During GIET, heart rate (HR), oxygen uptake (2mean and 2mean), metabolic equivalents (METs) in 2mean, and blood glucose and lactate concentrations (La−) were measured. Furthermore, jogging/running speeds (S) at specific La−, fat and carbohydrate oxidations (FATox and CHOox), and energetic contributions  were calculated. The percentages of HRmax, relative 2mean, 2mean, and METs in 2mean were all lower in A than they were in GP. FATox values were lower in GP than in A, while CHOox and La− were higher in GP than in A. Negative correlations between La− and FATox were also observed in both groups. Contributions of WOxi, WGly, and WPCr were higher in GP than in A during GIET. Moreover, values of WGly, and WPCr were significantly lower and higher, respectively, in male GP than in female GP. Furthermore, S at specific La− were higher in A than in GP. It is suggested that an individualized low-intensity recovery exercise program be established, to achieve increased metabolic flexibility and oxidative capacity (aerobic base), such as public health improvements and a greater volume of higher exercise intensities; this is the type of exercise that elite athletes worldwide mostly perform during their training period and progression. This may prevent cardiac/metabolic diseases in GP.Metabolic flexibility includes the ability to perform fat and carbohydrate oxidation, as well as oxidative capacity, which is associated with mitochondrial function, energetic contributions, and physical health and performance. During a session of graded incremental exercise testing (GIET), we investigated metabolic flexibility, the contributions of three energy systems, and performances of individuals with different metabolic characteristics. Fifteen general population (GP;  Physical inactivity has been shown to cause non-communicable diseases such as metabolic diseases ,2,3. Ins−1) between 136.5 and 238.8 watts than moderately active individuals and individuals with metabolic syndrome. Furthermore, the blood lactate concentrations (La−) of professional endurance athletes remained <1 mmol∙L−1 during the given exercise intensities, while the La− of the other groups were between 5.97 and 6.38 mmol∙L−1 during the same exercise intensities. The reduced levels of La− in athletes imply increased La− elimination occurring alongside adenosine triphosphate (ATP) re-synthesis, lipid oxidation, gluconeogenesis, and decreases in both glucose and total carbohydrate use [In an incremental cycling test, San-Millán et al.  reportedrate use ,11.− and oxygen uptake (2) levels, and fat and carbohydrate oxidation using stoichiometry in different individuals, which are related to indirect indicators of metabolic flexibility during incremental cycling tests [2 and La− values during different exercises in mathematical models [Lactate is the main energy source, a main gluconeogenic precursor, a regulator of intermediary metabolism, and a signal molecule ,10,12,13ng tests ,16,17. Fl models ,19,20,21l models . To dateTherefore, this study aimed to investigate energetic contributions including gender differences, metabolic flexibility, and performance parameters, and to compare how these abilities differ between the general population and athletes, during graded incremental jogging/running tests. These evaluations are expected to support an efficient method of individualized training prescriptions to improve physical performances in order to prevent cardiac/metabolic diseases in the general population, as they have proven useful worldwide in enhancing physical health and performance in elite athletes .n = 30). Fifteen physically active male and female individuals from the general population  and 15 national-level male and female half-marathon and triathlon athletes  participated in this study. The sample size was calculated and considered, based on previous studies ,21,23,24− measurements. The initial jogging speed was 1.5 m∙s−1, which was increased to 0.5 m∙s−1 every five minutes. The GIET was stopped when La− exceeded 4 mmol∙L−1 after each jogging/running speed in all participants [− and glucose levels for all steps were analyzed using an enzymatic–amperometric sensor chip system . Heart rate (HR) data were recorded using a Polar H10 sensor . The average HR value over the last 30 s of each step was estimated for statistical analyses [max) were calculated using previously described methods [−1 La− were analyzed using a previously suggested mathematical model of interpolation [2, 2mean, metabolic equivalents; METs in 2mean [2mean) was measured breath-by-breath, using a mobile gas analyzer MetaMax 3B . The gas analyzer was calibrated with calibration gas , and the turbine volume transducer was calibrated with a 3-L syringe .The graded incremental exercise test (GIET) was conducted on a treadmill  with 5-min steps interspersed with 30 s breaks between steps for Laicipants ,17,25. Canalyses . The per methods ,26. The polation ,17,27. DV˙O2mean , and car2 and 2 production were used to calculate metabolic flexibility as well as fat (FATox) and carbohydrate (CHOox) oxidation, using stoichiometric equations as described in previous studies [During GIET,  studies ,24,29:FAOxi), glycolytic (WGly), and phosphagen (WPCr) systems, were estimated by measurements of 2 during GIET, La− after each step of GIET, and the fast phase of excess 2 after exercise [Energetic contributions in kilojoules (kJ), such as contributions in the oxidative  [The Wody mass . The diftracted) ,20,21.PCr was calculated by considering 2 during the interval between graded incremental steps and the fast component of excess post-exercise after the last step of GIET [off 2 kinetics were fitted by mono-exponential and bi-exponential models using OriginPro 2021 . The slow component of the bi-exponential model was negligible. Therefore, the post-exercise 2 values were fitted to a mono-exponential model, and WPCr was obtained by calculating the amplitude and time constant of the exponential area [Oxi, WGly, and WPCr) in kJ. The contribution of the three energy systems was indicated as a percentage (%) related to total energy expenditure.The W of GIET ,20,21. Tial area ,19,20,21ial area . The tot−1 steps, because the GP group was able to perform their test until the 3.5 m∙s−1 steps. After the ND test, HR at 3.0 m∙s−1; La− at 1.5 and 3.0 m∙s−1; % of HRmax at 3.0 m∙s−1; relative 2mean at 2.0 m∙s−1; METs in 2mean at 1.5 and 2.0 m∙s−1; relative 2mean at 1.5, 2.0, and 3.0 m∙s−1; FATox at 3.0 and 3.5 m∙s−1; WPCr in kJ at 1.5 m∙s−1; WGly in kJ at 1.5 and 3.0 m∙s−1; WPCr in % at 2.0 m∙s−1; WGly in % at 1.5, 2.0, 2.5, and 3.0 m∙s−1; and WOxi in % at 2.0 m∙s−1 steps were compared, using a Mann-Whitney-U rank test (non-parametric test). The remaining data were analyzed using an unpaired t-test (parametric test). In addition, the average values up to 3.5 m∙s−1 steps during GIET of the three energy systems and the total energy demand between males (n = 7) and females (n = 8) in GP and A were statistically compared, using the Mann-Whitney-U rank test. The alpha level of significance was set at p < 0.05 for all tests. The effect size  was estimated for parametric and non-parametric tests. The thresholds for small, medium, and large effects were 0.2, 0.5, and 0.8, respectively (parametric test), and 0.1, 0.3, and 0.5, respectively (non-parametric test) [− and FATox in GP and A.All parameters were analyzed using GraphPad Prism 9.4.0. . Data are presented as mean ± standard deviation (SD) and standard error of the mean (S.E.M.). The normal distribution (ND) of all data was conducted using the Shapiro−Wilk test. During GIET, both groups were compared statistically up to 3.5 m∙sic test) . Further2mean, 2, and blood glucose between both groups were not significantly different (p > 0.05). Only the value of HR at 3.0 m∙s−1 was significantly higher in GP than in A  . The relative values of 2mean from 1.5 to 3.0 m∙s−1 were also significantly lower in A than in GP . Moreover, the values of METs in 2mean from 1.5 to 3.0 m∙s−1 were significantly lower in A than in GP . Lastly, the relative levels of 2mean from 1.5 to 3.5 m∙s−1 were significantly lower in A than in GP   . Furtherctively) .− at 1.5, 2.0, 2.5, 3.0, and 3.5 m∙s−1 were significantly higher in GP than in A  , while the values of CHOox at 2.5, 3.0, and 3.5 m∙s−1 were significantly higher in GP than in A  (Lactively) A. Moreovctively) B,C.− and FATox in GP and A  = 32.96; p < 0.0001, r = −0.661; 95%CI: −0.7715–−0.5119; R2 = 0.436; F (1.74) = 57.43; p < 0.0001, respectively) (Moderate negative correlations and linear regressions were observed between Lactively) D,E.−1 La− were significantly higher in A than GP, while the HR values from 1.5 to 4.0 mmol∙L−1 La− were significantly higher in GP than A  (The jogging/running speeds from 1.5 to 4.0 mmol∙Lctively) A–H.−1 showed that absolute (kJ) WGly in male GP was significantly lower than that in female GP , while WPCr (kJ) in male GP was significantly higher than that in female GP . Moreover, similar results were found in the relative (%) energetic contributions  There were no significant differences in energetic contributions between males and females in A and total energy demand between males and females in GP and A (p > 0.05)  A–T.PCr in kJ at 1.5 m∙s−1 was significantly higher in GP than A , while WGly in kJ from 1.5 to 3.5 m∙s−1 was significantly higher in GP than A . WOxi in kJ showed no significant difference between both groups. Furthermore, the relative energetic contributions (%) showed that only WPCr at 2.0 m∙s−1 was significantly lower in GP than A . Moreover, WGly in % from 1.5 to 3.5 m∙s−1 showed significantly higher values in GP than A . WOxi at 2.0 m∙s−1 was significantly higher in GP than A  while WOxi at 3.0 m∙s−1 was significantly lower in GP than A   A,B.−1 steps during GIET were compared between males and females in GP and A. To our knowledge, this study is the first to compare the FATox and CHOox, combining three energy system contributions while including gender differences during jogging/running GIET between both groups. Compared with GP, lower percentages of HRmax, relative 2mean and 2mean, and METs in 2mean were observed in A. The values of La−, CHOox, WOxi, WGly, and WPCr were found to be higher in GP than in A, including lower and higher mean absolute and relative values of WGly and WPCr were found in male GP than in female GP, respectively, while FATox was higher in A than in GP. Both groups showed predominant usage of the oxidative energy system and moderate negative correlations between La− and FATox.Physiological variables, metabolic flexibility, and three energy systems during the graded incremental jogging/running test were compared between GP and A. Moreover, the mean energetic contributions up to 3.5 m∙s−1 only. However, the values of HR in A during GIET tended to be lower than in GP, and the HR values at certain La− in A were lower than in GP. Furthermore, the percentages of HRmax, relative 2mean and 2mean, and METs in 2mean were all lower in A than in GP, thus indicating that athletes performed at a relatively lower exercise intensity compared to GP during the same absolute exercise intensity. These phenomena might be associated with so-called “athlete’s heart” [2-related parameters mentioned above.There was a significant difference in HR at 3.0 m∙ss heart” . Cardiovs heart” . For inss heart” ,32,33. Ts heart” ,34,35,36− levels were lower in A, in which, in turn, FATox was higher than it was in GP during GIET. This is attributable to the greater lactate elimination in A than in GP [− and FATox during exercise represents an alternative approach to reflecting mitochondrial function and metabolic flexibility in ambulatory and clinical settings [−, while the uptake of free fatty acids can be inhibited by accumulated malonyl-Coenzyme A (CoA), which decreases carnitine palmitoyltransferase 1 (CPT1) activation, beta (ß)-oxidation in mitochondria, and gluconeogenesis [ß-oxidation in mitochondria inhibits pyruvate dehydrogenase (PDH). However, the tricarboxylic acid cycle is substituted, to enable continued anaplerosis and pyruvate carboxylase (PC), because inhibited PDH is the major anaplerotic enzyme that immediately synthesizes oxaloacetate from pyruvate [+ handling [− and FATox support these mechanisms and the differences in mitochondrial function between both groups [Gly compared with A during GIET. Moreover, different values of WOxi in % were affected by higher WPCr and WGly in kJ. However, there were no differences in absolute WOxi and WTOTAL between both groups. The predominance of energetic contributions during GIET was WOxi between both groups, including males and females in GP and A. The values of WOxi were indicated to be over 90% up to 3.5 m∙s−1 steps during GIET in GP and A. However, relative WOxi (%) in A was progressively decreased with increasing exercise intensity from 4.0 to 4.5 m∙s−1 because of the increased anaerobic contributions, such as the increased lactate production rate (a reduction in WOxi of only 8.6% from the first to the last stage). These phenomena were also confirmed in a previous study by Bertuzzi et al. [Gly and higher WPCr than females in GP; there were no significant differences in WOxi. However, a higher trend toward WOxi was observed in male GP than in female GP, while males and females in A showed no significant differences, although there were similar tendencies to GP among the three energy systems , were mostly performed (approximately ≥ 80%) by elite athletes such as runners and marathoners, to improve their performances during preparatory, pre-competitive, and competitive periods [−), which can enable greater volumes at zones 2 and 3 and protect against cardiac/metabolic diseases [In previous studies, the volume of individualized low-intensity exercises (ILIE), such as high-volume training (HVT)  is necessary, to compare CHOox in different individuals. Moreover, direct parameters regarding mitochondrial function were not measured. Therefore, further studies are needed to investigate carbohydrate availability during higher exercise intensity. Indeed, studies should examine how the same interventional approach of the abovementioned training prescription in elite athletes affects metabolic flexibility, by conducting an analysis of proteomics and metabolomics and the energetic contributions in GP and in individuals with cardiac/metabolic diseases.The current study has some limitations. Higher exercise intensity , which are associated with mitochondrial function, cardiovascular fitness, and metabolic syndrome. To achieve physical health benefits in terms of increased FATox and La− elimination, along with improved oxidative capacity such as endurance performance, it is recommended that aerobic base training (baseline) as ILIE within a recovery domain (zone 1) be performed, as this is predominantly performed by elite athletes worldwide during training periods, supports higher exercise intensities (permissive of a higher volume), and may protect against risks of cardiac/metabolic diseases.Our findings show higher percentages of HR


This study aims to evaluate the mechanical properties of carbon fiber-reinforced reactive powder concrete (CFRPC) after exposure to cryogenic temperature. The mechanical properties of plain RPC and CFRPC with carbon fiber volume contents of 0, 0.5%, 1.0%, and 1.5% were examined after exposure to 20 °C, −5 °C, −15 °C, and −25 °C for 72 h. The effect of fiber contents and exposure temperatures on the cubic and axial compressive strength, splitting tensile strength, elastic modulus, and peak strain were systematically reported and analyzed. The results showed adding carbon fiber to RPC could significantly enhance the strength and slightly improve ductility performance. Additionally, CFRPC with 1.0% fiber content showed the best mechanical properties. The maximum increases in cubic and axial compressive strength and tensile strength were 26.0%, 25.7%, and 21.8%, the elastic modulus was 13.2%, and the peak strain was 13.0% over the plain RPC. Additionally, all mechanical properties continued to degrade with decreasing temperature. After exposure to −25 °C, the cubic, axial compressive strength, and tensile strength of CFRPC degraded to 82.2–84.9%, 80.7–87.5%, and 72.7–73.7% of the normal temperature strength, respectively. In addition, the linear relationship equation between the discount factor of each mechanical property and the temperature was established. Finally, the equation for the stress–strain ascending curve of CFRPC described by a quadratic polynomial was proposed, which fitted well with the experimental results. The cryogenic temperature, one of the most typical extreme environments, will directly threaten the safety of concrete structures in service and lead to premature deterioration of the material. According to GB50176-2016 ,9, whileNowadays, reactive powder concrete (RPC) is widely used because of its good mechanical properties and durability ,11,12,13Therefore, it is necessary to develop other fiber-reinforced materials, considering that SF has high self-weight, poor corrosion resistance ,26, and Based on the current research results, it can be seen that most of the research has focused on the performance evaluation of SF and PPF reinforced RPC at room temperature and high temperature, while the exploration of the performance of CFRPC is very limited. In particular, there is a gap in the study of the performance of CFRPC after cryogenic temperatures. Therefore, tests on the mechanical properties of CFRPC with various CF volume contents after exposure to cryogenic temperatures were conducted in this work. The cubic and axial compressive strengths, splitting tensile strength, elastic modulus, and peak strain were all measured. The equation for expressing the reduction factor of each mechanical index with the temperature was derived based on experimental data. Finally, the equation for the CFRPC stress–strain ascending curve is found. As an exploratory study, the results in this paper will help to understand the mechanical behavior of CFRPC after exposure to common cryogenic temperatures and provide detailed material performance data for the structural design of CFRPC in severe cold areas.Specimens were prepared with the following ingredients: Harbin Yatai Group produces ordinary Portland cement of grade 42.5 (Chinese cement grading system). The details of cement are provided in First, 100 mm × 100 mm × 100 mm cubic specimens were used for compressive strength testing and splitting tensile strength testing following CECS13:2009 . The axiThe three-step mixing technology provided in reference  was adopAs shown in ηcu,T is introduced.ηcu,T is the cubic compressive strength reduction factor. fcu,T is the cubic compressive strength after exposure to cryogenic temperature T. fcu,20 is the cubic compressive strength at 20 °C. ηcu,T ≤ 1 represents the degradation degree of cubic compressive strength after exposure to cryogenic temperature T.To describe the deterioration degree of the cubic compressive strength with temperature, a coefficient fcu after various exposure temperatures. ηcu,T with temperatures. After exposure to cryogenic temperatures, the cubic compressive strength continued to degrade with decreasing temperature. When compared to 20 °C, the strength of −5 °C, −15 °C, and −25 °C reduced by 10.2–12.7%, 15.1–22.3%, and 12.8–14.4%, respectively. Meanwhile, the plain RPC strength was more severely degraded than CFRPC. After exposure to −25 °C, the plain RPC showed the lowest strength of 65.5 MPa, which was 77.7% of the strength at 20 °C. While for CFRPC1, CFRPC2 and CFRPC3, the strengths were 74.3 MPa, 82.5 MPa, and 76.8 MPa, respectively, reducing to 82.2%, 84.9%, and 83.2% of the normal temperature strength. Based on two considerations, microstructural degeneration is the root cause of its strength degradation [radation . FirstlyOn the other hand, CF addition significantly improved the cubic compressive strength of RPC after different exposure temperatures. CFRPC1, CFRPC2 and CFRPC3 showed 7.5–13.4%, 15.3–26.0% and 9.5–17.3% higher than the plain RPC, respectively. This is due to the high tensile strength and elastic modulus of CF. CF that are evenly distributed in the concrete can be bonded with the matrix to generate a network structure that improves stiffness and toughness. CF can restrain the initial cracks and microcracks in the specimen, inhibiting premature cracking and allowing the strength of the base material to be fully utilized. However, due to their thin and short length, the CF cannot prevent the formation and development of large cracks. This is the main reason why CF-reinforced RPC specimens still exhibit brittle characteristics at failure. In addition, CFRPC2 with 1.0% CF showed the best cubic compressive strength. From 20 °C to −25 °C, the strength was 97.2 MPa, 85.6 MPa, 84.7 MPa, and 82.5 MPa, respectively, which was 15.3%, 16.3%, and 17.3%, and 26.0% higher than the plain RPC. When the CF content is less than 1.0%, the fracture resistance and strength enhancement of fibers is not obvious, and the specimen strength is poor. When the CF content was more than 1.5%, due to the excessive fibers produced by the “pellet effect”, the formation of weak interface areas, increasing the internal defects of concrete, which in turn leads to an unsatisfactory reinforcement effect. As a result, 1.0% is advised as the appropriate CF volume content, which is consistent with the reference .ηcu,T can be more visually expressed in ηcu,T than CF content. For broad application, a linear function is utilized to describe the relationship between ηcu,T and T, as expressed by Equation (2). R2 = 0.965, the linear fitting formula is highly accurate.The effect of temperature and CF content on The addition of CF changes the failure mode, as shown in fc of CFRPC after different exposure temperatures are given in ηc,T with T is given in ηc,T is the axial compressive strength reduction factor and is calculated in the same way as Equation (1). The axial compressive strength decreased as the temperature dropped. The strengths at −5 °C, −15 °C and −25 °C were 7.7–14.3%, 11.0–16.8% and 12.5–22.8% lower than at 20 °C, respectively. Meanwhile, the plain RPC strength was more severely degraded than CFRPC. After exposure to −25 °C, the plain RPC showed the lowest strength of 63.7 MPa, which was 77.2% of the strength at 20 °C. While for CFRPC1, CFRPC2 and CFRPC3, the strengths were 71.3 MPa, 80.1 MPa and 76.6 MPa, respectively, reducing to 80.7%, 87.5%, and 86.1% of the normal temperature strength. The degradation of axial compressive strength, like that of cubic compressive strength, is caused by microstructural degeneration after cryogenic temperatures. Meanwhile, CF enhanced the axial compressive strength. When compared to plain RPC, the strengths of CFRPC1, CFRPC2, and CFRPC3 were enhanced by 7.2–11.9%, 10.9–25.7%, and 7.9–20.3%, respectively. Furthermore, CFRPC2 with 1.0% CF performed better in axial compressive strength compared to other fiber content. From 20 °C to −25 °C, the strengths of CFRPC2 were 91.5 MPa, 84.5 MPa, 81.4 MPa, and 80.1 MPa, respectively, which were 10.9%, 19.5%, 18.7%, and 25.7% higher than plain RPC.The axial compressive strengths ηc,T is more significant, as shown in ηc,T and T. The correlation coefficient is R2 = 0.911 with high accuracy.The effect of temperature on As shown in fc/fcu is to 1.0. It is assumed that both CFRPC axial compressive strength and cubic compressive strength obey normal distribution, and the coefficient of variation of the two strengths is not significantly different by calculation. Therefore, fc,k and fcu,k also conform to the relationship of Equation (4).The ratio of axial compressive strength to cubic compressive strength is 0.97 can be obtained from Equation (4). It is much larger than 0.76 for ordinary concrete and 0.82 for high-strength concrete. It means that the higher the strength of concrete, the closer The splitting tensile failure mode of specimens was brittle, as shown in fts of CFRPC after various exposure temperatures. ηts,T with T. ηts,T is the reduction factor of splitting tensile strength and calculated in the same way as Equation (1). As with the compressive strength, the splitting tensile strength decreased significantly with the drop of temperature. The strengths at −5 °C, −15 °C, and −25 °C decreased by 8.0–10.3%, 16.3–22.7%, and 23.5–27.3%, respectively, compared to 20 °C. After exposure to −25 °C, the plain RPC had the lowest splitting tensile strength of 5.2 MPa, which was 76.5% of the strength at 20 °C. For CFRPC1, CFRPC2 and CFRPC3, the strengths were 5.5 MPa, 5.9 MPa, and 5.6 MPa, respectively, reducing to 73.3%, 73.7%, and 72.7% of 20 °C strength. The reason for large strength deterioration is the intensified cryogenic temperature damage and the continuous development of internal cracks in specimens. A comparison of the compressive strength showed that the tensile strength degraded more severely, due to a greater sensitivity to structural microcracks caused by the decrease in temperature. Moreover, CFRPC splitting tensile strength was higher than RPC after different cryogenic temperatures, indicating a positive effect of CF. CFRPC1, CFRPC2, and CFRPC3 improved by 5.5–13.1%, 13.5–21.8%, and 14.8–7.7% over the plain RPC, respectively. This is due to CF excellent crack resistance, which contributes to the ultimate tensile strength of concrete. Furthermore, CFRPC2 with 1.0% CF showed the highest splitting tensile strength. From 20 °C to −25 °C, 17.6%, 19.7%, 21.8%, and 13.5% increased, respectively.Tensile strength is another important mechanical property of concrete. ηts,T than fiber content. As a result, the linear function of Equation (5) is employed to characterize the relationship between temperatures and ηts,T. R2 = 0.920, the linear fitting formula is highly accurate.The contour lines from As shown by Equation (6), the specification GB 50010-2010  gives thηE,T is defined as the elastic modulus reduction factor and calculated in the same way as Equation (1). Additionally, the relationship between ηE,T and T is described by Equation (8), which fitted the test results well.Ec and the axial compressive strength fc of CFRPC after exposure to cryogenic temperatures can be obtained by non-linear regression as followsFrom the experimental results of this paper and references ,50, it cEquations (10) and (11) are the fitted equations for the elastic moduli of SFRPC  and RPC ηε,T is introduced as a peak strain reduction factor to express the degradation of peak strain due to low temperature, calculated in the same way as Equation (1). Additionally, the linear relationship between ηε,T and T is expressed as follows:Peak strain, to some extent, indicates concrete ductility. In general, the higher the peak strain, the greater the material’s ductility . It can As shown in The curves of the peak strain of SFRPC , RPC 5050 at norx = ε/εp,T and ordinate y = σ/σp,T, where ε and σ denote stress and strain, and εp,T and σp,T denote peak strain and peak stress after cryogenic temperature. CFRPC stress–strain curves after various cryogenic temperatures had the same geometric properties as those at normal temperatures. Additionally, both of them may be described by the same equation. Therefore, the compressive stress–strain equation for an ascending curve was fitted with a quadratic polynomial, as follows:Polynomials are mostly used to express the equation of the stress–strain ascending curves for RPC and fiber-reinforced RPC, as shown in x = 0 and y = 0 into account, α = 0.Taking the boundary constraints x = 1 and y = 1, we obtainWith the boundary constraints α1 as α, the equation is obtainedNoting It is clear from Equation (16) that different curves can be formed for different values of α.x = 0, α is as follows:E0,T at the curve’s zero point to the secant modulus, EP,T at the curve’s highest point. From the shape of stress–strain curves measured in tests, it is clear that E0,T ≥ EP,T, so α ≥ 1.When α = 1.35 is taken and replaced into Equation (16). As illustrated in Equation (18), the equation for the compressive stress–strain curve of CFRPC after cryogenic temperatures is found. In Based on the findings of the tests in this study, Adding CF to RPC can significantly enhance its strength and slightly improve ductility performance. CFRPC with 1.0% fiber volume content showed the best mechanical properties. The maximum increase in cubic and axial compressive strength and tensile strength is 26.0%, 25.7%, 21.8%, the elastic modulus is 13.2%, and the peak strain is 13.0% over the plain RPC.After exposure to cryogenic temperatures, strength and ductility continued to degrade with decreasing temperature. After exposure to −25 °C, the plain RPC showed 77.7%, 77.2%, and 76.5% lower cubic, axial compressive strength, and tensile strength than those of the normal temperature, respectively. While for CFRPC, the cubic, axial compression, and tensile strength degraded to 82.2–84.9%, 80.7–87.5%, and 72.7–73.7% of normal temperature strength, respectively.fc and fcu, the exponential function relationship between fts and fcu, and the root function relationship between Ec and fc, εp and fc were defined. Each equation agreed well with the test data. The mechanical properties and their relationship equations in this study can provide a basis for the research and engineering application of CFRPC in severe cold regions.Equations to express the linear relationship between the discount factor of cubic compressive strength, axial compressive strength, splitting tensile strength, elastic modulus, and peak strain with the exposure temperature were established in this paper. Moreover, the linear relationship between The compressive stress–strain ascending curves of CFRPC with different CF contents and after different cryogenic temperatures showed similar linear characteristics. Comparative analysis showed that, as the temperature decreased, the peak point shifted to the right and downwards, and the ultimate stress, peak strain gradually decreased. Cryogenic temperatures caused degradation of the strength and ductility of CFRPC. Finally, a quadratic polynomial equation expressing the stress–strain ascending curve was proposed for CFRPC at room temperature and after exposure to cryogenic temperatures, which fitted the test results well.In this study, the mechanical properties of CFRPC after exposure to cryogenic temperatures were investigated. The following conclusions can be drawn from the experimental results:


Tetrapleura tetraptera as well as seeds of Monodora myristica have largely focused on phytochemical properties of aqueous and methanolic and ethanolic extracts. To supplement existing information, the chemical composition, antibacterial efficacy (tested against Escherichia coli and Staphylococcus aureus), and antioxidant capacity  radical scavenging, ferric reducing power, and total antioxidant capacity) of essential oil and oleoresin extracted from T. tetraptera fruit and M. myristica seeds cultivated in Southeast Nigeria, were studied. Essential oil and oleoresin were respectively extracted by steam distillation and aqueous maceration. By way of gas chromatograph mass spectrometry (GC–MS) analysis, the chemical compounds from essential oil and oleoresin from M. myristica and T. Tetraptera samples totaled 6 and 5, as well as 27 and 16, respectively. Besides the oleoresin of M. myristica and the essential oil of T. tetraptera showing some resistance against S. aureus, the oleoresins seemed highly susceptible to E. coli—all of which demonstrated concentration-dependence to the antibacterial inhibition zone. Scavenging DPPH radical, reduction power activity, and total antioxidant capacity increased with essential oil and oleoresin extracts' concentrations, which positions M. myristica and T. tetraptera spices as very promising for food preservation, especially against autoxidation and microbial spoilage.Specific to the West African sub-region, previous studies involving fruit, stem, and bark of  Spices are among such particularly large groups of edible plants with beneficial natural ingredients usually added to foods. Furthermore, the capacity to influence both aroma and taste of foods is largely owed to the presence of essential/volatile oils that comprise terpenes and terpenoids along with various aliphatic hydrocarbons, acids, alcohols, etc.2, able to control food spoilage and prolong shelf life3. In addition to essential oils, spices show potential health benefits through antimicrobial, antioxidant, antidiabetic, anti-inflammatory, anti-viral, and antiprotozoal capacities. Additionally, the chemical composition and medicinal properties could be influenced by genetics and the type of extraction method3. In addition to their abundance from rural to urban areas, spices from African flora have helped in the discovery of novel drugs useful in biomedicine, which have helped to promote health and tackle diseases such as cancer, tumors, etc. Examples of indigenous spices in Africa include black pepper (Xylopia aethiopica), West African pepper (Piper guineese), Mentha piperita, Ocimum gratissimum, Tetrapleura tetraptera and Monodora myristica. Unlike the exotic types, most of the above-named spices are wholly used and continue to receive research attention given the reported phytochemical constituents, antioxidant, and antimicrobial capacities7.Globally, edible plants continue to be of research interest given their natural compounds and medicinal properties that are capable of improving health and preventing/combating diseasesT. tetraptera is a deciduous perennial flowering medicinal plant of West African origin belonging to the pea family(Fabaceae), and commonly known as aridan and yanayan amongst the Yoruba and Urhobos respectively in Nigeria and prekese in Ghana5. The essential oil present in T. tetraptera provide a distinctive pleasant fragrance and aroma, making its dry fruit a popular seasoning spice in South-eastern Nigeria5. Ghanaians believe that T. tetraptera fruits contain multivitamins, useful in the management of jaundice, inflammation, convulsion, fever, and leprosy8. The phytochemical constituents of T. tetraptera fruits provide medicinal attributes demonstrated by molluscicidal, antimicrobial, antioxidant, anticonvulsant, anti-inflammatory, antimalarial, antidiabetic, and anticancer properties11. Microbiological studies show the extract of T. tetraptera exhibits excellent antimicrobial activity against Gram-positive and Gram-negative bacteria12. On the other hand, the M. myristica, also known as African/calabash nutmeg, is a perennial plant of the family Annonaceae, which thrives in the tropics of West Africa and the Caribbean. Common names of this plant in Nigeria include ehuru, ariwo, ehiri, airama, and awerewa, lubushi6. The seed is rich in oil and is of great value due to its medicinal and nutritional qualities3. The fruits and seeds are used as stimulants, stomachic, against headaches, and sores, and as a natural insect repellent. The essential oil contains compounds like α-phellandrene, α-pinene, myrcene, limonene, and pinene. The pleasant aroma, like nutmeg, makes this spice useful in the preparation of traditional dishes in Nigerian communities6. Spice seed extracts are believed to possess both antioxidant7 and antimicrobial properties3. M. myristica could be effective in the treatment of stomach aches, febrile pains, eye diseases, and hemorrhoids13, while Tetrapleura tetraptera helps to tackle diabetes mellitus, arthritis, hypertension, epilepsy, and asthma5.On one hand, the T. tetraptera and M. myristica are among such common indigenous spices in Nigeria that are under-utilized/-valued, despite their sharp aroma and flavor that is highly perceivable by the sense of smell4. However, specific to West Africa sub-region, previous studies involving fruit, stem, and bark of T. tetraptera as well as seeds of M. myristica have largely focused on phytochemical properties of aqueous and methanolic and ethanolic extracts. There is a need, therefore, for further investigations into the essential oil and oleoresin of this T. tetraptera and M. myristica, specifically their chemical composition, antioxidant, and antibacterial properties and such studies would require respective activities of steam distillation maceration technique, which would help unravel their potential/relevance in the various communities in southeast Nigeria where they serve as a natural preservative to prevent food spoilage. To supplement existing information, this current work investigated the chemical composition, antibacterial efficacy, and antioxidant capacity of essential oil and oleoresin extracted from T. tetraptera fruit and M. myristica seeds cultivated in Southeast Nigeria. Specifically, the essential oil and oleoresin respectively extracted by steam distillation and aqueous maceration were subsequently subject to analytical tests in adherence to the relevant institutional guidelines. Furthering the knowledge and understanding underpinning the capacities of these extracted essential oil and oleoresin to tackle food spoilage challenges is warranted to help consolidate the product development potential of both T. tetraptera fruit and M. myristica seeds.Both T. tetraptera fruit and M. myristica seeds would thrive specific to the context of chemical composition, antibacterial efficacy (tested against Escherichia coli and Staphylococcus aureus), and antioxidant capacity  radical scavenging, ferric reducing power (FRAP), and total antioxidant capacity (TAC). Additionally, the analytical measurements were carried out independently using the different essential oil and oleoresin samples obtained from individual batches of T. tetraptera and M. myristica seeds. Importantly, the analytical measurements conducted were in adherence to the relevant guidelines prescribed by the Department of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. Furthermore, all chemicals and reagents used in this work, which were procured from reputable registered chemical retailers, were of analytical grade standard.Figure T. tetraptera fruit (approx. 1000 g) and M. myristica seeds (approx. 800 g) were procured as two separate batches from a local market in Owerri Imo State, in the Southeast of Nigeria. Furthermore, Fig. M. myristica seeds , and then oven dried using a Memmert UN30  oven at 55 °C for 72 h. The M. myristica seeds were cracked manually to recover the nibs, whereas the fruits of T. tetraptera were cut into small pieces, and both were kept in ambient conditions until required for further processing.Fresh eds Fig. a and T. its Fig. b. To furM. myristica seeds, and small pieced cut T. tetraptera fruit samples were individually ground into coarse and fine particles using an attrition type mill K-Tron type T-35volumetric feeder (K-Tron Corp. Pitman NJ USA). The coarse particles were kept for essential oil extraction, whereas those of fine powder was for oleoresin extraction. On one hand, essential oil extraction involved the steam distillation method as described by Chemat et al.15 with slight modification. Briefly, the conventional type steam distillation apparatus with a cylindrical Pyrex body supported by Teflon grid at its lower end, with a rheostat-controlled heating facility, was used. The essential oil was dried over anhydrous sodium sulfate (Na2SO4) and stored in airtight amber bottles at 4 °C until used. On the other hand, the oleoresin extraction involved the maceration technique modified from Fernández‐Ronco et al.16. For each spice, 200 g of finely ground spice was macerated in 400 ml of distilled water in a 1000 ml conical flask and tightly corked. This was shaken vigorously at 30-min intervals for 3 h, after which the supernatant was decanted and the residue was filtered out. After extraction, the supernatant from each spice was collected and evaporated at 60 °C in a stifling air oven  for 8 h. The oleoresins were stored in airtight amber bottles and kept in the refrigerator until used.Already cracked The chemical composition of the crude essential oil and oleoresin samples was determined following the routine method employed in the laboratory of the Department of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. This involved the gas chromatograph mass spectrometry (GC–MS) analysis, specifically the Agilent 6890 gas chromatograph  equipped with an on-column automatic injector, flame ionization detector, and HP 88 capillary column (100 m × 0.25 μm film thickness). The carrier gas was helium at a constant flow rate of 1.0 mL/min. The detector was maintained at 250 °C and both injectors at 220 °C with integrator chart speed at 2 cm/min. The initial column temperature was set at 100 °C (hold time =  ~ 2 min) to the final temperature of 180 °C at a rate of 50 °C/min, the volume injected was 1.0 μL and the split ratio was 50:1. Total chromatogram was auto-integrated by Shem-Station, and emergent chemical constituents were identified by comparison with both published mass spectral database (NIST02.L) and available literature data.17 was used. The microorganisms (0.1 mL) from previously prepared diluents were spread on previously prepared nutrient agar in standard Petri dishes in triplicates. Wells of 2 mL diameter and 0.5 mL depth were created using a sterile bore. The essential oil and oleoresin samples were introduced using a sterile micropipette, after which Petri dishes were stored at 37 °C for 24 h in an incubator to observe and measure the zone of inhibition (mm) of microbial growth caused by the aforementioned substances.The agar well diffusion method according to Balouiri, Sadiki, and Ibnsoudaaureus ATCC 27840 and Escherichia coli 0157, routinely used for the antibacterial assay, were obtained from the microbiology department of the University of Nigeria Nsukka, Nigeria. Substrates were refrigerated at 4 °C for 5 days and the growth and survival of the pathogens were monitored using the Plate colony-counting method. Using 1.0 mL from each pathogen Escherichia coli, and Staphylococcus aureus, suspended to a density of 105, were inoculated into a flask with nutrient agar. Extracts diluted in 0.1% buffered peptone water to appropriate concentrations, subsequently mixed thoroughly, and aseptically poured onto a petri dish. Colonies were manually counted periodically within 5 days of incubated storage at 37 °C.Staphylococcus 18 with slight modifications. This involved dissolving ~ 25 mg of DPPH in 100 mL methanol, and thereafter storing at 20 °C till needed. From the working solution obtained from the DPPH stock solution by methanol, 1 mL was added to 100 μL of various concentrations of test samples, incubated for 45 min, thoroughly shaken, and then left in the dark under ambient conditions. Ascorbic acid served as the standard, scavenging effect was calculated by Eq.  as described by Shah et al.3+ to Fe2+ was assessed by the method of Hinneburg, Dorman, and Hiltunen19 with slight modifications. A 2-mL aliquot of sample was added to 2.5 mL of sodium phosphate buffer  containing potassium ferricyanide [K3Fe (CN)6] . After the reaction mixture had incubated at 50 °C for 25 min, then 2.5 mL of trichloroacetic acid (10%) was added and centrifuged at 3000 rpm for 10 min. Subsequently, the supernatant (5 mL) was mixed with 2.5 mL of water, and 0.5 mL of 0.1% aqueous FeCl3, followed by an absorbance reading at 700 nm. Gallic acid was used as a standard reference compound.The ability of essential oil and oleoresin to reduce Fe20 with slight modifications. An aliquot (~ 0.1-mL) of the sample was mixed with 1 mL reagent solution , thereafter covered and incubated in a water bath at 85 °C for 75 min. When samples had been cooled, the absorbance was measured at 765 nm. The ascorbic acid was used as a standard reference compound. The TAC was estimated using the Eq.  of the essential oil and oleoresin was determined by the phosphomolybdate method of Jan et al.The data of three replicates from different samples were subjected to factorial approach analysis of variance (ANOVA). Results were represented as means ± standard error, with mean separation conducted using Fisher’s least significant difference (LSD) per measured variable. The probability level was statistically significant at p < 0.05 (95% confidence). Minitab 2018 software  was used to run the data.M. myristica and T. tetraptera are shown in Tables M. myristica, the identified chemical compounds predominantly monoterpenes respectively totalled 6 and 5 in essential oil and oleoresin. Chemical compounds in essential oil would include p-cymene at 50.58% as peak, followed by α-phellandrene at 32.09%, with α-pinene at 8.71% as least, whereas in oleoresin would include, 2-acetyl cyclopentanone (85.42%) as predominant, with α-phellandrene (6.40%) as least. Only δ-cadinene (1.56%) appeared in the sesquiterpene but very little. Previous reports about different extracts of M. myristica specific to Nigeria obtained variations in chemical constituents. For example, a report of seed oil of M. myristica from southwest Nigeria obtained major constituents like α-phellandrene epoxide (3.02%), carvacrol (2.09%), and δ-cadinene 21, as well as p-cymene (31.5%), α-phellandrene (18.1%), α-pinene (6.1%), β-pinene (5.1%)22. Elsewhere also, essential oil of M. myristica obtained by steam distillation showed chemical constituents like α-phellandrene (50.4%), α-pinene (5.5%), myrcene (4.35%), and germacrene-d-4-ol (9.0%)23, whereas another seed oil reported elsewhere showed chemical constituents like germacrene-d-4-ol (25.48%), tricyclo [5.2.1] dec-2-ene (13.35%), δ-cadinene (11.09%) and linalool (15.10%), and from their stem barks with chemical constituents like γ-cadinene (31.31%), α-elemene (17.98)13. In a neighbouring country of Chad and Cameroon, the essential oils extracted by hydrodistillation from fruits of Xylopia aethiopica  A. Rich, Xylopia parviflora (A. Rich) Benth.) and M. myristica (Gaertn) showed α-phellandrene  as a major constituent24. Specific to T. Tetraptera, the chemical compounds of essential oil and oleoresin respectively totalled 27 and 16 (Tables N-(triphenylphosphoranylidene)- (7.78%), 4-dibenzofuranamine (7.08), etc. On the other hand, the oleoresin comprised monoterpenes, for example, γ-terpinene (25.63%), linalool (20.74%), caryophyllene (9.68%), β-pinene (9.38%), 2-carene (6.98), γ-elemene (5.47%), etc. Terpenes were not found in the essential oil of T. tetraptera. The essential oil extracted from T. tetraptera fruits via hydro-distillation by Erukainure et al.4 largely showed acetic and carboxylic acid, with little traces of terpenes. Probably, the presence of linalool might account for the plant’s peppery nature, whereas both γ-terpinene and α-thujene might account for the flavor/fragrance. Elsewhere, the root and stem ethanol extract of T. tetraptera spice respectively obtained 70.14%25 and 32.01%10. Polyphenols like eugenol, quercetin, rutin, and tyrosol, according to Moukette et al.1, were considered 5.88–10.79-fold more concentrated in the water and ethanolic extracts of the fruits compared to the barks. Furthermore, α-phellandrene and α-pinene has been identified as a major chemotype for M. myristica seeds, whereas citral and linalool as major chemotypes of T. tetraptera22. Such factors as environmental influences, experimental conditions, place of origin, post-harvest handling of the fruits, adaptive metabolism of plants, and plant part analyzed might be responsible for the variations in the chemical compounds that have been detected in M. myristica and T. tetraptera samples at this current work.The main chemical compounds of essential oil and oleoresin of anone 85.2% as preT. tetraptera would exhibit very promising antimicrobial activity against Gram-positive/-negative bacteria12. Table M. myristica and T. tetraptera, which appeared concentration-dependent to the bacterial pathogens. Whereas oleoresin obtained the highest susceptibility to E. coli, those of M. myristica together with the essential oil of T. tetraptera showed some resistance to S aureus. Considering that the inhibition zone qualitatively deciphers the potentials of an antibacterial agent, the essential oil of M. myristica at concentration of 75 µg/mL obtained the highest zone (16 mm) against E. coli whereas the essential oil from T. tetraptera obtained the least activity. Elsewhere, inhibition zones of 8 mm have been reported and 13 mm for 2.5 mg/mL and 5.0 mg/mL, respectively, of essential oil M. myristica against E. coli3, whereas 11 mm for 2 mL also of M. myristica essential oil against E. coli26. Another study showed T. tetraptera stem extract against two Salmonella strains, with a remarkably high inhibition zone (> 20 mm diameter)10, besides the absence of carvone component believed as responsible for the weak activity of oleoresins28. Comparatively, the promising antibacterial activity of essential oil from M. myristica might be dependent on factors like chemical composition and its solubility3. The extraction process, cell wall composition of microbial entity, environmental conditions, chemotype of the plant parts used (which often influences biosynthetic pathways), as well as other physiological properties might also potential factors that contribute to the differences in the antibacterial activity.As mentioned earlier in this paper, there is evidence that the extract of 29 and promising in controlling pathogenic/spoilage microbial entities in foods. The survival of the E. coli and S. aureus population involving different concentrations of Mondora myristica and Tetrapleura tetraptera essential oil and oleoresin can be seen in Table E. coli and S. aureus population appeared significantly higher (p < 0.05) in the essential oil above those of oleoresin. Within three (3) days of storage, specific to concentrations 25 and 50 µg/mL of essential oil from T. tetraptera, the pathogen population increased markedly (p < 0.05) at 2.15 × 105 and 1.53 × 105 CFU/mL (E. coli) and 2.5 and 2.03 × 105 CFU/mL (S. aureus), respectively. Within 3 days of storage, however, no bacteria proliferated in T. tetraptera oleoresin as well as M. myristica essential oil and oleoresin. By day 5, the T. tetraptera essential oil respectively showed 7.05 and 6.10 × 105 CFU/mL for E. coli and S. aureus  from 1.5 × 104 CFU/mL at 25 µg/mL for T. tetraptera oleoresin. Gram-positive bacteria are believed to reveal more susceptibility to antibacterial compounds compared to Gram-negative bacteria30, given the lipoproteins and lipopolysaccharides present in the cellular wall/barrier-like structure that restricts the entry of hydrophobic compounds31. In essential oil, however, presence of terpenes should provide the major antibacterial capacity. More so, some simple phenols might appear in the form of phenylpropanoids32. Besides essential oils and oleoresins of this current work being concentration-dependent, their complex mix of several (minor) chemical components would assert multifunctional synergistic effects34.Natural compounds (terpenes inclusive), which are secondary metabolites from plants, are responsible for the defense against pathogensr p < 0.0 in the eM. myristica and T. tetraptera spices can be seen in Table T. tetraptera essential oil. The oleoresin of T. tetraptera showed the better DPPH radical scavenging activity across samples’ concentrations. For example, the highest concentration (640 µg/mL) of T. tetraptera oleoresin obtained maximum scavenging power (93.46%) that appeared significantly higher (p < 0.05) compared to the others, which specifically demonstrates enhanced antioxidant properties. Previous reports show roughly 29–37.7% scavenging powers of M. myristica seeds could concur with maximum concentration of 120 µg/mL35, contrasting 10–41% when concentration is maximum at 400 µg/mL7. Lower DPPH scavenging has been recorded at T. tetraptera fruit and bark ethanolic and hydroethanolic extracts36. Typically through the reduction of DPPH, the radical scavenging compounds  act like an antioxidant during lipid oxidation, given the ability to donate an electron or hydrogen molecule37. Of all reactive oxygen species, the OH radical appears the stronger, and able to react with most biological molecules found in living cells. Among effective defences of living body against various diseases is the removal of hydroxyl radicals38.The DPPH radical scavenging activity of essential oils and oleoresins (%) at different concentrations in M. myristica and T. tetraptera spices can be seen in Table M. myristica of this study. Specifically, M. myristica essential oil obtained the peak highest reducing power with mean absorbance value of 54.69 at concentration of 640 µg/mL. Despite the higher values at concentrations higher than 100 µg/mL7, the ethanol extract of T. tetraptera fruit peel (at similar concentrations higher than 100 µg/mL) might possess the lesser reducing power4, although the higher reducing power for ethanolic and hydroethanolic extracts of fruit and bark could resemble1. Acting as a defense mechanism against lipid peroxidation, the antioxidants in plant materials would bind to metal ions by reducing transition metals such as Fe2+ or Cu+33. Further, the reducing power of essential oils and oleoresins take place largely because the reductones can transfer electrons or hydrogen atoms that break the free radical chain4. The total antioxidant capacity (TAC) of essential oils and oleoresins (%) at different concentrations in M. myristica and T. tetraptera spices can be seen in Table M. myristica of this study. The TAC of both essential oils and oleoresins can be said to be dose dependent. M. myristica essential oil obtained the peak TAC value (15.75) at 640 µg/mL concentration, significantly higher (p < 0.05) than that of 360 µg/mL . T. tetraptera oleoresin obtained the second-highest absorbance values, while M. myristica oleoresins had the least absorbance levels. The TAC recorded in this current work seemed higher than those of M. myristica35, including those of aqueous and ethanolic extracts of fruits, pulps, and seeds of T. tetraptera8. Nonetheless and for emphasis, the TAC assay is based on the ability of a reducing agent (antioxidant) to reduce molybdenum (VI) ions to molybdenum (V) giving a green phosphomolybdate (V) complex at 765 nm39.The reducing power activity (%) of essential oils and oleoresins at different concentrations in M. myristica and T. tetraptera spices found in the Southeast of Nigeria, both essential oil and oleoresin were secured, respectively, by steam distillation and maceration. Very useful compounds were obtained, through which some considerable antibacterial and antioxidant potentials were exhibited. Essential oil of M. myristica appeared to have enhanced antibacterial and antioxidant activity compared to the others. The T. tetraptera, however, seemed less effective to inhibit both E. coli and S. aureus populations. The extraction process, environment, and genetics would be crucial influences on the natural compounds found in each spice’s essential oil and oleoresin, which would in turn strongly impact (both M. myristica and T. tetraptera spices’) antibacterial and antioxidant capabilities, and hold great promise in food preservation, especially against the autoxidation and microbial aspects of (food) spoilage. Considering the findings of this current work, the direction of future investigations should be to establish the purity of these chemical constituents of essential oil and oleoresin obtained from M. myristica and T. tetraptera spices, and their corresponding cytotoxic activities, which could possibly help better the understanding that underpin their pharmacological and other associated/related effects.In this study and from


Nicotiana tabacum var. Virginia fresh (VFL) and dry (VDL) leaf extracts. The extracts were loaded in the phytosomes by a solvent displacement method. The physical and chemical characteristics and stability of phytosomes were evaluated by dynamic light scattering and phytochemistry, respectively. The in vitro antioxidant activity and intracellular reactive oxygen species reduction of phytosomes and/or extracts were investigated by the DPPH and ABTS radical scavenging assays, FRAP assay, and DCFH-DA fluorescent probe. The cytotoxicity and anti-inflammatory activity of VDL and VFL phytosomes were studied by an MTT and a nitric oxide assay, respectively. Here, we first reported the total phenolic content in the dry leaf extract of N. tabacum var. Virginia was significantly greater than that of the fresh leaf extract. The HPLC analysis results revealed that VDL and VFL extracts contained 4.94 ± 0.04 and 3.13 ± 0.01 µg/mL of chlorogenic acid and 0.89 ± 0.00 and 0.24 ± 0.00 µg/mL of rutin, respectively. The phytosomes of the VDL and VFL extracts displayed stable size, polydispersity index, zeta potential values, and good chemical stability. VDL and VDL phytosomes showed higher phenolic and flavonoid contents which showed stronger DPPH and ABTS radical scavenging effects and reduced the intracellular ROS. The results suggested that the phenolic compounds are the main factor in their antioxidant activity. Both VDL and VFL phytosomes inhibited nitric oxide production induced by LPS, suggesting the anti-inflammatory activity of the phytosomes. The shape memory gel containing VDL and VFL phytosomes had good physical stability in terms of pH and viscosity. The VDL and VFL phytosomes dispersed in the shape memory gels can be considered as a promising therapeutic delivery system for protecting the skin from oxidation and reactive oxygen species.Oxidative stress is one of the major causes of skin aging. In this study, the shape memory gels containing phytosomes were developed as a delivery system for  Skin aging can be caused by internal and external factors . The intNicotiana tabacum var. Virginia, especially phenolic compounds and flavonoids, were investigated. Although these phytochemicals have good antioxidative activity due to their ability to bind free radicals with the hydroxyl functional group (-OH) in the chemical structure, most effective natural extracts have limited water solubility [Antioxidants resist or slow down the aging of the skin. Several formulations have been developed to deliver antioxidants to prevent or delay the deterioration of skin cells. Antioxidants in nature are usually phenolic compounds, flavonoids, and tannins. In this study, the biological activity, and phytochemical constituents of tobacco dry leaf extract (VDL) and fresh leaf extract (VFL) from lubility ,9,10,11.Centella asiatica extract, and grape seed (Vitis vinifera L.) extract [The phytosome is a type of drug delivery system. The phytosome encapsulates natural bioactive constituents by forming hydrogen bonds with the polar head of the phytosome. The phytosome and bioactive compound complexes are formed in which the phospholipids’ head group is anchored. In contrast, the fatty acid chains of phospholipids encapsulate the polar part of complexes to form a lipophilic surface . Phytoso extract ,16,17. PN. tabacum var. Virginia dry and fresh leaf extracts for preventing skin aging caused by oxidative stress. Therefore, the total phenolic content and total flavonoids and the antioxidant activities of ethanol extract of N. tabacum var. Virginia dry and fresh leaf were investigated. The VDL and VFL phytosomes were successfully formulated with good colloidal stability. We found that the storage temperature and phytosome formulation including phospholipids, cholesterol, and poloxamers played significant roles in the stability of the VDL and VFL phytosomes. Pearson correlation coefficients revealed the very high correlation between the total phenolic content and antioxidant activity of the extract and phytosomes. The effective and non-toxic concentrations of VDL and VFL phytosomes in reducing inflammation and intracellular reactive oxygen species in keratinocytes were reported. The gels containing phytosomes were developed to make phytosomes feasible for topical application. The method to prepare the shape memory gel containing phytosome developed in this study was simple and was achievable for scaling up commercially.Natural antioxidants, whether in the form of raw extracts or chemical constituents, are extremely effective at preventing the damaging processes caused by oxidative stress . DespiteThe average particle size and stability of the VDL and VFL phytosomes with and without cholesterol or poloxamer, and VDL and VFL nanoparticles are shown in w/w of cholesterol was used and was considered optimal for VDL and VFL phytosome formulations. In the absence of 0.1% w/v poloxamer 407, the VDL and VFL phytosomes were also significantly larger than the phytosomes stabilized with poloxamer. Poloxamer was assumed to be attached to the phytosome by adsorption. Minnelli et al. showed that combining poloxamer 407 with liposome increased liposome stability by hindering liposome aggregation, shielding the colloidal surface with the hydrophilic portions of the polymer, and decreasing the fusion of phosphatidylcholine multilamellar vesicles [The results showed that the particle size significantly increased in the absence of cholesterol in both VDL and VFL phytosome formulations. The importance of cholesterol in liposomes was reported. The solidity of the liposomes depended on the content of cholesterol. Numerous studies used cholesterol as a stabilizer of liposomes because it helped the packing of phospholipid molecules, reduced bilayer permeability, prevented liposome aggregation, and increased the rigidity and resistance to shear stress of the lipid bilayer . Here, 0vesicles . The sizvesicles . The colvesicles .The polydispersity index of VDL phytosome with cholesterol, VDL phytosome without cholesterol, VDL phytosome without poloxamer, and VDL nanoparticles were 0.136 ± 0.019, 0.128 ± 0.024, 0.169 ± 0.040, and 0.164 ± 0.027, respectively . The polThe phytosome and nanoparticle size distribution can be articulated through the polydispersity index value. The nanoparticles with PDI values less than 0.3 are considered monodisperse, while a PDI value higher than 0.7 indicates a polydisperse system . All VDLThe zeta potential values of VDL phytosome with cholesterol, VDL phytosome without cholesterol, VDL phytosome without poloxamer, and VDL nanoparticles were −22.57 ± 0.23 mV, −24.93 ± 0.49 mV, −40.07 ± 0.86 mV, and −35.97 ± 01.36 nm, respectively . The zetZeta potential results showed that all VDL and VFL formulations exhibited a negative charge with values ranging from 22.57 ± 0.23 mV to −40.07 ± 0.86 mV, and −44.23 ± 01.86 mV to 76.93 ± 1.71 mV, respectively. The negative charge value was due to the presence of phosphate and carbonyl groups of phosphatidylcholines. Compared with phytosomes coated with poloxamer, a more negative charge of phytosomes and nanoparticles was observed with phytosomes prepared without poloxamer. This could be because poloxamer adsorption on the phytosome or nanoparticle surface forms a coating layer, shielding the negative surface charge and shifting the plane of shear away from the particle surface. Overall, phytosomes containing cholesterol and poloxamer encapsulating VDL and VFL were the best formulation with optimal size, PDI, and zeta potential values. Both formulations were recommended to store at 30 °C.Phytosomes of herbal extracts have been developed and characterized. Tiwari et al. developed herbal extract-loaded phytosomes. They showed that the encapsulation of herbal extract in the phytosomes did not change the chemical structure due to an FTIR analysis . DireitoN. tabacum var. Virginia was significantly greater than that of the fresh leaf extract.The total phenolic content in the VDL and VFL ethanolic extracts using the Folin–Ciocalteu reagent was expressed in terms of the gallic acid equivalent. The standard curves plotted between the absorbance, and gallic acid reacted with the Folin–Ciocalteau reagent are shown in The standard curves of quercetin and EGCG reacted with the aluminum chloride reagent were presented in 50 values obtained from the DPPH method were 5.40 µg/mL, 17.39 µg/mL, 19.74 µg/mL, and 4.82 µg/mL for gallic acid, quercetin, ascorbyl glucoside, and EGCG, respectively  linkages that can form hydrogen bonds to yield its high mechanical strength. Combining hydrophobic polyurethane with butylene glycol generates a swellable copolymer network that is robust and durable . PolyureN. tabacum var. Virginia fresh and dry leaf extract to the skin. The phytosomes with different formulars were fabricated and the physical stability was compared. Both phosphatidyl choline and cholesterol played an important role in the physical stability of the phytosomes but did not affect the encapsulation efficiency of the phenolic compounds. VDL and VDL phytosomes had higher phenolic and flavonoid contents and displayed stronger DPPH and ABTS radical scavenging effects, suggesting that phenolic compounds are the main factor in their antioxidant activity. VDL and VFL phytosomes reduced the intracellular ROS and inhibited the NO production induced by LPS. The viscosity and pH of gels containing VDL and VFL phytosomes were not significantly changed after the six heating–cooling cycles of the accelerated stability study and after 1 month storage at 4 °C, 30 °C, and 45 °C.The phytosomes composed of phosphatidyl choline and cholesterol coated with poloxamer were successfully developed and suitable for the delivery of Gallic acid, Griess reagent, DPPH , TPTZ -s-triazine, and ABTS ) were purchased from Sigma-Aldrich, St. Louis, USA. Absolute ethanol, dimethyl sulfoxide, sodium bicarbonate, sodium nitrate, acetic acid, and sodium hydroxide were purchased from RCI Labscan, Bangkok, Thailand. Iron (III) chloride hexahydrate and 37% hydrochloric acid were purchased from Qrec, New Zealand. The Folin–Ciocalteu phenol reagent, aluminum chloride, sodium acetate trihydrate, ferrous sulfate heptahydrate (99% purity), and potassium persulfate were obtained from Loba Chemie, Mumbai, India. Quercetin (98% purity), epigallocatechin (EGCG) (98% purity), ascorbyl glucoside, phosphatidylcholine, cholesterol, poloxamer 407, and polyurethane-62 were purchased from Chanjao Longevity Co., Ltd., Bangkok, Thailand. Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco .w/v poloxamer 407 at a rate of 1 mL/h with a stirring speed of 700 rpm. The obtained phytosome was named “VDL phytosome” or “VFL phytosome” [w/o cholesterol)” and “VFL phytosomes (w/o cholesterol)” were prepared by the following method, where cholesterol was not included. Tobacco leaf extract (VDL or VFL) in 95% ethanol  was mixed with phosphatidylcholine (30 mg in 1900 µL 95% ethanol). The mixture was added dropwise into 15 mL of 0.1% w/v poloxamer 407 at a rate of 1 mL/h with a stirring speed of 700 rpm. To prepare “VDL phytosome (w/o poloxamer)” and “VFL phytosome (w/o poloxamer)”, mixtures of VDL or VFL  with phosphatidylcholine (30 mg in 1900 µL), 95% ethanol, and cholesterol (2 mg in 200 µL acetone) were infused into 15 mL of de-ionized water at a rate of 1 mL/h with a stirring speed of 700 rpm.Tobacco leaf extract (VDL or VFL) in 95% ethanol  was mixed with phosphatidylcholine (30 mg in 1900 µL 95% ethanol). Cholesterol (2 mg) in acetone solution (200 µL) was added to the above solution and mixed thoroughly. Then, the mixture was added dropwise into 15 mL of 0.1% ytosome” . “VDL ph“VDL nanoparticles” and “VFL nanoparticles” were prepared by the infusion of VDL or VFL extracts  into 0.1% poloxamer 407 solution at a rate of 1 mL/h with a stirring speed of 700 rpm ,55,56. TThe freshly prepared VDL and VFL phytosomes or nanoparticles were measured for the size, PDI, and zeta potential values using the Zetasizer . The colloidal stability of VDL and VFL phytosomes or nanoparticles was studied by storing phytosomes or nanoparticles at 4 °C, 30 °C, and 45 °C for 0.5, 1, 2, and 3 months. At the end of the incubation time, the particle size, PDI, and zeta potential of VDL and VFL phytosomes and nanoparticles were analyzed.v/v, 100 µL) was added to the wells, mixed well, and incubated for 4 min at room temperature. Then, the sodium carbonate solution  was added to the mixture and incubated in the dark for 60 min at room temperature [VDL and VFL extracts and phytosomes and gallic acid standard solution (3.9–125 µg/mL) were placed in a 96-well plate (50 µL/well). A Folin–Ciocalteau reagent  was added to the wells, mixed well, and incubated for 5 min at room temperature. Then, the aluminum chloride solution  was added to the mixture and incubated for 6 min at room temperature [The VDL and VFL extracts and phytosomes, quercetin standard solution (7.8–500 µg/mL), and EGCG standard solution (7.8–1000 µg/mL) were placed in a 96-well plate (50 µL/well). Sodium nitrate , VDL and VFL phytosomes , gallic acid solution (3.9–2000 µg/mL), quercetin solution (3.9–2000 µg/mL), EGCG solution (3.9–2000 µg/mL), and ascorbyl glucoside solution (3.9–2000 µg/mL) were added to 96-well plates (100 μL/well). The DPPH solution (0.1 mM) was added to the samples (100 µL/well). The mixtures were incubated in the dark at room temperature for 30 min . Then, tThe VDL and VFL extracts , VDL and VFL phytosomes , gallic acid solution (3.9–2000 µg/mL), quercetin solution (3.9–2000 µg/mL), EGCG solution (3.9–2000 µg/mL), and ascorbyl glucoside solution (3.9–2000 µg/mL) were added to 96-well plates (20 μL/well). The ABTS•+ solution (0.1 mM) was added to the samples and standard solutions (180 µL/well) . The mixThe VDL and VFL extracts , VDL and VFL phytosomes , gallic acid solution (3.9–2000 µg/mL), quercetin solution (3.9–2000 µg/mL), EGCG solution (3.9–2000 µg/mL), and ascorbyl glucoside solution (3.9–2000 µg/mL) were added to 96-well plates (20 μL/well). The FRAP reagent, consisting of the acetate buffer , ferric chloride (20 mM), and TPTZ (10 mM) mixture at a ratio of 10:1:1, was added to the sample solutions (180 µL/well). The mixture of samples and FRAP reagent was then incubated at 37 °C for 30 min before reading the absorbance at a wavelength of 595 nm  . The FRA18 guard column. The mobile phases were (B) acetonitrile and (C) 0.085% phosphoric acid in water using the following gradient elution: 10% B in C to 30% B in C for 15 min; 10% B for 5 min before each analysis, and the flow rate was set at 0.7 mL/min with the controlled temperature at 25 °C. The UV detector was set at the wavelength of 326 nm for the chlorogenic acid analysis and 356 nm for the rutin analysis, and the injection volume was 10 µL for every sample and reference standard [HPLC separation was achieved on the HPLC  equipped with the 1260 Infinity II quaternary pump, 1260 Infinity II autosampler, 1260 Infinity II multi-column thermostat, and 1260 Infinity II PDA detector. The separation was completed in an ACE 5 C18-AR column  with a Cstandard ,39.2 at 37 °C for growing. Cells were subcultured by incubating with 0.25% trypsin-EDTA every three days.HaCaT and RAW264.7 cell lines were obtained from Dr. Kanokwan Kiettisin and Dr. Natthachai Duangnin, respectively. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were incubated in 5% CO3 cells/well. Prior to an MTT assay, cells were allowed to adhere for 24 h. VDL and VFL extracts and VDL and VFL phytosomes were added to the cells and were incubated in 5% CO2 at 37 °C for 24 h. After treatment for 24, 48, and 72 h, an MTT solution in the culture medium (0.5 mg/mL) was added to each well and incubated for 2 h at 37 °C [50 values were calculated using GraphPad Prism v.7.0 . Cell viability was calculated using the following equation.In 96-well plates, HaCaT cells were seeded in a culture medium at 8 × 10at 37 °C . The for6 cells/mL). Cells were co-incubated for 2 h at 37 °C with 20 mM hydrogen peroxide and VDL or VFL phytosomes at different concentrations. Cells were washed three times with PBS, pH 7.4, followed by incubation with 10 μM DCFH-DA at 37 °C for 30 min [HaCaT cells were trypsinized and added into the microcentrifuge tubes (1 × 10r 30 min . The flu4 cells/well) were incubated with LPS (50 ng/mL) in the presence or absence of VDL and VFL phytosomes (15.6–500 µg/mL) for 18 h at 5% CO2, 37 °C. After incubation, an equal medium volume was mixed with the Griess reagent, consisting of 20 mg/mL sulfanilamide and 1 mg/mL N- and incubated for 2 h. The formazan product was measured at 550 nm using a UV-Vis spectrophotometer microplate reader .The viability of RAW264.7 cells after the 24 h incubation with VDL and VFL phytosomes (7.8–1000 µg/mL) was tested by the MTT assay. The anti-inflammatory effect of VDL and VFL phytosomes was determined from nitric oxide secretion against macrophage cells. RAW 264.7 cells  in de-ionized water (89.4%) at 50 °C with a stirring rate of 1200 rpm for 1 h. Then, glycerin (5%), phenoxyethanol (0.5%), and VDL and VFL phytosomes (0.1 %) were added to the gel.The pH and rheology of the shape memory gel containing VFL and VFL phytosomes were measured using a pH meter and rheometer  equipped with a plate and plate geometry, respectively . The phy® , where the donor chamber contained the PBS buffer (1000 µL). The total phenolic compounds released from extracts, phytosomes, and gels were collected at predetermined time intervals, i.e., 15 min, 30 min, 1, 2, 4, 6, 8, 12, and 24 h. The total phenolic contents were measured by the Folin–Ciocalteau method.The release profile of the VDL and VFL extracts, VDL and VFL phytosomes, and shape memory gels containing VDL and VFL phytosomes were evaluated by the method described by Liu et al. . Phytosop < 0.05, p < 0.01, p < 0.001, and p < 0.0001 were deemed statistically significant. Data were presented as the mean ± SD of the % cell viability (n = 3).The data were statistically analyzed using a one-way ANOVA, followed by the Newman–Keuls method as a post hoc test to determine the significance of differences . In all cases,


Pleural effusion can be caused by a wide range of benign and malignant conditions. Pleural biopsy and effusion cytology represent two key methods of pathological diagnosis. To compare the performance these two methods, a large cohort of matched pleural biopsy and effusion cytology with clinical follow‐up was reviewed.Pleural biopsies and effusion cytology specimens over a period of 18 years were retrieved. Cytology specimens collected within 7 days of pleural biopsy were matched. Reports were reviewed, and the cause for pleural effusion was determined by hospital disease coding and clinical data.n = 650). Malignant pleural effusion (MPE) was more common in older females (p < 0.001) and mostly due to lung cancer (n = 959), breast cancer (n = 64), and mesothelioma (n = 48). The inadequate/insufficient (B1/C1) rate of biopsy was higher than cytology (15.6% vs. 0.3%) but the rates for other diagnostic categories were similar. Biopsy and cytology showed a correlation coefficient of 0.315, improving to 0.449 when inadequate/insufficient (B1/C1) cases were excluded. The ROM for benign cytology (C2) was lower than biopsy (B2) (p < 0.001). Compared with biopsy, the diagnostic accuracy was higher in cytology overall and for metastatic carcinomas (p < 0.001) but lower for hematolymphoid malignancies (p = 0.014) and mesotheliomas (p = 0.002).Totally, 3026 cases were included. The leading cause of benign effusion was tuberculosis (These results suggest that effusion cytology may be better for confirming benignity and diagnosing carcinomatous MPE. In these cases, pleural biopsy may be withheld to reduce procedural risks. However, for suspected hematolymphoid malignancies and mesothelioma, biopsy should be considered. Effusion cytology is superior to pleural biopsy in excluding malignant pleural effusion and diagnosis of metastatic carcinomas. The underlying causes include a wide range of benign, reactive, infective, autoimmune, and neoplastic diseases.2A computerized search was performed in the department archives for pleural biopsies and effusion cytology specimens collected from February 2001 to February 2019. The reports were reviewed for patient demographical data and histologic/cytologic diagnosis. The diagnoses were classified into five‐tiered categories  .Pleural biopsies were paired with effusion cytology specimens by a hospital accession number unique to each patient in the region‐wide clinical management system. Only effusion cytology specimens collected within 7 days before or after the date of pleural biopsy were considered a match. When multiple cytology specimens were matched, the most high‐grade diagnosis was taken as the final diagnosis. This study was approved by the Joint Chinese University of Hong Kong—New Territories East Cluster Clinical Research Ethics Committee with an exemption of requirement of written consent , specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), accuracy, and risk of malignancy (ROM) were calculated. The 33.1n = 650/1686, 38.6%), parapneumonic effusion , and heart failure . Metastatic lung cancer was the leading cause of MPE , followed by metastatic breast cancer  and mesothelioma  (Table p < 0.001). Effusions due to hematolymphoid malignancies were observed in the youngest age group (average 55.6 years) compared with metastatic carcinomas which occurred in the oldest age group (average 69.6 years). MPEs were more common in older (p < 0.001) and female (p < 0.001) patients. There was a preponderance of male patients for mesothelioma  , with an average age of 65.6 years. Each pleural biopsy was on average matched with approximately three cytology specimens, and there were 245 cytology specimens matched to more than one pleural biopsy  B1, 1890 (62.5%) B2, 100 (3.3%) B3, 50 (1.7%) B4, and 513 (17.0%) B5 diagnoses, respectively. The biopsies were paired to 9 (0.3%) C1, 2029 (67.1%) C2, 247 (8.2%) C3, 175 (5.8%) C4, and 566 (18.7%) C5 cytologic diagnoses, respectively, from a total of 22 (0.4%) C1, 4159 (67.1%) C2, 471 (7.6%) C3, 344 (5.6%) C4, and 1198 (19.3%) C5 effusion cytology specimens . Considering B2/C2 diagnoses as negative and B5/C5 as positive, the Sp, Sn, PPVs, NPVs, and accuracies were 0.998, 0.495, 0.994, 0.742, and 0.782 for biopsy, and 0.998, 0.570, 0.996, 0.791, and 0.835 for cytology. Alternatively, when B4/C4 diagnoses were also considered positive, the Sp, Sn, PPVs, NPVs, and accuracies were 0.996, 0.517, 0.991, 0.724, and 0.786 for biopsy and 0.997, 0.634, 0.993, 0.791, and 0.845 for cytology. Both conditions resulted in a significant difference in accuracy (p < 0.001) favoring cytology  but lower in the mesothelioma  and hematolymphoid malignancy  groups compared with biopsy. Adding B4 and C4 as positive test results showed similar results  and benign than malignant diagnoses (p < 0.001). These demographic differences were also reported in cohorts of pleural effusion without paired biopsy and cytology,p < 0.001),p < 0.001).n = 27/1212) followed the lung  and the breast  as the third most common primary site contributing to MPEs caused by metastatic carcinomas. The distribution of primary tumors was similar to previous studies, including a cohort of Asian demographic.n = 501/959), breast and gynecological cancers in the cohort resulted in an overall predisposition to female patients for MPEs caused by metastatic carcinoma   was malignant on the follow‐up . As the cohort was collected prior to the publication of International System for serous fluid cytopathology, fluid volume was not considered for the diagnosis of insufficiency. Specimens were considered inadequate/insufficient when no mesothelial or lesional cells were included. The rate of inadequacy for pleural biopsy reported in the literature was variable, ranging from 7% to 32%,up Table .p = 0.014) and mesothelioma (p = 0.002)  Table . The majn = 21/37) and 76.5% (n = 26/34) of biopsy cases included for analysis in hematolymphoid malignancy and mesothelioma subgroups. This is higher than the proportion of cytology specimens with immunocytochemistry performed . Immunocytochemistry and other ancillary tests provide additional information for cytologic interpretation, but still have limitations in differentiating reactive lymphoid hyperplasia from low‐grade lymphomas,Further review of biopsy and cytology reports of the hematolymphoid malignancy and mesothelioma subgroups from Table n = 13/14), greater than that of cytology .For the accuracy in typing of malignant lesions (carcinoma vs. mesothelioma vs. hematolymphoid), analysis was limited by the small number of cases. In addition, a significant number of reports lacked diagnostic classifiers, only issuing a diagnosis of “malignancy” or “malignant cells.” When mesothelial lineage was specified in the report, both cytology and biopsy were very accurate (specific ROMs = 1) Table . The diaConsiderations in performing concurrent pleural biopsy at the time of thoracentesis is crucial in the management of pleural effusion. Factors include the availability of resource, likelihood of underlying disease determined by clinical impression and previous workup results, and the added risk of complication for pleural biopsy. For example, bedside blinded pleural biopsy is more commonly performed alongside diagnostic thoracentesis in tuberculosis endemic area for the exclusion of tuberculous pleuritis.The techniques in pleural biopsy have undergone significant improvements over the period in which data were collected for this cohort. The performance of pleural biopsy has been improved by prebiopsy ultrasound localization of pleural abnormality and real‐time image guidance.5This large cohort of paired pleural biopsy and effusion cytology specimens demonstrated that the inadequate/insufficient (B1/C1) rate was higher in biopsy than cytology, while that of the other diagnostic categories were similar. Biopsy and cytology showed fair to moderate concordance depending on whether inadequate/insufficient (B1/C1) specimens were included. The malignant (B5/C5) and suspicious (B4/C4) categories had similar ROMs. The ROM for atypia in biopsy (B3) was higher than that of cytology (C3). Cytology showed better performance in terms of ROM for the benign category (B2/C2) and overall diagnostic accuracy. Subgroup analysis showed that cytology was more accurate in carcinomatous MPEs but not for hematolymphoid malignancies and mesotheliomas. These results suggest that for patients with a high pretest probability of MPE, particularly when metastatic carcinoma is suspected, diagnostic thoracentesis alone may be sufficient as the initial step. However, for workup of hematolymphoid malignancies and mesothelioma, biopsy should be considered.2020.289).This study was approved by the Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee (reference number: The authors have no funding to declare.The authors declare that there is no conflict of interest regarding the publication of this paper.The abstract of this manuscript has been accepted for presentation in Pathology Update 2022.Ivan K. Poon – conceptualization, data curation, investigation, methodology, formal analysis, and writing—original draftRonald C. K. Chan – conceptualization, methodology, formal analysis, and resourcesJoseph S. H. Choi – software, formal analysis, and visualizationJoanna K. M. Ng – data curation, investigation, and methodologyKatsie T. Tang – data curation and investigationYolanda Y. H. Wong – data curation and investigationKa Pang Chan – validation and writing—original draftWing Ho Yip – validationGary M. Tse – methodology, formal analysis, and writing—review and editingJoshua J. X. Li – conceptualization, data curation, investigation, methodology, formal analysis, and writing—review and editing.


The differences in antioxidant activity between the investigated localities were statistically significant for both types of extract. For example, in 2013 in the locality of Zbojné, the FRAP in NCBs was 76.62 µmol·L−1·g−1 DM and in CBs was 138.27 µmol·L−1·g−1 DM, while in the Miľpoš locality, in NCBs there was 232.66 µmol·L−1·g−1 DM and in CBs there was 1178.98 µmol·L−1·g−1 DM. The differences in the antioxidant activity between the studied years in the case of NCB extracts were not statistically significant. In the case of CB extracts, significant differences between the evaluated years were found. Statistics by ANOVA confirmed that CB extracts prepared from berries in the year 2013 showed significantly higher activity compared to CB extracts from berries from the years 2012 and 2014. Based on the Pearson we found a negative correlation coefficient between the FRAP assay and the content of total polyphenols in NCB extracts . In contrast, CB extracts showed a positive correlation coefficient .In the present study, the seasonal variability of the in vitro ferric reducing ability of plasma (FRAP), total phenols, and terpene hydrocarbon content in 70% ethanol extracts were evaluated. The samples of crushed (CBs) and non-crushed ripe juniper berries (NCBs) collected at five localities in North-East Slovakia during the years 2012–2014 were compared. The method of preparation of the extract influenced the amount of dry matter (DM) in the extracts. In the CB extracts were statistically higher contents of DM (from 13.91 ± 0.11 g·L It is well known that the environmental conditions have an influence on the development of the natural components in plant matrices. The aim of the study is to compare the quality and the potential antioxidant activity of the extracts in different localities and different years.Free radicals are formed during normal cell metabolism . An imbaP values . Plants P values . Plant eP values , which hP values . Juniperrm shrub ,10,11,12rm shrub ,12,13,14lant age ,16. The lant age . The astlant age . Juniperlant age , as welllant age . The bralant age . The suplant age . This stwww.shmu.sk; accessed on 25 October 2014). In 2012, the highest average precipitation was measured in the Zbojné locality (808 mm) and the lowest in the Hôrka locality (550 mm) in the Presov self-governing region. A similar observation was made in 2013, when the highest precipitation was in Zbojné (815 mm) and the lowest in Hôrka (676 mm). The changes were noted in third year of research in 2014, when the highest precipitation was in two localities, Miľpoš and Lačnov (880 mm), following the Kamienka locality (836 mm), and the Hôrka (774 mm) and Zbojné (752 mm) localities noted the lowest amount of precipitation. Generally, in the first year of our research, the lowest amount of precipitation in all five localities (691 mm) was noted, while the highest amount of precipitation was noted in the third year, 2014 (810.5 mm)  .Average temperatures in different localities were observed as follows. In 2012, the average highest temperature was noted in the Zbojné locality (8.3 °C) and the lowest in the Hôrka locality (6.9 °C). As was described in the precipitation, an identical pattern is noted in temperature; in 2013, again the highest average temperature was noted in Zbojné (8.6 °C) and the lowest in Hôrka (7.0 °C). A similar pattern continues also in the third year, 2014, when in Zbojné was the highest average temperature (9.5 °C) and the lowest was in Hôrka (8.2 °C) . There w−1 DM) and the lowest in the Hôrka locality (8.49 GAE mg.g−1 DM) in NCBs. In the same year, the highest amount of phenols in CBs was noted in extracts from Hôrka (18.77 GAE mg.g−1 DM), and the lowest was from extracts from Miľpoš (9.82 GAE mg.g−1 DM). The rest of the three localities noted a similar amount (11.13–11.67 GAE mg.g−1 DM). In the second year (2013) of our experiment, the highest content of phenols in NCBs was evaluated in extracts from Hôrka (20.41 GAE mg.g−1 DM) and the lowest in extracts from lačnov (9.12 GAE mg.g−1 DM). In the extracts from CBs, a higher content of phenols was evaluated in an almost similar amount in three localities—Kamienka, Miľpoš, Lačnov —and, at the lower end, almost the same content was evaluated in Hôrka and Zbojné (7.11 and 7.84 GAE mg.g−1 DM). In the last year of our observation, the highest amount of phenols in NCBs was evaluated in the Hôrka locality (42.23 GAE mg.g−1 DM) while the lowest was in the Zbojné locality (14.97 GAE mg.g−1 DM). Three other localities had similar contents of phenols: Kamienka (20.6 GAE mg.g−1 DM), Lačnov (21.64 GAE mg.g−1 DM), and Miľpoš 23.4 GAE mg.g−1 DM). In the extracts from CBs, the highest amount of phenols was evaluated in extracts from Lačnov (12.53 GAE mg.g−1 DM), and a similar amount was also measured in two other localities: Miľpoš (11.58 GAE mg.g−1 DM) and Hôrka (10.7 GAE mg.g−1 DM). A lower amount of phenols were evaluated in Zbojné and Kamienka (8.92 and 6.87 GAE mg.g−1 DM).The evaluation of phenol content in non-crushed juniper berries (NCBs) and crushed juniper berries (CBs), as well as terpene hydrocarbon content in crushed berries, were evaluated for each locality and for three years. Generally, the higher number of phenols was noted in the extracts from non-crushed juniper berries. Each year, the significant differences were evaluated between localities . In 2012The results of antioxidant activity and dry matter for extracts of non-crushed (NCB) and crushed berries (CB) are shown in p < 0.001) as well as between the studied years (p < 0.001). Statistics by ANOVA confirmed that CB extracts prepared from berries in the year 2013 showed significantly higher activity compared to CB extracts from berries from the years 2012 and 2014.The number of extracted substances subsequently influenced the antioxidant activity of individual extracts, which was significantly higher in CB extracts compared to NCB extracts . General−1.g−1 DM) method and the content of total polyphenols (GAE mg.g−1 DM) in NCB ethanol extracts. In 2012, there was a correlation coefficient r = −0.531, in 2013 r = −0.349 and in 2014 r = 0.224. Opposite to these results, in CB extracts a positive correlation coefficient between antioxidant activity and total polyphenols was evaluated. In 2012, it was r = 0.843; in 2013, it was r = 0.742; and in 2014, it was r = 0.617. Correlation coefficients were decreasing in the research years from 2012 to 2014. Correlation coefficients between antioxidant activity and the content of terpene hydrocarbons ranged from r = 0.504 in the year 2012, until negative coefficient in 2013 (r = −0.684) and in 2014 (r = −0.220)  .J. communis plant is not only a rich source of nutrition but also is rich in aromatic oils and their concentration varies in different parts of the plant . The fruit berries contain essential oil (0.5% in fresh and 2.5% in dry fruit), invert sugars (15–30%), resin (10%), catechin (3–5%), organic acid, terpenic acids, leucoantho-cyanidin besides a bitter compound (Juniperine), flavonoids, tannins, gums, lignins, wax, etc [J. communis L. plant parts include 5-Ocaffeoylquinic and quinic acids, catechin, epicatechin, amentoflavone, quercetin, luteolin, apigenin, and naringenin and their derivatives [J. communis in as different origins as Turkey, Slovakia, Canada, Australia, Portugal, and Romania [−1 GAE and an antioxidant activity of FRAP of 0.24 mmol Fe2+.g−1 in water extracts of juniper berries [−1 of total phenolic and showed an antioxidant FRAP activity of 3.77 mM TE mL−1 [−1 DM and a FRAP activity of 6.82 mmol.100 g−1 DM. A high ferric reducing ability of plasma assays was reported [Juniper oxycedrus. A scientific study [−1. For the components of the essential oil limonene and borneol, the activity was 37.4 mmol.kg−1 resp. 28.7 mmol.kg−1. The reducing abilities of the components of volatile oils are lower than those of volatile oils. In addition to the terpenes, other biologically active compounds may also contribute to ferric reduction and in electron scavenging. For example, for β-pinene, the FRAP value 6.5 µM Fe.mg−1 was found [The wax, etc ,22,23. Jwax, etc ,11. The ivatives . The tot Romania ,27,28,29ugars 15–%, resin  TE mL−1 . In wate TE mL−1  found a reported  in methaic study  evaluateas found . The antas found . Another−2.s−1 [Juniperus excelsa berries. They found a negative correlation coefficient of r = −0.692 for berry extracts in 95% ethanol.The influence of abiotic stress on the content of total polyphenols and antioxidant activity is known. By testing two varieties of barley, it was confirmed that drought as an abiotic stress caused an increase in antioxidant activity compared to control samples. The same tendency was also observed with intensive irradiation of FAR = 800 µmol.m−2.s−1 . The inf−2.s−1 . The con−2.s−1 . The num−2.s−1  and r = −2.s−1 , respect−2.s−1 . A study−2.s−1  Stankov The juniper ripe berries were collected in five localities  in the Prešov self-governing region (PSGR)  which isPrecise locality characterization is described in a previous publication . The sumWhole dried galbuli as non-crushed berries (NCBs) and berries crushed in a porcelain mortar as crushed berries (CBs) were macerated in 100 mL of 70% ethanol. The extractions were carried out with occasional stirring for 72 h at room temperature. The obtained extracts were filtered over a filter KA 1-M . The dry matter (DM) content was determined by using filtrated extracts drying in Petri dishes in an oven at 105 °C for three hours, each extract in triplicate.All chemicals used were of the highest quality: 70% ethanol . Double distilled water (DDW) was used for preparation of solutions. Absorbances of solutions of various test assays were determined in 1 cm quartz cells with a Shimadzu type UV-1800 spectrophotometer .v/v). Each diluted extract (0.1 mL) was sequentially well-mixed with 0.2 mL of FCR, 2 mL of DDW, and 1 mL of Na2CO3 saturated solution . After incubation in the dark at room temperature for 90 min, the absorbance of the reaction mixture was determined at 765 nm with a Shimadzu UV-1800 spectrophotometer. The blank sample was prepared in a similar way by replacing the extract with the same volume of 70% ethanol. The phenolic content was calculated as Gallic acid equivalents (GAE) per mL of extract based on a standard curve of Gallic acid (Merck). All the determinations were carried out four times for each extract. All the solutions were used on the day of preparation.The total phenolic content of the ethanol extracts of the leaves was determined with the Folin-Ciocalteu reagent  according to a procedure described by , with slThe terpene hydrocarbon content of CB extracts was determined by gas chromatography with a Carlo Erba GC 6000 Vega Series 2 equipped with an ICU-600 programme controller, an EL 580 flame ionization detector (FID), a Spectra Physics SP 4270 integrator, and a SolGel-WAX GC Capillary Column . An injector was heated to 200 °C, and a flame ionization detector was heated to 300 °C. The column temperature was maintained at 50 °C for 5 min and then programmed to increase to 200 °C at a rate of 5 °C/min and held at this temperature for 10 min; the injection volume was 1 µL; the split ratio was 1:20; nitrogen was used as a carrier gas (1.5 mL/min). The identification of monoterpene and sesquiterpene hydrocarbons was performed on the basis of the co-injection of some commercially available standards. Quantification was conducted with the peak area values obtained from GC-FID, using sabinene as an external standard. The content of terpene hydrocarbons was expressed as milligrams of sabinene equivalents per gram of the extract dry matter.−1 for dissolving of 10 mmol L−1 TPTZ . Aqueous solutions of iron (II) sulfate heptahydrate in the concentration range of 0–900 mmol L−1 were used for calibration at 600 nm (r = 0.9997). The assay was performed manually at room temperature. The solutions were pipetted into test tubes in duplicates. Solutions were mixed well and the absorbance at 700 nm was recorded after 5 min with the spectrophotometer. A Gallic acid solution with a concentration of 10 mmol L−1 was used for comparison. The FRAP of the samples (in µmol L−1) was calculated by the formula: FRAP = (Sample–Blank) × 500 (Standard–Blank). All the determinations of FRAP in the samples were performed at least four times. All the solutions were used on the day of preparation. The FRAP assay, which provides fast reproducible results, measures the ability of an antioxidant to reduce the ferric tripyridyltriazine (Fe+3-TPTZ) complex and produce the ferrous tripyridyltriazine (Fe+2-TPTZ) complex, which is blue in color. The details can also be found in our previous publications [The working FRAP reagents and other reagents were prepared according to Benzie and Strain (1996). The only change was in the increase of hydrochloric acid concentration to 50 mmol Lications ,42.The statistical software Statgraphics 5.0  and multifactorial analysis of variance (ANOVA) method and an LSD (Least Significant Difference) of 95% were used for statistical analysis. Correlation analysis was performed by using the Excel software.The study revealed relatively high antioxidant activity as determined by the FRAP method. Significantly higher activity was found in CB extracts compared to NCB extracts. It is related to the number of extracted substances, which was higher in CB extracts. Statistically significant differences in antioxidant activity by the FRAP method between the investigated localities could be caused by environmental conditions. These could affect the accumulation of secondary metabolites involved in antioxidant activity. Statistically significant differences in the activity of CB extracts between the evaluated years were also found. Differences in the correlation between antioxidant activity and total phenolic content compared to the literature were found. The differences between the results in this study and reports in the literature are probably due to the application of different extraction methods, methodologies for the detection of phenols, and the determination of antioxidant activity. Other substances, which were not determined in this research, probably also participated in the antioxidant activity. The study shows that there are natural products, which are safe for practical use, for example in food industry. Our findings shall be considered in the search for new products which will prolong the quality of specific food products or in prolonging their shelf life.