Chromatographic separation was performed on a Restek Rxi-5MS capillary column (30 m × 0.32 mm × 0.25 μm), using 1-naphthol as internal standard (IS). MS detection of these compounds and IS was performed at m/z 178, 208, 208, and 144. Intra- and interday precisions of all compounds of interest were less than 10%. The recoveries are situated in the range of 92.4–105.2%. Pharmacokinetics of methyl eugenol confirmed to be one-compartment open model, α-asarone and β-asarone was two-compartment open model, respectively. The method will probably be an alternative to simultaneous determination and pharmacokinetic study of volatile ingredients in Acorus tatarinowii Schott.A sensitive and specific gas chromatographic-mass spectrometry with selected ion monitoring (GC-MS/SIM) method has been developed for simultaneous identification and quantification of  Acorus tatarinowii Schott is officially listed in the Chinese Pharmacopoeia and widely used for fighting ailments such as epilepsy, digestive disorders, cerebrovascular disease, stroke, senile dementia, and diarrhea [Acorus tatarinowii Schott dose-dependently suppressed the intensity of APO-induced stereotypic behavior and locomotor activity and extended the duration of sleeping induced by pentobarbital [Chinese medicines have played an important role in clinical therapy in China, Korea, and Japan attributed to high pharmacological activity and few complications. As a classic medicinal plant, the dry rhizome of diarrhea –5. Recenbarbital , 7. Inhabarbital . α-asarone, β-asarone, and methyl eugenol proved to be main bioactive ingredients in volatile oil of Acorus tatarinowii Schott in previous report [β-Asarone has significant pharmacological effects on cardiovascular and central nervous system, while α-asarone possesses hypocholesterolemic, hypolipidemic, neuroprotective, and antiepileptic activities [Acorus tatarinowii Schott due to outstanding pharmacological activities. The development of sensitive and rapid approach for simultaneous determination of these compounds was owing to increasing interest in the literature. High performance liquid chromatography (HPLC), gas chromatography (GC), and GC with mass spectrometric detection (MS) were the main assays for analyzing α- and β-asarone in the medicinal plant and plasma in previous studies [Low polar or nonpolar compounds including s report . β-Asarotivities –16. Methtivities , 18. The studies –23. HPLC studies , 25. α-asarone, β-asarone, and methyl eugenol of Acorus tatarinowii Schott in biological samples, which produced issues of inaccurate evaluation of the medicinal plant and limitation of the pharmacokinetic study. This work was designed to develop a sensitive and valid method for determining α-asarone, β-asarone, and methyl eugenol of Acorus tatarinowii Schott in rat plasma. The work also aimed to apply the proposed assay in pharmacokinetic study of the three compounds after oral administration of volatile oil extracted from Acorus tatarinowii Schott.To our knowledge scope, few methods were found to be feasible for simultaneous determination of α-Asarone (>98%) and 1-naphthol (>99%) were purchased from Sigma-Aldrich . Methyl eugenol (>99%) and β-asarone (>95%) were purchased from J&K Scientific Ltd . All other chemicals and reagents used in the experiment were of analytical grade unless stated specially. Acorus tatarinowii Schott was purchased from Xi'an Medical Company in China and identified by Professor Minfeng Fang in Northwest University.Acorus tatarinowii Schott was obtained by steam distillation of dry material of the plant. Acorus tatarinowii Schott (100.0 g) was put into 800 mL of distilled water and distilled for 8 h. The oil was collected from the condenser, dried by anhydrous sodium sulphate. The content of the oil in Acorus tatarinowii Schott was calculated to be 1.03% (v/w), with a determination of 0.50% methyl eugenol, 74.01% β-asarone, and 11.41% α-asarone by GC-MS analysis through area normalization method. The volatile oil of μg/mL with acetone.A stock solution of 1-naphthol (1.0 mg/mL) for IS was prepared in acetone and diluted to a concentration of 2.0 α-asarone, β-asarone, and methyl eugenol was prepared by dissolving the appropriate amount of the standards in acetone. Mixed standard solution was prepared using the above solutions to give concentrations of 50.0 μg/mL for α-asarone, 50.0 μg/mL for β-asarone, and 50.0 μg/mL for methyl eugenol. All the solutions were stored at 4°C in amber glass tubes. Stock solution of μm film thickness, Shimadzu, Japan) using nitrogen as carrier gas at 2 mL/min in a constant flow rate mode. The GC oven temperature was initially increased from 60°C to 120°C at a rate of 5°C/min, then elevated at a rate of at 2°C/min up to 150°C, and then raised to 240°C at a rate of 10°C/min, giving a total run time of 36 min. The temperatures of injector, interface, and ion source were 250, 280, and 230°C, respectively. Detection was operated by selected ionmonitoring (SIM) mode .GC-MS analyses were performed with a Shimadzu GC-MS QP2010 . Chromatographic separation was achieved on a Restek Rxi-5MS capillary column . In the SIM mode, the peaks of Six healthy male Sprague-Dawley rats, weighing 250–280 g, were supplied by the Guangdong Laboratory Animals Monitoring Institute . The rats were housed in a standard animal holding room with normal access to food and water and 12 h light cycle. The animals were fasted overnight prior to dosing, with water allowance ad libitum. All animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethics Committee. Drug-free rat plasma samples were obtained from the postorbital venous plexus veins of anesthetized animals and collected into heparinized tubes. All the blood samples were centrifuged for 10 min at 8000 rpm to yield the supernatant stored at −20°C until analysis.μL of plasma was spiked with 10 μL of 2.0 μg/mL IS solution. An aliquot of 40 μL 10% (V : V) trichloracetic acid water solution and 600 μL acetonitrile was used to remove the proteins in the plasma through vortexed for 2 min followed by the centrifugation of 10 min at 8000 rpm. The supernatant was collected in a clean tube for next use, while the residue was extracted by 300 μL acetic ether for three times. The acetic ether layer and the above supernatant were collected together and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 100 μL of acetone for further GC-MS analysis.In a 1.5 mL Eppendorf tube, 100 α-asarone, β-asarone, and methyl eugenol was investigated by replicate analyses of calibration solutions with different concentration ranging from 5 to 5000 ng/mL and evaluated by linear regression. The limit of detection (LOD) was calculated by sequential diluting the analytes until the ratio of signal to noise equals 3 : 1. The limit of quantitation (LOQ) was considered as ten times of the signal to noise ratio.The linearity of μg/mL. The intraday precision and accuracy were determined within one day by analyzing the QC samples (n = 6). The interday precision and accuracy were determined on five separated days using the same QC samples. Precision was reported as the relative standard deviation (RSD). Accuracy was expressed as the relative error (RE).Quality control (QC) samples to determine the accuracy and precision of the method were independently prepared by dissolving the standard in plasma at low, medium, and high concentrations of 0.025, 0.25 and 2.5 The stability of analytes in the plasma was evaluated using the QC samples in triplicate. Test conditions included 4 freeze-thaw cycles and room temperature stability . The stabilities were assessed by analysis of the RSD of measured samples.μg/mL. In order to test the matrix effect on the ionization of the analyte, that is, the potential ion suppression or enhancement due to the matrix components, the drugs at three concentration levels were added to the extract of 0.1 mL of blank plasma, evaporated and reconstituted with 0.1 mL of acetone; the corresponding peak areas (A) were compared with those of the drugs standard solutions evaporated directly and reconstituted with the same solvent (B). The ratio (A/B × 100) % was used to evaluate the matrix effect. By the same method, the matrix effect of internal standard was also evaluated.The extraction recovery of the three drugs was determined by calculating the peak areas obtained from blank plasma samples spiked with analyte before extraction with those from blank plasma samples, to which analytes were added after extraction. According to the guidance of USFDA, recovery experiments should be performed at three concentrations . This procedure was accordingly repeated for five replicates at three concentrations of 0.025, 0.25, and 2.5 α-asarone, β-asarone, and methyl eugenol in rats. Volatile oil of Acorus tatarinowii Schott was administered orally to the rat in a single dose of 0.2 g/kg. Blood samples were collected in heparinized tube at 0, 5, 10, 20, 30, 60, 90, 120, 180, 300, 480, 720, 960, and 1200 min after dose. All the samples were extracted and analyzed under the proposed condition.The GC-MS method was successfully applied in the pharmacokinetic studies of Cmax) and the corresponding time (tmax), the half-life of absorption (t1/2), and so forth were used to decrypt the pharmacokinetic properties of α-asarone, β-asarone, and methyl eugenol. Statistical analysis of the biological data was performed by the Student's t-test. All results were expressed as arithmetic mean ± standard deviation (SD). The Microsoft Excel Program Drug and Statistics 2.0  was employed to process and calculate the pharmacokinetic parameters. The parameters of the area under curve (AUC), the maximum plasma concentration  in ratios of 15 : 1 was found to be efficient to accomplish the extracting task. The recoveries of analytes were more than 90%. This solvent was accordingly used to prepare the plasma samples in the present work. In order to quantify drugs in a plasma sample, it is often necessary to disrupt the protein-drug binding for the production of the drugs-free form. Acetonitrile and trichloracetic acid were separately evaluated for extracting α-asarone, β-asarone, and methyl eugenol in plasma samples, respectively, shown in r) was calculated to be y = 6.326x − 0.020 and r = 0.999 for α-asarone, y = 6.469x − 0.027 and r = 0.997 for β-asarone, and y = 5.874x + 0.014 and r = 0.999 for methyl eugenol, using weighted least squares linear regression (the weighing factor was 1/C). For each drug, six calibration curves containing six concentrations  were linear within the concentration range of 5–5000 ng/mL. The CV of the three drugs at each level varied from 2.0 to 12.3. And the relative bias of α-asarone, β-asarone, and methyl eugenol from the theoretical value varied from −3.0 to 4.6. As a result, the calibration curves of the three compounds exhibited good linearity within the experimental range. The LOD for α-asarone, β-asarone, and methyl eugenol was calculated to be 1.9, 1.8, and 1.5 ng/mL. The LOQ of the tested drugs was 4.7, 4.9, and 4.2 ng/mL.The linear regression analysis was constructed by plotting the peak area ratio of the three analytes to IS against the concentrations of α-asarone was 7.08%, 3.24%, and 4.85%, respectively, and that of the interday analysis was 4.80%, 4.42%, and 7.99%, with an accuracy (RE) within −4.74 to 4.04%. The precision for β-asarone of the QCs was determined as 4.84%, 6.34% plus 4.99% for intraday and 6.20%, 3.75%, and 6.01% for interday. The accuracy was found ranging from −6.03 to 2.93%. For methyl eugenol, the intraday precisions were 5.18%, 6.69%, and 4.11%, while the interday precisions were 8.10%, 7.68%, and 4.74%. The accuracy of methyl eugenol ranged between −7.62 and 5.15% for the three levels of QC samples. The precision and accuracy of the present method conform to the criteria for the analysis of biological samples according to the guidance of USFDA, where the precision (RSD) determined at each concentration level is required not to exceed 15%.The data from QC samples were calculated to estimate the intraday and interday precision and accuracy of the method. The results are presented in The stabilities were demonstrated by analysis of the RSD of measured samples. The results indicated that analytes were stable at 2, 4, 8, 12, and 24 h at room temperature and during multiple freeze-thaw cycles (≥95.6%). The three compounds were believed to have good stability in 24 h at room temperature and during three freeze-thaw cycles.α-asarone from rat plasma were 99.2 ± 7.0, 95.3 ± 3.1, and 100.7 ± 4.9% at concentration levels of 0.025, 0.25, and 2.5 μg/mL, respectively, and the mean extraction recovery of IS was 93.6 ± 3.1%. The values for recoveries of β-asarone using the proposed method were 102.9 ± 5.0, 94.0 ± 6.0, and 95.1 ± 4.8%. For methyl eugenol, the extraction recoveries were calculated to be 105.2 ± 5.4, 92.4 ± 6.2, and 96.5 ± 4.0%. In terms of matrix effect, all the ratios (A/B × 100)% defined as in The results are presented in α-asarone, β-asarone, and methyl eugenol following single oral dose of 0.2 g/kg volatile oil of Acorus tatarinowii Schott. Mean plasma drug concentration-time curve of the three compounds in single dose study was shown in α-asarone and β-asarone was in line with two-compartment open model. Nevertheless, after oral administration of the volatile oil, the Cmax of the three drugs were calculated to be 0.021, 0.53, and 2.47 μg/mL, with the tmax values of 10.00, 11.25, and 13.75 min. The AUC (0 ~ ∞) were determined as 2.49, 56.67, and 407.52 mg/L· min, while the values of AUC (0 ~ t) were 2.13, 368.43, and 47.51 mg/L· min. The ratios of AUC t) for the three drugs were all less than 120%, which indicated that the proposed blood collecting time was feasible to evaluate the pharmacokinetic behaviors of the three compounds.The present method was applied to the pharmacokinetic investigations of α-asarone, β-asarone, and methyl eugenolin rat plasma is described in this work. The method combines the excellent power of separation performed by GC to the high sensitivity of MS detection system operating in SIM mode and it has the properties of high specificity, precision, accuracy, and stability. The method has been confirmed to be successful in pharmacokinetics study of α-asarone, β-asarone and methyl eugenol in Acorus tatarinowii Schott, which probably provides an alternative to monitor the three drugs in preclinical study.A novel GC-MS/SIM method for simultaneously qualitative and quantitative analysis of


Total time spent in sedentary, light, moderate and vigorous physical activities per day was 406±63 min , 244±56 min (30%), 75±18 min (9%) and 82±30 min (10%). Average total daily distance travelled to and from school was 7.5±3.0 km (0.8–13.4 km). Mean daily energy expenditure, activity-induced energy expenditure and physical activity level was 12.2±3.4 MJ. day−1, 5.4±3.0 MJ. day−1 and 2.2±0.6. 70.6% of the variation in 2 = 54.7%) and body mass index . Energy expenditure and physical activity variables did not predict variation in The relative importance of genetic and socio-cultural influences contributing to the success of east Africans in endurance athletics remains unknown in part because the pre-training phenotype of this population remains incompletely assessed. Here cardiopulmonary fitness, physical activity levels, distance travelled to school and daily energy expenditure in 15 habitually active male (13.9±1.6 years) and 15 habitually active female (13.9±1.2) adolescents from a rural Nandi primary school are assessed. Aerobic capacity ( The success of east African athletes in middle- and long-distance running over the past 40 years raises many questions about why and how this region has produced so many successful runners in endurance events ranging from 800 m to the marathon. As is also the case in Ethiopia . kg−1. min−1; . kg−1. min−1; . kg−1. min−1; . kg−1. min−1 vs. 55.1 ml. kg−1. min−1 of more active individuals). As has been observed with Kenyan athletes, the running economy of untrained Kenyan boys (range 179–246 ml. kg−1. km−1) tends to be superior to that reported for untrained Caucasian boys of the same age In terms of physiological factors, maximal oxygen uptake  gave verbal informed consent to participate in the study, which was approved by the Institutional Research Ethics Committee, Moi University, Eldoret, Kenya. This was achieved and witnessed by the principle investigators  with the help of the children’s teachers to provide information and answer questions. Written informed consent was not collected from parents due to illiteracy. Agreement by verbal consent was documented next to each child’s name. This process was in line with the approval received from the local ethics committee.Thirty Kenyan children known by the games teacher to have good cardiopulmonary fitness levels were selected to take part in the study. None of the subjects took part in formal or taught athletic training but all were habitually active. All commuted to and from school five days a week, four times a day  over very undulating terrain. This was completed on foot, as always, with a mixture of walking and running. The number of participants was based on previous related studies as well as budget constraints given the use of the relatively expensive doubly labelled water method to objectively assess DEE and as such, subject numbers are in line with previous studies The age of each subject was reported in years. Height was measured to the nearest 1 cm and mass to the nearest 100 g, whilst barefoot and wearing the school uniform (a light shirt and either shorts or a skirt). Each child completed two maximal exercise tests as well as a week of simultaneous assessment of physical activity levels and DEE using accelerometry and doubly labelled water. Distance travelled to school  was objectively measured using a global positioning system (GPS) device . Each exercise test was separated by at least 7 days and did not occur during the period of assessment of activity levels and DEE. All testing took place at an altitude of approximately 2400 m. The mean ambient temperature during testing was 22.5±1.8°C and mean relative humidity 55.9±8.8%.. hr−1. Subjects then went immediately into a slow jog at 8 km. hr−1 for three minutes before returning back to a three-minute walk at 6 km. hr−1. After each succeeding three-minute walking-rest bout, subjects then continued with a three-minute run that was 2 km. hr−1 faster than the previous attained speed until volitional exhaustion. The running test was terminated when the subject stopped or when the subject began to consistently fall behind the rear wheel of the bike despite verbal encouragement. Tests were considered acceptable if maximum heart rate was either ≥95% of the predicted value (220 minus age in years) or if the subject showed signs of intense effort including breathlessness, sweating, facial flushing, unsteady gait or difficulty maintaining pace as is customary in paediatric exercise testing Subjects performed two identical maximal running tests on an outdoor running track. The track was constructed to traditional 400 m track dimensions with the accepted constraints and imperfections of doing so in rural Kenya. As such the track, surfaced primarily with irregular tree bark chip, contained significant undulations (≤5% gradient) especially around the bends. A discontinuous incremental protocol involving three-minute bouts of exercise followed by three-minute bouts of active recovery was used. Such protocols are generally well tolerated by children and typically produce reliable and valid 2, Cosmed, Italy). The gas analysis system was calibrated before and after each exercise test using ambient air and a gas of a known concentration , the turbine flow meter using a three litre syringe, and time-delay by an experimenter breathing synchronously into the device. The device was worn over the shoulders of each individual so that the battery and gas analyser lay on the anterior and posterior aspects of the child’s thorax, respectively. This device has been shown to yield accurate and repeatable measurements of rate of oxygen uptake  with similar previous levels of use. The device was attached to the right hip of each subject using an elasticized belt and adjusted to ensure a tight fit. Each child was instructed to wear the device at all times except when sleeping and when bathing. The recording epoch was set at 15 s and data reduction and analysis is described below. The monitoring of activity levels was considered acceptable if a minimum of 12 h of recordings took place per day for at least six days (to include at least one Saturday or Sunday). Distance travelled to school was measured on a single occasion by asking each child to wear the GPS device along their usual route to and from school during their lunch break. Data was then multiplied by two in order to reach the total distance travelled to and from school each day.2O18 and 0.12 g 2H2O per kg body mass Doubly labelled water was used for the objective assessment of DEE over the same seven days used to quantify physical activity levels. Doubly labelled water was supplied by the Energetics Research Group, part of the Institute of Biological and Environmental Sciences at the University of Aberdeen,  where subsequent analysis was also completed. The protocol involved the oral consumption of doubly labelled water with 0.15 g H−1 loaded exercise bout to allow comparison with Kenyan pupils measured by Larsen et al. Accurate running speeds were calculated by averaging GPS data across each three minute running bout for each child (data not included). Cardiopulmonary variables from the last 30 s of each exercise bout were edited by removing data values that fell outside of three standard deviations from the mean  and averaged and plotted on the same axes. The http://www.R-project.org). Briefly, R was programmed to re-integrate data collected in 15 s epochs to 60 s; edit the data to exclude periods where it was likely that the accelerometer was not being worn Accelerometer data were analysed using algorithms developed in R  as would be expected in this circumpubertal age range.The ages and physical characteristics of this population are presented in . hr−1 (6 km. hr−1), 7.6±0.6 km. hr−1 (8 km. hr−1), 9.4±0.7 km. hr−1 (10 km. hr−1), 11.0±0.6 km. hr−1 (12 km. hr−1), 12.5±0.7 km. hr−1 (14 km. hr−1), 14.4±0.6 km. hr−1 (16 km. hr−1), 16.2±0.8 km. hr−1 (18 km. hr−1) and 18.2±0.1 km. hr−1 (20 km. hr−1). Final running velocity ultimately achieved was between 14.3–18.3 km. hr−1. The boys reached higher maximal running velocities than the girls on average (16.5±1.4 vs. 15.8±0.9 km. hr−1). The . kg−1. min−1 and the girls 61.5±6.3 ml. kg−1. min−1. . min−1 (2.02±0.49 for the boys and 1.96±0.32 for the girls). This corresponded to an average of 51.5±3.7 ml. kg−1. min−1 or 70.0±6.5% of . kg−1. min−1 or 75.2±5.8% of . hr−1 was 9.4 km. hr−1 when analysed using GPS. The mean running economy of the girls and boys at this pace was 256.7±29.3 ml. kg−1. km−1 and 270.7±21.0 ml. kg−1. km−1, respectively.Mean 3 CPD over an average recording period of 14±4 hours per day. The daily mean time spent in sedentary activity was 406±63 minutes or 50% of total recording time. Total time spent in light physical activities was 244±56 min ; moderate activity, 75±18 min (9%); vigorous, 82±30 min (10%) and therefore MVPA, 156±33 min (19%). The cumulative daily distance travelled to school by each sex is presented in Accelerometer data for the group is presented in . day−1, 5.4±3.0 MJ. day−1 and 2.2±0.6 respectively (2 = 0.86) and therefore BMI was used as our best-fit model. Hence 70.6% of the variance in −1 min−1 or 7.1% of the mean DEE recorded in this cohort was also at the highest end of the physical activity continuum with mean DEE, AEE and PAL for the Kenyan adolescents of 12.2±3.4 MJectively . In regrhe mean  .. EnergyBoys achieved significantly higher . kg−1. min−1, respectively. Comparing . kg−1. min−1; a difference of 32% . kg−1. min−1 and the town boys averaged 50.2 ml. kg−1. min−1. kg−1. min−1; . kg−1. min−1; . kg−1. min−1 for boys and 36.9 and 53.6 ml. kg−1. min−1 for girls . kg−1. min−1) and using allometric scaling, bodymasŝ0.75 (ml. kg0.75. min−1) to allow comparison with the relevant literature. Whilst no relationship between activity levels, energy expenditure and In this study, 15 Kenyan boys and 15 Kenyan girls completed two maximal exercise tests on an undulating outdoor running track. On both occasions, aerobic variables including is study . The imp. kg−1. min−1 or 70.0±6.5% of their . kg−1. min−1 or 75.2±5.8% of their The anaerobic threshold of the subjects in this study occurred at a high percentage of their . kg−1. km−1, respectively. The boys in Larsen et al. . kg−1. km−1 and were therefore more efficient runners than this sample. This may be accounted for by two study limitations. The first being the undulating nature of the outdoor running track used in the present study and the second, the varying nature of running speed as reflected in the recorded GPS. Such variations in gradient and running velocity will have resulted in failure to maintain exact work rates and may partially account for the lower running economy observed in our tests. Our subjects also had a lower mean age (13.9 vs. 16.6 years) and a wider range of ages (10–16 years vs. 15.2–18.4 years) compared to those studied by Larsen et al. The mean running economy recorded for boys and girls in the current study was 270.7±21.0 and 256.7±29.3 mlThe differences observed between the Kenyan village adolescents and their urban and European peers in previous studies has often been attributed to greater physical activity level observed in the rural population, especially regular walking and running to and from school . day−1) and AEE accounted for 44% of the DEE. Other qualifiers of physical activity intensity and distance travelled to school were not related to variation in DEE, AEE or PAL. Higher PALs, DEE and AEE were found compared to Western populations, which opposes the concept of a ceiling of energy expenditure The Kenyan boys and girls spent 40% of their recorded time engaged in light and moderate activity and only 10% of their recorded time engaged in vigorous intensity activity. Although this breakdown is largely consistent with previous findings in other populations The evidence relating habitual physical activity to 2 = 0.18; p<0.05) between Trowbridge et al. Cardiopulmonary fitness is a complex phenotype to study. Undoubtedly inter-individual variation in 2, Cosmed, Italy) used is sometimes unreliable, particularly when used outdoors and requires frequent and careful calibration. Whilst calibration was conducted frequently by accustomed users of the device and whilst the device had been validated by the team, indoor and outdoor, in the U.K. A limitation of the current study is that growth, maturity status and lean body mass were not assessed. It was evident, subjectively, that the adolescents participating in this study were both pre-pubescent and at varying stages of puberty as one would expect in a mixed sample of girls and boys between the ages of 10 and 17. Thus in this study we can only compare the chronological ages of each child, which were only deduced by self-report. Another caveat when interpreting the data was the impossibility of quantifying and maintaining exact work rates throughout the discontinuous, incremental protocol that we used. This was due to method of pacing and an undulating track surface. Despite this, incremental rises in The generalisation of such data in this population is of course unknown without further study. Given previous published studies We describe a population of Kenyan adolescents of both sexes who have demonstrated an enormous capacity for physical activity. Physiological markers of cardiopulmonary fitness, activity levels, distance travelled daily to school and DEE were quantified.


Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) at different stages of organogenesis, cultured in vitro on a few variants of Murashige and Skoog (MS) medium, containing different concentrations of plant growth regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (from 0.1 to 3.0 mg/l) and in extracts from overground parts of plants growing in vivo. Six of 12 analysed compounds were detected in all extracts: chlorogenic, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. Total contents of the examined metabolites in biomass of shoot-differentiating callus culture cultivated on six MS medium variants were dependent on concentrations of growth regulators in the media and ranged from 14.90 to 60.05 mg/100 g d.w. Total contents of the compounds in biomass extracts from undifferentiating callus culture maintained only on two of six MS medium variants were higher and amounted to 74.54 and 78.24 mg/100 g d.w. Maximum total contents of phenolic acids in both types of in vitro cultures were greater than in fruits (55.73 mg/100 g d.w.) and leaves (4.55 mg/100 g d.w.) of plants gowning in vivo. Chlorogenic acid and salicylic acid were the main compounds identified in biomass extracts of shoot-differentiating callus cultures , while chlorogenic acid (max 38.43 mg/100 g d.w.) and protocatechuic acid (max 20.95 mg/100 g d.w.) prevailed in the extracts from undifferentiating callus cultures. Other compounds dominated in fruits, namely p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.). This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.The contents of free phenolic acids and cinnamic acid were determined using an HPLC method in methanolic extracts from biomass of  Schisandra chinensis (Turcz.) Baill (Chinese magnolia vine) is an East Asian species which currently is very popular in Europe as an adaptogenic plant [Schisandrae chinensis fructus in the newest edition of European Pharmacopoeia 7th and in the National Pharmacopoeias of European countries e.g. in Polish Pharmacopoeia 9th [ic plant . We can oeia 9th , 3. Thisoeia 9th . It was oeia 9th –7. This oeia 9th , 6, 8.Schisandra genus [Other groups of active substances of the fruits were demonstrated to belong to organic acids , sugars, phytosterols (e.g. stigmasterol), tannins, volatile oil compounds , vitamins (e.g. C and E), and elements Cu, Mg, Ni and Zn , 5, 6. Tra genus .S. chinensis maintained in our laboratory have a potential to accumulate some lignans. Our studies analysing the contents of two main compounds of this group—schisandrol A and schisandrol B—revealed that biomass from shoot-differentiating callus culture could be a potential biotechnological source of these metabolites [In vitro cultures of abolites . MaximumS. chinensis carried out in other scientific centres focused on possibility to use enzymatic potential of the cells cultured in vitro for biotransformation of hydroquinone into phenolic glycosides. Few papers dealt also with micropropagation of this plant species, mainly by somatic embryogenesis. One report addressed the problem of validation of lignans assays in material from in vitro cultures [Lignans present in the plant species under study possess a unique structure, and their biogenesis has not been elucidated in detail, yet, though its initial steps are known to belong to the shikimic acid pathway, involved also in biogenesis of phenolic acids. These facts prompted us to undertake analysis of this group of compounds both in biomass cultured in vitro and in plants growing in vivo. The exceptional value of biological activity of this group of metabolites strengthened our decision of their investigation. Phenolic acids, either cinnamic or benzoic acid derivatives and depsides, like chlorogenic, and rosmarinic acids possess, e.g. anti-inflammatory, cholagogic, spasmolytic, hypolipemic, anti-aggregatory and immunostimulating actions –14. Manycultures –23.S. chinensis as a potential biotechnological source of the biologically active phenolic acids.The present study aimed to investigate the accumulation of free phenolic acids and cinnamic acid in biomass from in vitro cultures at different stages of differentiation cultivated on several variants of Murashige and Skoog (MS) medium  containiS. chinensis have not been studied so far in terms of the biochemical potential for production of free phenolic acids. Until now the native plant has not been studied for the contents of these metabolites, too. We found only interesting information, about the high amounts of cinnamic acid—precursor of one group of phenolic acids, in leaves of these plant species [Twelve compounds were determined by an HPLC method in methanolic extracts from biomass cultured in vitro and in the extracts from overground parts of plants growing under natural conditions . In vitro cultures of  species .S. chinensis (Turcz.) Baill. (Schisandraceae) from the Rogów Arboretum—Warsaw University of Life Sciences, Forest Experimental Station in Rogów (Poland); for details, see Szopa and Ekiert [The in vitro cultures were established from leaf buds of d Ekiert . After 42, LF-40W lamp, daylight, Piła) at 25 ± 2 °C for 4 weeks (three series).The shoot-differentiating callus culture was maintained on six solid MS  medium vPlant material harvested in Poland in 2010, analysed for comparison, comprised fruits and leaves of the plants growing under natural conditions .p-coumaric, ferulic, p-hydroxybenzoic, and vanillic acids (Fluka).The dried biomass from both types of in vitro cultures collected after 4-week growth cycles (three series) and plant material—fruits and leaves (from 0.2 to 0.5 g)—were extracted twice with boiling methanol for 3 h. In the methanolic extracts, chromatographic quantification of 11 phenolic acids and cinnamic acid was performed with the HPLC method according to Tian et al. . SeparatDry biomass of shoot-differentiating callus culture increased 2.1–6.1-fold during 4-week growth cycles on the tested six variants of MS medium. High over fivefold biomass increments were observed on the medium variants containing cytokinin (BAP) and auxin (NAA) at concentrations of 2 and 2, 2 and 1, and 3 and 1 mg/l of the medium.p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. None of the extracts under analysis contained caffeic, cinnamic, ferulic, rosmarinic, sinapic and vanillic acids  and p-hydroxybenzoic acid (about 4–4.5 mg/100 g d.w.) were smaller. Salicylic acid was accumulated at the lowest amounts  were also favourable for biomass growth (five- to sixfold increase in dry biomass). These variants of MS medium can be proposed as universal, both “productive” and “growth-promoting” media. Undifferentiating callus cultures were tested only on two MS medium variants containing 3 mg/l BAP and 1 mg/l NAA, and 2 mg/l BAP and 2 mg/l NAA. Total contents of phenolic acids obtained on these media were 1.24- and 1.63-fold greater, respectively, than in the extracts from shoot-differentiating callus cultures  than in undifferentiating callus culture (below 3 mg/100 g d.w.) [S. chinensis biomass cultured in vitro [Two media proposed now as beneficial for the production of phenolic acids in  g d.w.) . As indiin vitro .Arnica montana, Eleutherococcus senticosus) [Chlorogenic acid was accumulated in shoot-differentiating callus culture at marked amounts, and in undifferentiating callus culture, its contents were maximal Table . This coticosus) . Therefoticosus) –18.p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.) as dominating metabolites. Chlorogenic acid content was very low (below 2 mg/100 g d.w.). Leaves turned out to be a very poor source of phenolic acids . Two of these compounds dominated in biomass extracts from in vitro cultures, too. Maximum total contents of phenolic acids in biomass from in vitro cultures were 1.4 and 17.2 times higher than in overground parts of plants—fruits and leaves, respectively (Table S. chinensis in vitro cultures can be proposed as a rich potential biotechnological source of biologically active phenolic acids and a good model for further studies on optimization of production of these metabolites.Extracts from overground parts of plants growing in vivo contained the same compounds under study as biomass from in vitro cultures Table . ExtractS. chinensis in vitro cultures to produce the phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plant growing in vivo, too.The results presented for the first time the biochemical potential of cells from


CAT) is a risk factor for primary angle closure glaucoma (PACG) in the Saudi population.To Investigate whether the g.4760C>T polymorphism in the promoter region of the catalase gene  utilizing Taq-Man® assay. The association between different genotypes and various clinical indices important for PACG was also investigated.p= 0.568). Interestingly, there was a trend of association between the type of the variant  and one important parameter for PACG, which is visual acuity. The visual acuity increase was; 0.62 (±0.74), 0.88 (±0.88) and 1.27 (±0.95) in patients carrying the “C/C”, “C/T” and “T/T” genotypes respectively, which was statistically significant in both ANOVA and pairwise individual T tests  when compared to controls.The distribution of different genotypes was comparable between both study groups. The genotype “C/C” was predominant among cafses; 94 (68.1%) and controls; 289 (71.7%). Heterozygous genotype “C/T”, was present in 41 (29.7%) of cases and 103 (25.6%) of controls, where the homozygous variant genotype was present in only 3 (2.2%) of cases and 11 (2.7%) of the controls. The distribution of variant allele was similar in both study groups (This variant is possibly associated with visual acuity in PACG patients and thus had the potential to be used as a parameter for assessing PACG severity. PLEKHA7 (P=5.33×10(−12)), rs3753841 in COL11A1 (P=9.22×10(−10)) and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (P=3.29×10(−9)) were reported , while that for controls was 61.1 (±10.8) years, median 60 and range [40–93]. Comparing both age means, there were no statistically significant difference between cases and controls  and controls 125 (15.5%) compared to the wild type “C” allele [229 (83.0%)] and [681 (84.5%)] in cases and controls respectively, however, the alleles distribution had no statistically significant association with encountering the disease;   and controls; 289 (71.7%). The heterozygous variant genotype “C/T” was present in 41 (29.7%) of the cases and 103 (25.6%) of the controls, whereas the homozygous variant genotype “T/T” was only present in 3 (2.2%) and 11 (2.7%) in cases and controls respectively. There was no association between having the heterozygous genotype “C/T” and getting closed angle glaucoma; OR: 1.22 [95% CI: 0.773 - 1.197; % and conp = 0.336). A similar trend was observed in mean (±SD) duration between getting closed angle glaucoma and presentation to the hospital, intraocular pressure (IOP) and logMAR visual acuity. The visual acuity increase was; 0.62 (±0.74), 0.88 (±0.88) and 1.27 (±0.95) in “C/C”, “C/T” and “T/T” groups respectively, which was statistically significant in both ANOVA and pairwise individual T tests . More findings on other clinical indices at presentation which were not statistically significant are presented in Table Examining the likelihood of an association between genotypes and important glaucoma clinical indices, showed that, the mean (±SD) age at onset increased with change in genotype (homozygous variant is worse than heterozygous and heterozygous is worse than wildtype genotype); 49.0 ±22.7), 57.9 (±12.2) and 62.4 (±8.9) for “C/C”, “C/T” and “T/T” respectively. This increase was however, statistically insignificant in ANOVA . This indicates that neither the variant genotype nor the variant allele is a risk factor for PACG in this population. It’s possible that the link between mitochondrial abnormalities and/or oxidative stress and glaucoma are not linked through the catalase pathway and that other oxidative stress pathway(s) may be involved. In any case, the exact mechanism of how oxidative stress contributes to glaucoma pathogenesis remains speculative. Additionally, glaucomatous optic neuropathy implies loss of RGCs, including their axons, and a major tissue remodeling, especially in the optic nerve head. Mechanisms leading to glaucomatous optic neuropathy are not yet clearly understood. We previously investigated mitochondrial abnormalities in a group of patients with PACG and found absence of mitochondrial abnormalities in those patients [The results presented in Table patients . We suggpatients , in contpatients .p = 0. 022, 0.031 and 0.039 respectively). Other clinical indices listed in Table Interestingly, when we investigated the potential association between the presence of a certain genotype and different clinical indices, important for PACG, we found an interesting trend corresponding to various clinical indices and severity of the variant . The mean (±SD) age at onset increased with increase in variation severity (homozygous variant is worst than heterozygous and heterozygous is worst than wildtype), but statistically insignificant. On the other hand, the visual acuity increase was; 0.62 (±0.74), 0.88 (±0.88) and 1.27 (±0.95) in “C/C”, “C/T” and “T/T” groups respectively, which was statistically significant in both ANOVA and individual T tests (There are few limitations to this study: i) the study was conducted on the Saudi population only and thus need to be carried out in other populations to establish the link between this variant and various clinical indices; 2) at this point, modifiers for this catalase SNP leading to glaucoma are not known and thus we will not know for sure if this SNP alone can influence the disease severity; 3) We cannot exclude other SNPs at the same locus, which may be associated with POAG and 4) Considering the fact that the frequency of the homozygous (T/T) variants was low  it is also plausible that more individuals are needed for such case–control genetic association study.In summary, we showed a possible association of the catalase polymorphism (g.4760 C>T) with the visual acuity, which is an important clinical indices for PACG. Thus, this SNP can be potentially used as a marker for PACG severity.CAT: Catalase; c/d: Cup-to-disk ratio; IOP: Intraocular pressure; KAUH: King Abdulaziz University Hospital; LogMAR: Logarithm of the minimum angle of resolution; PACG: Primary angle closure glaucoma; POAG: Primary open angle glaucoma; PCR: Polymerase chair reaction; ROS: Reactive oxygen species; RGCs: Retinal ganglion Cells; SNP: Single nucleotide polymorphism; SOD: Superoxide dismutase; TM: Trabecular meshwork.The authors declare that they have no competing interests.KKA supervised the overall study, writing the manuscripts and analysis of the data; TAZ and TS carried out the technical work in the laboratory; AM statistical analysis of the data and writing the manuscripts; EAO and SAO patient's requirements and reviewing the files for clinical information. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/84/prepub


Octocorals are the second most important faunistic component in many reefs, often occupying 50% or more of the available substrate. Three species of octocorals from two families were studied in Eilat (Gulf of Aqaba), comprising the zooxanthellate Ovabunda macrospiculata and Heteroxenia fuscescens (family Xeniidae), and Sarcophyton sp. (family Alcyoniidae). They were maintained under normal (8.2) and reduced (7.6 and 7.3) pH conditions for up to 5 months. Their biolological features, including protein concentration, polyp weight, density of zooxanthellae, and their chlorophyll concentration per cell, as well as polyp pulsation rate, were examined under conditions more acidic than normal, in order to test the hypothesis that rising pCO2 would affect octocorals. The results indicate no statistically significant difference between the octocorals exposed to reduced pH values compared to the control. It is therefore suggested that the octocorals' tissue may act as a protective barrier against adverse pH conditions, thus maintaining them unharmed at high levels of pCO2.Increasing anthropogenic  The continuing rise in carbon dioxide emissions has already led to its increased concentration in the earth's atmosphere, from a level of 280 ppm in the pre-industrial era, to 385 ppm at present, which is the highest recorded in the last 650,000 years . We tested the hypothesis that their biological features would be affected by the declining pH, and studied its effects on: (1) zooxanthellae density of O. macrospiculata (colonies), H. fuscescens (colonies and primary polyps), and Sarcophyton sp.; (2) chlorophyll concentration; (3) ratio of sclerite weight to tissue weight in O. macrospiculata; and (4) pulsation rate of O. macrospiculata polyps. As octocorals constitute an ecologically conspicuous benthic component on coral reefs , and a control pH 8.2 (pCO2 = 387 μatm), corresponding to the ambient Eilat seawater  (29°30′N 34°55′E), and the octocoral colonies were collected by SCUBA 2009–2010) from the reef adjacent to the IUI at a depth of 8-12 m. Following 2 weeks acclimatization in a flow-through seawater table, the colonies were transferred to the experimental system (see ahead). To obtain –2010 froO. macrospiculata (n = 100 colonies), H. fuscescens (n= 15 adult and 70 primary polyps), and Sarcophyton sp. (twenty-one 3 × 3 cm fragments), were equally divided among the two pH treatments (7.3 and 7.6) and the control (8.2). The colonies and primary polyps were placed in transparent 6 L PVC containers, supplied with an air stone , and positioned under 400 W metal halide lamps, supplying ∼200 μmol quanta m−1s−1, 10 h light: 14 h dark regime.The selected species, 2 gas stored in a cylinder through seawater to reach the desired pH. A pH electrode  was located in each tank and connected to a pH controller  in order to control the gas flow. A pH deviation within the tank triggered the computer to activate the solenoid in order to either increase or decrease the flow of CO2, as necessary. The pH data were recorded using monitoring software . The seawater temperature was maintained at ∼25°C, using a combination of an array of 150W BluClima aquarium heaters  and an air-conditioner in the laboratory. Water flow in the tanks was maintained by power heads .For the experimental system, seawater was pumped from a depth of 30 m at the IUI reef into 1000 L tanks, where the pH was regulated. The pH values  were achieved by bubbling pure COO. macrospiculata , one for H. fuscescens , and one for Sarcophyton sp. . After polyps were removed from the colonies of O. macrospiculata in order to determine the biological features, the colonies were returned to the IUI reef, whereas those of H. fuscescens were repeatedly sampled. Primary polyps of H. fuscescens and Sarcophyton sp. fragments were sacrificed for the measurements. The octocorals were deliberately deprived of food.Three experiments were conducted for Sarcophyton sp. At different time points (see Results), samples comprised of six randomly selected polyps from each colony of O. macrospiculata, five from H. fuscescens (n = 2–8 colonies), and a fragment from Sarcophyton sp. (n = 5–7 fragments), were placed in filtered seawater . Each sample was separately homogenized , and its total volume was determined to a precision of 0.05 mL and centrifuged  for 5 min at 5000 rpm and 4°C, in order to separate the algal cells from the tissue, where applicable. A sample of 100 μL was removed from the supernatant for protein determination of the tissue using Quick Start Bradford Protein Assay Kit . Optical density was read at 595 nm using an ELISA reader , and the concentration was calculated according to the Quick Start Bovine Serum Albumin Standard Set  attached to a microscope . The dinoflagellates were manually counted in the photos, using ImageJ © program (Cell Counter application), and multiplied by 10,000 to obtain their total amount in a field size of 0.1 × 0.1 cm surface × 0.01 cm depth. In order to obtain the chlorophyll concentration of the zooxanthellae, the remaining content of each sample was centrifuged again, 1 mL of cold acetone 90% (4°C) was added to the pellet, and it was incubated at 4°C for 18 h in the dark. Chlorophyll a concentration was determined using spectrophotometry , following Jeffrey and Humphrey . Finally, clean dinoflagellate-cells were obtained and a sample of 50 Humphrey .O. macrospiculata (tissue and sclerites) was dried overnight and then weighed using analytic balance . Their sclerites were obtained by dissolving the tissue with 10% sodium hypochlorite, followed by repeated rinsing in double-distilled water (DDW), and then a wash with 95% alcohol . Six polyps were randomly chosen from each video per colony and the number of pulses of the polyps per 1 min was counted and averaged (±SD). In the reef, the pulsation of colonies was determined by underwater video recordings, using a Canon PowerShot G9 camera, Japan . Each colony was photographed three times for 1 min (n = 5 colonies), and the number of pulses of six randomly chosen polyps per 1 min was calculated.The possible effect of decreased Analysis of variance (ANOVA) was performed on the data using SPSS 15.0  and STATISTICA 8 . Log transformation was conducted on part of the data in order to achieve normal distribution (see Results). Results are expressed as mean ± standard deviation (SD).O. macrospiculata and H. fuscescens, due to a technical fault in the experimental system.Throughout the experiments, little mortality was noted among the octocoral colonies and primary polyps, and they maintained their normal appearance with no visible signs of stress. Data for pH 7.3 are not available for the second experiment, February–May 2010, on −1) in O. macrospiculata was not significantly affected over time by pCO2 in the two experiments, with no such differences between treatments and control on each of the sampling dates of both experiments . The results of the first experiment (April–May 2009) ranged between 4.176± 1.416  and 1.647± 4.776  cells per mg protein; and of the second one (February–May 2010), between 2.386± 1.236  and 6.196± 3.306  cells mg−1. In colonies of H. fuscescens (February–May 2010), the average number of zooxanthella-cells per tissue protein was not significantly affected by pCO2 (P = 0.06). The colonies featured 1.796± 6.195  to 2.576± 4.835  algal cells per mg protein. Similarly, the primary polyps of H. fuscescens in October–November 2009 were not affected over time by pCO2 (P = 0.11) and the results ranged between 1.016± 4.755  and 2.996± 1.246  cells mg−1. The average number of zooxanthella-cells per tissue protein of Sarcophyton sp.  was also not significantly affected by pCO2 (P = 0.59) and the results ranged between 1.776± 2.515 (pH 8.2) and 2.106± 1.106 (pH 7.3) cells mg−1.The average number of zooxanthella-cells per tissue protein (cells mgμg cell−1) in O. macrospiculata was not significantly affected over time by pCO2 in either the April–May 2009 or February–May 2010 experiments, with no such differences between treatments and control on each of the sampling dates of both experiments . The results of the first experiment (April–May 2009) ranged between 2.05−6 ± 6.89−7  and 3.64−6 ± 2.44−6  μg chlorophyll per algal cell; and that of the second (February–May 2010) between 1.24−5 ± 7.34−6  and 5.46−6 ± 1.61−6  μg cell−1. Similar results were obtained for colonies of H. fuscescens (February–May 2010) (P = 0.15). The primary polyps of a H. fuscescens (October–November 2009) were also significantly unaffected by pCO2 (P = 0.17), for which the results ranged between 1.53−6 ± 2.49−7  and 2.37−6 ± 8.91−7μg cell−1 . The average chlorophyll content per zooxanthella-cell (μg cell−1) of Sarcophyton sp.  was also not significantly affected by pCO2 (P = 0.18), and the results ranged between 1.16−5 ± 3.50−6 (pH 8.2) and 1.32−5 ± 1.16−6 (pH 7.3) μg cell−1.The chlorophyll content per zooxanthella-cell . The results of the first experiment (April–May 2009) ranged between 0.018 ± 0.026  and 0.557 ± 0.378 ; and of the second one (February–May 2010) between 0.092 ± 0.043  and 0.367 ± 0.211 .Average ratio between sclerite weight and polyp weight (tissue and sclerites) of O. macrospiculata did not indicate significant differences between pH treatments and control in both the 2009 and 2010 experiments .Similarly, the average weight of polyps per colony of O. macrospiculata was not significantly affected over time by pCO2, with no such differences between treatments and control on each of the measurement dates (P = 0.32). Similarly, there was no significant difference between the pulsation rate of colonies in the reef and in the experimental pH system . The results ranged between 21.39 ± 18.06  and up to 36.83 ± 3.73 pulses min−1 , with a pulsation rate of reef colonies averaging 38.20 ± 3.48 pulses min−1.The pulsation rate of pCO2. The results reveal that the octocorals remained statistically unaffected under such conditions over a period of up to 5 months. The number of zooxanthellae per tissue protein in colonies of O. macrospiculata  following exposure to lower pH. However, other studies found contradicting patterns of change, such as Krief et al. . Similarly, Anthony et al. (A. intermedia (Heron Island), whereas Reynaud et al. (S. pistillata (at ∼25°C). Overall, this suggests species-specific responses, but also that different experimental conditions, such as light intensity and temperature, may yield different patterns of biological responses to increased pCO2 , which they explained as compensation for the recorded decrease in algal cells. Crawley et al. (A. formosa (Orpheus Island) with no change in zooxanthellae density, even at pH 7.55, probably due to the short exposure time (4 days) and low light (110 μmol m−1 s−1). Photo-acclimation is a dynamic and immediate process reflected in rapid changes in chlorophyll within 2–4 days, whereas changes in zooxanthellae density may occur over a longer period of up to 40 days  coordination, and even the tentacular pinnules degenerated. In this study, variation in pulsation between colonies was demonstrated, but no such difference was found between pH treatments and control. Moreover, no difference was found between the system and the reef, which might indicate that the system itself does not exert additional pressure on the corals in term of pulsation. O. macrospiculata is a passive suspension feeder  Most experimental studies on the effect of ocean acidification on marine organisms have only lasted from a few hours to several days, whereas the present study was run for 5 months. This further strengthens our suggestion that octocorals might be able to acclimate and withstand rising levels of ocean acidification, even under conditions that are far beyond what is expected to occur by the end of the present century . The variable responses among taxa as a reaction to elevated pCO2 reflect differences in their ability to regulate their internal pH, and the extent to which their tissues can avoid changes in their response, including their mineral composition (Ries et al. pCO2 on octocorals.Numerous experimental studies have been performed on the possible effects of ocean acidification on marine biota, including the calcification response of stony corals and coccolithophores (e.g., Kleypas et al.


The calculation of two-phase flow boiling heat transfer of R22 in channels is required in a variety of applications, such as chemical process cooling systems, refrigeration, and air conditioning. A number of correlations for flow boiling heat transfer in channels have been proposed. This work evaluates the existing correlations for flow boiling heat transfer coefficient with 1669 experimental data points of flow boiling heat transfer of R22 collected from 18 published papers. The top two correlations for R22 are those of Liu and Winterton (1991) and Fang (2013), with the mean absolute deviation of 32.7% and 32.8%, respectively. More studies should be carried out to develop better ones. Effects of channel dimension and vapor quality on heat transfer are analyzed, and the results provide valuable information for further research in the correlation of two-phase flow boiling heat transfer of R22 in channels. R22 is still widely used in chemical engineering and refrigeration and air-conditioning industries. Calculation of flow boiling heat transfer coefficients of R22 is important for design, development, and assessment of the systems and equipment using R22. A number of correlations for two-phase flow boiling heat transfer were proposed, and their applicability to R22 remains an issue. On the other hand, the study of R22 flow boiling heat transfer can provide useful information for revealing the influence of refrigerants on flow boiling heat transfer coefficients and for better understanding mechanisms of flow boiling heat transfer.2s to 286 kg/m2s, and heat fluxes from 10.6 kW/m2 to 17.0 kW/m2. They compared their measurements with the correlations of Shah [Greco and Vanoli  investig of Shah , Gungor  of Shah , Chen [52s, evaporating pressures ranging from 3 to 12 bar, and heat fluxes from 11 to 21 kW/m2. The experimental data are discussed in terms of the heat transfer coefficients as a function of the vapor quality. The experimental results indicated that, with increasing pressure, the nucleate-boiling contribution to the heat transfer coefficient increases mainly because of the corresponding decrease of the wall superheat required to form a stable nucleus.Greco and Vanoli  measured2s, heat fluxes from 2.5 to 20 kW/m2, and vapor qualities form 0.1 to 0.89. They found that the heat transfer characteristics were dominated by nucleate boiling and that heat transfer coefficients of R407C were considerably lower than those of R22.Wang and Chiang  reported2, mass fluxes from 424 to 742 kg/m2s, saturation temperatures at 12°C, and qualities from 0.06 to 0.84. The comparison of the measurements of R22 with the correlations of Gungor and Winterton [Shin et al.  examinedinterton  showed tinterton  correlat2s, heat fluxes of 10, 20, and 30 kW/m2, temperatures of 0 and 5°C, and qualities from 0.05 to 0.97. The measurements were analyzed and compared using seven previous heat transfer coefficient correlations proposed by Shah [Oh and Son  investig by Shah , Gungor  by Shah , Liu and by Shah , Oh et a by Shah , Wattele by Shah , and Yan by Shah . The res by Shah  gave the by Shah  and Watt by Shah . 2s, heat fluxes from 10.4 to 27.9 kW/m2s, saturation temperatures from −7 to 15.8°C, and vapor qualities from 0.04 to 0.9. They compared their measurements of pure refrigerants with the correlations of Jung et al. [Choi et al.  conducteg et al. , Gungor g et al. , and theg et al.  correlatg et al.  correlat2 in horizontal stainless steel tubes with IDs  of  1.5 mm and 3.0 mm, obtaining the local heat transfer coefficients with heat fluxes from 10 to 40 kW/m2, mass fluxes from 200 to 600 kg/m2s, a saturation temperature of 10°C, and quality up to 1.0. They believed that nucleate boiling heat transfer was the main contribution, particularly at the low quality region. They compared the measurements with the correlations of Wattelet et al. [Choi et al.  examinedt et al. , Jung ett et al. , Kandlikt et al. , Tran ett et al. , Shah [2t et al. , and Che2s, heat fluxes from 1.89 to 40 kW/m2, and saturation temperatures from −5.2 to 15.3°C. They selected three heat transfer correlations of Shah [Wattelet et al.  conducte of Shah  to compa2s, and heat fluxes from 26.5 to 2.5 kW/m2. The comparison of the new database with the correlations of Gungor and Winterton [Col  collecteinterton , Liu andinterton , Kandlikinterton , and Wojinterton  showed t3H8, and CO2 in horizontal 0.5 mm, 1.5 mm, and 3.0 mm ID stainless steel tubes. The experimental data ranges were heat fluxes from 5 to 40 kW/m2, mass fluxes from 50 to 600 kg/m2s, saturation temperatures from 0 to −15°C, and qualities up to 1.0. The measurements were compared with correlations of Gungor and Winterton [Oh et al.  performeinterton , Jung etinterton , Shah [2interton , Chen [5interton , Kandlikinterton . It was 2, mass fluxes from 50 to 500 kg/m2s, and vapor qualities up to 1.0. They investigated effects of these physical parameters on the flow boiling heat transfer coefficients and evaluated two correlations of Jung and Radermacher [Jabardo and Filho  performeermacher  and Kandermacher  with theThe previous brief review shows that most examinations of the correlations of flow boiling heat transfer coefficients of R22 were only based on the authors' own experimental data, and thus big differences existed among the evaluation results. In this paper, 1669 data points of flow boiling heat transfer of R22 are collected from 18 papers, and 26 existing flow boiling heat transfer correlations are evaluated based on the database. The evaluation results provide valuable information for developing new heat transfer prediction methods.2s, heat fluxes from 1.9 to 57.5 kW/m2, vapor qualities from 0.006 to 0.982, saturation temperatures from −15.65 to 35°C, saturation pressures from 1.01 to 13.55 bar, and tube IDs from 1.5 to 13.84 mm. All the experiments were conducted with horizontal single circular smooth tubes. Dh < 3 mm) and other 1512 data points in conventional channel region (Dh ≥ 3 mm) according to the Kandlikar-Grande method [4 with most falling within 100–3  ×  104. 5 with most falling within 2000–5  ×  105.From 18 published papers from 12 independent laboratories, 1669 data points of two-phase flow boiling heat transfer of R22 are obtained . The expe method .  FigureWith the database of the 1669 data points, 26 correlations of flow boiling heat transfer coefficients are evaluated, including those of Liu and Winterton , Fang 2, Shah 2, Gungor The top 5 correlations which have an MAD ≤35% against the database  are desc(1)  Liu-Winterton  Correlat. Liu and Winterton [hlsp, is the convective boiling heat transfer coefficient calculated by the Dittus and Boelter [hnb is the nucleate boiling heat transfer coefficient calculated by the following Cooper [ε is the surface roughness (μm) and is set a value of 1 μm if unknown, and the factors S and F are calculated by  interton  proposed Boelter  correlatg Cooper  correlat(2)  Fang  Correlat. Fang [2 database of 2956 experimental data points with a new dimensionless number Fa.at. Fang  proposedConsider(3)  Wattelet et al.  Correlat Wattelet et al. [hlsp, is calculated with   Gungor-Winterton  Correlat. Gungor and Winterton [hlsp, is calculated with htp=SS+FF2hsp,l(5)  Bertsch et al.  Correlat Bertsch et al. developed a composite correlation from a database of 12 different fluids based on the Chen [  Bertschnb is calculated with (k denotes either liquid or vapor.Considerted with (12)hsp=With the database of the 1669 data points from 18 papers, 26 existing correlations of flow boiling heat transfer coefficients are assessed. The prediction deviations of the 26 correlations are listed in ConsiderDh ≥ 3 mm as conventional channels, 200 μm ≤Dh < 3 mm as minichannels, and 10 μm ≤ Dh < 200 μm as microchannels. Kew and Cornwell [Rel0.5 = 200 as the conventional-to-micro/minichannel criterion. Harirchian and Garimella [0.5 · Relo to classify channel dimensions.Channel dimensions have effects on flow boiling heat transfer. A number of channel transition criteria were proposed. Kandlikar-Grande  proposedCornwell , Cheng eCornwell , Ong andCornwell , and FanCornwell  classifiCornwell  proposedarimella  used theDh, Bd, Bd · Rel0.5, and Bd0.5 · Relo are shown in Figures Dh, which shows that it seems there is a transition taking place around Dh = 3 mm. Rel0.5 = 200, which coincides with the Li and Wu [0.5 · Relo. There are no data with Bd0.5 · Relo < 160, and thus the Harirchian and Garimella [Dh < 3 mm, Bd < 4 and when Dh ≥ 3 mm, Bd ≥ 4. Thus, the number of data in minichannel region with the Kandlikar-Grande method [The variations of the Nu number with i and Wu  result. arimella  conclusie method  is the se method .For two-phase flow boiling of R22, the variation of heat transfer coefficients with vapor qualities is different for different channel dimensions. For the 1.77 mm tube , the heaFrom x ≤ 0.3, the Bertsch et al. [x ≤ 0.7, except for the Fang [x < 1, except for the Bertsch et al. [x ≤ 0.3 than in the range of 0.3 < x ≤ 0.7, while the Fang [x ≤ 0.3 than in the range of 0.3 < x ≤ 0.7. All the top 5 correlations have the largest MAD in the range of 0.7 < x < 1, although the Fang [From h et al.  correlatthe Fang  and the the Fang  correlatthe Fang  correlath et al.  correlath et al.  correlath et al.  and Berth et al.  correlatthe Fang , Wattelethe Fang , and Gunthe Fang  correlatthe Fang  correlatThe 26 existing correlations for flow boiling heat transfer are evaluated against the 1669 data points of two-phase flow boiling heat transfer of R22 collected from 18 published papers from 12 independent laboratories with the statistical calculation method.Among the 26 correlations, the top two correlations are those of Liu and Winterton and Fang, with the MADs of 32.7% and 32.8%, respectively. It indicates the existing two-phase flow boiling heat transfer correlations cannot predict the heat transfer coefficients of R22 well. Therefore, more researches are needed to better understand the mechanism of flow boiling heat transfer to obtain more accurate values of the heat transfer coefficients.There are 6 correlations having the MAD ≤35%. Following the Liu-Winterton and the Fang correlations are those of Wattelet et al., Gungor and Winterton , and BerThe statistics show that channel dimension has important effects on two-phase flow heat transfer of R22. This work characterizes channel dimensions with the channel size method, the Bd-type method, and the multidimensionless parameter method. Using these methods, all the top 5 correlations perform better for conventional channels than for minichannels. More investigations should be made to obtain better channel transition criterion to characterize channel dimensions.The two-phase flow boiling heat transfer coefficients of R22 vary with vapor qualities. All the top 5 correlations have the worst prediction in the range of 0.7 to 1.0 because of dry-out phenomena, indicating that no correlation can predict the dry-out region. The dry-out effects on flow boiling heat transfer of R22 play an important role in developing a better correlation, and thus more research efforts should be made to understand dry-out mechanism for two-phase flow boiling heat transfer of R22. 2 database of 2956 experimental data points has an MAD of 15.5% for that database. However, its MAD for the R22 database of the present work reaches 32.8%.A number of the correlations do not work well for R22, which may be because they were not developed for R22. For example, the Fang correlation developed from the CO