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

Essential oils are complex mixtures isolated from aromatic plants which may possess antioxidant and anti-inflammatory activities of interest in thye food and cosmetic industries as well as in the human health field. In this work, a review was done on the most recent publications concerning their antioxidant and anti-inflammatory activities. At the same time a survey of the methods generally used for the evaluation of antioxidant activity and some of the mechanisms involved in the anti-inflammatory activities of essential oils are also reported.

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Antioxidant and anti-inflammatory activities of essential oils: a short review.

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 A resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 A away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

/pdf/crystal-structure-of-agaricus-bisporus-mushroom-tyrosinase-5egq24ti7m.pdf

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.

Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste

SUMMARY Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organism9s physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes.

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

The monoterpene D-limonene and its metabolites have been shown to exert chemopreventive and chemotherapeutic effects against different tumours in animal models and clinical trials. However, it is unknown whether these compounds modulate the inflammatory response in RAW 264.7 macrophage cells. The present study was therefore designed to elucidate the pharmacological and biological effects of D-limonene on the production of pro-inflammatory cytokines and inflammatory mediators in macrophages. The results indicate that D-limonene is an effective inhibitor of lipopolysaccharide (LPS)-induced NO and prostaglandin E(2) production in RAW 264.7 cells. These inhibitory effects of D-limonene included dose-dependent decreases in the expression of iNOS and COX-2 proteins. To evaluate the inhibitory effects of D-limonene on other cytokines, we also measured TNF-alpha, IL-1beta, and IL-6 levels in the cell supernatants of LPS-stimulated RAW 264.7 macrophages by enzyme-linked immunosorbent assay. In these assays, D-limonene decreased the expression of TNF-alpha, IL-1beta, and IL-6 in a dose-dependent manner. To assess the suitability of D-limonene for cosmetic applications, we also performed 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays on HaCaT keratinocytes. D-limonene did not display any cytotoxicity in these assays. From these results, we suggest that D-limonene may be considered a potential anti-inflammatory candidate.

Limonene suppresses lipopolysaccharide-induced production of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines in RAW 264.7 macrophages.

S-Adenosyl-l-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and l-methionine. Despite extensive research with many organisms, its role in Streptomyces sp. remains unclear. In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis. The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from l-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein. The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media. Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters. In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell. From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp.

Accumulation of S-Adenosyl-l-Methionine Enhances Production of Actinorhodin but Inhibits Sporulation in Streptomyces lividans TK23

Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase (iNOS) is known to be responsible for the vasodilation and hypotension observed during septic shock and inflammation. Thus, inhibitors of iNOS may be useful candidates for the treatment of inflammatory diseases accompanied by the overproduction of NO. In this study, we prepared alcoholic extracts of Jeju plants and screened them for their inhibitory activity against NO production in lipopolysaccharide (LPS)-activated macrophages. Among the 260 kinds of plant extract tested, 122 extracts showed potent inhibitory activity towards NO production by more than 25% at a concentration of 100 µg/mL. Plants such as Malus sieboldii, Vaccinium oldhamii, Corylus hallaisanensis, Carpinus laxiflora, Styrax obassia, and Securinega suffruticosa showed the most potent inhibition (above 70%) at a concentration of 100 µg/mL. The cytotoxic effects of the plant extracts were determined by colorimetric MTT assays and most plant extracts exhibited only moderate cytotoxicity at 100 µg/mL. Therefore, these plants should be considered promising candidates for the further purification of bioactive compounds and would be useful for the treatment of inflammatory diseases accompanying overproduction of NO.

/pdf/inhibition-of-nitric-oxide-production-in-lipopolysaccharide-297qcfr4t3.pdf

Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages by Jeju plant extracts.

Despite its beneficial role in host defense mechanisms, excessive nitric oxide (NO) production by activated macrophages has been implicated in several inflammatory diseases. To clarify the mechanisms of the anti-inflammatory activities of Sargassum micracanthum, we evaluated whether extracts of S. micracanthum could modulate the production of NO by activated macrophages. S. micracanthum were extracted with 80% EtOH. The extract was then successively partitioned with hexane, CH 2 Cl 2 , EtOAc, BuOH, and water. The results indicate that the hexane and CH2Cl2 fractions of S. micracanthum extract were effective inhibitors of LPS-induced NO and prostaglandin E2 (PGE2) production in RAW 264.7 cells. The inhibitory effects of the hexane and CH2Cl2 fractions of S. micracanthum were accompanied by dosedependent decreases in the production of iNOS and COX-2 proteins and iNOS and COX-2 mRNA expression. To test the inhibitory effects of S. micracanthum fractions on other cytokines, we also performed ELISA and RT-PCR assays for TNF- , IL-1s, and IL-6 in LPSstimulated RAW 264.7 macrophage cells. In these assays, the hexane and CH2Cl2 fractions of S. micracanthum produced dose-dependent decreases in the production and mRNA expression of TNF- , IL-1s, and IL-6. To test the potential application of S. micracanthum extract as a cosmetic material, we also performed MTT assays on human dermal fibroblast cells, as well as primary skin irritation tests. In these assays, S. micracanthum extracts did not induce any adverse reactions. Based on these results, we suggest that S. micracanthum extracts may be considered potential anti-inflammatory candidates for topical application.

Suppression of pro-inflammatory cytokines, iNOS, and COX-2 expression by brown algae Sargassum micracanthum in RAW 264.7 macrophages

To identify new active cosmetics ingredients of natural origin, we screened 299 parts of 263 plant species collected from Jeju Island, the southernmost island of the Korean Peninsula. Plant parts were investigated for their elastase and tyrosinase inhibitory activity for the purpose of identifying anti-aging and skin-whitening ingredients with the potential for use as raw materials in cosmetics. In the anti-elastase inhibition assay, 3 extracts, including Aesculus turbinata, Taxillus yadoriki, and Cornus walteri, showed high inhibitory activity (inhibition concentration ( I C ) 5 0<50 /vg/mL). The I C 5 0 of A. turbinata and T. yadoriki was 43.1 /vg/mL and 36.4 /vg/mL, respectively; C. walteri showed the highest elastase inhibition activity ( I C 5 0 = 26.1 /vg/mL). In the tyrosinase inhibition assay, 4 extracts, including C. walteri(139.2 vg/mL), Maackia fauriei (149.3 /vg/mL), Toxicodendron succedaneum (142.3 /vg/mL), and Sophora flavescens (41.6 v g/mL), showed significantly greater tyrosinase inhibition activity than the positive controls Distylium racemosum (145.9 /vg/mL) and arbutin (180.3 /vg/mL). However, they showed lower activity compared to the positive controls Morus alba (11.9 vg/mL) and Morus bombycis (22 v g/mL). These results suggest that medicinal plants possessing several biological activities may be potent inhibitors of the processes involved in pigmentation increases and aging. Further investigations will focus on in vivo assays and the chemical identification of the major active components responsible for anti-aging and whitening.

http://www.ejobios.org/download/screening-of-elastase-and-tyrosinase-inhibitory-ctivity-from-jeju-island-plants.pdf

Chang-Gu Hyun

Papers

Limonene suppresses lipopolysaccharide-induced production of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines in RAW 264.7 macrophages.

Accumulation of S-Adenosyl-l-Methionine Enhances Production of Actinorhodin but Inhibits Sporulation in Streptomyces lividans TK23

Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages by Jeju plant extracts.

Suppression of pro-inflammatory cytokines, iNOS, and COX-2 expression by brown algae Sargassum micracanthum in RAW 264.7 macrophages

Screening of elastase and tyrosinase inhibitory activity from Jeju Island plants