An abscopal response (AR) is a rare phenomenon defined as a distant response outside of the radiation field. It opens up the perspective of “in situ” vaccination of cancer. This phenomenon is rare and its mechanisms are unknown. In metastatic melanoma (MM), the approach regarding the efficacy of immunotherapy is to not use immunotherapy as a tool for enhancing radiation response but rather as one that needs to be integrated into immunotherapy to potentiate the specific effects of immunotherapy. The aim of our retrospective study was to investigate the incidence of the AR and its impact on therapeutic outcomes in a homogeneous population of patients with MM and a control group, to identify the factors associated with the AR. AR in metastatic melanoma seems highly prognostic of overall survival although it is a rare phenomenon. Factors associated with AR have been identified.p < 0.01) and not reached vs. 3.2 months, respectively. No grade ≥2 toxicity was reported. Patients who developed an AR were more likely to be treated with immunotherapy . In multivariate analysis, they had a higher number of irradiated metastases treated concomitantly  and a higher rate of mild infections during RT . Conclusions: AR in metastatic melanoma seems to be highly prognostic of overall survival, although it is a rare phenomenon. It may be promoted by multiple concomitant treatments with RT and immunotherapy and by acute inflammatory events such as infection.Objective: To evaluate the incidence of the abscopal response (AR) in patients with metastatic melanoma requiring palliative radiotherapy (RT). Patients and methods: Patients treated for metastatic melanoma between January 1998 and February 2020 in four oncology departments were screened. Patients with progression under immune checkpoint inhibitors or without ongoing systemic treatment, and requiring palliative RT were considered. The AR was defined as an objective response according to RECIST and/or iRECIST for at least one non-irradiated metastasis at distance (≥10 cm) from the irradiated lesion. Primary endpoint was the rate of AR. Secondary endpoints were overall survival (OS), progression-free survival (PFS), local control (LC) of the irradiated lesion, and toxicity as assessed by CTCAE v5. Results: Over the period considered, 118 patients were included and analyzed. Fifteen patients (12.7%) had an AR. With a median follow-up of 7.7 months , median OS and PFS after RT were significantly longer in patients with an AR compared to those without: 28 vs. 6.6 months ( Radiotherapy can be used to convert a tumor into an “in situ’’ vaccine . The mecSimultaneously, the interest in one of the holy grails of radiation oncology was reborn. Does the abscopal response (AR) exist, i.e., could the local radiation-induced tumor response generate a distant, out-of-field tumor response? Since the first description of the AR in 1953 by Dr. RJ Mole , only 46MM is recognized as one of the most immune-sensitive oncologic diseases and patients with MM may thus be the most likely to show an AR . The aimRetrospective data extraction was performed between January 1998 and February 2020 at the University Hospital in Brest (France), the Regional Hospital in Quimper (France), the Francois Baclesse Cancer Center in Caen (France), and the Institut de Cancérologie de l’Ouest . All consecutive patients with MM and requiring palliative RT were considered. Inclusion criteria were: histologically proven MM, disease progression under immunotherapy according to the Immune Response Evaluation Criteria in Solid Tumors (iRECIST) or disease progression without systemic treatment , RT for at least one primary or metastatic site, and the presence of at least one non-irradiated metastasis which was (i) ≥10 cm away from the irradiated target volume and (ii) assessable according to iRECIST criteria. A cut-off of 10 cm was arbitrarily set to avoid biases arising from potential low-dose radiation responses. Exclusion criteria were: initiation of a new systemic treatment, irradiation of brain metastases, and previous or concomitant treatment with BRAF inhibitors.All RT schedules were accepted. The RT-target irradiation volume was defined as the planning target volume (PTV) covered by at least 95% of the prescribed dose. The following patient characteristics were recorded at the beginning of the RT: Eastern Cooperative Oncology Group Performance Status (PS), American Joint Committee on Cancer 8th edition (AJCC 8th) TNM , RT totaIndividual immune-related events (irE) were defined as acute events occurring during RT only: documented infectious episodes, clinical symptoms of acute inflammation , and/or biological inflammatory syndrome with elevation of inflammation parameters including at least C-reactive protein (CRP) > 30 mg/L). Clinical acute inflammation was classified as (i) light clinical inflammation and CRP <40 mg/L, (ii) moderate clinical inflammation and CRP <40 mg/L, and (iii) severe clinical inflammation and CRP >50 mg/L . 18F-FDG-PET/CT) are performed.Usual follow-up after RT in participating centers is performed by an onco-dermatologist every 3 months and a radiation-oncologist every 6 months. Clinical examination, blood samples, and either a computed tomography (CT) or a 18F-Fluorodeoxyglucose positron emission tomography with CT  or partial response (PR) according to iRECIST ,17. All t-test or the Wilcoxon test was used for continuous variables, according to their distributions. PFS and OS were defined as the time between the first day of RT and either local, regional, or distant progression or death, respectively. Survival  was described by means of Kaplan–Meier curves and compared using log-rank tests in univariate analysis.The chi-square test or Fisher’s exact test was used to analyze and compare patients’ clinical characteristics at baseline between groups for categorical variables. Student OS and PFS were analyzed with a multivariable Cox model considering AR as a time-dependent binary variable, since its value changed over time among patients who experienced an abscopal effect.A secondary analysis used another Cox model to identify risk factors for developing AR over time. For the dosimetric variables, the ROC curve was built to identify the best value associated with the occurrence of AR within the first 30 days after the beginning of RT. For each variable and each model, the proportional hazard hypothesis and the log-linearity for continuous variables were assessed using Schoenfeld residuals and graphical visualization, respectively.Statistical analyses were performed using R software-© 2009–2019 RStudio, Inc.Overall, 118 patients treated with RT for melanoma between January 1998 and February 2020 in four French oncology institutions were included. Median age at the beginning of RT was 66.5 . PS was ≤1 in 70.4% patients .n = 80) received concomitant antiPD1 +/− antiCTLA4 immunotherapy during RT, including nivolumab (n = 47), pembrolizumab (n = 16), ipilimumab (n = 11), and nivolumab + ipilimumab (n = 6). Median time between the first injection of immunotherapy and RT was 2.9 months . Patients had primary and secondary resistance to immunotherapy before RT started in 42.5% and 57.5% of cases, respectively (Sixty-seven percent of patients (ectively . The medectively .p < 0.01) (An AR was observed after RT in 15 patients (12.7%) after a median time following RT of 1.7 months  and persisted over a median time of 11.9 months  . With me < 0.01) . Median p = 0.007) and concomitant immunotherapy  significantly improved OS (p < 0.01). OS was significantly longer in patients who received immunotherapy [12.6 (95%CI: 5.8–19.3) versus 2.9 months (95%CI: 1.1–4.8)] (p < 0.01)) ]  and in t< 0.01)) .p < 0.01) and concomitant irE (HR 403.45 (95% CI 13.83–11769.39), p < 0.01) were significantly associated with the occurrence of an AR  irradiated metastases (HR 16.85 (95% CI 2.16–131.49) of an AR .For dosimetric parameters, the optimal values associated with a higher chance of developing an abscopal response at 30 days was estimated at 19 Gy (AUC = 0.706) delivered in 3.5 fractions (AUC = 0.779) of 5.25 Gy (AUC = 0.796) over 4.5 days (AUC = 0.849) ,Figure 4In our multicenter cohort including 118 patients with MM treated with palliative RT, 15 patients (12.7%) developed an AR. In comparison, other studies found evidence for AR in 34.3%  of cases in an analysis of 10 nonrandomized studies of metastatic melanoma patients treated with RT and ICI between 2014 and 2019 [The occurrence of AR in our study was associated with significantly improved oncologic outcomes, including OS rates in multivariate analysis. By definition, an AR correlates with effective distant control; interestingly, it also resulted in better OS rates. Our results are in accordance with the literature: in a prospective study that aimed to induce an abscopal effect in 41 stable or progressing patients treated with systemic treatment for solid tumors, the median OS was 20.98 months (95% CI 11.05–30.96) in abscopal responders versus 8.33 months in others (95% CI 5.03–13.29). This study, however, did not include patients with metastatic melanoma, but mostly lung and breast cancer [p < 0.01). These observations—also coherent with the literature—corroborate such a frequently adopted strategy [With a median follow-up of 7.7 months, median OS (10.2 months) after the occurrence of antiPD1 +/− antiCTLA4 immunotherapy resistance was poor in our cohort, which is in agreement with the literature . Immunotstrategy . The usestrategy  has beenstrategy .Such AR-associated improved outcomes justify investigating how AR might be provoked. The interplay between immune environment and AR has already been described  and numeActivation of the interferon pathway may also come from the dose of radiotherapy and fractionation dose. In fact, in preclinical data regarding 8 Gy, there is more induction of IFN than at higher doses, such as 20 Gy or 30 Gy. Repeating the dose of 8 Gy three times seems to be the optimal formula for activating a type I IFN response and upregulates the expression of MHC-I. ,33. At hAmong other agents involved in the immune response against external injuries, lymphocytes are less likely to be involved as they are known to be radiosensitive and thus strongly depleted within radiation fields. This understanding has led to the idea that blood should be considered an organ at risk because RT-induced lymphopenia is associated with poorer survival in multiple tumor types . Lung caIn contrast, dendritic cells, while particularly stimulated during infections , are capAll these hypotheses merit further exploration but are currently not robust enough to be reproducible and need to be documented as accurately as possible. Infection-driven immune-related events can barely be distinguished from treatment-related immune events. As our study was retrospective, the parameters used to monitor inflammation have limitations. Although CRP has relatively good sensitivity for detecting inflammatory processes, it remains non-specific. Concentrations of 10–40 mg/l are generally found in cases of moderate inflammation or viral infections, and typically rise to 50–200 mg/l in cases of severe inflammation or bacterial infections , withoutA number of irradiated sites ≥ 2 at the same time also appeared to be a favorable factor for the AR in our study, as suggested in the literature , but notOur study has several limitations, with a possible selection bias inherent to its retrospective design. First, clinicians do not always report infections occurring during radiation therapy. Second, the small proportion of AR patients makes the analysis less robust but remains consistent with the rarity of the phenomenon. Lastly, the follow-up modalities and retrospective data recording may have influenced the results of the analysis.The abscopal response in metastatic melanoma patients seems highly prognostic. Although a rare phenomenon, it may be provoked with multiple concomitant radiation treatments with an acute immune-related event during radiotherapy. A biological substrate is urgently needed to corroborate our hypothesis.


In this study, we aim to evaluate whether thoracic ultrasound (TUS) and tracheal amylase (TA) alone or in combination can predict the development of ventilator-associated pneumonia (VAP) in neurocritical patients. Consecutive adult patients with neurocritical disease with normal chest radiographs who required intensive care unit admission and mechanical ventilation between March 2015 and July 2018 were included. TUS and Amylase levels were measured during the first 24 hours and repeated 48 hours after orotracheal intubation. Forty-three patients with a median age of 34 years (17–82) were included. TUS had a sensitivity of 100% and specificity of 96.3% as a predictor of VAP within the first 48 hours when nonpattern A was observed. TA levels > 200 UI/L in the first 48 hours had a sensitivity of 87.5%, and specificity of 63% as a predictor of VAP. Moreover, no benefit of TUS plus TA compared to TUS alone as a predictor of VAP was found. The identification of abnormal TUS patterns in the first 48 hours of orotracheal intubation is a significant predictor of VAP in neurocritical patients. In addition, previous studies have demonstrated that patients diagnosed with VAP have an increased length of intensive care unit (ICU) stay and related healthcare costs.–8VAP is a nosocomial infection that affects patients who receive mechanical ventilation (MV) for > 48 hours. A combination of clinical criteria, such as the Clinical Pulmonary Infection Score (CPIS), has been proposed for the diagnosis of VAP using X-rays as the imaging modality. However, CPIS has shown poor diagnostic performance.Risk factors for VAP, such as microaspiration of oropharyngeal secretions or gastric contents in neurocritical patients, are key to its pathogenesis.,10 histidine-rich glycoprotein, pentraxin 3, procalcitonin,,14 breath octane, acetaldehyde, and C-reactive protein. Procalcitonin, C-reactive protein, sTREM-1, and HRC have proven to be predictors of VAP; however, most are markers of inflammation, making them nonspecific.,13,14,16Currently, there are no validated biomarkers for VAP diagnosis. Several biomarkers have been studied to predict the severity and prognosis of VAP, such as interleukin-18 (IL-18) and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1),,5,17 This marker is inexpensive and easily measured and could be an attractive marker for predicting the development of early VAP.Recently, the α-amylase concentration in tracheal secretions and bronchoalveolar lavage has been reported as a potentially useful marker for diagnosing microaspiration and bacterial pneumonia. and few studies have proven the utility of TUS as a tool predictor in detecting lung changes prior to VAP diagnosis.In the last decade, there has been an increase in thoracic ultrasound (TUS) evaluation in the diagnosis of VAP with almost 100% sensitivity,Currently, there are no clinical scores or biomarkers to predict the occurrence of VAP.This study aimed to evaluate whether TUS alone or in combination with tracheal amylase (TA) is a significant predictor of VAP in patients with neurocritical disease.The study was approved by our ethics committee on January 28, 2016 (NM16-00001) and was conducted in accordance with the Declaration of Helsinki. All the patients provided informed consent to participate in the study.We prospectively recruited patients aged > 18 years with neurocritical diseases who were admitted to the ICU, between January 2016 and July 2018, were on MV for < 48 hours in order to protect the upper airway, and presented with normal chest X-ray. Patients suspected of having a respiratory tract infection, chronic infections , chronic use of steroids, neurogenic pulmonary edema, or pulmonary contusions (thoracic trauma and chest X-ray infiltration) were excluded. All the patients were treated using a standard ICU protocol to prevent pneumonia.,7 VAP was defined as a pulmonary infection that developed in-hospital care 48 hours after intubation and MV. VAP was diagnosed by an Intensive Care medical team composed of Internal Medicine, Pulmonology, and Critical Care, using the major CDC criteria or more than 6 points in the CPIS criteria. The Intensive Care medical team carried out its VAP diagnostic approach blinded to the TUS results.All patients were followed up for the next 30 days to determine who developed VAP with the Centers for Disease Control and Prevention (CDC) and CPIS. Tracheal suctioning was routinely performed using an open tracheal suction system with a 14 French endotracheal tube and 10 mL of sterile normal saline instilled into the trachea before endotracheal suctioning.Amylase levels were measured 24 and 48 hours after intubation using an enzymatic method .TUS was performed in a semi-upright position during the first 24 hours and repeated 48 hours after orotracheal intubation using a Z6 ultrasound equipment  with a low-frequency convex transducer (5 Hz). The thoracic posterior segments were not assessed because most patients could not adopt a lateral or sitting position. Only one pulmonologist with 6 years of experience in TUS, was blinded to the clinical data, and follow-up was performed on all TUS.Four points per hemithorax  were evaluated.The following ultrasound parameters were recorded as pulmonary findings: normal lung pattern (lung sliding and A lines), abnormal TUS pattern .To prevent VAP clinical staff followed the guideline recommendations, such as keeping the patient’s bed in a semifowler position (between 30 and 45º), taking off the ventilator once the patient no longer required it, and maintaining good hygiene.Other variables analyzed were hospitalization days, difficult orotracheal intubation according to the American Studies Association Task Force, and severity of neurocritical disease using the Glasgow score, Sequential Organ Failure Assessment Score (SOFA) score, and Acute Physiology and Chronic Health Disease Classification System (APACHE) II score.In descriptive statistics, general and specific variables were analyzed, obtaining measures of central tendency and dispersion, and determining normality with the Kolmogorov–Smirnov test.P < .05. Statistical analyses were performed using SPSS Software .In inferential statistics, categorical variables were analyzed using Pearson’s Chi-square test. Statistical significance was set at Forty-three patients with neurocritical disease met the eligibility criteria. Their median age was 34 years (17–82), and 35 were male (81.4%). The most common neurocritical diagnoses were subdural hematoma in ten patients (23.25%), intraparenchymal hemorrhage in 9 patients (20.93%), and subarachnoid hemorrhage in 7 patients (16.25%). Twenty-eight patients (65.1%) required decompressive craniectomy, and there was no significant difference between patients who developed VAP and those who did not. Difficult orotracheal intubation was identified in ten patients (23.3%), with no increase in the development of VAP compared to no difficult intubation. Prophylactic antibiotics were administered to 22 patients (51.1%) at the discretion of the neurosurgeon based on the infection risk associated with skull fractures; the most common antibiotic was cephalothin in 21 patients (48.8%).P = .037) . Emesis or gastric residue > 500 mL was presented in 6 patients (37.5%) who developed VAP versus 2 patients (7.4%) in the non-VAP group, with statistical significance (7) Table . The lenP = .59). The median SOFA score in the first 24 hours was 6 points (3–16) in the VAP group versus 6 (0–14) in the non-VAP group, without a statistically significant difference (P = .49) and the median SOFA score at 48 hours was 6.5 points (4–14) in the VAP group versus 7 (0–14) in the non-VAP group, without a statistically significant difference (P = .44) (See Table P = .54).VAP was identified in 16 patients (37.2%) of the total population with neurocritical diseases. The severity of neurocritical disease was evaluated using the Glasgow score, SOFA score, and APACHE II score; and the median Glasgow score in the total population was 7 points (3–15). In 36 patients (83.7%) the Glasgow score was less than 8 at the time of ward admission and required urgent intubation. Glasgow score higher than 13 points were identified in 4 patients (9.3%) with brain tumors and intubation was performed in the surgical room. There was no association between Glasgow score and urgent intubation with the development of VAP  was identified in the first 24 hours in 7 patients (43.7%) who developed VAP with a sensitivity of 43.8%, specificity of 96.3%, positive predictive value (PPV) of 87.5%, and negative predictive value (NPV) of 74.3%.Non-A pattern or abnormal TUS . The TA obtained less than 48 hours had a median of 904 (30–7941) in patients who developed VAP versus non-VAP 111  with statistical significance (P = .022) , and thus predicts a more efficient development of VAP. In contrast, Zhou et. al. reported the superiority of TUS and a different biomarker  combination with a sensitivity and specificity of 81% and 86% respectively, over TUS alone  for VAP diagnosis, but was not proven to be a predictor of VAP. probably because of the high prevalence of atelectasis and the TUS limitation in differentiating between inflammatory and noninflammatory consolidations in these previous studies.In our study, TUS alone and in combination showed high sensitivity (100%). Although TUS alone remained highly specific, it was not the same for the combination of TUS and amylase (96.3% vs 59.3%) after 48 hours. In contrast, previous studies on TUS reported low specificity (60%) and high sensitivity (100%),The identification of abnormal TUS patterns  in the first 48 hours of MV in neurocritical patients predicted the development of VAP before diagnosis using the classic criteria (CPIS or CDC) with a PPV of 94.1%. Moreover, when TUS was normal at 48h of MV , the NPV was 100%, with a high accuracy of 98.15%., besides it is a great tool requiring less than 5 minutes, no radiation exposure, and it can be performed at the patient’s bed. Although, the overall prediction of VAP using TUS focuses on detecting lung changes before the diagnosis of VAP has been proven but it has been evaluated in a few studies.We already know the great accuracy of point-of-care TUS for the diagnosis of VAP with almost 100% sensitivityP = .037). In addition, prophylactic antibiotics were administered in the surgical room to 22 patients (51.1%) most of whom had traumatic brain injuries at the discretion of the neurosurgeon based on the risk of meningeal infection. Unfortunately, the information about this topic remains controversial; however, some studies are in favor of prophylactic antibiotics, but other studies did not identify any benefit and could be a risk factor for resistant infections.,25We evaluated whether other classic risk factors for VAP were associated with its development in addition to abnormal TUS, such as difficult Intubation, time of intubation, points in GCS, and prophylactic antibiotics, but there is no relation with the development of VAP, besides solely emesis or gastric residue > 500ml was associated with the development of VAP  in diagnosing microaspiration.The other important factor that we incorporated in our study is TA because according to recent studies, median values of α-amylase in “tracheal” secretions could vary widely from 0 U/L in a small pilot study to over 22,000 U/L in patients with documented aspiration or VAP.Furthermore, compared to other markers and imaging studies, α-amylase and TUS are cheaper and easier to perform. However, the combination of TUS and α-amylase did not result in better VAP development than TUS alone. Our study had some limitations including its small sample size, the fact that not all patients received prophylactic antibiotics, and the fact that it was performed only in neurocritical patients. In addition, the imaging study was performed blindly by an expert, and we did not compare our results with those of another operator. This factor could probably have affected the results because there would be heterogeneity between each operator’s interpretations. However, adds to the knowledge of the relevance to detected lung changes with TUS before the appearance of VAP, by a no-radiation study bedside with high positive and negative predicted values and could be performed in less than 5 minutes, however, further studies are necessary to confirm these findings and see their scope.An abnormal TUS pattern alone in the first 48 hours of orotracheal intubation is a significant predictor of VAP in neurocritical patients; however, we did not identify the utility of the combination of TA and TUS in predicting VAP.Conceptualization: Juan O. Galindo-Galindo, Jorge M. Llaca-Díaz.Data curation: Juan O. Galindo-Galindo, Mario A. Ataxca-Gonzalez, Jorge M. Llaca-Díaz, Erick J. Rendón-Ramírez.Formal analysis: Perla R. Colunga-Pedraza, Samantha P. Peña-Lozano, Erick J. Rendón-Ramírez.Investigation: Roberto Mercado-Longoria, Perla R. Colunga-Pedraza, Jorge M. Llaca-Díaz, Erick J. Rendón-Ramírez.Methodology: Roberto Mercado-Longoria, Juan O. Galindo-Galindo, Mario A. Ataxca-Gonzalez, Jorge M. Llaca-Díaz.Project administration: Roberto Mercado-Longoria, Erick J. Rendón-Ramírez.Writing—original draft: Perla R. Colunga-Pedraza, Erick J. Rendón-Ramírez, Erick J. Rendón-Ramírez.Writing—review and editing: Perla R. Colunga-Pedraza, Samantha P. Peña-Lozano, Erick J. Rendón-Ramírez.


About two thirds of people with chronic pain report problems sleeping. We aimed to evaluate the effectiveness of non-pharmacological sleep interventions for improving sleep in people with chronic pain.We conducted a systematic review of non-pharmacological and non-invasive interventions to improve sleep quality or duration for adults with chronic non-cancer pain evaluated in a randomised controlled trial. Our primary outcome of interest was sleep; secondary outcomes included pain, health-related quality of life, and psychological wellbeing. We searched the Cochrane Library, MEDLINE, Embase, PsycINFO and CINAHL from inception to April 2020. After screening, two reviewers evaluated articles and extracted data. Meta-analysis was conducted using a random effects model. Risk of bias was assessed with the Cochrane tool.p < 0.00001). The effect size was only slightly reduced in meta-analysis of 3 studies at low risk of bias. The difference between groups was lower at 3 and 6 months after treatment but still favoured cognitive behavioural therapy for insomnia. Pain, anxiety and depression were reduced post-treatment, but evidence of longer term benefit was lacking. There was no evidence that sleep hygiene interventions were effective in improving sleep and there was some evidence in comparative studies to suggest that cognitive behavioural therapy for insomnia was more effective than sleep hygiene.We included 42 trials involving 3346 people randomised to 94 groups, of which 56 received an intervention targeting sleep. 10 studies were of fair and 32 of good methodological quality. Overall risk of bias was judged to be low in 11, high in 10 and unclear in 21 studies. In 9 studies with 385 people randomised, cognitive behavioural therapy for insomnia showed benefit post-treatment compared with controls for improved sleep quality, standardised mean difference − 1.23 (95%CI -1.76, − 0.70; Numerous other interventions were evaluated in small numbers of studies, but evidence was insufficient to draw conclusions about effectiveness.Cognitive behavioural therapy for insomnia is an effective treatment to improve sleep for people with chronic pain, but further high-quality primary research is required to explore combined CBT content that will ensure additional improvements to pain, quality of life and psychological health and longer-term maintenance of benefits. Primary research is also needed to evaluate the effectiveness of interventions for which insufficient evidence exists.CRD42019093799.PROSPERO registration number: The online version contains supplementary material available at 10.1186/s12891-022-05318-5. While chronic pain may be a primary complaint or secondary to an underlying disease, it is now recognised as a health condition in its own right  requirinThe relationship between sleep and pain is bidirectional –27. ReduThe protocol was registered prospectively with PROSPERO CRD 42019093799) , and the9093799 ,All of our studies of sleep problems in people with chronic pain are fully supported by patient involvement. This includes regular discussions during development, conduct and reporting of research.Population: People aged ≥18 years with chronic non-cancer painIntervention: Non-pharmacological and non-invasive intervention to improve sleep quality or durationComparison: Comparator of standard care, no treatment, attentional, or wait list controlOutcomes: Primary outcomes of sleep quality and duration, secondary outcomes of other sleep outcomes, pain, health-related quality of life, and psychological wellbeing, and a primary harm outcome of adverse events. Follow up post-treatment and at 3 and 6 months after end of treatment if reportedStudy: Evaluation in a randomised controlled trialEligible studies reflected PICOS criteria:We searched MEDLINE, EMBASE, PsycINFO, Cochrane Library, and CINAHL from inception up to 8th April 2020. The search strategy as applied in MEDLINE is included in Supplement Table Results of searches were imported into Endnote and duplicates removed. After an initial screen by one reviewer to remove clearly off-topic studies, all titles and abstracts were screened independently by two reviewers. Potentially relevant articles were acquired and independently assessed by two reviewers for eligibility with disagreements resolved in discussion with a third reviewer.One reviewer extracted data from eligible studies into Excel and a second reviewer checked this. Extracted data comprised: country; dates of recruitment; setting; inclusion and exclusion criteria; participant characteristics ; intervention and comparator content, timing, duration and intensity; assessment times; outcome measures; and information on intervention fidelity. We contacted study authors for clarification relating to review eligibility and for missing data.Risk of bias was assessed independently by two reviewers using the Cochrane tool , specifi2). Forest plots were generated. Risk of bias as a potential source of heterogeneity was considered in sensitivity analyses. If pooling of outcome data was not appropriate, a narrative synthesis was reported. In interpreting the outcomes from this review we consider effect sizes as described by Cohen: small, SMD = 0.2; medium, SMD = 0.5; large, SMD = 0.8 [We conducted meta-analyses with Review Manager 5.4 software to compare outcomes across studies with similar interventions and outcome measures. For continuous data, if outcomes were measured identically across studies, an overall mean difference (MD) with 95% confidence intervals (CIs) was calculated. If continuous outcomes were measured differently across studies, overall standardised mean differences (SMDs) and 95% CIs were calculated and presented alongside measures of heterogeneity . The improvement was sustained but reduced at 6 months in 3 studies with data. In 3 studies at low risk of bias, the benefit for CBT-I over control was slightly reduced, SMD -1.01 , p = 0.01 and heterogeneity remained high (I2 74%). Exploration of effectiveness in relation to a specific condition was only possible for 2 studies at unclear risk of bias including 79 people with fibromyalgia suggesting no benefit for CBT-I, SMD -0.57 , p = 0.2, but heterogeneity was high (I2 65%).At the end of intervention, overall questionnaire assessed sleep quality in 9 studies with 385 participants was improved in people receiving CBT-I compared with untreated controls, SMD -1.23 , Waking after sleep onset, as measured by actigraphy, improved in people receiving CBT-I post-treatment compared with controls and this was also apparent up to at least 6 months in those studies using sleep diaries and polysomnography. Sleep onset latency was improved up to 3 months after CBT-I when assessed by questionnaire, but not with actigraphy or polysomnography. Sleep efficiency was improved up to 6 months after CBT-I compared with control when measured in sleep diaries or by polysomnography, but not by actigraphy. Total sleep time measured by diary, actigraphy or polysomnography was not improved in those receiving CBT-I compared with controls. There were no improvements in diary recorded sleep awakenings or the Epworth Sleepiness Scale in people receiving CBT-I compared with control. Adverse events were assessed in 5 studies , 45–47. p = 0.02), but this was not apparent in 3 studies at low risk of bias, SMD -0.19 , p = 0.36, or sustained at 3 months or longer , and depression SMD -0.57 , p = 0.08 with high heterogeneity (I2 65%). IN 1 study exclusively including people with fibromyalgia, anxiety and depression were reduced in the Group receiving CBT-I compared with controls.No studies reported health-related quality of life outcomes. For psychological health, information from studies was mainly limited to post-treatment with benefit suggested for anxiety SMD -0.54 , In 2 studies, a further comparison was made between CBT-I and CBT solely for pain (CBT-P). One was a small pilot study with 11 people with chronic pain randomised to the 2 interventions . In a laIn 1 study with 132 people with chronic non-cancer pain randomised, a brief educational intervention incorporating sleep hygiene and cognitive behavioural strategies was compared with wait list controls . The stuCBT-I was compared with a sleep hygiene intervention in 4 randomised trials , 50–52. p = 0.40 but heterogeneity was high (I2 61%). Excluding the study at unclear risk of bias removed heterogeneity and there was an improvement in overall sleep quality after CBT-I compared with sleep hygiene, SMD -0.53 , p = 0.01 [Data on overall sleep quality was available for 3 studies , 50, 51.p < 0.0001 [p = 0.0002. Improvements in pain and health-related quality of life were not evident at longer follow up. Evidence relating to psychological health was limited to 2 studies at low risk of bias with 97 patients randomised. There was no benefit for CBT-I compared with sleep hygiene for anxiety, SMD -0.32 , p = 0.12 with no heterogeneity, but depression was reduced, SMD -0.61 , p = 0.006 with slight heterogeneity (I2 11%).In 3 studies, results for pain outcome were similar in direction to sleep quality , 50, 51 p = 0.05, and MD − 61.58 min , p = 0.006, respectively. In the 1 study at low risk of bias, the difference in sleep quality was smaller and in 2 studies with data, the benefit relating to sleep quality was not sustained at 3 and 6 months. In 2 studies with 299 people exclusively with fibromyalgia, there was no evidence for a difference in sleep quality, SMD -0.88 , p = 0.18 but heterogeneity was high (I2 94%).In 4 studies with 432 participants randomised, cognitive behavioural therapy focusing on insomnia and pain (CBT-IP) was compared with no treatment, wait list or attentional control , 53–55. All 4 studies reported a pain outcome post-treatment Fig.  44, 53––53–55. T2 75%).In 4 studies with 415 people randomised, CBT-IP was compared with a control condition that included cognitive behavioural therapy for pain (CBT-P) with no specific focus on sleep , 54, 55.In 1 study at unclear risk of bias due to limited reporting of methods with 95 people with fibromyalgia randomised, hypnosis additional to CBT-IP was evaluated . CompareIn 1 study, 299 people with chronic pain were randomised to a 7-week course of acceptance and commitment therapy based stress management, or to a control discussion group of similar intensity and duration, or to group-based exercise . The stup = 0.008, and this was consistent at 3 [In 2 studies including 239 people with fibromyalgia pain, a mindfulness-based intervention was compared with waiting list controls , or CBT ent at 3 , and 6 ment at 3 . For paiIn 1 study with 53 people with fibromyalgia pain, group and individualised applied relaxation was compared with a sleep hygiene-based educational intervention, and a wait list control . Risk ofFurther to the studies comparing sleep hygiene intervention with CBT-I , 50–52, p = 0.39 and this was consistent in the study at low risk of bias. There was no difference in 1 study with sleep measures at 6 months [p < 0.001, but not trouble falling asleep or staying asleep, waking up at night, or trouble staying asleep. Benefit was not maintained at 3 or 6 months. In one study including 53 people with fibromyalgia, the authors reported that sleep quality was improved in the group receiving group-based exercise compared with controls, p = 0.051 [In 5 studies with 697 people randomised, exercise programmes were compared with usual care or an attentional control , 60–63. 6 months . One stu = 0.051 , 60.p < 0.00001 [p = 0.039 [n = 1) and increase in knee pain (n = 1).Pain in 3 studies was reduced in the exercise group compared with controls, SMD -0.52 ,  0.00001 , 60, 61, = 0.039 , 60. In  = 0.039 , 60. The = 0.039 . Adverse = 0.039  with 2 eIn a feasibility study with 60 people with chronic low back pain randomised, a walking intervention was compared with supervised exercise and controls . Risk ofIn 2 studies, different exercise modalities were compared , 65.In a pilot study, 40 people with multiple sclerosis pain were randomised to a clinic-based moderate-intensity aerobic exercise programme or low-intensity home-exercise programme . ModeratA course of aquatic biodance was compared with a course of stretching exercises in 1 study with 76 people with fibromyalgia pain . The ranp = 0.32 and this was consistent in the study at low risk of bias. No heterogeneity was evident. Also in the study at low risk of bias, sleep efficiency measured by questionnaire and diary was improved in people receiving Tai Ji Quan [In 2 studies with 90 people randomised, Tai Ji Quan was compared with an education intervention , 67. Chr Ji Quan , but thePhysical therapy modalities were evaluated in 6 studies.Two studies described treatment comparisons including hydrotherapy , 69. In n = 2) and chlorine hypersensitivity (n = 1).A course of hydrotherapy was compared with conventional physiotherapy in 1 study at low risk of bias with 50 people with fibromyalgia randomised . Post-trA course of massage therapy was compared with relaxation therapy in 1 study with 30 people with low back pain . ReportiIn 1 study with 89 people with fibromyalgia pain randomised, a course of manual therapy was compared with no treatment . The stuIn 1 study including 60 people with fibromyalgia, a physical therapy programme with hot pack, ultrasound, transcutaneous electrical nerve stimulation and low power laser was compared with an untreated control group . Risk ofA course of pompage was compared with controls in 1 study with 23 people with fibromyalgia pain randomised . All parAuricular point acupressure was compared with sham auricular point acupressure in 1 study including 61 people with chronic low back pain . Risk ofIn 1 study, visor delivered bright light treatment was compared with sham therapy in 19 people with fibromyalgia pain randomised in a crossover trial . The stuIn 1 study, 68 people with pain from rheumatoid arthritis were randomised to a course of foot reflexology or control . Risk ofOne study compared transcranial stimulation with sham in 16 patients with musculoskeletal pain . This waIn 1 study at low risk of bias with 30 people with fibromyalgia pain randomised, a period of sleep on a magnetic mattress pad was compared with sham . Sleep, We identified 42 randomised trials evaluating sleep interventions for people with chronic pain. CBT interventions provided the largest evidence base with CBT-I interventions demonstrating benefit post-treatment compared with controls for improved sleep quality, however evidence for a longer-term sustained benefit was lacking. Evidence in people with specific conditions was limited due to primary study limitations and statistical heterogeneity. Numerous interventions were evaluated in small numbers of studies, but evidence was insufficient to draw conclusions on effectiveness.Findings from meta-analysis found that CBT-I (9 studies) and CBT-IP (4 studies) demonstrated a medium to large (− 0.79) effect compared with control for sleep quality at post-treatment. Differences between groups at 6 months was slightly reduced with a medium effect size for CBT-I and was not sustained for CBT-IP. CBT-I showed a small improvement in pain outcome post-treatment but this was not sustained. However, there was high heterogeneity in studies which should be considered when interpreting results. In 4 studies comparing CBT-IP with CBT-P only, no differences in pain outcome were found. Due to the active control it is therefore only possible to infer that adding insomnia specific content to CBT-P does not have an additional impact on pain outcomes. Our findings regarding the impact of CBT-I interventions on sleep quality reflect the existing literature. A recent systematic review of CBT-I therapies in patients with chronic non-malignant pain showed significant treatment effects immediately post-treatment for global measures of sleep . ConditiOur results demonstrate that improving sleep for people with chronic pain is possible, and that CBT approaches have the strongest evidence base. Poor sleep has a negative impact on optimism, sociability, and psychosocial functioning . Poor slEvaluation of the longer-term effectiveness of sleep interventions for people with chronic pain is lacking. Although evidence suggests that CBT interventions improve sleep immediately post treatment, effects reduce over time. In addition, most studies had follow-up data collected at 3 months or less post-intervention, with 9 studies reporting 6-month outcomes, and 2 studies reporting 12-month outcomes. Understanding longer-term effectiveness of these interventions is crucial for people with chronic pain. Due to the nature of the condition, individuals with chronic pain may experience disturbed sleep for many months and years, therefore effective interventions need to have sustained effects.Assessment of outcomes within the trials included in this review varied considerably, and this was particularly notable for the secondary outcomes. This heterogeneity limits comparison between studies, particularly for health-related quality of life and psychological wellbeing, because outcome measures assess different aspects of these constructs, for example general mood assessment versus specific anxiety or depression measures. The issue of heterogeneity across randomised trials is well established and initiatives such as COMET have been addressing this through the development of core outcome sets , 91. CorNine of the 42 studies in this review provided data on adverse events with no serious adverse events reported , 75, 79.This review has strengths and limitations that should be considered when interpreting the findings. The method used in this review was robust and systematic, following Cochrane guidance . The revCBT approaches have the potential to be an effective treatment to improve sleep for people with chronic pain, but further high-quality primary research is required to explore refinements that will ensure parallel improvements to pain, quality of life, psychological health and maintain all benefits in the long term. Individuals who experience depression and pain catastrophising may particularly benefit from sleep interventions. As CBT is an established treatment approach, future work should focus on how best to deliver these interventions, for instance by exploring any difference between online or face-to-face delivery or differences between delivery professionals. Importantly, future research could focus on how best to facilitate equal access and outcomes in underserved populations.Primary research is also needed to evaluate the effectiveness of interventions including mindfulness, aquatic exercise and hydrotherapy, Tai Ji Quan, manual therapy, physical therapy programmes, acupressure, foot reflexology and magnetic mattress pads. Individuals who experience chronic pain could benefit from interventions that address sleep, and research is needed to assess any impact of sleep interventions on pain.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


The aim of this study was to evaluate the effect of different intensities of tillage on the physiological characteristics and productivity of plants in crop rotation. Five tillage practices (DP—deep ploughing (22–24 cm); SP—shallow ploughing (16–18 cm); SH—shallow harrowing (8–10 cm); DH—deep harrowing (14–16 cm); and DD—direct drilling) were investigated in a long-term experiment in Dotnuva. The crop rotation was as follows: winter oilseed rape → spring wheat → spring barley → field pea → winter wheat. The simplification of conventional tillage negatively affected the photosynthetic indices of the majority of the crop rotation plants. The most favorable conditions for the photosynthetic processes in the plants were identified in the deep-ploughing treatment. The photochemical activity was negatively influenced and leaf senescence was accelerated under direct drilling. Direct drilling significantly decreased the grain yield of winter oilseed rape, spring wheat, and spring barley by 10.5%, 12.8%, and 17.2%, respectively, compared to deep ploughing. The grain yield of winter wheat was similar under deep ploughing and direct drilling; conversely, under shallow ploughing, shallow harrowing, and deep harrowing, the yield tended to decrease compared to deep ploughing. The tillage system influences the physical, chemical, and biological properties of soil ,2,3. SimTillage promotes root development . The extHowever, the impact of tillage intensity on crop physiological traits under field conditions in the Boreal region has not yet been investigated and understood. We hypothesized that by evaluating the photosynthetic activity of foliage and the differences in its parameters depending on the tillage method applied under field conditions, it would be possible to determine the most favorable conditions for photosynthetic processes in terms of tillage intensity. Our aim was to study the impact of different intensities of tillage on plant physiological traits under crop rotation.p ≤ 0.01) and tillage system   ( ≤ 0.01) . Growth p ≤ 0.01.GS (factor A) was responsible for 74.9%, 70.9%, 45.7%, and 63.6% of the total variance in Fv/Fm in winter oilseed rape, spring wheat, field pea, and winter wheat, respectively. In spring barley, the influence of GS was lower (14.7%) but still statistically significant at The dynamics of the SPAD and Fv/Fm values measured at the five different growth stages, averaged across tillage types, are provided in Fv/Fm values varied according to the GS . In fielIt was found that the influence of tillage intensity (factor B) on the physiological indices was not very strong and significant in only a few tested cases . TillageThe effect of interactions between factors A × B on SPAD and Fv/Fm was insignificant in all tested cases.In terms of SPAD, winter crops reacted to tillage intensity more strongly than spring crops . In compField pea was the most sensitive of the spring crops to the simplification of the tillage. All simplified-tillage treatments had a negative effect on SPAD for field pea. For DD, SPAD significantly decreased by 4.5% in comparison to DP. The application of DH also significantly decreased SPAD, by 3.7% compared to DP. SP and shallow harrowing (SH) decreased SPAD by 3.2 and 2.6%, respectively, compared to DP.In spring wheat, SPAD significant decreased by 3.2% under DH in comparison with DP.The influence of simplified tillage on Fv/Fm was negligible, with one exception: in spring wheat, Fv/Fm significant decreased by 4.3% under DD compared with DP .In this study, leaf senescence was evaluated through the dynamics of the SPAD and Fv/Fm indices in the final GSs of the tested crops. We found that in the final GSs, i.e., after the flowering stage (BBCH 69), the photosynthetic indices decreased in all of the tested crops . In compThe response of the spring crops to tillage intensity differed to that of the winter crops. In spring wheat and field pea, DD increased SPAD for most of the measurements in the final growth stages compared with DP . After tThe evaluation of the green-leaf number and loss dynamics in the final growth stages of the crops showed that the spring crops’ response to tillage simplification was less pronounced than that of the winter crops . AccordiIn the winter crops, DD had the most pronounced negative influence on leaf senescence compared with DP. The application of SH in winter oilseed rape also decreased the number of leaves at BBCH 77 and BBCH 79. In winter wheat under SP, we also recorded noticeable leaf senescence from BBCH 73 until BBCH 85 compared with DP.Increasing the efficiency of the photosynthesis process and its adaptation to changeable environmental conditions is an important agronomic objective ,23. UndeThe maximum quantum efficiency of PSII photochemistry (Fv/Fm) is mostly used to identify the efficiency of the photosynthetic apparatus . It is aLeaf senescence is an integrated response to age, developmental status, and environmental conditions . Leaf seThe effects of tillage on crop yield has been widely investigated ,3,5,8,37We found that tillage simplification (SH and DH) significantly enhanced the oil content of winter oilseed rape, but that SH, DH, and DD had a negative and significant effect on the protein content of winter wheat. DH and DD significantly decreased the protein content in both spring wheat and spring barley compared with DP. Our findings were in line with those of Ali et al.  and KuliTillage is an important factor affecting soil moisture, soil temperature, and nutrients ,21,28,39There is still lack of knowledge on the effect of tillage on plant physiological indices and their relationship with productivity. Some studies indicate that soil compaction under minimal tillage negatively influences the physiological traits of maize, including chlorophyll content . Kulig ep ≤ 0.01) and positively correlated with grain yield, and the strength of this relationship was similar under all tillage intensity levels. On the contrary, other researchers found that the correlation between SPAD and wheat grain yield was not significant [We ascertained a correlation between physiological traits, crop yield, quality indices, and soil temperature averaged across crop rotations under different tillage intensities . The datnificant  or was anificant .p ≤ 0.01) and positively correlated with protein content under DP, SP, and DH tillage. The correlation between SPAD and TGW was negative and significant (p ≤ 0.01) in all cases.We found that SPAD was significantly (p ≤ 0.01) relationship was found with HLM under all tillage regimes except DD.Fv/Fm was not correlated with crop yield, protein content, or TGW; however, a significant (p ≤ 0.01) with soil temperature in most of the tested cases. Under all tillage intensity levels, no relationship was found between Fv/Fm and soil temperature. Increasing air temperature and soil temperature and changes in precipitation patterns as a consequence of climate change affect crop production in agricultural ecosystems . Muñoz-REndocalcari-Epihypogleyic Cambisol. The loam soil was close to neutral ; medium rich in humus ; rich in available phosphorus ; and rich in available potassium .A long-term field experiment was carried out at the Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry in Central Lithuania (55°23′50″ N and 23°51′40″ E) over five growing seasons, 2012–2016. The soil of the experimental site was classified as an The field experiment was arranged in four blocks (replications) with five tillage treatments of different intensities: DP—deep ploughing (22–24 cm); SP—shallow ploughing (16–18 cm); SH—shallow harrowing (8–10 cm); DH—deep harrowing (14–16 cm); and DD—direct drilling. Each tillage plot was 10 m wide and 21 m long. The crop rotation was as follows: winter oilseed rape—spring wheat—spring barley—field pea—winter wheat. The crop husbandry details are provided in Leaf chlorophyll index (SPAD) was measured non-destructively with a portable Minolta SPAD 502 Chlorophyll Meter . The measurements were made in the middle part of fully expanded, randomly selected leaves of 40 plants per treatment (10 plants per plot x 4 blocks). SPAD measurements were carried out from 10 am until 2 pm  on clear days five times for all crop-rotation plants . Growth To obtain the maximum quantum efficiency of PSII photochemistry (Fv/Fm), a multi-functional pulse-modulated handheld chlorophyll fluorometer  was used to measure chlorophyll-α fluorescence in vivo. Fv/Fm was read directly on the chlorophyll fluorometer after a short period of adaptation to the dark . Leaves A total of 3 plants in each plot were randomly selected and marked with a bright strip. The same leaves were assessed at each measurement timepoint until they withered. The green-leaf number and loss dynamics were evaluated. SPAD and Fv/Fm were measured in the 1st, 2nd, and 3rd leaves from the top. The data were collected during the final growth stage of the crops .The soil temperature was measured using a digital long-stem thermometer . The soil temperature at the 5 cm soil layer was measured at the same growth stages  and time2. Grain/seed yield as t ha−1 was adjusted to 14% moisture content for cereals and field pea and to 9% moisture content for winter oilseed rape.The plots were harvested at complete maturity with a plot harvester . The harvested area totaled 36.9 mThe thousand grain weight (TGW) was counted with a Contador seed counter  from four samples of 250 seeds per plot. Protein content and oil content from each plot were measured using an Infratec 1241 grain analyzer .p ≤ 0.05 and p ≤ 0.01 probability levels. Standard statistical procedures were used for calculating simple correlation coefficients among physiological traits, yield, quality indices, and soil temperature under different tillage systems. The statistical analysis was carried out using STAT ENG software for Excel version 1.55 from the statistical data-processing package SELEKCIJA.Two-way ANOVA was used to determine the effects of growth stage and tillage system on the physiological indices. Fisher’s test was also used. Statistical significance was evaluated at the Rainfall and mean air temperature data over the five growing seasons  are provided in HTC > 1.6 indicates excessive irrigation; HTC = 1.0–1.5 optimal irrigation; HTC = 0.9–0.8 weak drought; HTC = 0.7–0.6 moderate drought (arid); HTC = 0.5–0.4 heavy drought; and HTC < 0.4 very heavy drought .Rainfall differed between the growing seasons, and the amount of rainfall totaled 370 mm (HTC = 1.6), 288 mm (HTC = 1.2), 350 mm (HTC = 1.6), 191 mm (HTC = 1.0), and 382 mm (HTC = 1.3) in the 1st, 2nd, 3rd, 4th, and 5th seasons, respectively.The results of the five-year study revealed that the simplification of traditional tillage had a varied influence on the photosynthetic indices of most of the crop-rotation plants. Direct drilling (DD) tended to reduce the Fv/Fm values in field pea, winter wheat, winter oilseed rape, and spring wheat leaves. Under shallow ploughing (SP), in most tested cases, Fv/Fm decreased in winter wheat, spring wheat, and winter oilseed rape. In terms of SPAD, winter crops responded more strongly to tillage simplification than spring crops. Compared to deep ploughing (DP), all simplified-tillage systems significantly reduced the SPAD values in winter wheat. In winter oilseed rape, direct drilling (DD) and shallow harrowing (SH) significantly reduced SPAD, while shallow plowing (SP) and deep harrowing (DH) decreased SPAD. Compared to DP, SP and DD significantly reduced SPAD in field pea. In spring wheat, DD had a significant positive effect on SPAD values. DD accelerated the senescence process in winter crops.The differences in the photosynthetic activity indicators depending on the applied tillage types suggested that the most favorable conditions for photosynthetic processes in plants were provided by DP.In comparison with DP, DD application significantly decreased the grain yield of winter oilseed rape, spring wheat, and spring barley by 10.5%, 12.8%, and 17.2%, respectively; meanwhile, the grain yield of field pea decreased under DD, but the difference was insignificant. The grain yield of winter wheat was similar under DP and DD, whereas SP, SH, and DH tended to decrease the yield compared with DP.


Arthrospira platensis contains abundant bioactive ingredients, which are beneficial for animal health. This study was conducted to investigate the effects of A. platensis on pigmentation, antioxidant capacity, and stress response after air exposure of fish. A total of 120 yellow catfish Pelteobagrus fulvidraco  were divided into three tanks per treatment and fed diets supplemented with 0 g kg−1 A. platensis (CON) and 20 g kg −1 A. platensis (AP) for 65 days. The results indicated that dietary A. platensis had no effects on the growth of yellow catfish. The AP diet significantly reduced lactic acid (LD) and cortisol levels stimulated by air exposure stress (p < 0.05). Dietary A. platensis significantly increased plasma superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities and glutathione (GSH) contents, and the relative expression levels of sod and cat, to protect against oxidative stress caused by air exposure (p < 0.05). The AP diet significantly improved the relative expression level of nrf2 (nuclear factor erythroid-2 related factor 2), while the relative expression level of keap1 (kelch-like ECH associated protein 1) was downregulated, and the protein levels of liver Nrf2 were significantly increased after air exposure stimuli (p < 0.05). Dietary A. platensis significantly increased skin lutein contents, increased skin redness, yellowness and chroma (p < 0.05), and improved body color abnormalities after oxidative stress caused by air exposure stimuli. Skin yellowness was associated with lutein contents and the expression levels of some antioxidant genes to varying degrees. Overall, dietary A. platensis could be utilized as a feed additive to activate the antioxidant response, as well as alleviate oxidative stress and pigmentation disorder induced by air exposure.In aquaculture, fish are often exposed to several stress conditions, which will cause oxidative disorder and bring about health and quality problems.  Stress is a general term that applies to a situation in which a person or an animal is subjected to a challenge that may result in real or symbolic danger to its integrity . In aquaAir exposure is a common acute stressor with a subsequent physiological disturbance in aquaculture since handling procedures during animal husbandry and the production cycle require exposing fish to air environments . Air expArthrospira platensis, a blue-green alga that is gaining worldwide popularity as a food supplement, contains high contents of protein, polyunsaturated fatty acids, vitamins, and minerals [A. platensis is plenitudinous in antioxidant compounds such as carotenoids, phycocyanin, and tocopherols [A. platensis can enhance antioxidant and immunity abilities as well as ensure skin pigmentation in fish [minerals . In addiopherols . In prev in fish ,16,17.Pelteobagrus fulvidraco is a commercially important fish species in aquaculture in East and South Asia because of its excellent meat quality and perfect flavor [A. platensis-supplemented diet and exposed to air as an experimental stressor causing oxidative stress. In addition, we also investigated the effect of A. platensis on the pigmentation of fish and detected the correlation between skin color and skin lutein content and oxidative stress. We are expecting that dietary A. platensis can reduce the stress responses and bring welfare to fish during fish farming.Yellow catfish t flavor ,19. Howe−1 diet) and isolipidic (80 g crude lipid kg−1 diet) diets were designed as practical diets, using white fishmeal, soybean meal, and rapeseed meal as blended protein sources (A. platensis) and an A. platensis group . All ingredients were completely mixed with appropriate water and then made into pellets using an SLP-45 laboratory granulator . The diets were oven-dried at 60 °C for 12 h and stored at 4 °C until used for experimentation. Moisture, crude protein, crude lipid, and lutein contents of diets were analyzed following the same procedures as the previous study [v/v/v/v) and analyzed by HPLC.Two isonitrogenous  were randomly selected, batch weighed, and placed into 6 fiberglass tanks . The experimental fish were hand-fed to apparent satiation twice daily (8:30 and 16:30) for 65 days. Each tank was provided with continuous aeration. During the experiment, the water temperature was maintained at 30.5 ± 1.0 °C. Total ammonia-nitrogen was maintained at <0.1 mg L−1, dissolved oxygen at >6 mg L−1, and residual chloride at <0.01 mg L−1. The light period was from 8:00 to 20:00, and the light intensity was approximately 2.20–3.70 μmols−1m−2 (at water surface).All experimental animal care protocols were approved by the ethics committee of the Institute of Hydrobiology, Chinese Academy of Sciences. Yellow catfish were obtained from the Dengjia State fish farm . Two weeks before the feeding trials, all fish were temporarily domesticated in a fiberglass cylinder (1500 L) and fed twice a day at 08:30 and 16:30. Feeding trials were conducted in an indoor recirculating system. At the beginning of each trial, the fish fasted for 24 h. Healthy and similarly sized fish  and then measured coloration and sampled for blood, liver, kidney, dorsal, and abdominal skin tissues. Blood samples were collected from the caudal vein with heparinized syringes. After centrifugation , plasma was collected and stored at −80 °C for further analysis. After blood sampling, the liver, kidney, dorsal, and abdominal skin were dissected on ice and stored at −80 °C.At the end of the feeding trial, the experimental fish were batch-weighed. Three fish from each tank were randomly selected and anesthetized with 80 mg LAt the end of the trial, three fish were randomly selected from each tank for a stress challenge. Fish were exposed to air for 5 min and then sampled for blood, liver, and kidney. The methods of plasma collection and sample storage were the same as those for normal sampling (without air exposure).2 + b*2)1/2 [The dorsal and abdominal skin color parameters of the fish were measured by a Konica Minolta CR-400 tristimulus colorimeter . The L* value represents lightness , a* represents the red/green dimension, and b* represents the yellow/blue dimension, the value of chroma C is the distance from the lightness axis (L*) and starts at 0 in the center, in accordance with the recommendations of the International Commission on Illumination . Chroma  b*2)1/2 . Fish frv/v), the lutein standard  was diluted into 100, 50, 20, 10, 5, 2, 1, 0.5, 0.1 µg/mL with the same solvent system. The obtained solution was used for HPLC  analyses immediately after passing through a 0.22 µm membrane filter. The HPLC was equipped with a Waters YMC Carotenoid C30 column ; the mobile phase consisted of solvent A (methanol: methyl-tert-butyl ether: H2O = 81:15:4) and solvent B (methanol: methyl-tert-butyl ether = 600:90). The gradient procedure was performed at a flow rate of 1 mL/min. The total run time was 40 min, and the injection volume was 10 µL.The extraction of lutein from the skin was determined by following the method described previously . Skin saThe contents of lactic acid (LD), cortisol, glucose (GLU), malondialdehyde (MDA), and reduced glutathione (GSH) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were tested using commercial kits .® ND-2000 UV–Vis Spectrophotometer . The total RNA was then reverse-transcribed with an M-MLV FirstStrand Synthesis Kit . The polymerase chain reaction (PCR) primer sequences for sod, gpx, gr, cat, nrf2, keap1, and the reference gene β-actin, which were designed based on the cDNA sequences of yellow catfish, are shown in ® Green I Master Mix  was used to perform quantitative RT–PCR. Each sample was run in duplicate, and the relative expression was calculated [Total RNA was extracted from the liver and kidney using TRIzol reagent according to the manufacturer’s instructions . The quality of total RNA was evaluated by 1% agarose gel electrophoresis. The purity and concentration were assessed by a NanoDroplculated .Liver tissues were lysed with RIPA lysis buffer  containing protease inhibitor cocktail and phosphatase inhibitor cocktail . Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked for 1 h with 5% milk in TBST buffer and then incubated overnight at 4 °C with Nrf2  and β-actin  antibodies. β-actin was used as an internal reference protein. Horseradish peroxidase-labelled secondary antibodies were used to generate a chemiluminescent signal that was detected by ImageQuant LAS 4000 mini  and quantified using ImageJ software .p < 0.05.All data were statistically analyzed with SPSS 19.0 and subjected to one-way ANOVA. Before any statistical analysis, normality and homoscedasticity assumptions were confirmed. Duncan’s multiple range test was used to detect the significance of differences in mean values among different treatments. The results are presented as the mean ± standard error (SEM). The Pearson’s correlation coefficient was calculated to analyze the significance of linear relationships between skin yellowness (b*) and the studied variables. The significance difference level was set at The survival rate of fish was 100% in both groups during the feeding trial; there were no significant differences in final body weight (FBW), specific growth rate (SGR), feeding rate (FR), or feed efficiency (FE) in either the CON or AP groups . At the p < 0.05, p < 0.05, Stress response markers such as LD, cortisol, and glucose were significantly increased after air exposure stress in fish fed the CON diet (p < 0.05). The contents of plasma GSH were significantly decreased in the CON group after air exposure stimuli (p < 0.05), while the AP diet significantly improved GSH contents to protect against oxidative stress caused by air exposure (p < 0.05). The MDA content showed no obvious changes.To test whether dietary AP affected fish antioxidant capacity, we analyzed antioxidant-related parameters in plasma. As shown in sod and gpx were significantly downregulated in the CON group after air exposure stimulation (p < 0.05), and there were no obvious differences in the relative expression levels of liver gr and cat between CON and AP groups after air exposure stimulation.As presented in nrf2 in the liver was significantly downregulated in the CON group after air exposure stimulation (p < 0.01), while the AP diet significantly improved the relative expression level of nrf2 to protect against oxidative stress caused by air exposure stimuli , while the AP diet significantly reduced the relative expression level of keap1 to protect against oxidative stress caused by air exposure  but did not significantly affect the relative expression levels of gr in both groups after air exposure stimulation (The relative expression levels of mulation .nrf2 in the kidney was significantly downregulated in the CON group after air exposure stimuli (p < 0.01), while the AP diet significantly improved the relative expression level of nrf2 to protect against oxidative stress caused by air exposure stimuli , while the AP diet significantly reduced the relative expression level of keap1 to protect against oxidative stress caused by air exposure , and air exposure led to a significantly decreased abdominal skin redness value (p < 0.05). Skin yellowness and chroma were significantly higher in catfish fed the AP diet at the end of the feeding trial (p < 0.05). In the CON group, the abdominal skin yellowness and chroma values of fish were significantly increased after air exposure, while the abdominal skin yellowness and chroma values of fish were significantly decreased in the AP group after air exposure (p < 0.05), and fish fed the AP diet had clearer black spots and saturated yellow coloration both before and after air exposure.As shown in p < 0.05, p < 0.05, sod and gr relative mRNA levels, while dorsal skin yellowness presented a very positive and significant correlation with liver sod and gr relative mRNA levels , while positive but nonsignificant correlations were observed between dorsal skin yellowness and kidney gpx relative mRNA levels  of dry matter and protein up to 70% and 90%, respectively [A. platensis in yellow catfish diets.The yellow catfish is an omnivorous freshwater fish , which, us study . The preformance . We demoectively . The aboA. platensis supplementation was effective against air exposure-induced stress responses by reducing plasma cortisol and lactic acid levels. In addition, the reduction of stress markers was dose-dependent on the A. platensis supplement [Cortisol is regarded as a primary physiological stress response in fish , and glupplement .A. platensis increased the activities of SOD and GPX and GSH contents to protect against oxidative stress caused by air exposure stimuli. A. platensis enhanced antioxidant enzyme activities to protect fish from stress [A. platensis might work to activate antioxidant response [The antioxidant enzymes SOD and GPX and the nonenzymatic antioxidant GSH play important roles in free radical scavenging to protect against oxidative damages . In our m stress ,33. β-caresponse ,35,36,37nrf2 transcription is inhibited by binding to keap1; when the keap1/nrf2 complex is activated, nrf2 is translocated into antioxidative factors in the nucleus, where it can regulate antioxidant enzymes, such as sod, cat, and gpx [nrf2 after air exposure were efficiently upregulated by A. platensis supplementation. Concurrently, the expression levels of sod and cat were obviously activated to improve the antioxidant status of yellow catfish, which is in accordance with a previous study [A. platensis involves various proteins, which could have biological activities to induce nrf2 stabilization and antioxidative enzymes [A. platensis, polyphenol, has been found to modulate Nrf2-mediated antioxidant events [A. platensis can activate the Nrf2 signaling pathway and may be advantageous for its use as an antioxidant nutritional supplement in aquatic animals.Nrf2 plays a critical role in antioxidative defense responses . Usually and gpx ,40,41. Ius study . A. plat enzymes . Additiot events ,44. HencA. platensis supplementation enhanced the body color of fish at the end of the experimental trial in accord with previous studies [A. platensis is considered a carotenoid-producing organism, and lutein and zeaxanthin are mainly found in A. platensis [A. platensis can also enhance the natural mucus layer, which is responsible for maintaining the glowing appearance of skin in fish [A. platensis is a proper color enhancer. studies ,46. It i studies . A. platlatensis ,49. The  in fish . The preA. platensis addition promoted increased lutein contents in the skin, and we demonstrated that lutein is mainly deposited in the skin of yellow catfish earlier [In this study,  earlier . This st earlier .Silurus glanis showed more obvious behavioral and physiological responses to short-term stress induced by a combination of air exposure and novel environmental stressors than pigmented fish [A. platensis improved body color abnormalities after oxidative stress caused by air exposure stimuli, and skin yellowness was associated with the expression levels of some antioxidant genes to varying degrees. The results may suggest that the antioxidant components (such as lutein) in A. platensis successfully help fish defend against body color disorders caused by oxidative stress.Skin color change has the potential to be a useful real-time indicator of stress . Our rested fish . In this−1 dietary A. platensis had no negative effects on the growth of fish but could reduce LD and cortisol levels, increase the activities of antioxidant enzymes, upregulate the expression levels of certain antioxidant-related genes, and regulate the Nrf2 signaling pathway, which aimed to protect against oxidative stress caused by air exposure stimuli. In addition, A. platensis could alleviate body color disorder caused by air exposure stress by increasing skin lutein contents. In terms of cost-benefit, the inclusion of 20 g kg−1 dietary A. platensis can enhance the ability of antioxidants and improve the body color of yellow catfish, as a result, the risk of suffering disease and farming losses will be reduced and market value will be enhanced. Moreover, in our previous study, the A. platensis can replace fishmeal in the diets of yellow catfish [A. platensis could be a potential protein source to replace fishmeal in the future.Our overall results demonstrated that 20 g kg


Senna occidentalis (L.) Link has been used worldwide in traditional treatment of many diseases and conditions including snakebite. In Kenya, a decoction from the plant roots taken orally, is used as a cure for malaria. Several studies have demonstrated that extracts from the plant possess antiplasmodial activity, in vitro. However, the safety and curative potency of the plant root against established malaria infection is yet to be scientifically validated, in vivo. On the other hand, there are reports on variation in bioactivity of extracts obtained from this plant species, depending on the plant part used and place of origin among other factors. In this study, we demonstrated the antiplasmodial activity of Senna occidentalis roots extract in vitro, and in mice.S. occidentalis root were tested for in vitro antiplasmodial activity against Plasmodium falciparum, strain 3D7. Cytotoxicity of the most active solvent extracts was determined using 3--2,5-diphenyltetrazolium bromide (MTT) assay and the curative potency in Plasmodium berghei infected mice evaluated by Rane’s test.Methanol, ethyl acetate, chloroform, hexane and water extracts of P. falciparum, strain 3D7, in vitro, with polar extracts being more active than non-polar ones. Methanolic extracts had the highest activity (IC50 = 1.76) while hexane extract displayed the lowest activity (IC50 = 18.47). At the tested concentrations, methanolic and aqueous extracts exhibited high selectivity index against P. falciparum strain 3D7 (SI > 10) in the cytotoxicity assay. Further, the extracts significantly suppressed the propagation of P. berghei parasites (P < 0.05) in vivo and increased the survival time of the infected mice (P < 0.0001).All of the solvent extracts tested in this study inhibited the propagation of Senna occidentalis (L.) Link root extract inhibits the propagation of malaria parasites in vitro and in BALB/c mice.The online version contains supplementary material available at 10.1186/s12906-023-03854-8. Malaria continues to be a public health challenge particularly in Sub-Saharan Africa , 2. TherP. falciparum malaria in various parts of the world. However, Plasmodium malaria resistance to current first line drugs, including artemisinin, has been reported [Currently, the World Health Organization (WHO) recommends combining artemisinin and its derivatives with other antimalarial drugs in combination therapy , 9, 10. reported , 10–14. Senna occidentalis (L.) Link is a widely used plant in traditional medicine throughout the world [Medicinal plants are a rich source of untapped antimalarial activities , 4, 15. he world . This plhe world –21. In the world . The plahe world –25.Senna occidentalis root decoction is taken orally three times daily for 3 to 4 days by the Luhya, Digo and Duruma communities for treatment of malaria [S. occidentalis extract is active against P. falciparum. In one of the studies, S. occidentalis inhibited the growth of chloroquine sensitive (strain 3D7) and resistant (strain Dd2) P. falciparum with half-maximal inhibitory concentration (IC50) values of 65 μg/ml and 110 μg/ml respectively [P. falciparum (IC50 < 3 μg/ml), in vitro. In yet another study, S. occidentalis extract showed in vitro activity against chloroquine sensitive P. falciparum, strain 3D7, (IC50 = 48.80 μg/ml) and the resistant parasites, stain INDO, (IC50 = 54.28 μg/ml) [P. falciparum (3D7 strain) with an IC50 value of 3.37 μg/ml. The variation in IC50 values obtained in these studies could be due to differences in geographical origin of the plant, parts of the plant used, season of the year when plant material collection was done, methods of preparation and storage [P. falciparum, in vitro and P. berghei, in vivo.In Kenya,  malaria –28. Prevectively . In a diectively , an etha8 μg/ml) . In a mo8 μg/ml) , an extr storage , 32, 33, storage –36. ThisApproval to conduct this study was granted by the Institute of Primate Research (IPR) Institutional Scientific and Ethics Review Committee (ISERC/02/18). The plant material used in this study was collected according to the WHO guidelines on Good Agriculture and Collection Practices (GACP) for medicinal plants . The hanSenna occidentalis roots were collected from Migori county , Kenya in the months of August and September. This plant was identified by Mr. Jonathan Ayayo, a taxonomist of the National Museums of Kenya (NMK), and a voucher specimen (38/81) deposited at East African Herbarium of NMK for future reference. Further, the plant name was verified with http://www.theplantlist.org on 10/05/2022.The collected plant roots were air dried in the shade, ground into powder and stored in airtight plastic containers at 4 °C until extraction.Hexane, chloroform, ethyl acetate and methanol, in their absolute forms, as well as distilled water were used for extraction by maceration. For organic solvents, the plants root powder was macerated separately with the solvents for 48 hours in an orbital shaker and a filtrate obtained (Whatman No. 1 filter paper). For water, the roots powder was soaked in double distilled water for 24 hours. In addition, a decoction was prepared by boiling the roots powder in double distilled water for 30 minutes at a mean temperature of 95 °C . FiltratS. occidentalis roots extract, .standard procedures were used to screen the extracts for the presence of saponins, tannins, alkaloids, flavonoids and sterols [Plant secondary metabolites are excellent predictors of their bioactivity potential . In orde sterols .Senna occidentalis root extracts were tested in vitro for antiplasmodial potency using P. falciparum, strain 3D7 obtained from Kenya Medical Research Institute (KEMRI) repository. A modified version of a previously used method [2, 5% O2 and 5% CO2. The culture was refreshed every 48 hours at 3% haematocrit.d method  was utilDilutions of the extract and standard drug were carried out in the same manner as described , with miP. falciparum malaria parasites by S. occidentalis root extract was assessed as described [. Non-infected RBCs as well as DMSO were also included in the assay as controls. The plates were incubated at 37 °C. After 72 hours, thin smears were prepared and parasites quantified by light microscopy. Percentage parasitemia suppression was computed and 50% inhibitory concentration (IC50) values determined for each extract [Growth inhibition of escribed , with mi extract , 46.S. occidentalis root extract, Vero cell line (sourced from KEMRI) was grown to confluent monolayer in Minimum Essential Medium Eagle (MEM) containing sodium bicarbonate and l-glutamine. The growth medium was supplemented with 1% pen-strep (Sigma), 1% HEPES (Gibco) and 10% Fetal bovine serum (FBS). The assay was performed as previously described [2 incubator to allow the cells achieve a layer of > 90% confluence. The cells were exposed to 100 μl of the extracts for 48 hours, in concentration ranges of 250–3.91 and 25–0.39 μg/ml for the extracts and pyrimethamine, respectively. Pyrimethamine and DMSO (0.4%) exposed, as well as untreated cells were included as controls. Following 48 hours’ incubation period, 10 μl of MTT reagent (5 mg/ml) was added aseptically to each well, tapped gently to mix and incubated for 3 hours at 37 °C. All media was then aspirated from the wells and 100 μl DMSO added. Optical density (OD) readings as obtained by an ELISA reader were then used to compute the extracts’ 50% cytotoxicity concentration (CC50) and selectivity index (SI) as described elsewhere [To assess the in vitro cytotoxicity of escribed , 48. Brilsewhere .In this study, male and female inbred BALB/c mice aged 8–9 weeks were used. The rodent facility at IPR provided the animals. They were housed in standard Macron type II cages within 12 hours’ dark/light cycle at 23 °C. Food and water was provided ad libitum according the IPR’s Animal Science Department standard operating procedures.Plasmodium berghei, strain ANKA (sourced from KEMRI) was retrieved from the IPR repository and maintained in mice [P. falciparum infection [P. berghei and the human malaria parasites possess conserved genes that have over time allowed their use in Peters’ model of antimalarial drug efficacy testing [ in mice  for use nfection . Despite testing .P. berghei in mice [6P. berghei infected erythrocytes and assigned randomly to 5 experimental groups; A, B, C, D and E (5 mice per group). At approximately 5% parasitemia (day 5 post-infection), treatment was started. Groups A and B were treated with 200 mg/kg and 100 mg/kg of the methanolic extract, respectively. Group C was treated with 200 mg/kg of the aqueous extract. Group D was treated with 1 mg/kg pyrimethamine . Group E (the placebo group) was administered with 1% DMSO in phosphate buffered saline (vehicle). The treatment was administered orally for 4 consecutive days. The oral route was chosen based on documented ethnomedical usage of the plant [Senna family [A modification of the method by Ryley and Peters (1970) was used to evaluate the curative potency of the extract against  in mice , 54. Miche plant . The miche plant  as well a family .P. berghei growth inhibition in mice was determined on days 6, 8, 10 and 12 post infection as described previously [The effect of the extract on eviously . Tail bleviously ;\documenTo assess the effect of the extract on survival time of the infected mice, the number of days that each mouse lived from the day of parasite inoculation to death was recorded in a 30 days’ period. The following formula was used to determine the mean survival time of each group ;\documenThe collected data was recorded as means ± standard error of the means (M ± SEM). In vivo parasite suppression was analyzed through Ordinary One-Way ANOVA followed by Tukey’s multiple comparisons test at 95% confidence level . Log rank analysis was used to compare survival time of mice in the different treatment groups. Graph Pad prism  was used for the analyses.S. occidentalis roots were screened for presence of secondary metabolites. All extracts contained saponins, tannins, flavonoids, alkaloids and sterols. Aqueous decoction provided the highest percentage yield (7%) whereas hexane maceration provided the least yield (2%). The characteristics and yields of the extracts are as shown . Methanol, ethyl acetate, chloroform, hexane and aqueous extracts of S. occidentalis were tested for growth suppression potency against P. falciparum. The use of solvents with differing polarity would enable evaluation of antiplasmodial activity of both polar and nonpolar metabolites of the plant root. The results  whereas hexane extract exhibited maximum IC50 value (IC50 = 18.47). The parasite growth suppression levels are as shown  on days 6, 8, 10 and 12, relative to the phosphate buffered saline (placebo) group. At a dose of 200 mg/kg body weight, the inhibitory activity of methanol extract was not significantly different from that of aqueous extract (P > 0.05), except on day 6 post infection when the methanolic extract depicted higher activity (P = 0.03). The suppressive activity of 100 mg/kg methanol extract was not significantly different from that of 200 mg/kg aqueous extract (P > 0.05). Likewise, the suppressive activity of 100 mg/kg methanolic extract was not significantly different from that of 200 mg/kg extract of the same solvent (P > 0.05). This suggests using 100 mg/kg methanolic extract as optimum for use in mice. Parasite multiplication increased steadily in the placebo group until day 8 post-infection when it began to lag. This was indicative of reticulocyte depletion resulting in fewer cells available for malaria parasite infection [S. occidentalis roots suppress P. berghei propagation, in vivo and that the aqueous extract is as effective as the methanolic extract at the tested concentrations. The parasite suppression levels for the different treatment groups are as shown in Table P. berghei in mice treated with the extract and the controls.The nfection , 61. TheS. occidentalis extract on the survival of P. berghei infected mice was assessed by monitoring survival time of the extract-treated animals for 30 days, post- infection. The extract prolonged the survival time of the infected mice in the range of 4 to 6 days, which is approximately 0.3- to 0.5-fold extension of survival time relative to the placebo-treated animals. Treatment with the standard drug, pyrimethamine, resulted in 100% survival of the infected mice. When compared to the placebo group, the increase in survival time of the extract treated mice was significant (P < 0.0001). The mean number of days survived by the infected animals post infection is as shown in Table The effect of S. occidentalis root extract inhibits malaria parasites growth in vitro and in mice. Our findings augment various studies that have illustrated the antimalarial potency of medicinal plants, as reviewed [Natural products, especially medicinal plants, have been used to treat malaria or manage conditions associated with it in various parts of the world , 7. We sreviewed –64. The reviewed , 28. Thireviewed . It is areviewed , 66.S. occidentalis for evaluation of antimalarial potential, the plants roots powder was extracted with methanol, ethyl acetate, chloroform, hexane and water. Preliminary phytochemical analysis of the extracts revealed the presence of biologically important secondary metabolites, some of which have been linked to antimalarial activities of plant extracts [S. occidentalis extracts as antimalarials. A bioactivity guided extract fractionation will reveal the actual molecules containing antiplasmodial activity.To obtain polar and nonpolar root extracts of extracts , 67, 68., against P. falciparum. This malaria parasite species is the foremost contributor to the global burden of malaria [S. occidentalis roots is polarity dependent with extracts from polar solvents showing higher activity than non-polar ones. This suggests that during extraction, the bioactive principles of the plant roots get localized more in polar solvents than non-polar ones. Extraction efficiency has been shown to be dependent on the method and solvent of extraction [S. occidentalis are mainly polar.In this current study, the extracts were found to be active, in vitro malaria , 4, 9. Otraction . Differetraction . OverallS. occidentalis root extract showed no toxicity to Vero cells and was highly selective of P. falciparum. This is suggestive of possible safety of the extracts when used in in vivo models of antimalarial efficacy testing.In this current study, S. occidentalis root extract was evaluated for antiplasmodial activity in situ using the P. berghei mouse model of antimalarial drug efficacy testing. In this in vivo assay, use of human malaria parasites , just like in the in vitro test, would have been more appropriate. However, we did not have access to an animal model for P. falciparum antimalarial drug efficacy testing. Alternatively, our laboratory has established protocols for in vivo antimalarial drug efficacy testing using P. berghei in a mouse model. The structure, life cycle and physiology of P. berghei compare with those of the human malaria parasites [P. berghei mouse model would provide valuable information regarding the in vivo antiplasmodial potential of the extract under investigation in the current study. The effect of S. occidentalis extract on P. berghei parasitemia was determined by monitoring parasite propagation in infected BALB/c mice upon treatment with the extract. The extract significantly suppressed P. berghei parasitemia in mice and increased the survival time of the infected animals. The longer survival time of mice treated with the extract corresponded with reduced parasitemia, suggesting that the extract may play a role in reducing the pathologic effects of the infection. A positive correlation between parasite density, disease severity and mortality due to malaria has been described [S. occidentalis extracts in a prophylactic, as well as non-human primate model for a more complete preclinical testing.Drug interaction with malaria parasites is heavily influenced by the host biological system , 70. In arasites . Accordiarasites , 73. As escribed , 75. In Plasmodium parasites by chelating nucleic acids of malaria parasites [Plasmodium parasites, hence kills malaria parasites [S. occidentalis root extract in suppressing P. falciparum and P. berghei parasites growth.The presence of secondary metabolites in the extract investigated herein may explain the growth suppressive activities of the extract against malaria parasites observed in this study. Plant secondary metabolites have been shown to induce malaria parasite death through diverse mechanisms. For instance, flavonoids have been shown to possess antioxidant potential and kill arasites , 76. It arasites . More stSenna occidentalis (L.) Link root extract has remarkable antimalarial activity, in vitro and in mice. In addition, the results show that polar extracts of S. occidentalis are more potent than non-polar ones, implying that polar extracts can be developed further into antimalarials. We recommend the isolation and identification of the bioactive entities and the establishment of the antimalarial mechanism of action of the extract.Overall, our findings demonstrate that Additional file 1. Characteristics of Senna occidentalis roots extract and the percentage yields.Additional file 2. Growth inhibitory activity of Senna occidentalis root extracts against Plasmodium falciparum, in vitro.