Malaria still constitutes a major public health menace, especially in tropical and subtropical countries. Close to half a million people mainly children in Africa, die every year from the disease. With the rising resistance to frontline drugs (artemisinin-based combinations), there is a need to accelerate the discovery and development of newer anti-malarial drugs. A systematic review was conducted to identify the African medicinal plants with significant antiplasmodial and/or anti-malarial activity, toxicity, as wells as assessing the variation in their activity between study designs (in vitro and in vivo).Key health-related databases including Google Scholar, PubMed, PubMed Central, and Science Direct were searched for relevant literature on the antiplasmodial and anti-malarial activities of African medicinal plants., and combined in vitro and in vivo, respectively. The most frequently investigated plant species were Azadirachta indica, Zanthoxylum chalybeum, Picrilima nitida, and Nauclea latifolia meanwhile Fabaceae, Euphorbiaceae, Annonaceae, Rubiaceae, Rutaceae, Meliaceae, and Lamiaceae were the most frequently investigated plant families. Overall, 248 (34.3%), 241 (33.4%), and 233 (32.3%) of the studies reported very good, good, and moderate activity, respectively. Alchornea cordifolia, Flueggea virosa, Cryptolepis sanguinolenta, Zanthoxylum chalybeum, and Maytenus senegalensis gave consistently very good activity across the different studies. In all, only 31 (4.3%) of studies involved pure compounds and these had significantly (p = 0.044) higher antiplasmodial activity relative to crude extracts. Out of the 198 plant species tested for toxicity, 52 (26.3%) demonstrated some degree of toxicity, with toxicity most frequently reported with Azadirachta indica and Vernonia amygdalina. These species were equally the most frequently inactive plants reported. The leaves were the most frequently reported toxic part of plants used. Furthermore, toxicity was observed to decrease with increasing antiplasmodial activity.In total, 200 research articles were identified, a majority of which were studies conducted in Nigeria. The selected research articles constituted 722 independent experiments evaluating 502 plant species. Of the 722 studies, 81.9%, 12.4%, and 5.5% were in vitro, in vivoCochlospermum planchonii (Bixaceae) conducted to date. There is, therefore, the need to scale up anti-malarial drug discovery in the African region.Although there are many indigenous plants with considerable antiplasmodial and anti-malarial activity, the progress in the development of new anti-malarial drugs from African medicinal plants is still slothful, with only one clinical trial with The online version contains supplementary material available at 10.1186/s12936-021-03866-0. Plasmodium, transmitted through the bite of an infected female Anopheles mosquito, cause malaria, including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi. Among these species, P. falciparum is the most virulent, responsible for the highest morbidity and mortality. It is also the predominant species in sub-Saharan Africa (SSA), a region with the highest number of malaria cases and deaths in the world. According to the World Health Organization (WHO), there were 228 million cases, and 405,000 malaria attributed deaths in 2018 [Malaria still constitutes a major public health menace, especially in tropical and subtropical countries. Various species of  in 2018 .Malaria can be treated using chemotherapy but there is widespread resistance to many of the drugs. The first case of resistance to artemisinins was reported in Cambodia in 2006 and has then spread to most of South-East Asia , 5. The In the past 50 years, natural compounds including plant products, have played a major role in drug discovery and have provided value to the pharmaceutical industry . For insReviews of the antiplasmodial and anti-malarial activities of medicinal plants are needed to drive research into the discovery and production of new anti-malarial drugs. Only a few reviews of the antiplasmodial or anti-malarial activity of medicinal plants have been published in the scientific literature –16. Thes50, ED50, LD50, and parasite suppression rate) of medicinal plants used in Africa to treat malaria. The current study conforms to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines [The literature was reviewed in search of scientific articles reporting antiplasmodial activities , the extraction solvent used, IC50 or ED50 values, parasite suppression rate, isolated compounds, interaction with known malarial drugs (whether synergistic or antagonistic), and toxicity. Articles that did not report antiplasmodial or anti-malarial activity of medicinal plants as well as review articles were excluded. The entire selection process is presented in Fig. The search was limited to studies published in English or containing at least an abstract written in English until May 2020. The titles and abstracts were subsequently examined by two reviewers, independently  to identify articles reporting the antiplasmodial activity of medicinal plants. In the case of any discrepancy in their reports, a third reviewer was brought in to resolve the issue. Relevant papers were equally manually cross-checked to identify further references. The following data were extracted from the selected articles by the reviewers: plant species, plant family, place of collection of plant, parts of the plant used, type of study  and reporting parasite suppression rate.In this study, antiplasmodial activity pertains to studies performed in vitro using different strains of 50 < 5 µg/ml, good 5 µg/ml ≤ IC50 < 10 µg/ml, and moderate 10 µg/ml ≤ IC50 < 20 µg/ml [For in vitro studies, the antiplasmodial activity of an extract was considered very good if IC20 µg/ml . For in 20 µg/ml . AntiplaPlasmodium used to assess the antiplasmodial or anti-malarial activity of the medicinal plants equally varied between studies. Furthermore, the extraction solvent, as well as the extraction yield of the plants in the different studies, was not the same, which may have accounted for the variation in the antiplasmodial and anti-malarial activities for the same plants but in the different studies.The level of risk of bias for the study was likely to be high mainly because of differences in the studies and the methods used to determine the antiplasmodial or anti-malarial activity. The stains of The PRISMA flowchart Fig.  presentsFor this review, the evaluation of the individual plant species was considered as an independent study, so it is common for one article to have more than one study depending on the number of plant species evaluated. In all, there were 722 independent studies. Five hundred and ninety-on 81.9%) of the independent studies were in vitro (Table .9% of thFabaceae 47 (6.5%), Euphorbiaceae 45 (6.2%), Annonaceae 37 (5.1%), Rubiaceae 37 (5.1%), Rutaceae 37 (5.1%), Meliaceae 30 (4.2%), and Lamiaceae 12 (1.7%). Five hundred and two (502) plant species were investigated in this study. Of them, the most investigated were: Azadirachta indica, Zanthoxylum chalybeum, Picrilima nitida, and Nauclea latifolia. The most frequent parts of the plants tested were the leaves, roots, root barkss, stems, and the whole plant. A majority of the studies used the crude extracts of the plants compared to pure compounds (95.7% vs. 4.3%). In descending order, methanol 322 (44.7%), dichloromethane 207 (28.7%), ethanol 103 (14.3%), water 85 (11.7%) and ethyl acetate 62 (8.6%) were the most frequent extraction solvent used.From 722 studies, the most frequent plant families studied included 50 values < 5 µg/ml or suppression rate of ≥ 50% at 100 mg/kg body weight/day), 241 (33.4%) reported good activity and 233 (32.3%) reported moderate activity. For the in vitro studies, a majority 228 (38.6%) reported very good activity; 206 (34.9%) reported good activity and 187 (31.6%) reported moderate activity. Meanwhile for the in vivo studies, a majority 19 (21.1%) reported moderate activity, 16 (17.8%) reported very good activity and 13 (14.4%) reported good activity. For studies reporting both the in vitro and in vivo activity, a majority of 17 (42.5%) reported only moderate activity, 13 (32.5%) studies reported very good activity and 10 (25.0%) reported good activity. Among the plants with very good activity, only one species demonstrated very good activity both in vitro and in vivo  of the studies reported activity that was very good  divided by the total number of studies that assessed the activity of that plant species (denominator).Among the studies, the most frequent plant species demonstrating very good antiplasmodial activity were: Azadirachta indica and Vernonia amygdalina were the most frequently reported inactive species  of the studies used the crude extract of the plants. The antiplasmodial and/or anti-malarial activity was significantly higher (p = 0.044) in studies using pure compounds compared to those using crude preparations.Azadirachta indica and Vernonia amygdalina. Plant families harboring the most toxic species were Lamiaceae, Anacardiaceae, Moraceae, Meliaceae, Asteraceae, and Fabaceae. Approximately 33% of the plants tested demonstrated some toxicity in vitro and 26.7% had some degree of toxicity in vivo. Among plants with very good, good, and moderate antiplasmodial activity, 17.8%, 28.3%, and 35.4% had some degree of toxicity, respectively. The leaf was the plant part with the most frequently reported toxicity. Albino mice and Vero E6 cells were the most commonly used assays for the assessment of the toxicity of the plants.Out of the 198 plants evaluated in toxicity assays, 52 (26.3%) were found to demonstrate some degree of toxicity. The most frequently reported plants with toxicity were Cochlospermum planchonii (Bixaceae) has been conducted so far. This reinforces the need for basic and clinical research in the region. Van Wyk [Resistance to the frontline anti-malarial drugs is increasing and is now a global concern. With this rising rate of resistance, there is a need to accelerate research into the discovery and development of new anti-malarial drugs. Unfortunately, from this study, it is evident that the progress into the discovery of a new anti-malarial drug in Africa is slothful. Despite a considerable number of plant species that have demonstrated significant antiplasmodial activity in vitro, fewer plants have been evaluated in vivo and only one clinical trial with  Van Wyk  had also50 values of < 20 µg/ml were considered as the cutoff of significant anti-malarial activity. This cutoff is considered the minimum to qualify as a first-pass “hit” in anti-malarial drugs screening [Euphorbiaceae, Fabaceae, Rubiaceae, and Annonaceae. However, the plant families containing the most active plants were Apocynaceae, Celestraceae, and Rutaceae. This finding suggests that more emphasis should be given to plants in these families for anti-malarial drug discovery. Besides, the most investigated plant species were Azadirachta indica, Nauclea latifolia, Picralima nitida, and Zanthoxylum chalybeum. Alchornea cordifolia, Flueggea virosa, Crytolepis sanguinolenta, and Zanthoxylum chalybeum were the only plant species with consistently very good antiplasmodial and anti-malarial activities between studies. This is very surprising that no clinical trial using any of these plants has been conducted. Further studies on these plant species should be performed.This review revealed research articles from 31 African countries. Most of the articles were from Nigeria. This is suggestive that Nigeria is leading the podium in research on anti-malarial drug discovery and development, deservedly so, because she is probably the most affected country in the world. It is noteworthy that South Africa which is generally more technologically advanced than Nigeria had very few (8) articles. The African region is the most affected in the world recording the greatest number of cases and malaria attributed deaths. However, the distribution of malaria in Africa is not even, with sub-Saharan Africa harboring disproportionately the greatest number of cases. This is suggestive that research to identify new anti-malarial drugs may be related to the burden of the disease, thus the government policy to control the disease. There is, therefore, the need for policy-driven research into new anti-malarial all across the African region. In this review, ICcreening . Five huUvaria acuminate decreased from good activity in vitro to inactive in vivo. However, a few studies show that plant activity could also increase from in vitro to in vivo. For example, Ngbolua et al. [Vernonia ambigua increased from in vitro to in vivo analysis. Other examples include studies by Muthaura et al. [Boscia angustifolia, Kweyamba et al. [Commiphora Africana, and Ajaiyeoba et al. [Annona senegalensis. This suggests that plants could still have significant anti-malarial activity in vivo although they failed to in vitro. Most investigators usually progress to in vivo studies only when they observe significant antiplasmodial activity in vitro. This may explain the findings of a smaller number of in vivo studies in the current study. The investigation of the anti-malarial activities of plants should continue in vivo despite the dismal performance of the plants in vitro.This study revealed that overall, a majority of the plants investigated had very good antiplasmodial activity in vitro. That activity decreases as you move to in vivo in most studies, with a majority of plants demonstrating only moderate activity. For example, Gathirwa et al.  showed ta et al.  showed ta et al.  using Boa et al.  using Coa et al.  using AnSenna occidentalis, Adansonia digitata, Acanthospermum hispidum, Rotheca myricoides, Anogeissus leocarpus, Annona muricata, Ageratum conyzoides, Albizia coriaria, Ekebergia capensis, Flueggea virosa, Lippia javanica, Maytenus senegalensis, Morinda lucida, Picralima nitida, Trichilia emetica, Vernonia amydalina, and Vernonia colorata. The factors that could have accounted for these differences may include differences in the extraction solvent thus the extraction yield and extracted metabolite. With dichloromethane, mainly the apolar metabolites are extracted. In contrast, with methanol, from polar to moderate apolar metabolites are extracted.The current study revealed substantial inter-study variation in the antiplasmodial activity of several plant species. For example, considerable variation in the antiplasmodial activity was observed for Acacia nilotica was moderate in South Africa and very good in Sudan. There was also variation between the different assay types. For example, the activities of Vernonia ambigua [Annona senegalensis [Alchornea cordifolia, and Zanthoxylum chalybeum, were observed to be consistently very good between studies. These plant species should be exploited further for their antiplasmodial activity. The activities of the plants were equally observed to increase with the isolation of the active compounds thus reinforcing the need for research into identifying the active compounds of African medicinal plants. The marked difference in the antiplasmodial activity of the crude extract of Artemisia annua and the pure compounds points out the issue that even the compounds which show only low potency and may be discarded from the initial screen for further development may still have active components with therapeutic potential [Plasmodium used may also be another factor accounting for the inter-study variation observed; studies using chloroquine-sensitive strains of the parasite like P. falciparum 3D7, D6, NF54 tend to report higher antiplasmodial activity compared to studies using chloroquine-resistant strains like P. falciparum W2, Dd5, K1 or D10.Most (95.7%) of the studies used crude extract for their investigation and rarely the pure compounds  has been shown to demonstrate some degree of toxicity, but betulafolientriol oxide isolated from the plant was very active with little or no toxicity against human kidney epithelial cells [This study revealed that only a few (26.3%) of the plants demonstrated some degree of toxicity. The families hosting the most toxic plant species were e former  is one o engleri , Celtis grifolia , Ficus patyhylla , Gutenberdifolia , Helchry cymosum , Microglyrifolia , Opilia idifolia , QuassiaAfricana , Rumex assinicus , Clausen anisota , Icacinagalensis , Abutilodiflorum , and Lannfurthii . The isoal cells . There wThis study, however, is limited in that the analyses may have been compounded by the substantial inter-study variation in the methodologies used by different independent studies for the extraction of plant material, the overall extraction yield, the diversity of extracted metabolites as well as the geographical variations in the different sites used in the plant collection. However, the study has provided important baseline data that may be exploited by researchers in the field for the discovery and development of new anti-malarial drugs.Cochlospermum planchonii (Bixaceae). The study also revealed considerable inter-study variation in the antiplasmodial activities of the plants, however, the activity of some plants including Alchornea cordifolia, Azadirachta indica, and Zanthoxylum chalybeum was consistently very good. The study demonstrates a relationship between antiplasmodial activity and toxicity whereby the toxicity of the plants decreases as the antiplasmodial activity increases. Besides, the active compounds were identified in just a handful of the plants. Therefore, there is a need for a policy-driven approach in the discovery and development of new anti-malarial drugs to subvert the rising resistance to the frontline anti-malarial drugs in the world.This study has revealed the slothful progress in the discovery and development of new anti-malarial drugs from African medicinal plants. Despite the encouraging activities demonstrated by the plants in vitro, fewer plants have been evaluated in vivo and just one clinical trial has been conducted so far with Additional file 1: Table S1. In vitro and in vivo studies reporting inactive antiplasmodial or antimalarial activity. Table S2. List of active compounds identified from plants.


N = 123), Chinese school children (N = 1072), and Malay/Chinese university students (N = 1119). In school children, four factors with eigen values > 1 explained 50.2% of the variance, with several items cross-loading on more than two factors and three items failing to load on any factor. Among 19 tested models, CFA revealed excellent fit of a unidimensional six-item IAT among ED women and university students (χ2(7) = 8.695, 35.038; p = 0.275, 0.001; CFI = 0.998, 981; TLI = 0.996, 0.960; RMSEA = 0.045, 0.060; SRMR = 0.0096, 0.0241). It was perfectly invariant across genders, academic grades, majors, internet use activities, nationalities , and Malay/Chinese female university students vs. Spanish women with anorexia nervosa, albeit it was variant at the scalar level in tests involving other EDs, signifying increased tendency for IA in pathological overeating. The six-item IAT correlated with the effects of internet use on academic performance at a greater level than the original IAT , indicating superior criterion validity. The six-item IAT is a robust and brief measure of IA in healthy and diseased individuals from different cultures.Internet addiction (IA) is widespread, comorbid with other conditions, and commonly undetected, which may impede recovery. The Internet Addiction Test (IAT) is widely used to evaluate IA among healthy respondents, with less agreement on its dimensional structure. This study investigated the factor structure, invariance, predictive validity, criterion validity, and reliability of the IAT among Spanish women with eating disorders (EDs,  The use of the internet for socialization and gaming has dramatically increased among children, adolescents, and young adults during the last two decades as a result of the expansion of internet technology ,2. ExcesThe current coronavirus disease 2019 (COVID-19) pandemic has been associated with an increase in the prevalence and severity of IA among the general population, especially youth and individuals with poor social support ,8. This Griffiths’ addiction components model proposes that both drug addiction and addictive behaviors, including IA, comprise a set of six distinct common components: salience, mood modification, tolerance, withdrawal, conflict, and relapse . In factIA is associated with poor academic/work performance, dysphoric mood, dysfunctional sleep, loneliness, and deficient real world social networks ,17,18. IThe Internet Addiction Test (IAT), developed by Kimberly Young, is one of the most widely used measures to diagnose IA ,26. The The IAT has been translated and validated into more than 20 languages other than English , includiVariations in the structure of the IAT can be accounted, in part, by the method of extraction. Obviously, studies reporting more than two-factor structures used exploratory factor analysis (EFA) or principal component analysis ,41,43, wA few studies have evaluated invariance of the IAT across different groups such as gender  and AsiaMeasurement invariance implies psychometric equivalence of a construct across groups, i.e., it has the same meaning to those groups ,47. SeveEating disorders (EDs) commonly develop in young groups, especially among adolescent girls and young women . EDs areThe state of being a student may be associated with distress due to academic demands; concerns about supporting tuition, housing, and other essentials for living; as well as concerns about attaining proper employment in the future . Apart fProlonged and repeated internet use is associated with reduced physical activity and increased consumption of fast food because of shrinkage of time allowed for cooking healthy food (containing fibers), which favor frequent snacking, food preoccupation, and loss of control eating ,70,71. IWomen with EDs frequently join online self-help groups and digital ED interventions, and those with higher addictive tendencies may develop IA ,72. GiveTo fill the gap, the current study aimed to examine the construct structure and invariance of the IAT among healthy school children, women with EDs, and university students. The structure of the IAT has been extensively revised among women with EDs based on item loadings, item-total correlations, and Griffith’s addiction component model. The resulting short version was examined for invariance across ED subtypes in the same sample. Because IA is reported to correlate with all ED subtypes , we hypoThis study evaluated the structure and invariance of the IAT across healthy and diseased groups from different cultural backgrounds. The current analyses integrated publicly accessible datasets involving a sample of school children from Hong Kong , a samplN = 1072, mean age = 12.4 ± 2.0 years, males = 62.9%) comprised children from elementary (N = 462), junior (N = 357) and high schools (N = 235). The Human Research Ethics Committee of the Education University of Hong Kong approved the data collection protocol [This study comprised a clustered random sample of school children, herein referred to as the “school children sample”, which was obtained from nine schools in Hong Kong during the period between May and July 2017. Out of 1121 pupils attending on the survey date, whose parents had previously signed an informed consent, 1097 pupils (97.9%) agreed to participate—they completed an assent form. The authors of the original dataset had removed twenty-five responses because they were outliers . The finprotocol .Problematic use of the internet was assessed by the IAT, a 20-item measure with responses rated on a five-point Likert scale  . In geneIn SPSS, age was described by mean ± SD while numbers and percentages were used to describe other characteristics of the sample. EFA with maximum-likelihood extraction and varimax rotation was used as an initial step of evaluating the structure of the IAT as noted by the number of factors with eigen values > 1. This is because it allows items to load freely on the corresponding factors without implying any constraints. To ensure that the sample size is adequate for EFA, the analysis included Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity. Significance was considered at a probability of 0.05, two-tailed.N = 123) of Spanish females (mean age = 27.3 ± 10.6 years) consecutively treated for EDs at the outpatient and inpatient departments of the General University Hospital of Ciudad Real between February and November 2018. Participants were included if they were >12 years of age and agreed to sign an informed consent. For patients below 18 years, their guardians signed informed consent. Patients with physical or mental disabilities were excluded. Reported diagnoses of EDs were AN , bulimia nervosa , binge EDs , and EDs not otherwise specified . On average, participants had a diagnosis of EDs for 10.2 ± 8.0 years, and they have been in treatment for an average of 8.1 ± 6.5 years. The average body mass index was 22.2 ± 8.4, with a significant difference between patients with AN (group1) and patients with other EDs (group2) t(73.0) = −6.77, p = 0.001. Around half the participants  had a history of psychiatric hospitalization while 48.8% had never been hospitalized. Less than half the participants  reported tobacco smoking. Depression was the only reported psychiatric comorbidity; it co-occurred in 29.3% of the participants. Most participants were single . Few participants had only elementary school education , more than half the sample  had high school education, and the rest of the participants had a university degree. The Ethics and Clinical Research Committee of Ciudad Real (Spain) approved data collection (ref. 2017C/123) [This study is based on a convenient sample (17C/123) .Data were collected from eligible participants through a self-administered questionnaire that comprised a section inquiring about participants’ sociodemographic and clinical characteristics such as age, weight, height, ED subtypes, treatment history, comorbidities, and smoking status .The Spanish version of the IAT , which hThe validated Spanish version of the Bergen Facebook Addiction Scale (BFAS)  was usedThe dataset was checked for missing data, and one incomplete response (data on psychiatric comorbidity) was deleted, resulting in a final sample size of 123. The normality of the IAT was examined by Shapiro-Wilk and Kolmogorov-Smirnov tests of skewness and kurtosis. Because AN was the most dominant diagnosis and other EDs were less presented, we created two major categories of EDs for subgroup analysis: patients with AN (group 1) and patients with other EDs (group 2). These two groups were established based on the fact that body mass index was significantly lower in group 1 than in group 2 .In a second stage, we used CFA with maximum likelihood method and bootstrap based on 2000 random samples and a 95% confidence interval (95% CI) to examine different structures of the IAT commonly reported in previous studies ,33,34,432) was used to reflect global data-fit to the models [A non-significant chi square index  are invariant across ED groups. Invariance was evaluated at the configural, metric, scalar, and strict levels as described above ,48. To in groups . CriticaReliability of the IAT and the six-item IAT was evaluated by Cronbach’s alpha , correctN = 1120, mean age = 21.1 ± 1.6) from both sexes and all grades obtained from Univerisiti Teknologi Malaysia (UTM). A detailed description of the sociodemographic and internet use characteristics of the participants is presented in This study included a purposive sample of undergraduate students , academic , and internet use data , and the effect of internet use on academic performance, which was assessed by a single question: rate the extent to which internet use affects your academic performance on a scale from 1  to 5 (extremely negative effect).One response was removed from the dataset because of missing data (the IAT). Based on study 2, we used CFA to examine the structure Pawlikowski et al. (2013)’s 12-item IAT  Model 1. In addiTo examine criterion validity in this sample, we used Spearman’s rho test to correlate the scores of the 12-item IAT and the six-item IAT with time spent online, years of internet use experience, and perceived effect of internet use on academic performance.Exploratory Factor Analysis of the Internet Addiction Test2(190) = 6175.11, p < 0.001) indicated adequacy of the sample size and appropriateness of participant-to-item ratio for EFA. While items 5, 7, and 9 had loadings below 0.3, several items significantly loaded on two or three factors  and Bartlett’s test . All these models held configural invariance among ED groups, but did not hold scalar and metric invariance, except for Model 11 . ExaminaThe IAT, the 12-item IAT, and the six-item IAT demonstrated excellent reliability in all the samples as noted by high values of coefficient alpha . All corCFA involving correlating few error terms  revealedThe results of multigroup CFA in the student sample uncovered invariance of the 12-item IAT and the six-item IAT at the configural, metric, scalar, and strict levels across groups of gender, nationality, major, academic grade, and types of internet use activities .Invariance analysis across groups of female students and women with different EDs shows configural invariance in all models . HoweverThe reliability of the IAT and the 12-item IAT was very good while that of the six-item IAT was good . The totGiven that the psychometric properties of the IAT have always been tested among students (in 20 out of 25 studies) and healthy young adults [Our results suggest impure factorial structure of the IAT. EFA in the school children sample uncovered the presence of four factors, with three items failing to load on any factor and numerous items with high cross-loadings . LikewisAlthough items 5, 7, and 9 did not load on any factor in the school children sample, they were not a source of noise in the structure of the IAT in the ED sample  and increased incidence of sleep problems  . TherefoApart from its superior fit and invariance, the six-item IAT enjoys additional excellent psychometric properties. Because multiple replicate items contribute to scale reliability, eliminating items is usually associated with a decrease in scale reliability unless it involves heterogeneous items ,91. DespThis study has the merit of being the first to examine the IAT in a clinical sample and to develop and test various psychometric properties of the six-item IAT in culturally-diverse clinical and healthy samples. Shorter versions of the IAT were invariant across Malay and Chinese students, who both live in the same part of the world—south-east Asia. However, examining invariance of these short versions of the IAT across Asian healthy female students and European women with EDs may not be standardized enough to judge invariance of the 12-item/six-item IAT in these populations. Therefore, future studies are encouraged to evaluate the effect of cultural variations on the properties of the six-item IAT both among healthy and diseased groups. This study has also other limitations. Test-retest reliability is important to present the psychometric soundness of a new scale; however, it was not possible to conduct that test, given the nature of the used data. In all the samples, IA was not diagnosed by a clinician based on the diagnostic criteria found in the DSM-5. Thus, we were not able to examine whether the six-item IAT could differentiate between healthy individuals and those with an internet use disorder. Because this research is based on public data, some important details on how the research was conducted in the original study are lacking. For example, the recruitment method and specific sampling strategy taken to obtain the university student sample are not described , which mAlthough the six-item IAT was invariant across gender among university students, we could not examine invariance across gender in the ED sample because it comprised females only. Invariance of the IAT across gender groups may not hold among ED patient population. This is because EDs are more frequent and more comorbid among women  who alsoUsing already existing data, this study is the first to examine the psychometric properties of the IAT in a clinical sample (women with EDs) and compare them across healthy students from different cultures. The new scientific knowledge attained in this study showing that the IAT expressed four-factor structures in school and university students, but it is best described by a bifactor structure or a two-factor structure among women with EDs. Both genders as well as healthy and ED women had the same global conceptualization of IA. However, excessive eating was associated with increased intensity of IA “tolerance, salience, and sleep loss”. Therefore, researchers developing interventions to address IA among patients with EDs should pay more attention to ED subtypes characterized by overeating, eating at night, and poor sleep. Extensive revision of the scale resulted in a unidimensional six-item IAT, which is capable of detecting the domains of IA according to Griffiths’ addiction component model. The six-item IAT expressed better psychometrics compared with the parent scale and the 12-item IAT. Thus, it may be reliably used for prompt capturing of IA in research and clinical practice, particularly during the current COVID-19 outbreak, which has witnessed an expansion in internet use and IA. Its brevity may support comprehensive assessments by allowing test batteries to include more measures, which may promote efforts for reducing comorbidities and enhancing recovery. To maximize the utility of this brief scale, more investigations of its psychometric properties in larger samples with more representation of ED subtypes as well as in more diverse clinical conditions and healthy groups from different cultures are needed.


Background: Psoriasis is a polygenic multifactorial immune-mediated skin disease associated with comorbidities. As one-third of adult psoriasis starts during childhood, early detection of these comorbidities might help to mitigate their impact on future health.Objectives: To investigate the risk for cardiovascular events and their relationship with psoriasis severity among Iraqi children and adolescents.Patients and methods: A prospective, case-control, cross-sectional study on 150 patients with psoriasis and 150 age and sex-matched individuals. The study was carried out at the Department of Dermatology/Basra Teaching Hospital from December 2018 to December 2020. Psoriasis severity was assessed by PASI (psoriasis area severity index) score, and in both groups, blood pressure and body mass index (BMI) were measured. Laboratory tests including fasting blood sugar (FBS) and lipid profile were also done.th plus 5 mm of mercury applied to BP levels for boys and girls by age and height percentile charts), none of the control group was hypertensive, and the difference was statistically significant . A significantly higher proportion of the psoriatic patients had abnormal lipid profiles compared with the control group, 62% versus 30% (p<0.05), 15.3% versus 6.7% had elevated cholesterol (p<0.05), 24.7% versus 8% had raised low-density lipoprotein , 18% versus 8.6% had low high-density lipoprotein , and 12.6% versus 6% had elevated very-low-density lipoprotein (VLDL) and triglyceride , 8% patients had elevated FBS (more than 100 mg per deciliter) versus 2.6% (p<0.05), and metabolic syndrome in 65 versus 2% (p<0.05). These changes were related to the severity of psoriasis.Results: More patients were overweight and obese in the psoriatic group compared to the control group (26.7% and 40% versus 11% and 8%), 5.3% of psoriatic patients who had stage 2 hypertension (defined as any blood pressure [BP] measurement higher than 99Conclusions: Pediatric psoriatic patients in our population may have an atherogenic lipid profile with an increased prevalence of risk factors for cardiovascular diseases, especially those with moderate to severe psoriasis. Psoriasis is a chronic, genetically determined, relapsing, proliferative, and inflammatory disease of the skin, characterized by well-defined erythematous plaques with large adherent white silvery scales . It is aPatients with pediatric psoriasis appear to have a higher prevalence of obesity (BMI more than 95th percentile) and adiposity (BMI more than 85th percentile) when compared to those children without psoriasis . There iFollowing the release of screening guidelines for comorbidities in pediatric psoriasis by the Pediatric Dermatology Research Alliance , many stAn analytical cross-sectional, case-control study was designed to recruit children with psoriasis and age and sex-matched children with dermatological diseases other than psoriasis or inflammatory dermatosis, like a wart, molluscum, viral exanthema, vascular lesions during the period extended from December 2018 to December 2020. Because the study was conducted in a hospital, the control sample was taken from pediatric patients who were there for other reasons and not from family members, neighbors, or classmates. The study was conducted at the Dermatological Outpatient Department in Basra Teaching Hospital, Basra City, Southern Iraq. Patients aged less than18 years with any type of psoriasis and no other coexisting inflammatory dermatosis were excluded. While patients aged more than 18 years, with any type of inflammatory dermatosis or autoimmune diseases other than psoriasis, or on medications like systemic steroid, retinoid, or other immune suppressant therapy for at least six weeks before enrollment and presence of metabolic diseases like diabetes or hypertension that was diagnosed before the onset of psoriasis were excluded.Ethical clearance was obtained from the council of the College of Medicine, University of Basrah, and Basrah Teaching Hospital Directorate to perform the study . Informed consent also was taken from all participant's parents.2) expressed in a unit of kg/m2, then the calculated BMI was applied on the chart for BMI percentile for boys and girls and for age, to find the corresponding percentile of each participant (85th percentile to 94th percentile defined as overweight by CDC and >95th percentile defined as obese). By appropriate pediatric-sized cuff sphygmomanometer (Cuff width must cover two-thirds of the distance from shoulder to elbow), and according to the International Pediatric Hypertension Association, blood pressure was categorized into three stages. The value of blood pressure was applied on the percentile for sex by age for height as follows: prehypertensive = 90th centile up to 120 mmHg, stage 1 hypertension = 95th centile and upward, and stage 2 hypertension = 99th centile + 5 mmHg. FBS and serum lipid profile  were measured using the Arccthitec 4000 analyzers  and values interpreted as high when: FBS>100 mg/dl, cholesterol>200 mg/dl, LDL>160 mg/dl, HDL<40 mg/dl, VLDL>40 mg/dl, TG>150 mg/dl.After careful interrogation and obtaining a detailed history, the age of participants was subcategorized into three groups: <6 years (preschool age), 6-12 years (primary schooling age), and those between 12 and 18 years (intermediate and secondary schooling age). Type of psoriasis was diagnosed by physical examination, and severity of the disease was assessed by using Psoriasis Area and Severity Index (PASI) score and classified as mild (score less than 7), moderate (score 7-15), and severe (more than 15) . All parThe sample size was calculated using the statistical formula for a qualitative variable of a cross-sectional study with the precision error of 5% and type 1 error of 5%. At least, 138 participants were needed for each group. At the time of data analysis, 150 participants were allocated for each group. Data were applied to excel sheet; SPSS version 26  was used for analysis includes descriptive statistics ; tests of significance (chi-square and Fischer exact test) was used for the analysis of qualitative variables. Means and standard deviations were used to present data of continuous variables. ANOVA and the post-hoc test were used test to determine the significant differences between quantitative variables such as age and duration, P-value < 0.05 was considered statistically significant.The results showed that the mean (SD) age of patients was 10.22 (6.8) years . Family history of psoriasis among the first relative was documented in 77 (51%). The majority of psoriatic patients were within the age group of 6-12 years (68%), and Psoriasis Vulgaris was the most common variant and was seen in 106 , and the difference was statistically significant . According to the International Pediatric Association, hypertension categorization of blood pressure, pre-hypertension status, first stage hypertension, and second stage hypertension were more noticeably observed in the psoriasis group than the control group, and the difference was statistically significant . Accordingly, psoriatic patients were associated with an increased risk of hypertension, Odd ratio = 2.3  levels of FBS and total cholesterol in both groups were within the normal range; however, they were statistically significantly higher in psoriatic patients than in the control group . While the mean (SD) level of LDL, HDL, VLDL, and TG were within normal range and no statistical difference between both groups for LDL and HDL , borderline for VLDL , and significant statistical difference regarding TG  and obesity BMI (>95th percentile) were more prevalent in a psoriatic group than in the control group and this finding was consistent with Phan et al. study in Turkey 2016 , and a cAccording to the International Pediatric Hypertension Association, the current study demonstrates that pre-hypertension status, first and second stage hypertension were observed more frequently in psoriatic patients than controls, findings were lower than Muhe et al. study  (OR =5, We demonstrated that the mean serum level of FBS in psoriatic patients and in the control group were both within the normal range. However, it was statistically significantly higher in psoriatic patients than in the control group. Additionally, the frequency and percentage of elevated serum levels of FBS above the normal range were more prevalent in the psoriasis group than in the control group, indicating that children with psoriasis are at an increased risk of developing diabetes mellitus. The findings were in line with the study by Augustin et al.  which reIn general, a high rate of hyperlipidemia and metabolic syndrome was demonstrated among psoriatic children than controls, which was consistent with many published studies ,26. HoweThe study has many limitations. First, it was a cross-sectional type, which has an inherent weakness regarding the causal inference between the exposure to risk and outcomes. To overcome this, we tried to eliminate any patients who had a history of comorbidities prior to the development of psoriasis as well as those who were on systemic medications that were known to cause comorbidities. Because the study was primarily focused on cardiovascular risk factors, we excluded additional comorbidities from assessment, such as polycystic ovaries, arthritis, and non-alcoholic liver disease.In summary, children and adolescents with psoriasis are more likely to be obese, hypertensive, dyslipidemic, and suffer from metabolic syndrome. The prevalence of these factors was positively related to the severity of psoriasis. During the baseline visit, these patients should be screened for overweight and obesity, type 2 diabetes, hypertension, and dyslipidemia, and those with abnormal initial results should be closely monitored, as early intervention may help to prevent problems in adulthood.


Acupuncture has a long history in China and is currently widely used in clinical practice. However, there is no large sample data confirming the effectiveness of acupuncture in treating stroke and its sequelae. This article presents a study protocol for a multicenter, randomized, controlled trial for evaluating the efficacy of acupuncture in treating post-stroke dysphagia.A randomized controlled trial will be conducted in three hospitals in Heilongjiang, Changchun, and Beijing. A total of 252 patients with post-stroke swallowing ability will be randomly divided into two groups; specifically, the experimental group, which will receive acupuncture treatment, and the control group, which will undergo rehabilitation training for 2 weeks. The main results will be evaluated using the standard swallowing function scale, videofluoroscopic swallowing examination, and functional magnetic resonance imaging. The secondary observation indexes will include surface electromyography signal analysis and the modified Barthel index. Measurements will be obtained before treatment, as well as 1 week, 2 weeks, and 1 month after treatment.This trial could clarify the effectiveness of acupuncture in stroke; moreover, it will determine whether acupuncture can improve swallowing function.Chinese Clinical Trial Registry (ChiCTR), ChiCTR2000030994. Moreover, the stroke burden has been increased by general population growth and the increasingly aging population.Stroke, which is also known as cerebral apoplexy, is characterized by high morbidity, mortality, recurrence, and disability rates; moreover, it has the second-highest mortality rate worldwide. Post-stroke damage to the brain, cerebellum, or brain stem can impair swallowing function. Swallowing difficulties cause several problems, including aspiration and malnutrition, which can cause further complications. Among them, pneumonia is the main mortality cause in patients with stroke; moreover, there is a need to address socioeconomic issues, such as long-term medical expenses.,7Stroke can cause several sequelae, including motor dysfunction, sensory dysfunction, cognitive function, and swallowing dysfunction. Approximately 65% of patients with acute stroke present dysphagia.,9 However, there remains no high-quality and effective evidence clearly showing acupuncture-mediated recovery of stroke and swallowing disorders.There has been rapid development and progress in modern rehabilitation technology, swallowing training, transcranial magnetic stimulation, electrical stimulation, and other methods, which allow significant improvements in post-stroke swallowing disorders. Acupuncture is an important treatment method in traditional Chinese medicine for rehabilitation. It has a definite effect, simple and easy operation, and less toxicity and side effects. It is a common clinical method for stroke treatment, including scalp acupuncture, nape acupuncture, tongue acupuncture, body acupuncture, and other needle methods. Currently, there have been numerous acupuncture studies on stroke treatment, including post-stroke swallowing dysfunction. Numerous clinical reports have indicated that acupuncture can significantly improve swallowing function.Consequently, the proposed study will use commonly used rating scales, surface electromyography (sEMG) signals, and functional magnetic resonance imaging (fMRI) to examine the acupuncture efficacy in the treatment of swallowing dysfunction in patients with stroke.22.1The study objective is to formulate a TCM acupuncture rehabilitation program for post-stroke dysphagia . The central regulation mechanism underlying dysphagia treatment using TCM rehabilitation will be analyzed from the perspective of post-treatment brain function changes using resting fMRI and self-comparative analyses.2.2This will be a multicenter, randomized controlled study conducted at the Second Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Changchun University of Traditional Chinese Medicine, and China Rehabilitation Research Center from December 1, 2018, to December 31, 2021. Patients with post-stroke dysphagia who meet the inclusion criteria will be divided into the experimental and control groups using a central randomization system. The experimental and control groups will receive acupuncture treatment and conventional rehabilitation training, respectively. The primary results will be obtained using the standardized swallowing assessment (SSA), videofluoroscopic swallowing study (VFSS), and fMRI. The secondary observation indicators will include surface electromyography (sEMG) analysis and modified Barthel index (MBI). The follow-up indicators include stroke-specific quality of life scale (SS-QOL) and the SSA. Measurements will be obtained before treatment, as well as 1 week, 2 weeks, and 1 month after treatment. fMRI will be only performed in the subject unit. Forty-two participants will undergo pre- and post-treatment fMRI assessment to analyze the central regulatory mechanism underlying swallowing function remodeling in the rehabilitation program of TCM. Figure 2.3The included participants will meet the following Western Medicine and TCM diagnostic criteria.2.3.1Regarding the diagnostic criteria for cerebral infarction/cerebral hemorrhage, a confirmed diagnosis will be obtained through brain computed tomography (CT) or MRI, with reference to the Chinese guidelines for diagnosis and treatment of acute ischemic stroke and cerebral hemorrhage, at the Cerebrovascular Disease Section in Chinese Neurology Branch of Medical Association in 2014. The diagnosis for dysphagia will refer to the diagnostic criteria for post-stroke dysphagia rectified during the sixth National Academic Conference on Cerebrovascular Disease.2.3.2Regarding the diagnostic criteria for cerebral infarction/cerebral hemorrhage, a confirmed diagnosis will be obtained through brain CT or MRI, with reference to the management guidelines for common diseases in internal medicine of TCM (ZYYXH/T19-2008). Dysphagia diagnosis will refer to the diagnostic criteria for post-stroke dysphagia rectified during the sixth National Academic Conference on Cerebrovascular Disease.2.4The inclusion criteria will be as follows: clinical diagnosis of first-episode unilateral cerebral hemisphere infarction or cerebral hemorrhage, with confirmation using CT or MRI; meeting the diagnostic criteria for dysphagia; showing abnormal expressions of the oral and pharynx stages upon VFSS evaluation; having stroke and dysphagia duration within 3 months; being conscious with stable vital signs and a mini-mental state examination score of ≥ 21; age 45 to 75 years; and having provided informed consent.2.5The exclusion criteria will be as follows: having a non-unilateral lesion; having a history of stroke, cerebral tumor, brain impairment, and other neuropsychiatric problems; having a history of dysphagia; having other cognitive disorders, aphasia, or serious cardiopulmonary and renal complications; and being unable to cooperate with examination and treatment.2.6The criteria for drop-out cases will be as follows: voluntary withdrawal from the study; being unsuitable for further assessment due to severe adverse reactions, severe complications, or condition deterioration; poor compliance that impedes the provision of consent for study participation; or incomplete treatment evaluation without rule compliance and with insufficient observation data.Regarding the disposal of drop-out cases, researchers should adopt home visits, phone, letters, etc to communicate with the participants, record the last treatment time, and extensively perform evaluation. After obtaining the drop-out reasons, they will be added to the case report form. All observation data from drop-out cases will be collected for analysis by the project teams. The researcher should record details regarding drop-out information and enroll more than one treatment course to be included in the efficient statistics.2.7Participants shall be discontinued during the trials in case of occurrence of serious adverse reactions, severe complications, or condition deterioration. Moreover, the clinical trials will be suspended according to the doctors’ judgment, if required.Regarding management of discontinued cases, the doctor will take some relevant clinical disposals after discontinuation. Subsequently, the doctor will perform a detailed investigation of the reasons and retain the obtained observation data. The last obtained result will be used as the final result and included in the analysis of efficacy and adverse reactions.2.8These will include cases with violation of inclusion or exclusion criteria; patients included in the wrong treatment group; and patients who withdraw from the trials, use prohibited drugs or treatments in combination, and with insufficient data.2.92.9.1The patients will be randomly divided into the experimental and control groups (both n = 126). Eighty-four cases will be obtained in the Second Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Changchun University of Chinese Medicine, and China Rehabilitation Research Center.2.9.2The experimental group will undergo acupuncture at the appropriate neck acupoints as follows:Nape cluster acupuncture treatment of the oral stage will employ the bilateral Fengchi, Gongxue, swallowing 2, Jiachengjiang, Xiaguan, Yingxiang, Lianquan, Waijinjin, Waiyuye, and Tongzhong acupoints.Nape cluster acupuncture treatment of the pharynx stage will employ the bilateral Fengchi, GongXue, swallowing 2, Tiyan, Zhiqiang, swallowing 1, Phonation, Lianquan, Zhifanliu, Waijinjin, Waiyuye and Tongzhong acupoints.The acupoints and acupuncture methods are described below. The Fengchi acupoint is at the same level as the Fengfu point, at the depression between the trapezius and sternocleidomastoid muscles. The needle tip will be slightly tilted downward and inserted 2 cm deep into the Adam's apple. The GongXue point is 2 cm below the FengChi point and flushes with the lower lip. The needle will be inserted 2 cm deep into the opposite lip. The swallowing 2 point is located at the flat chin lip groove and posterior edge of the sternocleidomastoid muscle. Here, the needle tip will be directly inserted approximately 1.5 to 2 cm into the opposite side and retained for 30 minutes. The JiaChengJiang point is on the face, 1 inch adjacent to the Chengjiang point. The XiaGuan point is ventral to the tragus and mandibular condyle at the depression formed by the zygomatic arch and mandibular notch. The Yingxiang point is located half an inch from the alar margin midpoint in the nasolabial groove.The Lianquan point is located at the hyoid depression above the Adam's apple, with the needle being inserted 3 cm into the tongue base, twisted for 10 to 15 s, and then removed. The Waijinjin and Waiyuye acupoints are 1 cm on each side of the Lianquan point, with the needle being inserted 2 to 3 cm into the tongue base, twisted for 10 s, and then removed. The Tongzhong point (Juquan point) is at the middle of the tongue, with the needle being inserted 0.1 cm into the tongue and then removed. For the Fengchi, Gongxue, Jiachengjiang, Xiaguan, and Yingxiang acupoints, the needles will be retained in the insertion point for 30 min.The Tiyan point, the needle will be directly inserted forward and downward at 1 to 1.5 cm at the depression in front of the mastoid and the lower earlobe edge. The Zhiqiang point is located between the hyoid bone and thyroid cartilage notch, with the needle being directly inserted approximately 0.3 to 0.5 cm, twisted for 10 to 15 s, and then removed. The swallowing 1 point is located at 0.5 cm adjacent to the midline between the hyoid bone and laryngeal node, with the needle being inserted approximately 0.5 cm laterally along the skin, twisted for 15 s, and then removed. The phonation point is located 0.5 cm below the midline of the Adam's apple; specifically, between the thyroid and cricoid cartilages. The needle will be inserted approximately 0.5 cm along the skin, twisted for 10 to 15 s, and then removed. The Zhifanliu point is located 1 cm behind the pronunciation point, the posterior edge of the cricoid cartilage, and the cricopharyngeal muscle. Regarding operation, the needle will be obliquely pierced 0.5 cm inward along the skin and removed after twisting for 10 to 15 s.The control group will undergo swallowing function rehabilitation training. A tongue muscle rehabilitation device will be used to perform tongue muscle activities in the directions of front and back, up and down, left and right, oblique down, etc. Moreover, the patients will perform training involving mouth opening and closing, chewing, frowning, smiling, cheek puffing, and the Mendelssohn technique to promote swallowing. The lingual and soft palates will be stimulated using ice and acid.2.9.2.11.Jaw, face, and cheek exercisesThe participant's mouth will be maximally opened, held for 5 s, relaxed for 3 s, and repeated 5 times. Moreover, the participants will close the lips, puff their cheeks, hold for 5 s, relax for 3 s, and repeat 5 times. Air will be rapidly moved around the left and right cheeks by puffing the cheeks 10 times. The participants will bare their teeth for 5 s for five sessions with 3-s relaxation intervals. Moreover, the participants will purse their lips for 5 s, relax for 3 s, and repeat the procedure five times. Finally, they will perform chewing movements ten times with 20-s intervals in between.2.Lip exercisesThe participants will clench their teeth and pronounce Yi, hold for 5 s, and relax for 3 s for a total of five sessions. Moreover, the participants will purse their lips and pronounce Wu, hold for 5 s, and relax for 3 s for a total of five sessions. Next, the participants will alternately pronounce Yi and Wu for 3 s each and repeat the pronunciation twice in each session. The participants will complete three sessions and relax for 5 s. With tongue depressors on both lips, the participants will forcefully pull out the tongue depressor, resist with closed lips, hold for 5 s, and relax for 2 s, with the procedure being repeated five times. Regarding the blowing exercise, the participants will blow five times and breathe evenly for two cycles after each expiration, with a 20-s interval break.3.Tongue exercisesThe participants will remove the tongue as far as possible out of the mouth, hold for 5 s, and retract and relax for 3 s, with this procedure being repeated ten times. Moreover, the participants will lift the tongue to the back of the front teeth, hold for 5 s, and relax for 3 s, with this procedure being repeated ten times. Furthermore, in each session, the participants will extend the tongue tip to the left and right lip angle for 2 s, with this procedure being repeated three times. The participants completed three sessions and relaxed for 5 s. Moreover, the participants will lick their lips with the tongue tip ten times with 20-s break intervals.4.Velopharyngeal closure exercisesAn ice cotton stick will be used to stimulate the palatopharyngeal arch; simultaneously, the participants will pronounce “a” thrice, relax for 3 s, and repeat the procedure five times with 10-s break intervals.5.Respiratory exercisesThe participant will breathe deeply by the nose while lifting the shoulders, and subsequently breathe out and relax. Each session will involve two respiratory cycles. The participants will complete three sessions with even breathing and a 5-s rest after each session. A 10-s break will be taken.6.Mendelsohn's techniqueHere, the patients will be instructed to swallow empty air. The therapist will push the patient's throat using the hand to promote swallowing and the procedure will be repeated five times, with a 10-s break.2.9.2.21.Tongue trainingThe participant will extend the tongue from the mouth as much as possible, maintain it there for 5 s, and then retract and relax for 3 s, with the procedure being repeated 10 times. Moreover, they will open the mouth, lift the tongue to the back of the front teeth, maintain it there for 5 s, relax for 3 s, with the procedure being repeated 10 times. Additionally, in each session, the participants will extend the tongue tip to the left and then the right lip corner thrice for 2 s. Further, the participants will complete three sessions with 5-second relaxation breaks. Finally, the participants will lick the lips with the tongue tip and repeat 10 times, with a 10-s break.2.Velopharyngeal closure training:To stimulate the velopharyngeal arch with an ice cotton swab will be used, and participant will simultaneously be instructed make a sound three times, relax for 3 s, repeat five times, and hold a break for 10 s.3.Breathing training:The patients will be instructed to deeply inhale through the nose while lifting the shoulders; subsequently, they will shrink the mouth, exhale, and relax. Each session will be comprised of two breathing cycles. The participants will complete three sessions while breathing and resting for 5 s after each session. A 10-s break will be provided in between.4.Mendelssohn manipulationThe patient will swallow empty air, with the therapist pushing the patient's throat using the hands to promote swallowing. The procedure will be repeated five times with a 10-s break.5.Direct feeding training.We will adopt empty, interactive, and side swallowing methods using viscous and uniform food for feeding training.2.102.10.1The primary outcome indicators will be the SSA and VFSS, while the secondary outcome indicators will be the sEMG signal analysis and MBI. The common evaluation index will be the SS-QOL while the follow-up indicators will be the SS-QOL and SSA.2.10.2Measurements will be obtained before the intervention, 1 week after intervention, and 2 weeks after intervention. The follow-up time point will be 1 month after the end of the intervention.2.11After the research plan is formulated by the project undertaking unit and approved by experts, it will be reported to the ethics committee of the project undertaking unit for approval. Each participating unit will report to the ethics committee to review its feasibility. Before study enrollment, the patients will be informed regarding the research background, purpose, benefits, and risks. The patients will be enrolled after providing informed consent. The adherence of the subjects was improved by scientific informed consent, active health education, humanistic care and long-term follow-up plan,.2.12To ensure the research quality and data validity, we will conduct unified and standardized training for the study researchers, formulate clinical research SOPs, and ensure that the clinical research consistently follows the operating procedures. Regular monitoring will be used to ensure the study's authenticity and reliability.2.13To establish a database, all data will be double-entered using computer software to compile a data entry program. The data manager will ensure the completeness and integrity of the data in the case report form report entered into the computer. The data will be blindly reviewed; moreover, after correct confirmation, the data will be locked by the main researchers and statistical analysts.2.14The main researchers of each unit will be assigned different user names and passwords to establish a central random system and data collection system. Before treatment, the patient information will be entered into the central random system; moreover, the patient will be screened according to the inclusion and exclusion criteria. Patients who meet the criteria will be randomly divided into the control and experimental groups.2.15In this study, due to the specificity of clinical acupuncture research, it is difficult to implement blind method. But it can be assessed by people who are not involved. We plan to assign specialized doctors from rehab centers as reviewers, and they will not be told how the subjects are grouped.2.16Referring to Liu Zhishun's “clinical study on acupuncture treatment of chronic moderate and severe dysphagia in stroke,” the effective rate of traditional rehabilitation is 80%, the estimated effective rate is 93.3%. According to the preset parameters, the effectiveness of the two groups were calculated with PASS 8.0 software, and 97 samples were needed for each group. At the same time, abscission and elimination were considered. The abscission rate was 30% and each group needed 126 samples. Therefore, 252 subjects were expected to be included in this study.2.17A third-party medical statistics professional  will perform statistical analysis. After the subject research plan and case report form are reviewed by experts, a third party will formulate a statistical analysis plan and determine the statistical software to use.1.Full Analysis Data Set (FAS): A total of 252 random numbers were designed in this study to exclude subjects in a minimum and reasonable manner according to the principle of intentionality analysis. This data set includes all subjects receiving treatment. Missing data for key indicators are carried forward to the closest observation (LOCF).2.Per-Protocol Data Set: those who have good compliance (who have received at least 2/3 or more of the courses of treatment), whose main indicators of observation are not missing, who have basically not violated the Protocol, and who meet the inclusion criteria without any exclusion indicator constitute the current PP population. Missing data in this data set is not carried forward, but is treated as missing data.3 Clinical patients are commonly in the oral, pharyngeal, or both stages. The mouth stage is the chewing food process to form a bolus, with the tongue moving the bolus back and forth before entering the pharynx. Dysfunction involves weakness of the facial muscles, including the chewing, labial, and tongue muscles, which impedes food bolus formation and smooth delivery to the pharynx. The pharyngeal stage refers to the process of the food bolus entering the oropharynx into the esophagus. This stage involves abnormal pharyngeal muscle and laryngeal function, which often causes aspiration, pharynx retention, and hoarseness.Dysphagia refers to swallowing dysfunction. The SSA includes 10 items divided into four levels: independent, less dependent, moderately dependent, and completely dependent. The VFSS allows dynamic assessment of the swallowing function of the mouth, pharynx, and upper esophagus using TV fluoroscopy. The VFSS is considered the “gold standard” for evaluating swallowing function. It is widely used for the comprehensive evaluation of the patients’ oral, pharyngeal, and esophageal swallowing conditions. The sEMG allows evaluation of the physiological process of swallowing. It is a simple and reliable screening that allows preliminary identification of dysphagia and has advantages of accuracy, rapidity, and non-invasiveness. fMRI is a safe, non-invasive, and non-radiation imaging method with high temporal and spatial resolution. Anatomical and functional images can be simultaneously obtained in a single imaging session.The SSA is divided into three parts: clinical examination, observation of patients swallowing 5 mL of water, and observation of patients swallowing 60 mL of water. It has a total score of 46, with the lowest possible score being 17. A higher cumulative score of each item indicates worse swallowing function. Some studies have reported that SSA is more feasible and suitable for evaluating dysphagia. This large-scale, multicenter, randomized controlled trial will employ neck acupuncture to assess its treatment effect on post-stroke swallowing dysfunction.Acupuncture is commonly applied in stroke rehabilitation; however, there remains no high-quality evidence regarding acupuncture efficacy. There are numerous acupuncture types, including scalp, body, nape, and latch acupunctures, as well as awn needles, which are all used for the treatment of post-stroke swallowing dysfunction. Neck acupuncture can directly act on the throat, as well as stimulate hypoglossal and recurrent laryngeal nerves. Fengchi and other acupoints can stimulate the occipital artery, occipital vein, vertebral artery, and vertebral vein, as well as promote blood circulation.Conceptualization: Qiang Tang, BiYing Liang, Luwen Zhu.Investigation: Qiang Tang, BiYing Liang, Runyu Liang, Shiqiang Zhang.Supervision: Luwen Zhu.Writing – original draft: Qiang Tang, BiYing Liang.Writing – review & editing: Qiang Tang, BiYing Liang.


Thunnus obesus) skin collagen have been successfully studied. The hydrolysates  were the result of the hydrolysis of collagen using alcalase, neutrase, and savinase. The peptide fractions  were the fractions obtained following ultrafiltration of the hydrolysates. The antioxidant activities of the hydrolysates and peptide fractions were studied using the DPPH method. The effects of collagen types, enzymes, and molecular sizes on the antioxidant activities were analyzed using profile plots analysis. The amino acid sequences of the peptides in the fraction with the highest antioxidant activity were analyzed using LC-MS/MS. Finally, their bioactivity and characteristics were studied using in silico analysis. The hydrolysates and peptide fractions provided antioxidant activity (6.17–135.40 µmol AAE/g protein). The lower molecular weight fraction had higher antioxidant activity. Collagen from pepsin treatment produced higher activity than that of bromelain treatment. The fraction from collagen hydrolysates by savinase treatment had the highest activity compared to neutrase and alcalase treatments. The peptides in the PBN and PPS fractions of <3 kDa had antidiabetic, antihypertensive and antioxidant activities. In conclusion, they have the potential to be used in food and health applications.The hydrolysates and peptide fractions of bigeye tuna ( Bioactive peptides are protein fragments that provide nutrition and positive benefits for body functions . An arraVarious proteins can act as sources of bioactive peptides ,19,20,21Research on bioactive peptides from collagen by-products of the fisheries industry is important for the following reasons. First, because it produces bioactive compounds that have the potential to be utilized in food, pharmaceuticals, or cosmetic products. Second, because it can provide added value for the fisheries industry. Third, because it is a form of concern in managing environmental impacts that may arise from the by-products of the fisheries industry.Collagen peptides can be prepared by a two-step hydrolysis process of the by-products of the fisheries industry . HydrolyThe study of the bioactive properties of a peptide can be carried out either in vitro, in vivo, and in silico. Research on peptides in vitro and in vivo can be quite expensive, requires a long time and is painstaking work. The use of web-based in silico methods is able to facilitate the research of bioactive peptides ,35,36,37Tuna is classified as a fish in high demand worldwide. Indonesia is one of the largest tuna-producing countries in the world . Tuna isThunnus obesus), are still limited. Therefore, the objectives of this study were to study the hydrolysates and peptide fractions of bigeye tuna skin collagen as antioxidant compounds; to explore the effects of several factors  on the antioxidant activities produced; to analyze the amino acid sequences of the peptides in the fractions with the highest antioxidant activity; and to explore the bioactivities and characteristics of the peptides in the fractions with the highest antioxidant activity by in silico studies.Comprehensive studies on bioactive peptides from collagen by-products of the tuna fishery industry, especially from collagen of the skin of tuna bigeye (Thunnus obesus) was obtained from PT Maluku Prima Makmur, Ambon, Indonesia. Other materials were 1-butanol, sodium hydroxide, acetic acid  100%, pepsin , sodium chloride GR for analysis, ethanol absolute for analysis, methanol gradient grade for liquid chromatography, L(+)-ascorbic acid, buffer solution (di-sodium hydrogen phosphate) pH 7.00 (20 °C), purchased from Merck ; bromelain from pineapple stem , alcalase or protease from Bacillus licheniformis (≥2.4 U/g), 2,2-diphenyl-1-picrylhydrazil, bovine serum albumin, purchased from Sigma-Aldrich ; neutrase 0.8 L (0.8 AU-N/g), savinase 16 L (16 KNPU-S/g), purchased from novozymes A/S ; distilled water, purchased from IKA Pharmindo Putramas Co. .The skin of tuna  is the amount of enzyme that catalyzes the reaction of 1 µmol of substrate per minute. One Novo Protease Units (NPU) is the amount of enzyme that hydrolyzes casein at such a rate that the initial rate of formation of peptides/minute corresponds to 1 micromole of glycine/minute, meanwhile 1 KNPU (Kilo NPU) equals 1000 NPU. One Anson unit (AU) is approximately equal to 3 KNPU. Baseline lipase, amylase, and free and total protease activity levels were determined for each sample in accordance with procedures specified by the Fédération Internationale Pharmaceutique (FIP) and the European Pharmacopoeia (PhEur). Results were converted from FIP/PhEur units to United States Pharmacopeia (USP) units. For protease, 1 FIP/PhEur unit = 62.5 USP units.Thunnus obesus) skin, which was a fishery by-product. The skin was from Ambon and brought to the research laboratory in Bogor. The skin was stored in the freezer (−28 °C). The skin was cleaned, cut into small pieces (0.5 × 0.5 cm2), and then lyophilized for 14 h using a TFD5503 Bench-Top freeze dryer . The lyophilized skin was used for collagen extraction.The skin was prepared according to the method . The skiv/v) butyl alcohol solution. Soaking was carried out in the refrigerator at 4 °C for 24 h. The ratio of skin and soaking solution was 1:10 (w/v). The soaking solution was changed every 12 h. At the second stage, the pre-treated skin was extracted using dilute acetic acid (0.5 M) containing 0.1% protease (w/v). The proteases used were bromelain and pepsin. Extraction was carried out at 4 °C (in the refrigerator) for 72 h. The ratio of pre-treated skin to extraction solution was 1:40 (w/v). During the pre-treatment and extraction processes, the solution must be shaken frequently. The suspension was then centrifuged using a refrigerator centrifuge Hermle Z 32 HK  at 6000× g for 30 min at 4 °C. The supernatant was separated from the precipitate. The supernatant was then salted-out by sodium chloride to a final concentration of 2.0 M. The precipitate was collected by centrifugation at 6000× g for 30 min at 4 °C and then dialyzed with cold distilled water using a dialysis membrane, Spectra/Por® Dialysis Membrane MWCO 6–8000, Regenerated Cellulose . The obtained wet collagen was then lyophilized for 14 h using TFD5503 Bench-Top freeze dryer , so that dry collagen (freeze dry collagen) was obtained. Collagen obtained from skin extraction using acetic acid and bromelain was called bromelain soluble collagen (BSC). The collagen obtained from skin extraction using acetic acid and pepsin was called pepsin soluble collagen (PSC).Collagen extraction was carried out according to the method in . The fir® Ultra-15 Centrifugal Filters Ultracel®-3K and 10 K, regenerated cellulose) .Hydrolysates and peptide fractions were prepared according to the method in . The hydg in a Hermle Z 32 HK . The resulting supernatant was a hydrolysate. Three hydrolysates were produced from each collagen.Hydrolysate preparation was carried out as follows. A total of 200 mg of freeze dry collagen was dissolved in 10 mL of solution A (20 mg of an enzyme in 50 mL of phosphate buffer pH 7) in a 50 mL covered Erlenmeyer. The mixture was homogenized and hydrolyzed at pH 7, temperature 50 °C for 6 h in a SW22 Shaking Water Bath . A shaking water bath was used to obtain the homogeneity condition of the mixture and temperature. Then, the enzyme was inactivated. Enzyme inactivation was carried out in boiling water for 10 min. The mixture was cooled, then centrifuged for 30 min at 6000× g for 30 min. Two peptide fractions were obtained, which were sized above and below 10 kDa. Then, the peptide fraction <10 kDa was replaced into a 3 kDa ultrafiltration tube. The filtration process using a 3 kDa ultrafiltration tube was carried out in the same way as the 10 kDa tube ultrafiltration process. In the ultrafiltration process with a 3 kDa tube, two peptide fractions were obtained with the molecular sizes 3–10 and <3 kDa. Thus, each hydrolysate produced three peptide fractions.The preparation of peptide fraction was as follows. A total of 10 mL of the hydrolysate was placed into a 10 kDa ultrafiltration tube, then centrifuged by a refrigerator centrifuge Hermle Z 32 HK  at 6000× Analysis of protein concentrations for hydrolysates and peptide fractions were carried out using the method in . The abs−1). Approximately 100 µL of the solutions were added with 100 µL of DPPH (125 µM in ethanol). The mixtures were homogenized and incubated for 30 min at room temperature and protected from light. Ascorbic acid was used as a positive control. The absorbances were determined at 517 nm using a microplate reader . The same procedures were conducted to measure the antioxidant activities of the peptide fractions. Antioxidant activities for the hydrolysates and peptide fractions were reported in µmol AAE/g protein.The antioxidant activities of the hydrolysates and peptide fractions were determined using the scavenging method against DPPH radicals according to the method . Each hythe SPSS program, (2) input data in the variable view, (3) set the data view, (4) took multivariate profile plots, (5) clicked continue, (6) clicked plot, set the dependent variables and the fixed factors, (7) clicked ok, (8) saved, and (8) opened the SPSS output.The antioxidant activities obtained at point 2.4 were then also studied statistically analyzed using the analysis of profile plots in the SPSS program. These analyses were conducted to see the influence of several factors on the antioxidant activities produced. The factors studied were collagen types , collagen hydrolyzing enzymes , and molecular sizes . The procedure for analyzing profile plots was as follows: (1) opened The PBN and PPS fractions <3 kDa were compounds that had the highest antioxidant activity of all the tested compounds using the scavenging method against DPPH radicals at point 2.4. Amino acid sequence analysis was performed on the PBN and PPS fractions <3 kDa. The amino acid sequences of the peptides in the PBN and PPS fractions of <3 kDa were studied according to the method in . The amiPrediction of the bioactivities of peptides in the PBN and PPS fractions of <3 kDa was studied using a web-based in silico application that can be accessed on the BIOPEP website . The anaThe study of the characteristics of bioactive peptides in the PBN and PPS fractions of <3 kDa was explored using a web-based in silico analysis that could be accessed on the PepDraw website . The stuData are presented as the mean ± standard deviation (SD). The data were conducted using Microsoft Excel 2016  for the one-way ANOVA and IBM SPSS Statistics Version 20 for Windows  for the profile plots analysis.Thunnus obesus) skin using dilute acetic acid (0.5 M) containing bromelain and pepsin. The collagen extracted using dilute acetic acid and bromelain was called bromelain soluble collagen (BSC). The collagen extracted using dilute acetic acid and pepsin was called pepsin soluble collagen (PSC). The isolation process was carried out in two stages, pre-treatment and collagen extraction. Pre-treatment aimed to remove the non-collagenous part, so that pure collagens were expected to be obtained. The yield and chemical characteristics of these collagens (BSC and PSC) have been described in our previous study [Collagens used for the preparation of hydrolysates and peptide fractions in this study were collagens obtained from the extraction process of bigeye tuna  by hydrolyzing the collagens (BSC and PSC) using three types of proteases, namely alcalase, neutrase, and savinase. The hydrolysates were dissolved as the supernatant. HBA, HBN, and HBS were BSC hydrolysates, which were hydrolyzed by alcalase, neutrase, and savinase, respectively. And, HPA, HPN, and HPS were PSC hydrolysates, which were hydrolyzed by alcalase, neutrase, and savinase, respectively. Thus, there were six hydrolysates resulting from the hydrolysis of BSC and PSC using these three proteases. In other words, each collagen produces three hydrolysates.The peptide fractions were obtained from the semi-purification process of hydrolysates using two 10 and 3 kDa ultrafiltration membranes. PBA, PBN, and PBS were the ultrafiltrated fractions of HBA, HBN, and HBS, respectively. PPA, PPN, and PPS were the ultrafiltrated fractions of HPA, HPN, and HPS, respectively. Three peptide fractions with different molecular sizes were produced for each hydrolysate . Thus, there were 18 peptide fractions obtained from the ultrafiltration of six hydrolysates using two ultrafiltration membranes of 10 and 3 kDa.The protein content (protein concentration) of hydrolysates and peptide fractions were analyzed using the Lowry method. The results are summarized in The antioxidant activities of the hydrolysates and peptide fractions were tested using the scavenging technique against 2,2-difenil-1-picrylhydrazil (DPPH) radicals. The resulting antioxidant activities are summarized in Antioxidant activities were then studied concerning the effects of three factors, namely the collagen types (BSC and PSC), the enzymes used to hydrolyze the collagens , and the molecular sizes . The effects of these factors on antioxidant activities were studied statistically using IBM SPSS Statistics 20 and expressed as profile plots. The resulting profile plots are shown in The PBN and PPS fractions <3 kDa were molecules that showed the highest antioxidant activity among all samples with in vitro studies using the DPPH method. The amino acid sequences of the peptides in the PBN and PPS fractions of <3 kDa were analyzed using LC-MS/MS . The amino acid sequences of the peptides in the PBN and PPS fractions of <3 kDa are summarized in It can be seen that the PBN and PPS fractions <3 kDa may consist of several peptides, one peptide was for PBN fraction <3 kDa, and 12 peptides were for PPS fraction <3 kDa. All peptides in the PBN and PPS fractions of <3 kDa obtained from the result of LC-MS/MS analysis possessed amino acid residues of 10 to 23 amino acids. The molecular mass varies from 1063.5031 to 2624.1539 Da.Prediction of bioactive properties of the peptides present in the PBN and PPS fractions <3 kDa has been studied using in silico analysis available on the BIOPEP website. The results of the prediction of bioactive properties for the peptides in the PBN and PPS fractions of <3 kDa can be seen in The characteristics of each of these bioactive peptides were studied using in silico analysis available on the PepDraw website. The results were then studied statistically for the maximum, minimum, first quartile (Q1), second or median quartiles (Q2), and third quartile (Q3) values. The characteristics of the bioactive peptides in the PBN fraction of <3 kDa are summarized in The number of bioactive peptides characterized by amino acid residues at C-terminal and N-terminal present in the PBN and PPS fractions <3 kDa is summarized in Thunnus obesus) using dilute acetic acid and bromelain, as well as using dilute acetic acid and pepsin. The collagen obtained was called bromelain soluble collagen (BSC) for collagen extracted using dilute acetic acid and bromelain, and pepsin soluble collagen (PSC) for collagen extracted using dilute acetic acid and pepsin.Collagens were successfully extracted from the skin of bigeye tuna (Both bromelain soluble collagen and pepsin soluble collagen provide high collagen yields (data not shown here). Reports on the collagen extraction with bromelain by other researchers are still limited . The higEach protease enzyme works specifically. The bromelain used in this study was stem-bromelain (Sigma-Aldrich), which can be suspended in acetate buffer, pH 4.5 (1 mg/mL), producing a cloudy white suspension. Stem-bromelain is a cysteine endopeptidase with broad specificity for protein cleavage. Stem-bromelain works efficiently on synthetic substrates containing Arg-Arg ,60. PepsThunnus obesus) through a two-stage hydrolysis process. The first step is the enzymatic hydrolysis stage of the skin into collagen. The second step is the enzymatic hydrolysis stage, which converts the collagen into hydrolysates.Hydrolysates were obtained from the skin of bigeye tuna  were further hydrolyzed using three protease enzymes, namely alcalase, neutrase, and savinase. Alcalase (Sigma-Aldrich) is a serine endoproteinase with broad specificity for native and denatured proteins. Alcalase is active under alkaline conditions, the optimum pH is between 6.5 and 8.5 and has an optimal temperature of 60 °C. Neutrase (Novozymes) is an enzyme with metallo-endoprotease activity that hydrolyzes internal peptide bonds. Savinase (Novozymes) is serine endoproteinase that hydrolyzes internal peptide bonds. Thus, these enzymes will cleave the peptide bonds in the collagens (BSC and PSC) at certain positions, resulting in hydrolysates (collagen hydrolysates) with certain properties.After obtaining the hydrolysates, the next step is the preparation of the peptide fractions, which were obtained through a semi-purification process of the hydrolysates (collagen hydrolysates) by ultrafiltration technique using 10 and 3 kDa membranes, which separates the peptides based on differences in molecular weight. The choice of membrane size was adjusted to the bioactive target in this study. Small peptide fractions are expected to have bioactive properties with higher activity than the large peptide fractions.All the hydrolysates and peptide fractions obtained were clear and odorless. These results are in agreement with previous researchers, who also stated that hydrolyzed collagen was neutral and colorless .Oxidation is an essential reaction in all living organisms. The formation of free radicals and other reactive oxygen species (ROS) is unavoidable during the process of oxidative metabolism. These reactive radicals play an important role in signal transduction. However, excessive free radicals can cause destructive effects on living tissues and foodstuffs  ,64.Antioxidants work by donating electrons or hydrogen to free radicals. Antioxidants can also stop chain reactions by removing singlet oxygen, deactivating metal catalysts and removing radical initiators or intermediates ,66,67. SStudies on antioxidant peptides have been reported. These antioxidant peptides were found to have relatively low molecular weight, simple structure, high activity, easy absorption, better stability in different situations, and no harmful immune reactions .Thunnus obesus) using a scavenging technique against 2,2-difenil-1-picrylhydrazil (DPPH) radicals. This scavenging technique against DPPH radicals was chosen because it is one of the commonly used techniques in analyzing the antioxidant activity of a compound in vitro. The principle is the DPPH provides maximum absorbance at a wave-length of 517 nm. The presence of antioxidant properties in the hydrolysates and peptide fractions was shown by a decrease in absorbance or a decrease in the intensity of the purple color. In the ascorbic acid solutions of 0.00, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00 mg/L, which were used as positive controls, the color change was clearly visible, as purple turned into yellow. Meanwhile, the color change was not as intensive for the hydrolysates and peptide fractions.In this study, we tested the antioxidant activities of hydrolysates and peptide fractions of collagen from the skin of big eye tuna (The two highest antioxidant activities were produced by the PBN fraction <3 kDa and PPS fraction <3 kDa (wherein these fractions consisted of peptides <3 kDa). This result is consistent with previous studies, which also found that small peptides gave better results than large peptides ,33.In addition, for future research, several test methods can be applied to produce more comprehensive observations of the antioxidant activity study. The selection of the analytical method, the solvent used, the concentration of the test compound, the concentration of the radical compound, and the incubation time also need to be considered.The antioxidant activities obtained by scavenging technique against DPPH radicals were studied statistically and expressed as profile plots. The factors observed were the collagen types, the enzymes used to hydrolyze collagen into hydrolysates and peptide fractions, and the molecular sizes.Thunnus obesus) using the same method, except for the enzymes used, namely bromelain for BSC and pepsin for PSC. The two collagens gave different effects on antioxidant activity even though they were obtained from the same skin, namely from bigeye tuna skin. The estimated marginal average value of the antioxidant activity of PSC is higher than that of BSC. This shows that in producing antioxidant compounds, overall, PSC gives better results than BSC.The types of collagens used in this study were bromelain soluble collagen (BSC) and pepsin soluble collagen (PSC). These collagens were obtained from the skin of bigeye tuna , the peptide fraction >10 kDa , the peptide fraction 3–10 kDa , and the peptide fraction <3 kDa .The results of statistical analysis expressed as profile plots showed that the peptide fraction <3 kDa gave the highest antioxidant activity, which was shown from the estimated marginal average value. As we all understand, the smaller the size of the molecule, the less the steric hindrance the compound has, making it easier to react with other compounds. Thus, the peptides in the peptide fraction <3 kDa were easier to react with the DPPH radicals, compared to the peptides in the larger peptide fraction.Interestingly, the hydrolysates gave a better effect on the resulting antioxidant activities compared to the peptide fractions 3–10 kDa and the peptide fractions >10 kDa. This may be due to the different types of peptides in the hydrolysates.However, for the semi-purified peptide fraction, the small peptide fraction (the peptide fraction <3 kDa) gave better antioxidant activity than the large peptide fraction (the peptide fractions 3–10 and >10 kDa). In general, antioxidant activity was found to be inversely related to molecular size. For the peptide fraction <3 kDa, the order of antioxidant activity was PBN > PPS > PPN > PPA > PBS > PBA.A comprehensive study on the effect of these factors on the antioxidant activity produced, especially for the antioxidant activity of hydrolysates and peptide fractions from collagen by-products of the fishery industry, has not been reported. However, our results are consistent with previous studies ,33 that The combination of in vitro and in silico methods can increase the discovery of bioactive peptides for further applications in food and health . This coIn the PBN fractions of <3 kDa, one peptide with a residue length of 23 amino acids was identified according to the results of the LC-MS/MS analysis. From the in silico results with the BIOPEP program, 16 dipeptides from this peptide sequence were indicated to possess bioactive properties. The sixteen dipeptides were PW, FP, LF, VG, GR, DA, GS, HG, DY, YV, AD, HF, SL, WC, TV, VH. These 16 dipeptides were grouped as antidiabetic peptides 81.25%, antihypertensive peptides 56.25% and antioxidant peptides 6.25%. Among these peptides, there were those predicted as multifunctional peptides, namely 25% as antidiabetic and antihypertensive ; and 6.25% as antidiabetic and antioxidant (PW). The identified antidiabetic peptides were alpha-glucosidase inhibitors 6.25%, dipeptidyl peptidase III inhibitors 12.50%, and dipeptidyl peptidase IV inhibitors 62.50%. Antihypertensive peptides were identified as ACE inhibitors.In the PPS fractions of <3 kDa, 12 peptides with residue lengths of 10–20 amino acids were identified according to the results of LC-MS/MS analysis. From the in silico results with the BIOPEP program, there were 83 peptides from the peptide sequences that have bioactive properties. The eighty-three peptides were KD, IY, GPP, LK, GAA, PGP, PP, EA, AD, WM, SM, PF, YG, GE, PR, GPRG, GPRGP, GPRGPP, GPR, GP, PG, GPGG, LSP, IG, GS, NY, MW, VF, FP, NK, GEP, KG, GT, TP, RG, ILP, IL, AP, GA, VG, GL, MG, QG, GD, EV, PQ, VGP, DM, MGP, AG, GG, TG, GRP, RP, GR, NG, DG, GPA, AA, AGP, QGP, GV, GAGP, SP, SL, WC, DR, PN, SW, YD, KK, EP, NP, PPG, PS, LP, DN, NV, PK, QL, VI, PA, GPAG. These 83 peptides were grouped as 73.49% antidiabetic peptides, 61.45% antihypertensive peptides, 6.02% antioxidant peptides and 15.66% peptides with other bioactive properties . Among these peptides, there were those that function as multifunctional peptides, namely 34.94% as antihypertensive and antidiabetic ; 2.41% as antioxidant and antihypertensive ; 1.20% as chemotactic, antithrombotic, and antiamnestic (PGP); 2.41% as antithrombotic, antihypertensive, antidiabetic, and antiamnestic ; 1.20% as antithrombotic, and antiamnestic (GPGG). The identified antidiabetic peptides had activity as alpha-glucosidase inhibitors in the amount of 3.61%, dipeptidyl peptidase III inhibitors in the amount of 7.23%, and dipeptidyl peptidase IV inhibitors in the amount of 62.65%. Antihypertensive peptides were identified as ACE inhibitors.Bioactive peptides in the PBN and PPS fractions of <3 kDa, namely antidiabetic peptides, antihypertensive peptides, antioxidant peptides, and peptides with other bioactive properties, generally have the N terminal of a non-polar (hydrophobic) amino acid. This finding is in agreement with previous researchers, who also linked bioactive properties with hydrophobicity . The bioResearch on bioactive peptides from the by-product of the tuna fishery industry has been successfully carried out using an integrated method. This combination research is considered cheaper, easier and faster in the research for bioactive peptides.Further hydrolysis using alcalase, neutrase and savinase, as well as their fractionation, showed the best antioxidants in the PBN fraction (Bromelain-Neutrase Peptide Fraction) of <3 kDa and PPS fraction (Pepsin-Savinase Peptide Fraction) of <3 kDa. The collagen types, enzymes, and molecular sizes affect the antioxidant activities produced. The in silico test showed the peptides in the PBN and PPS fractions of <3 kDa both had antidiabetic, antihypertensive, and antioxidant activities. Several bioactive peptides in the PBN and PPS fractions of <3 kDa were found to have multifunctional properties. The bioactive peptides in the PBN and PPS fractions of <3 kDa generally have the N-terminal of a non-polar (hydrophobic) amino acid.