Effects of the Kampo medicine Yokukansan for perioperative anxiety and postoperative pain in women undergoing breast surgery: A randomized, controlled trial.

Yokukansan (YKS) is a traditional Japanese herbal (Kampo) medicine prescribed for anxiety. In this randomized controlled trial, we compared the subjective assessment of anxiety using questionnaires and its objective assessment using salivary alpha-amylase concentrations in YKS and control (CNT) groups of women undergoing breast surgery. The trial was registered at the University Hospital Medical Information Network Clinical Trials Registry (registration number: UMIN000028998), and the investigators were blinded to drug administration. One hundred patients who underwent breast cancer surgery were allocated to either the YKS or the CNT group. Finally, 35 and 42 patients in the YKS and CNT groups were analyzed, respectively. The YKS group received two 2.5 g doses of the medication before sleeping on the night before surgery and 2 h before inducing anesthesia, while the CNT group did not receive medication preoperatively. Patients answered two questionnaires, the Hospital Anxiety and Depression Scale and the State-Trait Anxiety Inventory, pre-and postoperatively as subjective anxiety assessments. As an objective anxiety indicator, salivary alpha-amylase levels were measured the day before, directly before, and the day after surgery (T3). In the YKS group, salivary alpha-amylase scores directly before operation were significantly lower than those on the day before surgery and at one day postoperatively (F [2,150] = 3.76, p = 0.03). Moreover, the Hospital Anxiety and Depression Scale-Anxiety and State-Trait Anxiety Inventory-Trait scores were significantly more improved postoperatively in the YKS group than in the CNT group (difference in Hospital Anxiety and Depression Scale-Anxiety: YKS, mean -2.77, 95% confidence interval [-1.48 --4.06], p <0.001, and CNT, -1.43 [-0.25--2.61], p = 0.011; and difference in State-Trait Anxiety Inventory: YKS group, -4.23 [-6.95--1.51], p = 0.0004; and CNT group, 0.12 [-2.36-2.60], p = 0.92). No side effects were associated with YKS. YKS may reduce perioperative anxiety in patients undergoing surface surgery.

Introduction

Perioperative anxiety is associated with unfavorable physiological responses, such as tachycardia and hypertension [1]; it has been shown to prolong postoperative pain [2] and increase the risk of postoperative delirium [3]. Previous studies have shown that preventing preoperative anxiety improves surgical outcomes, including postoperative pain management [4] and recovery from anesthesia [5]. Benzodiazepines effectively reduce perioperative anxiety; however, they are associated with undesirable sedative effects [6]. Furthermore, antidepressants and other anxiolytics are unsuitable for perioperative administration due to their ineffectiveness and side effects, including nausea, thirst, and drowsiness [7].

Postoperative pain is among the most unpleasant symptoms associated with surgery. Severe postoperative pain may increase chronic postsurgical pain (CPSP) incidence. Accordingly, perioperative pain management is considerably important for both patient satisfaction and CPSP prevention. Since postoperative pain is exacerbated by perioperative anxiety, improving anxiety may improve CPSP.

Yokukansan (YKS) is a traditional Japanese herbal (Kampo) medicine that contains seven herbal extracts: Atractylodes Lancea Rhizome, Poria Sclerotium, Cnidium Rhizome, Uncaria Hook, Japanese Angelica Root, Bupleurum Root, and Glycyrrhiza. It is prescribed as a safe and effective medication for treating anxiety symptoms, such as irritability, restlessness, and insomnia. YKS has also been reported to effectively suppress chronic pain in several experimental settings [8] and improve pain disorders, such as headaches [9] and neuropathic pain [10]. Although the mechanism of action of YKS remains unclear, its anxiolytic-like effects may be due to its pharmacological effects on serotonin (5-hydroxytryptamine [5-HT]) and glutamate-mediated nervous system functions [11]. Furthermore, YKS has shown significantly better outcomes than diazepam in the modified Observer’s Assessment of Alertness/Sedation Scale in patients who underwent colon surgery [12]. However, no effects were observed on preoperative anxiety and postoperative delirium in patients undergoing highly invasive major surgeries in a study assessing the subacute effects of YKS on preoperative anxiety, that is, the effects of three doses daily for 7 days [13]. Moreover, postoperative delirium may be associated with the duration of surgery, postoperative rest, hypothermia, and intraoperative blood loss [14], which may have influenced the aforementioned study results. The acute effects of two doses of YKS preoperatively and that of YKS on perioperative anxiety and postoperative pain in patients undergoing surface surgery remain unclear.

We designed a randomized controlled trial (RCT) to compare the subjective assessment of anxiety using the Hospital Anxiety and Depression Scale (HADS) and State-Trait Anxiety Inventory (STAI) questionnaires with an objective assessment using measured salivary alpha-amylase (sAA) concentrations in YKS-treated and control groups of women undergoing breast surgery. The visual analog scale (VAS) for pain and quality of recovery (QoR)-15 scores was also measured to compare subjective pain and perioperative recovery quality assessments between both groups.

Materials and methods

Study setting and patients

This was a single-blind RCT in which the investigators were blinded to drug administrations. This study was approved by the institutional review board of the Niigata University Medical and Dental Hospital (reference number: 1031190049), and written informed consent was obtained from all enrolled patients. The trial was registered at the University Hospital Medical Information Network Clinical Trials Registry (registration number: UMIN000028998; principal Investigator: Hiroshi Baba; date of registration: July 10, 2017 [https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000032363]), and Japan Registry of Clinical Trials (registration number: CRB3180025; principal Investigator: Hiroshi Baba; date of registration: July 2, 2019 [https://jrct.niph.go.jp/latest-detail/jRCT1031190049]) with some modifications: the objective parameter of perioperative anxiety (sAA) was set as the primary outcome and questionnaire-assessed anxiety and perioperative pain (HADS, STAI, QoR-15, and VAS) were set as secondary outcomes. The trial was registered before initiation and patient enrollment. This manuscript adheres to the applicable CONSORT guidelines.

The study included 91 women aged 20–60 years who had been diagnosed with breast cancer and underwent partial or total mastectomy under general anesthesia at the Niigata University and Niigata Cancer Center Hospital between July 2017 and May 2020. Exclusion criteria were an American Society of Anesthesiology-Physical Status (ASA-PS) >4; a body mass index (BMI) >30 kg/m2; preexisting pain anywhere in the body; prescribed analgesics, opioids, antipsychotic drugs, or Kampo medicine; allergy to any drugs or food; psychological problems; central nervous system dysfunction; inability to understand Japanese; and the need for axillary lymph node clearance by intraoperative rapid pathological examination due to positive sentinel lymph node metastasis.

Participants were randomly assigned to the treatment (YKS) or control (CNT) groups using a computerized dynamic allocation algorithm written by the Division of Psych Statistics of Niigata University. Allocation was adjusted by minimization for procedure and age.

Intervention

One day preoperatively (T1), the age, BMI, surgical history, and ASA-PS of all eligible patients were recorded. On the night before surgery, HADS, STAI, and QoR-15 questionnaire surveys were conducted, and sAA was measured.

Patients in the YKS group received two doses of 2.5 g Tsumura Yokukansan Extract Granules (TJ-54, Tsumura & Co., Tokyo, Japan) before sleeping on the night before surgery and 2 h before inducing anesthesia, and 2.5 g of YKS contains 1.083 g of a dry extract of mixed herbal medicines in the following proportions: Atractylodes Lancea Rhizome 19.5%, Poria Sclerotium 19.5%, Cnidium Rhizome 14.6%, Uncaria Hook 14.6%, Japanese Angelica Root 14.6%, Bupleurum Root 9.7%, and Glycyrrhiza 7.3%. Patients in the CNT group only received water. sAA was measured again immediately preoperatively and immediately upon arrival to the operating room (T2). All eligible patients were induced with propofol-based general anesthesia, and only remifentanil was used as an intraoperative narcotic. One day postoperatively (T3), sAA was measured; VAS was assessed; HADS, STAI, and QoR-15 questionnaires were conducted again. The questionnaire was administered by an anesthesiologist who was not involved in the division of study groups and was not informed to which group the patients were assigned. The study outline is shown in Fig 1.

Fig 1

CONSORT flow diagram.

We measured sAA as an objective indicator of anxiety since psychosocial stress increases its release, reflecting the activity of the sympathetic-adrenal-medullary (SAM) system [15]; consequently, this system is associated with anxiety and arousal [16]. sAA was measured using a hand-held monitor (COCORO meter, NIPRO, Osaka, Japan) consisting of a disposable test strip and a monitor. The test strip contains 2-chloro-4-nitrophenyl-4-o-b-D-galactopyranosylmaltoside [17], which is hydrolyzed by alpha-amylase [18]. The hydrolyzing reaction proceeds until the substrates are completely consumed. The monitor quantifies the amylase activity by assessing the reaction time. sAA quantity has been suggested to reflect stress-related physiological changes and is used as an index for psychological stress, including anxiety, fear, and frustration [19].

We assessed HADS and STAI scores as subjective anxiety indicators pre-and postoperatively. HADS scale is a self-administered screening test developed by Zigmond et al. [20] for patients with physical illnesses. It is considered as a purely psychological assessment that excludes the influence of physical symptoms. This questionnaire consists of 14 items; 7 were related to depression (HADS-D), and 7 were related to anxiety (HADS-A), with values ranging from 0 to 21, and higher scores indicating more severe anxiety and depression. STAI is a self-reported measure of relatively stable individual differences in anxiety [21]. The test consists of two subscales: the S-scale, which evaluates anxiety as a transitory state, and the T-scale, which evaluates anxiety as a personality trait, with each subscale comprising 20 items. The S-scale evaluates how the respondent feels at a particular time due to stress owing to a perceived threat, while the T-scale evaluates how the respondent anticipates feeling during a hypothetical stressful situation. STAI is assessed on a scale of 20 to 80 points for both the T- and S-scales.

QoR questionnaires measure the recovery quality on multiple scales through patient-reported simple assessments of perioperative outcomes, including well-being, emotion, physical function, patient assistance, and pain [22]. In this study, we chose the QoR-15, which was previously utilized for breast cancer surgery [23]. QoR-15 was calculated by asking answers scored on a scale of 0 to 10, with the total score ranging from 0 to 150.

The 100-mm VAS score was used to measure subjective pain intensity and was assessed only once postoperatively at T3 (Fig 1). The VAS in this study had a 10-cm-long black line that was shown to the patient, with the far-left corresponding to “no pain” and the far-right corresponding to “the greatest pain imaginable”; this scale was used to indicate how much pain the patients were currently experiencing.

General anesthesia was induced by propofol target-controlled infusion (TCI) (target blood concentration 4.0 μg×ml-1), remifentanil continuous infusion (0.5 μg×kg-1 min-1), and a 0.6-mg×kg-1 bolus injection of rocuronium to facilitate the insertion of the ProSeal Laryngeal Mask Airway (Teleflex Medical Japan, Tokyo, Japan). Anesthesia was maintained by propofol TCI, and the rate of remifentanil infusion was adjusted in the range of 0.05−0.5 μg×kg-1×min-1. Heart rate and blood pressure were maintained within 20% of their baseline values.

All breast tissues were removed during total mastectomy, whereas some breast tissues with tumors were removed during partial mastectomy. The surgeon also removed one sentinel lymph node in the latter operation. Lymph nodes were subsequently tested by intraoperative rapid pathological examination to evaluate whether they contained any cancer cells. If sentinel node metastasis was negative, the wound was closed, and the study continued; if metastasis was positive, the patient was excluded as axillary lymph node clearance was required.

For postoperative analgesia, 0.001% of epinephrine containing 1% lidocaine was injected into the surgical site directly before incision, and 1000 mg of acetaminophen was administered intravenously at the time of wound closure. If the patient complained of pain after awakening, 100 mg of tramadol was administered intravenously, and if the pain was still severe, 50 mg of diclofenac was additionally administered.

We used perioperative sAA to objectively measure anxiety and the HADS and STAI scores as subjective measures of anxiety. Changes in sAA were considered primary outcomes, and those in HADS, STAI, QoR-15, and VAS were considered secondary outcomes.

We determined our sample size based on the following criteria using G* power version 3.1.7 [24]. The mean (standard deviation) sAA score in a previous study was 37 (12). A difference in the sAA score of 15 points was considered statistically significant, and the sAA score for pain on postoperative day 1 was 25. Thus, a power analysis using a type I error estimate of 5% and a power of 80% indicated that a sample size of 40 patients per group was needed to detect this difference. Therefore, we decided to enroll 50 patients in each arm, considering possible dropouts.

For continuous variables, data were expressed as mean (standard deviation) after evaluating normality. Differences in values between pre- and postoperative assessments for each participant were expressed as mean [95% confidence interval] values. sAA scores were log-transformed before the test to obtain a normal distribution. A Bartlett test was performed to confirm the equivalence of variances before analysis.

sAA measures data at three-time points. Therefore, the analysis of variance (ANOVA) performed on sAA measurements had two factors, that is, intervention (YKS and CNT) and time (pre-, directly post-surgery, and next day). The remaining data were measured at only two time points: pre-surgery and next day, as measurements could not be performed immediately after postoperatively.

Therefore, we described the ANOVA for sAA and other measures separately. Additionally, a repeated measures ANOVA was used to assess differences between HADS and STAI scores as intervention×time interaction effects. A multiple comparison analysis for intervention × time interaction effects were performed using Holm’s modified sequentially rejective Bonferroni procedure. Significant and insignificant results were then followed up with a generalized eta squared estimation of effect size specifically designed for a repeated measures ANOVA. Confidence intervals for effect sizes were calculated using the bootstrap method. We used Welch’s t-test to compare demographic data and VAS scores and Fisher’s exact test to compare categorical data. All tests were two-tailed, with an alpha level of 0.05 indicating statistical significance. All analyses were conducted using R version 4.0.0 [25] and R function “anovakun” version 4.8.5. Data were visualized using violin plots [26], as implemented in Graphpad Prism 8 for macOS (Graphpad software, San Diego, CA). QoR-15 was not used in this analysis because a serious error was discovered at the time of analysis after measurement was completed, making the values of scale scores and total scores unreliable. Moreover, as dropout occurred after data acquisition, the sample size did not reach the calculated sample size. Hence, a post-hoc power analysis was conducted using G* power version 3.1.7.

Results

Recruitment and baseline characteristics

The participant flowchart is shown in Fig 1. The remaining 35 patients in the YKS group and 42 patients in the CNT groups were analyzed. Demographic characteristics are presented in Table 1. There were no significant differences in age, BMI, ASA, implementing facilities, and surgical procedure between both groups.

Table 1

Patient demographic details.

	YKS (n = 35)	CNT (n = 42)	P-value
Age, year (SD)	49 (6.2)	48 (5.7)	0.72
BMI (SD)	20.9 (3.3)	21.7 (2.8)	0.09
ASA (1:2)	21:22	21:23	0.25
Facility (university: cancer center)	22:21	21:27	0.88
Procedure (partial resection: total mastectomy)	22:21	21:27	0.88

YKS, Yokukansan; CNT, control; SD, standard deviation; BMI, body mass index; and ASA, American Society of Anesthesiologists

Data are presented as mean (standard deviation) in each group and were analyzed using a t-test (age, BMI), and Wilcoxon rank sum test (ASA, implementing facilities, and surgical procedure).

Effects of YKS on perioperative anxiety and postoperative pain

sAA scores were significantly lower in the YKS group than in the CNT group at T2 (YKS, 0.88 (0.42); CNT, 1.14 ± 0.49; F [2,150] = 3.76; p = 0.03; and gη2 = 0.017). Moreover, the sAA score at T2 was significantly lower than those at T1 and T3 in the YKS group (T1, 1.14 (0.55); T2, 0.88 (0.42); T3, 1.07 (0.54); T1 vs. T2: p = 0.03; T2 vs. T3: p = 0.1; and T1 vs. T3: p = 1.0), yet there were no significant differences in the sAA scores of the CNT group between all timepoints (T1, 1.08 (0.50); T2, 0.14 (0.49); T3, 1.20 (0.49); T1 vs. T2: p = 1.0; T2 vs. T3: p = 1.0; and T1 vs. T3, 0.43; Fig 2). Results of the post-hoc power analysis showed slight high power (0.708) but did not reach the recommended power of 0.8.

Fig 2

Salivary-α-amylase activity.

Comparison of salivary-α-amylase activity in the Yokukansan (YKS) (a) and control (CNT) (b) groups by violin plots. Plot width is scaled to data distribution. Horizontal bars represent medians, and dashed lines represent 25th and 75th percentiles. The vertical axis represents logarithmically converted values. Data were analyzed by a two-way repeated-measures ANOVA and pre-specified follow-up tests of primary outcomes. Using a modified sequentially rejective Bonferroni test, a primary outcome with a p-value <0.05 was considered statistically significant.

Regarding the HADS-A score, there was an interaction effect between groups and time, with the YKS group showing a tendency towards higher scores than the CNT group; however, this difference was not statistically significant (F [1,75] = 3.08; p = 0.083; gη2 = 0.01, as assessed by a two-way ANOVA). HADS-A scores were reduced postoperatively in both groups. However, the corresponding decline was greater in the YKS group than in the CNT group (YKS: T1, 7 [6-10]; T3: 6 [3-7]; T3-T1 difference, -2.77 [-1.48–-4.06]; p <0.001; and CNT: T1, 6 [5–9.25]; T3, 5 [3–8.25]; T3-T1 difference, -1.43 [-0.25–-2.61]; p = 0.011, as assessed by Holm’s multiple comparison test, Fig 3).

Fig 3

Hospital Anxiety and Depression Scale-Anxiety (HADS-A) score.

Comparison of anxiety levels at T1 and T3, as assessed by the HADS-A score in the Yokukansan (YKS) and control (CNT) groups by violin plots. The vertical axis represents the score, and the plot width is scaled to data distribution. Horizontal bars represent medians, and dashed lines represent 25th and 75th percentiles. Data were analyzed by a two-way repeated-measures ANOVA. Using a sequentially rejective Bonferroni test, a primary outcome with a p-value <0.05 was considered statistically significant.

Regarding the STAI-T score, there was also an interaction between groups and time, with the YKS group showing higher scores than the CNT group at T1 and T3 (F [1,75] = 7.26; p = 0.009; gη2 = 0.011, as assessed by a 2-way ANOVA). The STAI-T score significantly decreased postoperatively in the YKS group, but not in the CNT group (YKS: T1, 47 [41-53]; T3, 44 [36-48]; T3-T1 difference, -4.23 [-1.51–-6.95]; p <0.001; CNT: T1, 45.5 [36.75–52.25]; T3, 44 [36.75–54.5]; T3-T1 difference, 0.12 [-2.36–2.60], p = 0.92, as assessed by Holm’s multiple comparison test, Fig 4).

Fig 4

State-Trait Anxiety Inventory-Trait (STAI-T) score.

Comparison of anxiety levels at T1 and T3, as assessed by the STAI-T score in the Yokukansan (YKS) and control (CNT) groups by violin plots. The vertical axis represents the score, and the plot width is scaled to data distribution. Horizontal bars represent medians, and dashed lines represent 25th and 75th percentiles. Data were analyzed by a two-way repeated measures ANOVA. Using a sequentially rejective Bonferroni test, a primary outcome with a p-value <0.05 was considered statistically significant.

Contrarily, there were no significant differences in HADS-D, STAI-S, and VAS scores between both groups (Table 2). The possible side effects of a single dose of YKS include appetite loss, nausea, diarrhea, and rash. We investigated the presence of such side effects when the corresponding anesthesiologist visited the patient’s room to measure postoperative amylase and collect questionnaires. There were no adverse events.

Table 2

Results of all questionnaires.

	YKS (n = 35)		CNT (n = 42)		P-value
	T1	T3	T1	T3	group	time	interaction
HADS-Anxiety	7.74 (2.84)	4.97 (2.58)	7.48 (4.00)	6.05 (3.69)	0.55	<0.01	0.08
HADS-Depression	4.71 (2.82)	5.17 (3.16)	6.10 (4.50)	6.64 (4.45)	0.09	0.11	0.88
STAI-state	47.26 (8.48)	39.51 (8.33)	50.33 (11.06)	41.93 (10.71)	0.17	<0.01	0.75
STAI-trait	46.83 (9.75)	42.60 (9.01)	45.36 (11.36)	45.48 (11.71)	0.76	0.01	0.01
VAS (pain)		1.4 (0.4971)		1.36 (0.49)	0.70

YKS, Yokukansan; CNT, control; HADS, Hospital Anxiety and Depression Scale; STAI, State-Trait Anxiety Inventory; and VAS, visual analog scale

Data are presented as the mean (standard deviation) in each group. Data were analyzed using a t-test for VAS and a two-way repeated measures ANOVA for the remaining parameters.

Discussion

This study evaluated the effects of YKS on perioperative anxiety and postoperative pain using sAA, HADS, STAI, and VAS scores. Preoperative YKS administration significantly reduced thepreoperative sAA scores, but not one day postoperative scores. Furthermore, YKS affected HADS and STAI scores.

In a previous study, STAI and HADS were compared as measures of perioperative anxiety and were shown to correspond with anxiety on VAS [27]. In this study, two of these three indicators were used, and in both groups, postoperative HADS-A scores were lower than preoperative scores, possibly reflecting reassurance among patients upon the completion of surgery. However, the decrease in the HADS-A score was larger in the YKS group than in the CNT group. Postoperative STAI scores were also reduced in both groups; however, postoperative STAI-T scores significantly decreased only in the YKS group.

To the best of our knowledge, this is the first superiority trial to investigate the effects of YKS on anxiety using both subjective and objective rating scales. Our results suggested that two doses of YKS produced immediate preoperative physiological and psychological changes and confirmed that YKS effectively reduces perioperative anxiety.

YKS is used as an anxiolytic for irritability, restlessness, and insomnia and may have the pharmacological properties of antidepressant drugs. This effect may be explained by basic research showing that YKS affects the serotonin system in nervous tissue [11, 28]. Further, a previous large-scale study has shown that antidepressants improved trait anxiety [29]; our result indicative of an improvement in STAI-T scores after YKS administration was consistent with the result of the aforementioned study.

Perioperative anxiety may prolong postoperative pain [2], which is one of the most unpleasant postoperative symptoms. CPSP, defined by the International Association for the Study of Pain as pain lasting >3 months postoperatively [30], is known to increase in patients with severe postsurgical pain. Moreover, depression, anxiety, satisfaction with postoperative pain management, and mental and physical stress are psychological risk factors for CPSP development and severity [31-33]. Currently, perioperative pain management is considered important for both patient satisfaction and CPSP prevention. Therefore, controlling perioperative anxiety and postoperative pain is crucial for CPSP prevention.

St. John’s wort, valerian, kava, and passionflower are widely used for anxiety as alternatives to benzodiazepine; however, they have been associated with adverse effects, including increased bleeding and drug interactions [34]. There are no reports of drug interactions with YKS, and a previous study has suggested that YKS improves behavioral and psychological symptoms of dementia without serious side effects [35]. Hence, we hypothesized that YKS might be more beneficial than the aforementioned drugs. It is generally believed that the number of herbs included in a traditional Japanese prescription is directly proportional to the duration it takes for it to be effective. YKS contains seven herbs and is usually administered for a long duration. However, our results showed that YKS is effective even when taken for a short period, reducing the risk of side effects and patient’s medication intake.

Basic research suggests that the pharmacological actions of YKS affect serotonin (5-HT) and the functions of the glutamate-mediated nervous system [11, 28, 36]. Serotonin is a neurotransmitter involved in the descending pain inhibitory system, weakening pain perception [37]. Moreover, excessive glutamate in the excitatory synaptic cleft is believed to be responsible for pain hypersensitivity [38]. Furthermore, the activation of the NMDA glutamate receptor is believed to be involved in hyperalgesia. YKS conventionally acts as an antagonist of NMDA receptors by binding to them and thus potentially treating neuropathic pain [39].

Although several clinical reports have suggested an analgesic effect of YKS, no relevant RCTs have been performed to confirm the same result. To the best of our knowledge, this is the first prospective RCT to investigate such effects. Contrary to our expectations, the effects of YKS on postoperative pain could not be demonstrated in this study. A possible reason for the lack of significant differences might have been the floor effect, with relatively low values in all patients. It may be necessary to investigate more invasive types of surgery to detect significant antinociceptive effects of YKS perioperatively. Moreover, the possibility that CPSP may be suppressed by the anxiolytic effect of YKS when taken postoperatively for a longer time remains to be elucidated.

This study has several limitations. First, it was not double-blinded. We could not form a placebo group because a placebo pill with the flavor and bitterness of Japanese herbal medicine is not available and would be difficult to produce. Conventionally, Japanese herbal medicines have a distinctive bitter taste and flavor. Sucrose and lactose, which are usually used as placebos, are different from Japanese herbal medicines, and people are familiar with the taste and thus cannot be used as placebos. Therefore, we deemed double blindness impossible, meaning that the patients were aware of their group. Thus, study results may not reflect the direct effects of YKS as the mere reassurance provided by its administration may have reduced anxiety and pain. Second, sAA may reflect stress-related physiological changes, though not anxiety specifically. However, as the psychological state of perioperative patients is often treated as “anxiety,” we considered anxiety as a stress response based on Lazarus’ psychological stress model [40]. Third, the number of subjects for analysis was less than 40 in the YKS group. In this study, more than 40 participants were initially assigned to each group, but after excluding dropouts, 42 and 35 participants in the YKS and CNT groups, respectively, were finally included in the analysis. The post-hoc power analysis yielded a high power of 0.708 but failed to reach the recommended power of 0.8. However, we attempted to provide reliable evidence from a small sample size by simultaneously reporting effect sizes and significant differences. Hence, this study was limited to 77 patients, and further large-scale investigations using placebos are needed to determine the effects of YKS on anxiety and pain.

Conclusions

This RCT compared perioperative anxiety and postoperative pain in YKS-treated and control groups of women undergoing breast surgery. Our results suggested that YKS may reduce perioperative anxiety in patients undergoing surface surgery. Currently, there is no standard treatment for suppressing perioperative anxiety. While larger, double-blind, placebo-controlled studies are necessary, this study justifies further research into the potential of YKS for reducing perioperative anxiety.

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8 Jun 2021

PONE-D-21-08399

Effects of the Kampo medicine Yokukansan for perioperative anxiety and postoperative pain in women undergoing breast surgery: A randomized, controlled trial

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: YKS is used for Neurosis, insomnia, night cry in children, and peevishness in children in Japan. The authors have applied YKS for anxiety in the patients undergoing surgery. This randomized controlled study showed the effect of traditional Japanese medicine Yokukansan (YKS) for reducing perioperative anxiety in the patients undergoing surface surgery.

This study has novelty of YKS on the treatment in clinical setting. However, there are some lacks of study information. I comments some points as follows.

Introduction

The author described the side effects of benzodiazepines or antidepressants as follows in the Introduction section.

Benzodiazepines effectively reduce perioperative anxiety; however, they are associated with undesirable sedative effects [6]. Furthermore, antidepressants and other anxiolytics are not suitable for perioperative administration due to their ineffectiveness and significant side effects [7].

Please describe the detail of the dis advantage of these medicines.

Method

A detailed description of the intervention chemicals is needed. The amounts of crude drugs included in YKS extract should be explained in the Method section.

This study design seemed open label study. If not, the method of investigator blinding should be explained.

TJ-54 is indicated for the relief of the following symptoms of those patients with delicate constitution and nervousness: Neurosis, insomnia, night cry in children, and peevishness in children in Japanese National Health Insurance System. In the present study, YKS has prescribed for anxiety. Was YKS prescribed on the Japanese National Health Insurance System or administered with research funds? Please clarify how intervention was performed.

Result and interpretation

Patients can understand their intervention with or without YKS. Placebo effects is highly affected to the result. Please discuss more about these limitation.

The author showed the result of no side effects in the present study. However, there is no explanation about the evaluation of adverse event and side effect. Please add the explabation how the investigator evaluate adverse event and side effect.

Please add the detail data of QoR-15.

Reviewer #2: This is a well-written report of a clinical trial. From a statistical point of view, the investigators used minimization on two variables to randomize (?). The question mark indicates that it is not clear if they used a deterministic minimization (Taves) or a randomized design (Pocock-Simon). Please clarify. Also, if there are only 2 variables, why use minimization? Why not just stratify? Please explain. The sample size computation is appropriate, but they were not able to achieve the requisite sample size. In the discussion, I would like to see a post-hoc power analysis using the observed parameter estimates, rather than the guesses in the sample size section. Finally, since it was not double-blinded, how was the outcome assessed? Who administered the questionnaires? Was it someone who was unaware of the "treatment"?

Reviewer #3: The authors have shown the efficacy of yokukansan on anxiety using both subjective and objective rating scales. We think that it is very interesting study; however, the problems listed below will need to be addressed before the paper is suitable for publication.

Major points

Although the authors used changes in sAA, HADS, and STAI as primary outcomes, it would be best to use only one thing as the primary outcome. Further, the sample size was for sAA in this study. If you decide on a primary outcome other than sAA, the sample size will become quite different.

The authors planned for yokukansan to be taken two times before sleep on the night before surgery and 2h before the induction of anesthesia. Was the duration of the first two times the same in this study? Further, why did you decide to take yokukansan 2hr before the induction of anesthesia?

Minor points

1 In the analysis of HADS, is there a statistically significant difference between the two groups over time?

2 The figure legends should be written after the references, please check that the formatting follows the journal’s instructions for authors.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Shin Takayama

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: 210531 Kainuma Reviewers comments for PLOS one.pdf

Click here for additional data file.

7 Jul 2021

Reviewer #1: YKS is used for Neurosis, insomnia, night cry in children, and peevishness in children in Japan. The authors have applied YKS for anxiety in the patients undergoing surgery. This randomized controlled study showed the effect of traditional Japanese medicine Yokukansan (YKS) for reducing perioperative anxiety in the patients undergoing surface surgery.

This study has novelty of YKS on the treatment in clinical setting. However, there are some lacks study information. I comment some points as follows.

Introduction

The author described the side effects of benzodiazepines or antidepressants as follows in the Introduction section.

Benzodiazepines effectively reduce perioperative anxiety; however, they are associated with undesirable sedative effects [6]. Furthermore, antidepressants and other anxiolytics are not suitable for perioperative administration due to their ineffectiveness and significant side effects [7].

Please describe the detail of the disadvantage of these medicines.

We thank reviewer #1 for the valuable comments. We have added specific examples of the disadvantages of using antidepressants and other anxiolytics in the introduction as follows (P5, L2–5): “Benzodiazepines effectively reduce perioperative anxiety; however, they are associated with undesirable sedative effects [6]. Furthermore, antidepressants and other anxiolytics are not suitable for perioperative administration due to their ineffectiveness and side effects, including nausea, thirst, and drowsiness [7].”

Method

A detailed description of the intervention chemicals is needed. The amounts of crude drugs included in YKS extract should be explained in the Method section.

We have added extensive details on yokukansan as follows (P9, L7-10): “Patients in the YKS group received two doses of 2.5 g Tsumura Yokukansan Extract Granules (TJ-54, Tsumura & Co., Tokyo, Japan) before sleeping on the night before surgery and 2 h before inducing anesthesia, and 2.5 g of YKS contains 1.083 g of a dry extract of mixed herbal medicines in the following proportions: Atractylodes Lancea Rhizome 19.5%, Poria Sclerotium 19.5%, Cnidium Rhizome 14.6%, Uncaria Hook 14.6%, Japanese Angelica Root 14.6%, Bupleurum Root 9.7%, and Glycyrrhiza 7.3%.”

This study design seemed open label study. If not, the method of investigator blinding should be explained.

As reviewer #1 indicated, this clinical study is a prospective open label trial in which the assessor was single-blinded, as shown in head of the method section (P7, L9-10).

TJ-54 is indicated for the relief of the following symptoms of those patients with delicate constitution and nervousness: Neurosis, insomnia, night cry in children, and peevishness in children in Japanese National Health Insurance System. In the present study, YKS has prescribed for anxiety. Was YKS prescribed on the Japanese National Health Insurance System or administered with research funds? Please clarify how intervention was performed.

In this study, YKS was prescribed on the Japanese National Health Insurance System.

Result and interpretation

Patients can understand their intervention with or without YKS. Placebo effects is highly affected to the result. Please discuss more about these limitations.

We completely agree with the notion of reviewer #1. Since this is an open-label clinical trial, the influence of the placebo effect cannot be excluded. We also were not able to exclude the possibility of the fact that the patients took the medication affected their amylase levels or questionnaire results. Based on the results of this study, a more rigorous, double-blind study should be conducted.

Several other studies used sucrose or lactose powder as placebo medication, but since many Japanese people understand the unique flavor of Kampo medicine, even if these were used as placebo medicine, patients would immediately know that this was not Kampo medicine and would not be blinded. In other words, we thought it was less legitimate to use sucrose or lactose as placebo medication and decided to have the control group drink only water as an open-label study. We have added this opinion in the limitations section (P26 L9-P27L1).

The author showed the result of no side effects in the present study. However, there is no explanation about the evaluation of adverse event and side effect. Please add the explanation how the investigator evaluates adverse event and side effect.

The possible side effects of a single dose of yokukansan include appetite loss, nausea, diarrhea, and rash. We investigated the presence of such side effects during the visit of the anesthesiologist to the patient’s room for measuring postoperative amylase levels and collecting questionnaires. There were no adverse events (P21 L5-L8).

Please add the detail data of QoR-15.

Thank you for your advice. Based on your suggestions, we reviewed the data in detail to perform a reanalysis and found that there was a serious mistake in the QoR-15 questionnaire; therefore, we deemed analyzing the corresponding data to be inappropriate Since all outcomes were pre-registered, it was clearly stated that QoR-15 was excluded from the analysis. QoR-15 was described as a measured item in the methods section. We have added the corresponding statements in the methods section (P15 L14-P16L1).

Reviewer #2: This is a well-written report of a clinical trial. From a statistical point of view, the investigators used minimization on two variables to randomize (?). The question mark indicates that it is not clear if they used a deterministic minimization (Taves) or a randomized design (Pocock-Simon). Please clarify.

We are glad to receive positive comments from reviewer #2. In this study, our allocation algorithm was based on the Pocock & Simon method, and we used three variables for dynamic allocation: age, HADS, and institution. We did not use stratification because we wanted to make grouping as simple as possible.

Also, if there are only 2 variables, why use minimization? Why not just stratify? Please explain. The sample size computation is appropriate, but they were not able to achieve the requisite sample size. In the discussion, I would like to see a post-hoc power analysis using the observed parameter estimates, rather than the guesses in the sample size section.

Moreover, following your suggestion, we tested the post-hoc power analysis using G* power version 3.1. The result of the power analysis was 0.708, which was deemed to be somewhat a high-power result; however, the analysis did not reach the recommended power of 0.8. As described in the main text, although we reached the predetermined number of cases, there were more dropouts than expected. We have added the results of the post-hoc analysis to the text and added a description in the limitation section about the fact that the power of the test was less than 0.8 (P18 L4-5).

Finally, since it was not double-blinded, how was the outcome assessed? Who administered the questionnaires? Was it someone who was unaware of the "treatment"?

The questionnaire was administered by an anesthesiologist who was not involved in the allocation of the study groups and not informed to which group the patients were assigned (P9 L15-P10L2).

Reviewer #3: The authors have shown the efficacy of yokukansan on anxiety using both subjective and objective rating scales. We think that it is very interesting study; however, the problems listed below will need to be addressed before the paper is suitable for publication.

We thank reviewer #3 for the positive comments. We will answer the issues raised by reviewer #3 as much as possible as follows:

Major points

Although the authors used changes in sAA, HADS, and STAI as primary outcomes, it would be best to use only one thing as the primary outcome. Further, the sample size was for sAA in this study. If you decide on a primary outcome other than sAA, the sample size will become quite different.

Thank you for your advice. As you suggested, we set sAA as the primary outcome and questionnaire results as secondary outcomes. We calculated the sample size using differences in sAA as already shown in the sample size calculation (P13 L12-15).

The authors planned for yokukansan to be taken two times before sleep on the night before surgery and 2h before the induction of anesthesia. Was the duration of the first two times the same in this study? Further, why did you decide to take yokukansan 2hr before the induction of anesthesia?

In a previous study (Evid Based Complement Alternat Med. 2014;2014: 965045, ref. #12), YKS was administered once 5 h before surgery. However, in cases in which surgery is conducted in the morning, YKS is orally administered very early in the morning, two hours before prohibiting water. However, since the previous study used drugs that were non-inferior to benzodiazepines, we aimed to administer a slightly higher dose of YKS to clarify its effect in this superiority study. Nevertheless, since prolonging the duration of medication would increase the number of dropouts, we started medication administration after the corresponding patient was admitted to the hospital to ensure that the medication was administered twice.

Minor points

1 In the analysis of HADS, is there a statistically significant difference between the two groups over time?

We found that there were no significant differences in the main effect, but there were significant differences in the interaction.

2 The figure legends should be written after the references, please check that the formatting follows the journal’s instructions for authors.

The journal guidelines verify that figure legends should be included immediately after the citation of the corresponding figure in the text. Please see the corresponding guidelines below:

• Figure captions are inserted immediately after the first paragraph in which the figure is cited. Figure files are uploaded separately.

22 Oct 2021

PONE-D-21-08399R1

Effects of the Kampo medicine Yokukansan for perioperative anxiety and postoperative pain in women undergoing breast surgery: A randomized, controlled trial

PLOS ONE

Dear Dr. Kamiya,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the discussion with Reviewers, the authors revised as follows.

Changes in sAA were considered as primary outcomes, and those in HADS, STAI, QoR-15, and VAS were considered as secondary outcomes.

However, in the UMIN registration, the authors declare that Primary outcomes as “subjective evaluation items: HADS( Hospital anxiety and depression scale)” and “objective evaluation items: salivaly amylase activity” and Secondary outcomes as “STAI(State Trait Anxiety Inventory)”, QoR (quality of recovery) score”, and “VAS score of pain”.

This study is designed clinical trial, if you change this point, amendment of trial design is needed.

Reviewer #2: (No Response)

Reviewer #3: Major points

Although the authors used changes in sAA, HADS, and STAI as primary outcomes, it

would be best to use only one thing as the primary outcome. Further, the sample size

was for sAA in this study. If you decide on a primary outcome other than sAA, the

sample size will become quite different.

OK

Thank you for your advice. As you suggested, we set sAA as the primary outcome and

questionnaire results as secondary outcomes. We calculated the sample size using

differences in sAA as already shown in the sample size calculation (P13 L12-15).

The authors planned for yokukansan to be taken two times before sleep on the night

before surgery and 2h before the induction of anesthesia. Was the duration of the first

two times the same in this study? Further, why did you decide to take yokukansan 2hr

before the induction of anesthesia?

In a previous study (Evid Based Complement Alternat Med. 2014;2014: 965045, ref.

#12), YKS was administered once 5 h before surgery. However, in cases in which

surgery is conducted in the morning, YKS is orally administered very early in the

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

morning, two hours before prohibiting water. However, since the previous study used

drugs that were non-inferior to benzodiazepines, we aimed to administer a slightly

higher dose of YKS to clarify its effect in this superiority study. Nevertheless, since

prolonging the duration of medication would increase the number of dropouts, we

started medication administration after the corresponding patient was admitted to the

hospital to ensure that the medication was administered twice.

OK

Minor points

1 In the analysis of HADS, is there a statistically significant difference between the two

groups over time?

We found that there were no significant differences in the main effect, but there were

significant differences in the interaction.

OK

2 The figure legends should be written after the references, please check that the

formatting follows the journal’s instructions for authors.

The journal guidelines verify that figure legends should be included immediately after

the citation of the corresponding figure in the text. Please see the corresponding

guidelines below:

•Figure captions are inserted immediately after the first paragraph in which the figure is

cited. Figure files are uploaded separately.

OK

The author responded appropriately to reviewers comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Shin Takayama

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Nov 2021

Reviewer #1: In the discussion with Reviewers, the authors revised as follows.

Changes in sAA were considered as primary outcomes, and those in HADS, STAI, QoR-15, and VAS were considered as secondary outcomes.

However, in the UMIN registration, the authors declare that Primary outcomes as “subjective evaluation items: HADS　(Hospital anxiety and depression scale)” and “objective evaluation items: salivary amylase activity” and Secondary outcomes as “STAI (State Trait Anxiety Inventory)”, QoR (quality of recovery) score”, and “VAS score of pain”.

This study is designed clinical trial, if you change this point, amendment of trial design is needed.

Response

We thank reviewer #1 for pointing out the ambiguity in the manuscript. In this clinical study, we needed re-registration with the Japan Registry of Clinical Trials (jRCT). In the modified registry, we set the value of sAA (objective outcome of perioperative anxiety) as the primary outcome, and questionnaire-assessed anxiety and perioperative pain (HADS, STAI, QoR-15, and VAS) were set as secondary outcomes. The jRCT has been reviewed by the secretariat of the Central Clinical Research Review Committee of Niigata University, which is a third-party organization and has a stricter protocol. This research was conducted in accordance with the research protocol registered in jRCT, which is generally consistent with the research protocol registered in UMIN.

We have added the description about registration in jRCT and described primary and secondary outcomes in the materials and methods section (P8L109 – P9L113).

Reviewer #2: (No Response)

Reviewer #3: Major points

・Although the authors used changes in sAA, HADS, and STAI as primary outcomes, it would be best to use only one thing as the primary outcome. Further, the sample size was for sAA in this study. If you decide on a primary outcome other than sAA, the sample size will become quite different.

Response: Thank you for your the valuable suggestion. Accordingly, we set sAA as the primary outcome and questionnaire results as a secondary outcome. We calculated the sample size using differences in sAA as already shown in the sample size calculation (P14 L191-195).

OK

・The authors planned for yokukansan to be taken two times before sleep on the night before surgery and 2h before the induction of anesthesia. Was the duration of the first two times the same in this study? Further, why did you decide to take yokukansan 2hr before the induction of anesthesia?

Response: In a previous study (Evid Based Complement Alternat Med. 2014;2014: 965045, ref. #12), Yokukansan was administered once 5 hours before surgery. However, in cases where surgery is conducted in the morning, YKS was orally administered very early in the morning, two hours before prohibiting water. However, since the previous study used drugs that were non-inferior to benzodiazepines, we aimed to administer a slightly higher dose of Yokukansan to clarify its effect in this superiority study. Nevertheless, since prolonging the duration of medication would increase the number of dropouts, we started administration of yokukansan after hospital admission of patients to ensure that the medication was administered twice.

OK

Minor points

1 In the analysis of HADS, is there a statistically significant difference between the two groups over time?

Response: We found no significant differences in the main effect, but there were significant differences in the interaction.

OK

2 The figure legends should be written after the references, please check that the formatting follows the journal’s instructions for authors.

The journal guidelines verify that figure legends should be included immediately after the citation of the corresponding figure in the text. Please see the corresponding guidelines below:

Response: Figure captions have been inserted immediately after the first paragraph whereeach figure is cited as mentioned in the submission guidelines. Figure files are uploaded separately.

OK

The author responded appropriately to reviewers’ comments.

Response: We thank reviewer #3 for the effort in assessing our rebuttal letter.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

12 Nov 2021

Effects of the Kampo medicine Yokukansan for perioperative anxiety and postoperative pain in women undergoing breast surgery: A randomized, controlled trial

PONE-D-21-08399R2

Dear Dr. Kamiya,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Johannes Fleckenstein

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

16 Nov 2021

PONE-D-21-08399R2

Effects of the Kampo medicine Yokukansan for perioperative anxiety and postoperative pain in women undergoing breast surgery: A randomized, controlled trial

Dear Dr. Kamiya:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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PLOS ONE