Olive plants exhibited high sensitivity to light quality and intensity. Higher red/blue ratios or lower light intensities stimulated plant growth and biomass mainly as a consequence of a higher internodal elongation rate, not affecting either the total number of nodes or shoots. In comparison to fluorescent illumination, LED lighting improved leaf area and biomass, which additionally was positively correlated with light intensity. Stomatal frequency was positively, and pigments content negatively, correlated with light intensity, while no clear correlation was observed with light quality. In comparison with fluorescent lamps, LED illumination (particularly the 70/30 red/blue ratio with 34 µmol m−2 s−1 PPF intensity) allowed optimal manipulation and improved the quality of in-vitro micropropagated olive plants.Light-emitting diodes (LEDs) are useful for the in-vitro micropropagation of plants, but little information is available on woody species. This work compares the effects of light quality and intensity on the growth and development of micropropagated olive plants from two different subspecies. Illumination was provided with fluorescent and LED lamps covering different red/blue ratios  or red/blue/white combinations, as well as different light intensities (30, 34, 40, 52, 56, 84, 98 and 137 µmol m Olea europaea, L.) is a woody crop belonging to the Oleaceae family. It is a medium-sized tree of about 4 to 8 m high, depending on the variety. The Olea europaea species is found in the Mediterranean area [Olive  lighting has clear benefits for optimizing the performance of plant growth chambers, including smaller size, longer life, lower temperature and the option to regulate single wavelength emission ,18,19. Iilifolia  and rapeilifolia , culturerophylla , Cedrelafissilis , Pinus sii-regis , has beeOlea europaea plants, nor on the specific benefits of LED lighting. The aim of this study was to evaluate to what extent light quality and intensity regulate developmental, morphological, anatomical, physiological and photosynthetic parameters of micropropagated olive plants. For this purpose, two wild olive genotypes belonging to two different subspecies of Olea europaea were micropropagated under different illumination regimes supplied by both fluorescent and LED lighting, and the following parameters were measured: total stem length, internodal length, number of nodes, number of shoots, shoot and callus biomass, total leaf area, number of leaves, stomatal size, stomatal frequency and content of photosynthetic pigments. LED illumination facilitates the use of different light spectra to induce different developmental responses, for example, shoot multiplication, internode elongation, rooting, hardening, or even flowering in different plant species ,34,35,36A clear variation of plant growth was observed in micropropagated olive plants treated with different light regimes . p ≤ 0.001) were observed when light intensity was compared with the total stem length  induced significantly shorter stems (−2s−1 PPF light intensity (R7B3-34 treatment) in both olive genotypes. A similar response was observed when the effect of the R6B4W-98 treatment on total stem length (−2s−1 PPF) were compared in the R9B1-56 and R7B3-52 treatments, respectively, the 90% red light present in the R9B1-56 treatment determined significantly longer stems , the higher light intensity in the R7B3-52 treatment (52 μmol mer stems B and shoer stems D than thm length B and intm length D was comer stems B and inter stems D than th−2s−1 PPF) resulted in the shortest internodes and the lowest stem height (europaea subspecies (Plants subjected to daylight white fluorescent illumination (137 μmol mm height  throughom height . The strbspecies . p ≤ 0.001) between the light intensity (PPF) and the dry biomass was observed in in-vitro grown plants  with total leaf area (−2s−1 PPF respectively), did not determine differences in either leaf area  and/or high red:blue ratios  enhanced internodal elongation, facilitating the propagation of uninodal explants. Different problems have been associated with the propagation of olive shoots containing short internodal segments: the easy chance to damage axillar meristems, the contact of leaves with the medium and the difficulty to adequately embed the explant into the agar medium. These problems frequently cause necrosis and give rise to undifferentiated structures in micropropagated olive explants. This undesirable growth is induced by high light intensity or low B:R ratios . A negative effect of fluorescent lighting on shoot growth in comparison with LED illumination has also been reported in other plant species [−2s−1 impairs in-vitro culture of olive plants. Previous reports also observed a decrease of dry weight in plants grown in-vitro at high light intensity [−2s−1 PPF) [2 concentration in the vessel, while this limitation is counteracted by sucrose supply in the medium [The internodal elongation rate of micropropagated olive plants, but not the total number of nodes, was highly sensitive to both light quality and intensity . Thus, l species ,26,47,48ntensity ,50. Thiss−1 PPF)  since ite medium .The stimulation of stem elongation in response to a higher red/blue ratio has also been described in other micropropagated species. Conversely, blue light is known to inhibit stem elongation ,55,56,57Light quality/intensity can be used to regulate leaf growth in-vitro. We observed a positive correlation between light intensity and both leaf area and leaf fresh weight A,B. This2 concentration. Our results show that stomatal frequency in micropropagated olive is induced by higher light intensity , 20 gL−1 mannitol and 6 gL−1 agar. The pH of the medium was adjusted to 5.7 before autoclaving at 121 °C for 20 min. Six explants were incubated per jar under sterile conditions, avoiding direct contact of axillary buds with the agar culture medium. Jar lids were sealed with breathable sealing film  and incubated in the growth chamber for 69 days. Growing conditions were 23 ± 2 °C and a 16 h light photoperiod, under the lighting conditions described in Plantlets of two wild varieties belonging to different subspecies of −2s−1 or µE (R9B1-56); 80%/20% (R8B2-30) with 30 μmol m−2s−1 PPF; 70%/30% with 34 μmol m−2s−1 PPF (R7B3-34); 70%/30% with 52 μmol m−2s−1 PPF (R7B3-52); and 60%/40% with 40 μmol m−2s−1 PPF (R6B4-40). Red/Blue/White illumination was obtained with intercalated red, blue and white 14.5 W/m LED strips: Red/Blue 70%/30% plus white LED with 84 μmol m−2s−1 PPF (R7B3W-84); and Red/Blue 60%/40% plus white LED with 98 μmol m−2s−1 PPF (R6B4W-98). When white LED lighting was supplemented to the R/B lighting, a 50% white vs. 50% R/B was used. The R/B ratio was also adjusted within the white LEDs lighting in order to equal it to that of the R/B lighting in the corresponding treatment (R7B3 in the R7B3W-84 treatment and R6B4 in the R6B4W-98 treatment). The fluorescent lighting consisted of daylight (6500 °K) tubular fluorescent lamps with 137 μmol m−2s−1 PPF (TFL-137). The resulting spectral patterns are given in Light treatments, which included seven types of LED quality/intensity combinations and one fluorescent light, are summarized in 2s−1 light intensities are similar enough not to involve significant differences in plant responses; (ii) R8B2-30 vs. R7B3-34, given that 30 vs. 34 μmol/m2s−1 light intensities are similar enough not to involve significant differences in plant responses; (iii) R6B4-98 vs. R7B3-84, given that 98 vs. 84 μmol/m2s−1 light intensities are similar enough not to involve significant differences in plant responses.For a given number of LED chips, changes in the light quality (R/B ratio) inherently lead to changes in the light intensity (photon flux). To overcome the problem that two variables are assigned to a single treatment, different approaches have been addressed. On the one hand, in order to compare the effect of different light intensities without modifying light quality, we have compared plant responses to the following treatments: (i) R7B3-34 vs. R7B3-52 vs. R7B3-84; (ii) R6B4-40 vs. R6B4-98. The R7B3-34 vs. R7B3-52 comparison was possible by increasing the number of chips without modifying the R/B ratio. The additional treatment R7B3-84 was applied through supplementation with white LEDs. The same approach was used with the R6B4-98 treatment to make possible the comparison of the R6B4-98 vs. the R6B4-40 treatments. On the other hand, in order to compare the effect of light quality (different R/B ratios) without modifying the intensity (so that a single variable is assigned to a single treatment) we have compared the plant responses to the following treatments: (i) R9B1-56 vs. R7B3-52, given that 56 vs. 52 μmol/mFor the quantification of vegetative development, the following parameters were quantified during the course of explants growth: plant height, stem length, number of shoots, number of nodes, and number of leaves. After 69 days of growth, plantlets were harvested and fresh weight (FW) was obtained. Dry weight (DW) and water content (WC) was calculated after drying the explants at 70 °C for 72 h. WC was determined in leaves obtained from six plants, using all leaves per plant according to the following equation:After removing plantlets from the jars and before oven incubation, the leaves were excised and scanned in an Epson Stylus DX4000 multifunction printer  to obtain total leaf area through pixel quantification with the Software ‘Medición de Hojas v1.0′ .n = 12). After filtering, 1 mL of the suspension was mixed with 2 mL acetone, and chlorophyll-a (Chl a), chlorophyll-b (Chl b) and carotenoid (Cx + c) content (μg g−1 FW) was determined using a spectrophotometer  measuring the absorbance at 663 nm, 645 nm and 470 nm, respectively, according to Arnon (1959) [Photosynthetic pigments were extracted from fully expanded leaves using 0.1 g of fresh plant material in 10 mL of 80% aqueous acetone (n (1959) .Chlorophttp://www.zeiss.com/ (accessed on 20 September 2021), Carl Zeiss Microscopy GmbH, Jena, Germany) as described in [Ten samples from three fully expanded leaves, each leaf belonging to a different plant in a total of three plants per treatment, were used. Analysis of abaxial leaf cells was carried out in epidermal impressions, performed as described in . Number ribed in .p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Non-significant (ns) differences were indicated when p > 0.05. Multiple comparisons of means were analyzed by Tukey’s HSD (honestly significant difference). A multiple range test was calculated using the Statistical Analysis System ; StatPoint Technologies, Warrenton, VA, USA). In the graphics where the correlation between the photosynthetic photon flux and different growth/developmental parameters were plotted, the regression lines and the coefficient of determination (R2) were obtained using the corresponding function in Excel .Unless otherwise specified, the data analysis was carried out from 12 single-plantlet replications within each treatment. The data from all experiments were subject to analysis of variance (ANOVA) and levels of significance are indicated in the figures by asterisks: * Olea europaea showed better growth and development under LED illumination in comparison to fluorescent lamps. Seventy percent red and thirty percent blue LEDs with a PPF of 34 μmol m−2s−1 of light irradiance was the optimal lighting treatment for olive micropropagation in terms of lower energy consumption, higher height and biomass, accumulation of photosynthetic pigments, optimal manipulation of in-vitro explants, lower leaf area and minimal stomatal density.The aim of this study was to evaluate the effect of light quality and intensity on olive micropropagation, also comparing specific responses to LED vs. conventional fluorescent illumination. We concluded that two different subspecies of


Changes (p < 0.05) in all measures were observed after the LIST, and the greatest changes were observed after the six than after the three LISTs followed by one LIST . Many of the variables did not recover to the baseline for 5 d after six LISTs. These suggest that fixture congestion induces greater muscle damage and performance decline than a single match.The present study aimed to compare changes in muscle damage and performance parameters after playing single versus multiple soccer matches to examine fixture congestion effects on performance. Twelve elite female university soccer players performed single, three and six consecutive 90-min bouts of the Loughborough Intermittent Shuttle Test (LIST) with ≥12-weeks between conditions in a pseudo-randomized order. Heart rate, blood lactate, rating of perceived exertion and covering distance in each LIST were examined. Changes in several types of muscle damage  and performance measures  were taken before each LIST, 1 h, and 1–5 d after the last LIST. The total distance covered during the LIST was shorter ( Playing a soccer  match induces muscle damage that is represented by a combination of delayed onset muscle soreness and prolonged decreases in muscle function ,2,3,4. SJulian et al.  defined Some studies have demonstrated that residual fatigue accumulated over three to four successive soccer matches and performance is adversely affected in junior and young male players ,9,10,11.Therefore, the aim of the present study was to investigate changes in indirect markers of muscle damage and performance variables after multiple (three and six) simulated soccer matches (90-min LIST) performed by elite female university soccer players in comparisn to a single LIST condition. In the present study, the same players performed a LIST or three or six LISTs on consecutive days, and performance in the LIST, several indirect markers of muscle damage, and soccer-specific performance measures were taken before every LIST, one hour, and one to five days after the last LIST. The six LIST conditions were included as a possible worst-case scenario. It was hypothesized that the recovery and changes in muscle damage and performance variables would be slower and greater in the order of six LISTs, three LISTs, and the single LIST.2max) test three to four days before the first session to calculate the speeds (55% and 95% VO2max) applied during the LIST. To examine the effects of the LISTs on muscle damage and performance, MVC-KE and maximal voluntary isometric contraction torque of knee flexors (MVC-KF), muscle soreness of the knee extensors (SOR-KE) and flexors (SOR-KF), plasma CK activity, CMJ, 30-m dash, 30-m timed hop, balance, agility t-test, 6 × 10-m shuttle run and YYIR1 were measured one to two days before the 1st LIST (baseline) and one hour after each LIST, and one to five days after the last LIST  were compared in six female soccer players. Based on the effect size of 1 for a difference in MVC-KE change post-match between the two conditions, it was estimated that at least 11 participants were necessary for each condition, with an alpha level of 0.05 and power (1 − β) of 0.80 . ConsideTwelve elite female university soccer players who were selected for the Chinese Taipei Team of the 2018 Asian University Football Tournament provided informed consents to participate in the present study, which had been approved by the Research Ethics Committee of National Taiwan Normal University, Taiwan . The study was conducted in conformity with the policy statement regarding the use of human subjects by the Declaration of Helsinki. None of them had injuries during the period of the data collection and completed the three conditions. They played 17–20 official matches during the experimental period of 40 weeks and had training sessions five days a week (2.5–3 h per session). The present study was performed when the players had not had an official match for more than five days before the 1st LIST, and each condition  was preceded by at least five days of rest. All of the players had experienced three official matches in a row, and some of them had played an official match every day for five days.2max) and maximal heart rate (HRmax). An automatic gas analysis system  was used to measure oxygen consumption. Participants warmed up at a speed of 6.4 km/h on a gradient of 1% for 5 min, and the speed was increased by 1.6 km/h every 3 min while a gradient of 1% was kept [2max, running speeds corresponding to 55% and 95% VO2max were calculated. Thereafter, the participants performed and practiced the running corresponding to 55% and 95% VO2max on the treadmill for 5 min for each intensity.The participants performed an incremental continuous run test on a treadmill  until the point of volitional fatigue to determine maximal oxygen uptake , sub-maximal and maximal voluntary isometric contractions at 90° and 30° of knee flexion for knee extensors and flexors, respectively, on an isokinetic dynamometer , 30-m dash, 30-m timed hop, balance, agility r 20 min .Heart rate (HR), rating of perceived exertion (RPE), blood lactate (LA) and distance covered during LIST.During each LIST, heart rate (HR) was recorded by a Polar HR monitor ; rating of perceived exertion (RPE) was assessed with a 6–20 Borg’s scale ,21; and MVC torque of the dominant knee extensors (MVC-KE) and flexors (MVC-KF) were adopted from our previous studies, and the details were described elsewhere ,25,26,27CMJ was considered to be a marker of muscle damage, according to previous studies ,30,31, aMuscle soreness was quantified by a visual analog scale that had a 100-mm continuous line with “not sore at all” on one end (0 mm) and “very, very sore” on the other end (100 mm). Muscle soreness of the dominant knee extensors and flexors was assessed, separately, when each participant performed a full squat of dominant knee extensors and flexors, respectively ,19,36.Approximately 5 mL of venous blood was withdrawn by a standard venipuncture technique from the cubital fossa region and centrifuged for 10 min to extract plasma, and the plasma samples were stored at −80 °C until analyses. Plasma CK activity was assayed spectrophotometrically by an automated clinical chemistry analyzer  using a commercial test kit  ,37.t-test, 6 × 10-m shuttle run and YYIR1 tests) were performed indoors, and the order of measures was 30-m dash, balance, 30-m timed hop, agility t-test, 6 × 10-m shuttle run and YYIR1 tests with 5–10 min rest between tests. All participants had performed these tests regularly (eight times a year) for one year before the present study in a project by the Sport Administration, Ministry of Education (Taiwan); thus, they were familiar with the tests. However, performing all tests over consecutive days had not been done before the current study.Before the tests, all the participants performed warm-up exercises consisting of jogging, specific mobility exercises and stretching routines, and three 10-m sprints in 10–15 min. All measures . Each participant stood 0.3 m behind the starting line, started on a verbal signal being time-activated when the player crossed the first pair of photocells, and was instructed to run as fast as possible to complete the 30-m distance . ParticiThe 30-m timed hop test was adapted from a previous study . The 30-The present study used a test of eyes closed on unstable surface (ECUS) as a measure of balance ability using the Biodex BioSway Portable Balance System . Each participant was instructed to maintain her feet on the platform for 30 s for each test and had a 30-s rest between tests. Based on the study by Collado-Mateo et al. , a heel-t-test by Semenick [An adapted version of the Semenick ,41 was uThe 6 × 10-m shuttle run was carried out using the same telemetric photoelectric cells placed at the start line and 10 m. Each participant stood 0.3 m behind the start line, started on a verbal signal being time-activated when the participant crossed the first pair of photocells, and was instructed to run as fast as possible to complete the 10-m distance and then returned to the starting line for six repetitions. Players completed two runs interspersed by a 5-min recovery period, and the best time was used for further analysis.Endurance was assessed through the YYIR1, commonly used to assess endurance performance in soccer players . KeepingData were verified by a Shapiro–Wilk test for the normality and a Levene test for the homogeneity of variance assumption. All dependent variables before the single LIST condition, the 1st LIST of the three LIST and six LIST conditions were compared by one-way ANOVA. Changes in HR, RPE, lactate concentration, total distance covered during the LIST were compared among the trials and conditions by two-way repeated measures ANOVA. Changes in each dependent variable before, 1 h after, and 1–5 d after the 1st, 3rd and 6th LIST for the single LIST, three LIST and six LIST conditions, respectively, were compared by two-way repeated measures ANOVA.2 was calculated as a measure of effect size, which was considered as ~0.02 = small effect; ~0.13 = medium effect; and >0.26 = large effect [d effect size (ES) was calculated by the method of Thalheimer and Cook [n = 36) and the magnitudes of change in peak muscle soreness of KE and KF as well as peak CK after a single LIST, three LISTs and six LISTs. Similarly, the correlation analyses were performed for the magnitude of the decreases in MVC-KE, MVC-KF or CMJ and the magnitude of changes in the 30-m dash, 30-m timed hop test, balance, agility t-test, 6 × 10 m shuttle run and YYIR1 at 1 d after the single LIST, three LISTs and six LISTs (n = 36). A significant level was set at p ≤ 0.05. The data were presented as mean ± SD.The normalized changes in muscle damage and performance measures from the baseline at 1 h and 1–5 d after the 1st LIST for the single, three and six LIST conditions were obtained and were compared by two-way repeated measures ANOVA. When a significant interaction effect was found, a Tukey’s post-hoc test was performed, and ηe effect . Cohen’sand Cook  based onand Cook . Pearson2max) of the participants were 21.0 ± 1.1 (19–23) y, 159.1 ± 4.6 (152–165) cm, 56.2 ± 6.7 (45–65) kg, 14.4 ± 4.3 (7.3–21.5)%, and 54.1 ± 4.4 (47.9–59.2) mL/kg/min, respectively. No significant differences in the baseline MVC-KE , MVC-KF , muscle soreness of KE  and KF , plasma CK activity , CMJ , 30-m dash , 30-m timed hop test , balance , agility t-test , 6 × 10-m shuttle run , and YYIR1  were evident among the single LIST, three LIST and six LIST conditions . Before the 1st LIST, no significant differences among the conditions were found for HR , RPE , and LA concentration .The mean ± SD (range) age, height, body mass, percentage of body fat, and maximal oxygen consumption , HR , RPE , and post-exercise LA  in the 1st LIST were found among the three conditions , RPE  and LA  during the 1st LIST were similar among the conditions (p < 0.001) in the 2nd to 6th LISTs  for six LISTs (η2 = 0.418), and the 2nd  and 3rd LISTs (9883 ± 339 m) for three LISTs (η2 = 0.580) when compared with the 1st LIST of all conditions , HR , and RPE  before the 2nd to 5th or 6th LIST of the six LISTs were significantly greater than those before the 1st LIST . This was also the case for the baselines of LA , HR  and RPE  before the 2nd and 3rd LISTs of the three and six LISTs when compared with the 1st LIST .The baselines of LA (p < 0.001) after six LISTs  and three LISTs  than a single LIST, and the decreases were greater (p < 0.01) after six LISTs than three LISTs . The recovery of MVC-KE and MVC-KF was slower (p < 0.01) after six LISTs  and three LISTs  than a single LIST, and after six LISTs than three LISTs .p = 0.001, η2 = 0.641) than a single LIST, and the decreases were greater after six LISTs than three LISTs  without differences between three LISTs and a single LIST . The recovery of CMJ height was slower (p < 0.001) after six LISTs (η2 = 0.748) and three LISTs (η2 = 0.346) than a single LIST, and after six LISTs than three LISTs .The magnitude of decrease in CMJ height was greater after six LISTs  and three LISTs  was significantly greater than that after a single LIST, and after six LISTs than three LISTs . The extent of increases in plasma CK activity after six LISTs (η2 = 0.738) and three LISTs (η2 = 0.745) was significantly greater (p < 0.001) than that after a single LIST, and the increases were greater after six LISTs than three LISTs .Muscle soreness of KE and KF after six LISTs , 30-m timed hop , balance , agility t-test , 6 × 10-m shuttle run , and YYIR1  after six LISTs and three LISTs were significantly greater (p < 0.001) than that after single LIST, and the changes were greater (p < 0.001) after six LISTs than three LISTs .As shown in p < 0.001) correlations were evident between the magnitude of decrease in MVC-KE torque at 1 d after a single LIST, three LISTs and six LISTs; CMJ height at 1 d after a single LIST, three LISTs and six LISTs (r = 0.907); peak muscle soreness of KE (r = −0.606) and KF (r = −0.762) as well as peak CK activity (r = −0.875). Significant (p < 0.001) correlations were also evident between MVC-KF torque and CMJ height (r = 0.880), peak muscle soreness of KE (r = −0.653) and KF (r = −0.873) and peak CK activity (r = −0.849). Peak CK activity was also correlated (p < 0.001) with peak soreness of KE (r = 0.708) and KF (r = 0.838).Significant (p < 0.005) among MVC-KE (−19 ± 7%), MVC-KF (−22 ± 7%), CMJ (−10 ± 5%), balance (16 ± 8%), 30-m dash (6 ± 3%), 30-m timed hop (12 ± 6%), agility t-test (7 ± 4%), 6 × 10-m shuttle run (5 ± 2%) and YYIR1 (−18 ± 8%). As shown in n = 36) were correlated (p < 0.001) with the changes in 30-m dash (r = −0.762), 30-m timed hop (r = −0.793), balance (r = −0.793), agility t-test (r = −0.821), 6 × 10-m shuttle run (r = −0.822), and YYIR1 (r = 0.806) at 1 d after a single LIST, three LISTs and six LISTs. This was also the case for the normalized changes in MVC-KF and 30-m dash (r = −0.854), 30-m timed hop (r = −0.795), balance (r = −0.799), agility t-test (r = −0.831), 6 × 10-m shuttle run (r = −0.882) and YYIR1 (r = 0.891). The normalized changes in CMJ at 1 d after a single LIST, three LISTs and six LISTs (n = 36) were also correlated (p < 0.001) with the changes in 30-m dash (r = −0.770), 30-m timed hop (r = −0.803), balance (r = −0.836), agility t-test (r = −0.851), 6 × 10-m shuttle run (r = −0.889), and YYIR1 (r = 0.886) at 1 d after a single LIST, three LISTs and six LISTs  varied  were similar to those of North European elite female players reported in the previous studies [The level of the female players used in the present study was considered to be high, since their physiological characteristics , and linear sprint time (ES = 0.4 to 0.6), CK activity in the blood (ES = 0.4), well-being  and muscle soreness (ES = 0.6–1.3) remained substantially impaired at 3 d after an official soccer match. When compared to simulation protocols, a match format (11 versus 11 players) induced greater increases in CK activity in the blood (ES = 1.8 versus 0.7), IL−6 (ES = 2.6 versus 1.1), monocytes (ES = 1.71 versus 0.95) and neutrophil counts (ES = 2.06 versus 1.04) and muscle soreness (ES = 1.5 versus 0.7). The authors concluded that a period of 3 d post-match-play would not be long enough to completely restore homeostatic balance, and the recovery period post-soccer match was not a “one size fits all” situation [Significant changes in muscle damage and performance markers were observed after the single LIST, and none of the measures returned to the baseline levels until 4–5 d post-LIST , Table 1Silva et al.  stated tAlthough it is rare or unlikely that professional soccer players are involved in several official matches in a row, it is possible that players have three  or four t-test (−5%), 6 × 10-m shuttle run (−4%), and YYIR1 (−19%) all decreased. The magnitude of the decrease in some of the variables appeared to be greater in the present study when compared with that of the previous study [In the present study, 1 h after three LISTs, the 30-m dash (−5%), 30-m timed hop (−11%), balance (−16%), agility us study . It shouus study . As showAs shown in The mechanisms underpinning the greater extent of muscle damage and impaired performance measures following multiple LISTs were not investigated in the present study. As discussed above, a single soccer match leads to acute fatigue and muscle damage characterized by a prolonged decline in maximal muscle strength and muscle soreness, which could require at least three days to fully recover , Table 1n = 4) might have had more difficulty than those who had a single LIST (n = 4) or three LIST condition (n = 4) first. Sixthly, the present study did not assess the effects of muscle damage on skill performance, which is assumed to be affected and influence a game performance. Furthermore, previous studies [There are several limitations in the present study. Firstly, the participants of the study were female university soccer players, thus the results of the study may not reflect male, youth and professional players. Secondly, the changes in ovarian hormone status of these female players during the study were not recorded, and the possible effects of the hormones on the outcome measures were not controlled. Thirdly, the LIST does not include kicking a ball, tackles, maximal jumps, changes of directions, and direct contacts with opposing players, and the LIST was performed on the wooden floor of an indoor sport hall in the present study. Thus, this may be different from actual matches played on a grass pitch. Fourthly, previous studies showed that aerobic performance measurements such as YYIR1 affected the recovery of other tests such as short-sprint tests  after a soccer match ,58. In t studies ,60 reporIn conclusion, multiple LISTs resulted in greater muscle damage and impaired performance than a single LIST, and the extent of muscle damage was associated with the extent of decline in performance measures. The findings of the present study suggest that playing multiple soccer matches can impair the performance of elite female players; thus strategies to minimize muscle damage and enhance recovery are important for the players to perform better in a tournament.


Pedobacter jejuensis TN23 was isolated from soil from Terra Nova Bay, Victoria Land, Antarctica. The assembled draft genome size is 4,795,808 bp, and it contains a total of 4,095 genes with 3,970 coding sequences, including genes putatively involved in the degradation of chitin. Pedobacter jejuensis TN23, a Gram-negative strict aerobe . The concentration and purity were determined using a Qubit 2.0 fluorometer , and 1 μg of genomic DNA was processed to the next step. A Nextera DNA Flex library prep kit  was used to prepare the DNA library for sequencing following the manufacturer’s protocol. The sequencing was performed using the Illumina MiSeq platform and generated 1,567,670 reads (2 × 250-bp paired-end format).The genomic DNA of N50 value of 316,667 bp, a GC content of 35.8%, and a genome coverage of 196.13×. Gene prediction and annotation were carried out using the NCBI Prokaryotic Genome Annotation Pipeline  and one chitin disaccharide deacetylase (D7004_02630) related to chitin degradation. While the optimal temperature of most chitin deacetylases in fungi is 50 to 60°C  under the accession number


Toosendanin (TSN) is a triterpenoid compound mainly used as an ascaris repellant. Recent studies have shown that it possesses antitumor effects in many types of tumor cells. However, the effects of TSN on glioma cells have rarely been reported.Different assays were performed to investigate the effects of TSN on the different glioma cell lines including U87MG and LN18. The assays included colony formation, wound healing, and transwell assays. Furthermore, Hoechst 33342 staining, flow cytometry, and western blotting analysis were performed to investigate the apoptotic activities of TSN. Finally, the results were confirmed using a xenograft tumor model that comprised of nude mice.In vitro, the CCK-8 and colony formation assays showed that TSN effectively inhibited glioma cell proliferation. Moreover, the inhibitory effects on glioma cell migration and invasion were demonstrated through the wound healing and transwell assays, respectively. Hoechst 33342 staining, flow cytometry, and western blotting assays demonstrated the significant effect of TSN in the apoptosis induction of glioma cells. Furthermore, the anti-glioma effect of TSN was exerted through the inhibition of the PI3K/Akt/mTOR signaling pathways as demonstrated by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model confirmed the suppressive effect of TSN on tumor growth in vivo.Our results suggest that TSN is a promising chemotherapeutic drug for patients with glioma. Gliomas are tumors originating from brain glial cells such as astrocytes, oligodendrocytes, and ependymal cells. Among its different types, glioblastomas (GBM) are the most common intracranial tumor with the highest degree of malignancy, accounting for approximately half of the incidence, i.e., 3–4 per 100,000 , 3. In aToosendanin (TSN) is a tetracyclic triterpenoid derived from the Chuan neem seed or blast. It is mainly used as an ascaris repellent; additionally, it has biological activities including anti-botulism, neuromuscular junction inhibition, and central nervous and respiratory system effects . A largeTherefore, this study aimed to explore the anti-tumor effects of TSN on glioma cell lines (U87MG and LN18) in vivo and in vitro and potentially elucidate the underlying mechanisms.TSN (purity of approximately 99%) was purchased from Chroma Biotechnology Company . TSN was dissolved in dimethyl sulfoxide (DMSO), which was diluted with intact cell culture medium to achieve the concentration required for the experiment. The final concentration of DMSO was < 0.1% to avoid side effects.Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher . Penicillin/streptomycin (100×) and phosphate-buffered saline (PBS) were purchased from Shanghai Basal Media Technologies Co., Ltd. . Cell counting Kit-8 (CCK-8), BCA Protein Assay Kit, Annexin V-FITC Apoptosis Detection Kit, Hoechst Staining Kit, Cell Cycle and Apoptosis Analysis Kit, electrochemiluminescence (ECL) kit, and Tris-buffered saline with Tween-20 (TBS-T) were purchased from Beyotime Biotechnology Company . 740Y-P was purchased from TargetMol . Primary antibodies against cleaved caspase-3, Bax, cyclin D1, p21, matrix metalloproteinase-2 (MMP2), phosphoinositol 3-kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) were purchased from Cell Signaling Technology . Primary antibodies against PARP-1, phosphorylated PI3K (p-PI3K), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), matrix metalloproteinase-9 (MMP9), Bcl-2, cleaved caspase-9, p27, CDK-4, and CDK-6 were purchased from Abcam . Secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) were obtained from Cell Signaling Technology .2 at 37 °C.Human GBM cell lines U87MG, LN18, U251, LN229, and SVG p12were purchased from American Type Culture Collection (ATCC) and preserved in our laboratory at the Translational Medicine Institute of the First Hospital of Jilin University. All cell lines were cultured and maintained in DMEM supplemented with 10% FBS and antibiotics, including 1% streptomycin (100 μg/mL) and 1% penicillin (100 U/mL) in a humidified atmosphere of 5% CO3 cells per well. The cells were incubated overnight, which allowed adherence to the wall at 37 °C. The original cell culture medium was replaced with fresh medium containing different concentrations of TSN. After 24, 48, and 72 h of drug treatment, 10 µl CCK-8 solution was added to each well (avoiding air bubbles). Incubation was extended for 1.5 h, after which the optical density (OD) value was measured and recorded at 460 nm.The cell suspensions of U87MG, LN18, U251, LN229, and SVG p12cells were seeded into 96-well plates at a density of 7 × 102 humidified incubator. The culture medium containing the desired drug concentrations was added to each well. After 24 h, the medium was removed, and fresh medium was added to each well every 3 days for approximately 2 weeks. When colony formation was visible, they were fixed with ice-cold paraformaldehyde for 20 min and then stained with crystal violet for 20 min. Colonies with > 50 cells were observed under a microscope.U87MG and LN18 glioma cells were seeded into 6-well plates at a density of 800 cells per well, which were cultured overnight in a COU87MG and LN18 cells were inoculated into 6-well plates and cultured until the cell density reached approximately 80–90%. A sterile 200 μL was used to vertically scratch the plate surface and achieve similar width of cell wounds. Fresh serum-free medium containing different concentrations of TSN was added after discarding the original culture medium. The scratch area of each group was recorded separately by an inverted microscope at 0 and 48 h. The measurement of the wound healing area was performed using Image J software .4 cells per well were seeded in the upper chamber using 180 µL of serum-free medium; then, 700 µL medium containing 10% FBS was added to the lower chamber. After conventional incubation for 16 h, the residual matrix glue and cells in the upper chamber were lightly removed with cotton swabs; the chamber was then fixed with methanol for 15 min. The chamber was removed, the membrane was dried in air, and then stained with 1% crystal violet for 30 min. Finally, the invasive cells were observed, photographed, and quantified in three random fields under an upright microscope .The surface of the transwell upper chamber was evenly coated with the matrix and precooled serum-free solution at a ratio of 1:9 for 6 h. Then, 70 μL of serum-free medium was added to hydrate the basement membrane for 30 min. U87MG and LN18 cells treated with the indicated concentration of TSN with a density of 1 × 10U87MG and LN18 cells treated with TSN for 48 h were collected and fixed with 70% pre-cooled ethanol at − 20 °C for at least 24 h. Cells stained with propidium iodide (PI) and RNase for 20 min were detected by flow cytometry .4 cells per well. After the cells were adherent to the wall, different concentrations  of TSN were added to each well. After 24 h, the cells were stained with Hoechst 33342 (1 ug/mL) for 15 min under light-proof conditions and observed under a fluorescence microscope at 200× magnification; then, the number of apoptotic cells in each field was counted and analyzed.U87MG and LN18 cells were seeded in 6-well plates at a density of 5 × 105 cells/mL. After treatment with the corresponding TSN for 48 h, the cells with 1 × binding buffer (100 μL) were incubated with PI (5 μL) and Annexin V-FITC (5 μL) at room temperature for 20 min. Subsequently, 1 × binding buffer (400 μL) was added, and the apoptosis level of glioma cells was detected using flow cytometry. The apoptosis rate was analyzed using FlowJo software .The Annexin V-FITC/PI cell apoptosis detection kit was used to observe the effect of TSN on the apoptosis of human glioma cells. U87MG and LN18 cells were seeded in a 6-well plate at a concentration of 4 × 10After glioma cells were treated with different concentrations of TSN for 48 h, the cells were lysed to extract the total protein. The protein concentration was determined using a BCA kit. A total of 30 mg of protein was loaded per lane onto an SDS-PAGE gel (8–10%). The protein in the gel was transferred to the PVDF membrane using a wet transblotting system . The membrane was blocked in 5% skimmed milk powder for at least 1 h. It was left to incubate overnight with the appropriate primary antibody at 4 °C on a shaker. Subsequently, the membranes were incubated with the appropriate secondary antibody at room temperature for 2 h. The exposure of chromogenic protein bands with ECL luminescent solution was photographed and analyzed using a gel imaging system.7 cells/mouse) was inoculated subcutaneously into the right armpit of nude mice. When the tumor size reached approximately 180 mm3, the mice were randomly divided into two groups with five mice in each group. The mice from the experimental and control groups were intraperitoneally injected with 1.0 mg/kg TSN and the same volume of saline, respectively, every day for consecutive 21 days.5-week-old, male, specific pathogen-free (SPF)-level, athymic, immunocompromised (BALB/c nude) mice were obtained from Charles River Laboratories . Nude mice were reared carefully in the SPF animal laboratory of The First Hospital of Jilin University. Under sterile conditions, a 100-μL suspension of U87MG cells  and shortest diameters (S) of the tumors were recorded. The tumor volume was calculated according to the standard formula: tumor volume = 0.5 × L × SThe organs were fixed with 4% formaldehyde for 24 h, embedded in paraffin, cut into 4 pieces-μm thick sections, and dehydrated. The sections were then stained with H&E. In addition, some tumor slices were immunostained with the, Ki-67 (1:500) antibody, and p-Akt (1:100) antibody. Digital images were observed and captured under a standard optical microscope.All experimental results were obtained from at least three independent experiments using GraphPad Prism 8.3 . Data are presented as the means ± standard deviation (SD). Student’s t-test was used to analyze data between groups, while one-way ANOVA was used to analyze the differences between multiple groups. Statistical significance was set at p < 0.05.Glioma cells were treated with different concentrations of TSN  to evaluate the effect of TSN. The CCK-8 assay results showed a dose- and time-dependent decrease on the viability of glioma cells when TSN concentration increased ; furthermore, it showed the limited toxicity of TSN on normal human astrocytes. In addition, we found that the U87MG and LN18 cell lines were more sensitive to TSN; therefore, we selected them as the research objects in the follow-up experiment.Second, the wound healing, transwell, and western blotting assays confirmed the effect of TSN on migration and invasion. Migration and invasion of malignant tumor cells is a complete and complex process . StudiesThird, Annexin V-FITC/PI double staining, Hoechst 33342 fluorescent staining, and western blotting assay were used to detect apoptosis. The results showed that TSN effectively induces apoptosis, which involves the rate of apoptosis, changes in apoptotic nuclei, and the expression level of related apoptotic proteins. Apoptosis is known as a form of programmed cell death, and its inhibition leads to unlimited growth of tumor cells . PreviouIn addition, flow cytometry results showed that the TSN treatment group had significantly higher cell ratio in the G0/G1 phase of the cell cycle compared to the control group. P21 and p27 are CDK inhibitors; these bind to cyclins-CDKs complex in the G1 phase, which negatively regulates the cell cycle through the inhibition of the G1/S transition phase of the cycle, which prevents cell proliferation and tumor formation , 32. AddFurthermore, recent studies have found a close relation between the activation of the PI3K/Akt/mTOR pathway and the occurrence, development, prognosis, and drug resistance of tumor cells , 35. TheTo further confirm the anti-glioma cancer effect of TSN in vivo, we established a xenograft tumor model of nude mice by transplanting U87MG cells. The results confirmed the inhibitory effect of TSN on the transplanted tumor with limited side effects on the major organs of the nude mice. Likewise, western blotting with immunohistochemical results showed that TSN had a significant effect on cell proliferation, migration, invasion, apoptosis, cell cycle arrest, and PI3K/Akt/mTOR signaling pathway in vivo.Our current research showed that TSN plays a crucial role in the anti-tumor effect on glioma cells in vivo and in vitro through interference of the proliferation, invasion, migration, apoptosis, and cycle distribution, mediated through the inhibition of the PI3K/Akt/mTOR signaling pathway. Collectively, these findings provide evidence that TSN is a potential therapeutic agent for glioma.


Theobroma cacao L.), native to French Guiana. No data were available regarding its chemical composition and biological properties; therefore, a study was necessary, using Forastero as a reference. To exemplify biological activities of the cacao species, cocoa extracts were evaluated by antioxidant  and anti-inflammatory assays. Our results showed that raw Guiana presented equivalent DPPH and FRAP activities, but a 1.3-fold higher antioxidant activity (1097 ± 111.8 μM ET/g DM) than Forastero (838.5 ± 67.8 μM ET/g DM) in ORAC assay. Furthermore, the impact of fermentation  on Guiana cocoa beans composition and health properties was also studied. Indeed, fermentation, a key step necessary to obtain the taste and color of chocolate, is generally known to alter bean composition and modulate its health benefits. At six days, the fermentation process led to a nearly 25% lower antioxidative capacity in various assays. Moreover, in inflammation-induced macrophage assays, Guiana and Forastero unfermented extracts induced a 112% stimulation in TNF-α production, and a 56.8% inhibition of IL-6 production. Fermentation altered the cocoa composition by diminishing bioactive compounds, which could be responsible for these biological activities. Indeed, after six days of fermentation, compounds decreased from 614.1 ± 39.3 to 332.3 ± 29 mg/100 g DM for epicatechin, from 254.1 ± 14.8 to 129.5 ± 20.7 mg/100 g DM for procyanidin B2 and from 178.4 ± 23.5 to 81.7 ± 2.9 mg/100 g DM for procyanidin C1. The similar composition and the equivalent or higher antioxidant activity of Guiana leads us to propose it as an alternative to Forastero.Guiana is a little-known and endemic variety of cocoa ( Theobroma cacao L., belonging to the Malvaceaes family and Theobroma genus, is a plant native to Neotropical rainforests with more than 10 known existing varieties [Cocoa designation arieties . Guiana arieties ,4.The chemical composition of several varieties of cocoa has already been studied. High fat contents account for more than 40–57% of the bean weight, and the majority of lipids, including stearic, oleic, palmitic and linoleic acids, were found in varying amounts depending on variety . SterolsCocoa is also well-known to stimulate the immune system by presenting anti-inflammatory (AI) activities. Indeed, the immune system is based on two complementary pathways: the first is an innate non-specific response, and the second is an adaptative response involving specific reactions to antigens. The innate response is characterized by an inflammation process. In many pathologies, particularly metabolic, the inflammatory state can become chronic and harmful for the cellular tissues and their functions. Thus, the use of anti-inflammatory agents may be of great interest. The cocoa anti-inflammatory effects have already been underlined due to its polyphenol’s effects on mediators such as tumor necrosis factor alpha (TNF-α) and interleukin 1 (IL-1) and 6 (IL-6) . These cConsidering chocolate as the most consumed food in the world, its basic ingredient (cocoa beans) must undergo several stages of processing in order to acquire its typical aroma. Among these, fermentation and roasting play a major role in the formation of flavors. However, these steps also induce chemical modifications, engendering alterations in seed composition and biological properties ,17,18. TGuiana and Forastero cocoa pods were harvested in Sinnamary, French Guiana, from the CIRAD cocoa station (GPS data: 5°20′05.8″ N 52°57′14.9″ W). The pods were discarded, and only cocoa beans (with shells) were used for the next steps. They were divided into 2 groups: those to be unfermented and those to be fermented at different degrees of fermentation. In the case of the unfermented group, the pulp of fresh beans was removed by pectinase enzyme  without physical alteration of the bean. For the other group, beans were fermented for 2, 4 and 6 days in plastic boxes. The choice of these fermentation times was based on the fact that the samples taken on the second day were at the alcoholic fermentation stage, and those taken on the fourth day were at the acetic fermentation stage. The choice to stop the process at 6 days of fermentation is explained by the fact that this corresponds to the maximum duration traditionally chosen to obtain the Forastero commercial cocoa. Then, each group was dried in oven at 60 °C, cooled with nitrogen, and ground into a spray powder.g, 4 °C, 10 min) and filtrated. This procedure was repeated five times and all supernatants were collected, then evaporated to measure fat content. The fat-reduced cocoa powder was then extracted following the same procedure with a mixture of acetone/water/formic acid   three times. Acetonic extracts were evaporated, then dissolved in 50% methanol  at a concentration of 40 g/L. Finally, CEs were stored in the dark at −20 °C until analysis. These extractions were realized in triplicates.The Cocoa extract (CE) was realized as follows. Freeze-dried cacao powder was degreased by maceration with n-hexane  overnight. Following this, this mixture was immerged in an ultrasonic bath for 10 min at 130 kHz. Then, the mixture was centrifuged  were mixed. After 2 h in the dark at room temperature, the absorbance of the blue color was measured at 750 nm against a blank sample with a UV–VIS 50 Bio Varian spectrophotometer. A calibration curve with gallic acid , as standard was produced. The TPC was expressed as the milligram equivalent of gallic acid per gram of dry matter (mg GAE/g DM). All TPC measurements were performed in triplicate.The total polyphenol content (TPC) of the CE was determined spectrophotometrically according to Arnous et al. (2002), with some modifications . BrieflyThe total Flavonoid content (TFC) was quantified after derivation with dimethylaminocinnamaldehyde (DMACA)  in 1N HCl acid methanolic medium , which conducted to a colored carbonium ion at 640 nm. The assay was realized following the method of Arnous et al. (2002) with some modifications . In a he50 and TE concentrations were determined with the following equations:DPPH assay was performed according to the method described by Brand-Williams et al. . This prAll DPPH measurements were performed in triplicate.v/v/v): 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) at 10 mM , FeCl3  at 20 mM in 40 mM hydrochloric acid (HCl) , and 300 mM sodium acetate buffer , (pH 3.6). Readings of the absorption maximum at 593 nm were taken after 30 min in dark conditions at room temperature using a UV–VIS 50 Bio Varian spectrophotometer. The Fe2+ solution was used to perform the calibration curves. Results were expressed as μmol equivalents of Fe2+ per gram of dry matter (μmol Fe2+ Equation/g DM). All FRAP measurements were performed in triplicate.This procedure is based on the ferric reducing ability of each standard solution according to Benzie and Strain, with some modifications . BrieflyThe ORAC assay was carried out according to a procedure described by Morel et al. (2012), with some modifications . For ORA2.Murine macrophage cells J774-A1 were grown in RPMI 1640 medium  containing 10% heat-inactivated fetal bovine serum , and 1% of penicillin/streptomycin  in a humidified incubator at 37 °C and 5% of CO2. Then, a pre-treatment of 100 μL of CE proceeded for 24 h. The cells were incubated with 50 μL of MTS/PMS for 4 h at 37 °C. The amount of MTS formazan product was determined by measuring absorbance using a micro-plate reader (Molecular Devices) at a test wavelength of 540 nm.The 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)  and phenazine methosulfate (PMS)  reduction assay was based on the following procedure. Murine macrophages were plated at a density of 0.6 million cells/mL on 96-well plates in 100 μL Hanks’ balanced salt solution (HBSS) , for 16 h at 37 °C, 5% CO2. After forming a cellular mat by adhesion, cells were pre-treated with CE  for 4–24 h. Cell inflammation was induced with 100 μL lipopolysaccharide (LPS) Escherichia coli serotype 55B5  at 100 ng/mL and murine interferon gamma (IFNγ)  at 10 UI/mL for 18 h. The supernatant of the pre-treated macrophages (at 4 and 24 h) was harvested. The quantification of NO was obtained by measuring the absorbance of 100 μL of cell culture supernatant revealed with Griess’s reagent (100 μL) at 540 nm with a micro-plate reader (Molecular Devices). The NO inhibition (%) was determined spectrophotometrically with a calibration curve of NaNO2 (1.56 to 100 μM). All NO measurements were performed in triplicate.In 24-well plates, 500 μL of murine macrophages at 1 million cells/mL were plated during 2 h at 37 °C, 5% COThe supernatant of the pre-treated macrophages (4 and 24 h) was harvested and stored at −80 °C until analysis. Tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) of the 4 and 24 h pre-treated samples were quantified via ELISA assay kit (eBioscience). Samples for TNF-α analysis were measured with the diluted supernatant (1/10) for all the samples, and for IL-6, supernatants were diluted by 1/10 for unstimulated and 1/50 for stimulated samples with a micro-plate reader (Molecular Devices) at 540 nm. With calibration curves (0 to 1000 pg/mL of TNFα or 0 to 500 pg/mL of IL-6), results are expressed as the % stimulation of TNF-α production and % inhibition of IL-6 production. All cytokine measurements were performed in triplicate.® 5 C18-column . The used solvents were (A) water–acetonitrile–formic acid  and (B) acetonitrile–formic acid  ). The analysis was made with the following gradient: 0.1–4 min for 5%B, 4–15 for 5%B, 15–75 for 13%B, 75–85 for 35%B, 85–95 for 100%B, 95–95.5 for 100%B, and 95.5–105 for 5%B. Flowing at 0.7 mL/min, 10 μL of CE was injected at 10 g/L and analysis was conducted with a DAD detector at 280 nm. Results of the quantification of theobromine , caffeine , epicatechin, and procyanidins A1, A2, B2 and C1  were expressed in mg of compounds per 100 g of dry matter (mg/100 g DM). All quantifications were performed in triplicate.Analyses of CE compositions were performed by liquid chromatography using apparatus equipped with a quaternary pump, diode array detector, Varian 445 LC-DAD column compartment temperature control on an ACEp ≤ 0.05).Statistical significance was determined using non-parametric analysis (Mann–Whitney U test) using STATA\IC-version 12. Significance was accepted at the 5% level (v/v), fermentation induced a significant impact. After six days of fermentation, extraction yield increased in 1.9% and 2.1% in Guiana and Forastero varietals, respectively. No varietal difference was noted whatever the process used.Due to possible interferences of carotenoids and apolar compounds such as lipids in AO assay measurements , defattiAll results dealing with TPCs, TFCs and antioxidant activities are shown in If no varietal differences were noticed in the TFC assays , a significant varietal difference was noticed in TPCs .2+/g DM) with unfermented Forastero. However, unfermented Guiana presented a 1.3-fold higher activity (1097 ± 111.8 μM ET/g DM) than unfermented Forastero (838.5 ± 67.8 μM ET/g DM) in the ORAC assay.In vitro AO activities cannot be determined with a single method; therefore, we have selected a few with various AO mechanisms to do so. FRAP induces an electron transfer, while DPPH induces a radical electron transfer and ORAC induces a proton-transfer mechanism. Unfermented Guiana presented equivalent DPPH activity (294.9 ± 17.9 μmol ET/g DM) and FRAP activity , that could cause their death. We pre-treated macrophages with Guiana and Forastero CEs with only unfermented samples those and fermented for two and six days. These fermented samples were chosen because they represented the two stages of the fermentation process: alcoholic and acetic fermentation, respectively. The three fermentation times were analyzed at three concentrations: 12.5, 25 and 50 μg/mL.LPS/IFNγ stimulation is well known to cause a significant production of NO, TNF-α or IL-6, by inducing inflammation in macrophages. The impact of CE on this production could be determined with the ratio of cytokines produced with or without CE before LPS/IFNγ stimulation.In the case of NO production in our study, no impact was observed with a 24 h pre-treatment of CE at different concentrations. Moreover, fermentation might not modulate NO production either; the slightly stimulating rate, which was noticed for the CE fermented for six days, was not significant to confirm an impact. This trend was noticed for both varieties in Conversely, a stimulation of the TNF-α production was noticed after a 24 h pre-treatment with the Guiana CEs at all concentrations . Indeed,As described below, LPS/IFNγ stimulation caused a significant accumulation of IL-6 in the culture medium. Unlike TNF-α, CE pre-treatment at different concentrations significantly inhibited the LPS/IFNγ-induced IL-6 production . Indeed,This inhibition appeared after 4 h of pre-treatment, whereas no actions in TNF-α at this same incubation time were noticed . FurtherA varietal difference was noted between Guiana and Forastero, despite a very small difference in their lipid rates. This characteristic was also noticed in another comparative study involving Guiana (52.1%) and Forastero-type cocoa “Amelonado” (53.2%) from French Guiana . In our v/v), an Indonesian cocoa has demonstrated an extraction yield of 12.95% [Extracted with ethanol  has an equivalent capacity.By comparing our results with those of the literature we observed that Guiana has an equivalent DPPH activity to two blends of Brazilian cocoa hybrids which have AO capacities of 324.06 ± 51.7 and 363.3 ± 120.1 μmol TE/g DM ,31. For 2+/g DM , Guiana Caffea canephora) demonstrated a DPPH capacity of 116 ± 15 μmol TE/g DM [Caffea arabica) demonstrated DPPH capacities of 83 ± 18 and 1627.09 ± 158.7 μmol TE/g DM) [Caffea benghalensis and Caffea Liberica also demonstrated high DPPH activities of 1691.4 ± 153.3 and 2212.8 ± 204.1 μmol TE/g DM, respectively [2+/g DM, respectively) to Guiana [Commonly compared to another widely consumed daily food, unfermented Robusta green coffee  ,35. Otheectively . Compareo Guiana . ConsequNo varietal difference was observed in FRAP and DPPH assays, but Guiana has a higher ORAC capacity than Forastero. DPPH and ORAC are both based on antiradical mechanisms, but the DPPH• radical is a non-natural radical and cannot be found in organisms. In contrast, ORAC is based on AAPH use which, in the presence of oxygen, leads to the formation of peroxyl radicals ROO•. These radicals are naturally produced in living biological organisms. Consequently, Guiana demonstrated a better action on a more realistic test, which may indicate that Guiana is more effective than Forastero. The difference in the AO capacities for both cocoa varieties could be explained by a different composition, because the correlation between AO capacity and polyphenols compounds has already been demonstrated. For example, Todorovic et al. (2015) showed that cocoa powder, which presented a three-fold higher TPC than white chocolate, has 17-fold and 11-fold higher DPPH and ORAC activities, respectively . In our R2 value of 0.98 for FRAP, 0.89 for DPPH, and 0.81 for ORAC, suggesting that a good correlation exists between total phenolic contents and AO capacities. In the literature, this correlation was observed after six days of fermentation: Forastero hybrids TSH565 and ICS60 TPCs decreased by 35% and 20%, while DPPH activity decreased also by 25% and 8%, respectively [Fermentation induces a degradation of compounds within cocoa beans, particularly polyphenols. Indeed, polyphenol oxidase and glycosidase would be responsible for the conversion of polyphenols into quinones, which will complex with other cellular compounds. Additionally, the acidic conditions of the medium induce hydrolysis of polyphenols . With anectively . FurtherAnalysis of our chromatograms showed that we identified the main compounds commonly found in cacao species. Indeed, methylxanthines and phenols as procyanidins and epicatechins have been found . MethylxNo significant differences were identified in the epicatechin and procyanidins contents between Guiana and Forastero. As for methylxanthines, polyphenol contents could be variable, depending on factors such as variety or geographical origin. Indeed, for Forasteros from Nigeria, Cameroon, Ivory Coast, Ghana, and Ecuador, epicatechin contents were between 151 ± 6 and 451 ± 20 mg/100 g DM and procyanidin B2 were between 68.4 ± 15 and 157 ± 16 mg/100 g DM . A signiOne of the aims of fermentation is the reduction in bitterness of the bean; this could be possible due to the external diffusion of methylxanthines 30%) and polyphenols (20%), caused by the increase in permeability of cells of the seed . In our % and polWith epicatechin contents of 614 ± 0.39 mg/g DM and procyanidin contents (dimer and trimer) of 512 ± 0.21 mg/g DM, the valorization of Guiana into chocolate could be a good alternative to obtain new sources of antioxidants. For Guiana cocoa beans, after six days of fermentation, we observed a loss of 46% and 49% for epicatechin and procyanidins, respectively. Bordiga et al. (2015) have demonstrated that the transformation of fermented beans to chocolate induced an average loss of epicatechin by 81.8 ± 8.1% for African cocoa . Consequ−6 μM for unfermented Guiana and 6.11 × 10−6 μM for unfermented Forastero. These small amounts of epicatechin in our analysis could explain the absence of NO production modulation. Moreover, we used a pre-treatment of 24 h, whereas Ramiro et al. (2005) used a simultaneous action of CE and epicatechin with inflammatory stimuli (added at the same time).In the literature, polyphenols seemed to be responsible for AI activities in macrophages by modulating key inflammation-regulators such as TNF-α, IL-1-β and IL-6 in vitro and in vivo . AccordiEven though most of the time cocoa could inhibit inflammatory cytokines such as TNF-α, some authors have reported the stimulating capacities of TNF-α production, which would confirm that cocoa has a pro-inflammatory potential. For example, CEs which were incubated for 72 h in another inflammatory cell model  have demonstrated a TNF-α production stimulation after the initiation of inflammatory process by phytohemagglutinin (PHA). Indeed, in stimulated PBMC, stimulation rates of TNF-α production were noticed, with a maximum of 128% for hexamers and a minimum of 1.5% for monomers . Moreover, in non-stimulated PBMC, CE also induced a stimulation in TNF-α production by 42–183% with phenolic fractions  with a maximum of 412% for hexamers . ConsequIn our study, we determined the procyanidin contents (dimers and trimers) in both unfermented cocoa samples at 50 μg/mL. Guiana presented a maximum of 0.7 μg/mL of dimer B2 and 0.5 μg/mL of trimer C1, whereas impacts were noticed at 25 μg/mL of fraction cocoa polymers in the study of Mao et al. (2002). We used a 24 h pre-treatment to determine the impact of CEs on TNF-α production, whereas Mao et al. started the inflammatory stimulation at the same time as the CE treatment. Other authors have also applied pre-treatments instead of simultaneous treatments and demonstrated stimulating effects. For example, cells pre-treated with short-chain flavonoid fractions (SCFFs—monomers to pentamers) and those with long-chain flavonoid fractions (LCFFs—pentamers to decamers) for 16 h before stimulation, demonstrated TNF-α stimulations of 36% and 90%, respectively .Moreover, fermentation, which was demonstrated to diminish the polyphenol content, particularly epicatechin and procyanidins, seemed to improve the stimulating capacity on TNF-α production. This trend could reinforce the hypothesis that polyphenols are not the only responsible for this potential. Otherwise, TNF-α, which is often illustrated as a negative cytokine, could play a role in immunocompromised metabolisms. TNF-α demonstrated an antitumoral potential by inducing the apoptosis of dangerous cells, but also by inducing the resolution of inflammation through triggering cell recruitment ,51.−6 μM for an unfermented sample and 49% ± 0.1 for 2.7 × 10−6 μM for a sample fermented for six days. These appear to be enough to induce an inhibition of IL-6 production, although the possibility of a cocktail effect with other compounds should not be overlooked.Unlike the previous two actions, Guiana has shown an anti-inflammatory action on the IL-6 production. This anti-inflammatory capacity has already been found in other studies. Indeed, with human monocytes pre-treated for 1 h with CEs, and then stimulated, Zeng et al. (2011) noticed an inhibition of IL-6 production by 24 ± 5% (0.1 μg/mL) to 65 ± 7% (10 μg/mL) . As for Consequently, in our CE, procyanidins seemed to be responsible for the TNF-α production stimulation, and monomers such as epicatechin seemed to be responsible for IL-6 production inhibition. The procedure seemed to also have an impact, and various actions could depend on the fact that CE was used before or simultaneously with the pro-inflammatory stimuli. These opposite immunomodulatory capacities could be due to the balanced relationship between IL-6 and TNF-α .Streptococcus pyogenes [Indeed, an IL-6 deficit contributes to a suppression of high TNF-α levels in infected mice by pyogenes . This copyogenes . This mopyogenes . AdditioTo the best of our knowledge, this is the first time that Guiana biochemistry and health benefits have been characterized. The few existing studies involving this variety only focused on its agronomic performances or genetic distinctions with other varieties. Our work highlighted the in vitro AO capacity of Guiana and showed that it would be as efficient as other cocoa varieties. Polyphenols were correlated with these AO activities. Guiana presented a similar phenol content (mainly epicatechins or procyanidins) when compared to Forastero. A varietal difference could explain methylxanthine content differences, because Guiana contains less caffeine and more theobromine than Forastero. This characteristic, leading to a lower astringency, could induce a fine aroma and a reduction in the fermentation process time. Moreover, because fermentation is also responsible for a 30% decrease in AO potential, this reduction in process time could potentially maintain the benefits of Guiana, while having its characteristic flavors.Furthermore, Guiana has demonstrated a better inhibition in IL-6 production than Forastero, but also a stimulation in TNF-α production at two pre-treatment times. This is the first time that Guiana has been demonstrated to show opposite actions on inflammatory assays. This behavior shows that cocoa is a complex matrix which presents different attributes depending on how it is used (preventive or curative aims). Fermentation could modulate this immunomodulatory capacity by improving the TNF-α stimulation of Guiana CEs. Indeed, further studies might be conducted to allow a better comprehension of these AO and AI capacities. Moreover, study using fractions of compounds could be a good strategy to study bioguiding and synergic effects.


Hydrogen peroxide (H2O2) production, lipid peroxidation, protein, phenolic content and antioxidant enzyme activities were investigated. Increased H2O2 formation was detected even at the lowest BPA treatments from the beginning of the experiment and both the enzymatic and non-enzymatic antioxidant defense mechanisms were activated upon application of BPA. Elevated H2O2 levels that were detected as a response to increasing BPA concentrations and incubation time, led to the decrease of protein content on the 4th day even at the two lower BPA concentrations, and to the increase of the lipid peroxidation at the highest concentration. However, on the 6th day of BPA exposure, protein content did not differ from the control, indicating the ability of both the enzymatic and non-enzymatic mechanisms (such as superoxide dismutase (SOD) and phenolics) to counteract the BPA-derived oxidative stress. The early response of the protein content determined that the Low Effect Concentration (LOEC) of BPA is 0.3 μg L−1 and that the protein content meets the requirements to be considered as a possible early warning “biomarker” for C. nodosa against BPA toxicity.The effects of environmentally relevant bisphenol A (BPA) concentrations (0.3, 1 and 3 μg L EDC c 0.087–5.25 μg L−1 μg L−1)  and micr μg L−1) , where tL−1) [−3 , and theL−1) [−3 . For exawastes 1–0 μg L−1 L−1) [−3 ,23,24,252−, hydrogen peroxide, H2O2, singlet oxygen 1O2) [One interesting route of BPA toxicity is the production of reactive oxygen species (ROS)  ,22,23,26gen 1O2) ,17,26, cgen 1O2) ,26 or ingen 1O2) ,23.Cymodoceanodosa (Ucria) Ascherson 1870, one of the five most widespread Mediterranean seagrass species [C. nodosa has the ability to act as a “bioindicator” organism of many anthropogenic-derived pressures [C. nodosa, for the ecological status assessment of coastal and transitional waters have also been established [Aquatic plants could be used as bioindicators of BPA toxicity . In this species  and belo species , can act species . C. nodoressures , such asressures ,33,34,35ressures ,33,34,35ablished ,37.C. nodosa plants [C. nodosa cell cytoskeleton (actin filaments and microtubules) and the inhibition of various plant part elongation [C. nodosa leaf photosynthetic machinery was further investigated. It has been stated that BPA-derived H2O2 generation  seemed to be a key factor, triggering oxidative damages on epidermal leaf cells but simultaneously inducing retrograde signaling, also conferring tolerance to BPA [−1) C. nodosa intermediate leaf blades, ROS generation (H2O2), lipid peroxidation, protein and phenolic content, and antioxidant enzyme activity systems were investigated. The main goal of this study was to assess the impact of environmentally relevant BPA concentrations [C. nodosa leaf blade oxidative stress parameters , and the alterations in the cell membrane permeability (lipid peroxidation) and their protein contents. In addition, the toxic BPA effect levels were evaluated, and the possible sensitive stress “biomarkers” were identified as early warning indicators of BPA risk in aquatic environment.Concerning the impact of BPA to seagrasses, the only relevant data existing is that reported for a plants ,23,25. Iongation ,25 act atrations  on C. no2O2 in the epidermal cells of intermediate C. nodosa leaves increased with increasing incubation time and BPA concentrations, as indicated by the H2DCF-DA fluorescence (2O2 was detected even at the lowest BPA treatment (0.3 μg L−1) from the beginning of the experiment  values, which is calculated at all BPA treatments and each incubation time, was significantly higher than in the control  (Levels of Hrescence . H2O2 waperiment b. H2O2 p< 0.001) .2O2 generation (max CTCF ratio) was observed at the 3 μg L−1 in the beginning of the experiment  . Howeverperiment .−1), on the 4th day, was significantly (p < 0.05) increased compared to the control (1.6–2.0-fold) and the higher solution (3 μg L−1) and continued to be slightly elevated (p > 0.05) on the 6th day. At the highest treatment (3 μg L−1), SOD activity was equal to (p > 0.05) or lower (p < 0.05) than the control and the two lower treatments in the experiment (Superoxide dismutase activity (SOD) at the two lower BPA solutions (0.3 and 1 μg Lperiment a.−1 was higher (p < 0.05) compared to the control and the lowest BPA treatment (0.3 μg L−1), at the beginning of the experiment (2nd day), whereas at 0.3 μg L−1 BPA, its activity was elevated (p < 0.05) relative to 1 μg L−1 on the 6th day (Ascorbate peroxidase (APX) activity at 1 and 3 μg L 6th day b.−1 BPA was lower (p < 0.05) than the 3 μg L−1 on the 2nd day, and at the 0.3 and 1 μg L−1 BPA treatments, it was lower (p < 0.05) than the control (p < 0.05) and the 3 μg L−1 BPA treatment on the 4th day (Protein content at 1 μg L 4th day c.−1 was higher (p < 0.05) than the control and the two lower BPA treatments, at the start of the experiment, whereas it was lower (p < 0.05) than the control and the lowest BPA solution on the 4th and 6th day  content, at the 3 μg L 6th day d.−1) was elevated (p < 0.05) compared to the control and the highest solution from the beginning (1st and 2nd day) and at the end of the experiment (8th day) , but this increase was significant (p < 0.05) on the 4th day , and on the 4th day (p > 0.05) as well  a–d. Mann8th day) e. At 1 μ 4th day e. Finall as well e.Lemna gibba and Ceratopyllum demersum and the seagrass Cymodocea nodosa. In L. gibba, at higher than the environmentally relevant concentrations, BPA affected its growth and survival, photosynthetic activity , and sugar and starch contents [C. demersum, BPA caused protein and photosynthetic pigment reductions [−1) were within the environmentally relevant concentrations . Spect plants ,7,8. The. nodosa ,23,25, aC. nodosa [C. nodosa, BPA (i) disrupted the cell cytoskeleton  at 0.03 and 0.1 μg L−1 BPA, respectively [50 values: 0.137, 0.029 and 0.025 μg L−1, respectively [2O2 increase (positive H2DCF-DA staining) firstly at 0.5 μg L−1 BPA exposure [2−, hydrogen peroxide, H2O2, etc.) generation [C. nodosa, under BPA exposure, a decrease of the photosynthetic parameters ΦPSII and ΦNPQ, and an increase of ΦNO were observed, which supported the role of H2O2 in the C. nodosa response against BPA toxicity [C. nodosa intermediate leaf blades, BPA induced H2O2 overproduction even at the lowest concentrations used (0.3 μg L−1), and the shorter duration of exposure  reexposure ,40,41,42neration ,26,43. Iexposure ; Table 1reatment ; Table 1olerance ,46,47,48s .2O2 is relatively stable and a compound with high longevity [2O2 overproduction exceeds the antioxidant defense capacity can create oxidative stress in mitochondria, chloroplasts and peroxisomes [C. nodosa leaf blades the activity of SOD, an enzyme which catalyzes the dismutation of O2− radical to H2O2 and O2  on the 4th and up to 6th day, following an increase of H2O2 production pattern (see CTCF ratio). In contrast, at the highest treatment concentration (3 μg L−1), SOD activity was lower or equal compared to the control and the other BPA solutions used , and could be attributed to the fact that high BPA doses are able to overwhelm the self-defense system, resulting in functional and structural cell damages, which seems to be also the case in C. nodosa leaves, since BPA damaged the structural integrity of chloroplasts, dictyosomes and endoplasmic reticulum [2O2 to H2O [−1 on the beginning of the experiment and at 0.3 μg L−1 on the 6th day (2O2 production (2O2) could be possibly activated. For instance, in a previous study CAT and APX activities showed dissimilar trends, due to the unavailability of their common substrate [Hongevity . Therefooxisomes , and canoxisomes . In C. n2 , was inons used a. The obs    , indicat values) d. The dees) , which was then overstimulated at the end of the experiment (8th day), may indicate an activation of an antioxidant protective mechanism  and the non-enzymatic mechanism (see high phenolic content at 1 μg L−1 on 4th day) may mitigate the overproduction of H2O2 and lead to the recovery of SOD activity, and of protein and phenolic contents, on the 6th day, at the control levels  are highly dynamic and variable among seagrasses, as they are modulated by external environmental or internal cellular factors . HoweverCymodocea nodosa (Ucria) Ascherson 1870 collected, during June from the eastern coast of the Gulf of Thessaloniki, Northern Aegean Sea , were kept for 24 h in seawater under laboratory conditions  to acclimatize. Seawater used in the experiment was also collected from the sampling area and had a BPA concentration below detection limit (DL = 10 ng L−1) [The seagrass  ng L−1) .−1 bisphenol-A  solutions in filtered seawater (0.45 μm Whatman GF/C) and filter seawater (used as a control). It should be noted that attention was paid to the selection of leaf shoots without epiphytes, while only the first intermediate leaves were used for our experiments, as the adult leaves are usually covered with epiphytes. BPA solutions were changed every two days, to adjust to the original levels out of an abundance of caution, although BPA in seawater remains stable for 35 days [2O2 production measurements. Additionally, each day from each treatment, intermediate leaves from another nine shoots (three per aquarium) were selected for the estimation of antioxidant enzyme activities and protein content, and another nine shoots for the estimation of lipid peroxidation, and other nine shoots for the estimation of the total phenolic compound contents.Seagrass consisting of horizontal rhizome, vertical rhizomes, roots and leaf shoots were incubated in non-BPA-based polypropylene (PP) copolymer (PPCO) constantly aerated aquaria, containing 20 L of 0.3, 1 and 3 μg L 35 days . The exp 35 days  with nea2DCF-DA, Sigma) in dimethyl sulfoxide (DMSO) [BPA treated and untreated intermediate leaf blades at each exposure time were incubated with 2′, 7′-dichlorofluoresce in diacetate (He (DMSO) . Τhe obse (DMSO) .w/v) polyvinyl pyrrolidone (PVPP). The homogenates were centrifuged at 16.500× g for 30 min at 4 °C. Total protein content (mg g−1 wet weight) was determined in the supernatants, based on the principle of protein-dye binding (Coomassie Brilliant Blue G-250), using bovine serum albumin (BSA) as standard [At each BPA concentration at the 2nd, 4th and 6th day, three subsamples were implemented. Each subsample  were grounded in liquid nitrogen and afterwards homogenized in 3 mL of 50 mM sodium phosphate buffer (pH 7.8), containing 0.1 mM EDTA and 2% (standard .−1 protein) was determined under UV light at 560 nm, based on the inhibition rate of nitro blue tetrazolium (NBT) to photochemical decline [For the quantification of SOD activity, a reaction mixture (5 mL) of 50 mM potassium phosphate, 0.1 mM EDTA, TritonX-100 0.025% (pH 7.8), 13 mM methionine and 0.075 mM nitro blue tetrazolium (NBT) were used; in aliquots (50 μL) of the extracts 0.002 mM riboflavin was added. SOD activity  was measured using the extinction coefficient 2.8 mM−1 cm−1.APX activity was estimated according to a modified method . Three sg for 10 min, at 4 °C. The supernatants (1.25 mL) were combined with 3.75 mL of 0.5% thiobarbituric acid (TBA), diluted in 20% TCA, heated at 95 °C for 30 min, and centrifuged for 3 min. MDA concentration (nmol g−1 wet weight) was measured spectrophotometrically  at 532 and 600 nm, using the extinction coefficient of 155 m−1 M−1 cm−1.MDA content was determined according to the method described in , in trip−1 dry weight) was determined in the extracts, with a modified Folin-Ciocalteu method [v/v), 250 μL distilled water and 2 mL sodium carbonate 0.7 M. The mixture was incubated in a water-bath for 30 min at 37 °C and the absorbance was measured at 760 nm with a spectrophotometer . Gallic acid was used as a standard. The Folin-Ciocaltefreu formulation, is known to be less affected by interfering compounds, and to avoid precipitation, give greater color, less variation and better recovery [Phenolic compounds were analyzed in 3 freeze dried subsamples  s . Differential interference contrast (DIC) microscopy was applied, and imaging acquisition was performed with the AxioVision SE64 4.8.3 or the ZEN. Blue 2.3. software following the manufacturer’s instructions as already reported .®25.0.Analyses on raw and ln-transformed data indicated unequal variances and severe violation of the normality assumption. Mann-Whitney U-test analysis was carried out to determine differences in the response parameters among BPA concentrations and between BPA concentrations and the control at each incubation time; these approaches consider average values, 54 values for CTCF, and for enzyme activity, MDA, and protein contents, the three subsamples at each BPA concentration and time. The above statistical analysis was carried out using IBM SPSS2O2 formation was detected in intermediate C. nodosa leaves even at the lowest BPA treatment, from the beginning of the experiment, causing the antioxidant defense mechanisms to be activated, even at environmentally relevant concentrations. ROS overproduction resulted in protein content decline at the two lower BPA exposures, on the 4th day and lipid peroxidation, at 3 μg L−1 of BPA, at the beginning of the test. The enzymatic and non-enzymatic antioxidant defense mechanism  effectively scavenges excess ROS, on the 4th day, re-establishing protein content to control levels on the 6th day. Therefore, this early warning response of the protein content determines the LOEC of BPA to be 0.3 μg L−1, and additionally, it meets the requirements for protein content to be considered as a possible early warning ‘biomarker’ for C. nodosa against BPA toxicity. However, it should be noted that biochemical parameters in ecotoxicological studies should be studied in combination as they are interrelated.In summary, our results demonstrated that H


Background:Klebsiella pneumonia has emerged as a critical pathogen causing severe clinical problems, such as pneumonia and sepsis. Meanwhile, intensified drug resistance induced by antibiotic therapy necessitates discovering novel and active molecules from Traditional Chinese Medicine (TCM) for treatment.Methods and results: In this study, the isolated Bergenin monohydrate showed an anti-inflammatory effect in Klebsiella-infected mice. We initially investigated the anti-inflammatory effects and cytoprotection against oxidative stress in vitro and in vivo. Interestingly, a specific dose of Bm can effectively ameliorate lung injury and suppress the expression of inflammatory cytokines such as TNF-α, IL-6, IL-1β and PEG2. Moreover, Bm was also shown to reduced the levels of MPO, MDA and increased SOD and GSH activities. Moreover, we assessed the intracellular signaling molecules including p38, ERK, JNK, IκB, NF-κB-p65 by western blotting and verified through MAPK and NF-κB pathways inhibition experiments. These results reveal that Bm executed its effects via the classical MAPK signaling pathway and NF-κB pathway.Conclusion: Given its underlying anti-inflammatory effect, Bm may be used as a promising therapeutic against Klebsiella-induced infection, thus providing a benefit for the future clinical therapy of pneumonia and medicine design. Klebsiella pneumoniae (Kp), a nonmotile, facultatively anaerobic, gram-negative, rod-shaped bacterium, can cause acute lung injury in humans and is considered an urgent health concern  has thousands of years of history in Asian countries. TCM studies have contributed to pharmacodynamic and pharmacokinetic development by generations of practitioners and researchers . To dateneumonia . Notablyneumonia . This neneumonia .Over the last few years, our laboratory has set out to screen effective TCM molecules to treat Kp infection. Intriguingly, we found that Bergenin monohydrate (Bm) has multiple biological activities, suggesting its anti-inflammatory and immunomodulatory properties. Here, we confirmed that Bm exhibits a protective effect against Kp-induced cell damage and was effective in Kp-induced mice. In addition, we uncovered the involvement of the NF-κB and MAPK signaling pathways in the activity of Bm. This study might provide a potential reference for exploring therapeutic candidates derived from Chinese medicinal herbs to treat Kp infection.The Kp strain was purchased from the China Center of Industrial Culture Collection (CICC-10870). Bergenin monohydrate was obtained from Chengdu Herbpurify Co. Ltd., Sichuan, China, with a certified purity of ≥98. RAW 264.7 cells and WI-38 cells were obtained from our laboratory, and MEM and DMEM were provided by Procell Life Science and Technology Co. Ltd., Wuhan, China. The assay kits and instructions were purchased from Jiancheng Bioengineering Institute, Nanjing, China. The NF-κB pathway inhibitor (BAY 11-7082) and MAPK inhibitor (SB203580) were purchased from Sigma (Sigma-Aldrich). IκB, p-IκB, p68, p-p65, ERK1/2, p-ERK1/2, p65, p-p65, JNK, and p-JNK antibodies (rabbit antibodies) for western blot analysis were purchased from Beijing Boaosen Biotechnology Co., Ltd. Secondary antibodies (IgG-HRP) were obtained from Zhongshan Jinqiao Biological Technology Co., Ltd. Unless otherwise specified, the other materials were from Sigma-Aldrich (United States).A total of 90 six-week-old BALB/c mice weighing 18–20 g were used in this study. All mice were fed ad libitum, kept in a pathogen-free environment, and maintained in groups of 5 per cage in a specific pathogen-free (SPF) animal room. All animal experiments, including any relevant details, were performed under the applicable guidelines and regulations of Experimental Animal Care and were approved by the Animal Welfare and Research Ethics Committee  20170005).2, pH 7.0–7.4 and 37°C. Bacterial supernatant was collected for MTT, ROS. The rest of the Kp bacterial sample was utilized for cells experiments and animal experiment.The methods for bacterial growth and preparation were described in a previous study by our lab . Kp was 7 CFU/mL, and100 μL drops of the dilution were added to each well. Following incubation at 5% CO2, pH 7.0–7.4, and 37°C for 24 h, we observed the turbidity of the broth. Transparency of the samples indicated that bacterial growth was inhibited at the corresponding concentration of the drug, and the lowest drug concentration that could inhibit bacterial growth was obtained. This concentration was considered the minimum inhibitory concentration (MIC).Double-gradient dilution was applied to obtain a series of Bm solution concentrations, which were then sequentially added to the wells of a 96-well culture plate . The Kp 2 at 37°C, RAW 264.7 cells were cultured and adjusted to 2 × 104 cells/well, seeded into 96-well plates and incubated for 24 h. Bm was dissolved in DMEM. No significant treatment effect of Bm was found for Kp-infected RAW 264.7 cells in our experiments. Thus, RAW 264.7 cells were first administered Bm at different concentrations  and a positive control drug  for 1 h and then exposed to KP for 24 h.After incubation in Dulbecco’s modified Eagle’s medium (DMEM) in 5% CO8 cells/well and incubated in 6-well plates for 24 h. After that, the cells were treated with Bm at different concentrations  in DMEM for 1 h. Then, the cells were infected with Kp for 24 h and washed twice with PBS. Subsequently, 50 μL DCFH-DA was added to the cells for 30 min without light. DCF fluorescence intensities were detected spectrophotometrically at excitation and emission wavelengths of 485 and 535 nm, respectively.Cultured RAW 264.7 cells were adjusted to 2 × 107 CFU, 0.2 ml), Kp + Bm  groups, Kp + DEX  group, Bm was diluted in saline. In the mouse model of Kp lung infection, Kp was administered intranasally, and the mice were treated with Bm via intraperitoneal injection 1 h before Kp infection. After the experiments, all the mice were sacrificed via carbon dioxide asphyxiation. Afterward, lung tissues were rinsed and fixed for further analysis, bronchoalveolar lavage fluid (BALF) was prepared for the following experiments. All vitro experiments were repeated at least three times, and all animal experiments were repeated twice for under identical conditions.Ninety BALB/c mice were kept under SPF conditions and randomly divided into six groups:control group , Kp group  staining. Images were acquired under a light microscope . Comparison of the staining results was performed by research team members who were blinded to the grouping information.Harvesting of protein lysates from RAW 264.7 cells and mouse lung tissues was performed according to the present study. Then, the same quantity of proteins (30 µg/lane) were subjected to electrophoresis in a 12% polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% FBS at room temperature for 1 h. The blots were then separately incubated with primary murine anti-human β-actin , IκB , phospho-IκB , p68 , phospho-p68 , p65 , phospho-p65 , ERK1/2 , and phospho-ERK1/2  antibodies overnight at 4°C. β-Actin was used as an endogenous control and for normalization. The membranes were washed with TBST for 10 min and then incubated with anti-mouse IgG secondary antibody (1:100) for 30 min at room temperature. Finally, the membranes were visualized using an enhanced chemiluminescence (ECL) agent.2. When the density of the WI 38 cells increased to 80%, we seeded them into 96-well plates at 1 × 105 cells/well, incubated them for 24 h, and added SB203580 and BAY 11–7,082 at two different doses (10 and 20 μmol/L). After 1 h, 10 μg/ml Bm was added and incubated with the cells for another 1 h, and then, the plate was exposed to Kp for 24 h. The cytotoxic effects of these two pathway inhibitors were determined.To verify that Bm attenuates the inflammatory response via the MAPK and NF-κB pathways in WI 38 cells, we cultured WI 38 cells in MEM containing 10% FBS, 1% P/S and 1% GlutaMax in an atmosphere of 95% air and 5% COp < 0.05 and “**” for p < 0.01.All the data were collected and analyzed using SPSS 22.0 software (IBM). The experimental groups were compared via one-way analysis of variance (ANOVA), and an LSD test was applied for multiple comparisons. In the western blotting analysis, densitometry analyses were quantified with ImageJ software. Statistical significance was defined as “*” for The MIC of Bm was greater than 256 μg/ml, which indicated that Bm had no considerable effect on Kp growth . In addiFurthermore, Bm protected RAW 264.7 cells from Kp infection , and theHistological changes in lung tissues were assessed by H&E staining . There wELISA measurement of TNF-α, IL-6, IL-1β and PEG2 contents after Kp treatment for 48 h.As indicated in As illustrated in Bm treatment restored the MPO, MDA, SOD and GSH levels in Kp-infected mice.As shown in We first examined the effect of a MAPK pathway inhibitor (SB203580) and NF-κB pathway inhibitor (BAY 11-7082) on WI38 cell viability. SB203580 and BAY 11-7082 at concentrations up to 20 µM barely altered cell viability, but at concentrations above 20 μM, they were cytotoxic . Within in vivo studies confirmed a similar trend in p38, ERK, and JNK phosphorylation as observed in the in vitro experiments (As shown in the western blotting results, compared with the control and Bm groups after infection with Kp, the Kp group displayed higher levels of p38, ERK, and JNK phosphorylation . There weriments . Hence, Klebsiella pneumonia is a crucial type of bacteria in the genus Klebsiella and accounts for more than 95% of Klebsiella infections (fections . Kp infefections . Increasfections . TCM monfections .in vitro and in vivo experiments showed that Bm can suppress the expression of major inflammatory cytokines in Kp-infected cells and ameliorate lung injury in Kp-infected mice. ROS measurements in RAW 264.7 cells revealed a dose-dependent protective effect of Bm. Histological changes and pulmonary bacterial count results indicated that Bm reduced Kp bacteria levels while improving lung tissue histopathology. Bm is the main medicinal component of Ardisia japonica, and a large number of studies have shown that Ardisia japonica can be employed clinically to treat lung injury diseases, such as tuberculosis (Klebsiella pneumonia.In the present study, both rculosis  and promrculosis . GSH andrculosis . In our TNF-α, IL-6, IL-1β and PEG2 proinflammatory cytokines, including TNF, IL-1, IL-6 and IL-8, are small molecular peptides that can regulate a variety of physiological functions and increase the expression of proinflammatory cytokines . These iTo explore further pathway mechanisms, we investigated whether Bm regulates the MAPK and NF-κB signaling pathways to execute protective and anti-inflammatory actions. Western blotting and ELISA results revealed that the levels of the proinflammatory cytokines TNF-α, IL-6, IL-1β and PEG2; the MAPK subfamilies p38, ERK, and JNK; and the NF-κB subfamilies IκB and NF-κB-p65 exhibited a downward trend in a dose-dependent manner with Bm treatment after Kp infection. Both the MAPK and NF-κB signaling pathways are regarded as classical and significant anti-inflammatory pathways . The patin vitro are unable to mimic that in vivo environment with hypoxia, hypoglycemia, and other metabolic changes (in vitro, they can actually inhibit the growth of pathogens by changing the microenvironment in vivo (The study had a few limitations. First of all, Bm has not been found an obvious inhibition effect for Kp growth and protective effect for Kp-infected Raw cells, but in animal experiments, it demonstrated a good anti-inflammatory effect. This phenomenon attracted our attention. By reviewing the available literature, we have found that conditions  changes , althoug in vivo . HoweverIn conclusion, our results demonstrated a dose-dependent protective role of Bm in Kp-infected cells and mice through modulation of inflammatory cytokine expression. Bm exerted anti-inflammatory action by regulating Iκ-Bα and p65 phosphorylation and regulated the MAPK signaling pathway by inhibiting p38, ERK, and JNK phosphorylation after Kp infection. Thus, our findings provide evidence that Bm may be a promising therapeutic agent for Kp prophylaxis.