Aulacorthum solani raised on soybean (Glycine max) at 10 constant temperatures between 2.5 and 30 °C, and described the relationship between temperature and several critical biological characteristics using mathematical models. We found that A. solani could survive and reproduce on soybean at temperatures ranging from 5 to 27.5 °C. High fecundity was observed at temperatures from 12.5 to 20 °C. The lower developmental threshold and thermal constant for this species’ nymphal stages were estimated to be 5.02 °C and 131.2 degree-days, respectively, using a linear model. The upper developmental threshold was estimated to be 33.9 °C using the Lactin-2 model. The optimum temperature for nymphal development was estimated to be 26.9 °C. The maximum total fecundity was estimated as ca. 76.9 nymphs per adult at 18.1 °C. The daily fecundity sharply increased at earlier adult ages, and slowly decreased thereafter until final parthenogenesis occurred, over a range of temperatures from 12.5 to 25 °C. The maximum daily fecundity was estimated to be ca. 6.1 nymphs per adult per day for a 5.2 day old of adult at 21.3 °C using an age- and temperature-dependent model of adult fecundity. In terms of life table statistics, the intrinsic rates of increase and the finite rate of increase were both highest at 25 °C, while the net reproductive rate was highest at 20 °C.We investigated several characteristics of the development and reproduction of the aphid  Aulacorthum solani (Hemiptera: Aphididae), is a highly polyphagous insect. The number of host plants on which it can feed is as high as 540 identified species  from 33 orders and 82 families throughout the world; in Korea, it has been recorded to feed on 26 (identified) and up to a maximum of 30 plant species (including unidentified ones) from 10 orders and 13 families [Glycine max L. (Fabaceae), in Korea and Japan. It mainly feeds on the backsides of soybean leaves, and causes reduced yields of soybean through virus transmission and directly sucking out leaf fluids, as well as severe leaf yellowing [The foxglove aphid, A. solani is a species that is better adapted to low temperatures than insects in other orders, like many other aphid species [The aphid can develop through both holocyclic and anholocyclic life cycles. The insect overwinters as eggs in the holocyclic cycle, or as nymphs or adults in the anholocyclic cycle on various host plants ,7,8,9. A species . Tempera species . Many st species ,13,14.A. solani at multiple temperatures (ranging from low to high ones), derived general relationships between temperature and its development, and constructed life tables for the species when reared on lettuce (Lactuca sativa L.)  [Viola × wittrockiana)  [A. solani on soybean at multiple temperatures between 10 and 35 °C, and did not perform statistical analyses on their data [Manduca sexta L. larvae reared in diurnally fluctuating temperatures had significantly higher optimal temperatures and maximal growth rates than larvae reared in constant temperatures. Considering constant temperature has the advantage of allowing direct effects of temperature on the development, longevity, and fecundity of insects to be measured.A few studies have described experiments examining the effects of temperature on the development and reproduction of eraceae) ,17,18,19olaceae) . Howevereir data . Kingsoleir data  pointed A. solani on soybean at different temperatures; (2) describe the generalized relationships between temperature and biological traits, development, and fecundity in this species using mathematical models commonly used in arthropod species; and (3) construct life tables for this species to understand the biological characteristics when reared on soybean. We used the traditional age-specific life table and the age–stage, two-sex life table developed by Chi and Liu [A. solani populations are parthenogenetic, we applied the age–stage, two-sex life table to precisely present the stage differentiation among individuals and then used the life tables to project the population growth at different temperatures. The results of this study can support future studies of this aphid’s population phenology, adaptation to climate change, and performance on different soybean cultivars.Therefore, in this study, we used a wider range of temperatures, from 2.5 to 30 °C, in order to (1) investigate the developmental characteristics of  and Liu  and Chi  and Liu . AlthougA. solani were collected from soybean fields in Suwon, Korea, in 2014, and non-parasitized clones were selected through individual rearing in the laboratory. The clones were combined and reared successively on seedlings of the soybean cultivar Taegwangkong in an acrylic plastic cage (27.5 cm width × 30 cm depth × 40 cm height) in a plant growth chamber. The environmental conditions of the chamber were maintained at 20 ± 3 °C temperature, 15:9 h (light:dark) photoperiod, and 60% ± 15% relative humidity. During successive rearing processes, a small proportion of alate form was observed depending on density.Nymphs and adults of Several apterous adults in the rearing colony were transferred to soybean leaves in Petri dishes (50 mm diameter × 15 mm height) held in the same rearing conditions, and then nymphs newly born on the next day (after ca. 24 h) were used for rearing experiments at different temperatures. For adult experiments at 30 °C only, late-instar nymphs from the rearing colony were transferred to soybean leaves in other Petri dishes, and the apterous adults that emerged the next day were used. Experiments were conducted during the period from 2015 to 2017.Ten constant temperatures  were examined, with aphids in each temperature treatment held at the same photoperiod (15:9 h = light:dark). A low-temperature incubator  was used to rear aphids at temperatures from 2.5 to 15 °C, while a four-chamber incubator  was used for rearing at all other temperatures. Each neonate nymph was reared on a single leaf of soybean (cultivar Taegwangkong) within a polystyrene Petri dish (50 mm diameter × 15 mm height). The petiole of the leaf was wrapped in moistened cotton wool, and small drops of distilled water were applied to the cotton wool every day to maintain the leaf’s freshness. Each leaf was exchanged with a new one at 4–7 day intervals. Thirty replicated nymphs were used for each temperature. The occurrence of developmental milestones such as molting, death, and reproduction was recorded daily until death. Neonate nymphs produced from adults were removed immediately after being counted. For the adult experiment at 30 °C, thirty apterous adults were treated without examination of their nymphal stages.α = 0.05) were conducted using the PROC GLM function in the program of SAS Ver. 9.1 [Mean values and standard deviations (SD) for the duration of the total nymphal period, each instar period, adult longevity, pre-reproductive period, reproductive period, total fecundity, and daily fecundity during the reproductive period were calculated. One-way analyses of variance and comparisons of significant differences among different temperatures via Tukey’s HSD test , while those of the three-dimensional model (Equation (8)) were estimated with TableCurve 3D Ver. 4.0 .A. solani nymphs was modeled using both a linear model and the non-linear Lactin-2 empirical model. Nymphal development rates, r(Tc), at a given temperature (in °C), cT, were calculated as the reciprocals of the individual nymphs’ developmental periods (days) during the stage; these values were then averaged for each temperature. In the linear model [x-axis) and development rate (y-axis) was linear, were used to estimate the model’s parameters. In the linear model, the lower development threshold (°C) (LDT) and degree-days (DD) above the LDT required for the completion of development  of the nymphal stages were calculated as −b/a and 1/a, respectively, based on the parameters in the linear equation as presented below.The relationship between temperature and the development rate of ar model  , ΔT is the temperature range over which fast physiological breakdown occurs near UDT, and λ is a parameter that is derived to make the development curve intersect the abscissa at a suboptimal temperature; the formula for this model is shown below.In the nonlinear Lactin-2 model (Equation (2)) , data frTopt), at which the maximum development rate was predicted to occur, was calculated as LT − ΔT. The LDT was obtained as the approximate Tc value when r(Tc) = 0; this calculation was performed with the parameters estimated in Equation (2) using Microsoft Mathematics Version 4.0.In this model, the optimal temperature (F(x) is the cumulative proportion of nymphal development completed at a normalized time, x, corresponding to development time divided by the mean developmental period, and η and β are fitted parameters, as follows.A two-parameter Weibull equation (Equation (3)) was used to model inter-individual differences in the completion of development ,27. In tr(Ac), at a particular temperature, T (in °C), during the adult stage was calculated as the mean value of the reciprocals of all individual longevities. The temperature-dependent aging rate was fitted with an Eyring model (Equation (4)) previously used for Rhopalosiphum padi (Hemiptera: Aphididae) [a and b are fitted parameters as shown below.Aging rate, hididae) ,29,30, wf(T) is the number of total nymphs laid by one female at each temperature, LT and MT are the lower and higher temperatures for oviposition, and a and m are empirical constants of the equation.The relationship between total fecundity per female and temperature was estimated using Equation (5), proposed by Briere et al. .(5)f(T)The relationship between cumulative fecundity ratio and the normalized adult age (=adult age/mean adult longevity) was fitted with the two-parameter Weibull model used for describing the completion of nymphal development (Equation (3)) above.s(x), and the normalized adult age, x, was described with a model (Equation (6)) previously used for Carposina sasaki (Lepidoptera: Crambidae) [γ is the normalized age at 50% cumulative survival rate and δ is a fitted parameter, as follows.The relationship between cumulative survival ratio, ambidae) , in whicy, and adult age, x, at the same temperature was described with an adjusted nonlinear model (Equation (7)) previously used for Myzus persicae (Hemiptera: Aphididae) [p1 is the total fecundity, p2 is the mean pre-reproductive period, and p3 is a speed constant, as follows.The relationship between daily fecundity, hididae) , in whicT, adult age, x, and daily fecundity, F(x)T, was described with a model (Equation (8)) previously used for a mite species, Hypoaspis miles (Mesostigmata: Laelapidae) [ϵ, θ, κ, ρ, ν, and ψ are fitted parameters, as shown below.The relationship between temperature, lapidae) , in whicxl) (Equation (9)) and the survival rate (xjs) were defined to be the probability that a newly born individual would survive to age x and stage j, respectively. The xjs provides the beginning age x of the survival curve of stage j, last age x of the survival curve of stage j, mortality of stage j, and death age of individuals that died in stage j. The death, the beginning, and end of a stage can be provided.k is the number of stages.Life tables were constructed using the methods described by Jandricic et al. , Birch  and Soutxb) (Equation (10)) was calculated to take individuals of different stages at age x into account. The age–stage-specific fecundity (xjf) is the number of nymph eggs produced by a female adult at age x.The age-specific fecundity (R0) was calculated as rm) was obtained with a Euler equation, ∑[erm × x); this calculation was performed using Microsoft Excel . The finite rate of increase (λ), generation time (G) (in days), and doubling time (DT) (in days) were calculated as erm, Loge(R0)/rm and Loge(2)/rm, respectively.Net reproductive rate  is calculated using the Euler–Lotka formula (Equation (11)) with age indexed from Day 0 [The life history data of e theory  and the e theory  using the theory . The intom Day 0 , as follR0)/rm and ln(2)/rm, respectively. The statistical differences among different temperatures were analyzed with a paired bootstrap test at the 5% significance level [A. solani at different temperatures using the TIMING-MSChart program [j from time t to t + 1.The generation time (G) (in days), and doubling time (DT) (in days) were calculated as ln at 30 °C. Reproductive rates were high at all temperatures. The length of the pre-reproductive period decreased with increasing temperature from 5 to 25 °C, but then increased slightly at 27.5 °C, and then decreased again at 30 °C. The reproductive period was the longest at 7.5 and 12.5 °C, and it decreased with increasing temperature within the upper range of the temperatures tested. Total fecundity was significantly higher from 12.5 to 20 °C compared with that at lower and higher temperatures. Mean daily fecundity during the reproductive period was the highest at 20 °C and 25 °C, with 3.8 and 3.6 nymphs produced per adult per day, respectively, at these temperatures . Notablyradj2) values, which were ≥0.99 for both models. The data for inter-individual variation in developmental completion were fit well by the model tested (Equation (3)), with a high radj2 value of 0.90 (LDT and the degree-days (DD) above LDT required to complete nymphal development  were determined to be 5.02 °C and 131.2 DD, respectively, based on the linear development model (Equation (1)) . The LDT of 0.90 . The modradj2 value of 0.88 ) and cumulative fecundity (Equation (3)) models applied to the normalized ages of adults fit the observed values well, with high radj2 values.Aging rate increased exponentially with increasing temperature, and its generalized model (Equation (4)) achieved a high  of 0.88 . HoweverA. solani are presented in xl) is the sum of xjs at each age x and is the simplified version of xjs. The curves sharply dropped at an earlier age from 15 °C to 27.5 °C. The daily fecundity at temperatures from 10 to 27.5 °C sharply increased at earlier ages, and slowly decreased thereafter until the final parthenogenesis occurred, while at lower temperatures of 5 and 7.5 °C, daily fecundity values were relatively stable across ages. The highest age-specific fecundities were 0.70 (81 d), 1.04 (67 d), 1.82 (40 d), 2.90 (36 d), 3.43 (22 d), 5.73 (14 d), 4.82 (9 d), and 3.30 (9 d) offspring at 5, 7.5, 10, 12.5, 15, 20, 25, and 27.5 °C, respectively. The peaks of xm and xmxl tended to increase with increasing temperature from 5 °C to 20 °C. The model (Equation (7)) used to generalize the relationship between adult age and daily fecundity fit the observed data well at temperatures between 12.5 and 25 °C, with a high radj2 value over 0.9. The performance of this model at lower (5~10 °C) and higher temperatures (27.5 and 30 °C) than those in this range, however, was relatively poor (p2) in the model were shorter than the observed periods at all temperatures. Although the p2 parameter of the model at 25 °C gave a negative value, it was close to zero. The maximum daily fecundity was estimated to be ca. 6.1 nymphs per adult for a 5.2 day old of adult at 21.3 °C in the age- and temperature-dependent daily fecundity model (Equation (8)) ) .rm) and the finite rate of increase (λ) from both analyses were highest at 25 °C . Doubling time (DT) was shortest at 25 °C (2.07 and 2.05 days). Net reproductive rate (R0) was highest at 20 °C (77.49 and 78.34 nymphs), and the temperature with the second-highest R0 value was 12.5 °C (73.19 and 73.56 nymphs) (The intrinsic rate of increase ( nymphs) . The genThe population growth and stage structure at different temperatures based on the age–stage, two-sex life table is presented in A. solani on different host plants, including eggplant ), fennel ), lettuce, pea ), pansy, potato ), and soybean, are as follows: 48.7–63.15 days at 5 °C, 16.7–28.0 days at 10 °C, 16.9–19.2 days at 12.5 °C, 10.3–13.4 days at 15 °C, 7.2–8.9 days at 20 °C, 6.5–7.4 days at 25 °C, 6.3 days at 27.5 °C  decreased significantly at 25 °C compared to at 20 °C, at which the highest fecundity and R0 values were found  over which we found higher fecundities was similar to that found in other studies, which was 16–22 °C and 12.5–20 °C on lettuce, and 10–20 °C on pansy ,17,20. T °C over re found . Similarat 25 °C ; on pansA. solani on soybean in the study described in Kim et al. [Acythosiphon pisum had been heat-stressed for a period of about 4 h at 39 °C as young (1 day) or as older (5 day) nymphs, A. pisum without pea aphid secondary symbiont (PASS) or pea aphid rickettsia (PAR) failed to reproduce, though not necessarily to survive. Although we did not identify the facultative bacterial secondary symbionts of A. solani in this study, it may be speculated there is a relationship between development and the facultative symbionts of A. solani, because the species could not develop the adult stage at 30 °C and 35 °C but they reproduced at the same temperature.Several characteristics of the adult biology of m et al.  were quim et al.  than thom et al. . Fecundim et al.  found thA. solani to show the advantage of using the age–stage, two-sex life table in revealing the stage structure of insect populations.Life table parameters  are appropriate indicators for evaluating the effect of host plants on the development, survival, and fecundity of different populations. The intrinsic rate of increase of this study was higher than that of De Conti et al. , but lowA. solani on soybean at 10 different temperatures, and described the relationship between temperature and this species’ biology. Our results indicated that A. solani can survive and reproduce on soybean over a temperature range from 5 to 27.5 °C. High rates of reproduction occurred from 12.5 to 20 °C. The estimated LDT, UDT, and Topt for the nymphal development of this species were ca. 5.02 °C, 33.7 °C, and 26.9 °C, respectively. The estimated total fecundity was ca. 76.9 nymphs per adult at 18.1 °C. Most models and their parameters used in this study fitted the observed data well. These models and the life table statistics derived herein should, however, be evaluated under conditions of fluctuating temperatures in the field or in field-simulating conditions before they are applied in future population dynamics studies.We investigated several characteristics of the development and reproduction of


A stable isotope-labeled microsphere technique was used to determine OBF. Cardiac and arterial pressures were monitored with fluid-filled pressure catheters, and CO was measured by thermodilution. Two groups were treated with either LS (n = 7) or SNP (n = 7) during the last hour of hypothermia and throughout rewarming. Two groups served as hypothermic (n = 7) and normothermic (n = 6) controls. All hypothermia groups had significantly reduced CO, oxygen delivery, and OBF after rewarming compared to their baseline values. After rewarming, LS had elevated CO significantly more than SNP (66.57 ± 5.6/+30% vs. 54.48 ± 5.2/+14%) compared to the control group (47.22 ± 3.9), but their ability to cause elevation of brain blood flow (BBF) was the same (0.554 ± 0.180/+81 vs. 0.535 ± 0.208/+75%) compared to the control group (0.305 ± 0.101). We interpret the vasodilator properties of LS and SNP to be the primary source to increase organ blood flow, superior to the increase in CO.Rewarming from hypothermia is often challenged by coexisting cardiac dysfunction, depressed organ blood flow (OBF), and increased systemic vascular resistance. Previous research shows cardiovascular inotropic support and vasodilation during rewarming to elevate cardiac output (CO). The present study aims to compare the effects of inodilatation by levosimendan (LS) and vasodilation by nitroprusside (SNP) on OBF and global oxygen transport during rewarming from hypothermia. We used an  SNP vasdilation , and in actility .2) are essential factors in the treatment of critical care patients  and oxygen transport , Levosimendan (n = 7), Nitroprusside (n = 7), and Normothermic control (n = 6).The main aim of this study was to investigate if the beneficial effects of LS and SNP on cardiac function translate into improved organ perfusion during hypothermia and rewarming. Four experimental groups were included: control  by 30% compared to historical controls . On averThe animals were kept at 37°C for 5 h. After 2.5 h, the animals received a 10-min bolus infusion of 2.0 ml/h glucose (5%), followed by a 0.5 ml/h infusion lasting throughout the remaining experiment.Arterial pressure was obtained with a fluid-filled 22G cannula inserted in the left femoral artery and connected to a fluid manometer. Left ventricular pressure was obtained with a fluid-filled catheter inserted into the right carotid artery and advanced to the left ventricle under pressure guidance and connected to a manometer.BL) and, after that, hourly till final recording (37°C5h). Heart rate (HR) was calculated based on the femoral pressure signal, and SVR, stroke volume (SV), and cardiac index (CI) were calculated using the following formulas: SVR = MAP/CO, SV = CO/HR, and CI = CO/9.83*[body weight(2/3)].CO was measured with the thermodilution method, by injecting 0.15 ml of precooled (5°C) 0.9% saline through the jugular vein . The rapTo investigate organ blood flow, we applied the stable isotope microsphere technique . A volum2, pCO2, oxygen saturation (SaO2), pH, hemoglobin (Hb), hematocrit, and lactate. 0), during rewarming at 30°C (30°CRW), and after rewarming (37°CRW). At 37°CRW, a venous blood gas was also sampled. In the normothermia group, arterial blood gases were analyzed at 37°CBL, and finally, after 5 h, also venous blood gases were analyzed. Arterial and venous blood oxygen content (CaO2 and CvO2), DO2, and oxygen consumption (VO2) were calculated using the following formulas: CaO2 and CvO2 = (Hb × 1.34 × SaO2/100) + (pO2 × 0.0031 × 7.5), DO2 = CaO2 × CO and VO2 = CO × (CaO2 – CvO2).Arterial blood was analyzed for pOperature . In the Levosimendan was purchased from Orion pharma as SIMDAX® (2.5 mg/ml). On the day of the experiment, it was diluted in 5% glucose . DilutioNitroprusside was purchased from Hospira as NITROPRESS® (25 mg/ml). On the day of the experiment, it was diluted to 0.125 mg/ml (1:200) in 5% glucose .All statistical analysis were performed using SigmaPlot 13.0 (SAS).The sample size was calculated with three independent sample size analysis. The first two used the expected difference between myocardial blood flow and mean arterial pressure after rewarming in the three group thermic groups. The third used the expected change in myocardial blood flow after rewarming compared to baseline.Based on these sample size analyses, we concluded that a total of 27 animals were needed to attain a statistical power >0.8.Post hoc analysis and all group comparisons were performed using a Holm–Sidak method.Hemodynamic results were analyzed with a two-way repeated measure ANOVA analysis. Post hoc analyses were performed using a Holm–Sidak method. Between-group analyses at baseline, during, and after rewarming were done using a one-way ANOVA. Post hoc analyses were performed using a Holm–Sidak method.Within-group comparisons of organ blood flow and blood gas data were done using repeated measure one-way ANOVA. BL, blood flow in the brain, stomach, right, and left kidney was reduced in all groups at 30°CRW. No difference in blood flow of other organs was found between the three groups.RW, the control and LS-treated groups demonstrated significant reductions in blood flow to all organs, except for the liver, compared to 37°CBL Similar results were found in the SNP group. However, heart blood flow was not reduced at 37°CRWAt 37°CRW, both LS and SNP elevated brain blood flow compared to the control group. Blood flow in the heart was elevated in the SNP-treated group compared to both the control group and LS. Blood flow in the stomach was elevated in the SNP group compared to the control group. No difference was found between the three groups in kidneys or liver blood flow.At 37°CBL).RW), all three groups, independent of intervention, showed depressed MAP, CI, CO, and HR when compared to 37°CBL.At 30°C during rewarming . SVR was elevated only in the control group at 37°CRW compared to 37°CBL. At 37°CRW, MAP, CI, and CO were all depressed in all three groups, while SV was depressed only in the SNP and control groups compared to 37°CBL.Compared to 37°CRW, CO, CI, and SV were significantly higher and SVR significantly lower in the LS-treated group compared to the control. Compared to the control group, SNP significantly elevated CO and CI and reduced MAP and SVR. CO, CI, and MAP were significantly higher with LS than SNP.At 37°CBL.BL, Hb), hematocrit and lactate were significantly elevated at 30°CRW in all groups. SaO2 was elevated in the control group, and pCO2 was elevated in the SNP group. Further, DO2, pH, base excess, and RW. In addition, SaO2 was decreased in the control group.Compared to 37°CRW, DO2 was significantly higher in the LS group than in the control. Further, plasma lactate levels were elevated in the SNP group compared to both control and LS groups. No other between-group differences were found at 30°CRW.At 30°CRW Hb, hematocrit, pO2, and lactate were elevated in all groups compared to 37°CBL. Further, DO2, pH, base excess, and RW. In addition, SaO2 was reduced in the control group.At 37°CRW, DO2, and VO2 were significantly elevated in the LS-treated group than in the SNP and control. No differences were found between groups in other variables at 37°CRW.At 37°CThe main findings of this study are that LS and SNP equally improved blood flow to the brain, despite elevating cardiac output to different levels. When comparing the effects of two different pharmacologic interventions during rewarming, vasodilation by SNP vs. inotropic support plus vasodilation by LS, we find that, although LS is superior to SNP to restore global hemodynamic function, OBF is equally or better preserved after intervention with SNP. This finding indicates that increased vascular resistance is a central element in the complex pathophysiology of cardiac dysfunction and reduced OBF after rewarming from hypothermia.The aim of this experiment was to verify if the documented effects that both LS and SNP to elevate OBF during normothermic conditions –20 are vA mismatch between brain blood flow (BBF) and cerebral metabolic rate of oxygen during hypothermia is a much-discussed topic , 26–29. Aortic pressure and coronary resistance strongly regulate myocardial blood flow (MBF) by myocardial metabolic demand . While MInvestigators have demonstrated a reduced MBF during hypothermia with spontaneous circulation , 40–43. independent vasodilation but normalized after rewarming  following rewarming , 48. As In contrast to the RBF, Broome et al. found SNP to have a vasodilating effect on stomach blood flow (SBF) during angiotensin II-induced vasoconstriction . In the The physiological message this study brings to the clinical table is to focus on the mismatch between organ perfusion/microcirculation and global perfusion in the hypothermia/rewarming setting. Routine bedside intensive care lacks tools to assess organ perfusion and microcirculation changes, continually and accurately, in response to treatment. This highlights the need to be cautious in translating variables related to global circulation into changes that are important for organ blood flow and microcirculation, in this case, during rewarming from hypothermia.This experiment, describing both LS and SNP's pharmacologic effects to alleviate post-hypothermic circulatory dysfunction and OBF in a rodent model, may encourage further research in large animal models before clinical applications can be suggested.2 and serum lactate are present in the SNP group, although not significantly. Other investigators have toned down the importance of cyanide poisoning by SNP, also during hypothermia and cardiopulmonary bypass. Interestingly enough, the SNP-treated animals, despite possible cyanide toxicity, had improved hemodynamic and blood flow parameters compared to the control group (SNP is a known cyanide donor , 53. Enzol group , 53.From critical care reports, we know that efforts to elevate OBF will improve end-organ function and patient survival. The present findings indicate potential beneficial effects on organ function by combining cardiac inotropic support and reducing peripheral organ vascular resistance –17. In mThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Norwegian Animal Research Authority.BH has contributed with research ideas, protocol development, experimental work, data analysis, and manuscript. TK has contributed with experimental work and data analysis. ED has contributed with research ideas, data analysis, and manuscript. TT has contributed with research ideas, protocol development, data analysis, and manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mobile phase A was buffer (50 mM KH2PO4) and mobile Phase B was acetonitrile at a flow rate of 1.0 mL/min. The column temperature was kept at 30°C, and the injection volume was 20 μL. All analytes were separated simultaneously at a retention time (tr) of 3.93, 5.84, 7.06, 8.76, and 9.67 min for DPM, DBM, PTM, FSM, and APL, respectively, with a total run time of less than 15.0 min. The proposed method was validated according to ICH guidelines with respect to accuracy, precision, linearity, limit of detection, limit of quantitation, and robustness. Linearity was obtained over a concentration range of 12.0–240.0, 12.0–240.0, 20.0–200.0, 6.0–240.0, and 10.0–200.0 μg/mL DPM, DBM, PTM, FSM, and APL, respectively. Interday and intraday accuracy and precision data were recorded in the acceptable limits. The new method has successfully been applied for quantification of all five drugs in their injection dosage form, infusion samples, and for evaluation of the stability of investigated drugs in mixtures for endovenous use. The results of the stability study showed that mixtures of DPM, DBM, PTM, FSM, and APL in 5% glucose or 0.9% sodium chloride injection were stable for 48 hours when stored in polypropylene syringes at 25°C.A simple, fast, and validated HPLC method was developed for the simultaneous quantization of five cardiovascular agents: dopamine (DPM), dobutamine (DBM), phentolamine (PTM), furosemide (FSM), and aminophylline (APL) either in infusion samples or in an injection dosage form. The proposed method was achieved with a 150 mm × 4.6 mm, 5.0  Congestive heart failure (HF) is an important health care problem across the world . The preDopamine DPM, , 2-3, 4, 4- was supplied by Shenzhen Yihao Technology Development Co., Ltd. . Water (HPLC -grade) was prepared in our laboratory using a flow water purification system . Acetonitrile (HPLC-grade) was purchased from Fisher Scientific International . The marketed formulations of drug used in this study were DPM hydrochloride injection 20 mg/2 mL , DBM hydrochloride injection 20 mg/2 mL , PTM mesylate injection 20 mg/2 mL , FSM injection 20 mg/2 mL , and APL injection 20 mg/2 mL . The commercial solution of 0.9% sodium chloride injection was purchased from Kelun Pharmaceutical Co., Ltd. .The reference standards of DPM, DBM, PTM, FSM, and APL were procured from the National Institute for Control of Pharmaceutical and Biological Products  and stored at 4°C. Potassium dihydrogen phosphate KHThe HPLC system used for the optimized method was a UltiMate3000 LC (Thermo Fisher) consisting of a column oven, a quaternary pump, an automatic injector, and a diode array detector. The system was controlled by Chromeleon 7.2 software .μm particle size) was used as the stationary phase. Mobile phase A was 50 mM KH2PO4 buffer, and mobile phase B was acetonitrile with simple gradient program  was delivered at a flow rate of 1.0 mL/min. The mobile phase was prepared daily and filtered with a 0.45 μm membrane filter . The column temperature was set at 30°C. And, samples were analyzed at a wavelength of 280 nm and injected at 20 μL injection volume.A SinoChrom ODS-BP column (4.6  mm × 150  mm i.d. and 5.0 The standard stock solutions (0.6 mg/mL) of DPM, DBM, and FSM and (1.0 mg/mL) of PTM and APL were prepared by dissolving an accurately weighed 6 or 10 mg of each reference standards separately in 10 mL of deionised water in a 10 mL volumetric flask. The solutions were kept at 20°C until use.μg/mL for DPM, 12.0 to 240.0 μg/mL for DBM, 20.0 to 200.0 μg/mL for PTM, 6.0 to 240.0 μg/mL for FSM, and 10.0 to 200.0 μg/mL for APL).For the linearity studies, a 6-point calibration curve was prepared by diluting the working standard solution with deionised water and the range of this calibration curve  six consecutive times in the same day. Interday variability was realized by using the same quality control samples for six successive days. The relative standard deviation (RSD) values were calculated for the integration area and considered to be the measure of precision. The limit of detection (LOD) for each marker compound was determined at signal-to-noise ratios (S/N) of 3. For limit of quantification (LOQ), the ratio considered was 10 : 1 with RSD % value less than 10% [n = 3, at each level of 50, 100, and 150% levels) into infusion samples in triplicate. In this work, the mean recovery of the DPM, DBM, PTM, FSM, and APL concentration was 100 ± 2% for acceptance. The forced degradation studies were performed to prove the specificity of the HPLC method. The DPM, DBM, PTM, FSM, or APL samples were prepared with distilled water and later diluted with distilled water, aqueous hydrochloric acid, aqueous sodium hydroxide, or aqueous hydrogen peroxide solution for 5 h at 60°C. The chromatogram obtained for the degraded preparation was compared with a chromatogram obtained from the standard curve to determine whether or not any degrading peaks were produced and any changes in concentration, retention time, and peak shape of DPM, DBM, PTM, FSM, and APL. Robustness of the developed method was evaluated with respect to small deliberate alterations in the flow rate (1.0 ± 0.1 mL·min−1), the column temperature (30 ± 1°C), and the buffer concentration (50 ± 2 mM).Method validation of quantitative analysis was performed on parameters such as linearity, precision, recovery, and specificity of the five drugs. The linearity of response was performed using plotting peak areas against the concentration of the injected standard. Method precision was evaluated based on intraday and interday variability. Intraday variability was conducted by injecting the triplicate determination of quality control samples , DBM (0.12 mg/mL), PTM (0.10 mg/mL), FSM (0.12 mg/mL), and APL (0.05 mg/mL), respectively. The solution was filtered through a 0.45 n = 3). The initial concentration of the five cardiovascular agents was defined as 100%, and subsequent sample concentrations for the drugs in the mixtures were reported as the percentage of the initial concentration. Drug stability was defined as the remaining 90% of the initial value of each drug.This study was carried out to mimic as closely as possible a projected routine use of the five cardiovascular agents. Commercially available DPM, DBM, PTM, FSM, or APL were mixed in 50 mL polypropylene syringes of 5% glucose or 0.9% sodium chloride injection at concentrations of 3.0 mg/mL, 3.0 mg/mL, 1.0 mg/ml, 4.0 mg/ml, and 10.0 mg/mL, respectively. All admixtures were investigated at room temperature (25°C) over 48 hours with light protection or light exposure. The stability studies were evaluated in triplicate for each type of infusion media and storage condition. The physical parameters such as color change, presence of turbidity, and precipitate were evaluated qualitatively whenever samples were withdrawn. At each time point, the pH values for each admixture were determined with a precision pH meter . The concentrations of DPM, DBM, PTM, FSM, or APL were determined by the above described HPLC-DAD method. All samples from each syringe were analyzed in triplicate . The precision and accuracy data for both inter- and intraday analysis of DPM, DBM, PTM, FSM, and APL in the quality control samples at three levels is depicted in Under the above described experimental conditions, the linearity of the proposed method was investigated by plotting the peak area DPM, DBM, PTM, FSM, and APL versus the concentration of each standard drug. The characteristic parameters for regression equations of the proposed HPLC method are given in The validated method was applied to the determination of DPM, DBM, PTM, FSM, and APL in the commercially available injection dosage form. The results are shown in The mean concentrations over time of DPM, DBM, PTM, FSM, and APL diluted with 5% glucose or 0.9% sodium chloride injection and packaged in polypropylene syringes are outlined in Tables The present investigation represents a simple and accurate HPLC method for the separation of DPM, DBM, PTM, FSM, and APL. The proposed method could be applied for the analysis of all five drugs in their injection dosage form, infusion samples, and for the evaluation of the stability of investigated drugs in mixtures for endovenous use. On the basis of the results of stability study, DPM, DBM, PTM, FSM, and APL mixed in 5% glucose or 0.9% sodium chloride injection were physically and chemically stable at 25°C for 48 hours (2 days) when stored in polypropylene syringes.


Panax ginseng C. A. Meyer  root and leaf have always been considered in the traditional theory as hot and cold properties, respectively.hot and cold properties of ginseng root and leaf from a thermodynamic viewpoint. To clarify the th day, serum T3 or T4, liver or brown adipose tissue (BAT) mitochondrial respiration were investigated. Thirty ICR male mice were randomly assigned to control (water), ginseng root group (GRP) and ginseng leaf group (GLP) with a concentration of 0.075 g/mL; the volume was 0.1 mL/10 g (body mass) per day by intragastric administration for 20 days. Ultra-Performance Liquid Chromatography (UPLC) was used to determine quality control through seven ginsenosides contained in ginseng root and leaf. Rest metabolic rate (RMR) and energy expenditure were monitored every 9 days by TSE System. At the 202/h) in GLP (47.95 ± 4.20) was significantly lower than control (52.10 ± 4.79) and GRP (55.35 ± 4.48). Mitochondrial protein concentration and respiration were significantly increased in GRP  and decreased in GLP  compared with control. The quality control of GRP and GLP were within requirements of 2015 China Pharmacopoeia. The RMR (mLOhot and cold properties of ginseng root and leaf are correlated with thermogenic capacity and mitochondrial function of BAT and liver, which deserve to further research. The  However, the underlying mechanisms of the properties in CHMs remain unclear, which in turn presents a barrier for the modernization of Chinese herbal medicine. Usually, people rely on the Mammals produce heat via the following processes  are regarded as the range of ambient air temperatures, over which the BMR or resting metabolic rate (RMR) is maintained constant at the basal level  is very important because of its capacity to enhance energy expenditure (EE) and regulate the energy balance, which is why it has received strong scientific interest over recent years , which are secreted by the thyroid gland, are known to increase metabolism and are the well-established principal endocrine regulators of both obligatory and facultative thermogenesis in human and animals  has been used for over 4000 years as a main constituent in many traditional Chinese medicine prescriptions for the prevention and/or treatment of diseases. In general, P. ginseng root has mainly been used due to its hot properties and important Tonifying-Qi function in treating ‘cold disease’, whose most obvious expression is that the body feels cold. Interestingly, the P. ginseng leaf, which is another part of ginseng, has been thought to have ‘cold’ properties and ability to let the body feels cold. According to Chinese Pharmacopoeia and some ancient books such as ‘New Compilation of Materia Medica’, which was first recorded by Yi-Luo Wu of the Qing dynasty, argue that the function of P. ginseng leaf has the function opposite to P. ginseng root. Until recently, scientists believed that the chemical composition of P ginseng leaf was similar to P. ginseng root and that both contained ginsenosides such as Re. Yet, it has been reported that ginseng root might be equally useful in improving cold tolerance in young and old animals  male mice (SPF), weighing 30–35 g and 8 weeks old, used in this study were bought in the Vital River Laboratory Animal Technology Co., Ltd. Beijing, China. Animals were housed in groups of 3–4 in plastic cages (30 × 15 × 20 cm) with sawdust bedding in the animal house of Institute of Zoology. Food  and water were provided P. ginseng (Chinese Ginseng) by Professor Sui-qing Chen from Henan University of Chinese Medicine.The ginseng root and ginseng leaf were bought from Tongrentang Chinese medicine pharmacy in July, 2017, and were identified as the root and leaf of 18 column ; 2) chromatographic conditions were: flow rate 0.3 mL/min; detection wavelength (λ) = 203 nm; sample volume 5 µL; column temperature 30 °C. With acetonitrile (B) and 0.1% phosphoric acid solution (D) as mobile phase, gradient elution: 0–3 min, 19% B; 3–5 min, 20% B; 5–12 min, 21% B; 12–13 min, 29% B; 13–25 min, 31% B; 25–32 min, 35% B; 32–35 min, 19% B; 35–41 min, 19% B.3) Reference Standard: ginsenoside Re (lot number: 1205A021); Rg1 (lot number: 20160811); Rb1 (lot number: 2016530); Rb2 (lot number: 530A021); Rf (lot number: 817A021); Rc (lot number: 20151201); Rd (lot number: 20160816) were purchased from Beijing Solarbio Science Technology Co., Ltd. (http://www.solarbio.com/default.php).In order to ensure the quality of the samples and the controllability of traceability, the ginseng root and ginseng leaf were examined by Ultra Performance Liquid Chromatography (UPLC) to determine the content of ginsenoside Re, Rg1, Rb1, Rb2, Rf, Rc, and Rd. The experimental conditions were as follows: 1) Column: Waters Acquity BEH CThe method of 2015 ‘China Pharmacopoeia’ fourth edition 9101  was used as a general reference for drug quality standard analysis to carry out the verification of guiding principles for determination of validation: 1) precision test: the RSD of the 7 main component peak areas were in the range of 0.80–0.91%; 2) repeated experiments: the chromatographic peak areas of 7 kinds of ginsenoside were in the range of 1.03–2.80%; 3) stability experiment: the RSD of 7 kinds of ginsenoside chromatographic peak area were in the range of 0.43–2.74%; 4) recovery: the recovery of 7 kinds of ginsenoside were in the range of 98.84–103.76%, the RSD value was between 1.23–2.72%, the results met the requirements.We used 75.0 g of ginseng root and ginseng leaf, respectively, adding 600 mL water that was boiled for 30 min and the remaining water extract was drained out. Next, 450 mL water was added and left to boil for 30 min, after which the remaining water extract was drained out again. All water extract was put together, reaching the constant volume of 1000 mL. Finally, 0.075 g/mL concentrations of water extract of ginseng root and ginseng leaf were obtained. Importantly, the water extract of ginseng root and ginseng leaf were divided in vacuum bag, each one was kept at −20 °C for one day. Afterwards, the extracts were warmed to room temperature and shaken when used.During experiment, the body mass was weighed at 08:00–10:00 every day at the same time. The intragastric administration was initiated with the volume of 0.1 mL/10 g (body mass). Cumulative body mass was calculated using the following formula: 2) and CO2 production (VCO2) were measured by an open-flow respirometry system  with an overall flow rate of 0.8 L/min and a sample flow rate of 0.39 L/min. RER were calculated as VCO2/VO2 , and as RERmin (minimum RER during the entire metabolic rate measurement) was defined using recordings where the average of two consecutive RER values was the lowest.Each metabolic rate measurement lasted for 3 h. No fasting before measurement. Food and water were not provided during the measuring. RMR was calculated for each mouse as the average of two lowest constant oxygen consumption records  at every temperature by using the data of last 2 h of metabolic rate measurement. Energy expenditure was recorded the same as RMR. RER was calculated using recordings of the average of two consecutive oxygen consumptions where the lowest was defined as RERad libitum at the end of experiment. Then, at the 20 day of experiment, the mice were sacrificed by CO2 overdose. The trunk blood was collected 1 h later, and was centrifuged at 4000 rpm for 10 min, serum T3 and T4 were measured by ELISA kit  according to the instructions. These kits were validated for mouse (reference). Intra- and inter-assay coefficients of variation were 11% and 9% for both.In order to eliminate the effect of food, the animals were fasted 8 h and water g at 4 °C for 10 min, and the supernatant was centrifuged at 10,600 g at 4 °C for 10 min. This was repeated twice and the final deposit was resuspended with 0.1 mL isolation medium.After collecting trunk blood, liver and inter-scapular-brown adipose tissue were dissected and weighted after fat or connective tissue were removed. Then the sample of liver was homogenized with ice-cold medium (5 mM Tris/HCL (pH 7.4), 250 mM sucrose and 2 mM EGTA), then centrifuged at 1000 −6 mol/L BSA, pH 7.2). The homogenate was centrifuged at 12096 g at 4 °C for 10 min. Then, the supernatant was discarded, the precipitation with medium was resuspend , centrifuged at 500 g at 4 °C for 10 min. The supernatant was then centrifuged at 8740 g at 4 °C for 10 min, and the last pellet was resuspended  with medium  and subsequently used for mitochondrial respiration. All medium was ice-cold.The BAT sample was weighed and then homogenized  with medium  at 30 °C, after which the ADP was added to determine state 3 respiration  with body weight as a covariate to decrease its potential effects on BMR. Pearson correlation was executed to examine possible associations between BMR and metabolism markers. Values are expressed as mean ± SE, The results of content determination (mg/g) of seven ginsenosides by Ultra Performance Liquid Chromatography (UPLC) were Rg1 (3.8482), Re (2.3623), Rf (1.0797), Rb1 (4.5249), Rc (1.9063), Rb2 (2.5465), Rd (0.7904) in ginseng root and Rg1 (13.0862), Re (32.5088), Rf (0), Rb1 (1.8325), Rc (5.4642), Rb2 (7.8677), Rd (20.7490) in ginseng leaves, respectively . All the = 33.898, p < 0.001), there was no significant difference among groups  = 2.516, p = 0.100). However, the interaction between days and groups was statistically significant  = 3.734, p = 0.002). The body mass at day 12 (p = 0.027) and 18 (p < 0.001) of the experiment significantly increased compared with the initial mice body mass.The results of Repeated Measure ANOVA showed that the body mass of mice significantly increased with treatment  = 2.224, p = 0.128), day 6  = 1.374, p = 0.270), day 12  = 2.185, p = 0.132). At the day 18 of experiment, the body mass in mice were significantly different between groups  = 4.205, p = 0.026). Moreover, the body mass of ginseng root group at day 12 (p = 0.048) and 18 (p = 0.007) of the experiment was significantly higher than in ginseng leaf group  = 2.722, p = 0.084), and day 12  = 3.150, p = 0.059) of the experiment had no significant difference. However there was significant difference of cumulative body mass at day 18  = 5.200, p = 0.012) of experiment. As the experiment continued, the body mass change rate quickly increased, especially in ginseng root groups which were significantly higher than the control group at day 6 (p = 0.046), day 12 (p = 0.018), and day 18 (p = 0.009) (The cumulative body mass change reflect the body mass change rate. The data showed that the body mass in ginseng root and ginseng leaf group increased more during the experiment. The cumulative body mass at day 6 (F= 0.009) .2/h) was observed at day 9  = 3.485, p = 0.045) and 18  = 6.795, p = 0.004) of experiments among groups (2/h) and body mass, we analysed this relationship with Pearson correlation. The results showed that the relationship between RMR (mLO2/h) and body mass had strong positive correlation  = 4.347, p = 0.047; Group, F = 3.288, p = 0.053), RMR  = 8.538, p = 0.007, Group, F = 3.114, p = 0.061) and EE  = 4.854, p = 0.037; Group, F = 3.234, p = 0.056) at day 0, day 9 and day 18 of the experiment. The results of multiple comparisons showed that there were significant differences between ginseng root and ginseng leaf group in body mass-independent of RMR (mLO2/h/g), RMR (mLO2/h) and EE  at day 18 of the experiment  with body weight as a covariate was performed. The main significant differences in body mass of different groups was as follows: RMR  = 0.224, p = 0.801,  = 0.011, p = 0.989,  = 2.403, p = 0.110) by one-way ANOVA. However, the T3/T4 ratio in ginseng root group was significantly higher compared to control group  = 0.011, p = 0.989, In addition, it has been reported that mg T4 in the serum can generate approximately 4200 kcal of thermogenesis. Consequently, we calculated it to predict the thermogenesis of T4. The data showed no differences among groups  = 1.008, p = 0.378). Nevertheless, the BAT wet mass in ginseng root group and the ginseng leaf group were lower compared to control group. Interestingly, there was a significant difference of mitochondrial protein concentration  = 23.185, p < 0.001) and total mitochondrial protein  = 34.363, p < 0.001) between groups. Moreover, we compared State 4 Respiration of BAT in different groups. The data showed that either in protein-specific  = 15.388, p < 0.001), mass-specific  = 29.144, p < 0.001) or total BAT activity  = 7.095, p = 0.003) were significantly different among group. All of the above were significantly higher in ginseng root group compared to control group or ginseng leaf group (multiple compare).There was no differences in wet weight of BAT among groups  = 15.116, p < 0.001) among groups in the liver. Moreover, we also analysed liver’s State 3 and 4 Respiration in different groups. The data showed significant difference among groups either in protein-specific  = 46.574, p < 0.001 for State 3 Respiration; F = 9.401, p = 0.001 for State 4 Respiration), mass-specific  = 90.174, p < 0.001 for State 3 Respiration; F = 11.295, p < 0.001 for State 4 Respiration) or respiration control rates  = 8.932, p = 0.001).Following the same trend, there was a significant difference of mitochondrial protein concentration . Also, all of them in ginseng leaf group were lower compared to control group; however, the observed difference was not statistically significant except for the mass specific State 4 Respiration of BAT, mitochondrial protein concentration and mass specific State 3 Respiration of liver .2/h) against the BAT or liver mitochondria protein concentration, and mitochondria state 4 respiration. The results showed a positive significant correlation between RMR and BAT mitochondria protein concentration  that has a long-standing tradition of nearly 4000 years. Numerous scientists have explored these properties from the aspect of endocrinological, immunological, neurological , the results of body mass-independent RMR (mL O2/h/g) and RMR (mL O2/h) were significantly higher in ginseng root group than in ginseng leaf group. In particular, we found that the energy expenditure measured by TSE increased in ginseng root group and decreased in ginseng leaf group compared with control group; the observed difference between ginseng root and ginseng leaf group was statistically significant. This evidence provides direct support for the hot property of ginseng root and cold property of ginseng leaf. During the experiment, the RERRMR did not differ among groups, while RERmin was increased in ginseng root group compared to control group. This suggested that the ginseng root might induce the metabolic intensity of fatty tissue. We also detected the content determination (mg/g) of seven ginsenosides by UPLC method finding a very significant difference between the ginseng roots and leaves. It is possible that ginsenosides contribute to the above differences. These results are similar to those observed in North American ginseng root with reference to effectiveness of a particular active component that can significantly enhance the thermogenesis and cold resistance in rats  and Panax quinquefolius L. (American ginseng) have no effect on body temperature, VO2, VCO2, activity, food intake and energy expenditure after acute or chronic treatment with ginseng under normal conditions in mice  were positively correlated . The similar positive correlation was also found between liver mitochondrial protein concentration (mg/g) and RMR . We also found that liver contributed to some 20% of BMR, while metabolic intensity differences were linked with differences in oxidative capacities, mitochondrial densities and mitochondrial proton leaks  and RMR , which was consistent with the previous studies reporting that thyroid hormones are an important determinant of BMR , suggesting that thyroid is an important hormone for the RMR thermogenesis. Moreover, the thyroid might contribute to the BAT adaptive thermogenesis, since the relationship between T3/T4 and BAT total mitochondrial protein , BAT mitochondrial state 4 respiration  was positively correlated. However, we did not find the significant relationship between T3/T4 and RMR , liver mitochondrial protein  and liver mitochondrial state 4 respiration , which suggests that BAT may have an important role in the determination of hot/cold property for ginseng root and leaf. As is well known, the thermogenic capacity of BAT mostly depends on UCP1, which can make the proton gradient across the inner mitochondrial membrane dissipate (Endo and Kobayashi hot/cold property.At the hormonal level, the T3 and T4 concentrations in serum did not differ among groups; however, the T3/T4 ratio in the ginseng root group was higher than in control groups. More importantly, there was a significant positive correlation between T4 thermogenesis and RMR (mL Ocold and hot properties of herb medicines are correlated with body energy conditions and regulation, which confirmed our initial hypothesis. The cold herbs down-regulate, while hot herbs up-regulate thermogenesis. In particular, this is the first report that used BAT thermogenesis and mitochondrial respiration to study the traditional theory of hot and cold herbal drugs. Thanks to the physiological and biochemical analysis, this method may provide a unique insight in the research of herbal drugs, and can also be applied for research and study of other herbs.The above reported results showed that the


Nidularium procerum, an endemic bromeliad of Atlantic Forest vulnerable to extinction. Extracts of dry leaves obtained from in vitro-grown plants were recovered by different extraction methods, viz., hexanoic, ethanolic, and hot and cold aqueous. Chromatography–based metabolite profiling and chemical reaction methods revealed the presence of flavonoids, steroids, lipids, vitamins, among other antioxidant and antitumor biomolecules. Eicosanoic and tricosanoic acids, α-Tocopherol (vitamin E) and scutellarein were, for the first time, described in the Nidularium group. Ethanolic and aqueous extracts contained the highest phenolic content (107.3 mg of GAE.100 g−1) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, respectively. The immunomodulatory and antitumoral activities of aqueous extracts were assessed using specific tests of murine macrophages modulation (RAW 264.7) and 3--2,5-diphenyltetrazolium bromide (MTT) assay against adrenocortical carcinoma cell line, respectively. The aqueous extract improved cell adhesion and phagocytic activities and phagolysossomal formation of murine macrophages. This constitutes new data on the Bromeliaceae family, which should be better exploited to the production of new phytomedicines for pharmacological uses.This study reports the first phytochemical and biological characterization in treatment of adrenocortical carcinoma cells (H295R) of extracts from  Almost the entire family is native to the American continent, with the exception of Pitcairnia feliciana (A.Chev), an endemic specie of West Africa2. Due to their wide distribution and abundance in tropical habitats, bromeliads represent a very important ecological component in many communities, with a direct impact on richness and diversity of fauna and flora3.Bromeliaceae is a morphologically distinctive and ecologically diverse family, divided into eight subfamilies  based on morphological and molecular DNA data4. Among these, six species are classified as vulnerable, three endangered and seven critically endangered, indicating threatened ecosystems according IUNC criteria5. Nidularium is a genus with high vulnerability to extinction and Nidularium procerum Lindm is one of the most prevalent bromeliads found in the Atlantic Rain Forest6. It is a polymorphic specie, with varying appearance in response to the environment, especially the coloration of the leaves and bracts involved. The populations are mainly concentrated on the coast, where they develop in isolation or in groups of 2–10 individuals7.Bromeliads are also worldwide recognized for their ornamental value. In the past decades, it has become very popular as a garden plant, which increased the extraction pressures from natural populations. Brazil is the diversity center of Bromeliaceae, with 1,246 species cataloged to date, in which, 1,067 are endemic to the country8. More recent research has confirmed the beneficial effects of bromeliads supported by traditional medicine, such as improvement of digestive, diuretic and respiratory processes10. Other biological actions include relief of fever symptoms and diabetes mellitus11, as well as anti-inflammatory and anti-allergic proprieties, being able to inhibit the influx of pleural neutrophils and mononuclear cells in allergy-induced mice and, also, decrease the number of eosinophils by inhibiting PAF and eotaxin-induced eosinophil chemotaxis13. In addition, bromeliad extracts are reported and used as antitumoral agents. Bromelain and fastuosain — a complex natural mixture of proteolytic enzymes described in the group — was demonstrated to induce the apoptosis pathway of human epidermoid carcinoma and melanoma cells15. Phytochemical compounds present in bromeliads family were also shown to affect cell adhesion molecules involved in other pathways of carcinoma cells growth16.The bromeliad family has been used for centuries in Native American medicinein vitro plant tissue culture represents an ecological alternative to obtain competent explants (plant parts) under controlled conditions. This cultivation system allows the obtaining of several compounds with pharmacological interest without affecting natural population levels. In addition, micropropagated plants produce secondary metabolites at an early stage of growth17, which can be a way to provide rapid propagation of a large number of uniform plants, without being affected by adverse natural factors, such as climate, season, diseases and slow plant growth18. This allows for a technology alternative for rapid production of pharmacological compounds that can be utilized for medicinal purpose.In the current scenario of vulnerability caused by human exploitation, it is necessary to use alternative methods that allow cultivation of plant species. Thus, N. procerum. Amendoeira et al.12, Amendoeira et al.19 and Vieira-de-Abreu et al.13 reported that extracts of N. procerum have analgesic, anti-inflammatory and antiallergic properties with nontoxic activities, making it an attractive candidate for future drug development. However, the phytochemical composition of N. procerum remains poorly studied. Only a study conducted by Williams (1978)20 reported the flavonoid composition of leaves of Bromelia spp. including N. procerum, which observed the presence of quercetin. Here, crude extracts from the leaves of in vitro cultivated N. procerum were analyzed by diverse chemical analysis, including phytochemical screening by colorimetric tests, target compounds by gas chromatography assays (GC), chlorophyll quantification, total phenolic compounds and individual phenols by high performance liquid chromatography (HPLC), and antioxidant activity. In addition, for the first time, the immunomodulatory and antitumoral activities of aqueous extract of N. procerum leaves were assessed using specific tests of murine macrophages modulation and MTT assay against adrenocortical carcinoma, a cancer with rare treatment.Recent studies have revealed the pharmacological properties of in vitro cultivated N. procerum revealed the presence of some secondary metabolites, according to the solvent used  and ET extracts. HE showed the formation of yellow color, indicating the possible presence of a methyl group on carbon 1423. Finally, both aqueous extracts showed intense green color, referring to the presence of pyrocatecholic tannins (condensed tannins), formed by condensation of two or more flavanols, which are not hydrolyzed by acids, bases or specific enzymes23.The flavonoid group was observed in ET, HA and CA extracts, with the appearance of yellowish green and intense yellow, respectively, by Shinoda test. In addition, the ferric chloride assay confirmed the presence of tannins in these extracts. The ET showed a blueish black color, indicating the presence of pyrogallol tannins or hydrolysable tannin, which generates gallic acid or ellagic acid when hydrolyzed by acids, bases or appropriated enzymesN. procerum extracts are important due to their biological activities. Alkaloids, flavonoids, tannins and other biomolecules are known for their antioxidant, antifungal, anticancer, antiviral, anti-inflammatory and antiophidic activities26. Moreover, previous studies reported the presence of sterols, di and triterpenes, phenolic compounds, flavonoids, lignin, saponins, coumarins and cinnamic acids derivatives in other species grown under natural conditions of the Bromeliaceae family27.These secondary metabolites found in the 28. Steroids (β-cytosterol and stigmasterol) and lipids  were described in Bromelia Laciniosa Mart. ex Shult. & Schult.f, Neoglaziovia variegata Mez and Encholirium spectabile Schult. & Schult2, while cyclolaudenol triterpene was reported in Tillandsia fasciculata Sw. hexanoic extract29.GC-MS was carried out in order to describe and quantify the compounds found in a solvent polarity gradient, chosen according to permission of method. A total of 43 phytocompounds were found Table . These c30. Stigmasterol, in particular, has been investigated for its pharmacological potential, including cytotoxic, antioxidant, antitumoral, antimutagenic, among other herbal approaches to pathological states in principles and practice of phytotherapies31. Furthermore, to the best of our knowledge, it is the first time that some important compounds, already described in the literature in other plant species, were reported in the Nidularium group. These include α-Tocopherol (vitamin E) and the 5,6,7-trimethoxy-2-(4-methoxyphenyl)chromen-4-one flavone, also known as scuttelarein. Previously, α-Tocopherol was reported in four species of the family B. laciniosa (1.8% content in hexane leaves extract), N. variegate (1.5% content in hexane leaves extract), E. spectabile  and Ananas erectifolius (31.4 mg/Kg of fiber)32. The presence of vitamin E in N. procerum crude extracts increase the antioxidant and nutritional importance of the specie. In addition, scutellarein, previously found in Pitcairnia darblayana Sallier, Pitcairnia poortmanii André, Pitcairnia xanthocalyx Mart., Pitcairnia corallina Linden at André, Pitcairnia punicea Schiedw20 and Bromelia pinguin L. bromeliads33, were also related as anticancer agent against fibrosarcoma cells, by induction of cells apoptosis pathway34.The most commonly foun sterols in plants include campesterol, sitosterol and stigmasterolN. procerum was investigated in the same apolar extractors used in GC-MS assay, whereas in the polar phase, it was analyzed in aqueous solvents, used in biological tests. The major identified constituents were Linolelaidic acid methyl ester (C18:2 – Omega 6), Methyl palmitate , Cis-9-Oleic acid methyl ester (C18:1 – Oleic acid) and ɣ-Linolenic acid methyl ester (C18:3 – Gamma linolenic acid/Omega 6) 32 and reported for having anticancer and antiinflamatory potential37; while tricosanoic acid was present in hexane extract of leaves from Ananas cosmosus bromeliad, which demonstrated potential cytotoxic against tumoral cell lines38.Other minor constituents detected were stearic and palmitoleic acids, previously reported in 39, decreased risk of coronary heart disease, and protective action against stroke, age-related cognitive decline and Alzheimer disease41. Moreover, Omega-6 fatty acids has gained attention in medicine studies for showing potential in preventing sarcopenia, modulate cancer, atherosclerosis, obesity, immune function and diabetes42. This is the first time the lipid profile of N. procerum was described, improving the knowledge of its chemical compounds.Fatty acids play an important role in biological functions of living organisms, contributing to the prevention and treatment of some diseases. Diets with oleic, linolenic, linoleic and linoleic conjugates have been shown to reduce plasma cholesterol levels, in addition to affecting some physiological reactions, such as immune response and inhibition of tumor growthin vitro cultivated N. procerum showed a greater concentration of chlorophyll b (215.06 ± 14.8 µg.g−1 of fresh mass) than chlorophyll a (170.75 ± 18.5 µg.g−1 of fresh leaves). In comparison with Nidularium campo–alegrense Lem (56.4 ± 11.2 µg.g−1 fresh mass) and Aechmea ornate Baker (97.1 ± 11.2 µg.g−1 fresh mass) wild-type bromeliads43, N. procerum presented higher amounts of both chlorophylls. The photosynthetic mechanism of plants grown during in vitro culture is not completely active and the leaves have a reduced capacity to synthesize organic compounds44. Then, plants can compensate this failure with greater amounts of photosynthetic pigments, as they tend to increase its concentration, with reduced light intensity. Furthermore, bromeliads usually grow under the canopy and the leaves of shade plants often have higher content of chlorophylls than sun species45.Leaves of −1), when compared to N. campo – alegrense Lem (3.07 µg.g−1) and A. ornate Baker (2.94 µg.g−1) grown in normal conditions43, the higher amount of pigments can be related to the improvement of biological activity of the extracts. Chlorophyll compounds have been described as potential antioxidants with effective activity against lipidic peroxidation, DNA degradation and some cases of anemia47. Furthermore, recent works showed that chlorophyll derivatives, such as chlorophyllide, are also closely correlated to enhanced selectivity and improved cytotoxic activity against a range of carcinoma cells48.Despite the low ratio between chlorophylls a/b , followed by CA (96.82 mg of GAE/100 g) and HA (78.57 mg of GAE/100 g). Similar results (70.73 mg of GAE/100 g) were reported in fresh fruit extracts of wild-type 50. The aromatic benzene rings with substituted hydroxyl groups are responsible for their biological activity through the capacity to eliminate or absorb free radicals, and to chelate reactive oxygen species molecules formators. Furthermore, the effectiveness is generally proportional to the number of hydroxyl (OH) groups present in their aromatic rings51.In general, phenolics have gained attention due to their antioxidant, antimutagenic, anticancer and anti-inflammatory capacitiesThe phenolic profile of aqueous extracts was evaluated in HPLC, in order to identify some antioxidants present in the solvents applied in the biological tests. The compounds were identified and quantified by comparing their retention times and absorption spectrum data in ultraviolet, which presented UV-band characteristic for gallic acid, p-coumaric acid, rutin, daidzein, quercetin, trans-cinnamic acid and genistein Table .Table 4R−1)—a compound belonging to the flavonoids class of phenols. Other flavonoids were also detected, such as tannin gallic acid (2.6 µg.g−1), flavone rutin (3.4 µg.g−1) and flavonol quercetin (0.7 µg.g−1). With the exception of genistein, that is usually found in leguminous52, flavonoids are characteristic of the Bromeliaceae family, having been reported in Bromelia balansae Mez, E. spectabile Mart. ex Schult. & Schult.f among others54.The main component found was the isoflavone genistein  and cold aqueous extract (IC50: 0.29 ± 0.01 g.g−1 dry weight) was dose-dependent, inhibiting up to 90% of free radicals and lower than the standard Trolox (IC 50: 18.30 ± 0.1 g.g−1 dry weight). However, HA extract also showed the highest value of Trolox equivalent (21.4 mg of Trolox.g−1 dry wheight), than CA extracts (18.83 mg of Trolox.g−1 dry weight) in 2,2-azinobis-3-ethyl-benzoatiazolin-6-sulfonic acid (ABTS) assay. According to total phenolic content of N. procerum, HA showed a significant difference to CA , which is closely related to its higher antioxidant potential between the extracts.DPPH scavenging activity of hot aqueous  of HA extract elicited a significant raise (p < 0.05) of about 32% of its activity . On the other hand, CA extract showed no significant impact at 2 µg.mL−1, but increased the adhesion capacity at a higher concentration tested (1000 µg.mL−1) in over 28% of isolated macrophages. The adhesion alteration capacity of cells may be linked to the biological compounds present in HA extract. Fatty acids and polyphenols, especially flavonoids and tannins, can change properties of the plasma membrane, altering its fluidity capacity, as well as the distribution of adhesion molecules within the plasma membrane, such β1 (CD29) and β2  integrins63. β1 (CD29) and β2  are molecules involved in mediate the adherence of phagocyte cells to endothelium cell receptors.Many plant extracts have long been described as possessing anti-inflammatory and immunomodulatory actions. The first line of human body defense against invading pathogens is the innate immune system, through macrophage cells. In the present study, murine macrophages were assessed ion Fig. . The macity Fig. . On the −1 HA elicited a significant increase in the phagocytic capacity of macrophages (p < 0.05), while in CA extract, no significant effect was observed at all concentrations tested  can induce activities in phagocytic cells, by activating surface receptors, such Fc gamma , starting the “zippering” phagocytosis65 and also increasing the expression of complement receptors-CR1, CR3 and CR4-promoting the “sinking” phagocytosis66. Furthermore, unsaturated fatty acids have previously shown to improve phagocytosis ability67, and according to Table Cells treated with 10 µg.mLted Fig. . Similar−1 , raising ~25% of neutral red uptake in both HA and CA, at 20 µg.mL−1 Fig. . This he2O2) and superoxide anion (O2−), are generated in the first minutes of macrophage stimulation, during the so-called “respiratory burst”69. They are involved in the inflammation process, acting as efficient protectors; however, uncontrolled or excessive ROS production can further promote oxidative stress —a disruption in the redox balance system that contributes to damaging the body´s own cells and tissues70.Reactive oxygen species, such as hydrogen peroxide (H−1) was capable to inhibit 36% the production of hydrogen peroxide (H2O2) in macrophage cells  significantly reduced 38% of the production of superoxide anion , with maximum reduction (40%) of activity at 2 µg.mL−1. This may be related to the potential of some flavonoids, such as rutin and quercetin, present in higher concentrations in CA extracts (3.41 and 0.71 µg.g−1 respectively) than HA, in the inhibition of xanthine oxidase and phosphoinositide 3-Kinase γ enzymes71. Furthermore, the higher concentration of H2O2 in relation to superoxide anion radical could be involved through the formation of enzyme-flavonoids hydrogen bonds, inhibiting the antioxidant activity of some peroxidase enzymes, such as catalase72.HA extract  alpha, among others73. Plant extract compounds can also increase the expression of genes associated with the antioxidative system, such Cu/Zn-SOD, Mn-SOD, catalase, and GPx genes, suppressing oxidative stress by increasing antioxidant activity of enzymes74.Some molecules described in N. procerum extract can be promising to help fight inflammation and even maintain cell homeostasis under different conditions. Leaf aqueous extract of N. procerum was also shown to interfere in different functions of host response capacity against injuries, such the inhibition of lipid body formation, PGE2 and cytokine production of in vivo pleural leukocytes12. Taken together, these data indicate that the substances described in the leaves of N. procerum proved to be efficient in modulating significant responses mediated by macrophages. This can be a potential alternative as a therapeutic agent applied in the prevention and treatment of pathologies related to the immune system. In addition, previous studies demonstrated that plant-derived compounds are able to alter the immunosuppressive status of patients, increasing antitumor immunity, promoting the proliferation of immune cells and accelerating macrophage phagocytosis75. To the best of our knowledge, there is no study on the anti-tumor activity of N. procerum extract. Studies were also carried out to evaluate the cytotoxic activity of in vitro cultured N. procerum Lindm against H295R cell line, a carcinoma with rare, heterogeneous malignancy and a very poor prognosis76.The ability to modulate all macrophage parameters found in −1 to 1000 µg.mL−1 for 24 h are summarized in Fig. −1) and 34,4% -(HA at 250 µg.mL−1). On the other hand, there was no statistical difference among extracts and control in the viability of non-tumor cells (VERO)  cell lines exposed from 2 µg.mLRO) Fig. . The levA. comosus L.78, Tillandsia recurvata Baker79 and Bromelia fastuosa Lindl.15. The antitumoral activity was attibuted to cysteine proteinases  as well as flavonoids, including penduletin, cirsimaritin and HLBT-10079. Biological compounds are related to the suppression of some metastatic markers, resulting in regulation of mitogen activated protein kinase and protein kinase B80. Genistein, present in higher amounts in both HA and CA  from clusters previously micropropagated in vitro were used as explants and subcultured in vitro to elongation and rooting for 90 days, free of plant growth regulators, up to the formation of a complete explant -becoming available for extraction of leaves compounds, protocol adapted from Kim et al.80. Plantlets were removed from culture chambers and the leaves were cut into small pieces and 1 g of fresh leaf mass was macerated and extracted in 10 mL of specific solvent, over 24 hours, under 80 rpm agitation and 25 °C in a dark room. The extracts were filtered with Whatman n° 1 filter paper, lyophilized and stored at −20 °C for further characterizations and applications.Plants were stablished and multiplicated N. procerum compounds were carried out in different solvents, in order to detect the maximum range of substances extracted as summarized in the flowchart below . The analyses were performed based on the results obtained in preliminary phytochemical screening and finally focused on aqueous extracts used in biological tests. The antioxidant, immunomodulatory and antitumoral potential of the aqueous fractions were explored due to their lack of toxicity and the low-cost of the process.The chemical investigation of low Fig. . The anaN. procerum: hexane , ethanolic , hot aqueous (100 °C) and cold aqueous (25 °C). The screening was performed according to Iqbal et al.92. Identification of alkaloids was determined using Dragendorff, Mayer and Wagner´s test, reducing sugars using Fehling´s reagent, quinones by Bornträger´s test, saponins by permanent foam appearance, mucilage by gelatinous consistency after cooled, coumarins using Bajlet´s test, steroids/triterpenoids using Liebermann-Buchard´s test, resins by precipitation test, flavonoid by Shinoda´s test and tannins/phenols using Ferric Chloride´s test.The phytochemical tests were carried out in four different extracts from −1 up to 280 °C, which remained constant for 15 minutes, for a total of 47 min of analysis. Helium was the carrier gas used, at a 1 mL.min−1, 88.3kPa column press and split ratio of 1:40. The solvent cut off was 2.5 min. The mass spectrometry range was 30–500 (m/z), at an ion source temperature of 250 °C. The chemical compounds were identified by comparison of the mass spectra present in NIST98/2014 and Wiley 7 data library.The GCMS profiles of solvents with different polarity ranges  were obtained by electron impact with GC-MS-TQ Series 8040–2010 Plus (Shimadzu-Japan) equipped with a 95% PDMS and 5% Phenyl capillary column . The temperature program started at 50 °C, maintained for 2 minutes and raised at a flow of 7 °C.minPlus), a capillary column (SH-Rtx-Wax - Shimadzu: 30 m × 0.32 mm × 0.25 μm), flame ionization detector (FID) and split injection mode (1:10). The injector and detector temperatures were 240 °C and 250 °C, respectively. The oven temperature was programmed to start at 100 °C during 5 min, followed by an increase up to 240 °C at a rate of 4 °C.min−1 and maintained at this temperature for 5 min. The carrier gas was Helium at 32.5 cm3.min−1. The samples were prepared according to the official method (Ce 2–66) of the American Oil Chemist’s Society  to convert triacylglycerol and free fatty acid of samples into fatty acid methyl esters (FAMEs). FAMEs were identified by comparison with retention times of the standard mixture FAMEs . The quantification of fatty acids was conducted by area normalization procedure. Results were expressed as percentage of each individual fatty acid present in the sample.Fatty acid profile of CHL, HE, HA and CA extracts were analyzed using a Shimadzu chromatograph (GC 2010 93. The assays were carried out in triplicate and the results were expressed in μg.g−1 of fresh weight.Fresh leaves (5 g) were macerated in 10 mL acetone (P.A. VETEC). The solution was filtered with Whatman n° 1 filter paper and stored at −6 °C for five minutes. The absorbance was measured with spectrophotometry at 470, 662, 645, and 652 nm to chlorophyll a, b, relation between and chlorophyll a/b, respectively94 and the standard curve was performed using 0.39, 3.9, 7.8, 15.6, 31.2, 62.5 and 125 μg.mL−1 of gallic acid. The results were expressed in mg of gallic acid equivalent (GAE) in 100 g of fresh weight.Total phenolic contents of ET, HA and CA extracts were determined using Folin-Ciocalteu method−1 flow. The mobile phase was 2,5% acetic acid (solvent A) and methanol (solvent B). The elution gradient was carried out as follows: 90% A/10% B, 0–13 min; 75% A/25% B, 13–28 min; 15% A/85% B, 28–32 min; 10% A/90% B, 32–36 min. Chlorogenic acid, caffeic acid, ferulic acid, tocopherol, genistein, transcinnamic acid, catechin, rutin, p-coumaric acid, gallic acid, resveratrol and epicatechin (SIGMA) were used as standards. To obtain the calibration curve, all standard reagents were solved in mobile phase and used at 1, 2, 5, 8 and 10 ppm. The samples were microfiltered trough a hydrophilic membrane GV (Durapore) made of polyvinylidene difluoride (PVDF), with a pore size of 0,22 μm. The resulting chromatogram values were plotted and a linear equation was generated by calculating the average of triplicate runs for each compound. The equations were used to quantify the phenolic compound contents of the samples. The injection volume was 10 µL. All the assays were also performed in triplicate.The phenolic content of aqueous extracts was separated in HPLC using an Agilent Technology 1200 Series system, coupled to a diode array detector (DAD) at wavelengths 235, 260, 275, 280, 290, 311, 357, 370 nm and a scanning from 190 nm to 600 nm. A ZorbaxElipse XDB-C18  column was used at 0,7 mL min69. A Trolox (Sigma) standard solution was diluted from 0.25 to 25 mg/ml and used as positive control to the assay, mixing 200 μL of each concentration in 800 μl of 0.004% methanol solution of DPPH. After 30 min of incubation in absence of light at room temperature, the absorbances were read against blank at 517 nm using a SP-2000 spectrophotometer. The same protocol was used for the HA and CA treatments. DPPH solution was used as negative control with the solvent extraction. Tests were carried out in triplicate and the percentage of free radical inhibition was calculated by the following Eq. (blank is the negative control and Asample is the absorbance of extracts. The results were expressed in extract concentration producing 50% inhibition (IC 50%), calculated from the graph of the DPPH scavenging effect against the extract concentration.The antioxidant potential of the aqueous extracts was determined by their ability of quenching the free radical DPPH95. The ABTS radical had to be pre-formed by the reaction between 5 mL ABTS 7 mM (Sigma) with 88 μL of 140 mM potassium persulfate, stored in the dark at room temperature for 16 hours. The ABTS solution (1 mL) was previously diluted in 50 mL of ethanol P.A. (Alphatec) to obtain an absorbance of 0.700 at 734 nm. In absence of light, 10 μL of each aqueous plant extract were added to 500 μL ABTS solution. After 6 minutes, the absorbance was read in the spectrophotometer (SP 2000) at 734 nm. Distilled water was used as blank and as negative control. All measurements were carried out in triplicates. The scavenging capability of tests compounds was calculated using the following Eq.  supplemented with 10% fetal bovine serum (FBS - Gibco and 1% antibiotic solution (10.000 U.mL−1 penicilin and 10 mg.mL−1 streptomycin - Gibco®), maintained in a humidified atmosphere with 5% CO2 at 37 °C until 80–90% confluence was reached. The cells were divided at 105 cells/well in 96-well plate (Biofil) and exposed into the following experimental groups: cells without treatment (C), Hot Aqueous (HA) and Cold Aqueous Extract (CA), both at concentrations 2, 10, 20, 100 and 1000 μg.mL−1 for 24 h at same conditions of growing. The analyses were performed in 12 repetitions.Macrophage activity was assessed by its reactive oxygen species production - superoxide anion and hydrogen peroxide, cell adhesion, phagocytic efficiency and phagolysosomal formation2 incubator at 37 °C, with humidified air (95%) and CO2 (5%) until 80–90% confluence was reached. The cells were divided in 105 cells/wells in a 96-well plate (Biofil) and exposed into the following experimental groups: cells without treatment (C), Hot Aqueous and Cold Aqueous Extract, both at concentrations 2, 10, 20, 100 250, 500, 750 and 1000 μg.mL−1 for 24 h at the same growing conditions. The analyses were performed in 12 repetitions97.H295R and VERO cells were cultured in DMEM F-12 (Sigma Aldrich) medium supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic solution (Gibco). They were incubated in a COThe data were expressed as mean ± standard error and analyzed by One Way Anova followed by Tukey´s test using Graph Pad Prism 6 software. Differences between means at the 5% of confidence interval (p < 0.05) were considered significant.