Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicon and Carbowax 20M phases

Plant cell cultures: Chemical factories of secondary metabolites

Phenolic compounds and their role in oxidative processes in fruits

Tetrahydrocannabinol (THC) has been the primary focus of cannabis research since 1964, when Raphael Mechoulam isolated and synthesized it. More recently, the synergistic contributions of cannabidiol to cannabis pharmacology and analgesia have been scientifically demonstrated. Other phytocannabinoids, including tetrahydrocannabivarin, cannabigerol and cannabichromene, exert additional effects of therapeutic interest. Innovative conventional plant breeding has yielded cannabis chemotypes expressing high titres of each component for future study. This review will explore another echelon of phytotherapeutic agents, the cannabis terpenoids: limonene, myrcene, α-pinene, linalool, β-caryophyllene, caryophyllene oxide, nerolidol and phytol. Terpenoids share a precursor with phytocannabinoids, and are all flavour and fragrance components common to human diets that have been designated Generally Recognized as Safe by the US Food and Drug Administration and other regulatory agencies. Terpenoids are quite potent, and affect animal and even human behaviour when inhaled from ambient air at serum levels in the single digits ng·mL−1. They display unique therapeutic effects that may contribute meaningfully to the entourage effects of cannabis-based medicinal extracts. Particular focus will be placed on phytocannabinoid-terpenoid interactions that could produce synergy with respect to treatment of pain, inflammation, depression, anxiety, addiction, epilepsy, cancer, fungal and bacterial infections (including methicillin-resistant Staphylococcus aureus). Scientific evidence is presented for non-cannabinoid plant components as putative antidotes to intoxicating effects of THC that could increase its therapeutic index. Methods for investigating entourage effects in future experiments will be proposed. Phytocannabinoid-terpenoid synergy, if proven, increases the likelihood that an extensive pipeline of new therapeutic products is possible from this venerable plant.

LINKED ARTICLES
This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

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Taming THC: potential cannabis synergy and phytocannabinoid‐terpenoid entourage effects

Polyphenol oxidases in plants. Recent progress

A series of artemisinin-related endoperoxides was tested for cytotoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolium (MTT) assay. Artemisinin [1] had an IC50 value of 29.8 microM. Derivatives of dihydroartemisinin [2], being developed as antimalarial drugs (artemether [3], arteether [4], sodium artesunate [5], artelinic acid [6], and sodium artelinate [7]), exhibited a somewhat more potent cytotoxicity. Their IC50 values ranged from 12.2 to 19.9 microM. The presence of an exocyclic methylene fused to the lactone ring, as for artemisitene [9], led to higher cytotoxicity than 1. From the two epimeric 11-hydroxyartemisinin derivatives, the R form 12 showed a considerably higher cytotoxicity than the S form 13. Opening of the lactone ring of 1 dramatically reduced the cytotoxicity. The ether dimer 8 of 2 was the most potent cytotoxic agent, its IC50 being 1.4 microM. The variations in cytotoxicity between the structurally related compounds mostly correlated well with the theoretical capacity of radical formation and stabilization. In some cases lipophilicity or the presence of an electrophilic moiety seemed to have a determinant influence on cytotoxicity. The artemisinin-related endoperoxides showed cytotoxicity to EAT cells at higher concentrations than those needed for in vitro antimalarial activity, as reported in the literature.

Cytotoxicity of Artemisinin-Related Endoperoxides to Ehrlich Ascites Tumor Cells

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l-1 BAP, 0.05 mg l-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l-1 gibberellic acid, 0.5 g l-1 casein hydrolysate, 10 mg l-1 or 20 mg l-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

The sesquiterpenoid artemisinin (3) and its biosynthetic precursors arteannuic acid (1), arteannuin B (2) and artemisitene (4) can be separated and identified by combined gas chromatography/mass spectrometry both as a mixture of reference standards as well as in extracts of Artemisia annua L. From this study a generally applicable gas chromatographic method has been developed for the analysis of these sesquiterpenoids in A. annua. Analysis of plant parts revealed that the highest sesquiterpenoid concentrations were present in the leaves, buds and small green stems, while the main stem contained only small amounts. Low levels of arteannuic acid and arteannuin B were present in the side-roots. No sesquiterpenoids could be detected in the main root and none of the plant parts accumulated detectable quantities of artemisitene.

Analysis of artemisinin and related sesquiterpenoids from artemisia-annua l by combined gas-chromatography mass-spectrometry

The volatile constituents of Artemisia annua L. plants, grown in the field in The Netherlands from seeds of Chinese and Vietnamese origin, were investigated using GC and GC–MS (EI, NICI) analysis. The plants grown from Chinese seeds contained 4.0% (v/w) essential oil on a dry weight (DW) basis, those from Vietnamese seeds, 1.4% (v/w). More than forty compounds were identified. The principal component of the Chinese oil was artemisia ketone (63.9%); other major constituents included artemisia alcohol (7.5%), myrcene (5.1%), α-guaiene (4.7%) and camphor (3.3%). In the Vietnamese oil the main components were camphor (21.8%) and germacrene-D (18.3%); other important constituents were β-caryophyllene (5.6%), trans-β-farnesene (3.8%) and 1,8-cineole (3.1%). In the Vietnamese variety the terpenoid biosynthesis proceeded further towards sesquiterpenes, whereas in the Chinese predominantly monoterpenes were formed. This was also reflected in the artemisinin contents, found in dichloromethane extracts of the herbaceous plant material: 0.17% (DW) in Chinese and 1.00% (DW) in Vietnamese plants. We suggest that the differences in the essential oil composition may be ascribed to the existence of different A. annua chemotypes. In a dichloromethane extract of the roots several non-volatile sesquiterpenes were found. The main constituent was a new compound, which was identified tentatively as arteannuic alcohol.

Volatile constituents of Artemisia annua L. (Asteraceae)

In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferin into podophyllotoxin, maximally a 12.8 fold increase in content was found. Permeabilization using isopropanol, in combination with coniferin as a substrate, did not result in an extra increase in podophyllotoxin accumulation. Concentrations of isopropanol exceeding 0.5% (v/v) were found to be rather toxic for suspension growth cells of P. hexandrum. When coniferin was fed in presence of such isopropanol concentrations, β-glucosidase activity was still present, resulting in the formation of the aglucon coniferyl alcohol. In addition, podophyllotoxin was released into the medium under these permeabilization conditions. Entrapment of P. hexandrum cells in calcium-alginate as such or in combination with the feeding of biosynthetic precursors, did not improve the podophyllotoxin production. Cell-free medium from suspension cultures at later growth stages incubated with coniferin, resulted in the synthesis of the lignan pinoresinol.

Theo M. Malingré

Papers

Cytotoxicity of Artemisinin-Related Endoperoxides to Ehrlich Ascites Tumor Cells

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

Analysis of artemisinin and related sesquiterpenoids from artemisia-annua l by combined gas-chromatography mass-spectrometry

Volatile constituents of Artemisia annua L. (Asteraceae)

On the improvement of the podophyllotoxin production by phenylpropanoid precursor feeding to cell-cultures of podophyllum-hexandrum royle