Based on a procedure reported by Crombie and coworkers, a solution of olivetol and food-grade α-phellandrene in benzene was treated with p-toluenesulfonic acid monohydrate and the mixture was allowed to stir at room temperature for 1 h. Te solvent was removed in vacuo and the residue was purified by silica gel chromatography using a gradient elution to give H2CBD as a dark yellow oil. Spectroscopic data were in full agreement with the literature.Animals were randomly divided into 5 groups of 12 animals per group and received either vehicle , 2:1:17, a positive control or 8,9-dihydrocannabidiol via intraperitoneal injection 1h prior to administration of the convulsant agent pentylenetetrazole to achieve brain cannabinoid Tmax. PTZ was administered intraperitoneally 1h afer drug or vehicle treatment. Seizure activity was video recorded for 30min and video records blinded before ofine review and coding using a modified Racine scale .4,4-Dichlorodiphenyltrichloroethane was used as the internal analytical standard . HPLC grade n-hexane, acetonitrile, water and ascorbic acid were purchased from Sigma Aldrich UK and Fisher Scientifc. Stock standard solutions of CBD, H2CBD and DDT were prepared in acetonitrile and stored at −20 °C until use. These were further diluted in acetonitrile:water , to achieve calibration concentrations of 0.1, 0.2, 0.5, 1, 5, 10 μg mL−1 . Plasma samples were prepared for HPLC using a previously validated method. Briefly, DDT was added to 150 μL of rat plasma sample as internal standard and plasma proteins were precipitated by the addition of ice cold acetonitrile followed by water ,indoor grow shelves with 1min vortexing between additions. n-Hexane was added to each tube and following a 5min vortex, tubes were centrifuged at 1160 × g for 15min at 10 °C and the upper organic layer was carefully decanted by glass pipette and retained.
The organic layer was evaporated to dryness under a stream of nitrogen at room temperature and reconstituted in 150μL of the mixture of acetonitrile and water prior to HPLC analysis. For post-mortem brain analysis, brains were weighed and 1.5 x ice-cold solvent was added followed by homogenization for 1 min. DDT was added to each homogenized brain tissue as internal standard, samples were mixed and allowed to equilibrate overnight at −20 °C. Samples were then centrifuged at 3500 rpm for 15min and the top layer retained. Samples were dried by SpeedVac concentrator at room temperature and reconstituted in 150μL of the mixture of acetonitrile and water for HPLC analysis. An Agilent 1200 series HPLC equipped with a photodiode array detector was used for analysis. 30 μl of all samples were injected and separation was achieved using an ACE C18-PFP 150 mm 4.6 mm, 3 μm particle size column , protected by an ACE C18-PFP 3 μm guard cartridge. The mobile phase was a mixture of acetonitrile and water in a ratio of 62:38 . The flow rate was set at 1 mL min−1 and the column temperature was maintained at 55 °C. The absorbance of the compounds of interest was monitored at 220nm.Youth are regularly exposed to protobacco messaging through a wide variety of media channels, including static tobacco advertising on newspapers and magazines, retail outlets, the Internet,and on television or in the movies.Marketing activities of tobacco industry are a key factor in leading young people to take up tobacco, keeping some users from quitting, and achieving greater consumption among users.The 201257 and 201421 US Surgeon General reports concluded that tobacco industry promotional activities, including branding, imagery, event sponsorship, and marketing campaigns, cause the onset and progression to smoking among young people.
NCI’s smoking and health monograph, The Role of the Media in Promoting and Reducing Tobacco Use, had earlier found a causal relationship between tobacco marketing exposure and youth smoking. Even minimal exposure to tobacco advertising positively influenced youth attitudes and perceptions on smoking, as well as smoking intentions among youth.Causal effects of tobacco marketing on smoking may be stronger among youth than adults as youth are also more likely to be brand loyal. and are more susceptible to tobacco industry marketing.Youth susceptibility to smoking, experimentation, and current use varies by the Tobacco advertising influences youth smoking behavior at multiple levels.Tobacco advertising and promotion affect awareness of smoking, recognition of specific brands, attitudes about smoking, intentions to smoke, and actual smoking behavior among youth and contribute to reduced risk perceptions around tobacco use.Even with prohibitions on youth-targeted marketing, tobacco industry marketing directed at young adults, encourages use and increased consumption within the young adult population,and indirectly impacts youth smoking because youth consider young adults as aspirational role models.The WHO Framework Convention on Tobacco Control recognizes that the most effective strategy to protect public health would be to prohibit tobacco marketing entirely.Tobacco companies use advertising as a marketing technique to create positive imagery and associations with tobacco products, and to attach desirable characteristics, activities, and outcomes with tobacco product use.Branded merchandise helps to establish brand identity and brand loyalty among novice users, which is an integral part of the tobacco industry’s long term economic strategy. Indeed, the tobacco industry specifically targets young adults in clubs using branded promotions and merchandise.For youth, there is evidence that owning cigarette-branded or alcohol-branded items leads to progression to being an established smoker and initiation of drinking.
Among adults, young adults are significantly more likely that older adults to own cigarette-branded items and to be attracted to the advertising of a cigarette brand .In short, promoting products through branded merchandise is a particularly important strategy for companies and they seem to be heavily targeting youth and young adults, who appear to be more susceptible to it than older adults and are the demographic that is susceptible to initiation or escalation of product use. Brand sharing and brand stretching grant another access point for tobacco companies to subliminally advertise and market their products.In addition to using the cigarette package, tobacco companies place brand names and use other design techniques on the actual stick, which is rated by smokers as more attractive than cigarettes without these characteristics.Despite some restrictions in the USA, tobacco companies continue to advertise in magazines with significant youth audiences, and are more likely to advertise youth preferred brands in these magazines.Tobacco companies circumvent partial advertising restrictions by concentrating advertisements in magazines where youth audience composition is near or at the minimum threshold level, thereby still exposing a sizeable number of youth to tobacco ads. For example, in the United States even a 15% threshold, which was the FDA-proposed rule in 1996 for advertising in print media, would have exposed at least two million youth to tobacco industry advertising.Cigarette companies consolidate marketing expenditures for magazine advertisements to brands that are popular among youth, African Americans, and LGBTQ populations .In the 1990s most of the US state attorneys general sued the major cigarette companies alleging, among other things, that the source of pro-tobacco media. Current tobacco use is associated with exposure to static advertising and to on-screen smoking depicted in TV and in movies, both directly and through perception of peer use among youth and young adults.Tobacco advertising influences youth smoking behavior at multiple levels.Tobacco advertising and promotion affect awareness of smoking, recognition of specific brands,grow table attitudes about smoking, intentions to smoke, and actual smoking behavior among youth and contribute to reduced risk perceptions around tobacco use.Even with prohibitions on youth-targeted marketing, tobacco industry marketing directed at young adults, encourages use and increased consumption within the young adult population,and indirectly impacts youth smoking because youth consider young adults as aspirational role models.The WHO Framework Convention on Tobacco Control recognizes that the most effective strategy to protect public health would be to prohibit tobacco marketing entirely.Tobacco companies use advertising as a marketing technique to create positive imagery and associations with tobacco products, and to attach desirable characteristics, activities, and outcomes with tobacco product use.
Branded merchandise helps to establish brand identity and brand loyalty among novice users, which is an integral part of the tobacco industry’s long term economic strategy. Indeed, the tobacco industry specifically targets young adults in clubs using branded promotions and merchandise.For youth, there is evidence that owning cigarette-branded or alcohol-branded items leads to progression to being an established smoker and initiation of drinking. Among adults, young adults are significantly more likely that older adults to own cigarette-branded items and to be attracted to the advertising of a cigarette brand . In short, promoting products through branded merchandise is a particularly important strategy for companies and they seem to be heavily targeting youth and young adults, who appear to be more susceptible to it than older adults and are the demographic that is susceptible to initiation or escalation of product use. Brand sharing and brand stretching grant another access point for tobacco companies to subliminally advertise and market their products. In addition to using the cigarette package, tobacco companies place brand names and use other design techniques on the actual stick, which is rated by smokers as more attractive than cigarettes without these characteristics.Despite some restrictions in the USA, tobacco companies continue to advertise in magazines with significant youth audiences, and are more likely to advertise youth preferred brands in these magazines.Tobacco companies circumvent partial advertising restrictions by concentrating advertisements in magazines where youth audience composition is near or at the minimum threshold level, thereby still exposing a sizeable number of youth to tobacco ads.For example, in the United States even a 15% threshold, which was the FDA-proposed rule in 1996 for advertising in print media, would have exposed at least two million youth to tobacco industry advertising.Cigarette companies consolidate marketing expenditures for magazine advertisements to brands that are popular among youth, African Americans, and LGBTQ populations .In the 1990s most of the US state attorneys general sued the major cigarette companies alleging, among other things, that the companies were advertising to children.The litigation was resolved with the “Master Settlement Agreement,” in which the companies agreed to some restrictions on marketing to children. After the agreements was signed, the percent of total magazine advertisement spending for mentholated brands increased from 13% in 1998 to 76% in 2006, with an associated increase in youth mentholated cigarette smoking .The tobacco industry’s claims that marketing is only used for brand switching and increasing marketing share12 does not make economic sense.For such claim to be economically viable, the number of people switching brands between companies would have to exceed individual tobacco company marketing expenditures, which is unlikely because several brands are sold by a few cigarette companies. Despite claims made by RJ Reynolds in the US in the late 1980s that it did not directly target children, youth were more likely than adults to report previous exposure to RJ Reynolds’ Joe the Camel cartoon character advertising campaign and accurately associated such image with Camel cigarette brand name . Children also found cigarette advertisements that used Joe the Camel as more appealing than adults.The market share for youth use of Camel had also increased from 0.5% in 1988 to 33% in 1991 during the Joe the Camel campaign. Youth receptivity to tobacco marketing is a strong predictor for smoking initiation and consumption patterns independent of other important predictors of smoking behavior .The odds of initiating smoking among youth receptive to tobacco marketing are twice that compared to unreceptive peers .Longitudinal studies show increased odds of progression from initiation of smoking to established smoking among adolescents who both owned cigarette promotional items and had a favorite cigarette advertisement. In the United States, there are few regulations on tobacco industry marketing of other tobacco products , despite rising use among young adult populations. Among young adult bar patrons , marketing receptivity is associated with other tobacco product use, including smokeless tobacco, hookah, cigarillos, and e-cigarettes. Moreover, current smokers receptive to tobacco marketing are also more likely to be poly-tobacco users than among youth not exposed to similar ads.Youth who thought the ads were more effective were more likely to have a positive attitude toward e-cigarettes and greater intentions for future e-cigarette use. Adults in the United States are also influenced by e-cigarette advertising, with adults reporting greater intention to initiate e-cigarette use after exposure to ecigarette advertising.Similar observations on the effects of alcohol marketing on youth substance use behavior are noted in the literature.