Tobacco-specific N-nitrosamines in tobacco and mainstream smoke of Indian cigarettes

Different brands of Indian cigarettes were analysed, by gas chromatography-thermal energy analysis, for the presence of carcinogenic tobacco-specific N-nitrosamines (TSNA) in both tobacco and mainstream smoke. Preformed TSNA in cigarette tobacco ranged between 68 and 730 ng N-nitrosonornicotine (NNN)/cigarette, between 19 and 174 ng 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK)/cigarette and between 98 and 519 ng N-nitrosoanabasine (NAB) together with N-nitrosoanatabine (NAT)/cigarette. The amounts of NNN, NNK and NAB/NAT in mainstream smoke were 11-156, 7-73 and 17-146 ng/cigarette, respectively.     

Nicotine-derived N-nitrosamines (TSNA) and their relevance in tobacco carcinogenesis

Biochemical studies and bioassays support the concept that the increased risk for cancer of the oral cavity in snuff dippers and cancer of the lung, upper aerodigestive tract, and pancreas in smokers is most likely associated with the exposure to tobacco-specific N-nitrosamines. The doses of TSNA required to induce tumors in the oral cavity in rats and tumors in the lungs of rats and hamsters are comparable to the total doses of TSNA to which a long-term snuff dipper, respectively a cigarette smoker, are exposed over 4 decades. The carcinogenic NNN and NNK are metabolized to highly reactive electrophiles which react with nucleophilic centers of DNA and with proteins. The DNA-adducts are known to cause miscoding which can activate K-ras proto-oncogenes. K-ras oncogenes have been detected in adenocarcinomas of the lung of smokers. TSNA also form globin adducts in the blood of laboratory animals as well as of snuff dippers and smokers. Such adducts are currently being evaluated for their significance as biochemical markers of exposure to TSNA. These biomarkers may also serve as indicators for the possible endogenous formation of TSNA in tobacco chewers and smokers. Additional research needs in TSNA carcinogenesis include the development of modified methods for the reduction of TSNA in tobacco and smoke, inhibition of TSNA carcinogenesis by nutrients and micro-nutrients and the testing of chemopreventive agents.     

New brands of oral snuff

Snuff dipping is causally related to cancer of the oral cavity and pharynx. The most powerful carcinogens in snuff are nitroso compounds, particularly the tobacco-specific N-nitrosamines (TSNA). Concentrations of TSNA in snuff exceed the known concentrations of carcinogenic nitrosamines in any other consumer product by two to three orders of magnitude. During the last decade a gradual decrease in TSNA has occurred in the two leading snuff brands in the USA (about 90% of the market). Of two recently introduced snuff brands one has relatively low levels of nitroso compounds while the other contains the highest concentrations of nitrosamines ever reported in smokeless tobacco. This observation suggests that control of nitrosamines in snuff brands on the US market is desirable.     

Efficiency of supercritical fluid extraction for the production of non-volatile terpenoids from Taraxaci radix

Supercritical fluid extraction (SFE) is an extraction technique which exploits the solvent properties of fluids above their critical point. As a result supercritical fluid extraction was used to gain various active substances from plants. The use of SFE to obtain bioactive substances from medicinal plants over the past twenty years has been proved to be a viable alternative for the extraction of natural compounds. Dandelion (Taraxacum officinale Wiggers et Webers, Asteraceae) is one of the best known European medicinal plants, not only as a traditional medicine but perspective raw material for modern phytopharmaceuticals. From the characteristic principles our attention has been directed to triterpenes and phytosterols with anti-inflammatory activity, which were extracted with supercritical carbon dioxide. Designed experiments were carried out to determine the optimal extraction parameters. The products obtained by supercritical fluids extraction were compared to extracts prepared by traditional extraction method (Soxhlet) with n-hexane and ethyl alcohol solvents. The content of triterpenes and sterols was monitored after saponification by thin layer chromatography-densitometry. The products gained by supercritical fluid extraction were different from the traditional ones both in their appearance and composition. Triterpenes and their esters were extracted quantitatively by supercritical fluid extraction using CO2 as solvent and the extraction dynamic for triterpenes and phytosterols was different. Triterpenes had a higher concentration in the SFE product then in the extracts prepared by traditional methods.     

Identification and Assessment of Tumor-Promoting and Cocarcinogenic Agents: State-of-the-Art in Vitro Methods

Abstract

Chemical-analytical studies during the past 4 years led to several new observations on the formation of tobacco-specificN-nitrosamines (TSNA) and their occurrence in smokeless tobacco, mainstream smoke (MS), and sidestream smoke (SS) of American and foreign cigarettes. When snuff was extracted by means of supercritical fluid extraction with carbon dioxide containing 10% methanol, analysis of this material confirmed that the extraction with organic solvents had been partially incomplete.

Epidemiological studies in the northern Sudan showed a high risk for oral cancer for users of toombak, a home-made oral snuff. Toombak contains 100-fold higher levels of TSNA than commercial snuff in the U.S. and Sweden. The TSNA content in the saliva of toombak dippers is at least ten times higher than that reported in the saliva of dippers of commercial snuff. Biomarker studies have shown corresponding high levels of hemoglobin adducts with metabolites of NNN and NNK as well as for urinary metabolites of NNK. These data supported the epidemiological findings.

The analyses of MS of U.S. and foreign cigarettes smoked under FTC conditions revealed comparable data for the smoke of nonfilter cigarettes and filter cigarettes except in the case of low- and ultralow-yield cigarettes, which showed reduced TSNA yields. The MS of cigarettes made from Burley or dark tobacco is exceptionally high in TSNA, primarily because of the high nitrate content of those tobacco types. Taking puffs of larger volume and drawing puffs more frequently, practices observed among most smokers of cigarettes with low nicotine yield, results in high TSNA values in the MS. The formation of the lung carcinogen NNK is favored during the smoldering of cigarettes, between puffs, when SS is generated. Consequently, in most samples from indoor air polluted with environmental tobacco smoke (ETS), the highest concentration of an individual TSNA is that of NNK. When nonsmokers had remained for up to 2 h in a test laboratory with high ETS pollution, they excreted measurable amounts of NNK metabolites in the urine, indicative of the uptake of TSNA.     

Assay of vitamin A palmitate and vitamin E acetate in cosmetic creams and lotions by supercritical fluid extraction and HPLC

The use of supercritical fluid extraction (SFE) as an alternative to liquid extraction was examined for the isolation of vitamin A palmitate and vitamin E acetate from cream and lotion preparations. Investigation of the factors controlling the extraction efficiency in SFE indicated that vitamin recoveries were affected mainly by the extraction pressure and by the degree of sample dispersion. The vitamins were analysed by high-performance liquid chromatography after a 30-min extraction of the cosmetic product with supercritical carbon dioxide at 40°C and at a pressure of 250 atm. Compared with conventional liquid extraction SFE produced slightly lower recoveries (>91.6%) but afforded a more effective purification of the cosmetic matrices. Moreover, SFE minimized sample handling and the use of harmful solvents and provided mild extraction conditions for the analysis of the labile vitamins. Vitamin E acetate and vitamin A palmitate were assayed in commercial cream and lotion formulations using the proposed SFE technique.     

Analytical studies on tobacco-specific N-nitrosamines in tobacco and tobacco smoke

Chemical-analytical studies have led to the identification of approximately 3000 compounds in tobacco and 4000 in tobacco smoke. These include carcinogens in processed tobacco as well as tumor initiators, tumor promoters, cocarcinogens, and organ-specific carcinogens in tobacco smoke. The latter group includes N-nitrosamines, in particular those that derive from nicotine and other tobacco alkaloids, the TSNA. In vitro nitrosation of nicotine yields NNN, NNA, and NNK. Nitrosation of other tobacco alkaloids leads to the formation of NAT, and NAB. Our analytical studies using GC-TEA have led to the identification of seven TSNA in tobacco and tobacco smoke. In addition to NNN, NAT, NAB, and NNK, we also identified NNAL, iso-NNAL, and, most recently, iso-NNAC. Their levels range from 0.01 to 92 ppm in tobacco and from 6 to 530 ng/cigarette in tobacco smoke. The high levels observed in snuff are primarily due to fermentation and aging. Technological methods exist today to reduce the levels of TSNA in both tobacco and cigarette smoke.     

烟叶中茄尼醇的提取与分离

目的 对不同提取方法提取烟叶中茄尼醇的提取率及其粗提物质量分数进行分析,并将粗提物分离纯化,以期得到一种较优的提取分离茄尼醇的方法.方法 采用超临界CO2提取法、超声提取法和溶媒提取法提取烟叶中的茄尼醇,并将得到的粗提物分别经过硅胶柱色谱和结晶进行分离纯化.结果 烟叶中茄尼醇的提取,超临界CO2提取法优于超声波和溶媒提取法,茄尼醇提取率为94.5%,粗提物质量分数为33.8%,经硅胶柱色谱和结晶得到质量分数为93.4%.结论 超临界CO2提取结合硅胶柱色谱,制备的茄尼醇纯度高、收率高.     

远志超临界CO2萃取工艺研究

目的研究远志超临界CO2萃取工艺。方法采用正交试验方法,考察影响远志萃取的因素,建立了超临界CO2萃取最佳条件。结果远志超临界CO2萃取最佳工艺为：压力30MPa,温度45℃,流速25kg·h^-1,时间1．5h。结论用超临界CO2萃取远志可以较好的获得其脂溶性成分,提取物可以供进一步深入研究。     

Modifier effects on supercritical CO2 extraction efficiency of cephalotaxine fromCephalotaxus wilsoniana leaves

The effects of modifiers such as methanol, water, diethylamine in methanol (10 v/v %), and diethylamine in water (10 v/v %) were investigated at three different concentrations (1, 5, and 10 v/v %) of the modifiers in supercritical CO2 (SC-CO2) in order to enhance the supercritical fluid extraction (SFE) efficiency of cephalotaxine from Cephalotaxus wilsoniana leaves. Among the modifiers employed, methanol basified with diethylamine was found to greatly enhance the extraction efficiency relative to any other modifiers employed. The results suggest that cephalotaxine in plant matrices may be readily changed from SC-CO2-insoluble salt to SC-CO2-soluble free base by basified modifiers. In addition, SC-CO2 modified with basified methanol could enhance the extraction efficiency of cephalotaxine more than 30% when compared to the conventional organic solvent extraction.     

The molecular basis for induction of human cancers by tobacco specific nitrosamines

Cellular DNA damage that is misrepaired or not repaired, constitutes a necessary, although not sufficient prerequisite for induction of cancer. For carcinogenic oral snuffs with extremely high concentrations of tobacco specific nitrosamines (TSNA) the DNA adduct levels predicted from animal experiments exceed those found in "unexposed" individuals. On the other hand, and supported by extensive Swedish epidemiological data, no significant increase of TSNA-induced DNA damages can be anticipated in humans from the use of low-nitrosamine oral snuffs. The extrapolated adduct concentrations are orders of magnitude lower than those found in the corresponding human tissues, a discrepancy that is difficult to account for by species differences. Furthermore, in exposed subjects the observed increment in the background levels of pyridyloxobutyl(POB)-hemoglobin adducts - a relevant indicator for TSNA activation - lie in a range predicted by rodent data. When based on the same type of tissues this provides justification for extrapolating rates of TSNA induced adduct formation from animals to humans. A TSNA exposure that does not affect the background level of pro-mutagenic DNA lesions should be considered as "virtually safe". The high background concentrations of methylated and POB-DNA adducts in "unexposed" humans must be ascribed to other sources than tobacco.     

Comparison of solid-phase extraction and supercritical fluid extraction for the analysis of morphine in whole blood.

A comparative study of the quantitative determination of morphine in whole blood using solid-phase extraction (SPE) and supercritical fluid extraction (SFE) is described. Comparative studies were made of the two techniques for the extraction of morphine from authentic forensic blood specimens. Quantitative results indicate that morphine levels measured using SPE correspond well to morphine levels produced using SFE. The two techniques are therefore comparable, although SFE is faster and cleaner and extracts may be produced with higher analyte recoveries than with SPE. This paper presents a comparison of the two techniques and the morphine concentrations determined in blood.     

超临界CO_2萃取与水蒸气蒸馏法提取太子参挥发油化学成分气质联用研究

目的:分析比较采用超临界CO2流体萃取法与水蒸气蒸馏法提取的太子参提取物中挥发性化学成分的异同。方法:使用水蒸馏提取法和超临界CO2萃取技术从太子参中提取挥发性成分,用归一化法测定其百分含量。用气相色谱-质谱(GC-MS)计算机联用技术分离鉴定其中的化学组成。结果:太子参超临界CO2流体萃取物中初步鉴定了33种成分,主要成分为:亚油酸乙酯(28.70%)、n-十六酸(23.12%)、3-糠醇(5.51%)等;水蒸气蒸馏法提取挥发油初步鉴定了17种成分,主要成分为2-丙基呋喃(22.45%)、3-糠醇(19.78%)、3-乙基-3-甲基戊烷(19.47%)。结论:2种方法提取的挥发油化学成分差异较大,超临界CO2流体萃取法提取的挥发油能更真实、全面地反映太子参药材中的化学成分。     

Review on essential oil extraction from aromatic and medicinal plants: Techniques,performance and economic analysis

Essential oils extracted from herbal plants have many foods, cosmetic and medical industries applications. Present study discussed various conventional extraction techniques (steam distillation, hydro-distillation, hydro diffusion and solvent extraction) and advanced (non-conventional) extraction techniques (solvent free microwave extraction, subcritical extraction liquid and supercritical fluid extraction). Economic analysis, kinetics modelling, and GC-MS analysis of essential oil produced by various extraction techniques have also been presented. India is the biggest producer of Indian basil oil and Japanese peppermint oil, and USA is the major importer and exporter of EOs with 14% (US$390.9 m) of world imports and 17% (US$351.7 m) of world exports. Production cost for Steam distillation, Water distillation, solvent extraction and Supercritical fluid extraction varies from 15.85 - 76.50US$/kg, 7.05-86.4US$/kg, 8.35-8.53US$/kg and 6.71-42.69US$/kg, respectively. Second order model are consistent with experimental data as determination coefficient (R²) is higher than first order model. Non-conventional extraction methods are superior to conventional extraction methods in terms of low cost, time and energy saving, less solvent requirements, shorter extraction time, etc. From the techno-economic and environmental perspective, water distillation with full energy integration is the best method for rosemary oil. In contrast, supercritical fluid extraction is the best method for oregano oil.     

Supercritical carbon dioxide extraction of podophyllotoxin from Dysosma pleiantha roots

Supercritical fluid extraction (SFE) was utilized to extract podophyllotoxin from the roots of Dysosma pleiantha. The extraction was carried out in the temperature range of 40-80 degrees C and the pressure range of 13.6-34.0 MPa with supercritical carbon dioxide. However, podophyllotoxin was not sufficiently extracted by pure supercritical carbon dioxide. Methanol was used as modifier to enhance the extraction yield of podophyllotoxin. As a result, SFE produced a higher recovery of podophyllotoxin than conventional 95% ethanol extraction.     

Influence of extraction methods on the composition of essential oils

The aim of this work is to demonstrate our results on comparison of composition of essential oil fractions obtained by traditional steam distillation and supercritical fluid extraction. The plant materials for the various extraction methods were selected from the Lamiaceae, Apiaceae and Asteraceae families. For the supercritical fluid extraction (SFE) carbon dioxide was used as supercritical solvent. The extracts were collected by stage wise precipitation in two separators. The waxy product and extract rich in essential oil were collected in the 1st and in the 2nd separator respectively. The traditional water steam distillation (SD) was carried out in the special apparatus of the Hungarian Pharmacopoea (7th ed.). GC analysis was carried out on capillary silica fused columns coated with DB-1701 and the specific chiral columns coated with Rt-beta DEX m or Rt-beta DEX sm. Comparing the composition of steam distilled oils with that of volatile SFE fractions the following general characteristics were established. The SFE fractions were richer in monoterpene-esters and poorer in alcohols than the traditional essential oils (clary sage, lavander, moldavian dragonhead). Regarding the distribution of the monoterpene and sesquiterpene compounds, the SFE fractions contained sesquiterpenes in higher percentage than the distilled oils (Salvia fruticosa). Furthermore, the proportion of sesquiterpenes increased in SFE fractions collected successively with time (Salvia officinalis) similar to the ratio of oxygenated monoterpenes to monoterpene hydrocarbons (Rosmarinus officinalis). The phtalides of lovage (Satureja hortensis) did not show regular change during the supercritical extraction. In other cases it was verified that part of the mono- and sesquiterpenes were present originally in bound form (glycosides) in plants. Thus they appeared only in essential oil fractions after previous acidic treatment (Thymus, Origanum, Satureja species). During the super-critical extraction the azulenogene sesquiterpene lactones did not transform to azulenes (chamomile, yarrow), but SFE fractions of some Asteraceae plants contained sesquiterpene-gamma-lactones of unchanged structure.     

Supercritical fluid extraction-liquid chromatography method development for a polymeric controlled-release drug formulation

We have recently been involved in the development of a method for assaying the active component in a controlled-release drug formulation, which is composed of a drug substance covalently bonded to polymer matrix. The drug substance in the formulation is the active enantiomer of misoprostol, a synthetic analog of natural prostaglandins and the active ingredient in Cytotec^®. Our method development consisted of a systematic evaluation of dynamic, off-line supercritical fluid extraction (SFE) as sample preparation for the formulation assay. Extracts were analyzed with normal phase and reversed-phase HPLC methods. The reversed-phase system utilized postcolumn reaction to provide selective detection of the extracted prostaglandin sample components. Several SFE parameters were investigated to optimize the recovery of the drug substance from the formulation, including sample quantity, extraction cell volume, extraction duration, supercritical carbon dioxide modifier, temperature, pressure, and collection solvent. The SFE experiments were completed with a commercially available multicell extractor. Preliminary validation studies utilized a formulation made with radiolabeled drug to determine the recovery achieved under the optimized SFE conditions and assessed the precision of replicate determinations. Analysis was completed under the optimized conditions to quantitate levels of the active component and related compounds in lots of the experimental polymeric formulation and to determine the total weight per cent extracted.     

超临界流体萃取法测定川芎中藁本内酯含量的研究

系统研究了压力、温度、静态萃取时间、动态萃取量、改性剂加入量等因素对超临界流体萃取(SFE)中药川芎中藁本内酯的影响,确定最佳萃取条件为:压力27.6MPa,温度40℃,静态萃取时间3min,动态萃取量7ml,改性剂加入量0.1ml。并对超临界流体萃取的收集方法作了研究,发现固液收集法在收集效率和精密度方面比溶剂收集法效果好。并用离线的SFE RPHPLC对川芎药材中藁本内酯的含量进行了测定。     

超临界CO_2-分子蒸馏对螺旋藻有效成分的萃取与分离

目的　研究螺旋藻的化学成分并建立提取方法。方法　采用超临界CO2 分子蒸馏对螺旋藻化学成分进行萃取与分离 ,并对其提取物和蒸出物进行GC MS分析。结果　从超临界CO2 萃取物和蒸出物分别得到 2 0种和 13种化学成分。结论　超临界CO2 分子蒸馏技术对螺旋藻的提取分离是一种先进合理的提取方法     

Supercritical fluid extraction method development for extraction of an experimental HIV protease inhibitor drug from animal feed

Supercritical fluid extraction (SFE) was evaluated as a sample preparation procedure for the recovery of experimental drugs from animal feed preparations that are generated during long-term toxicology studies. A commercially available supercritical fluid extractor was utilized to develop and validate an off-line procedure for the recovery of an experimental HIV protease inhibitor drug from animal feed. Extracts were analyzed with a conventional reversed-phase HPLC method. Elements of the SFE method developed that are described include optimization of the system temperature and selection of the extraction media modifier. The study emphasized the performance of two-day precision and accuracy studies. Precision and accuracy studies were carried out with SC-52151 levels of 0.05, 0.1 and 1.0% (w/w) and used an internal standard quantitation format. Also, the study utilized a relatively large analytical scale extraction vessel size of 10 ml to accomodate 6 g animal feed samples.