MF worsened reaction time on the visuomotor task, while other secondary measurements remained largely ambiguous. Spectral power  was influenced by MF, while ERPs measured during the visuomotor task remained unaltered. The present study confirms that MF negatively impacts table tennis performance, specifically inhibitory stimuli during the visuomotor task. These findings also further augment our understanding of the effects of MF on human performance.Mental fatigue (MF) is a psychobiological state negatively impacting both cognitive and physical performance. Although recent research implies that some table tennis (TT) performance outcomes are impaired by MF, open skill sports such as TT require a more detailed overview of MF-related performance decrements. Moreover, research into MF and sport-specific psychomotor performance lacks the inclusion of brain-related measurements to identify MF mechanisms. Eleven experienced TT players participated in this randomized counterbalanced crossover trial. Participants were either required to perform an individualized Stroop task (MF condition) or watch a documentary (control condition). The primary outcomes were reaction time on a sport-specific visuomotor task and EEG activity throughout the trial. The subjective feeling of MF was significantly different between both conditions and confirmed that the MF condition induced the mentally fatigue state of participants ( In elite sport performance, athletes use both advanced cognitive and motor skills to win games, advance in world rankings and earn trophies. Moreover, in elite athletes, it is often differences in cognitive skills such as decision making , visuomoMF can be defined as a psychobiological state that emerges during prolonged demanding cognitive activity and results in an acute feeling of tiredness and/or decreased cognitive skills ,18. ReceIt is well established that MF impairs cognitive skills ,23 and pTherefore, the primary aim of the present study was to investigate whether MF impairs sport-specific visuomotor performance in TT players. We hypothesised that MF would worsen reaction time, based on knowledge gathered by similar studies ,20. A seA total of 11 experienced table tennis players  were included in this experiment. The table tennis level of participants ranged between A3 (elite) and C4 (trained). Participants were required to sleep a similar amount of time before each trial (at least 7 h), refrain from the consumption of caffeine and/or alcohol, and to avoid any vigorous physical activity the day before and the day of each visit. In addition, participants were asked to have a similar meal on the morning of each trial. The use of any kind of medicinal products during and between the trials was prohibited. If participants could not meet these standards, they were excluded from the study. Each subject gave written informed consent prior to the study. The present study protocol and its procedures were approved by the Research Council of the Vrije Universiteit Brussel.The study employs a randomized counterbalanced cross-over trial. Participants reported for the study on three separate occasions  on the same time of day (in the morning) and with a minimum of three days between sessions. Measurements were conducted in thermoneutral conditions  and every trial took about two hours to complete. It was not specified to participants which was the control, and which was the experimental procedure. Participants were told that the goal of the study was to examine the effect of two different cognitive tasks on their TT specific reaction time and where, as such, blinded for our predefined hypotheses.Participants were first invited to a familiarization trial where they had the chance to become acquainted with the different questionnaires , Matthews motivation scale, rating of perceived exertion (RPE), and National Aeronautics and Space Administration Task Load Index (Nasa-TLX)), the visuomotor task, and the experimental task . The participants familiarized themselves with the experimental condition by performing a Stroop max test, which was designed to individualize the difficulty level of the Stroop test during the experimental trial. The circumference of the head was also measured for fitting the EEG cap during the experimental and control condition.In the Stroop max task, four coloured words  were presented one at a time on a computer screen. The participants were required to indicate the colour of the word, ignoring the meaning of the word itself. If the ink colour was red, the button to be pressed was the button linked to the real meaning of the word. The presented word and its ink colour were randomly selected by the computer (100% incongruent), with all incongruent word–colour combinations being equally common. The Stroop max test of the familiarization trial consisted of 108 stimuli in total, divided into three cycles of 36 stimuli with an average interstimulus time of 1500 ms. The level of difficulty on this test changed depending on the stimulus presentation time , ranging from 1200 ms to 500 ms with a difference between levels of 100 ms. Before the actual max test, subjects performed a warm-up to get acquainted with the cognitive task. Afterwards, participants commenced the Stroop max test at the 1200-ms level. If the participant was more or equal to 85% accurate , they were allowed to proceed to the next level. If participants were less than 85% accurate, they had to repeat this level. Participants were allowed three attempts at a level and five attempts in total (in the case of multiple retries across levels). If a participant exceeded the amount of retries, the max test was concluded and the last level where the participant was able to meet the 85%-threshold was selected as their maximal cognitive performance level and was applied within the experimental trial.The experimental and control trial proceeded in a similar manner, with the only difference being the intervention task in the middle of the trial. At the beginning of each trial the EEG device and heart rate monitor were attached, and a baseline EEG measurement was performed while the participant remained seated. Following the baseline EEG measurement, participants performed the visuomotor task and the flanker task. Participants were then instructed to perform either the intervention or the control procedure. Immediately afterwards the flanker task and visuomotor task were again performed. Throughout the measurements, participants were required to fill in a number of questionnaires see .Flanker task: Participants were instructed to respond as quickly and accurately as possible to the direction of a target arrow while ignoring the direction of the other arrows on each side . Each array of arrows was focally presented in white text (font size 34) for 200 ms on a black background with a variable interstimulus interval of 1000, 1200, 1400, or 1600 ms. A total of 120 stimuli were given (15 cycles of 8 stimuli) with both right and left arrows appearing with equal probability. The total duration of the task was approximately 5 min. To assess performance on the flanker task accuracy and reaction time were collected. The aim of the flanker task was to make sure that participants were adequately familiarized and not already fatigued before the start of the intervention/control task .Intervention procedure: The Stroop task was chosen to induce MF in the participants of the present trial. The instructions of the task were the same as the ones given when performing the Stroop max task. The differences between this task and the Stroop max task were the duration and the task specifications. The Stroop task of the intervention procedure had a duration of 60 min, which translates to 1500 stimuli that were divided into four blocks, with 360 stimuli every block and 90 stimuli for every colour. The time that the stimuli were presented was based on the achieved level on the Stroop max task. In between the four blocks, 15 stimuli were not taken into account for the performance analysis. This allowed the researchers to pose the question: “how mentally fatigued do you feel on a scale from 0 to a 100, where 0 means not fatigued at all and 100 means extremely mentally fatigued?”. The participants had no knowledge of the existence of the four blocks and received no feedback relating to performance or time during the task.Control procedure: The control procedure consisted of watching a documentary of the same duration as the intervention task. Participants had the choice between all parts of Planet Earth, the complete collection . These documentaries were chosen based on their emotionally neutral yet engaging content. Every 15 min the level of MF was assessed verbally.https://www.fitlighttraining.com/, accessed on 5 October 2020), consisting of eight LED lights. Six of these lights were installed on a table tennis table in a specific manner, while one light was placed behind the participant on the preferred hand site . The inter-stimulus time of the task varied between 3–6 s, with each time randomly chosen for a total of 16 times. Total task duration was approximately 6 min. Accuracy  and response time  for each stimulus type (complex vs. simple stimuli) were calculated based on the data  collected by the respective software. Because of the motor simplicity of the present task as opposed to Van Cutsem et al. [The visuomotor task was designed in accordance to Van Cutsem et al. 2019) [19 [20]. site see . The lasFor EEG recording and data preparation, brain activity was continuously measured throughout the entire duration of the experimental protocol; 32 active gel-based Ag/AgCl electrodes  were attached to the heads of the participants, conforming to the international 10–20 system . SamplinFor ERP analysis, the N1, P2, N2, and P3b ERPs were assessed across all task data segments . All information concerning definitions, suspected latencies and electrode locations based on regions of interests (ROI) of the measured ERPs can be found in Spectral power analysis: Following the inverse ICA for the continuous EEG data set of interest, segments with a length of 4 s and with an overlap of 2 s were extracted . The resMultiple subjective outcome measures were administered to the participants. A pre-test checklist was evaluated at the beginning of each trial to check for any abnormalities which warranted exclusion . The M-VAS was used as a subjective manipulation check. This VAS scale consisted of a 10-cm line which was labelled at one end with ‘not at all mentally fatigued’ and at the other with ‘extremely mentally fatigued’ . Particip2) and Cohen’s d (d) were used as effect sizes. Ranges used for ηp2 were small = 0.01; medium = 0.06; large = 0.14. Ranges used for d were <0.2 = trivial; 0.2–0.6 = small; 0.6–1.2 = moderate; 1.2–2.0 = large; >2.0 = very large. The significance level was set at 0.05.All statistical tests were conducted using the Statistical Package for the Social Sciences, version 24 . All data are presented as means ± standard deviation (SD). Normality was checked using the Shapiro–Wilk test and visually confirmed with histograms. If data were not normally distributed, non-parametrical equivalents were used instead. Mauchly’s test was used to verify for sphericity. However, if sphericity could not be assumed, the Greenhouse–Geisser procedure corrected the significance of F-values. Two-way repeated measures (RM) (2 × 2) effect ANOVAs were conducted to assess the effect of condition (MF vs. CON) and time (PRE vs. POST) on RPE, Matthews intrinsic and success motivation scores, and accuracy and reaction time on the flanker task. A two-way RM (2 × 3) ANOVA was used to assess the effect of condition and time (first to third measurement) on the different subscales of the NASA-TLX. Another two-way RM (2 × 4) ANOVA was used to assess the effect of time (first to fourth block) and stimuli (meaning vs. color) on reaction time and accuracy of the Stroop task. A two-way RM (2 × 9) ANOVA was employed to investigated the effect of condition and time (first to ninth measurement) on the M-VAS and HR measurements. A three-way RM (2 × 2 × 2) effect ANOVA was used to analyse the effect of condition, time and stimuli (simple vs. complex) on the RT of the visuomotor task. A three-way RM ANOVA was adopted to assess the effect of condition, time and ROI on the amplitude and latency of the investigated ERP values during the flanker and Fitlight tasks. By including ROI as a factor, the amount of statistical testing required is significantly reduced. The effect of ROI was not added to the result section in order to reduce irrelevant information, as we were only interested in ROI effects if significant interaction effects were present. The effects of condition, time and ROI on transformed spectral power of theta (θ), lower alfa (α) and upper α bands during the flanker and Fitlight task were investigated using a three-way (2 × 2 × 7) RM ANOVA. If interaction effects between the investigated factors were seen in the three or two-way mixed ANOVAs, subsequent two-way mixed ANOVAs or paired sample t-tests, respectively, were employed to elucidate the main effects of condition and time. If no interaction effect was present, the main effects of condition and time were immediately interpreted using the aforementioned statistical tests and Bonferroni corrections. Partial eta square  = 5.607, p = 0.050, ηp2 = 0.445). Subsequent post hoc tests showed a significantly slower visuomotor reaction time from pre- (713 ± 23 ms) to post (764 ± 19 ms) in the MF condition  = 14.899, p = 0.006, ηp2 = 0.680) for all stimuli; specifically, in the inhibitory stimuli (t(7) = 5.199, p = 0.001, d = 1.84), a significantly slower visuomotor reaction time post-task in MF (816 ± 111 ms) compared to CON (745 ± 112 ms). A 2 × 2 × 2 RM ANOVA was conducted to assess the effect of condition, time and stimuli on Fitlight task performance. This analysis showed a significant interaction effect between time and condition  = 5.825, p = 0.024, ηp2 = 0.393). Post-hoc analysis showed a significant interaction between time and condition in the Cz, C3 and C4 electrodes  = 15.789, p = 0.003, ηp2 = 0.637) and the fusiform gyrus  = 6.556, p = 0.031, ηp2 = 0.421), while also showing a general effect of time  = 6.632, p = 0.030, ηp2 = 0.424) in the inferior/orbitofrontal cortex, with the pre-task energy (1.03 ± 0.49 μV2) being higher than the post-task energy (0.68 ± 0.12 μV2). Paired sample t-tests showed a decrease in energy between pre-task (0.65 ± 0.54 μV2) and post-task (0.28 ± 0.18 μV2) in the MF condition (t(9) = 2.712, p = 0.024, d = 0.94) in the P7, P8, PO9 and PO10 electrodes only.Upper α band: There was a significant interaction effect between time and ROI  = 1.828, p = 0.026, ηp2 = 0.411). Additional analysis of the different ROIs showed an effect of time  = 6.348, p = 0.033, ηp2 = 0.414), with a higher power pre-test (1.14 ± 0.26 μV2) compared to post-test (0.74 ± 0.15 μV2) in the F7 electrode, independent of condition.Lower α band: an interaction effect between time and ROI was observed  = 10.735, p = 0.007, ηp2 = 0.544). Distinct post-hoc tests examining all ROIs showed a significant effect of time  = 11.430, p = 0.008, ηp2 = 0.559; PRE = 1.91 ± 0.35 μV2; POST = 1.19 ± 0.27 μV2) in the F7 electrode, and an effect of condition  = 6.592, p = 0.030, ηp2 = 0.423; MF = 0.29 ± 0.04 μV2; CON = 0.38 ± 0.06 μV2) in the Fz, F3 and F4 electrodes.θ band: a significant interaction effect between time and ROI was found (p = 0.007), with MF causing a higher frustration compared to CON. The effects of time are also presented in the table, with an additional effect of time on frustration in the MF condition.F = 12.692, p = 0.005, ηp2 = 0.559) between condition and time in the intrinsic motivation scale was observed. Post hoc paired sample t-tests showed a significant difference between pre (22.09 ± 2.17) to post (19.27 ± 2.94) in the MF condition (t(10) = 3.492, p = 0.006, d = 1.05) and between MF and CON (19.55 ± 3.08) in the pre-task time interval (t(10) = 3.088, p = 0.011, d = 0.93). There were no significant effects for either time or condition on the success motivation scale.A significant interaction effect  = 8.711, p = 0.014, ηp2 = 0.466) and condition  = 11.011, p = 0.008, ηp2 = 0.524) in RPE measures, without a significant interaction effect. Post hoc Bonferonni tests showed a significant increase from pre (8.36 ± 0.50) to post  experimental/control task, and a significantly higher value for RPE in the control condition (9.59 ± 0.56) compared to the MF condition (8.32 ± 0.51).A 2 × 2 RM ANOVA showed a significant effect of time  = 5.106, p < 0.001, ηp2 = 0.422), with post hoc analysis showing that these significant effects can be contributed to the significant drop in HR in measures 4, 5, 6, 7 (during and right after the Stroop task) and 8 (right after the post-flanker task) compared to the first time point (at the beginning of the experiment).Analysis of HR values showed no significant effect of condition. However, there was a significant effect of time  = 263.402, p < 0.001, ηp2 = 0.701) was present. This interaction indicated that, in terms of the effect of time, the subjective experience of MF increased in both the MF  = 42.970, p < 0.001, ηp2 = 0.811) and the CON  = 13.497, p < 0.001, ηp2 = 0.574) condition. MF significantly increased during the Stroop task (from M-VAS 4 onward (first M-VAS administered during the Stroop task)), and also significantly decreased afterwards (comparison between M-VAS 7 (M-VAS administered immediately after the Stroop task) and 8 (M-VAS administered immediately after the post-flanker task)) (A significant interaction effect between condition and time (r task)) . In the r task)) /Table 4.F = 0.422, p = 0.587, ηp2 = 0.040) or stimuli  = 2.562, p = 0.141, ηp2 = 0.204). For accuracy variables, two Friedmann tests showed no effect of time regardless of type of stimuli. Four Wilcoxon signed rank tests (one for every block) showed significant differences in all four blocks between the type of stimuli, with a significantly higher accuracy when performing the simple stimuli compared to the complex stimuli.Analysis of reaction time on the different blocks of the Stroop task showed no effect of time  = 5.299, p = 0.047, ηp2 = 0.371), with the accuracy post-task (96 ± 1%) being higher than pre-task (95 ± 1%).There was no effect of condition or time on flanker reaction time see . There wSPA: There was no effect of time or condition on upper and lower α and θ frequency band during the flanker task.F = 6.724, p = 0.036, ηp2 = 0.490), with the amplitude of the MF condition (−1.05 ± 0.21 µV) being lower than the amplitude of the control condition (−1.29 ± 0.26 µV). No effects were found on the latency of the N1 variable. The opposite was true for the P2 ERP, showing an effect of time on latency  = 8.018, p = 0.025, ηp2 = 0.534), but no effects on amplitude. This effect of time consisted of a shorter latency pre-task (157 ± 4 ms) compared to post-task (161 ± 4 ms). Due to a significant three-way interaction effect between time, condition and ROI on amplitude of P3b  = 9.306, p = 0.015, ηp2 = 0.571), subsequent post hoc tests were carried out. These tests revealed a significant interaction effect between time and ROI within the MF condition  = 13.061, p = 0.001, ηp2 = 0.651) and a significant interaction effect between condition and ROI pre-task  = 6.318, p = 0.034, ηp2 = 0.474). Paired sample t-tests pointed to decreases in amplitude between pre-task (3.75 ± 1.99 µV) and post-task (1.40 ± 2.13 µV) in the MF condition (t(7) = 3.607, p = 0.009, d = 1.14) and between conditions (CON = 1.73 ± 1.52 µV) pre-task (t(7) = 2.743, p = 0.029, d = 1.14), with both effects only present in the fusiform gyrus. There were no effects of time or condition on P3b latency. There were no effects of time or condition on amplitude and latency of N2.ERP: There was a significant effect of condition on the amplitude of the N1 ERP  when examining the inhibitory stimuli, with subjects who experienced MF having a higher reaction time compared to the control condition. These results are in line with the previous study of Van Cutsem et al. , on whicResearch has suggested that elite athletes might have superior inhibitory control, which could provide them with a higher resistance to MF and its detrimental effects on performance . MoreoveThe present study investigated two major EEG outcomes, SPA  and ERPA drawback of the SPA technique is that the analysis provides only an average change in frequency over a given time period, which fails to utilise the most important benefit of EEG measurements, namely an excellent temporal resolution . StudiesSeveral authors also connect the effects of MF on performance to a decrease in motivation. MF causes decreased motivation, which in turn leads to less attention, and responses that are not automatic are therefore impaired ,61. StudA last measure frequently associated with MF is RPE, since an increase in RPE due to MF was seen in the original review by Van Cutsem et al. . This inAn interesting observation of this study was the increase in subjective feelings of MF in the control task over time. Mangin et al.  recentlyDefinitive evidence of the mentally fatigued state of participants can be found in the physiological manipulation checks, showing a decrease in N1 and P3b amplitude  due to condition effects. These results are consistent with the literature . As JacqAs a last confirmation of the mentally fatigued state, the performance of the different subjects was still negatively impacted by MF, as seen by the effects on the visuomotor task. Therefore, it can be concluded that MF was successfully induced. This study further emphasizes the necessary implementation of the three different types of manipulation checks physiological) in future MF studies .Some limitations concerning the present study should be mentioned. Some data were lost in the visuomotor task. This loss in data could have impacted the results, and should be taken into consideration when interpreting the present data. However, the confirmation that MF affects visuomotor performance, also determined by other studies , seems tAs mentioned before , more stThe present study provides further proof that the current visuomotor task is a valid way to assess visuomotor performance decrements due to MF. As Van Cutsem et al.  mentioneThe present study provides new proof that MF affects sport-specific psychomotor performance in healthy athletes, as shown in the review of Habay et al. . SpecifiThe present study provides clear evidence that the visuomotor performance of trained TT players is negatively impacted by MF, further adding to knowledge regarding the effects of MF on visuomotor performance ,20. Spec


The specimens identified belonged to the genera Chara, Nitella, and Tolypella, with predominance of the first genus to which five species were assigned though they presented some interesting aberrant taxonomic features: C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris. Based on our integrative study, the globally rare species C. globata was reported for the second time for the whole African continent. The genus Nitella was only represented by N. flagellifera, and based on the available literature, it is a new record for North Africa. Noteworthy, an interesting Tolypella sp., morphologically very similar to T. glomerata, was collected and characterized and finally designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, mainly based on its concatenated rbcL+ITS1 phylogenetic position. This study not only improved our understanding on the diversity, biogeography and autecological preferences of charophytes in Egypt, but it also broadened our knowledge on this vulnerable algal group in North Africa, emphasizing the need of more in-depth research work in the future, particularly in the less–impacted desert habitats.Present-day information available on the charophyte macroalgae in Egypt, including their phylogenetic affinities, remains largely incomplete. In this study, nine charophyte populations were collected from different aquatic biotopes across the Egyptian Western-Desert Oases and Sinai Peninsula. All populations were investigated using an integrative polyphasic approach including phylogenetic analyses inferred from the chloroplast-encoded gene ( Charophytes , including both extant and fossil members of the order Charales , constitute an ancient group of terrestrial autotrophic macroalgae, the ancestors of which invaded land and developed to the present-day land plants 450 million years ago ,2,3. EcoChara strigosa A.Braun and C. tomentosa L., have also been reported. Their study also included some Mediterranean endemic and endangered species, such as C. imperfecta A.Braun, C. oedophylla G.Feldmann  R.D.Wood), and C. vulgaris var. gymnophylla (A.Braun) A.Braun, besides the typical tropical taxa C. zeylanica Klein ex Willdenow and Lamprothamnium succinctum (A.Braun) R.D.Wood. In their study on the charophytes inhabiting wetlands of Numidia in north-eastern Algeria, Zouaïdia et al. [Chara braunii C.C.Gmelin, C. galioides A.P.De Candolle, Nitella gracilis (J.E.Smith) C.Agardh, and N. hyalina (De Candolle) C.Agardh as rare species, as well as Nitella batrachosperma C.Agardh  A.Braun ex Leonhardi), found in a very-clean water pool as a new record for Algeria. All the above-mentioned taxa posed conservation values for their habitats and are currently designated as rare species in the Maghreb countries [In North Africa, particularly in the Maghreb countries and starting from the late 19th century, the family Characeae attracted the attention of many researchers, and hence a wealth of information is available on this group of algae from the morphological and ecological standpoints for this geographic area ,31,32,33a et al.  reportedountries ,5,15, coChara and only one species each for the genera Nitella (N. opaca) and Tolypella (T. nidifica). They emphasized the necessity of more intensive surveying studies on the family Characeae in Egypt to have a complete picture on this group of algae and also advocated the need of taxonomic revision for certain species such as Chara diaphana (F.J.F.Meyen) R.D.Wood. Several years later, a few studies reported some cosmopolitan taxa sporadically, mainly from different desert biotopes [In Egypt, knowledge about the diversity and ecological preferences of the streptophytes, including the stoneworts, is still grossly limited ,35. The biotopes ,39,40,41biotopes ,43,44.The goal of this study was to identify morphotaxonomic diagnostic traits, phylogenetic affinities, and autecological preferences of charophyte populations collected from different Egyptian biotopes in the Western-Desert Oases and Sinai Peninsula to improve our current limited understanding on the biogeography and diversity of charophytes in Egypt and, generally, in North Africa.rbcL sequences of charophytes representing major genera  or strongly supported (Nitella and Lamprothamnium) generic clades. Chara was resolved only topologically as a sister of Lamprothamnium . Similarly, in the genus Nitella the new sequence was placed in the robust N. flagellifera clade. Only our Tolypella accession occupied unresolved position in a weakly supported clade. In the analyses with the concatenated data set that included 16 rbcL and ITS1 sequences of Tolypella, our sequence showed weak affinity to Tolypella sp. from Australia , Nitella (N. flagellifera), and Tolypella (Tolypella sp. PBA–1704) were identified and discussed from the standpoints of morphotaxonomy and ecological characterization. The worldwide rare species C. globata is herein reported for the second time in the whole African continent. Interestingly, N. flagellifera represents the first record for both Egypt and North Africa. An interesting Tolypella sp., morphologically similar to T. glomerata, is designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, mainly based on its concatenated rbcL+ITS1 phylogenetic position. Detailed descriptions, ecological preferences, and biogeography of all these taxa are given in the following. Hydrochemical characteristics of the habitats studied are provided in In the present study, seven taxa belonging to the genera Description: Plants green, dioecious, up to 40 cm tall, without incrustations A. Axes mDistribution in Egypt: This charophyte species has already been recorded in Egypt .Chara aspera population was found in a mineral spring-fed agricultural ditch in the Siwa Oasis. Hydrochemical conditions in this Saharan biotope were as follows: high water temperature (°C): 27.7; neutral pH: 7.32; high electrical conductivity (μS·cm−1): 6350; Na+ and Ca2+ were the major cations: 1140 and 276 mg·L−1, respectively; Cl− was the major anion: 2104 mg·L−1; low N, NO3− and NH4+: 460 and 68 µg·L−1, respectively; high P, TP and SRP: 260 and 55 µg·L−1, respectively (C. aspera in nitrate-rich brackish wetlands (NO3−: 400–840 μg·L−1), with moderate orthophosphate levels (30–48 μg·L−1). Additionally, Caisová and Gąbka [C. aspera has a wide range of ecological preferences in fresh and brackish calcareous waters, mainly in drainage canals and lakes.General distribution and ecology: Cosmopolitan species in Europe , Atlantiectively . In a sind Gąbka  and UrbaC. aspera population and the information provided by Wood and Imahori [C. aspera population in these groundwater-dependent biomes, but the plants were still genetically identical to other specimens of C. aspera from several European countries. Taxonomically, they showed that these plants were ecorticated and sterile, stipulodes were absent, branchlets with 5–10 segments, cortex of the branchlets rudimentary or missing, and eventually branchlets tipped with 2–3 ecorticated cells. Our observations and findings of Langangen et al. [C. aspera. Accordingly, Blindow et al. [Remarks: There is general agreement between the characteristics of our  Imahori ; however Imahori  on the cn et al.  confirm w et al.  pointed Description: Plants olive green to green, monoecious, 15–50 cm tall, without incrustations A or modeDistribution in Egypt: Previously recorded in Upper Egypt .Chara contraria is rather rare in the Mediterranean region and can be found in various biotopes. With respect to its conservation status, Langangen [−1): 760–2960; Na+ and Ca2+ were the major cations: 62–361 and 62–147 mg·L−1, respectively; Cl− was the major anion: 146–768 mg·L−1; low N, NO3−: 2320–5400 µg·L−1; NH4+: 57–924 µg·L−1; high P, TP and SRP: 150–1680 and 40–276 µg·L−1, respectively , the two species are also well separated genetically : 26.5; circumneutral pH: 6.85; high conductivity (μS·cm−1): 6280; low DO (mg·L−1): 3.4; Na+ and Ca2+ were the major cations: 1430 and 94 mg·L−1, respectively; Cl− and SO42− were the major anions: 1795 and 989 mg·L−1, respectively; low N, NO3−, NO2− and NH4+: 200, 120, and 270 µg·L−1, respectively; high P, TP and SRP: 135 and 100 µg·L−1, respectively  species preferring waters rich in sodium sulphates, and calcium/magnesium bicarbonates . So far,ectively .C. globata population fitted better the specimens recently described by Romanov et al. [C. globata should be transferred and assigned to the subsection Chara in the section Chara, instead of the section Grovesia having a triplostichous stem cortex, in terms of the taxonomic observations obtained , corresponding well to the section Chara [C. globata specimens investigated in the present study are mainly characterized by the presence of isostichous to tylacanthous diplo- to triplostichous stem cortex . In our opinion, the accurate taxonomic placement of C. globata is still problematic and more integrative studies are needed.Remarks: The diagnostic taxonomic features of the Siwa v et al.  than thev et al. ,53. Howev et al.  by the fv et al.  also proon Chara . On the m cortex F–I. Lingv et al.  was not v et al. ,60, who is study , as wellC. globata having been recently recorded for the first time in North Africa in Tunisia, its morphotaxonomic diagnostic traits were poorly revealed , and it could therefore be considered as one of the characteristic Chara populations not only for Egypt but also for North Africa and the Sahara–Arabian Desert in general. We also think that the only available records of C. hispida var. hispida f. polyacantha (A.Braun) R.D.Wood and C. hispida var. baltica (Bruzelius) R.D.Wood from the Siwa Oasis [C. globata. Although the morphotaxonomic traits of both taxa are not available in Corillion and Guerlesquin [C. globata. We predict that C. globata might be recorded in the future in the other Maghreb countries, particularly by applying combined morphological and phylogenetic approaches. Accordingly, it has been established that subtle species identification of members of the charophytes at the species and intraspecific level has nowadays become much easier thanks to the integrative polyphasic approaches, irrespective of the occurrence of populations showing marked phenotypic variability and developing so-called “phenoecodemes” as a result of the environmental and/or culture conditions [Our observations on the oospore wall ornamentation  coincide with the findings of Romanov et al. , and con 2e,f in ). In ourd Israel . From th Romanov , who as  Romanov ,61. It s Romanov  recordedwa Oasis  are misirlesquin  for an inditions ,29,44.Description: Plants dioecious, small, robust, moderately to heavily encrusted, parts of the plants red in color, up to 8 cm tall A. Axes sDistribution in Egypt: This charophyte species has been previously recorded in the Siwa Oasis .C. tomentosa was found in a brackish, calcium-rich, shallow marsh in the El-Dakhla Oasis. The main physical and chemical features of this Saharan biotope were: high temperature (°C): 27.2; slightly alkaline pH: 7.53; high conductivity (μS·cm−1): 7700; DO (mg·L−1): 7.1; Na+ and Ca2+ as major cations: 1072 and 654 mg·L−1, respectively; Cl− and SO42− as major anions: 2543 and 582 mg·L−1, respectively; NO3− and NH4+: 1765 and 114 µg·L−1, respectively; TP and SRP: 715 and 295 µg·L−1, respectively : 960–4470; Na+ and Ca2+ as major cations: 25–545 and 30–135 mg·L−1, respectively; Cl− was the major anion: 131–1235 mg·L−1; SO42−: 36–300 mg·L−1; HCO3−: 221–314 mg·L−1; NO3−: 200–400 µg·L−1; NH4+: 150–271 µg·L−1; TP and SRP: 20–100 and 17–48 µg·L−1, respectively .General distribution and ecology: Cosmopolitan species in all freshwater biotopes, and occasionally in brackish waters ,49. It tectively . In agreChara vulgaris; not to the variety longibracteata. The same observation has already been reported by Saber et al. [C. vulgaris population from the Springs of Moses on the Sinai Peninsula, where they noticed that the bract-cells and bracteoles were clearly longer (ca. 4–12 times) than the oogonia. Similarly, Muller et al. [Remarks: Strong resemblance of our population with the one described by Wood and Imahori . Althougr et al.  during tr et al.  considerDescription: Plants monoecious, diffuse, delicate, pale green in color, up to 18 cm tall. Axes moderately slender, 400–435 µm in diameter. Internodes 1–2 times as long as the branchlets A,B. BranDistribution in Egypt: This is the first record of this charophyte both in North Africa and in Egypt.N. flagellifera was found in a nutrient-rich agricultural ditch in the El-Dakhla Oasis, the Western Desert of Egypt. Main physical and chemical characteristics of this biotope were: high water temperature (°C): 31.5; alkaline pH: 8.13; high conductivity (μS·cm−1): 6670; low DO (mg·L−1): 3.8; Na+, K+ and Ca2+ as major cations: 1368, 136 and 110 mg·L−1, respectively; Cl− and SO42− as major anions: 2378 and 356 mg·L−1, respectively; NO3− and NH4+: 1570 and 86 µg·L−1, respectively; TP and SRP: 2750 and 421 µg·L−1, respectively . Cond Necchi  pointed igospira ,47. Howetral ray C,D, a unnus tree .Description: Plants monoecious, pale green to green, unencrusted, fragile, up to 22 cm tall, with few coarse heads. Axes moderately slender, 500–850 µm in diameter. Internodes 1–2 times as long as the branchlets, becoming shorter towards the apex, up to 5 cm long. Sterile and fertile branchlets different A–D. The Tolypella sp. PBA–1704 from a desert, freshwater wetland’ mainly based on its concatenated rbcL+ITS1 phylogenetic placement.Distribution in Egypt: This is the first record worldwide of this genetically distinctive charophyte. We therefore designated it with the working name ‘Tolypella sp. specimens only showed a weak affinity to Tolypella sp. from Australia based on the concatenated data set of 16 rbcL and ITS1 sequences. During the present study, Tolypella sp. PBA–1704 was found in a shallow meso–eutrophic wetland in the El-Dakhla Oasis, the Western Desert of Egypt. This biotope had the following hydrochemical characteristics: relatively-low temperature (°C): 19.2; slightly-alkaline pH: 7.64; medium-low conductivity (μS·cm−1): 360; low DO (mg·L−1): 3.3; Ca2+ as the major cation: 38 mg·L−1; SO42− and HCO3− as major anions: 88 and 82 mg·L−1, respectively; NO3− and NH4+: 421 and 19 µg·L−1, respectively; TP and SRP: 217 and 62 µg·L−1, respectively  cryptic species new to science or to recognize it as belonging to a wide genetic variability of T. glomerata.Remarks: In spite of the high morphotaxonomic similarities between our lomerata , it is aChara contraria could be sampled from Wadi El-Arbaeen. It is a mountain wadi located in the UNESCO world heritage site “Saint Catherine Protectorate”. The only water source in this mountain valley is the shallow aquifers that are typically recharged by heavy rainfalls [® RZT stereomicroscope , and a BEL® photonics biological light microscope , and the light microscopy (LM) micrographs were taken with a Canon Powershot G12 digital camera. The biometric data provided were based on a minimum of 20–25 measurements for each character per species. The oospores were treated with acetic acid to remove any lime–shell, washed with distilled water and cleaned from the spiral cells by adding 10% Triton X100, and then stored at 60 °C for at least 10 h [® 300 VP electron microscope  at 3.0–20.23 kV at the A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia. The terminology used to describe the oospore surface follows Urbaniak [® . Voucher specimens were deposited in the collections of the Phycology Unit (No. 341) of the Botany Department, Faculty of Science, at Ain Shams University, Cairo (Egypt), curated by Abdullah A. Saber, under the accession numbers PBA–1801, PBA–1603, PBA–1701, PBA–1604, PBA–1702, PBA–1601, PBA–1602, PBA–1703, and PBA–1704 for Chara aspera from the Siwa oasis, C. contraria from Wadi El-Arbaeen (South Sinai), C. contraria from El-Dakhla Oasis, C. globata from the Siwa Oasis, C. tomentosa from El-Dakhla Oasis, C. vulgaris from the Siwa Oasis, C. vulgaris from El-Farafra Oasis, Nitella flagellifera from El-Dakhla Oasis, and Tolypella sp. from El-Dakhla Oasis, respectively.During sampling campaigns conducted from October 2016 to May 2018 to unravel the hidden phycological diversity in the Egyptian Oases and other comparable habitats, the charophyte populations investigated in the present study were collected from different aquatic biotopes, including thermal mineral desert springs, agricultural ditches, and shallow wetlands in the Western Desert Oases  and a nutrient-rich artificial muddy pool in the mountain valley “Wadi El-Arbaeen” in the Sinai Peninsula, Egypt . The majainfalls . Charophainfalls ,53 and Kainfalls . The keyast 10 h . They weUrbaniak . All pho® Instruments Co., Ltd., Woonsocket, RI, USA). Dissolved Oxygen (DO) was measured in the field with a Lutron® YK-22DO  oxygen meter. Hydrochemical analyses followed standard methods [−, CO32−, HCO32−, and SO42−), major cations , and metals were estimated using ionic chromatography (ICS 1500 Dionex Corp.). Nutrients  were measured by molecular absorption spectrometry. Silicates (SiO2) were analyzed by the molybdosilicate method.In situ water temperature, pH, ion conductivity, and total dissolved solids (TDS) were measured with a calibrated HANNA HI 991301 m  analysis was carried out using PAUP 4.0b10 . Bayesiahttp://embnet.vital-it.ch/raxml-bb/; accessed on 2 May 2021; [The robustness of the ML trees was estimated by bootstrap percentages  and posay 2021; ).Nitella flagellifera is here recorded for the first time in Egypt and North Africa.This study improved and updated our understanding on the taxonomic status, species diversity and autecological niches of nine charophyte populations colonizing different biotopes in the Egyptian Western-Desert Oases (North Africa) and Sinai Peninsula. Tolypella sp. has been designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, based on its distinct position in rbcL+ITS1 placement from the morphologically similar T. glomerata. In spite of the fact that most Chara taxa we recorded are cosmopolitan and eurytopic [C. globata for the second time in North Africa. The surveys carried out in the present study have also made it possible to provide further information for some species already reported from Egypt, such as C. aspera, C. contraria, C. tomentosa, and C. vulgaris [An interesting urytopic , our intvulgaris ,36,44. Ivulgaris  on the cvulgaris  pointed In accordance with the SRP-based trophic system proposed by Lambert-Servien et al.  for the


Rumex rothschildianus is the sole member of a unique section of the genus Rumex, in the family Polygonaceae. This species is a very rare small dioecious annual, endemic to Palestine that is traditionally used as food and for the treatment of various diseases. Therefore, the current investigation aimed to screen the chemical constituents, antioxidants, anti-α-amylase, anti-α-glucosidase, antilipase, and cytotoxic effects of four solvents fractions of R. rothschildianus leaves.R. rothschildianus leaves was extracted in four solvents with different polarities. Several qualitative and quantitative phytochemical tests were performed to determine the components of the extracts. The colorimetric analysis was used for the quantitative determination of phenols, flavonoids, and tannins. In-vitro assays were performed to evaluate the extracts for antioxidant, anti-α-amylase, anti-α-glucosidase, and antilipase inhibitory activities, as well as cytotoxicity by MTS assay against cervical carcinoma cells line (HeLa) and breast cancer cell line (MCF7).Dried powder of R. rothschildianus leaves showed the most significant antioxidant activity, due to having the highest content of flavonoids and phenolics, with an IC50 value of 6.3 ± 0.4 μg/ml, compared to 3.1 ± 0.9 μg/ml for Trolox, and regarding lipase inhibition activity the acetone fraction showed the most potent activity with an IC50 value of 26.3 ± 0.6 μg/ml, in comparison with orlistat positive control IC50 12.3 μg/ml. The same extract was the most potent inhibitor of α-amylase and α-glucosidase, with IC50 values of 19.1 ± 0.7 μg/ml and 54.9 ± 0.3 μg/ml, respectively, compared to 28.8, 37.1 ± 0.3 μg/ml of acarbose, respectively. The hexane fraction showed 99.9% inhibition of HeLa cells and 97.4% inhibition for MCF7 cells.The acetone fraction of R. rothschildianus leaves might provide a source of bioactive compounds for the treatment of oxidative stress. Similarly, the hexane fraction indicates the promising antitumor potential of R. rothschildianus. Clearly, these initial indications need further purification of potentially active compounds, and ultimately, in-vivo studies to determine their effectiveness.The acetone fraction of  Plants have been used as therapies since ancient times. Roots, seeds, bark, leaves, and flowers have all been used for remedial purposes. In the present day, synthetic medicines are available and are effective in the treatment of a wide range of diseases; however, some people still prefer herbal medicines as they are viewed as being less harmful to the human body , 2. MediRumex rothschildianus Aarons. is the sole member of a unique section of the genus Rumex, in the family Polygonaceae. This species is a very rare small dioeciously annual, endemic in Palestine. It has a mean height of 45 cm, is characterized by erect stems holding radical petiolate leaves, which are short-hastate at the base and short-acuminate at the apex. Flowers have a diameter of 3–4 mm, while pistillate flowers are about 2 mm in diameter with a coriaceous membranous layer [Rumex spp. are widespread in different regions of Turkey, are represented by 22 species. Some of the most common species are R. patientia L., R. crispus L., R. acetosa L. R. caucasicus rech., and R. alpinus L. R. alpinus and R. caucasicus are perennial plants distributed in middle and eastern Anatolia at an altitude of 1000–3000 m above sea level. The Rumex genus has been widely used in traditional medicine in Turkey to treat disorders, such as constipation, diarrhea, and eczema [d eczema . The gend eczema .Rumex vesicarius L., such as carotenoids, tocopherols, polyphenols, flavonoids, and ascorbic acid, which have a role as antioxidants and natural detoxifying agents. The dietary intake of antioxidant phytochemicals, like carotenoids, phenolics, and flavonoids may protect against non-communicable diseases in humans, such as cancer, cardiovascular disorders, and other health problems related to oxidative stress [A variety of research has been carried out on Rumex species, such as antimicrobial activities being reported for some species. Some bioactive phytochemicals have previously been found in e stress , 8.Harmful free radicals are known to play an important role in many major health problems, such as cancer, cardiovascular disease, rheumatoid arthritis, cataracts, and Alzheimer’s disease, and other degenerative diseases related to aging. Antioxidants are beneficial components that neutralize these free radicals before they can attack cells, and hence they prevent damage to cell proteins, lipids, and carbohydrates. A variety of both natural and synthetic antioxidants have been proposed for the treatment of human diseases. Such interest in the role of antioxidants in human health has prompted research in the fields of food science and medicinal herbs, assessing the function of herbs as antioxidants. Antioxidant action includes free radical scavenging capacity, inhibition of lipid per̴oxidation, metal ion chelating ability and also reducing capacity , 10.Cancer is one of the most global health care problems. The development and discovery of novel anticancer medication remain extremely important due to various factors. These factors include treatments that may cause major side effects or can be rather expensive. Alternatives that are safer biologically and more affordable are still highly desirable –14.Several plant species are considered potential sources of bioactive molecules such as atropine from Belladonna leaves, cocaine from coca leaves, vincristine from Vinca plant, and many others which still play an important role in modern medicine , 16.R. rothschildianus leaves.Useful therapeutic effects can come from mixing secondary products present in medicinal plants. These compounds are mostly secondary metabolites, like alkaloids, steroids, tannins, flavonoids, and phenolic, which are synthesized and deposited in specific parts of these plants , 18. TheR. rothschildianus leaves were harvested from Western regions of Palestine, between February and March 2018. They were identified by Dr. Nidal Jaradat, from the Pharmacognosy Laboratory at An-Najah National University, under the voucher specimen code Pharm-PCT-2066. All chemicals were purchased from Sigma-Aldrich. A spectrophotometer-UV/Visible , filter papers , shaker device , rotavap apparatus , grinder , electronic-balance , freeze dryer - BT85  and cryo-desiccator  were used.R. rothschildianus leaves was extracted by adding solvents sequentially based on their polarity, beginning with the non-polar solvent hexane, and then acetone (a polar aprotic organic solvent), methanol , and finally distilled water (a polar protic solvent). For each extraction, about 25 g ground dried leaves were placed in 0.5 l hexane for 72 h in a shaker device at 100 rotations per minute at 25 °C. Firstly, the hexane was replaced with 0.5 L acetone, and then subsequently replacement involved equivalent volumes of methanol and water. Incubations in the solvents were as described above for hexane. Each organic fraction was filtered and concentrated under a vacuum on a rotary evaporator, while the aqueous fraction was dried using a freeze dryer. Finally, all crude fractions were stored at 4 °C [Dried powder of  at 4 °C , 20.The yield of each fraction was calculated using the following formula:R. rothschildianus leaves four fractions were carried out to identify active secondary metabolites. The qualitative results were expressed as (+) for the presence and (−) for the absence of bioactive phytochemicals [Phytochemical screening tests of hemicals , 21.2CO3 in a volumetric flask and adjusting the volume to 100 ml with distilled water. A standard reference solution  was prepared by dissolving 100 mg of gallic acid in distilled water to a final volume of 100 ml. From this, a serial dilution was performed to obtain solutions of gallic acid at 100, 70, 50, 40, and 10 μg/ml). The stock solutions of the fractions from leaves were prepared by dissolving 100 mg plant extract in distilled water, adjusted to a total volume of 100 ml. Reaction mixtures were prepared by mixing 0.5 ml of each fraction solution with 2.5 ml 10% Folin-Ciocalteu’s reagent, which was dissolved in water with 2.5 ml 7.5% sodium bicarbonate. The sample tubes were incubated for 45 min at 45 °C. Then, the absorbance of each was measured in a spectrophotometer at wavelength 765 nm. The working samples were prepared in triplicate for each analytic trial, from which the mean and standard deviation values were calculated [The procedure to determine TPC was based on that of Cheung et al. TPC was expressed in milligram of gallic acid equivalents per gram dry weight of leaves (mg GA/g dry weight). Freshly prepared 7.5% sodium carbonate solution was made by placing 7.5 g Nalculated .R. rothschildianus leaf fractions was assessed using a calibration curve of rutin (standard reference compound). Results were expressed as milligram of rutin equivalent per gram dry weight of leaves extract (mg RU/g dry weight). A calibration curve for rutin was established using serial dilutions generated from a stock solution of 100 μg/ml. To make the stock solution, 10 mg of rutin was dissolved in 10 ml of distilled water and then diluted to 100 ml. Subsequently, the stock solution was diluted to provide rutin at concentrations of 10, 30, 40, 50, 70, and 100 μg/ml. For working solution preparation, 0.5 ml of each fraction solution was mixed with 3 ml methanol, 0.2 ml 10% AlCl3, 0.2 ml 1 M potassium acetate and 5 ml distilled water, and then incubated at room temperature for 30 min. The previous steps were repeated for each of the fractions, after which, absorbance was measured at a wavelength of 415 nm. For a blank control, a working solution was set up with distilled water in place of the sample extract. The samples were prepared in triplicate for each analytic trial, from which the mean and standard deviation values were calculated [The TFC in the four lculated .R. rothschildianus leaf fractions, being the most commonly used procedure. Catechin was used as a reference compound to construct a calibration curve. A 100 μg/ml stock in methanol was prepared, from which a dilution series was generated to give catechin concentrations of 10, 30, 50, 70, and 100 μg/ml. A 4% solution of vanillin in methanol was prepared freshly. Stock solutions of the fractions at 100 μg/ml were prepared using methanol as a solvent. For the working solution, 0.5 ml of each fraction solution was mixed with 3 ml vanillin solution and 1.5 ml of concentrated HCl. The mixture was allowed to stand for 15 min, and then the absorbance at 500 nm was measured, using a working solution set up with methanol in place of the sample extract as a blank. All working samples were analyzed in triplicate, from which the mean and standard deviation values were calculated. Total tannin in each fraction was expressed in terms of catechin equivalents (mg of CAE/g dry weight of leaves) [The protocol of Sun et al. was used to determine TTC in the four  leaves) .R. rothschildianus leaves. A 1000 μg/ml stock solution of each plant fraction was prepared in methanol. In addition, a 1000 μg/ml solution of trolox was also prepared (the reference standard). A dilution series was prepared from the stock solutions for each fraction, giving six serial dilutions at 2, 5, 10, 20, 50, and 100 μg/ml. One ml of each extract dilution was mixed with 1 ml 0.002 g/ml DPPH in methanol. One ml methanol was added to give a final working volume of 3 ml. The DPPH solution was freshly prepared, as it was very sensitive to light. The blank control of the series concentrations was DPPH in methanol in a ratio of 1:2, without the addition of an extract. All working solutions were incubated at room temperature (25 °C) in the dark for about 30 min. Optical densities were then measured with a spectrophotometer at a wavelength of 517 nm. The following equation was used to calculate % DPPH inhibition for each plant fraction, with trolox as the standard compound:The free 2,2-diphenyl-picrylhydrazyl (DPPH) radical scavenging assay was used to measure antioxidant activity in the different fractions of B is the recorded absorbance of the blank solution, and Ats is the recorded absorbance of the tested sample solution [where, Asolution .Stock solutions of 500 μg/ml were made from each plant fraction in 10% DMSO. From these, a dilution series of five concentrations of 50, 100, 200, 300, and 400 μg/ml were made. A 1 mg/ml stock solution of porcine pancreatic lipase in Tris-HCl buffer was prepared freshly just before use. The substrate, p-nitrophenyl butyrate (PNPB) was prepared by dissolving 20.9 mg in 2 ml acetonitrile.For each working solution, 0.1 ml porcine pancreatic lipase was mixed with 0.2 ml plant fraction from each member of the dilution series. Tris-HCl was added to make the final volume of the working solutions 1 ml, and they were incubated at 37 °C for 15 min. After incubation, 0.1 ml p-nitrophenyl butyrate solution was added to each test-tube. The mixture was then incubated for a further 30 min at 37 °C. Pancreatic lipase activity was determined by measuring the hydrolysis of PNPB into p-nitrophenolate at 410 nm, using a UV spectrophotometer. The same procedure was repeated using orlistat as a standard reference compound. Percentage lipase inhibition by plant fractions was calculated with the following equation:B is the recorded absorbance of the blank solution, and Ats is the recorded absorbance of the tested sample solution [where, Asolution .2HPO4/NaH2PO4, 0.006 M NaCl, pH 6.9 to give finally stock solutions with concentrations of 1000 μg/ml. From these, the following dilutions were prepared of 10, 50, 70, 100, and 500 μg/ml, using 10% DMSO as the diluent. A 0.2 ml volume of 2 units/ml porcine pancreatic α-amylase was mixed with 0.2 ml plant fraction and was incubated for 10 min at 30 °C. After incubation, 0.2 ml of a freshly prepared 1% starch solution in water was added, and the tubes were then incubated for at least three more minutes. At this point, the reaction was stopped by the addition of 0.2 ml 3,5-dinitro salicylic acid (DNSA) color reagent and was diluted with 5 ml of distilled water, before being heated at 90 °C for 10 min in a water bath. The mixture was then cooled to room temperature, and the absorbance was measured at 540 nm. The blank control was prepared using the same quantities described above, but replacing the plant fraction with 0.2 ml buffer. Acarbose was used as a standard reference following the procedure described above. α-amylase inhibitory activity was calculated using the following equation:A100 mg of each fraction was dissolved in a few milliliters of 10% DMSO, and then further dissolved up to 100 ml in 0.02 M NaB: is the absorbance of the blank sample, and AT is the absorbance of the test sample [where, At sample .p-nitrophenyl α-D-galactopyranoside PNPG was added to the reaction mixture and incubated was extended for a further 20 min at 37 °C. The reaction was terminated by adding 0.1 M Na2CO3. The absorbance was recorded at a wavelength of 405 nm for all samples. Acarbose was used as a positive control at the same concentrations as the plant extracts. The results were expressed as percentage inhibition according to the following equation:A dilution series of fractions was made, to yield concentrations of 100, 200, 300, 400, and 500 mg/ml. The reaction mixtures contained 0.1 ml 1 U/ml α-glucosidase solution mixed with 0.2 ml of an extract dilution and 0.5 ml 100 mM phosphate buffer, pH 6. 8. The mixtures were incubated at 37 °C for 15 min. Then, 0.2 ml 5 mM B is the absorbance without enzyme inhibitor, and AS is the absorbance in the presence of the enzyme inhibitor [where, Anhibitor .2 at 37 °C. Cells were seeded at 2.6 × 104 cells/well in a 96-well plate. After 48 h, cells were confluent, the media was changed. Cells were then incubated with different leaf fraction concentrations ranging from 0.25–10 mg/ml of R. rothschildianus for 24 h. Cell viability was assessed with the CellTilter 96® Aqueous One Solution Cell Proliferation (MTS) Assay according to the manufacturer’s instructions . Briefly, at the end of the treatment, 20 μl MTS solution per 100 μl media was added to each well and incubated at 37 °C for 2 h. Absorbances were measured at 490 nm [HeLa and MCF7 cancer cell lines were obtained from ATCC, the cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin antibiotics, and 1% L-glutamine. Cells were grown in a humidified atmosphere with 5% COt 490 nm , 28.p-value was < 0.05. Data were compared using unpaired t-tests.All of the obtained results of the four studied plant fractions  were expressed as mean ± SD standard deviation; the result was considered significant when the R. rothschildianus aqueous fractions showed the presence of saponin, phenols, protein, starch, and flavonoids. The methanol extract showed the presence of complex polysaccharides, phenols, protein, and flavonoids, while phenols, tannins, and flavonoids were observed in the acetone fraction, and in hexane fraction phenols, tannins, and flavonoids, glycosides, terpenoids, and steroids were identified as shown in Table The results of the preliminary phytochemical tests on the R. rothschildianus fractions are presented in Table For the evaluation of TPC, TFC, and TTC, the absorption (Abs) values of several concentrations of the gallic acid, rutin acid, and catechin standards (STDs) were obtained, and regarding these points, three equations were obtained for each STD versus its concentrations to calculate the total phenol, flavonoid, and tannin contents of the hexane, acetone, methanol, and aqueous R. rothschildianus leaves, using trolox as a potent antioxidant standard reference, were expressed as percentage DPPH inhibition . Hexane and methanol fractions only recorded moderate IC50 values, equal to 39.8 ± 0.3 μg/ml and 60.3 ± 0.4 μg/ml, respectively; while the aqueous fraction was inactive.In this assay, the anti-obesity activity of fractions from ent Fig. . R. rothR. rothschildianus leaves extract was compared to that of acarbose, a potent α-amylase inhibitory agent . Hexane and methanol fractions only recorded moderate IC50 values, equal to 39.8 ± 0.3 μg/ml and 60.3 ± 0.4 μg/ml, respectively; while the aqueous fraction was inactive. Pancreatic lipase is a major enzyme involved in enterocyte triglyceride absorption. Therefore, its inhibition represents an important strategy in the management of obesity [Solanum̴ tuberosum), Brassicaceae (Brassica nigra and Raphanus sativus), Rosaceae  Maxim.), Ericaceae (Arctostaphylos uva-ursi (L.) Spreng. and Vaccinium myrtillus L.), and Fabaceae (Pisum sativum L. and Phaseolus vulgaris L.) [ obesity . Plants aris L.) .50 of 19.0 ± 0.7 μg/ml, compared to 28.8 ± 1.2 μg/ml for acarbose, the reference compound. This suggested that R. rothschildianus might be a powerful herbal remedy for diabetes. The aqueous fraction only showed moderate activity in this assay with an IC50 value of 45.7 ± 0.3 μg/ml, while hexane had an IC50 value of 354.8 ± 1.2 μg/ml. The methanol fraction was inactive against α-amylase. A possible explanation for the aqueous fraction being a good enzyme inhibitor was the presence of saponins. Earlier scientific investigations found that saponins were bioactive against diabetes [Corchorus olitorius exerts α-amylase and α-glucosidase inhibitory effects due to constituents, especially phenolic compounds like caffeic acid [The acetone fraction was the most potent inhibitor of α-amylase, with an ICdiabetes . The pot50 of 54.9 ± 0.3 μg/ml, compared with that of acarbose, the reference compound, at 37.1 ± 0.3 μg/ml. By contrast, the methanol extract fraction showed only moderate inhibition of α-glucosidase, with an IC50 of 251.2 ± 0.4 μg/ml, while the hexane and aqueous fractions were inactive in this assay. The third category of oral hypoglycemic agents includes α-glucosidase inhibitors. There are a variety of α-glucosidase inhibitors, such as acarbose and voglibose, which usually are found in plant sources. They show valuable stabilization of blood glucose levels after a meal and have been used clinically in the management of diabetes mellitus [The acetone fraction exerted the greatest inhibitory action on α-glucosidase with an ICmellitus , 37.The results of treatment of both cancer cells (HeLa and MCF7) with various concentrations in mg/ml for different extracts showed that the general activity against the HeLa cancer cell line was better than MCF7 cancer cell line. The hexane extract showed potent anticancer activity at 4 mg/ml concentration with inhibition percentage 98.9 and 97.4% against HeLa and MCF7 cancer cell lines, respectively, while methanol extract at the same concentration showed potent activity against HeLa and MCF7 cancer cell lines with 97.2 and 95.6% inhibition percentage. From the previous results, the hexane fraction exerted a significant cytotoxic effect on both HeLa and MCF7 cell, with inhibition percentages reaching 99 and 92.4% at 4 mg/ml of hexane fraction concentration, respectively. This was consistent with the cytotoxic effects of both terpenoids and steroids, which were found in the hexane fraction. Diosgenin, a naturally occurring steroid and triterpenoids found in some plants, has been shown to inhibit breast cancer , 39.R. rothschildianus leaves indicated that the acetone extract fraction had significant potential in providing phytotherapies for diabetes and obesity, based on its potent inhibition of lipase, α-amylase, and α-glucosidase. In addition, the acetone fraction also showed a significant free radical scavenging activity. On the other hand, the hexane fraction showed significant inhibition of both HeLa and MCF7 cell lines, which might be related to its high content of terpenes and steroids. These observations in this study might lead to further in vivo studies to develop new natural pharmaceutical formulations effective in the treatment of obesity, diabetes mellitus, and some cancers.The results from this study on


High performance liquid chromatography with ultra-violet detection (HPLC-UV) and gas chromatography–mass spectrometry (GC-MS) methods were developed and validated for the determination of chlorambucil (CLB) and valproic acid (VPA) in plasma, as a part of experiments on their anticancer activity in chronic lymphocytic leukemia (CLL). CLB was extracted from 250 µL of plasma with methanol, using simple protein precipitation and filtration. Chromatography was carried out on a LiChrospher 100 RP-18 end-capped column using a mobile phase consisting of acetonitrile, water and formic acid, and detection at 258 nm. The lowest limit of detection LLOQ was found to be 0.075 μg/mL, showing sufficient sensitivity in relation to therapeutic concentrations of CLB in plasma. The accuracy was from 94.13% to 101.12%, while the intra- and inter-batch precision was ≤9.46%. For quantitation of VPA, a sensitive GC-MS method was developed involving simple pre-column esterification with methanol and extraction with hexane. Chromatography was achieved on an HP-5MSUI column and monitored by MS with an electron impact ionization and selective ion monitoring mode. Using 250 µL of plasma, the LLOQ was found to be 0.075 μg/mL. The accuracy was from 94.96% to 109.12%, while the intra- and inter-batch precision was ≤6.69%. Thus, both methods fulfilled the requirements of FDA guidelines for the determination of drugs in biological materials. Chlorambucil (CLB) is a nitrogen mustard agent, chemically known as 4-(4-(bis(2-chloroethyl)amino)phenyl)butanoic acid . For manA review of the literature on the quantitative determination of CLB in biological materials revealed only a few methods from the 1980s to 1990s, i.e., liquid chromatographic methods with ultraviolet detection (HPLC-UV) ,7,8 and Valproic acid (VPA), a chemical molecule composed of two propyl groups combined with an acetic acid moiety , can be Due to the results from many experimental studies showing the ability of VPA to inhibit proliferation of different cancer cells, it was proposed as a candidate for the therapy of various types of cancer. Its antitumor activity could be based on inhibiting specific enzymes, i.e., histone deacetylases, up-regulating the expression of some types of G protein-coupled receptors and affecting Notch signaling activity . What isThe main goal of the present study was to develop reliable quantitative methods for the determination of CLB and VPA in plasma, as a part of our large project focused on the potent anticancer activity of CLB and VPA in combination. From the literature survey and our preliminary experiments, it became clear that it would not be possible to develop a simple and sensitive LC-UV or GC-UV method for the simultaneous determination of these drugs. It was because VPA is a compound without a strong chromophore that does not allow detection by conventional UV spectrometry above 210 nm . PreviouBearing in mind the high volatility of VPA, the GC-MS method seemed to be a suitable approach to increase the sensitivity of the assay. Thus, it was taken into account and used for the determination of VPA and CLB. Unfortunately, these preliminary GC-MS experiments showed extremely low volatility of CLB and a limit of quantification above 5 µg/mL . Since wThe chromatographic conditions were optimized over various experiments  to achieve a short run time of analysis and symmetrical peaks for CLB and mefenamic acid ). It was found that the RP18 end-capped column and a simple mobile phase containing acetonitrile, water and formic acid at pH 2.6 were sufficiently effective for separation the analytes in a short time of below 3.5 min, as well as for the reduction of the peak tailing . At the Six solutions containing CLB and I.S. at a concentration of 15 µg/mL were injected onto the column for estimation of the system suitability. Retention times, resolution and the peak area ratios (CLB to I.S.) were evaluated according to the obtained results. Finally, we obtained the relative standard deviation (RSD) of peak area ratios below 4.0%, the RSD of the retention times (both CLB and I.S.) below 1.0%, and resolution between the analytes above 4 , confirmWhat is more, the presented CLB retention time was much shorter than that of 12 min which was reported previously . MefenamThe selectivity of the method was shown as the responses of the blank plasma a, zero p2) equal to 0.9996. The weighed (1/x) linear regression equation y = (0.15501 ± 0.00123)x + (0.00244 ± 0.00141) was obtained (mean ± SD), where y was the peak area ratio of CLB to I.S. and x was the concentration of CLB in µg/mL. The calibration curves showed linearity over the concentration range of 0.075–15 µg/mL with the mean correlation coefficient , with an acceptable precision of less than 20%, and accuracy of 80–120% . For theWhile solid phase extraction (SPE) was used for the determination of CLB by the LC-MS/MS method, a limit of CLB quantification of 4 ng/mL was reported using 200 µL of plasma . In turnThe acceptance criteria for the intra-batch and inter-batch precision, set at 15% for all QC samples, were achieved . The intThe accuracy, expressed as recovery of CLB extracted from plasma in relation to nominal values, was in the range of 95.21–101.12% . Thus, tThe stability of CLB was determined as the percentage ratio of the measured CLB concentration to its initial concentration in two QC samples, i.e., 0.15 µg/mL and 15 µg/mL. The results from the short-term, long-term and freeze-thaw stability tests are presented in A carryover test was conducted by injecting a blank sample after injecting the sample with the highest concentration of CLB in the standard curve (15 µg/mL). The acceptance criterion of the carryover was that the peak in the blank sample should be less than 20% of the peak in the LLOQ concentration . As a reAn important aspect of the present study was that a relevant internal standard was used, tracking VPA as a main analyte during derivatization and extraction steps, and compensating for the possible matrix effect. Benzoic acid was found to be the best option for all these purposes. Similarly to VPA, it belongs to a group of carboxylic acids, but possesses a sufficiently distinct fragmentation pattern that allowed us to obtain clean MS chromatograms without significant interferences in the selective ion monitoring (SIM) channel. To optimize our experiments, a simple derivatization and extraction method was chosen from the literature  and optiWe realize that precolumn derivatization of the analytes can be considered as a difficulty by many researchers. However, it was shown to be an essential step in the presented method, visibly reducing the peak tailing and increasing sensitivity. What is more, the simple esterification with methanol followed by the simple extraction with hexane occurred sufficiently fast, and was reproducible and effective. In addition, our procedure was very cheap, not requiring any sophisticated reagents, and allowing sufficient sensitivity.m/z 159, 116, and 87) and three ions for benzoic acid , which were applied for quantitative determinations. It was found that the optimized GC-MS procedure in the SIM mode clarified the chromatograms and provided the single peaks of VPA and I.S. . It provided three SIM ions for VPA (and I.S. .The selectivity of the method was shown as the responses of (a) the blank plasma, (b) zero plasma containing I.S. and (c) the plasma sample containing VPA at an LLOQ level and I.S. Representative chromatograms demonstrating no significant interferences from endogenous substances around the acquisition times of the analytes were shown in n = 6). The weighed (1/x) linear regression equation y = (0.32003 ± 0.01209)x − (0.02029 ± 0.00279) (mean ± SD) was obtained, where y was the peak area ratio of VPA to I.S. and x was the concentration of VPA in µg/mL  versus concentrations of VPA, and were shown to be linear over the concentration range of 0.075–15.0 µg/mL, with the mean determination coefficients (r2) equal to 0.9987 (in µg/mL .The sensitivity of the method (LLOQ) was determined as the lowest concentration of VPA, within the range of linearity, with a signal-to-noise ratio of at least 10:1, with an acceptable precision of less than 20%, and accuracy of 80–120% . For theOne previously described HPLC method, based on the extraction of VPA with hexane from acidified plasma, evaporation and derivatization with 2-bromo-2´-acetonaphtone, offered the LLOQ of 0.05 µg/mL using 160 µL of plasma . When LCPrecision and accuracy were determined at four concentrations of VPA  on two different days, and the acceptance criteria for the intra-batch and inter-batch precision were achieved . The intThe accuracy, expressed as recovery of VPA after derivatization and extraction from plasma in relation to the nominal values, was in the range 96.07–106.61%, proving the method was adequately reliable and reproducible within the required analytical range. The matrix effect was evaluated by comparing the concentrations of VPA after extraction from plasma with those analyzed directly from methanol. The obtained results of 98.39–101.31% suggested that significant matrix effects were not seen in the present method. All these results are summarized in A carryover test was conducted by injecting a blank sample after injecting the sample with the highest concentration of VPA in the standard curve (15.0 µg/mL). As a result, no peak of VPA more than 20%  of the pThe stability of VPA was determined as the percentage ratio of the measured VPA concentration to the initial VPA concentration in two QC samples, i.e., QC 0.15 µg/mL and QC 15.0 µg/mL. The results from stability studies are shown in Finally, we could conclude that the elaborated GC-MS method with simple derivatization ensured the quantitative determination of VPA, with desired sensitivity, precision and accuracy. Consequently, it could be a good alternative to previously described LC-MS methods ,21,23,24Pure substances, CLB, VPA, mefenamic acid and benzoic acid, were purchased from Sigma-Aldrich . Human pooled plasma  was purchased from MP Biomedicals Inc. . Acetonitrile, methanol, hexane and formic acid for HPLC and GC-MS were obtained from Sigma-Aldrich or Merck KGaA . Sulfuric acid 98% and sodium bicarbonate for analysis were obtained from POCh . Deionized water was produced in our laboratory with a Simplicity UV Water Purification System from Merck-Millipore . A model of 375 centrifuge from MPW Med. Instruments , a drying oven SL115 from Pol-Eko Aparatura Sp. J. , and a pH-meter HI9024C from Hanna Instruments  were used. Nylon membrane filters (0.45 µm) and hydrophilic PTFE syringe filters (0.2 µm) from Merck were also applied.Statistica version 13.3 from TIBCO Software Inc.  and free GNU R computational environment version 3.4.0 were used for the statistical analysis.The elaborated methods were validated according to the international regulations recommended by the Food and Drug Administration Agency ,31. ValiThe selectivity of the methods was shown as the response of (a) the blank plasma samples without both drugs and respective I.S., (b) zero plasma samples without drugs but containing respective I.S., and (c) the plasma samples at the LLOQ concentration of CLB or VPA and I.S.Six point calibration curves were constructed by plotting the peak area ratios of CLB or VPA to respective I.S. (y) versus CLB or VPA concentrations (x). The slope, intercept and correlation coefficient r2 were calculated by weighted linear regression (1/x).The limits of detection (LOD) were determined from the SD of the intercept and the slope of the mean regression lines. The lowest limits of quantification (LLOQ) were determined by analyzing progressively lower concentrations of CLB or VPA under the procedures described below, using a serial dilution method. The obtained LLOQ values were based on the signal-to-noise ratio of 10:1 which was determined by visual inspection.The stability of all stock solutions was examined after storage for 3 weeks at 4 °C. In addition, stability of CLB and VPA in plasma was assessed for short-term, long-term and freeze-thaw storages. Two different QC samples at low and high concentrations, i.e., 0.15 µg/mL and 15 µg/mL of CLB or VPA, were used for all stability tests. The samples were analyzed five times for every storage condition. The short-term stability test was conducted by maintaining the QC samples at 23 °C for 24 h, while the long-term stability was determined by freezing QC samples at −20 °C for 2 weeks. The QC samples were also stored at −20 °C for 24 h and then thawed at 23 °C. These steps were repeated three times for estimation of the freeze-thaw stability. In addition, the processed QC samples after extraction from plasma were checked after being placed on the table (CLB) or in an autosampler (VPA) at 23 °C for 6 h, for estimation of the post-preparative stability.Carryover tests were conducted by injecting blank samples without the drugs after injecting the samples with the highest concentration of CLB or VPA in their calibration ranges (15 µg/mL).v/v) adjusted to pH 2.6 with formic acid, filtered using nylon membrane filters (0.45 µm) and degassed. The flow rate of the mobile phase was 1.0 mL/min, while the UV detection was set at 258 nm. All analyses were performed at room temperature 23 °C. Mefenamic acid was used as an I.S.The HPLC-UV method was performed using a model 515 pump, a Rheodyne 10 µL injector and a model UV 2487 detector, controlled by Empower 3 software, all from Waters UK . Separation was performed on a LiChrospher® 100 RP18 end-capped column  from Merck. The mobile phase was a freshly prepared mixture of acetonitrile and water , 0.075 mg/mL (C2 solution), 0.015 mg/mL (C3 solution), 0.0075 mg/mL (C4 solution), 0.0015 mg/mL (C5 solution) and 0.00075 mg/mL (C6 solution). A 100 µL volume of the C6-C1 solutions of CLB and 650 µL of the solution of I.S. (1.0 mg/mL) were added to 250 µL of plasma, to obtain CLB concentrations of 0.075 µg/mL, 0.15 µg/mL, 0.75 µg/mL, 1.5 µg/mL, 7.5 µg/mL and 15 µg/mL. The samples were thoroughly vortexed and centrifuged at 5000 rpm at 23 °C for 5 min. The supernatants were injected onto the HPLC system through hydrophilic PTFE syringe filters (0.2 µm).The stock solution of CLB was obtained in methanol at a concentration of 1.5 mg/mL and used for the QC samples at concentrations of 0.15 mg/mL (QC1 solution), 0.03 mg/mL (QC2 solution), 0.0015 mg/mL (QC3 solutions) and 0.00075 mg/mL (QC4 solution). These QC solutions were stored in the freezer at −20 °C and brought to room temperature for precision and accuracy, as well as recovery and stability studies.The QC samples in plasma were prepared by adding 100 µL volumes of QC4-QC1 solutions to 250 µL of plasma and spiking with 650 µL of the I.S. working solution (1.0 mg/mL). In this way, CLB concentrations of 0.075 µg/mL (LLOQ level), 0.15 µg/mL (a low level), 3 µg/mL (a medium level), and 15 µg/mL (a high level) were obtained. The samples were thoroughly vortexed and centrifuged at 5000 rpm at 23 °C for 5 min. The supernatants were filtered through hydrophilic PTFE syringe filters and injected onto the HPLC system. The intra-batch precision and accuracy were determined by analyzing the QC samples five times on the same day. The inter-batch precision and accuracy were determined by analyzing the QC samples on two different days.The recovery of CLB from plasma was assessed in the QC samples at low (0.15 µg/mL), medium (3 µg/mL) and high (15 µg/mL) levels in five replicates, and calculated by comparing the determined concentrations with the nominal values. The matrix effect was evaluated by comparing the peak area ratios of CLB to I.S. which were obtained for the samples extracted from plasma, with the peak area ratios of the analytes at the same concentrations analyzed directly.m/z 159, 116, and 87, and for benzoic acid (I.S.) at m/z 136, 105 and 77 were selected for the quantitative determinations. The mass spectrometer was tuned manually using perfluorotributylamine from Sigma-Aldrich, with m/z 69 and 502. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The operating parameters used were, the injector temperature 250 °C, source temperature 280 °C, MS transfer line temperature 300 °C and quadrupole temperature 150 °C. The starting oven temperature was 80 °C (1.2 min held) and it increased at a rate of 25 °C/min to 250 °C (2 min held). Injections were done in the split mode with the split ratio 10:1. Standard electron impact conditions (70 eV) were used. For collision induced dissociation (CID), ultra-high purity nitrogen (1.5 mL/min) was used and 25 V as a collision cell voltage was applied. Helium gas (2.25 mL/min) was also used as a quenching gas to eliminate the meta stable helium species. The chromatographic data were analyzed by MassHunter Data Analysis Reporting B.07.01 software from Agilent.A GC chromatograph 7890A equipped with a 7692AALS autosampler coupled to a 7000A triple quadrupole mass spectrometer from Agilent  was used. Separation was done using an HP-5MS UI column  from Agilent. Ionization was carried out by an electron impact (EI) ionization with SIM mode. Three SIM ions for VPA at The stock solution of VPA was prepared by dissolving the substance in methanol to obtain the concentration of 1.5 mg/mL. It was diluted with methanol to obtain working solutions at the following concentrations: 0.15 mg/mL (C1 solution), 0.075 mg/mL (C2 solution), 0.015 mg/mL (C3 solution), 0.0075 mg/mL (Q4 solution), 0.0015 (Q5 solution) and 0.00075 mg/mL (C6 solution). The stock solution of benzoic acid (I.S.) was prepared at a concentration of 1.0 mg/mL and diluted with methanol to obtain the working concentration of 0.05 mg/mL.v/v) were added to each sample and thoroughly mixed. Then, the vials were heated at 60 °C for 30 min. After cooling to room temperature, 1 mL volumes of saturated sodium bicarbonate solution and 1 mL volumes of hexane were added to each sample for extraction. The mixtures were vortexed and centrifuged at 5000 rpm at 4 °C for 5 min. Then, aliquots of 500 µL of the upper (organic) layers were transferred to GC-MS vials, filled up with hexane to an equal volume of 1.5 mL and taken for GC-MS analysis.To obtain VPA concentrations of 0.075 µg/mL, 0.15 µg/mL, 0.75 µg/mL, 1.50 µg/mL, 7.5 µg/mL and 15.0 µg/mL, aliquots of 100 µL of C6-C1 solutions and 650 µL of the I.S. working solution (0.05 mg/mL) were added to 250 µL aliquots of plasma. The samples were thoroughly vortexed and centrifuged at 5000 rpm at 4 °C for 5 min. Supernatant volumes of 200 µL were placed in chemically inert glass vials and subjected to derivatization and extraction procedures. Derivatization was carried out via simple esterification with methanol, according to the literature . In shorThe stock solution of VPA at a concentration of 1.5 mg/mL was prepared by independent weighing of the pure substance. Then, it was diluted to obtain the concentrations of 0.15 mg/mL (QC1 solution), 0.03 mg/mL (QC2 solution), 0.015 mg/mL (QC3 solution) and 0.0075 mg/mL (QC4 solution). These QC solutions were stored in the freezer at −20 °C and brought to room temperature before use.The QC samples at concentrations of 0.075 µg/mL (LLOQ level), 0.15 µg/mL (a low level), 3.0 µg/mL (a medium level) and 15.0 µg/mL (a high level) were prepared by adding 100 µL volumes of QC4-QC1 solutions to 250 µL aliquots of plasma, and spiking with 650 µL of the I.S. working solution (0.05 mg/mL). The samples were mixed and centrifuged at 5000 rpm at 4 °C for 5 min. Then, 200 µL of the supernatants were subjected to derivatization and extraction procedures described above, and taken for GC-MS analysis.The recovery of VPA from plasma was assessed in the QC samples at low (0.15 µg/mL), medium (3 µg/mL) and high (15.0 µg/mL) levels in five replicates, and calculated by comparing the measured concentrations with the nominal values. The matrix effect was evaluated by comparing the peak area ratios of VPA to I.S. obtained after derivatization and extraction from plasma, with those obtained for the samples derivatized directly from methanol. In short, the solutions of VPA at three concentrations, i.e., 0.15 mg/mL, 0.03 mg/mL and 0.0015 mg/mL were prepared. Next, the 100 µL volumes of each VPA solution and 650 µL of I.S. working solution (0.05 mg/mL) were mixed and filled up with methanol to an equal volume of 1 mL. Then, 200 µL volumes were taken for esterification followed by heating at 60 °C, extraction with hexane and finally GC-MS analysis, as described above.Accumulating data shows that VPA belongs to a group of epigenetic modifying agents and can effect many alterations in cancer cells, through modulation of multiple signaling pathways. Thus, VPA could be effective in some anticancer therapy, especially in combination with other anticancer drugs, e.g., CLB. Our in vitro study  confirmed potent anticancer activity of VPA in CLL cells, especially due to the enhancement of CLB activity when used in combination. Based on these results, we can conclude that the combined treatment with VPA and CLB might be a new alternative in the therapy of CLL. Thus, two simple quantitative HPLC-UV and GC-MS methods were elaborated and validated for further studies of CLB and VPA in cultured cells and plasma, and to examine pharmacological aspects of such a combined therapy. As was stated above, in the literature there is no method for the simultaneous determination of CLB and VPA in biological materials. What is more, there are a limited number of procedures for the determination of CLB, as well as a limited number of simple methods for the determination of VPA. As a consequence, their determination in plasma is an ongoing challenge for researchers. On the one hand, it was not possible for us to develop one simple method for the simultaneous determination of CLB and VPA with the required sensitivity, accuracy and precision, mainly due to large differences in their physico-chemical properties. On the other hand, the presented methods allow the determination of CLB and VPA in the same plasma samples, without any mutual interference. As far as the required sensitivity, linearity, precision, accuracy, matrix effect, carryover and stability are concerned, they could be good alternatives to previously described HPLC or LC-MS methods. In addition, we think that the GS-MS method as such, should be promoted on a large scale in medical institutions at all levels as a suitable method for bioanalytical studies. What is more, both methods could be easily adopted for monitoring of CLB and VPA in a variety of in vitro as well as ex vivo scenarios, e.g., cell cultures.


This study aimed to evaluate acute toxicities associated with irradiation between the X‐CSI  and P‐CSI  groups in children with brain tumors.Sixty‐two consecutive patients who received initial craniospinal irradiation (CSI) for brain tumors in our center between January 1, 2011 and May 31, 2021, were included in the study. Acute toxicities were retrospectively evaluated during CSI using Common Terminology Criteria for Adverse Events version 5.0. Maximum grades of fatigue, headache, insomnia, nausea, vomiting, dermatitis, constipation, abdominal pain, oropharyngeal mucositis, and hematological toxicities were evaluated.p < 0.001). The P‐CSI group had a lower incidence of more than grade 2 nausea  and vomiting , compared with the X‐CSI group. Multivariate logistic regression analysis with adjustments for potential confounding factors of doses of CSI showed that proton radiation therapy was associated with a marked reduced risk of more than grade 2 nausea and vomiting during CSI .Thirty‐six patients received X‐CSI, and 26 patients received P‐CSI. The median dose of CSI was 18.0 Gy in the X‐CSI group and 23.4 Gy  in the P‐CSI group . This result suggests that proton beam CSI reduces acute gastrointestinal toxicities associated with irradiation. Craniospinal irradiation (CSI) is usually indicated for medulloblastoma (MBL), embryonal tumor with multilayered rosettes, and some germ cell tumors, ependymoma, and atypical teratoid rhabdoid tumors.To reduce the dose to normal tissue at risk keeping the optimal dose for the target coverage, proton radiation therapy (PRT) has used as an alternative technique. Photons emit maximal energy near the body surface; this energy gradually decreases at deeper points in the body. Hence, photon radiation therapy (XRT) is closely related with various acute and late adverse events.PRT can reduce damage to the normal tissue and offers an obvious advantage in reducing dose of organ at risk compared with XRT.Although the number of patients receiving PRT is increased worldwide, there is limited data focused on the comparison of acute toxicities associated with CSI between XRT and PRT in children with brain tumors.This retrospective cohort study aimed to evaluate acute toxicities associated with irradiation between the X‐CSI  and P‐CSI  groups in children with brain tumors.22.1A total of 63 consecutive patients who received initial CSI for brain tumors in Kobe Children's Hospital and Kobe Proton Center between January 1, 2011 and May 31, 2021 were included in the study. The patients treated between January 1, 2011 and September 30, 2019, received CSI as XRT, while the patients treated between February 1, 2019 and May 31, 2021, received CSI as PBT. One patient was excluded because of discontinuation of CSI due to disease progression. Finally, we retrospectively analyzed data of 62 patients from the clinical records. Baseline patients' characteristics were obtained from their medical records.This study was approved by the ethics review committees of Kobe Children's Hospital and Kobe Proton Center.2.2Acute toxicities during CSI were retrospectively evaluated using Common Terminology Criteria for Adverse Events version 5.0. Maximum grade of fatigue, headache, insomnia, nausea, vomiting, dermatitis, constipation, abdominal pain, oropharyngeal mucositis, and hematological toxicities, including white blood cell (WBC) count decreased, anemia, and platelet count decreased, were evaluated. The observational period was from the first day of CSI to the next day after completion of CSI.2.32 on day 1), CDDP (90 mg/m2 on day 2), and CPM , (II) CDDP (90 mg/m2 on day 2), CPM (1000 mg/m2 on day 1), and etoposide (ETP) (100 mg/m2 on days 1–5), and (III) VCR (1.5 mg /m2 on day 1), CDDP (90 mg/m2 on day 2), CPM (1200 mg/m2 on day 1), and pirarubicin (40 mg/m2 on day 1). The second group included weekly VCR: VCR at a dose of 1.5 mg/m2 weekly until the completion of RT. The third group consisted of other regimens: (I) oral ETP (60 mg/m2 daily), (II) temozolomide (150 mg/m2 on days 1–5) and irinotecan (50 mg/m2 on days 1–5), and (III) topotecan (0.75 mg/m2 on days 1–5) and CPM (250 mg/m2 on days 1–5).To evaluate the association of concurrent chemotherapy with acute toxicities during CSI, we divided the regimens of concurrent chemotherapies into three groups due to the heterogeneity of the regimens. The first group consisted of cisplatin (CDDP) and cyclophosphamide (CPM) containing regimen.Methotrexate (MTX)‐containing intrathecal chemotherapy consisted of MTX and dexamethasone (DEX). The dose of these drugs were defined according to age as follows: MTX  and DEX .2.4All patients, other than one patient, received CSI in Kobe Children's Hospital. All patients received anti‐pneumocystis prophylaxis and antifungal therapy using oral sulfamethoxazole/trimethoprim. When the neutrophil count was <500/μL, granulocyte‐colony stimulating factor, and fluconazole were administered.All patients who received concurrent chemotherapy received granisetron (40 μg/kg dose) from day 1 to the last day of each regimen. All patients who received CDDP as concurrent chemotherapy received oral aprepitant or fosaprepitant.During CSI, granisetron, osmotic diuretics, and sodium alginate were administered at the physician's discretion.2.5Dmin (minimum dose) ≥90% of the prescribed dose (PD), and Dmax (maximum dose) <105% of PD; lens, Dmax <10 Gy (RBE). Table The radiation treatments were planned using a computed tomography (CT)‐based three‐dimensional treatment planning system. Each patient was immobilized using a custom‐made thermoplastic cast in the supine position; then, CT was performed. The target volumes and organs at risk were delineated on the CT images basically, and the CT magnetic resonance imaging fusion images were also used, if necessary. The clinical target volume (CTV) included the entire cranial and spinal meninges. The planning target volume was defined as the CTV plus a setup margin , and the vertebral bodies were included if the patient was aged <10 years to prevent scoliosis. The dose constraints were defined only for CTV and the lens: CTV, 2.6t‐test or Mann–Whitney U test was also used to compare continuous variables in patients' characteristics between the X‐CSI and P‐CSI groups. To compare the maximum grade of acute toxicities associated with irradiation, chi‐square test or Fisher's exact test was used. The logistic regression model was used to calculate the adjusted odds ratio (OR) with 95% confidence interval (CI) for the development of more than grade 2 nausea and vomiting associated with the modality of CSI. The multivariate logistic regression model was performed with adjustments for the potential confounding factors of the use of granisetron during CSI, regimens of concurrent chemotherapy, need sedation during CSI, and doses of CSI. For all models, the number of examined covariates was determined by the number of outcome events with 10 events required for one covariate. A p < 0.05 was considered statistically significant. Statistical analyses were performed using EZR , a modified version of R commander .In the present study, the primary outcome was to evaluate acute toxicities associated with irradiation between the X‐CSI and P‐CSI groups. Chi‐square test or Fisher's exact test was used to compare categorical variables in patients' characteristics between the X‐CSI and P‐CSI groups. Unpaired 33.1p = 0.22). There was no significant difference in female/male ratio between the two groups (p = 0.30). In both groups, MBL accounted for the most common as the primary disease (55.6% in the X‐CSI group vs. 73.1% in the P‐CSI group).Table p < 0.001). Eleven (30.5%) patients in the X‐CSI group and all patients in the P‐CSI group received the radiation with daily fractional dose of 1.8 Gy (RBE) (p < 0.001). There was no significant difference between the X‐CSI and P‐CSI groups in the number of patients who received subsequent RT (p = 1.00). Thirty‐five patients (97.2%) in the X‐CSI group and 25 patients (96.2%) in the P‐CSI group received CSI, followed by focal or whole brain irradiation.The median dose of CSI was 18 Gy (12.0–36.0) in the X‐CSI group and 23.4 Gy (RBE) (18.0–36.0) in the P‐CSI group (p = 0.44).In both groups, CDDP and CPM containing regimen was the most common concurrent chemotherapy (65.0% in the X‐CSI group vs. 75.0% in the P‐CSI group). There was no significant difference in the use of intrathecal MTX during CSI between the X‐CSI and P‐CSI groups .Acute toxicity profiles during CSI are summarized in Table p = 0.59). In patients who received irradiation with vertebral body sparing, there were no significant differences in WBC count decreased, anemia, and platelet count decreased between the X‐CSI and P‐CSI groups. There were also no significant differences in neutropenia, episodes of febrile neutropenia, and needs of transfusions between the X‐CSI and P‐CSI groups.Differences in the maximum grade of fatigue, headache, insomnia, constipation, abdominal pain, and oropharyngeal pain were not significant between the X‐CSI and P‐CSI groups Table . Acute hTo identify the risk factor of more than grade 2 nausea and vomiting, multivariate logistic regression analysis was performed with adjustments for the potential confounding factors of use of granisetron during CSI, sedation during CSI, regimens of concurrent chemotherapy, and doses of CSI. Table 4Herein, we compared the acute toxicity profiles between the X‐CSI and P‐CSI groups. In this study, we revealed that the incidence rates of more than grade 2 nausea and vomiting in the P‐CSI group were lower than in the X‐CSI group, although P‐CSI group had higher doses of CSI than the X‐CSI group. Nausea and vomiting are common side effects of RT.A previous study in adults showed that patients who received P‐CSI had significantly lower rates of acute gastrointestinal toxicities, including weight loss, nausea, vomiting, and anorexia compared with patients receiving X‐CSI.p = 0.02, Table In our center, patients who needed sedation during irradiation stopped eating 6 h before irradiation and drinking clear fluids 2 h before irradiation. For this reason, all patients who needed sedation could not eat during CSI. Sedation during CSI reduced the amounts of meals obviously. Hence, the incidence rates of anorexia and weight loss were not examined in the present study. In general, P‐CSI takes longer times to perform than X‐CSI. The number of patients who needed sedation during CSI was higher in the P‐CSI group than in the X‐CSI group,  and CPM (1000–1200 mg/m2 for 1 day or 3 days) and induces greater myelosuppression than other regimens, such as weekly VCR.However, in our study, there were no significant differences in WBC count decreased, anemia, and platelet count decreased between the X‐CSI and P‐CSI groups. This difference could be explained by the intensity of concurrent chemotherapy. In our study, 21 patients (58.3%) in the X‐CSI group, 15 patients (57.7%) in the P‐CSI group received CDDP and CPM containing regimen. This regimen consisted of CDDP . Because of the nature of the retrospective analysis, informed consent, or assent was waived and opt‐out system was presented.Suguru Uemura: Treatment of patients, conceptualization and study design, data collection and interpretation, writing‐original draft, and writing‐review and editing. Yusuke Demizu: Study design and methodology, data analysis and data interpretation, and writing of the manuscript. Daiichiro Hasegawa: Conceptualization, study design, methodology, data analysis, data interpretation, critically revised article. Tomoko Fujikawa: Treatment of patients and data extraction. Shotaro Inoue: Treatment of patients and data extraction. Akihiro Nishimura: Treatment of patients and data extraction. Ryunosuke Tojyo: Treatment of patients and data extraction. Sayaka Nakamura: Treatment of patients and data acquisition. Aiko Kozaki: Treatment of patients and data acquisition. Atsuro Saito: Treatment of patients and data extraction. Kenji Kishimoto: Treatment of patients, data extraction, analysis, and interpretation of the data. Toshiaki Ishida: Treatment of patients and data extraction. Takeshi Mori: Treatment of patients and data extraction. Jyunji Koyama: Treatment of patients, data extraction, and analysis and interpretation of the data. Atsufumi Kawamura: Treatment of patients, data extraction, and analysis and interpretation of the data. Yoshinobu Akasaka: Treatment of patients, data extraction, analysis, and interpretation of the data. Makiko Yoshida: Treatment of patients, data, analysis, interpretation of the data, and critically revised article. Nobuyoshi Fukumitsu: Treatment of patients, data acquisition, formal data analysis, and data interpretation. Toshinori Soejima: formal data analysis and data interpretation, and writing of the manuscript. Yoshiyuki Kosaka: Conceptualization, study design, methodology, formal data analysis, and data interpretation. All authors have approved the final manuscript.