Efficacy and Safety of Chinese Herbal Medicine Xiao Yao San in Functional Gastrointestinal Disorders: A meta-Analysis and Trial Sequential Analysis of Randomized Controlled Trials

Background and Aims: Functional gastrointestinal disorders are now named disorders of gut-brain interaction (DGBI) according to the Rome IV criteria, characterized by the interaction of gastrointestinal symptoms and dysregulation of central nervous systems. Xiao-Yao-San (XYS) is effective in the treatment of gastrointestinal symptoms in China, especially in patients with concurrent mood disorders. A meta-analysis was designed to evaluate the efficacy and safety of Xiao-Yao-San for FGIDs. Methods: We searched randomized controlled trials in seven databases from their inception till November 22, 2021. Pooled analysis included therapeutic efficacy, symptom score, Self-Rating Anxiety Scale (SAS) score, Self-Rating Depression Scale (SDS) score, and the recurrence rate. Conventional meta-analysis with random-effects model or fixed-effects model and trial sequential analysis (TSA) were performed. Results: A total of 48 RCTs were eligible for inclusion (n = 4,403). Meta-analysis results showed that XYS could improve the effective rate of FGIDs compared with western drugs [RR = 1.23; (95%CI, 1.19–1.27); p < 0.00001], and XYS combined with western medicine could also improve the effective rate [RR = 1.26; (95%CI, 1.21–1.33); p < 0.00001]. In addition, XYS could reduce the symptom score [SMD = −1.07; (95%CI −1.42, -0.72); Z = 6.03; p < 0.00001], SAS score [MD = −6.24; (95%CI −7.48, −4.99); Z = 9.81; p < 0.00001] and SDS score [MD = -6.70; (95%CI −8.18, −5.21); Z = 8.83; p < 0.00001] of FGIDs patients, and reduce the recurrence rate [MD = -6.70; (95%CI −8.18, −5.21); Z = 8.83; p < 0.00001]. XYS was safe in most cases and no serious adverse events were observed in any of the included trials. TAS showed adequate “information size” for the primary outcome, and further confirmed the efficacy of XYS in the treatment of FGIDs. Conclusion: XYS could improve symptoms and reduce recurrence rates in FGIDs patients, and XYS may be a potential candidate for the treatment of FGIDs. However, due to the limited quality of current studies, more long-term, randomized, double-blinded clinical trials are needed in future studies. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=284308, identifier CRD42021284308.


INTRODUCTION
Functional gastrointestinal disorders (FGIDs) are common disorders that are characterized by persistent and recurring gastrointestinal symptoms (Black et al., 2020). They occur as a result of abnormal functioning of the gastrointestinal tract. However, it is well recognized that, investigation reveals no underlying structural abnormality to explain these symptoms, and after several updates, they are now named disorders of gut-brain interaction (DGBI) according to the Rome IV criteria. According to the different gastrointestinal symptoms, they are divided into eight categories of 32 diseases (Drossman and Hasler 2016). Functional dyspepsia (FD), irritable bowel syndrome (IBS) and functional constipation (FC) are among the most widely recognized types of FGIDs. Their distribution differs by countries and geographic areas. For example, FD has a high prevalence of 10-40% in the West and low numbers (5-30%) in Asia (Enck et al., 2017). The global prevalence of IBS is around 11.2% (Enck et al., 2016), FC 15.3% (Barberio et al., 2021), leaving a considerable impact on patients, health-care systems and society as a whole because of repeated consultations, surgeries, prescriptions and over-the-counter medicine use, and impaired the quality of life and ability to work (Black et al., 2020).
The occurrence and development of FGIDs is the result of the interaction of physiological, psychological, and social factors. At present, the pathogenesis of FGIDs is not fully understood. The symptoms of FGIDs are closely related to dynamic disorders, visceral hypersensitivity, mucosal and immune function changes, intestinal flora changes, and central nervous system (CNS) dysfunction.
A retrospective analysis of 407 patients was conducted to study the relationship between three types of pathophysiological factors (visceral hypersensitivity, colonic transit abnormalities and psychological factors) and symptoms of IBS. It was found that these factors had cumulative effects on gastrointestinal and nongastrointestinal symptoms and quality of life in IBS patients (Simrén et al., 2019). At present, diet therapy, psychological therapy, behavioral therapy and other treatment methods gradually received attention, and the treatment of FGIDs has gradually shifted from single treatment to multidisciplinary treatment. A clinical trial of 188 patients with FGIDs found that comprehensive multidisciplinary clinical treatment (including gastroenterologists, dietitians, hypnotists, psychiatrists, and behavioral physiotherapists) was superior to gastrointestinal nursing in improving symptoms, psychological status, quality of life and reducing nursing costs for FGIDs. It was suggested that multidisciplinary treatment should be considered for FGIDs patients (Basnayake et al., 2020).
In this scenario, given the limitations of clinical treatment methods, Traditional Chinese medicine has become a choice for the treatment of FGIDs. A meta-analysis of 49 studies showed that Chinese medicine are well-tolerated and effective treatment for FGIDs (Tan et al., 2020). In traditional Chinese medicine theory, "liver stagnation and spleen deficiency" is one of the main causes of digestive tract symptoms. Xiao-Yao-San (XYS) is a traditional Chinese medicine prescription, which is used to treat the digestive system symptoms caused by this cause with a history of hundreds of years. It can also improve the subsequent adverse outcomes of mood disorders in patients of FGIDs.
Although XYS is widely used in clinical practice, its specific therapeutic effect on FGIDs is still not fully understood. There have been several randomized controlled trials (RCTs) which showed that XYS could produce good results in the treatment of FGIDs. However, there is still a lack of high-quality meta-analysis. Thus, we aimed to conduct a systematic review with meta-analysis to gather evidence on XYS in the treatment of FGIDs. The sample size was estimated by trial sequential analysis (TSA) to make a more objective evaluation of current studies and provide reference for future clinical medication and clinical research.
English. The specific random assignment method should be described, or the word random assignment should be mentioned. There was no restriction on the implementation of the blind method. 2) Participants in the studies were adults (age ≥ 18 years) with FGIDs diagnosed according to specific diagnostic criteria, regardless of gender or ethnicity. 3) The intervention measures of the experimental group were XYS or traditional Chinese medicine adjusted on the basis of XYS. The experimental group could be combined with the same western medicine as the control group. The form of traditional Chinese medicine was not limited, can be in the form of decoction, granule, capsule, tablet, powder and so on. 4) The intervention in the control group was routine treatment of FGIDs, and the type and dose of drugs were not limited. 5) The data in the original article was complete and extractable.
Exclusion criteria were as follows: 1) Duplicate detected or published studies. 2) The intervention in the experiment group included a combination of treatments that were not included in the criteria. 3) Formats without full text or results reported cannot be used for this meta-analysis.

Data Extraction
Two reviewers independently searched and abstracted the data and screened them according to inclusion and exclusion criteria. For studies that met the inclusion criteria, data were extracted independently by two reviewers. The following data was retrieved: title, author, date of publication, sample size, diagnostic criteria, baseline data, interventions, course of treatment, follow-up time, outcome evaluation indicators, results, and adverse events. Any discrepancy was discussed with the third party. The final data were reviewed by the corresponding author.

Methodological Quality Assessment
The quality of the study was graded independently by two reviewers according to the Cochrane collaboration tool. Its assessment includes random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other bias. The judgment of low risk, unknown risk, and high risk were given one by one according to the performance of the included literature in the above evaluation items. When two researchers disagree, a third-party researcher steps in to assess.

Statistical Analysis
Review Manager 5.3 was used for the meta-analysis. Relative risk (RR) was used for dichotomous variables, and standardized mean difference (SMD) was used for continuous variables as the combined statistic. Both were expressed with 95% confidence interval (CI). A statistical test for heterogeneity was conducted with the χ 2 test and inconsistency index statistic (I 2 (Higgins et al., 2003). If significant heterogeneity existed (I 2 >50% or p < 0.05), the pooled RR was evaluated by using a random effect model. Otherwise, a fixed-effect model was used (DerSimonian and Laird 1986). A statistical test for heterogeneity was conducted with the χ 2 test and inconsistency index statistic (I 2 (Higgins et al., 2003). If p > 0.10 and I 2 <50%, heterogeneity was considered acceptable, and fixed effects model was used to calculate the combined statistics. If the included study has high heterogeneity, sensitivity analysis or subgroup analysis should be performed to find the potential source of heterogeneity, and the random effects model should be used to calculate the combined statistics. The sensitivity analysis was carried out by deleting studies one by one, and the stability of the results can be determined at the same time.

Trial Sequential Analysis
Because of the increased risk of random errors resulted from sparsity data and repeated significance testing (Higgins et al., 2011), we conducted trial sequential analysis (TSA) for the primary outcomes to assess this risk using TSA program version 0.9.5.10 beta. The probability of class Ⅰ error was set as α 0.05, the probability of class Ⅱ error was set as β 0.2, and the statistical efficiency was 80%. The sample size was taken as required information size (RIS). The relative risk reduction rate (RRR) and control group event rate were set according to the results of meta-analysis.

Study Selection
According to the search strategy, a total of 552 articles were retrieved. After the duplicate articles were removed, the abstracts of the remaining 238 articles were browsed, filtered according to the inclusion and exclusion criteria, and a further 173 articles were excluded. Browse the full text of the remaining 65 articles and eliminate the articles with incomplete data and unqualified intervention measures. Finally, 48 articles were included for meta-analysis. The specific article screening process was shown in Figure 1.

Risk of Bias Assessment
All the inclusion trials were randomized, but some of them did not describe the specific randomization method, hence the evaluation was "unclear." Blindness was not described in any of the included studies. Complete details of the bias risk assessment for each included trial are shown in Table 3.  (Figure 3). The heterogeneity of each subgroup analysis was low, and the results were credible.   Fu (2016) Rome Ⅲ

Self-Rating Anxiety Scale
Four studies analyzed SAS score, including 272 subjects. The intervention methods of all the four studies were XYS combined with western medicine. The heterogeneity test results showed that I 2 <50% (I 2 32%, p 0.22), so the fixed effect model was adopted. The combined results showed that XYS combined with western medicine can effectively reduce the SAS score of patients with FGIDs [MD −6.24; (95%CI −7.48, −4.99); Z 9.81; p < 0.00001] ( Figure 5).

Self-Rating Depression Scale
SDS score was analyzed in four studies, including 272 participants. The intervention methods of all the four studies were XYS combined with western medicine. The heterogeneity test results showed that I 2 73%, so the random effect model was adopted. The combined results showed that XYS combined with western medicine can effectively reduce the SAS score of patients with FGIDs .62); Z 5.45; p < 0.00001] ( Figure 6A). The heterogeneity of the combined analysis was high, and the sensitivity analysis was carried out by removing the study. We found that the main source of heterogeneity was the study of Mai FY (Mai 2010), and the heterogeneity decreased after removing the study (I 2 0%, p 0.86). The results suggested that XYS combined with western medicine can reduce SDS score [MD −6.70; (95%CI −8.18, −5.21); Z 8.83; p < 0.00001] ( Figure 6B), which was the same as before, indicating that the results were relatively stable. The source of heterogeneity was related to the difference in clinical data collection.

Recurrence Rate
There were nine studies which followed up 602 participants for 6 months after treatment and calculated the recurrence rate. The combined results showed that the heterogeneity was low (I 2 0%, p 0.61), and the fixed effect model was adopted. The combined results showed that XYS could effectively reduce the recurrence rate of FGIDs patients [RR 0.23;(95%CI,; p < 0.00001] (Figure 7). Subgroup analysis showed that XYS [RR 0.33;(95%CI,; p < 0.0001] and XYS combined with western medicine treatment [RR 0.15;(95%CI,; p < 0.00001] (Figure 7) could both reduce the recurrence rate of patients. There was low heterogeneity between each subgroup (I 2 0%), so the results were reliable.

Trial Sequential Analysis
TSA was further performed based on the effectiveness of XYS in treating FGIDs. Analysis was made according to the different intervention methods and diseases of the treatment group (Figure 8).

Security Analysis
The safety of 19 trials (Jin 2007;Weng 2009;Gao and Liu 2010;Wei et al., 2010;Cui and Qian 2012;Zhu and Chen 2012;       Since most of the trials had no adverse events and could not be combined, the data were summarized in tabular form (Table 1).

DISCUSSION
This meta-analysis is the first systematic review and metaanalysis of RCTs of XYS in the treatment of FGIDs. The sample size of this meta-analysis was estimated through TSA in order to make a more objective evaluation of the current research and provide new evidence levels for patients, decision makers and doctors. The results of this study suggest that compared with western medicine treatment, single use of XYS or XYS combined with general drug treatment could increase the treatment efficiency of FGIDs and reduce symptom scores. Furthermore, the conclusion of the sample size was estimated by trial sequential analysis also confirmed the effect of XYS in the treatment of FGIDs. The current research has reached the expected sample size, and it is not necessary to expand the sample size for research. Rome IV clearly stated that FGIDs is a kind of "brain-gut interaction abnormalities" disease. Studies have shown that the probability of FD in patients with anxiety disorders is 7.6 times higher than that in patients without anxiety (Aro et al., 2015). The prevalence rates of anxiety disorder and anxiety in IBS patients were 39.1% and 23%, respectively, and the prevalence rates of depression disorder and depression were 28.8% and 23.3%, respectively (Zamani et al., 2019). Compared with normal people, constipation patients have higher anxiety and depression scores . Psychological changes often interact with gastrointestinal symptoms. A study followed up 2,885 randomly selected participants for 1 year and found that one third of people had mood disorders prior to FGIDs, and two thirds showed up FGIDs earlier than mood disorders (Koloski et al., 2016). In clinical treatment, the promotion of psychological factors on FGIDs cannot be ignored.
XYS has the effect of adjusting gastrointestinal function and improving mental and psychological abnormalities. XYS was recorded in the "Taiping Huimin Heji Jufang" (1078-1085 A.D.) in the Song Dynasty in China . It is composed of eight components: Radix Bupleuri, Radix Angelicae Sinensis, Radix Paeoniae Alba, Rhizoma Atractylodis Macrocephalae, Poria, Herba Menthae Haplocalycis, Rhizoma Zingiberis Recens and Radix Glycyrrhizae. The constituents of XYS analyzed by ultra-high performance liquid chromatography (UPLC) showed the representative constituents are paeoniflorin, ferulic acid, glycyrrhizic acid, liquiritin, and atractylenolide I (Su et al., 2021). XYS can improve gastrointestinal motility by increasing the level of motilin and 5-hydroxytryptamine (p < 0.05) and decreasing the level of somatostatin (p < 0.05) (Chen 2017). Studies have found that XYS may exert antidepressant effects by modulating inflammation-related receptors in advanced glycation end products (RAGE) to affect functional connectivity signals in the cingulate gyrus (Cg) and improve depressive-like behavior (Yan et al., 2021).
In this meta-analysis, a total of 48 studies were included in the meta-analysis, including 32 FD studies, 13 IBS studies and 3 FC studies. It could be seen that XYS was more commonly used in FD, while the related studies on XYS in the treatment of FC were rare. In the analysis of effective rate and symptom score, subgroup analysis was used according to different specific diseases, and the results were beneficial. The use of XYS alone and XYS combined with general drug treatment both lead to more positive results, suggesting that XYS could be used as an alternative to FGIDs regular therapy or as an additional treatment.
In addition, we also analyzed the SAS and SDS scores of FGIDs patients treated by XYS. In this systematic review, four studies analyzed the SAS and SDS of patients, using XYS combined with regular treatments. Although the results were positive, due to few included participants, future studies still need to be focused on investigating its effectiveness.
In 19 studies describing the safety of XYS, only 3 patients had adverse reactions, compared with 23 cases in the control group. All the adverse reactions were mild and tolerable. Since most of the experimental groups and the control group did not appear adverse reactions, meta-analysis cannot be carried out, nor can the above data explain the difference between the two groups. However, it suggested that XYS has a low probability of adverse reactions and is safe in clinical application. Although the adverse events were recorded in the 19 studies, no general safety indicators such as blood routine, urine routine, liver and kidney function were detected in patients.
FGIDs are chronic and difficult to cure. In the trials included in this meta-analysis, 9 studies followed up for 6 months after the end of treatment of FGIDs, and the low recurrence rate suggested that XYS could reduce the clinical recurrence rate of FGIDs, which also provided favorable evidence for XYS in clinical application. We conducted a TSA analysis of the effectiveness of XYS in the treatment of FGIDs, and the results showed that the cumulative sample size was sufficient to support the current meta-analysis. However, TSA analysis cannot solve the errors caused by methodological quality defects of included RCTs. Due to the general low quality of RCTs, it may affect the reliability of TSA results. Therefore, this result still needs to be treated with caution.
The following limitations should be paid close attention to in this study: First, some studies did not describe the specific random methods. Second, there was no detailed description of allocation concealment or blinding, which may be due to the large differences in the properties of traditional Chinese medicine compound and western medicine. Since it was difficult to implement blinding, it was undeniable that the lack of blinding would bring some bias. Third, the included studies reported inconsistent outcome indicators, which may lead to negative results unpublished, causing publication bias. Fourth, most of the included studies were single-center and smallsample studies, and most of the studies did not conduct long-term follow-up evaluation of the treatment effect of the subjects. Therefore, the results still need more rigorous multicenter, long-term, follow-up studies to verify. Fifth, all the participants included were Chinese, so the research conclusion has limited applicability to people of other ethnic groups. Sixth, there was evidence that the Low FODMAP diet and/or gluten Free diet can be beneficial for FGIDs patients (Bellini et al., 2020), but the studies included in this metaanalysis did not restrict patients' diets, which may lead to some bias. In future studies, attention should be paid to the possible influence of diet on these diseases.
However, future trials should address the limitations of existing methods. Many trials that contribute to results have an unclear risk of bias in sequence generation, allocation, concealment, and blinding.

CONCLUSION
Our study showed that XYS was effective in the treatment of FGIDs, including FD, IBS and FC, which could reduce the overall symptom score, SAS and SDS scores of patients and reduce the recurrence rate. No obvious adverse reactions were observed. TSA analysis confirmed our meta-analysis results. Therefore, XYS may be a potential candidate for the treatment of FGIDs. More randomized clinical trials focusing on the effect of XYS on FGIDs with long-term outcomes are warranted to support the clinical recommendation in future studies.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
QL wrote the manuscript and, TJ and XL conducted the databases search, and TZ conducted analysis and visualization. ZS was in charge of writing-reviewing. XS and YY took supervision. WW provided conceptualization. All authors reviewed the final manuscript.