Mental disorders cause substantial health-related burden worldwide. Mobile health interventions are increasingly being used to promote mental health and well-being, as they could improve access to treatment and reduce associated costs. Behavior change is an important feature of interventions aimed at improving mental health and well-being. There is a need to discern the active components that can promote behavior change in such interventions and ultimately improve users’ mental health.This study systematically identified mental health conversational agents (CAs) currently available in app stores and assessed the behavior change techniques (BCTs) used. We further described their main features, technical aspects, and quality in terms of engagement, functionality, esthetics, and information using the Mobile Application Rating Scale.The search, selection, and assessment of apps were adapted from a systematic review methodology and included a search, 2 rounds of selection, and an evaluation following predefined criteria. We conducted a systematic app search of Apple’s App Store and Google Play using 42matters. Apps with CAs in English that uploaded or updated from January 2020 and provided interventions aimed at improving mental health and well-being and the assessment or management of mental disorders were tested by at least 2 reviewers. The BCT taxonomy v1, a comprehensive list of 93 BCTs, was used to identify the specific behavior change components in CAs.We found 18 app-based mental health CAs. Most CAs had <1000 user ratings on both app stores  and targeted several conditions such as stress, anxiety, and depression . All CAs addressed >1 mental disorder. Most CAs  used cognitive behavioral therapy (CBT). Half  of the CAs identified were rule based  and the other half  were artificial intelligence enhanced . CAs used 48 different BCTs and included on average 15  BCTs. The most common BCTs were 3.3 “Social support ,” 4.1 “Instructions for how to perform a behavior,” 11.2 “Reduce negative emotions,” and 6.1 “Demonstration of the behavior.” One-third  of the CAs claiming to be CBT based did not include core CBT concepts.Mental health CAs mostly targeted various mental health issues such as stress, anxiety, and depression, reflecting a broad intervention focus. The most common BCTs identified serve to promote the self-management of mental disorders with few therapeutic elements. CA developers should consider the quality of information, user confidentiality, access, and emergency management when designing mental health CAs. Future research should assess the role of artificial intelligence in promoting behavior change within CAs and determine the choice of BCTs in evidence-based psychotherapies to enable systematic, consistent, and transparent development and evaluation of effective digital mental health interventions. Mental disorders affect approximately 18% of the global population , cause sCAs are computer programs designed to mimic human conversations . They inCAs often adopt a behavior change approach for mental health promotion, with cognitive behavioral therapy (CBT)  and behaAlthough there has been an increasing number of smartphone apps involving CAs for mental health , the capTherefore, to provide an overview and guidance for the development of future effective interventions , there iThis study aimed to systematically assess mental health and well-being CAs on Apple’s App Store and Google Play using the BCT taxonomy v1 . SpecifiIdentify the main characteristics of mental health CAsDescribe BCTs in the existing mental health CAsExamine the use of chosen BCTs in relation to the CA characteristics and types of mental health interventions delivered by the CAsAssess the technical aspects and quality of mental health CAs in terms of engagement, functionality, esthetics, and information using a validated scaleThe search, selection, and assessment of apps used an established methodology -40. The Information was extracted from the app store descriptions, app websites, and after testing the apps. The apps were evaluated based on the following assessment criteria that included four sections:General characteristics of the app and CA, as described on the app store product page, were evaluated, including the developer’s name, app store category, version number, user rating, cost (if any), number of downloads, country of origin, and target users. The features of the CAs [ the CAs  comprise the CAs , definedEvidence-based mental health interventions delivered by CAs, based on established clinical guidelines [idelines , a CBT cidelines , a CBT midelines , and comidelines ,46,47, widelines , as oppoidelines . The detBehavioral change techniques offered by the CA, based on the BCT taxonomy v1 [onomy v1  that desTechnical aspects and quality assessment of the app, based on the Health on the Net Foundation certification of mobile applications (mHONcode) [HONcode)  and MobiHONcode) ,52, wereHONcode) , the totmental health chatbot was also performed on April 29, 2022, and apps that were identified in the first 3 pages of the search results were added to enhance the rigor of the search. The inclusion and exclusion criteria for the apps are specified in A systematic search of the commercially available apps on Apple’s App Store and Google Play was performed on March 28, 2022, using 42matters , a mobilThe app selection process is illustrated in a flowchart . Two indApps were downloaded and assessed using an iPhone 13 (iOS 15.4.1), Samsung A52 , and Xiaomi Mi Max (Android 6.0.1) by at least 2 reviewers, including XL, LM, and 1 of 3 medical students who assisted with the project. If the apps were available on both platforms, both versions of the app were assessed and counted as 1 individual app. The data were extracted and tabulated using Microsoft Excel .To ensure consistency and comprehensiveness of the assessment, the research team created 3 user personas with information relating to mental health, including the user’s name, demographics, medical history, opening statements with CAs, and answers to common self-reported questionnaires . For example, 1 profile was of a user with moderate depression that included symptoms of sadness, insomnia, and anhedonia, with the user staying at home most of the time and not looking for a new job. These personas were used by the study team to select or generate user responses when interacting with the CA. The web-based BCT taxonomy training program  was compDescriptive statistics were used to analyze the CAs’ characteristics, evidence-based mental health features, number of BCTs, app store user ratings, and MARS ratings using Microsoft Excel. BCTs that were frequently observed in >75% of apps in each subgroup were included in the narrative analysis, following the definitions of common BCTs that ranged from 50% to 80% in previous app reviews ,32,55. TFor the classification of mental health and well-being topics, a free-for-use text analytics platform  was usedThe app search yielded 2657 apps (1616 from Android and 1928 from iOS) after removing duplicates . After tMore than half  of the included apps were available on both Android and iOS platforms . The aveMost CAs  were represented by a cartoon avatar, often representing an object or animal, whereas one-third  of the CAs had no visual representation. Half  of the CAs used a rule-based approach that delivered predetermined responses, whereas the other 9 CAs included AI algorithms that allowed free-text input from users. However, it was apparent that AI-enhanced CAs used rule-based dialogs for therapeutic delivery, although AI was primarily used for understanding user input, selecting conversation responses, and possibly for selecting the treatment approach but not delivering the therapeutic content, such as psychotherapeutic exercises or information about mental health. For example, in Talk to Poppy, although conversational starters with the CA varied from time to time, the delivery of health information by the CA remained fixed and did not change over multiple repeated interactions, suggesting a rule-based approach for the delivery of therapeutic content. Most CAs  were also part of multifunctional apps with other components such as human support and additional course content delivered not through CAs but via articles, stories, and motivational quotes for users, to cite a few.Most CAs were encouraging, motivating, and nurturing  or informal . Approximately half  of the CAs explicitly identified themselves as a CA and not a person. One gender-specific CA (Inwords) provided 9 different voice output options.Most CAs  engaged in multiple interactions with users to support the completion of long-term goals such as self-management of mental health conditions . AlternaAll 18 CAs aimed to improve mental health and well-being, including 2 apps  that addressed only general well-being. Most CAs targeted ≥1 mental health issues such as stress , anxiety , and depression  and often focused on all 3 mental health topics . General wellness topics included suggestions to reduce negative emotions  and advice to improve the quality of sleep , encourage personal growth , and manage relationships . Most CAs mentioned using evidence-based therapies, mainly CBT . Conversely, 2 apps  did not specify the therapeutic approach or techniques offered by the app.In general, CAs included an average of 3.33 (SD 1.69) therapeutic techniques. Most CAs  offered patient education about the symptoms, diagnosis, or treatment of mental health conditions. However, less than half  of the apps included information about specific mental disorders such as depression and anxiety disorders. Most CAs  also offered relaxation techniques such as breathing exercises, mindfulness, and muscle relaxation. Half of the apps included behavioral activation  and cognitive restructuring  techniques. However, only a few apps that offered cognitive restructuring  also included behavioral experiments to test the accuracy of users’ maladaptive thoughts or beliefs. None of the CAs used exposure techniques to challenge users to engage in anxiety-provoking activities. Apps also offered other behavioral strategies such as emotional agility exercises for emotional regulation (Woebot), scheduling worry time as part of CBT (InnerHour and Iona), and encouraging positive self-affirmations (Iona) as described by positive psychology. The other techniques offered by the apps included journaling , mood monitoring , and gratitude exercises . Two CAs (Happify and Magnify Wellness) included video games aimed at reducing users’ worry and stress .Approximately a fifth  of the apps included an in-app user community. In 1 app , users could chat with the members of the peer support network either individually or as a group. Moreover, 2 apps offered group chats with other users , and another app  shared users’ completed tasks with a few peers to encourage users. Some apps  also provided access to health professionals within the country of origin of the app .Few CAs included protocols to manage emergencies such as risk of suicide. In total, 2 CAs  were able to assess users at the risk of suicide by responding to user statements that included the words “suicide” or “suicidal thoughts.” Moreover, only approximately a third  of the apps included crisis helpline phone numbers limited to the country in which the app was developed or a limited number of countries. One app  provided a comprehensive list of crisis helpline numbers for 93 countries and regions .On average, the apps included 15.8 (SD 8.77) BCTs, ranging from 4 (Zifcare) to 30 BCTs Woebot; . In totaAI-enhanced CAs used a wider range of BCTs such as 5.6 “Information about emotional consequences” , 7.1 “Prompts/cues” , 1.4 “Action planning” , and 10.4 “Social reward”  compared with rule-based CAs.We observed that BCT 12.6 “Body changes” was commonly used in CAs targeting anxiety  and was demonstrated through breathing and relaxation exercises. CAs aimed at depression treatment used 2 additional BCTs. These were 5.6 “Information about emotional consequences”  as a justification for engaging in the suggested activities for depression and 12.6 “Body changes”  through relaxation, breathing, and stretching exercises.The use of BCTs in CAs also differed with respect to the therapeutic approach. CAs with CBT approaches commonly used 12.6 “Body changes”  and 5.6 “Information about emotional consequences” . CAs with mindfulness-based approaches also used the same additional BCTs 12.6  and 5.6 . In these CAs, BCT 12.6 was used in physical activity and relaxation exercises, whereas BCT 5.6 was used to explain the benefits of the suggested tasks. CAs offering positive psychology approaches commonly included 6 additional BCTs, such as BCTs 5.1, 5.4, and 5.6 relating to information about health and emotional consequences; 8.1 “Behavioural practice or rehearsal”; 12.4 “Distraction”; and 12.6 “Body changes” .Regarding the technical aspects, all CAs required a stable internet connection to function. Most apps  could send notifications to remind the users of scheduled activities and conversations with the CA, and most apps  also required users to sign up for an account. Only 1 CA  used phone sensors for additional data analytics by using the microphone to collect the user’s voice input for mood analysis.For the evidence base, only a few apps  were previously evaluated in studies such as qualitative research (Wysa and Happify) and randomized trials (Woebot), whereas other apps  reported ongoing studies.In general, apps showed high compliance with 5 (63%) out of the 8 mHONcode principles. Most apps clearly stated the qualifications of app development teams , provided disclaimers that the app does not replace health care providers’ advice , included the date of the last update , indicated funding sources or companies that commissioned the app , and had no advertisements . Notably, many apps  were either inconsistent or did not provide health references to the information included on the app or their websites. Finally, the majority of apps did not request for user consent before data collection  or check the use of the apps by minors , comprising engagement , functionality , esthetics , and information . The average MARS subjective score was 2.85 (SD 0.77), ranging from 1.38 to 4.38 . Figure This study systematically assessed the BCTs in 18 app-based mental health CAs in relation to their characteristics and quality. The CAs included 15 BCTs on average, of which the most commonly used were BCTs 4.1 “Instructions for how to perform a behaviour,” 3.3 “Social support ,” 11.2 “Reduce negative emotions,” and 6.1 “Demonstration of the behaviour.” Most CAs used similar BCTs across different therapeutic interventions and topics. CAs that incorporated AI used a larger number of BCTs than purely rule-based CAs.Of the common BCTs, only BCT 3.3 “Social support ” was frequently used in both short-term and long-term CAs. These findings suggest that the provision of social support may be one of the key functions of mental health CAs, as it was present even in short-term CAs that did not deliver therapeutic content over multiple sessions with the user. However, CAs that delivered interventions focusing on problem-solving or behavioral activation techniques did not offer emotional social support, but they offered other forms of support such as providing advice on where to seek social support and encouraging help seeking. This finding is in line with a previous review on the use of health apps for behavior change  and anotApproximately one-third  of CAs claiming to be based on CBT did not include core CBT concepts such as behavioral activation, cognitive restructuring, and behavioral experiments ,61. For A noteworthy finding was that AI-enhanced CAs included a greater variety of BCTs than rule-based CAs, an observation that has not been replicated in other similar studies. This finding suggests that the use of supervised machine learning and natural language processing techniques to predict, identify, and provide appropriate treatment options  may suppApproximately two-thirds of the CAs in this assessment had <500,000 Android downloads and <1000 user ratings, and only 2 CAs had >1,000,000 Android downloads and >20,000 ratings on both app stores. This contrasts with earlier reviews of mHealth apps that had >50,000 user ratings on average ,67,68. IAccording to the MARS assessment, the average mental health CA was highly functional, easy to learn and navigate, visually esthetic, appealing, and stylistically consistent. In contrast, it was lacking in engagement and interactivity as well as the quality of the information provided. This finding is consistent with previous assessments of mindfulness  and healWe used a search strategy for app search, selection, and assessment that was based on a systematic review methodology. The apps were evaluated based on a comprehensive list of criteria and a reliable procedure for identifying the active components of health interventions using the BCT taxonomy v1. The use of the web-based BCT taxonomy training program before data extraction also ensured consistency in the specification of BCTs across reviewers.However, this study has some limitations. First, although we aimed to retrieve all commercially available mental health apps with CAs through a systematic search strategy at the time of the search, owing to the expansive growth of digital health apps , relevanMost app-based mental health CAs were transdiagnostic and targeted several mental health issues such as stress, anxiety, and depression, which is reflective of a broad therapeutic focus. The provision of emotional support, instructions to perform a behavior, and strategies to reduce negative emotions were the most common BCTs identified in mental health CAs, supporting their role in the self-management of mental disorders. There were only a few highly used CAs, and most CAs were infrequently downloaded and used. Future CA developers should consider the quality of the information provided by CAs, user confidentiality, user access and appropriateness, and emergency management in the design of mental health CAs. There remains a need to clarify the role of AI in the provision of BCTs in CA-based interventions that target mental health promotion, and further research is required for the systematic identification of BCTs in evidence-based psychotherapies such that effective techniques can be effectively implemented in future mHealth interventions.


An electronic search was performed in nine databases. References from eligible studies were also selected to find more registers. A total of 28 studies met the eligibility criteria, and one more was recovered from the references of eligible studies, totaling 29 selected studies. The overall response rate, complete response, and partial response were higher in dogs treated with tyrosine kinase inhibitors than in dogs treated with vinblastine. The overall survival and progression-free survival of vinblastine-treated dogs were higher compared to tyrosine kinase inhibitors-treated dogs. Dogs with mutated KIT treated with tyrosine kinase inhibitors have longer overall survival and progression-free survival compared to those treated with vinblastine. It is important to consider the limitation of the study which should temper the interpretation of the results, videlicet, the extracted data lacked sample standardization and included variables such as animal characteristics, mutation detection methods, tumor characteristics, and treatment types which may have influenced the outcome of the study.The current gold standard treatment for canine mast cell tumors (MCT) uses vinblastine sulfate (VBL) as chemotherapy, although tyrosine kinase inhibitors (TKI) have recently been shown to be worthy candidates for treatment. This systematic review aimed to analyze the overall survival (OS), progression-free survival (PFS), overall response rate (ORR), and complete (CR) or partial response (PR) in dogs with MCT treated with TKI compared to standard VBL treatment. The systematic review was registered in the Open Science Framework (OSF) database under the identifier 10.17605/OSF.IO/WYPN4 (https://osf.io/, identifier: 10.17605/OSF.IO/WYPN4. Kinases are related to tumor development due to three main carcinogenic factors: their involvement in cell proliferation, cell survival, and tumor angiogenesis –7. TheseThe proto-oncogene c-kit encodes a receptor tyrosine kinase that plays an important role in mast cell growth and differentiation, and depending on the type of mutation in this proto-oncogene might cause ligand-independent activation of the c-kit. The incidence of mutations in the proto-oncogene c-KIT in canine mast cell tumor ranges from 14 to 26.2% and is primarily detectable in exons 8 and 11. Most mutations occur in exon 11 with an incidence of up to 64%, and ~50% of mutations are represented by internal tandem duplications –13. The 2 intravenously. These applications are completed weekly for 4 weeks, then bi-weekly . This systematic review was reported according to the preferred reporting items for Systematic Reviews and meta-analysis (PRISMA)   and regi(PRISMA) , 30 and (PRISMA) .The review was designed to answer the following question: “Is there evidence of overall response rate, overall survival, and progression-free survival of dogs with mast cell tumors treated with tyrosine kinase inhibitors?” following the PCC strategy for structuring, in which: P (population), C (concept), and C (context): (1) Population: dogs with mast cell tumors; (2) Concept: treatment with tyrosine kinase inhibitors (3) Context: alternative treatments for dogs with mast cell tumors compared to standard treatments; (4) Case studies with five or more individuals and observational, randomized, and non-randomized studies were included; (5) There were no restrictions on publication language or year. Studies in a language other than Portuguese and English were translated using tools available online and included in the selection.Exclusion criteria included: (1) Studies in which tyrosine kinase inhibitors were administered in dogs with other types of tumors; (2) Studies with drug treatments other than standard treatments or tyrosine kinase inhibitors; (3) Studies with variables other than the overall response rate, complete or partial response, overall survival, and progression-free survival; (3) Books, book chapters, case reports, case series with fewer than five individuals, conference papers, editorials, letters to the editor, literature reviews, and qualitative studies.Electronic searches were performed in the databases of BMC Veterinary, Embase, Latin American, and Caribbean Health Science Literature (LILACS), LIVIVO, and MedLine databases (via PubMed) and the Web of Science citation database. The EASY, Google Scholar, and Open Access Thesis and Dissertations (OATD) were used to partially capture the “gray literature.” These strategies were implemented to minimize selection publication bias. The MedLine search was constantly updated with electronic alerts, until June 2022. The search descriptors were selected according to the resources of MeSH , DCS , and Emtree (Embase Subject Headings). Several combinations among the descriptors were performed with the Boolean operators “AND” and “OR,” respecting the syntax rules of each database. TM software , where duplicates were automatically removed, and the remaining duplicates were manually removed. The remaining results were exported to Rayyan QCRI  for the study selection phase. The gray literature was manually analyzed, simultaneously and fully, with Microsoft WordTM 2010 .The results obtained were exported to EndNote WebBefore selecting the studies, two reviewers completed a calibration exercise, in which they discussed eligibility criteria and applied them to a sample of 20% of the retrieved studies to determine interexaminer agreement. After reaching an adequate level of agreement (Kappa < 0.81), the selection started.In the first phase, two eligibility reviewers (YNBC and LRS) methodically analyzed the titles and abstracts of the studies independently. Disagreements between examiners were analyzed and defined by a third examiner (PHRS). This phase excluded titles that were unrelated to the topic, as well as abstracts that did not meet the eligibility criteria. In the second phase, the full texts of the preliminary eligible studies were obtained and evaluated. The references of eligible studies were also selected to search for more registers. If the full texts could not be located, a bibliographic request was made to the library database (COMUT) and an e-mail was sent to the corresponding authors in order to retrieve the texts.The source of funding of the extracted studies was evaluated. These data are important because studies sponsored by the pharmaceutical industry are more often favorable to the sponsor's product compared to studies with other sources of sponsorship, and are related to risk of bias . The spoThe electronic search identified 1.555 results distributed in nine electronic databases, including the “gray literature.” After removing the duplicates, 1.144 results remained for the analysis. A careful reading of the titles and abstracts excluded 1.098 results.After reading the full texts, 15 studies with reasoning were excluded, and 3 studies were unavailable for retrieval see  online. The studies were published from 1999 to 2022 and conducted in nine different countries, with 17 studies in North America , 58, 61,Three types of tyrosine kinase inhibitors were identified: imatinib, toceranib, and masitinib. Six studies used these drugs alone –52, 54 aIn order to achieve an appropriate and comprehensive comparative analysis, a total of four distinct cohorts were established: (1) the VBL group, which was treated exclusively with vinblastine chemotherapy; (2) the TKI group, which was solely administered a tyrosine kinase inhibitor; (3) the Chemotherapy + TKI group, which received either vinblastine or other forms of chemotherapy in conjunction with a tyrosine kinase inhibitor adjuvant; (4) the Other + VBL group, which was subjected to vinblastine chemotherapy accompanied by a non-tyrosine kinase inhibitor adjuvant . These groups were also juxtaposed with surgical interventions and assessments of metastatic risk.In the TKI group included in the present analysis, outcomes were classified as positive, neutral, and negative, with each classification being dependent on the principal objectives and hypotheses of the respective investigations. The positive results were defined as those studies which demonstrated supportive evidence for the employment of any of the drugs investigated across the four groups, and which effectively proved the efficacy of the treatments in the management of mast cell tumors. The neutral results indicated no discernible differences when comparing the efficacy of one or more drugs evaluated in each study, and there is no scientific support to claim that one drug is more effective than the other. The negative results are those outcomes that contradicted the hypotheses of the investigation, indicating that the main drug being evaluated within the four groups was inferior to another form of therapy. The main findings of the selected studies are presented in Positive results were observed in 21 articles. The VBL group had better results when compared to surgery alone, and also presented effectiveness as an adjunct to surgery , 38, 42.n = 6) of the studies had sponsorship declarations by the drug manufacturers or with authors associated with an industry: three received financial support from Pfizer Animal Health (n = 12) of the studies did not present a sponsorship declaration and could not state whether there was financial support or not, and in only 19.0% (n = 4) studies the authors declared that there was no conflict of interest.Regarding the positive results, 28.5% , a mean was performed to obtain the total value. The values extracted individually from each group evaluated in the studies were also presented. To evidence the different methods of detection of mutation of the oncogene c-kit that may present variation in its sensitivity, the main methodologies of detection of each study that evaluated the mutation status were described, as well as the classifications of the mutation type  and the amplified and sequenced exons. These data are described in the The sponsorship status is shown in The mean follow-up of treatment responses was 785 days. In studies that had information on previous treatments, 51.9% of the animals received the tyrosine kinase inhibitor or vinblastine as the first line of treatment, while 48.03% received previous treatments. In studies that specified the type of previous treatment, it was observed that 28.5% received some type of surgical excision, 39.1% received chemotherapy, and 32.2% received radiation therapy.In dogs that received TKIs, in studies provide information, 257 dogs received treatment under the label, and 261 received the drug off-label: 87 dogs received Masitinib for presenting non-resectable mast cell tumors (Grade II or III) with confirmed mutated c-kit tyrosine kinase. No information was collected from three studies, as they did not present the mutation status of the c-kit; 170 dogs received Toceranib because they presented mast cell tumors with histological classification in Patnaik grade II or III, recurrent, with or without regional lymph node involvement. In one study only four patients underwent the histopathological examination, so it is undetermined whether other dogs received the drug under label. All 34 animals that received Imatinib were disregarded, considering that this drug is not labeled for dogs; therefore, the use of this drug in the dog is off-label.Among the 1.134 dogs included in the studies with reported gender, 499 (44%) were males and 635 (55.9%) were females. The mean age of the dogs in the selected studies was 9.1 years (7.6 ⊥ 16.6).n = 714, 67.4%), followed by grade III , grade I , and lastly undetermined tumors . This information was presented in 25 studies. In other publications, the histological degree was performed using the criteria of Kiupel (n = 133) were classified as high-grade malignancy and 136 (49.4%) with intermediate/low-grade malignancy. However, these criteria were only evaluated in nine studies, and 55.02% of tumors were classified as microscopic diseases and 44.7% as macroscopic diseases.Only 1,058 dogs had histological grades following the criteria of Patnaik . Most dof Kiupel  in whichn = 188) dogs presented tumors with a c-kit mutation and 71.2% (n = 466) had wild-type c-kit. In the studies that presented mutation prevalence data in exons, about 67.5% (n = 77) had mutations in exon 11, and 32.4% (n = 37) had a mutation in exon 8. Regarding the assignment of the standard kit, 16.2% expressed the kit type I pattern (perimembranous expression), 53.8% expressed standard kit II  and 29.9% had kit III patterns .Regarding c-kit, 29.7%  value was 50.36% (138/274) , 57, 59.The complete response (CR) of the TKI group value reached 26.49% 133/502)  , 54, 59./375 , 54Data on overall survival collected from all included studies were analyzed as follows: mean survival , mean survival according to tumor grading  , 63, andIn the TKI group, the mean overall survival of all dogs was 308 days (113 ⊥ 1.018). The mean overall survival in dogs with grade II mast cell tumors within this group was greater (369 days) when compared to grade III (278 days) . In a stIn the VBL group, the mean overall survival of the included studies was 524 days (101 ⊥ 1.374) –57, 60. The dogs in the Chemotherapy + TKI group that used VBL as a chemotherapy agent for adjuvant treatment had an overall survival of 893 days, and dogs undergoing palliative treatment survived for 218 days . Dogs with high-grade mast cell tumors had an overall survival of 563 days. The total mean obtained from these dogs was 559 days (555 ⊥ 563) .In the Other + VBL group, the mean overall survival was 728 days (209.5 ⊥ 1359). The dogs with measurable disease had a mean overall survival of 195 days and those with a non-measurable disease had a mean of 1.214 days , 43, 49.Progressive survival was collected from all included studies and was analyzed as follows: Mean progression-free survival , mean progression-free survival according to the line of therapies (first line or second line), type of chemotherapy treatment , tumor classification  , 63, dogIn the TKI group, it was possible to obtain a progression-free interval with a mean of 207 days 30 ⊥ 453)   , 56. Forin vitro and in vivo, decrease stem cell factor generation through fibroblasts and epithelial cells, and reduce inflammation /sequencing methods for the detection of somatic mutations in canine mast cell samples. This can be considered a limitation since data indicate that this methodology has limited sensitivity , 74. In In the current study, progression-free survival of dogs that underwent vinblastine treatment alone was shown to be longer compared to dogs treated with TKI, according to data presented earlier, in which dogs treated with VBL had higher OS compared to dogs treated with TKI. This is possibly due to the association with prednisolone, which has already been associated with a high PFS. This contradicts other previous studies with inhibitors, in which they demonstrated longer progression-free survival for animals undergoing TKI treatment . This isOther clinical parameters such as a good objective response rate and, consequently, lower adverse events could have influenced this survival , 59. In The median overall response rate, determined by tumor size or complete remission, varied according to the type of treatment, with a lower overall response rate observed when vinblastine was used alone compared to TKI treatment. This variability is possibly due to the type and aggressiveness of the mast cell tumor. Chemotherapy works mainly through cell ablation and tyrosine kinase inhibitors through growth limitation by inhibiting VEGFR2 and PDGFR, acting as an antiangiogenic agent and, as a result, causing tumor regression . Due to A contradiction was found in this study regarding the VBL group having better OS than the TKI group, and the TKI group had a greater clinical benefit of ORR compared to the VBL group. The relationship between response rate and survival is complex. Some authors suggest the lack of correlation between ORR and OS due to confounding factors, such as treatment crossing after progression, number of subsequent therapies, long post-progression survival in the first-line scenario, and non-cancer-related deaths . New tarn = 6) were sponsored and 57% (n = 12) did not present any declaration of conflict of interest. Data indicate that studies funded by industries more often present data favorable to the use of the product produced compared to studies not sponsored by industry   , 80. Anoarisons) .Assuming this, the results should be interpreted with caution, and more studies should evaluate the clinical efficacy of these drugs without financial support from the industries, or even the provision of information on the status of sponsorship, which was absent in most studies.It is important to emphasize that the data extracted from the studies did not present a sample standardization, that is, to obtain the general data, important characteristics about the patients, tumor, and type of treatment were not considered, which may interfere with the outcome of this study.In conclusion, vinblastine had a greater clinical benefit than TKI in terms of overall survival and progression-free survival. However, TKI is more efficient in mast cell tumors with c-kit mutated. Although, without more robust results to assure greater efficacy in these patients, TKIs should not be considered a frontline for mast cell therapy.The original contributions presented in the study are included in the article/YC performed the database searches and wrote the main manuscript text along with LS, PS, and CM. LP, TS, and MS were responsible for the process of searching and writing guidance of the manuscript. All authors contributed to the article and approved the submitted version.


Citrus genus, namely Aurantii Fructus Immaturus (Zhishi), Aurantii Fructus (Zhiqiao), Citri Reticulatae Pericarpium Viride (Qingpi) and Citri Reticulatae Pericarpium (Chenpi), are widely used for treating various cardiovascular and gastrointestinal diseases. Many ingredients have already been identified from these herbs, and their various bioactivities provide some interpretations for the pharmacological functions of these herbs. However, the complex functions of these herbs imply undisclosed cholinergic activity. To discover some ingredients with cholinergic activity and further clarify possible reasons for the complex pharmacological functions presented by these herbs, depending on the extended structure–activity relationships of cholinergic and anti-cholinergic agents, a simple method was established here for quickly discovering possible choline analogs using a specific TLC method, and then stachydrine and choline were first identified from these Citrus herb decoctions based on their NMR and HRMS data. After this, two TLC scanning (TLCS) methods were first established for the quantitative analyses of stachydrine and choline, and the contents of the two ingredients and synephrine in 39 samples were determined using the valid TLCS and HPLC methods, respectively. The results showed that the contents of stachydrine (3.04‰) were 2.4 times greater than those of synephrine (1.25‰) in Zhiqiao and about one-third to two-thirds of those of Zhishi, Qingpi and Chenpi. Simultaneously, the contents of stachydrine, choline and synephrine in these herbs present similar decreasing trends with the delay of harvest time; e.g., those of stachydrine decrease from 5.16‰ (Zhishi) to 3.04‰ (Zhike) and from 1.98‰ (Qingpi) to 1.68‰ (Chenpi). Differently, the contents of synephrine decrease the fastest, while those of stachydrine decrease the slowest. Based on these results, compared with the pharmacological activities and pharmacokinetics reported for stachydrine and synephrine, it is indicated that stachydrine can be considered as a bioactive equilibrist for synephrine, especially in the cardio-cerebrovascular protection from these citrus herbs. Additionally, the results confirmed that stachydrine plays an important role in the pharmacological functions of these citrus herbs, especially in dual-directionally regulating the uterus, and in various beneficial effects on the cardio-cerebrovascular system, kidneys and liver.Four Chinese herbs from the  Qi-regulating Chinese herbs + ; 1H NMR  δ: 4.07 , 3.70 , 3.53 , 3.29 , 3.10 , 2.48 , 2.33~2.24  and 2.23~2.11 ; 13C NMR  δ: 171.3 (C-8), 76.4 (C-2), 67.2 (C-5), 52.1 (C-6), 45.9 (C-7), 25.3 (C-3) and 18.6 (C-4). These NMR data are in accordance with those reported for stachydrine + , 150.0903 [M + H − H2O]+  which presented as base peak ion.3 is not a choline analog. After spraying with the improved Dragendorff reagent, the spots of compounds 1 and 2, two choline analogs, presented brown or reddish-brown color, while compound 3 showed an orange-red spot on the thin-layer plates. These indicated that the compounds represented by the spots are unlikely choline analogs if the color of the spots is red. A detailed discussion is given in The above indicated that compound Along with the identification of stachydrine and choline from the four Chinese citrus herbs Zhishi, Zhiqiao, Qingpi and Chenpi, it is known that three alkaloids, namely stachydrine, choline and synephrine, are extensively distributed in these herbs, and their proportions and contents in these herbs are different from each other. As we mentioned in the introduction, the decoctions or aqueous extracts of Zhishi and Zhiqiao connoted probable cholinergic activities, contrary to the adrenergic effects of synephrine. Simultaneously, these herbs have different pharmacological functions according to the theory of Chinese medicine. Thus, it was worth exploring whether there were some relationships between the complex pharmacological functions of these herbs and the contents and proportions of these three ingredients. To achieve this, the contents of these three ingredients were determined.As there is no chromophore in the structures of stachydrine and choline, it is unsuitable to perform their quantitative analyses using the HPLC-UV method. Based on the detection procedure of choline analogs and their separation effects on thin-layer plates, the TLC scanning (TLCS) method was considered for the quantitative analyses of stachydrine and choline. Meanwhile, the quantitative analysis for synephrine was established using the HPLC-UV method, referring to that described in the Chinese pharmacopeia .−1) with a relative standard deviation (RSD) of 0.08% and 2.0 μg (0.40 mg·mL−1) with an RSD of 0.18%, respectively, and those for choline were 0.5 μg (0.10 mg·mL−1) with an RSD of 0.08% and 0.8 μg (0.15 mg·mL−1) with an RSD of 0.18%, respectively. The repeatability tests showed that the RSDs for a strip of stachydrine and choline on a thin-layer plate were 0.34% and 0.48%, respectively. The precision tests indicated that the RSDs for six strips of stachydrine and choline on the same thin-layer plate were 1.20% and 3.55%, respectively, and those for the identical solutions of stachydrine and choline on six thin-layer plates were 4.05% and 4.15%, respectively. A good linearity correlation y = 6565.7x + 5522.7 (r = 0.9996)   between  0.9996)  was pres 0.9996) , and tha 0.9996) . These rSimilarly, the chromatographic peaks of synephrine presented good symmetry and can be well separated with a resolution of 4.06 from its nearest peaks, according to the procedure of HPLC analysis described in Using the above validated methods for the quantitative analyses, the contents of stachydrine, choline and synephrine in these citrus herbs were determined, and the results are shown in From p < 0.05 or p < 0.01), and those of choline in Qingpi (or/and Zhishi) are larger than in Zhiqiao and Chenpi (p < 0.05 or p < 0.01). Simultaneously, stachydrine is the largest of these three ingredients in Zhiqiao (p < 0.05 or p < 0.01).To make the content differences between these three ingredients clearer to explain the differences in the pharmacological functions of these herbs, the data in Citrus aurantium L. (or its cultivated varieties), and Qingpi and Chenpi are the dried peel from young (or immature) fruits of Citrus reticulata Blanco (or its cultivated varieties), it was further concluded that all three ingredients will reduce (p < 0.05) with the prolongation of growth time for Citrus genus plants, except for stachydrine in the herbs Qingpi and Chenpi. Among them, the contents of synephrine decrease the most rapidly, while those of stachydrine decrease the most slowly. Although all their contents reduce with the prolongation of growth time for these Citrus genus plants, the contents of synephrine are significantly higher than those of stachydrine and choline, except those of stachydrine are largest in the herb Zhiqiao. Moreover, since herbs Zhishi (or Zhiqiao) and Qingpi (or Chenpi) are derived from the fruits and the pericarps, the fact that synephrine and stachydrine distribute more in the exocarp than in the mesocarp could be further inferred from their decreasing speeds and degrees with the growth time of these Citrus genus plants, and this inference was also in accordance with a previous report [Since herbs Zhishi and Zhiqiao are the dried young fruits of s report . HoweverCitrus genus plants. Herbs Zhishi and Zhiqiao originate from the fruits of Citrus aurantium L. or its cultured varieties at different harvest times, and the herbs Qingpi and Chenpi originate from the peels of Citrus reticulata Blanco or its cultured varieties at different harvest times [N-methyl tyramine), with higher content and a wider distribution in these herbs, have various bioactivities in the digestive system, cardiovascular system and respiratory system, and which are mainly responsible for the pharmacological effects of these herbs [Zhishi, Zhiqiao and Qingpi on the in vitro and in vivo uteruses of both pregnant and non-pregnant rabbits. Moreover, some related investigations remain insufficiently clear, such as which components are responsible for the two-way regulating effects on gastrointestinal smooth muscle, showed by the decoction of Zhiqiao [These four herbs are derived from the fruits or peels of st times . Their dst times ,3,11. Mast times ,3. It wase herbs ,20. Howe Zhiqiao ,20.Considering that the decoctions or aqueous extracts of these herbs conceal some possible cholinergic activities, here, probable choline analogs in these herbs were detected and determined. The results from α1-adrenoreceptor and an antagonist of α2-adrenoreceptor, can constrict peripheral blood vessels, cerebrovascular and aorta, while stachydrine, an ingredient coexisting with synephrine in these citrus herbs, presents various cardio-cerebrovascular protections including rapid vascular relaxation, accelerating blood circulation, increasing coronary and myocardial blood flow, relieving myocardial necrosis, slowing heart rate and decreasing cardiac output, suppressing and ameliorating myocardial fibrosis, ameliorating cardiac hypertrophy and fibrosis. Some results were obtained from animal models induced by adrenergic receptor agonists  [Based on the extended structure–activity relationships of cholinergic and anti-cholinergic agents , choline Houtt.) . Based oα- and β-asarones), since the potassium bismuth iodide reagent colors these compounds to orange-red or brownish-red. This was also supported by the fact that compound 3 was found not to be a choline analog, and colored an orange-red spot c , assigned to three to nine hydrogens (one to three methyl groups). It is noteworthy that these analyses should eliminate the signal peaks from the deuterated methanol used for the NMR experiments or the possible residual methanol in the process of sample preparation. Thus, the deuterated solvents MeOH-d4 should be avoided to use for the NMR experiments as much as possible. Considering that these compounds contain a structural fragment of the quaternary ammonium, D2O is considered the preferred solvent.Moreover, possible choline analogs can be quickly identified by the combinational method of preparative TLC and Xiangyuan (dried fruits of Citrus wilsonii Tanaka) and Foushou (dried fruits of Citrus medica L. var. sarcodactylis Swingle) , Juhua (dried capitulum of Chrysanthemum morifolium Ramat.) and Chuanxiong (dried rhizome of Ligusticum chuanxiong Hort.)  , and thil fruits . Moreoveg Hort.) . This inAccording to the theory of Chinese medicine, these four citrus herbs have different pharmacological functions . To clarAs there is no chromophore in the structures of stachydrine and choline, it is unsuitable to perform their quantitative analyses using the HPLC-UV method, although this method was used for the quantitative analyses of stachydrine in Yimucao and choline in various plants. According to the detection procedure of choline analogs, stachydrine and choline can be perfectly isolated from their adjacent spots in these herbs . Thus, hZhishi (harvested in June) to Zhiqiao (harvested in July), and from Qingpi (harvested in July) to Chenpi (harvested in January of next year). However, the decreasing speed of stachydrine is slower than that of synephrine, which was also supported by the depth analyses for the ratio values of stachydrine and synephrine, comparing Zhishi (0.44 ± 0.16) with Zhiqiao (2.43 ± 1.03) (p < 0.05) and Qingpi (0.34 ± 0.14) with Chenpi (0.67 ± 0.33) (p < 0.05). Although they were collected from different plants, the changing trends of synephrine, choline and stachydrine in 39 batches of samples are certain according to statistical analyses -dependent methyltransferases with a universal methyl donor SAM [N-methyltransferases are responsible for the bio-syntheses of stachydrine and choline, while the sub-classified O-methyltransferases are responsible for that of multi-methoxy flavonoids [Citrus genus plants indicate there is a kind of intrinsic mechanism for simultaneously regulating both sub-classified enzymes with a similar effect. More probably, there are some physiological needs regulating the whole biosynthesis pathway involving SAM-dependent methyltransferases down in the fruits and peels of these citrus genus plants. Thus, it is worth further studying the physiological regulations of these Citrus genus plants to these components in their fruits and peels, which would be very helpful for clarifying the different pharmacological functions of these herbs and the regulation relationship of the two sub-classified enzymes in Citrus genus plants.It is noteworthy that some multi-methoxy flavonoids having various bioactivities in the cardiovascular system, such as nobiletin and tangeretin , are repse herbs ,79. Simuse herbs ,80, in aest time ,82, whicest time ,83,84. Aonor SAM ,86,87. DCitrus flavonoids exert multiple beneficial effects on cardiovascular and metabolic health through antioxidant, antidiabetic and anti-inflammatory activities, and by modulating lipid metabolism and adipocyte differentiation, etc. [It was reported that these citrus herbs mainly contain flavonoids, alkaloids, essential oils and coumarins ,15,88. Mon, etc. ,90. Simuon, etc. . ConsideC. aurantium flowers can reduce spontaneous motility and decrease the uterus contractions of pregnant rats, related to voltage-dependent calcium channels and without involving β-adrenoceptors and opioid receptors [Some reports indicated that aqueous extracts or the decoctions of these herbs can contract the in vitro and in vivo uteruses of both pregnant and non-pregnant rabbits, and relax the in vitro uterus of pregnant rats or mice ,93,94. Aeceptors . From Taeceptors ,48,49,51Citrus flavonoids confer cardiovascular protection via their antioxidant, antidiabetic, anti-inflammatory, anti-atherosclerosis and other biological activities [Citrus fruits should be encouraged in the diet for their potential neurological benefits [N-methyltyramine on the heart for non-drug purposes, which is probably one of the reasons why blood pressure sometimes presented an inapparent or transient raise and even decline after the oral administration of these herb decoctions to experimental animals [Traditionally, these herbs are used for the treatment of some cardiovascular disease, and have various beneficial effects on cardiovascular health, including antioxidant and anti-inflammatory, hypolipidemic, anti-thrombosis and anti-atherosclerosis effects, and cardio-cerebrovascular protection, etc. ,96,97. Rtivities ,90, and benefits . Howeverbenefits ,100,101.benefits ,103. Thu animals . Further animals .It was reported that the concentrated solutions (1.5 g dried herbs per milliliter) of these citrus herb decoctions have obvious in vitro antiplatelet aggregation activities in human platelets . SynephrQingpi, would play an important role in maintaining the function and health of the liver [Citrus flavonoids on the cardiovascular system mentioned above, they also have beneficial effects on gastrointestinal health and function [γ-aminobutyric acid and synephrine should be responsible for various functions of the central nervous system, such as sedation, hypnosis, anxiolytic, anticonvulsant and anti-depression [Furthermore, choline is already used for the treatment of nonalcoholic fatty liver disease ,108. Thuhe liver ,46. Morehe liver ,110. Besfunction ,14, and function ,112,113.function ,92. Simufunction ,113, whiN-methyl tyramine and γ-aminobutyric acid) and Citrus flavonoids, essential oil and coumarins, these can more scientifically and reasonably interpret the substance bases for the various pharmacological effects of these citrus herb decoctions. Conversely, the differences in the formation, content, water solubility, extractability and pharmacokinetic characteristics of these components would lead to efficient differences among these four Chinese citrus herbs.Taken together, stachydrine should play an important role in the pharmacological functions of these citrus herbs, especially it can dual-directionally regulate the uterus and has various beneficial effects on the cardio-cerebrovascular system, blood, kidney and liver. Simultaneously, as a pair of bioactive equilibrists, the cardio-cerebrovascular protection of stachydrine can counteract the possible cardiovascular risk brought out from synephrine, which is very beneficial for the safe use of these citrus herbs. Moreover, together with the pharmacological activities of alkaloids . Moreover, eight leaf and fruit samples of Citrus aurantium L. and its cultivated varieties, Citrus junos Siebold ex Tanaka, and Citrus reticulata Blanco ‘Zhangtouhong’ were collected from different habitats and growth ages and were identified by senior agronomist Yao Nie, and some fresh leaves and fruits of Xiangyuan were collected in Xinyu, China. The following Chinese herbs were purchased from Yifeng Pharmacy : Foshou, the dried fruits of Citrus medica L. var. sarcodactylis Swingle ; Xiangyuan, the dried fruits of Citrus wilsonii Tanaka; Huanglian, the dried rhizome of Coptis chinensis Franch ; Chuanxiong, the dried rhizome of Ligusticum chuanxiong Hort. ; Dafupi, the dried peel of Areca catechu L. ; Banxia, the processed products according to the legal process for the dried tuber Pinellia ternata (Thunb.) Breit; Juhua, the dried capitulum of Chrysanthemum morifolium Ramat.; Mahuang, or Ephedrae Herba; Duzhong, the dried bark of Eucommia ulmoides Oliv.; Kushen, the dried root of Sophora flavescens Ait.; and Gancao, or Glycyrrhizae Radix et Rhizoma.Ten dried samples  of Zhish samples  of ZhiqiReference standard choline chloride (No. C12799084) with a purity of 98% was purchased from Shanghai Macklin Biochemical Co., Ltd. , and stachydrine hydrochloride (No. PS012344) and synephrine (No. PS000966), each with purity of more than 98%, were purchased from Chengdu Push Bio-Technology Co., Ltd. .TLC silica gel plates were purchased from Qingdao Ocean Chemical Co., Ltd. , and HPTLC Silica gel 60 F254 was purchased from Merck KGaA . Bismuth subnitrate used for preparing the chromogenic reagent of TLC analyses was purchased from Aladdin Chemistry Co., Ltd. . Chromatographic-grade methanol  and sodium dodecyl sulfonate  were used for the HPLC analyses of synephrine. The ultra-pure water was prepared by the TST-UPB-10 ultra-pure water machine . Other chemicals  were purchased from China National Pharmaceutical Group Co., Ltd. .γ-aminobutyric acid and synephrine were taken as excluded controls. Two chromogenic reagents (Dragendorff’s and Wagner) and an improved Dragendorff reagent for alkaloids were selected for color reactions on the thin-layer plate. The stock solutions were prepared by the equal mixture of solution I  and solution II (8.00 g of potassium iodide dissolved in 20.0 mL of water), and 1.0 mL of the stock solution, 2.0 mL of acetic acid  and 10.0 mL of water were mixed as Dragendorff’s reagent before use. Then, 1 g of iodine and 10.0 g of potassium iodide were dissolved in 50 mL of water, and the Wagner reagent was prepared by transferring this solution into a 100 mL volumetric flask, subsequently supplementing it with 2.0 mL of acetic acid and the required amount of water to make a constant volume. The improved chromogenic reagent was prepared by adding a mixture of bismuth nitrite (0.82 g), potassium iodide (11.06 g) and 50% (v/v) phosphoric acid (90 mL) into a 100 mL volumetric flask to make a constant volume using water.Choline chloride was used as a positive control, and −1 for three reference solutions was prepared by dissolving and supplementing with 80% (v/v) ethanol to a constant volume of 25 mL.A total of 25.0 mg each of choline chloride, stachydrine hydrochloride and synephrine was transferred into a volumetric flask, and then a concentration of 1.0 mg·mLv/v), and centrifuged to obtain the supernatant. After filtering with a 0.45 μm microporous membrane, the sample solution for the decoction of each herb was obtained.For Chinese herb slices, 20.0 g of herb slices was placed into a decocting jar, and 250 mL of purified water was added to it, soaking for 30 min. Next, the mixture was decocted for 30 min on MSD-1-12 ceramic decocting equipment produced by Meisidi Craft Products Factory , and then the extracted liquid was poured out of the jar to obtain decoction I. Subsequently, another 250 mL of purified water was added to the decocting jar and the herb residue was decocted for 20 min, and the extracted liquid was poured out to obtain decoction II. Finally, decoctions I and II were put together to obtain the decoction of each herb. For the detection of choline analogs, 100 mL of the decoction was concentrated into a small amount of mixture under vacuum, which was then transferred to a 10 mL volumetric flask to make a constant volume with purified water. Then, 1000 μL of the suspension was transferred to a centrifuge tube with a pipette, diluting with equal volume of 80% ethanol (v/v) ethanol was added. After sonicating twice for 10 min each time in a DK-410T water bath sonicator with a frequency of 40 kHz, the mixture was filtered, and the residue was washed twice with 80% (v/v) ethanol. The filtrate was concentrated under vacuum, and the residual suspension was transferred into a volumetric flask to prepare 2 mL of the sample solution with 80% (v/v) ethanol.Fresh samples were cut into thin slices and then dried under 60 °C ambient temperature in a blast drying oven. All dried samples with the amount of 20 to 30 g were crushed into coarse powder and then passed through a 65-mesh sieve to obtain their corresponding powders. Then, 2.0 g of each powder sample was placed into a 50 mL Erlenmeyer flask with a stopper, to which 25 mL of 80%  and II , respectively. After drying, the thin-layer plates were visualized with Dragendorff, Wagner and improved Dragendorff reagents.According to the general procedure of TLC analysis, the reference solutions of choline, Zhishi, Zhiqiao, Qingpi and Chenpi were performed for discovering probable choline analogs. After this, other herbs from the Citrus genus, including Foushou and Xiangyuan, and some samples of the leaves from the plants of the Citrus genus were also detected for possible choline analogs. Moreover, to further verify the efficiency of the method for detecting choline analogs, the TLC analyses for sample solutions from some other Chinese herbs, including Huanglian, Juhua, Mahuang, Chuanxiong, Dafupi, Banxia, Duzhong, Kushen and Gancao, were also determined. Among these herbs, it was reported that herbs Chuanxiong and Huanglian contain choline or its analogs.According to the optimized procedure, the TLC analyses for sample solutions from Chinese herbs Zhishi mixed sample from 10 different producing areas was prepared. Using preparative TLC, probable choline analogs in the Chinese herb Zhishi were isolated. Solvent I  was used as the developing agent, and probable choline analogs were located by visualization of the slices of thin-layer plates immersed in the improved Dragendorff′s reagent.According to the sample solution process in 1 and 2 were identified based on the spectral analyses of their NMR and MS  from a TripleTOF 5600+ hybrid quadrupole time-of-flight mass spectrometer system .Compounds R and MS , combiniv/v/v) for 15 min, the spots were developed for a span length of 60 mm. Subsequently, the plate was taken from the chamber and dried, and then was immersed in the improved Dragendorff reagent for 2 to 4 s. After taking them from the stained jar for 20 min, the TLCS analyses were performed with the reflection absorption method for 50 min on a KH-3000 Plus TLC Scanner , and the measured/reference wavelengths for choline and stachydrine were 568/820 nm and 574/820 nm, respectively. The contents of choline or stachydrine in Chinese herbs from the Citrus genus were calculated from the mean peak areas of three bands of choline or stachydrine from reference and sample solutions.Reference and sample solutions were prepared according to the procedure described in In the above procedure, the measured/reference wavelengths and the chromogenic stability were simultaneously determined from the 10 min interval determination in 70 min for the absorption curves of choline and stachydrine bands on the TLC plate after being visualized, and the sampling volume was 5.0 μL. Depending on the typical chromatographic profile of TLC analyses for the sample solutions of Chinese citrus herbs, the resolution between choline (or stachydrine) and its nearest strip was calculated.According to the general procedure of methodology validation, the limit of detection (LOD), the limit of quantitation (LOQ), precision, repeatability, linearity and range were evaluated using the reference standard of choline or stachydrine. The reproducibility was assessed using the powder of Zhishi. The recovery was tested using the powder of Zhishi with a known content of choline or stachydrine, and the reference standard of choline or stachydrine.−1), and the sampling volume was 10.0 μL. Simultaneously, intra- and inter-plate precision were assessed by detecting choline or stachydrine solutions, respectively, on a thin-layer plate with six replicates and on six thin-layer plates. The linear correlation was established using seven sampling volumes (three replicates for each) at a concentration of 1.0 mg·mL−1 for choline or stachydrine solution. The reproducibility was evaluated by the quantitative determination of choline or stachydrine in Zhishi powder (No. 20101001) with six replicates, and the sampling volume was 2.0 μL. The recovery was tested from 9 samples prepared by adding an amount of choline or stachydrine into the powders of Zhishi (No. 20101001) with a known content of choline or stachydrine, including three different amounts for choline or stachydrine with three replicates for each.In detail, the LOD was defined as the lowest concentration of choline or stachydrine at which the signal-to-noise ratio was from 3 to 4, and the LOQ was defined as the lowest concentration of choline or stachydrine at which the signal-to-noise ratio was greater than or equal to 10 with a precision below 5%. According to the procedure of TLCS analysis in According to the validated procedure of TLC analyses, the quantitative analyses of choline or stachydrine in the 39 purchased samples, including 10 of Zhishi, 10 of Qingpi, 10 of Chenpi and 9 of Zhiqiao, were performed with the external standard method in triplicate for each sample. Three spots were sampled on the plate for each sample or standard solution. The sampling volume of each spot for the sample solutions of Zhishi was 2.0 μL, and that for the sample solutions of Zhiqiao, Qingpi or Chenpi was 3.0 μL. Simultaneously, the sampling volumes were 1.0 and 3.0 μL for the reference solutions of choline and stachydrine, respectively. After being visualized, each lane on the plates was scanned to obtain the peak areas of choline or stachydrine in samples and corresponding standards, and then the contents of choline and stachydrine in various samples were calculated.−1 stock solution for synephrine was prepared by dissolving 10.0 mg of synephrine into 80% (v/v) ethanol, and transferred into a 2 mL volumetric flask which was further supplemented with 80% (v/v) ethanol to a constant volume. From this stock, a concentration of 0.5 mg·mL−1 for standard solution was prepared by 10-fold dilution.A 5.0 mg·mLv/v) was used as the mobile phase of the isocratic elution, and the flow rate was set at 1.0 mL/min. The phosphate buffer was prepared by dissolving 0.60 g of KH2PO4, 1.00 g of sodium dodecyl sulfonate and 1.0 mL of glacial acetic acid into 700 mL of water, which was then transferred into a 1000 mL volumetric flask and subsequently supplemented with the required amount of water to make a constant volume. Moreover, the injection volume for all sample solutions and standard solutions was 10.0 μL. Depending on the typical chromatographic profile of HPLC analyses for sample solutions of Chinese citrus herbs, the resolution between synephrine and its nearest chromatographic peak was automatically calculated by the HPLC system.The concentrations of synephrine in sample solutions were determined with the external standard method, referring to the procedure of the Chinese pharmacopoeia . Briefly−1 were performed to evaluate the repeatability. Using a powder sample of Zhishi (No. 2010001), the stability of a sample solution in 24 h was evaluated, and the reproducibility was also assessed by the quantitative determination of synephrine in the powder sample with six replicates. The recovery was tested from nine samples prepared by adding an amount of synephrine into the powders of Zhishi (No. 20101001), including three different amounts of synephrine with three replicates for each.Similar to the validation of TLC analysis, the LOD was defined as the lowest concentration of synephrine at which the signal-to-noise ratio was from 3 to 4, and the LOQ was defined as the lowest concentration of synephrine at which the signal-to-noise ratio was greater than or equal to 10 with precision below 5%. The linear correlation was established using seven concentrations  of synephrine solution with three replicates for each. Six injections for a standard solution with a concentration of 0.50 mg·mL−1.All sample solutions used for the quantitative analyses of chlorine and stachydrine in t-test was used for the comparison of two groups. A paired t-test was selected when the number of data was the same (both n = 10), while two-sample equivariance was selected for the t-test when the number of data was different (n = 10 and 9). A p value less than 0.05 shows that the data difference between two groups is significant, and that less than 0.01 indicates that the data difference between two groups is very significant.All statistical analyses were performed using the Excel program of Microsoft Office 2016, and a bilateral Studies published in the past 10 years on the bioactivities of synephrine, chlorine and stachydrine were unsystematically searched using the Google Scholar search engine and the databases Medline, CNKI and RSC, using the keywords “pharmacological” or “activity” or “review”, and “synephrine” or “chlorine” or “stachydrine”. Furthermore, the relevant references in the obtained literature were also checked. Based on the literature results and the contents of synephrine, chlorine and stachydrine in these four Chinese citrus herbs, the pharmacological effects of these compounds were comprehensively analyzed, together with their contributions to the pharmacological effects of the four Chinese citrus herbs. Some reasonable explanations for the differences in the pharmacological activities of these four Chinese herbs have been presented here.To discover some ingredients with cholinergic activity and further clarify possible reasons for the complex functions presented by four Chinese citrus herbs, a simple and specific method was first established for quickly discovering possible choline analogs, and stachydrine and choline were discovered in these citrus herbs. Then, a TLCS method was first established for the quantitative analyses of stachydrine and choline, and the contents of both ingredients and synephrine in 39 herb samples were determined. The results showed that stachydrine and synephrine have commensurate contents in these herbs, and the contents of the two ingredients and choline in these herbs present similar decreasing trends with the delay of harvest time. However, the contents of synephrine decrease the fastest, while those of stachydrine decrease the slowest. Based on these findings, the pharmacological activities and pharmacokinetics reported for stachydrine and synephrine were compared, and the results indicated that stachydrine and synephrine can be considered as a pair of bioactive equilibrists in these citrus herbs, especially for their effects on the cardio-cerebrovascular system. Additionally, the results confirmed that stachydrine plays an important role in the pharmacological functions of these citrus herbs, especially in dual-directionally regulating the uterus, and in various beneficial effects on the cardio-cerebrovascular system, kidney and liver. Considering these findings, some more scientifical and reasonable interpretations would be concluded with the help of network pharmacology analyses performed for the main ingredients and the various pharmacological functions of these citrus herb decoctions, and which would indicate that the component differences in the formation, content, water solubility, extractability and pharmacokinetic characteristics can lead to the differences in the pharmacological functions of these citrus herbs. Furthermore, the quality markers of these herbs should be reevaluated based on this study, the results of which would present a good reference for the quality control of these herbs.


The increasing prevalence of NAFLD has been related to type 2 diabetes mellitus (T2D). However, the relationship between short-chain fatty acids (SCFAs) and NAFLD severity is ambiguous in T2D subjects. This study aimed to explore the association of SCFAs with the severity of NAFLD in T2D patients. (2) Methods: We employed echography to examine the severity of hepatic steatosis. The serum levels of nine SCFAs, namely, formate, acetate, propionate, butyrate, isobutyrate, methylbutyrate, valerate, isovalerate, and methylvalerate, were measured using gas chromatography mass spectrometry. (3) Results: A total of 259 T2D patients was enrolled in this cross-sectional study. Of these participants, 117 with moderate to severe NAFLD had lower levels of formate, isobutyrate, and methylbutyrate than the 142 without NAFLD or with mild NAFLD. Lower circulating levels of isobutyrate and methylbutyrate were associated with an increased severity of NAFLD. A relationship between NAFLD severity and circulating isobutyrate and methylbutyrate levels was found independently of a glycated hemoglobin (HbA1C) level of 7.0%. (4) Conclusion: Circulating levels of isobutyrate and methylbutyrate were significantly and negatively correlated with NAFLD severity in the enrolled T2D patients. SCFAs may be related to NAFLD severity in T2D patients. Non-alcoholic fatty liver disease (NAFLD) is a crucial global public health concern, with growing prevalence over recent decades ,2. In TaThe gut microbiota has frequently been linked to metabolic diseases such as NAFLD, diabetes, and obesity, and several studies have reported that dysregulation of the intestinal microbiota (gut dysbiosis) can cause NAFLD ,11. The A total of 259 patients with T2D were recruited between October 2016 and December 2017 in this observational research from a tertiary hospital in the south of Taiwan. T2D was defined by prescriptions for antidiabetic drugs, a history of diabetes, or blood glucose values, according to the criteria of the American Diabetes Association. The exclusion criteria were patients who consumed an average of >30 g/day of alcohol and the presence of autoimmune hepatitis or hepatitis B and C. All patients were asked to attend T2D education programs. The Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-G(II)-20160021) gave ethical approval for the study, which was conducted according to the Declaration of Helsinki, and all enrollees provided written informed consent.2. Blood and urine samples were collected after a 12 h fast for biochemical studies and to measure albuminuria, respectively.Demographic data, including a history of tobacco smoking and alcohol consumption, and clinical data were collected through interviews and medical records at enrollment. The medical records were also used to collect information on medication usage, including statins (one of antihyperlipidemic agents) and antidiabetic drugs such as sulfonylurea, Dipeptidyl peptidase 4 (DPP4) inhibitor, metformin, thiazolidinediones, and insulin. The patients recorded their dietary habits using a basic questionnaire. Body mass index (BMI) was recorded as kg/mA single operator blinded to the patients’ status performed echography to assess the severity of the NAFLD. Normal liver parenchyma was defined as that with equivalent or slightly greater echogenicity than the adjacent spleen and kidney. Lipid droplets in steatosis scatter ultrasound beams, resulting in the transducer receiving more echo signals and consequently causing the appearance of a hyperechoic or “bright” liver. Fat also reduces the strength of the beam, thereby decreasing its penetration into tissues. The brightness of the liver and/or dimming of the vessels and diaphragm can therefore be used to assess the severity of steatosis. To avoid inaccuracies caused by factors associated with acquisition, the brightness of the liver was estimated through comparison with the spleen or kidney as a standard reference . The degN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride-6% pyridine solution in 100% aqueous methanol. The mixture was reacted at 40 °C for 30 min. The solution was then diluted to 210 μL with 10% aqueous methanol. A 75 μL aliquot was mixed with 25 μL of the internal standard (IS) mix solution, and a 10 μL aliquot was injected for LC–MS/MS.The serum levels of nine SCFAs, namely, formate, acetate, propionate, butyrate, isobutyrate, methylbutyrate, valerate, isovalerate, and methylvalerate, were measured using liquid chromatography mass spectrometry (LC–MS/MS). Human serum (50 μL) was mixed with 20 μL of 3-Nitrophenylhydrazine hydrochloride (200 mM) in 100% aqueous methanol and 20 μL of 120 mM An ultraperformance liquid chromatography system  with tandem MS  and Xcalibur software 2.2  were used for the detection and quantification analysis. The LC–MS/MS system was equipped with an electrospray ion source and was run in positive mode. A volume of 10 μL was injected into an ultraperformance liquid chromatography column  equipped with a filter in front of the column. The flow rate was 300 μL/min and the column temperature was 40 °C.t-test or Mann–Whitney U analysis, as appropriate, and the chi-square test was used for categorical variables. To reduce the effect of possible confounding factors on the relationship between circulating SCFA levels and the severity of NAFLD, the covariates, including clinical data, laboratory data, and medication, were initially analyzed with univariate analysis. Then multivariate logistic regression models, including significant variables (p < 0.05) in univariate analysis, and other traditional covariates, such as habit of smoking, alcohol drinking, and medical disease, were adjusted to clarify the association between circulating SCFA levels and the severity of NAFLD. We further categorized the patients according to sex, obesity (defined as a BMI of 27 kg/m2), and a glycated hemoglobin (HbA1C) level of 7% to further evaluate the synergistic interactions between the SCFAs and sex, obesity, and sugar control in the severity of NAFLD. Statistical analyses were conducted using SPSS version 22.0 for Windows  and GraphPad Prism version 9.0 . Statistical significance was set at a two-sided p-value smaller than 0.05.The T2D participants were categorized on the basis of the severity of NAFLD to a no NAFLD and mild NAFLD group and a moderate and severe NAFLD group, and their baseline characteristics were compared. If continuous data are normally distributed, they are presented as a mean ± SD. If continuous data are non-normally distributed, they are presented as a median (interquartile range). Categorical data are presented as a percentage. Non-normally distributed continuous variables were log-transformed for normal distribution. Between-group differences in the continuous variables were compared using the independent Comparison of the demographic and clinical characteristics between the two groups divided by the severity of NAFLD is described in The age of the subjects with moderate to severe NAFLD ranged from 25.8 to 82.5 years, and the age of the subjects without NAFLD or with mild NAFLD ranged from 35.8 to 88.2 years. Therefore, the subjects with moderate to severe NAFLD were younger than the subjects without NAFLD or with mild NAFLD. The subjects with moderate to severe NAFLD also had a higher BMI than the subjects without NAFLD or with mild NAFLD. There were no significant differences in the proportion of smoking, drinking alcohol, or usage of sulfonylurea, DPP4 inhibitor, metformin, thiazolidinediones, insulin, or statin between the two groups. The moderate to severe NAFLD group had higher glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), triglyceride (TG), and HbA1C levels than those without NAFLD or with mild NAFLD.We investigated the serum levels of formate, acetate, propionate, butyrate, isobutyrate, methylbutyrate, valerate, isovalerate, and methylvalerate using LC–MS in the study patients. The T2D subjects with moderate to severe NAFLD had lower levels of circulating formate, isobutyrate, and methylbutyrate than the subjects without NAFLD or with mild NAFLD. Higher isovalerate levels were borderline significantly found in T2D subjects with moderate to severe NAFLD. There were no significant differences in circulating acetate, propionate, butyrate, valerate, or methylvalerate between the two groups .2; metformin usage; and serum GOT, GPT, hemoglobin (Hb), log-formed TG, and isovalerate levels were significantly and positively correlated with an elevated risk of moderate to severe NAFLD in T2D patients : 0.17, 95% confidence index (CI): 0.03–0.86) and methylbutyrate  levels had an increased risk of moderate to severe NAFLD. However, there was no significant relationship between circulating isovalerate and the severity of the NAFLD in the adjusted analysis.To explore the association between the circulating SCFA levels and the severity of NAFLD in T2D patients, the logistic regression model was used and clinical data, laboratory data, and medications were also analyzed . In univpatients . Age and2, and HbA1C of 7.0%, and the results of the relationships between circulating isobutyrate and methylbutyrate levels and the severity of NAFLD independently of glycemic control remained consistent  and GPR43 receptors in adipose tissue, the intestine, and the liver, or by directly acting locally without binding to their receptors . In addiIn this study, we also explored the interaction effect of glycemic control on the relationship between circulating isobutyrate and methylbutyrate levels and the severity of NAFLD in T2D subjects. Our results showed negative relationships between the severity of NAFLD and circulating isobutyrate and methylbutyrate levels independent of glycemic control. Previous studies have reported that SCFAs act as mediators between the gut microbiota and the pancreas directly through receptors on pancreatic cells or via the gut-brain-pancreatic axis . SCFAs hIn addition, we found that the circulating levels of isobutyrate and methylbutyrate were negatively correlated with the severity of NAFLD in the male patients. Whether there are sex differences in the association between SCFAs and NAFLD severity is unknown. A previous study suggested that men have lower SCFA levels and dietary fiber intake compared with women . Sex difSome studies have measured SCFA in feces because the concentration range of SCFAs in the colon is at the millimolar level. However, this may not accurately reflect direct interactions with related organs, because approximately 95% of colonic SCFAs are absorbed, and only the remaining 5% are excreted in the feces . BecausePrevious studies have suggested that antidiabetic drugs may influence gut microbiota composition and the biosynthesis of SCFAs . In thisThis study has several limitations. First, SCFAs are mostly derived from the microbial fermentation of dietary fiber. However, we did not have detailed information on dietary fiber intake. Nevertheless, we included the crude dietary habits of the patients to minimize the impact of diet on the relationship between SCFAs and NAFLD. Second, there were no nondiabetic individuals in this study to compare with the T2D patients. However, our study aim was to examine the relationship between circulating SCFAs and NAFLD severity in T2D patients, not in the general population. Thus, these findings were not influenced regardless of whether nondiabetic individuals were enrolled in this study. Third, this cross-sectional study recruited a comparatively small sample size of participants, and the causality cannot be identified. In addition, we did not estimate SCFAs in the stool to compare the differences with serum SCFAs. Further research is needed to explore the interactions among SCFAs in the serum or feces, microbiota, and the severity of NAFLD in T2D subjects. Finally, the biological mechanism is clearly unknown, and future in vitro and in vivo researches are necessary to investigate the pathophysiologic mechanism of SCFAs in NAFLD progression and other novel biomarkers in T2D patients.In conclusion, circulating levels of isobutyrate and methylbutyrate were significantly and negatively correlated with NAFLD severity in the enrolled T2D subjects. Apart from the usual metabolic determinants, SCFAs may be associated with NAFLD severity in T2D patients.