabisativine (323). In 1978, the spermidine alkaloid was isolated from the roots and leaves of a Mexican variant [112]. In 1977, the Mississippi group also identified anhydrocannabisativine (322) in 15 different Cannabis variants using TLC eluted with chloroform: acetone: ammonia (1:1:1) [113].

Conclusions
To date, there have been over 500 constituents of Cannabis sativa reported, out of which 125 are identified to belong to the cannabinoid-type compounds, with five new cannabinoids reported in the last 2 years. The other non-cannabinoid-type compounds are classified into various chemical classes, including alkaloids, flavonoids, non-cannabinoid phenols, and terpenes. This review discusses the chemistry, identification, isolation, and structural elucidation of these major classes of compounds, to provide an overview of their chemical structures and to better understand the complexity of the chemical profile of C. sativa.

Conclusions
To date, there have been over 500 constituents of Cannabis sativa reported, out of which 125 are identified to belong to the cannabinoid-type compounds, with five new cannabinoids reported in the last 2 years. The other non-cannabinoid-type compounds are classified into various chemical classes, including alkaloids, flavonoids, non-cannabinoid phenols, and terpenes. This review discusses the chemistry, identification, isolation, and structural elucidation of these major classes of compounds, to provide an overview of their chemical structures and to better understand the complexity of the chemical profile of C. sativa.