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Efficacy of MaZiRenWan, a Chinese Herbal Medicine, in Patients With Functional Constipation in a Randomized Controlled Trial
Accepted Manuscript Efficacy of MaZiRenWan, a Chinese Herbal Medicine, in Patients With Functional Constipation in a Randomized Controlled Trial Dr. Linda LD. Zhong, MD, PhD, Ms. Chung-wah Cheng, MPhil, Dr. Wai Kun, MD, Mr. Liang Dai, MPhil, Ms. Dong-dong Hu, MPhil, Ms. Zi-wan Ning, MPhil, Dr. Haitao Xiao, PhD, Dr. Cheng-yuan Lin, PhD, Ms. Ling Zhao, MPhil, Dr. Tao Huang, PhD, Dr. Ke Tian, PhD, Dr. King-hong Chan, MD, Dr. Ting-wa Lam, MD, Dr. Xiaorui Chen, MD, Dr. Chi-tak Wong, MD, Prof. Min Li, MD, Prof. Ai-ping Lu, MD, PhD, Prof. Justin CY. Wu, MD, Prof. Zhao-xiang Bian, MD, PhD
PII: DOI: Reference:
S1542-3565(18)30341-0 10.1016/j.cgh.2018.04.005 YJCGH 55782
To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 2 April 2018 Please cite this article as: Zhong LL, Cheng C-w, Kun W, Dai L, Hu D-d, Ning Z-w, Xiao H-t, Lin Cy, Zhao L, Huang T, Tian K, Chan K-h, Lam T-w, Chen X-r, Wong C-t, Li M, Lu A-p, Wu JC, Bian Zx, Efficacy of MaZiRenWan, a Chinese Herbal Medicine, in Patients With Functional Constipation in a Randomized Controlled Trial, Clinical Gastroenterology and Hepatology (2018), doi: 10.1016/ j.cgh.2018.04.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title page Title: Efficacy of MaZiRenWan, a Chinese Herbal Medicine, in Patients With Functional Constipation in a Randomized Controlled Trial
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Authors:
Dr. Linda LD Zhong1*, MD, PhD; Ms. Chung-wah Cheng1*, MPhil; Dr. Wai Kun1, MD; Mr. Liang Dai1, MPhil; Ms. Dong-dong Hu1, MPhil; Ms. Zi-wan Ning1, MPhil; Dr.
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Hai-tao Xiao1, PhD; Dr. Cheng-yuan Lin1, PhD; Ms. Ling Zhao1, MPhil; Dr. Tao Huang1, PhD; Dr. Ke Tian1, PhD; Dr. King-hong Chan2, MD; Dr. Ting-wa Lam3, MD; Dr.
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Xiao-rui Chen2, MD; Dr. Chi-tak Wong2, MD; Prof. Min Li1, MD; Prof. Ai-ping Lu1, MD, PhD; Prof. Justin CY Wu4, MD#; Prof. Zhao-xiang Bian1, MD, PhD#
1. Hong Kong Chinese Medicine Study Centre, Hong Kong Baptist University, Hong
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Kong, P. R. China
2. Department of Family Medicine & General Out-patient Clinics, Kowloon Central Cluster, Hospital Authority, Kowloon City, Hong Kong, P. R. China
R. China
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3. Department of Medicine, Queen Elizabeth Hospital, Hospital Authority, Hong Kong, P.
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4. Institute of Digestive Disease, Faculty of Medicine, The Chinese University of Hong Kong, P. R. China
*: Equal contribution.
Grant Support: This study was supported by the Health and Health Services Research Fund (HHSRF) from the Food and Health Bureau of Hong Kong (project no. 09101501).
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Abbreviations Used in This Paper: AE, adverse event; ANOVA, analysis of variance; CHM, Chinese herbal medicine; CSBM, complete spontaneous bowel movement; CI,
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confidence interval; FC, functional constipation; GMP, Good Manufactory Practice; HHSRF, Health and Health Services Research Fund; MZRW, MaZiRenWan; NHS, National Health Service; RCT, randomized controlled trial; SD, standard deviation; TCM,
Co-correspondence: Prof. Justin CY Wu, Institute of Digestive Disease, Faculty of
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#
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Traditional Chinese Medicine.
Medicine, The Chinese University of Hong Kong, P. R. China, Email address: [email protected], Telephone: 852 3505 3174, Fax: 852 2646 8915 and Prof. Zhao-xiang Bian, Hong Kong Chinese Medicine Study Centre, Hong Kong Baptist
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University. 3/F, Jockey Club School of Chinese Medicine Building, 7 Baptist University Road, Kowloon Tong, Hong Kong, P. R. China. E-mail address: [email protected],
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Telephone: 852 3411 2905, Fax 852 3411 2902.
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Disclosures: The authors declare that they have no competing interests.
Writing Assistance: We have asked Dr. Martha Dahlen for critical English editing.
Authors Contributions: All authors participated in the design of the study and performed the trial. Linda LD Zhong drafted and Chung-wah Cheng revised the manuscript. Justin CY Wu and Zhao-xiang Bian supervised and coordinated the clinical
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trial. Linda LD Zhong and Wai Kun participated in general trial coordination and recruited the participants. King-hong Chan, Ting-wa Lam and Xiao-rui Chen referred and recruited patients. Chung-wah Cheng, Liang Dai, Tao Huang and Ke Tian participated in
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randomization and statistical analysis. Dong-dong Hu, Zi-wan Ning, Cheng-yuan Lin, Ling Zhao and Hai-tao Xiao conducted all the quality control studies of MZRW. Min Li
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authors read and approved the final manuscript.
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and Ai-ping Lu gave critical advice on the design and implementation of the study. All
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Abstract Background & Aims: The Chinese herbal medicine, MaZiRenWan (MZRW), has been used for more than 2000 years to treat constipation, but it has not been tested in a
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randomized controlled trial. We performed a trial to evaluate the efficacy and safety of MZRW, compared with the stimulant laxative senna or placebo, for patients with
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functional constipation (FC).
Methods: We performed a double-blind, double-dummy, trial of 291 patients with FC
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based on Rome III criteria, seen at 8 clinics in Hong Kong from June 2013 through August 2015. Patients were observed for 2 weeks and then assigned randomly (1:1:1) to groups given MZRW (7.5 g, twice daily), senna (15 mg daily), or placebo for 8 weeks. Patients were then followed for 8 weeks and evaluated at baseline and weeks 4, 8 (end of
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treatment), and 16 (end of follow up). Participants recorded information on stool form and frequency, feeling of complete evacuation, and research medication taken. Data on individual bowel symptoms, global symptom improvement, and adverse events were
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collected. A complete response was defined as an increase ≥1 complete spontaneous bowel movement (CSBM)/week from baseline (the primary outcome). Secondary
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outcomes included response during the follow-up period, colonic transit, individual and global symptom assessments, quality of life measured with 36-item short form Chinese version, and adverse events.
Results: Although there was no statistically significant difference in proportions of patients with a complete response to MZRW (68%) vs. senna (57.7%) (P=.14) at week 8,
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there was a statistically significant difference vs. placebo (33.0%) (P<.005). At the 16-week timepoint (after the 8-week follow-up period), 47.4% of patients had a complete response to MZRW, 20.6% had a complete response to senna, and 17.5% had a complete
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response to placebo (P<.005 for MZRW vs. placebo). The group that received MZRW group also had significant increases in colonic transit and reduced severity of constipation, straining, incomplete evacuation, and global constipation symptoms compared with the
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groups that received placebo or senna in (P<.05 for all comparisons).
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Conclusion: In a randomized controlled trial of 291 patients with FC, we found MZRW to be well-tolerated and effective in increasing CSBM/week. MZRW did not appear to be more effective than senna, and might be considered as an alternative to this drug.
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ClincialTrials.gov no: NCT01695850.
KEY WORDS: alternative medicine, plant, Asia, functional bowel disorder
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(382 words-edited by CGH’s Science Editor)
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Background Constipation is a common chronic gastrointestinal disease with prevalence varying from 0.7% to 79% (median 16%) in the global population.1 Conventional drug therapies for
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constipation include fiber supplements, stool softeners or wetting agents, osmotic and stimulant laxatives, and, more recently, chloride channel activators, guanylate cyclase C activators, opioid receptor antagonists and serotonergic agonists.2 However, with the
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unsatisfactory response and immaturity and relatively high economic burden of new
herbal medicine (CHM).
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agents,3-5 many patients seek help from alternative medicine, mostly by taking Chinese
According to Traditional Chinese Medicine (TCM) theory, constipation can be divided into excessive and deficient Patterns based on the underlying etiology6,7. The former is
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characterized by the presence of heat or pathological accumulation of Qi (stagnation). The Chinese herbal compound formula, MaZiRenWan (MZRW), also called Hemp Seed Pills, was first recorded in the TCM classic, “Discussion of Cold-induced Disorders”
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(ShangHanLun). It comprises six herbs: Fructus Cannabis (HuoMaRen), Radix et Rhizoma Rhei (DaHuang), Radix Paeoniae Alba (BaiShao), Semen Armeniacae Amarum
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(KuXingRen), Fructus Aurantii Immaturus (ZhiShi) and Cortex Magnoliae Officinalis (HouPo). MZRW is marketed in the United States and many other countries as dietary supplement. Based on modern pharmaceutical studies, MZRW can stimulate intestinal mucosa, increase secretion, accelerate intestinal peristalsis and decrease water absorption6-9-12. From our previous studies of MZRW, the dose of 7.5g b.i.d. had better therapeutic effect than its lower dose (2.5g b.i.d) and recommendated dose (5.0g b.i.d)
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among 96 subjects. Furthermore, its efficacy was superior to placebo in a double-blinded, randomized, controlled trial (RCT) with 120 subjects13.
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Senna, due to its low cost, safety and ease of administration, is widely prescribed in clinical practice and recorded in Laxative Treatment Guideline 14-17. This study aimed to evaluate the efficacy and safety of a Chinese herbal medicine, MZRW, by comparing with
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stimulant laxative, senna, and placebo for patients with FC in excessive TCM syndrome.
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In reporting details of trial, we followed the recommendations of CONOSRT Extension for Chinese Herbal Medicine Formulas 2017 (CONSORT-CHM Formulas 2017).18
Methods
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Study Design
This trial was a multi-center, randomized, double-blinded, double-dummy, controlled trial conducted in the Lee Kee Memorial Dispensary (general outpatient clinic), the Li Ka Shing
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Specialist Clinic of Prince of Wales Hospital and six Chinese medicine clinics affiliated with the School of Chinese Medicine, Hong Kong Baptist University. After a 2-week
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run-in, eligible subjects were randomly assigned to the MZRW arm (MZRW plus placebo senna), the senna arm (placebo MZRW plus senna), or the placebo arm (placebo MZRW plus placebo senna). All subjects received eight weeks of treatment and eight weeks follow-up. Five visits, in total, were scheduled at week -2 (run-in), week 0 (baseline), week 4 (within treatment), week 8 (end of treatment) and week 16 (end of follow-up).
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The study protocol was approved by the Ethics Committee on the Use of Human Subjects for Teaching and Research (Approval no. HASC/10-11/16) in September 2011, registered at ClinicalTrials.gov (NCT01695850) in September 2012 and published in 2013
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(Supplemental file 1).19 The study design was based on the recommendations made in the 'Design of Treatment Trial for Functional Gastrointestinal Disorders’, as proposed by the Rome III Working Team20. The duration of treatment was based on our previous two
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clinical studies of MZRW13. Written informed consent was obtained from each subject
and approved the final manuscript.
Participants and Recruitment
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who agreed to participate. All the authors had access to the study data and had reviewed
The participants were recruited from the public through advertisements or press releases in
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local newspapers. Diagnosis of FC was based on Rome III criteria: 1) including two or more of the following in at least 25% of defecations: straining, lumpy or hard stools, sensation of incomplete evacuation, sensation of anorectal obstruction/blockage, manual
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maneuvers to facilitate, or less than 3 defecations per week; 2) rare presence of loose stools without the use of laxatives; 3) insufficient criteria for irritable bowel syndrome.20
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Excessive Pattern was defined as any three of the chief symptoms: 1) dry and hard stools; 2) difficult bowel movements; 3) abdominal distension, with or without tenderness; 4) belching; 5) dry mouth or halitosis; 6) red tongue with dry and/or yellow coating; 7) wiry pulse.6,7
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Subjects were recruited only if they fulfilled all of the followings: 1) the diagnostic criteria for FC; 2) the diagnostic criteria for Excessive Pattern; 3) aged 18 to 65 years; 4) complete spontaneous bowel movement (CSBM) ≤2times/week (CSBM was defined by feeling of
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complete passage of stool and without the use of laxatives or enemas within 24 hours);21 5) severity of constipation ≥3 points (on a 7-point scale); 6) total symptom score ≥8 points (on a 7-point scale for constipation-related symptoms); 7) normal colonic examination (barium
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enema or colonoscopy) within five years; 8) normal liver and renal function in blood test
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within three months.
In addition, patients were excluded if they had drug-induced or secondary causes of constipation, abdominal surgery within one year, taken other herbal medicine for constipation in the last three months, or severe diseases (e.g. cancer, acute asthma).
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Patients with a history of allergy to CHM, psychiatric or addictive disorders requiring medications with side effects of constipation, or women who were pregnant or
Interventions
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breast-feeding were also excluded from the study.
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Based on previous study result, a dose of 15g a day (7.5g, b.i.d) of MZRW granules was selected.13 The placebo granules were made from dextrin (76.03%), tea essence (23.61%), gardenin (0.02%) and caramel (0.34%).13 Patients were instructed to dissolve a sachet of granules in 150ml of hot water; and to take the solution orally twice daily, after breakfast and dinner. Both of them were prepared by PuraPharm Pharmaceuticals (Nanning, China). The entire manufacturing process was in strict compliance with the standards of Good
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Manufactory Practice (GMP) and Chinese Pharmacopoeia 2010.22 The composition of MZRW and placebo, and the authentication, production, pharmacology study and safety details are listed in Supplemental file 2. The senna tablets (trade name: Senokot)
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containing 7.5mg Sennoside B were manufactured by Reckitt Benckiser Company, UK.16,23 The placebo tablets were made of starch and pigment by Guangzhou Huahai Pharmaceuticals (Guangzhou, China). Patients were instructed to take two tablets at
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bedtime.
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Rescue medication/enema: Dulcolax tablets and Dulcolax suppositories were provided as rescue medicines to ensure bowel movements only in those patients without bowel movements for at least three consecutive days during the study.
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To ensure the success rate of blinding, the color, smell, taste, appearance and packaging of placebos were comparable to the interventions (see Supplemental file 3).
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Outcomes
Participants were requested to record their stool form and frequency, feeling of the
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completeness of evacuation, and research medication taken daily in the diary.
The primary outcome was the proportions of patients with a complete response during treatment. Participants with an increase ≥1CSBM/week from baseline were defined as complete response. The changes in colonic transit detected by using a commercially available radio-opaque Sitzmarks capsule (Konsyl Pharmaceuticals, US) was secondary
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outcome24-26. Slow transit constipation (STC) is defined as expulsion of less than 80% markers after 120 hours, i.e. more than 5 out of 24 markers remained. Other secondary outcomes included the complete response of CSBM during the follow-up period.
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Individual symptoms of severity of constipation, sensation of straining, incomplete evacuation, bloating, abdominal pain / cramping, nausea, and passing of gas) were self-assessed by subjects using a 7-point ordinal scale (0=not at all to 6=very severe) at
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every visit. Global symptom improvement was defined as the feeling of adequate relief of constipation in comparison with baseline with scores (0=markedly worse to 6=markedly
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better). The quality of life was measured by the 36-item Short Form (SF-36) Chinese version.27
Safety profiles of MZRW were assessed by determining important adverse events (AEs)
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reported or clinical laboratory evaluations. The success of blinding was evaluated for both investigators and patients, as which of MZRW, senna or placebo had been taken during the
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last visit.
Randomization and Blinding
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Block randomization was carried out in a 1:1:1 ratio according to the sequence generated with Random Allocation Software (Version 2.0), Isfahan, Iran. Research assistant assigned interventions according to the codes kept in opaque sealed envelopes with consecutive randomization numbers. Treatment assignments were not revealed and were blinded to patients and investigators (including statisticians) until the entire study was completed.
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Sample size calculation Based on the results of our previous placebo-control study, the proportions of a complete 13
Since there
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response of MZRW group and placebo groups were around 40% and 10%.
has been no RCT study of senna, we assumed the response rates for MZRW, senna and
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placebo were 40%, 20% and 10%, respectively.
Therefore, 82 patients per treatment group were deemed sufficient to achieve 80% power
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in detecting treatment differences, based on two-sided Chi-square test without continuity correction at a significance level of 0.025 (used to maintain the overall significance level at 5%). Further assuming a 15% dropout rate, a total of 291 patients (97 per arm) were recruited to ensure statistically significant results. The calculation was performed using
Statistical Analysis
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Study Size 2.0 software, London, UK.
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All efficacy and safety analyses were conducted according to the intention-to-treat (ITT) principle, as the ITT population included all patients who were randomized. Missing
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values were imputed by the last-observation-carried-forward method. The details of statistical analysis appear in Supplemental file 4.
Results
Patient Characteristics
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From June 2013 to August 2015, 843 potential subjects were screened; of these, 291 were randomized and assigned to MZRW, senna, or placebo groups (97 per arm). In total, 33 subjects withdrew from the study. Seven of them were lost to follow-up, 18 discontinued
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their interventions due to unsatisfactory effects, seven withdrew due to AEs and one was pregnant. For those who completed the study, 95.3% (246/258) were compliant with at least 80% of scheduled doses, which was determined by counting the medication returned.
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The flow diagram is shown in Figure 1. The baseline characteristics of the three groups
Primary Outcome Assessment
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were well balanced (P>.05) and are summarized in Table 1.
The proportion of complete response of MZRW group was comparable to senna group during treatment (68.0% vs. 57.7%, with P=.14), but both were significantly higher than
(P<.005).
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that of placebo group (33.0%, with P<.005), with significant inter-group difference
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Secondary Outcome Assessment Complete response and CSBMs
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The proportions of complete responses during the follow-up period were 47.4% in MZRW group, 20.6% in senna group, and 16.5% in placebo group (P<.005). The mean CSBM per week increased 2.2times (95%CI: 1.8, 2.6) in MZRW group, 2.0times (95%CI: 1.6, 2.3) in senna group and 1.2times (95%CI: 0.8, 1.6) in placebo group during treatment, with significant inter-group difference (P=.002). In contrast, the mean CSBM in the follow-up period increased 1.5 times (95% CI: 1.1, 1.9) in MZRW group, 0.6 times
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(95%CI: 0.3, 0.8) in senna group and 0.5times (95%CI: 0.2, 0.7) in placebo group, with significant inter-group difference (P<.0005) (Table 2). The weekly changes of complete response and CSBM are shown in Figure 2. Proportion of patients with weekly
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frequency CSBM ≥3 and an increase ≥1 from baseline among three groups is presented in Supplemental file 5.
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Colonic transit
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Before treatment, proportions of slow transit colonic (STC) patients (>5 out of 24 markers remained) were among 47.1% to 59.6%, without significant inter-group difference (P=.637) and post-hoc pairwise multiple comparison (P>.05). Significant change of STC proportion were found after 8-week treatment (P=0.006). Post-hoc pairwise multiple comparison of slow transit after 8-week treatment is as follows: MZRW
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vs. placebo: P=.002; MZRW vs. senna: P=.039; senna vs. placebo: P=.251. Proportion of STC subjects and no STC subjects were represented by percentage in Figure 3.
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Individual symptom assessment and rescue medicine
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In general, comparing outcomes with baselines for all three groups, scores for individual symptoms were lower after treatment and during follow-up period. MZRW was superior to senna and placebo in reducing the severity of constipation, sensation of straining, incomplete evacuation, and passing of gas (P<.05), while it was comparable to senna in managing abdominal bloating and abdominal pain/cramping (Table 2). The frequency on using rescue medicine per week was lower in the MZRW group than in both senna and
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placebo groups during treatment (P=.029 and .003, respectively) and the follow-up period (P=.007 and .001, respectively) (Table 2).
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Other secondary outcomes
The results of global symptom assessment and quality of life assessment are represented
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in Supplemental file 6.
Success of blinding
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With regard to participants, 34.5% (89/258; 35 in MZRW group, 26 in senna group and 28 in placebo group) correctly guessed their treatment. With regard to investigators, 55.4% (143/258; 57 in MZRW group, 49 in senna group and 37 in placebo group) correctly guessed. For subjects with correct answers, 68.5% (61/89) were based on the
intervention.
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efficacy of treatment, 22.5% (20/89) on the taste and 9.0% on the appearance of the
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Safety and adverse events
There were no significant differences in renal and liver function between and within the
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MZRW, senna and placebo groups at baseline and after treatments. Most subjects well tolerated the research medication, and no serious AEs reported (Supplemental file 7).
Discussion
The results from this study showed that both MZRW and senna had beneficial effects on increasing CSBM and higher proportion of complete response than placebo during
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treatment, and MZRW had even better than senna with regard to relieving most of constipation-related symptoms and improving the colonic transit during treatment and follow-up period. Why MZRW prolonged improvement in bowel movement even after
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treatment, i.e. during the follow-up period, might due to some specific metabolites, whose changes are significantly correlated with CSBM improvement. Our further research is
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focusing on MZRW’s mechanism28.
It has been reported in other studies of FC that placebo effects can range from 18.0% to
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37.5%29,30. In this study, we found the placebo effect was about 33.0% during treatment and 17.5% during follow-up, a rate consistent with those studies. But in our previous study with 120 patients reported placebo effect was 8.3%13. A number of factors could explain this variation, such as three-armed design (MZRW, senna and placebo), two types
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of medications (Chinese herbal medicine and conventional laxative) and larger sample sizes (291 vs. 120 subjects).31
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How to apply the randomized controlled clinical trial methodology in CHM research always challenges clinical investigators. Our comprehensive assessment of MZRW, from
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first identification of target intervention with a systematic review, determination of optimal dosage with a RCT, evaluation of its efficacy and safety with a placebo-controlled study, to further verification of therapeutic effects by comparison with standard conventional treatment, can be a reference study model for other CHM interventions. This evidenced-based approach with transparent and full disclosure of the composition, authentication, processing, production and quality control of MZRW
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following the recommendations of CONSORT-CHM Formula 2017 ensures the quality and reproducibility of intervention, as well as the study results.18
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The current study also investigated the efficacy and safety of senna by comparing with placebo. Results showed that senna was better than placebo in improving mean CSBM by 2.0/week (95%CI: 1.6, 2.3) vs. 1.2/week (95%CI: 0.8, 1.6) during treatment (P<.05). As
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being the first RCT comparing senna with placebo, the study results provide consolidated
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evidence of the efficacy of senna in the management of FC.
There are several limitations to this study. First, the majority of the study population was female. Second, although the Pattern concept was used to select eligible subjects, the changes in Pattern parameters were not quantitatively assessed nor calculated as an
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outcome. Thirdly, the sample size calculation was only based on MZRW and placebo; the effects of senna was assumed. If we want to investigate the superiority of MZRW as compared to senna, a larger sample size clinical trial would be required. Our results also
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suggest that combination therapy with both MZRW and senna is worthy of investigation. Fourthly, the population was all ethnic Chinese. There may have been a higher response
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rate to TCM due to preconceived cultural expectations or beliefs; the efficacy might be reduced, or different, in other ethnic populations.
In conclusion, MZRW is a well-tolerated and effective intervention for FC. It could be considered as an alternative remedy for FC. Further, the approach of assessing the efficacy and safety of MZRW by first having a systematic review, determining the
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optimal dose, followed by comparing with placebo and standard conventional treatment
Table Legends Table 1. Baseline characteristics of participants
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can be a reference for other CHM intervention studies.
Table 2. Comparison of treatment effect toward bowel movement, individual symptom
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Figure Legends
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assessment and rescue medicine used
Figure 1. FC = functional constipation; MZRW = MaZiRenWan; ITT analysis = intention-to-treat analysis, which consisted of all randomly assigned patients.
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Figure 2. a. Weekly CSBMs were recorded by patients in the stool and symptom diary. Error bars represented 95% confidence intervals. The MZRW group and senna group increased 2.2 and 2.0 mean CSBM/week from baseline during treatment, respectively
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(P>.05). The P values were calculated from two-way ANOVA analysis and t-test (*, P<.05, **, P<.01).
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b. Complete response was defined as an increase ≥1 CSBM/week from baseline. The P values were calculated from Fisher-exact test (*, P<.05; **, P<.01). CSBM = complete spontaneous bowel movement; MZRW = MaZiRenWan
Figure 3. The colonic transit was measured by using a radio-opaque capsule containing 24 markers. Slow transit constipation (STC) was defined as expulsion of less than 80%
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markers after 120 hours (>5 out of 24 markers remained). Proportion of STC subjects (cyan) and no STC subjects (purple) were represented by percentage. No significant differences among three groups were detected at baseline (pre-treatment, P=.637).
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Significant change of STC proportion were found after 8-week treatment (P=.006).
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MZRW = MaZiRenWan; ns=no significance.
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17. National Institute for Health and Care Excellence. Clinical Knowledge Summary: CKS Constipation Guideline. https://cks.nice.org.uk/constipation. Accessed May 18, 2017.
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18. Cheng CW, Wu TX, Shang HC, et al. CONSORT extension for Chinese herbal
Internal Medicine 2017;167:112-121.
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medicine formulas 2017: recommendations, explanation, and elaboration. Annual of
19. Zhong LL, Cheng CW, Chan Y, et al. Chinese medicine formula (Ma Zi Ren Wan) for functional constipation: study protocol for a prospective, double-blinded, double-dummy, randomized controlled trial. Trials 2013;14:366.
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20. Drossman DA, Corazziari E, Delvaux M, et al. Rome III: The Functional Gastrointestinal Disorders. McLean, VA: Degnon Associates, 2006. 21. Tarumi Y, Wilson MP, Szafran O, et al. Randomized, double-blind, placebo-controlled
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trial of oral docusate in the management of constipation in hospice patients. Journal of Pain and Symptom Management 2013;45:2-13.
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22. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China, 2010th ed. Beijing: People’s Medical Publishing House, 2010. 23. Ford AC, Suares NC. Effect of laxatives and pharmacological therapies in chronic idiopathic constipation: systematic review and meta-analysis. Gut 2011;60:209-218. 24. Konsyl Pharmaceuticals. Sitzmarks: the preferred colonic transit diagnostic test. http://www.sitzmarks.com (For Professionals). Accessed May 19, 2017.
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25. Park JM, Choi MG, Choi H, et al. Measurement of colonic transit using a delayed-release capsule containing radio-opaque markers. Scandinavian Journal of Gastroenterology 2008;43:545-550.
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26. Drossman D, Chang L, Chey WD, et al. Rome IV Functional Gastrointestinal Disorder-Disorder of Gut Brain Interaction, 4th ed. Raleigh, NC: Rome Foundation, 2016.
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27. Lam CL. Reliability and construct validity of the Chinese (Hong Kong) SF-36 for patients in primary care. Hong Kong Practitioner 2003;25:468-475.
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28. Huang T, Ning ZW, Hu DD, et al. Uncovering the mechanisms of Chinese Herbal Medicine (MaZiRenWan) for functional constipation by focused network pharmacology approach.
29. Lacy BE, Schey R, Shiff SJ, et al. Linaclotide in chronic idiopathic constipation
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patients with moderate to severe abdominal bloating: a randomized, controlled trial. PLos One 2015;10:e0134349.
30. Rao S, Lembo AJ, Shiff SJ, et al. A 12-week, randomized, controlled trial with a
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4-week randomized withdrawal period to evaluate the efficacy and safety on linaclotide in irritable bowel syndrome with constipation. American Journal of Gastroenterology
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2012:107:1714-1724.
31. Pollo A, Benedetti F. The placebo response: neurobiological and clinical issues of neurological relevance. Progress in Brain Research 2009;175:283-294.
(Total words: 3972)
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Supplemental files Supplemental file 1: Published study protocol
safety details of MaZiRenWan Supplemental file 3: Packaging and appearance of interventions Supplemental file 4: Statistical analysis
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Supplemental file 2: Composition, authentication, preparation, pharmacokinetics and
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Supplemental file 5: The proportion of patients with weekly frequency ≥3times CSBM
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and an increase ≥1 CSBM from baseline
Supplemental file 6: Results of other secondary outcomes
Supplemental file 7: Adverse events occurred in different groups
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Supplemental file 8. CONSORT – Chinese Herbal Medicine Formulas Checklist
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ACCEPTED MANUSCRIPT Table 1 Baseline characteristics of participants Characteristic
All Patients
MZRW
Senna
Placebo
(N=291)
(n=97)
(n=97)
(n=97)
Sex, n (%)
P value
0·071 263 (90·4)
86 (88·7)
93 (95·9)
84 (86·6)
Male
28 (9·6)
11 (11·3)
4 (4·1)
13 (13·4)
Age, mean (SD), y
45·4 (12·0)
45·5 (12·0)
45·1 (11·7)
Duration of FC, mean (SD), y
15·6 (11·5)
15·9 (11·4)
16·2 (11·7)
SBM per week, mean (SD)
1·8 (0·9)
1·9 (1·0)
1·7 (0·6)
CSBM per week, mean (SD)
1·8 (0·9)
1·9 (1·1)
1·7 (0·8)
CSBM < 3 per week, n (%)
256 (88.0%)
83 (85.6%)
Severity of constipation
4·5 (1·1)
4·5 (1·1)
Sensation of straining
4·1 (1·5)
4·2 (1·6)
Incomplete evacuation
3·6 (1·5)
Bloating
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Female
0·934
14·6 (11·4)
0·579
2·0 (0·9)
0·187
1·9 (0·9)
0·340
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45·7 (12·4)
90 (92.8%)
83 (85.6%)
0.204
4·6 (1·0)
4·4 (1·2)
0·566
4·1 (1·4)
4·0 (1·4)
0·673
3·9 (1·3)
3·7 (1·5)
3·4 (1·6)
0·081
1·0 (1·4)
1·2 (1·5)
0·9 (1·4)
1·0 (1·4)
0·504
Abdominal pain/cramping
0·4 (1·1)
0·3 (1·0)
0·5 (1·2)
0·3 (1·0)
0·608
Passing of gas
3·1 (1·5)
3·2 (1·8)
3·3 (1·3)
2·9 (1·3)
0·198
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Individual symptoms, mean (SD)
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SBM = spontaneous bowel movement; CSBM = complete spontaneous bowel movement; FC = functional constipation; MZRW = MaZiRenWan
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Individual symptoms were assessed on a 7-point scale, from 0 = not at all to 6 = very severe.
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Table 2 Comparison of treatment effect toward bowel movement, individual symptom assessment and rescue medicine used MZRW
Senna
Placebo
MZRW vs. Senna Differences
MZRW vs. Placebo
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Outcome
P Value
Differences
(95%, CI) Bowel movement [Mean Changes From Baseline (95% CI)]
a) Spontaneous bowel movement 2.2 (1.8 to 2.6)
2.0 (1.6 to 2.4)
1.2 (0.8 to 1.6)
0.3 (-0.2 to 0.8)
Follow up
1.6 (1.2 to 2.0)
0.6 (0.4 to 0.9)
0.5 (0.3 to 0.7)
1.1 (0.7 to 1.5)
b) Complete spontaneous bowel movement Treatment
2.2 (1.8 to 2.6)
2.0 (1.6 to 2.3)
1.2 (0.8 to 1.6)
0.4 (-0.1 to 0.9)
Follow up
1.5 (1.1 to 1.9)
0.6 (0.3 to 0.8)
0.5 (0.2 to 0.7)
1.1 (0.6 to 1.6)
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Individual symptom assessment [Mean Changes From Baseline (95% CI)]
a) Severity of constipation
0.190.9 (0.4 to 1.4)
Differences
3 Groups P Value
P Value
(95%, CI)
0.001
0.9 (0.3 to 1.4)
0.004
0.001
<0.00051.0 (0.5 to 1.4)
<0.0005
0.2 (-0.3 to 0.6)
0.498
<0.0005
0.151.0 (0.5 to 1.5)
<0.0005
0.8 (0.2 to 1.4)
0.006
0.002
<0.00051.0 (0.6 to 1.5)
<0.0005
0.1 (-0.4 to 0.6)
0.691
<0.0005
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Treatment
2.
(95%, CI)
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1.
P Value
Senna vs. Placebo
-2.4 (-2.8 to -2.1)
-1.7 (-2.0 to -1.4)
-1.0 (-1.4 to -0.7)
-0.8 (-1.2 to -0.3)
0.001
-1.3 (-1.8 to -0.8)
<0.0005
-0.5 (-0.9 to -0.1)
0.026
<0.0005
Follow up
-1.8 (-2.1 to -1.5)
-0.8 (-1.1 to -0.5)
-0.4 (-0.6 to -0.1)
-1.1 (-1.5 to -0.7)
<0.0005
-1.4 (-1.8 to -1.0)
<0.0005
-0.3 (-0.6 to 0.1)
0.095
<0.0005
b) Sensation of straining
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Treatment
-2.3 (-2.6 to -2.0)
-1.7 (-2.0 to -1.4)
-0.9 (-1.2 to -0.7)
-0.7 (-1.1 to -0.2)
0.001
-1.2 (-1.6 to -0.8)
<0.0005
-0.5 (-0.9 to -0.1)
0.014
0.003
Follow up
-1.7 (-2.0 to -1.4)
-0.8 (-1.0 to -0.5)
-0.5 (-0.7 to -0.2)
-1.0 (-1.3 to -0.6)
<0.0005
-1.0 (-1.4 to -0.7)
<0.0005
-0.1 (-0.4 to 0.3)
0.661
<0.0005
c) Incomplete evacuation
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Treatment
Treatment
-1.9 (-2.2 to -1.5)
-1.2 (-1.5 to -0.8)
-0.5 (-0.8 to -0.1)
-0.5 (-1.0 to -0.1)
0.012
-0.9 (-1.3 to -0.5)
<0.0005
-0.4 (-0.8 to 0.1)
0.088
<0.0005
Follow up
-1.5 (-1.9 to -1.1)
-0.6 (-0.9 to -0.2)
-0.1 (-0.4 to 0.2)
-0.8 (-1.2 to -0.4)
<0.0005
-0.9 (-1.3 to -0.5)
<0.0005
-0.1 (-0.5 to 0.2)
0.464
<0.0005
d) Bloating
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-0.7 (-1.0 to -0.4)
0.1 (-0.3 to 0.4)
-0.5 (-0.8 to -0.2)
-0.5 (-0.9 to -0.2)
0.006
-0.03 (-0.3 to 0.2)
0.929
0.5 (0.2 to 0.9)
0.004
0.005
Follow up
-0.7 (-1.1 to -0.4)
0.01 (-0.3 to 0.3)
-0.4 (-0.7 to -0.1)
-0.5 (-0.8 to -0.2)
0.001
-0.1 (-0.4 to 0.1)
0.469
0.4 (0 to 0.7)
0.028
0.004
-0.2 (-0.4 to 0.04)
0.398
0.04 (-0.2 to 0.3)
0.778
0.36
-0.2 (-0.4 to 0.1)
0.697
0.1 (-0.2 to 0.3)
0.727
0.475
e) Abdominal pain/cramping
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Treatment
-0.2 (-0.4 to 0.001)
-0.1 (-0.3 to 0.04)
-0.02 (-0.05 to 0.01)
-0.2 (-0.5 to 0.01)
0.150
Follow up
-0.2 (-0.4 to 0.03)
-0.1 (-0.3 to 0.04)
-0.03 (-0.1 to 0.004)
-0.2 (-0.4 to 0.03)
0.24
Treatment
-1.1 (-1.5 to -0.8)
-0.7 (-1.0 to -0.4)
-0.3 (-0.6 to 0.01)
-0.6 (-1.0 to -0.2)
0.004
-0.6 (-1.0 to -0.3)
0.001
0.01 (-0.4 to 0.4)
0.955
<0.0005
Follow up
-1.1 (-1.4 to -0.7)
-0.4 (-0.7 to -0.1)
-0.2 (-0.4 to 0.1)
-0.8 (-1.2 to -0.5)
<0.0005
-0.6 (-1.0 to -0.3)
<0.0005
0.2 (-0.1 to 0.6)
0.246
<0.0005
2.
*Rescue medicine frequency per week [Mean (SD)]
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f) Passing of gas
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Treatment
1.2 (2.0)
1.3 (4.0)
0.9 (1.1)
-0.1 (-0.6 to -0.5)
0.151
-0.6 (-1.8 to -0.5)
0.419
-0.4 (-1.3 to 0.1)
0.504
0.077
Treatment
0.8 (2.2)
1.2 (4.5)
0.8 (2.7)
-0.3 (-0.6 to -0.03)
0.029
-0.4 (-0.6 to -0.1)
0.003
-0.1 (-0.3 to 0.2)
0.683
0.002
Follow up
0.8 (2.0)
1.2 (4.7)
0.8 (2.5)
-0.4 (-0.7 to -0.1)
0.007
-0.4 (-0.6 to -0.2)
0.001
-0.03 (-0.3 to 0.3)
0.859
<0.0005
CI = confidence interval; MZRW = MaZiRenWan; SD= standard derviation
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Baseline
Individual symptoms including severity of constipation, sensation of straining, incomplete evacuation, bloating, abdominal pain/cramping and passing of gas, were assessed on a 7-point scale, from 0 = not at all to 6 = very severe and presented in mean (95% CI).
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*Rescue medicine (Dulcolax tablets or Dulcolax suppositories) was provided only in those patients without bowel movements for at least three consecutive days during the whole study.
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P values were calculated from Post-hoc pairwise multiple comparison
ACCEPTED MANUSCRIPT Figure 1 Study Flow Diagram
Assessed for eligibility (n= 843)
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Enrolment
Excluded (n=552) Not FC (n=238) Declined to participate (n=89) Not excessive constipation (n=179) Exams with significant abnormality (n= 46)
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Allocated to MZRW (n=97) Received allocated intervention (n= 97) Did not receive allocated intervention (give reasons) (n=0)
Allocated to Senna (n=97) Received allocated intervention (n= 97) Did not receive allocated intervention (give reasons) (n=0)
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Lost to follow-up (n=2) Discontinued intervention (n=7) 2 due to bloating and passing of gas 5 due to unsatisfactory effects
Analysis
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Follow-up
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Allocation
Randomized (n=291)
ITT analysis (n=97)
Lost to follow-up (n=2) Discontinued intervention (n=8) 2 due to stomachache and headache 1 due to pregnant 5 due to unsatisfactory effects
ITT analysis (n=97)
Allocated to Placebo (n=97) Received allocated intervention (n= 97) Did not receive allocated intervention (give reasons) (n=0)
Lost to follow-up (n=3) Discontinued intervention (n=11) 3 due to insomnia 8 due to unsatisfactory effects
ITT analysis (n=97)
ACCEPTED MANUSCRIPT Figure 2 Weekly changes of CSBM and responder rate
Baseline period
a
Treatment period
Follow-up period
**
**
**
**
** **
2.0 ** **
1.5
*
*
1.0 0.5
-2
-1
0
1
2
3
4
**
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-3
80 70 ** 60
* *
50
**
** *
40 30 20 10 0 -2
-1
0
1
2
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-3
5
**
3
6
7
*
4
5
8
**
9
10
11
12
** *
6
**
**
**
**
7
Week
8
9
10
11
13
**
14
**
**
15
16
Senna group Placebo group
**
**
**
MZRW group
**
**
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Responder rate (%)
**
*
*
0.0
b
**
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** **
2.5
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Change of CSBM from baseline
3.0
**
12
**
13
**
**
**
14
15
16
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Supplemental File 2: Composition, authentication, preparation, pharmacokinetics and safety details of MaZiRenWan (A) Composition and authentication of MZRW ingredients
MaZiRenWan (MZRW) is an herbal formula originally recorded in traditional Chinese medicine classic ‘Discussion of Cold-Induced Disorders’ (Shang Han Lun) (1). This
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formula has been commonly used to treat constipation in China for more than 2000 years. This formula comprised six herbs: Rhei Radix Et Rhizoma, Cannabis Frustus, Paeoniae Radix Alba, Magnoliae Officinalis Cortex, Aurantii Fructus Immaturus and Semen Armeniacae Amarum (2). To gain the quality of MZRW granules, the source and
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processing protocols of raw medicinal substance, as well as the operation of production should be highly standardized and critically controlled. Voucher numbers of individual specimens of MZRW ingredients were given and samples were kept at the Clinical Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China. Each herb was authenticated and tested by the Purapharm (Nanning) Pharmaceuticals Co. Ltd according to the methods recorded in
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corresponding monographs of Chinese Pharmacopoeia (2010) (3). Results of authentication were summarized in Supplement 2-Table 1.
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Chinese name (Pinyin) Huomaren
Medicinal
Source
Latin name Cannabis Frustus
Processing method
parts
Cannabis sativa L.
Dry seeds
Eliminating the foreign matter and pericarp
Radix
(DH)
Rhizoma
Et
Rheum tanguticum
Dry roots
Maxim,ex Balf.
Kuxingren
Armeniacae
Prunus
(KXR)
Semen Amarum
mandshurica
Dry seeds
(BS)
Alba
Radix
Paeonia
lactiflora
Pall.
chromatogram
with
reference herb
and cut into thick slices
total amount of aloe-emodin, rhein,
amount of aloe-emodin, rhein,
emodin, chrysophanic acid and
emodin, chrysophanic acid and
physcion
physcion
1. Thin layer chromatography
1. Identical
Breaking to pieces
Dry roots
Identical Contained
Eliminating the foreign matter
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Paeoniae
Thin layer chromatography Contained not less than 1.5% of the
(Maxim.) Koehne Baishao
Result
Eliminating the foreign matter
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Rhei
Methods / standards cited in the Chinese Pharmacopeia
(HMR) Dahuang
Authentication
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Ingredients
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Table 1. The name, source, processing method of each ingredient and its authentication report
and cutting into thin slices and
2. Contained not less than 3.0% of amygdalin Contained not less than 1.6% of the
2.2%
of
the
chromatogram
total
with
reference herb 2. Contained 6.1% of amygdalin Contained 3.0% of paeoniflorin
paeoniflorin
stir-bake
Magnoliae
Magnolia
(HP)
Officinalis Cortex
officinalis
Dry barks Rehd.et
Wils. Aurantii Fructus
(ZS)
Immaturus
Citrus aurantium L.
Dry fruits
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Zhishi
Scraping off the coarse bark and
Contained not less than 2.0% of the
Contained
cut into slivers
total amount of magnolol and
amount of magnolol and honokiol
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Houpo
3.2%
of
the
total
honokiol
Eliminating the foreign matter,
1. Thin layer chromatography
cutting
2. Contained not less than 0.3% of
into
thin
slices
stir-baking with bran
and
synephrine
1. Identical
chromatogram
with
reference herb 2. Contained 0.4% of synephrine
*Authentication methods and standards are referenced to the Pharmacopeia of the People’s Republic of China 2010, Vol. 1.
(B) Preparation of MZRW granules 250 kg raw medicinal materials in total were mixed by six herbs in proportion listed in Supplement 2-Table 2, and extracted with 8 times of boiling water (w/v) for 2.5 hours
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and filtered. The filtrate was concentrated to the density of 1.10~1.20 (60℃) under reduced pressure and then subjected to dry in spraying. Subsequently, the dried powders
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were thoroughly mixed with 70kg dextrin to gain 100 kg granules by dry method. Finally, the granules were packed into small quantities with sealed opaque aluminum sachets (7.5 g per sachet).
Table 2. The dosages of all ingredients for MZRW granule Composition (%)
Basic formulation (g)
Production formulation (kg)
Cannabis Frustus
35.7
892.9
89.29
Rhei Radix et Rhizoma
17.9
446.4
22.32
17.9
446.4
8.9
223.2
10.7
267.9
8.9
223.2
Paeoniae Radix Alba Magnoliae Officinalis Cortex Aurantii Fructus Immaturus
Each gram of MZRW granules is equivalent to 4.05 g of raw herbs.
(C) Preparation of placebo
21.7
5.4
10.9
22.32
5.4
10.9
44.64
2.7
5.4
26.79
3.3
6.5
2.7
5.4
44.64
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*
10.8
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Amarum
g/day
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Armeniacae Semen
g/sachet
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Ingredients
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Green tea was decocted with 10 times of water for 1.5 hours and filtered. The filtrate was then concentrated to sticky solution with a relative density of 1.10~1.20 at 60℃. Caramel (0.34kg) and gardenia yellow (0.02kg) dissolved in water were mixed with the green tea essence (23.61kg), followed by mixed with dextrin (76.03kg). The mixture was dried in vacuum and granulated. The granules were packed in sealed opaque aluminum sachets (7.5g per sachet).
(D) Quality report of MZRW granules
MZRW granules was manufactured in Purapharm (Nanning) Pharmaceuticals Co. Ltd with GMP standard. The granules used in this study were originated from one batch, and the qualities in appearance, taste, paeoniflorin identification using TLC, water content, particle size, dissolubility, loading difference, content of paeoniflorin, microbial
ACCEPTED MANUSCRIPT
limit, heavy metals limit and pesticide residue were determined according to the requirements of Chinese Pharmacopoeia (version 2010) for granules and quality standard of
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MZRW granules (as shown in Supplement 2-Table 3). In addition, under the accelerating storage condition of 40℃ and 75% relative humidity, the description, identification, determination of water, particle size, determination of dispersibility, assay and microbial limit test meet the product specification after storage for 3 months. Accelerated stability study was indicated that MZRW granules had good
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stability and the results were presented in Supplement 2-Table 4.
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Table 3. Quality report of MZRW granules
Description
Quality requirements
Results
Reference methods
Light yellow to brownish yellow in color, fragrant in smell,
Brownish yellow in color,
Chinese
acrid and slightly bitter in taste.
fragrant in smell, acrid and
2010, Vol. 1, Appendix I C)
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Test items
Pharmacopoeia
(Version
slightly bitter in taste. consistent distance travelled and color with spot of in the
The thin-layer chromatograms
chromatogram of paeoniflorin was observed.
were shown in S2-Figure 1.
Not more than 6.0%
Chinese
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Determination of
Pass
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Identification
Corresponding spot in chromatogram of test samples with
5.5%
water
Sum of weight of granules that cannot pass through sieve Particle size
No.1 and weight of powder that can pass through sieve NO.5 ≤ 15%
Determination of
Light turbidity is allowed and no foreign matters
Paeoniflorin no less than 0.70 mg/g
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Assay
The filling margin of error shall be plus or minus 5%
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Filling variation
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dispersibility
7%
Slight turbidity Comply
4.72mg/g The high performance liquid chromatograms were shown in
Pharmacopoeia
(Version
2010, Vol. 1, Appendix VI B) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix IX H) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix XI B) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix I C) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix I C)
Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix VI D)
S2-Figure 2.
Microbial limit test
Chinese
-Total Aerobic Count
2010, Vol. 1, Appendix XIII C)
Not more than 500 colony/g
≤10 colony/g
Pharmacopoeia
(Version
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and
Yeast
Not more than 100 colony/g
≤10 colony/g
Count -Escherichia coli
Not detectable
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-Mould
Not detectable
Heavy metal limit
Chinese
Not more than 150 ppm
3.596ppm
-Arsenic (As)
Not more than 41.67 ppm
0.157ppm
-Cadmium (Cd)
Not more than 97.22 ppm
0.014ppm
-Mercury (Hg)
Not more than 4.97 ppm
0.264ppm
-Lead (Pb)
Not more than 1.00 ppm
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Not more than 0.05 ppm
-Chlordane
Not more than 0.05 ppm
-Total DDT’s
Not more than 1.0 ppm
-Total BHC’s
Not more than 0.3 ppm
-Eldrin
Not more than 0.05 ppm
-Heptachlor
Not more than 0.05 ppm
-Hexachlorobenzene
Not more than 0.1 ppm
-Lindane
Not more than 0.6 ppm
-Quintozene
Not more than 1.0 ppm
2010, Vol. 1, Appendix IX B)
＜0.001ppm
Pesticide residues -Aldrin & Dieldrin
(Version
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-Copper (Cu)
Pharmacopoeia
Not detectable
Kong (Application form: Registration
Not detectable
of proprietary Chinese Medicines)
Not detectable
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0.00504ppm
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Chinese Medicine Council of Hong
Not detectable Not detectable Not detectable Not detectable 0.00106ppm
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Table 4. The test report of accelerated stability study for MZRW granules
Identification
T=1(Jan. 19, 2013)
Light yellow to brownish yellow in
Brownish yellow in color,
color, fragrant in smell, acrid and slightly bitter in taste.
T=3 (Mar. 19, 2013)
Brownish yellow in color,
Brownish yellow in color,
Brownish yellow in color,
fragrant in smell, acrid and
fragrant in smell, acrid and
fragrant in smell, acrid and
fragrant in smell, acrid and
slightly bitter in taste.
slightly bitter in taste.
slightly bitter in taste.
slightly bitter in taste.
Same fluorescent spots observed in
Same
the
observed
corresponding
position
of
reference paeoniflorin
Determination of
T=2 (Feb. 19, 2013)
Not more than 6.0%
fluorescent
spots
in
weight of powder that can pass
foreign matters
Assay
Paeoniflorin no less than 0.70 mg/g
Microbial
limit
test -Total
Aerobic
Not more than 500 colony/g
Count -Mould Yeast Count
and
Not more than 100 colony/g
in
the
Same
fluorescent
observed
in
spots the
reference paeoniflorin
reference paeoniflorin
reference paeoniflorin
5.4%
5.3%
4.4%
8%
7%
7%
Slight turbidity
Slight turbidity
Slight turbidity
4.48mg/g
4.20mg/g
4.12mg/g
≤10 colony/g
≤10 colony/g
≤10 colony/g
≤10 colony/g
Negative
Negative
7%
Slight turbidity
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dispersibility
observed
spots
reference paeoniflorin
4.72mg/g
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Light turbidity is allowed and no
the
fluorescent
corresponding position of
through sieve NO.5 ≤ 15% Determination of
in
Same
corresponding position of
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cannot pass through sieve No.1 and
observed
spots
corresponding position of
Sum of weight of granules that Particle size
fluorescent
corresponding position of
5.5%
water
the
Same
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Description
T=0 (Dec. 19, 2012)
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Months
Quality requirements
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Test items
≤10 colony/g
≤10 colony/g
≤10 colony/g
≤10 colony/g
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Negative
Negative
Negative
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-Escherichia coli
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Figure 1. The thin-layer chromatograms (TLC) of MZRW granules
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1-3: the sample of MZRW granules; 4: negative control sample (without Paeoniae Radix Alba); 5: paeoniflorin reference standard
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Figure 2. Typical chromatograms of assay for MZRW granule
A. Paeoniflorin reference standard ; B. The sample solution of MZRW granule; C. Negative control sample (without Paeoniae Radix Alba)
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(E) Toxicity of MZRW granules
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We have done the acute toxicity test of MZRW granules in our previous study (2). 60 ICR mice weighed 18-22g (half male and female) were randomized into 5 groups. MZRW granules was dissolved with distilled water and administered at 20ml/kg via gavage. Single dose at 75g/kg, 37.5g/kg, 19.25g/kg, 9.625g/kg, 4.8125g/kg, which was equal to 300, 150, 75, 37.5, 19.75 times of clinical dose, was given. Mice were under daily inspection for 7 days. No death was recorded by the end of the study.
1.
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(F) Study on major active compounds of MZRW granules in rat plasma Major component identification
The aforementioned report is generally an acceptable quality control for a Chinese medicine intervention; however, it does not fully cover the phytochemical feature of
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MZRW granules. A comprehensive chemical profile of poly-chemical species is sought for establishing higher standard of quality control. LC/MS was chosen for its sensitivity, broad capability and spectral sensitivity (4).
Analysis was performed on UPLC-Q-TOF-MS/MS (Agilent G6520 (USA)). (Supplement 2-Figure 3 & Supplement 2-Figure 4). Around 300 compounds were found in MZRW granules with 80 compounds can be predicted (Supplement 2- Table 5). 41 out of 80 were contributed by (DH), 6 by (HMR), 13 by (BS), 9 by (HP), 12 by (ZS) and
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4 by (KXR).
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Figure 3. LC/MS chromatograms of extraction of MZRW granules
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LC-MS referred to the published article (5).
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This figure showed 3D total ion chromatograms (TIC) of MZRW granules. UPLC-Q-TOF-MS/MS analysis was performed for the compound components, nearly 300 compounds were found. Analytical conditions of
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Figure 4. Ion-current chromatograms obtained in negative-ion mode
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This figure showed ion-current chromatograms of the chemical component extract screened using negative-ion mode. 80 compounds were identified.
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Peak No. RT
Observed mass
Ion species
Molecular
Error
Plant
formula
(mDa)
source
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Table 5. Chemical profile of MZRW granules Identification
0.735
225.0614
[M + HCOO]-
C6 H12 O6
-1.12
KXR
Inositol
2
1.61
169.0133
[M - H]-
C7 H6 O5
-0.93
DH;BS
Gallic acid
3
1.61
331.0659
[M - H]-
C13 H16 O10
1.52
DH
Gallic acid 3-O-β-D-glucopyranoside
4
1.634
125.0253
[M - H]-
C6 H6 O3
0.89
BS
1,2,3-benzenetriol
5
1.984
331.066
[M - H]-
C13 H16 O10
1.09
DH
Gallic acid 4-O-β-D-glucopyranoside
6
2.588
493.1199
[M - H]-
C19 H26 O15
0.1
DH
6-O-galloylsucrose
7
3.871
451.1234
[M - H]-
C21 H24 O11
-1.41
ZS
Hesperetin-7-O-β-D-glucoside
8
4.509
451.1234
[M + HCOO]-
C20 H22 O9
-1.32
DH
Piceatannol 3-O-β-D-glucopyranoside
9
4.661
577.1335
[M - H]-
C30 H26 O12
-1.72
DH
Procyanidin B-1
10
4.852
451.1235
[M - H]-
C21 H24 O11
-2.82
DH
(+)-catechin-5-O-β-D-glucopyranoside
11
4.956
289.071
[M - H]-
C15 H14 O6
-0.87
DH;BS
(+)-catechin
12
4.956
483.0767
[M - H]-
C20 H20 O14
-0.54
DH
1,6-di-O-galloylycerol-β-D-glucopyranoside
13
5.268
417.1387
[M + HCOO]-
C17 H24 O9
-1.46
BS
Paeonin A
14
5.528
456.1511
[M - H]-
C20 H27 N O11 0.47
KXR
Amygdalin
15
5.828
289.0711
[M + HCOO]-
C14 H12 O4
-0.63
DH
Piceatannol
16
5.979
729.1466
[M - H]-
C37 H30 O16
0.61
DH
Procyanidin B-1-3-O-gallate
17
6.131
340.1037
[M + HCOO]-
C14 H17 N O6
-0.13
KXR
Prunasin
18
6.51
609.1829
[M - H]-
C28 H34 O15
7.36
ZS
Neohesperidin
19
6.51
623.2
[M - H]-
C29 H36 O15
1.71
HP
Acteoside
20
6.541
449.1467
[M + HCOO]-
C21 H24 O8
1.23
DH
Desoxyrhaponticin
21
6.541
479.1565
[M - H]-
C23 H28 O11
2.54
BS
Albiflorin
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6.541
525.1636
[M - H]-
C24 H30 O13
2.1
BS
Mudanpioside E
23
6.541
609.1843
[M + HCOO]-
C27 H32 O13
2.54
DH
10R-chrysaloin1-O-β-D-glucopyranoside
24
6.554
119.0503
[M - H]-
C8H8O
0.01
HP
Benzeneacetaldehyde
25
6.744
639.1921
[M + HCOO]-
C28 H34 O14
-0.15
ZS
Poncirin
26
6.962
567.2092
[M - H]-
C27H36O13
0.81
ZS
Citrusin B
27
7.02
269.0448
[M - H]-
C15 H10 O5
-0.73
HMR;ZS
Apigenin
28
7.02
863.2018
[2M - H]-
C21 H20 O10
-0.26
DH
Emodin-8-O-β-D-glucopyranoside
29
7.081
441.0817
[M - H]-
C22 H18 O10
-1.04
DH
(-)-Epicatechin-3-O-gallate
30
7.081
445.077
[M - H]-
C21 H18 O11
-0.48
DH
Rhein-8-O-β-D-glucopyranoside
31
7.142
417.1184
[M - H]-
C21 H22 O9
-5.11
DH
Cassialoin
32
7.172
477.1395
[M - H]-
C23 H26 O11
-0.97
DH
Lindleyin/isolindleyin
33
7.264
525.1606
[M + HCOO]-
C23 H28 O11
-3.17
BS
Paeoniflorin
34
7.35
623.1973
[M - H]-
C29 H36 O15
-1.03
DH
Physcion-8-O-β-D-gentiobioside
35
7.411
631.1659
[M - H]-
C30 H32 O15
1.6
BS
Albiflorin; 4-O-(3,4,5-Trihydroxybenzoyl)
36
7.597
609.1816
[M + HCOO]-
C27 H32 O13
-0.6
DH
Cascaroside C
37
7.668
861.1833
[M - H]-
C42H38O20
1.22
DH
Sennoside A or Sennoside B
38
7.696
579.1717
[M - H]-
C27 H32 O14
0
ZS
Naringin
39
7.935
298.1075
[M - H]-
C17 H17 N O4
-1.65
HMR
N-trans-caffeoyltyramine
40
8.026
355.1022
[M + HCOO]-
C15 H18 O7
-1.55
DH
2-O-cinnamoyl-β-D-glucose
41
8.13
609.1823
[M + HCOO]-
C27 H32 O13
-0.22
DH
Cascaroside D
42
8.13
609.1824
[M - H]-
C28 H34 O15
-0.49
ZS
Hesperidin
43
8.398
189.0546
[M - H]-
C11 H10 O3
-1.06
DH
2,5-dimethyl-7-hydroxychromone
44
8.398
393.1182
[M - H]-
C19 H22 O9
-1.43
DH
Aloesone-7-O-β-D-glucopyranoside
45
8.428
431.0975
[M - H]-
C21 H20 O10
-0.76
DH
Kaempferol-3-O-rhamnoside
46
8.428
525.1595
[M + HCOO]-
C23 H28 O11
-2.09
BS
Mudanpioside I
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8.652
593.1917
[M - H]-
C34H30N2O8
-0.62
HMR
Cannabisin A
48
8.667
461.1086
[M + HCOO]-
C21 H20 O9
-0.23
DH
Chrysophaein
49
8.778
595.2073
[M - H]-
C34 H32 N2 O8 -1.36
HMR
Cannabisin B
50
8.877
315.1232
[M - H]-
C18 H20 O5
-0.61
HP
Honokitriol; (7R*,8R*)-form
51
9.615
461.1082
[M - H]-
C22 H22 O11
-0.47
DH
1-O-galloyl-6-O-cinnamoyl-β-D-glucose
52
9.645
639.1922
[M + HCOO]-
C28 H34 O14
-0.94
ZS
Neoponcirin
53
9.798
415.1019
[M - H]-
C21 H20 O9
0.47
DH
Chrysophanol-8-O-β-D-glucopyranoside
54
9.872
847.2097
[M - H]-
C42 H40 O19
4.51
DH
Sennoside C
55
9.949
313.0344
[M - H]-
C16 H10 O7
-1
DH
laccaic acid D
56
10.065 253.0496
[M - H]-
C15 H10 O4
-1.01
DH
Chrysophanol
57
10.065 831.2135
[2M - H]-
C21 H20 O9
-0.43
DH
Chrysophanol-1-O-β-D-glucopyranoside
58
10.385 517.0975
[M - H]-
C24 H22 O13
-1.16
DH
Emodin-8-O-(6'-O-malonyl)-glucoside/7-O-glucosyl-6''-malonyl genistein
59
10.39
[M - H]-
C18 H20 O4
-0.86
HP
Magnolignan A
60
10.449 629.1871
[M + HCOO]-
C30 H32 O12
-0.29
BS
6'-O-galloylalbiflorin
61
10.449 629.1871
[M - H]-
C31 H34 O14
-0.37
BS
Mudanpioside B
62
10.613 629.187
[M + HCOO]-
C30 H32 O12
-1.03
BS
Benzoylpaeoniflorin
63
10.707 723.2134
[M - H]-
C33 H40 O18
-0.51
ZS
Melitidin
64
10.745 285.0396
[M + HCOO]-
C14 H8 O4
-0.78
DH
1,8-Dihydroxyanthraquinone
65
10.745 285.0395
[M - H]-
C15 H10 O6
-1.05
DH
Citreorosein
66
10.916 283.0607
[M - H]-
C16 H12 O5
-0.47
DH
Physcion
67
10.916 445.113
[M - H]-
C22 H22 O10
-1.18
DH
Physcion-1-O-β-D-glucopyranoside
68
11.05
241.0858
[M - H]-
C15 H14 O3
-1.28
HP
Magnatriol B
69
11.7
297.0396
[M - H]-
C16 H10 O6
-0.9
DH
6-methyl-rhein
70
12.133 253.0865
[M - H]-
C16 H14 O3
-0.65
HP
Magnaldehyde D
71
12.193 515.1925
[M + HCOO]-
C26H30O8
0.11
ZS
Limonin
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299.1281
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12.245 283.0242
[M - H]-
C15 H8 O6
-0.49
DH
Rhein
73
12.558 269.0813
[M - H]-
C16 H14 O4
-0.66
ZS
Imperatorin
74
12.59
[2M + HCOO]-
C15 H12 O5
-0.68
ZS
Naringenin
75
13.379 269.045
[M - H]-
C15 H10 O5
-0.64
DH
Emodine
76
13.809 265.1243
[M - H]-
C18H18O2
0.81
HP
Honokiol
77
13.991 281.1182
[M - H]-
C18 H18 O3
-0.19
HP
Obovatol
78
14.055 265.1247
[M - H]-
C18 H18 O2
1.25
HP
Magnolol
79
16.084 327.2535
[M + HCOO]-
C18H34O2
-0.64
HMR;KXR Oleic acid
80
22.057 279.223
[M - H]-
C18H32O2
0.08
HMR
2. Quantification of ten active compounds in MZRW granules
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Linoeic acid
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589.1344
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Remission of constipation by ten major components in MZRW granules has been highlighted by recent literature and our pilot study. Quantification of these components, including rhein, emodin, aloe emodin, hesperidin, naringin, paeoniflorin, albiflorin, magnolol, honokiol and amygdalin (Supplement 2-Figure 5), was performed with
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appropriate reference compounds (Supplement 2-Figure 6 and Supplement 2- Table 6).
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Supplement 2-Figure 5. Chemical structures of biologically active components on constipation of MZRW granules
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Amygdalin Aloe emodin O
OH
O
OH
Rhein OH
O
OH
NC HO
CH 2OH CH3
COOH OH
O
O
OH
C 15H 10O5 exact mass: 270.0528
O
OH
OH
HO
OH
O
O
OH
H 3C
O
O H OH
O
O
C23H 28O11 exact mass: 480.1632
C 27H 32O14 Exact mass: 580.1792 OH OH O
O
HO
CH3 OH
O
C28H34O 15 Exact Mass: 610.1898
O
OCH3
honokiol OH
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OH
O
O
O CH3 O
C23H 28 O11 exact mass: 480.1632
magnolol OH HO
OH
O
HO
O
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Hesperidin HO
OH
O
O
HO
Alibiflorin
OH
OH
O
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HO
HO
O
O O
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OH
HO
Peaoniflorin
OH
O
HO
OH
OH
Naringin
O
HO
O
OH
C 20H 27 NO 11 Exact mass: 457.1584
C 15 H 18 O6 exact mass: 284.0321
CH 3 HO
OH
HO
O
C 15 H10O5 exact mass: 270.0528
OH
O
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HO
O
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Emodin
C 18H 18O2 Exact Mass: 266.1307
C 18H18 O2 Exact Mass: 266.1307
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Figure 6. Total ion chromatography of 10 mixed standard solution, MZRW solution and SRM chromatography for each compound and I.S.
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MZRW granules
Plant source
Amygdalin
11.03±0.15
KXR
Albiflorin
0.56±0.01
BS
Paeoniflorin
1.3±0.03
BS
Naringin
0.67±0.01
ZS
Hesperidin
12.25±0.13
ZS
Aloe emodin
1.53±0.02
DH
Rhein
18.23±0.10
DH
Emodin
6.15±0.03
DH
Honokiol
6.87±0.05
HP
Magnolol
1.24±0.02
HP
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Active compounds
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Table 6. The content of each compound in MZRW granules (mg/g, Mean± SD, n = 5)
3. The pharmacokinetics assessment of major compounds
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We elaborated the pharmacokinetic features in rats of these ten compounds when they were orally administrated in the form of MZRW granules. The concentration-time (C-T) profile of rhein was appeared with dual peaks, which may be probably due to the replenishment of rehein by metabolites of some similar anthraquinones and anthraquinone derivatives in this formula, and another possibility is because of enterohepatic circulation of these chemicals. The t1/2 of rhein, emodin and aloe emodin were more than 10 h,
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though they were rapidly absorbable (Tmax was less than half an hour). This well interpreted the both quick and long-lasting effects of MZRW. The C-T curve of amygdalin showed that it was detectable in rat plasma within 5min post-dosing; and a shorter Tmax (0.75 h) indicated that amygdalin was rapidly absorbed after oral administration. Elimination of amygdalin from plasma was also relatively fast, with the t1/2 values at approximately 3 h. Probably due to the similarity in chemical structure, albiflorin and paeoniflorin exhibited consistent tendencies in plasma C-T profiles and similar Tmax (both at 0.75 h) after oral administration of MZRW. The t1/2 values of these two compounds were (2.81±1.09) h and (5.90±1.36) h, respectively, suggesting a rapid elimination from plasma of the two compounds. Naringin was absorbed into the body with Tmax at 0.75 h and eliminated with t1/2 near 4 h after oral administration of MZRW. The rapid elimination may be due to the fact that orally administered naringin can be
ACCEPTED MANUSCRIPT
quickly metabolized into naringenin and naringenin glucuronide. The plasma concentration and AUC of magnolol were both higher than that of honokiol, while in MZRW the
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amount of magnolol (1.24 mg/g) was much lower than honokiol (6.87 mg/g). This data suggest that magnolol was better absorbed by the gastrointestinal tract than honokiol. Illustrating the pharmacokinetic profile of each compound can foster better understanding of the efficacy of MZRW, and can facilitate clinical study and quality evaluation for development of this formula into even more effective pharmaceuticals (5). Further clinical investigation about its pharmacokinetics in human is necessary in the future.
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References
1. Zhang B, Dong J, Zhou Z. Traditional Chinese Internal Medicine [in Chinese]. Shang Hai Science and Technology Press: Shanghai, 1985.
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2. Cheng CW, Bian ZX, Zhu LX et al. Efficacy of a Chinese herbal proprietary medicine (Hemp Seed Pill) for functional constipation. Am J Gastroenterol 2011;106(1):120-9.
3. Chinese Pharmacopeia Commission. Hemp Seed Pills. In: Pharmacopeia of the People’s Republic of China, Vol. 1, 2010 edn. China Medical Science Publisher: Beijing, 2010, pp 1093.
4. Wang G, Fu H, Ye W et al. Comprehensive characterization of the in vitro and in vivo metabolites of ziyuglycoside I in rat microsome, intestinal flora, excretion specimen and fresh tissues based on LC-Q-TOF/MS. J Pharm Biomed Anal 2016; (128):191-200.
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5. Hu DD, Han QB, Zhong LL et al. Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of
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Ma-Zi-Ren-Wan. J Chromatogr B Analyt Technol Biomed Life Sci 2015; 1000:136-46.
ACCEPTED MANUSCRIPT Supplemental File 3: Packaging and appearance of interventions
a
MZRW
Placebo
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c
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b
(I) Packaging and appearance of MaZiRenWan (MZRW) granules and its placebo a The one-week course package of MZRW granules (a zip lock bag contained 14 sealed aluminum sachets. is placebo granules).
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b The appearance of MZRW granules and placebo granules (Left side is MZRW granules and right side
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c The clear appearance of MZRW granules.
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a
c
Placebo
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Senna
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b
(II) Packaging and appearance of Senna and its placebo
a The 4-week course package of Senna and placebo (a plastic bottle contained 56 tablets). b The appearance of Senna and placebo (Left side is Senna tablets and right side is placebo tablets).
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c The appearance of Senna.
ACCEPTED MANUSCRIPT Supplemental file 4: Statistical analysis Data are presented by mean and standard deviation (SD), 95% confidence interval
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(95% CI) or percentages. Differences among three groups were assessed using one-way Analysis of variance (ANOVA) or Kruskal-Wallis non-parametric test for continuous variables, or using Chi-square test or Fisher’s exact test for categorical variables.
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Analysis of covariance (ANCOVA) was also applied with baseline as covariate if
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needed. Post-hoc pairwise multiple comparisons were made with LSD or Dunnett’s procedure if significant differences were found among three groups. Changes in the eight domains of SF-36 before and after treatment in three groups were calculated and the within-group comparisons were performed using paired t-test or Wilcoxon
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22.0.
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matched-pairs signed rank sum test. Analyses were carried out using SPSS version
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MZRW
Senna
MZRW vs. Senna$
Placebo
χ2
P Value
csbm>=1 than baseline and csbm>=3/wk (%)
MZRW vs. Placebo$
Senna vs. Placebo$
3 Groups
χ2
P Value
χ2
P Value
χ2
P Value
7.774
0.005
5.446
0.020
9.183
0.01
23.918
<0.0001
2.212
0.137
26.356
<0.0001
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Outcome
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Supplement File 8: The proportion of patients with weekly frequency ≥3times CSBM and an increase ≥1 CSBM from baseline
68 (70.1%)
65 (67.0%)
49 (50.5%)
0.215
0.643
Follow up
53 (54.6%)
29 (29.9%)
20 (20.6%)
12.167
<0.0001
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Treatment
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$: Post-hoc pairwise comparison of chi-squared test with applying the Bonferroni corrected alpha level (corrected α = 0.05/3 for three compared pairs)
*: P<0.05/3, vs. Placebo; #: P<0.05/3, vs. Senna.
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Supplemental file 6: Results of other secondary outcomes (A) Comparison of the treatment effect on changes of global symptom assessment among three groups
Global Symptom Assessment
MZRW
Senna
Placebo
MZRW vs. Senna
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MZRW vs.
3 Groups
Senna vs. placebo
placebo
(%)
(n=89)
(n=88)
(n=90)
P-value
P-value P-value
72.7%
45.6%
Treatment
Same
9.0%
26.1%
51.1%
Wk6
Worse
1.1%
1.1%
3.3%
Improved
91.0%
76.1%
51.1%
Treatment
Same
9.0%
21.6%
45.6%
Wk10
Worse
0.0%
2.3%
Improved
70.8%
34.1%
28.9%
Same
27.0%
64.8%
67.8%
Worse
2.2%
1.1%
3.3%
Follow up
0.004
<0.0005
0.001
<0.0005
0.013
<0.0005
0.001
<0.0005
<0.0005
<0.0005
0.505
<0.0005
3.3%
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End of
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89.9%
Within
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Improved
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P-value
Wk18
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MZRW = MaZiRenWan Global symptom improvement was assessed the participant’s subjective feeling of adequate relief of symptoms as “improved”, “same” and “worse”.
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P values were calculated from Fisher’s Exact test.
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Higher proportion of subjects in the MZRW group improved during the treatment period, with significant differences among three groups (P<.0005). Post-hoc
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pairwise multiple comparison results is as follows: MZRW vs. placebo: P<.0005 (Wk6 & 10); MZRW vs. senna: P=.004 (Wk6) and P=.013 (Wk10); senna vs. placebo: P=.001 (Wk 6 & 10). In the follow-up period, over 70% subjects of the MZRW group sustained this improvement, with significant inter-group
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difference (P<.0005). Post-hoc pairwise multiple comparison: MZRW vs. placebo: P<.0005; MZRW vs. senna: P<.0005; senna vs. placebo: P=.505.
(B) Comparison of different domains of SF-36 questionnaires within different groups
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MZRW (n=97)
Senna (n=97)
Pre-Treatment
Post-Treatment
Mean
Mean
SD
P-value*
SD
Placebo (n=97)
Pre-Treatment
Post-Treatment
Mean
Mean
SD
P-value*
SD
Pre-Treatment
Post-Treatment
Mean
Mean
SD
P-value*
SD
SF-36 18.2
88.2
10.5
0.588
85.6
15.3
86.3
11.1
Role-Physical
81.1
19.1
78.2
18.8
0.118
81.1
18.2
77.8
19.8
Bodily Pain
63.6
27.7
65.3
22.5
0.425
62.3
26.3
60.3
23.1
General Health
50.5
22.7
53.5
18.3
0.021
50.6
21.5
52.0
19.7
Vitality
50.6
14.4
52.9
18.0
0.035
49.2
15.1
51.4
17.6
Social Functioning
75.4
24.2
79.7
19.2
0.024
75.9
23.4
73.3
Role-Emotional
79.6
22.1
75.0
21.6
0.031
78.6
22.2
74.8
Mental Health
59.9
13.9
62.5
14.4
0.162
59.0
14.2
18.5
87.3
10.3
0.681
81.5
18.9
79.5
17.6
0.288
0.310
61.9
26.0
62.4
23.9
0.809
0.350
51.4
21.8
53.9
18.1
0.159
0.012
50.9
13.0
52.9
14.9
0.060
22.4
0.171
74.9
23.8
75.6
20.6
0.747
21.3
0.044
79.4
22.8
75.1
21.5
0.033
59.3
0.824
59.3
12.8
60.0
12.6
0.540
14.9
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#P value was from an analysis of covariance (ANCOVA) with baseline as covariate
86.6
0.039
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*P value was from an analysis of Paired-sample T-test
0.694
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87.1
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Physical Functioning
The eight domains of SF-36 questionnaires (physical functioning, role–physical, bodily pain, general health perceptions, vitality, social functioning,
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role-emotional, and mental health) were compared before and after treatments in three groups. We found that three groups had significantly decreased on the
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scores of role-emotional after treatment (P<.05). The MZRW group showed significant improvement on general health, vitality and social functioning (P<.05). The senna group also showed significant improvement on vitality, and worse on role-physical (P<.05). As life satisfaction is associated with number of doctor visits, it may be the reason the scores of role-emotional and role-physical after treatment.
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MZRW (n = 97)
Senna (n = 97)
Placebo (n=97)
Bloating and passing of gas Mild diarrhea Headache Dizziness
2 2 1 1
0 0 1 0
0 0 1 0
Abdominal pain Vomiting
3 0
3 1
0
1
Worsening of straining Worsening of sleeping quality
0
4
0
0
Discomfort of stomach
2
a
2
0 0 1
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Stomach ache
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Event
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Supplement file 7 Adverse events occurred in three group a
1 3
5
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There were no significant differences in any adverse event among three groups, as examined with Fisher Exact test.
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Item Section / Tropic
Reported Standard CONSORT Checklist Item
abstract 1a
Page Number
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No. Title,
Identification as a randomized trial in the title
Statement of whether the trial targets a TCM Pattern, a Western Page 1
and keywords
medicine–defined disease, or a Western medicine–defined disease with a
Structured summary of trial design, methods, results, and conclusions
Illustration the name and form of TCM formula used, and the TCM
1c
Page 4
Pattern applied, if applicable.
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(for specific guidance, see CONSORT for abstracts)
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specific TCM Pattern, if applicable 1b
Determination of appropriate keywords, including “Chinese herbal Page 5 medicine formula” and “randomized control trial”
2a
Scientific background and explanation of rationale
2b
Specific objectives or hypotheses
Methods 3a
3b
Pattern or western medicine disease with a specific TCM Pattern.
Description of trial design (such as parallel, factorial), including allocation ratio
Page 6-7
Whether the TCM formula targeting on western medicine disease, TCM Page 7
Page 7, 11
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Trial design
Statement with biomedical science approaches and/or TCM approaches
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objectives
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Introduction Background and
Important changes to methods after trial commencement (such as
N/A
eligibility criteria), with reasons Participants
4a
on
Extension for TCM Formulas
Eligibility criteria for participants
Statement of whether the formula targets a Western medicine–defined Page 8 disease, a TCM Pattern, or a Western medicine–defined disease with a specific TCM Pattern
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Page 7-8
5
The interventions for each group with sufficient details to allow replication, including how and when they were actually administered
Description(s) for different types of formulas should include the following:
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Settings and locations where the data were collected
5a. For fixed CHM formulas
(Page 6-7, 9)
2. Name, source, processing method, and dosage of each medical
(Page 9-10,
substance. Names of substances should be presented in at least 2
Supplemental
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1. Name, source, and dosage form (e.g., decoctions, granules, powders)
substances used should be specified.
& 2)
3. Authentication method of each ingredient and how, when, where, and
(Supplemental
by whom it was conducted; statement of whether any voucher specimen
File 2 P.6,
was retained, and if so, where they were kept and whether they are
Supplemental
accessible
File 2-Table 1)
4. Principles, rationale, and interpretation of forming the formula
(Page 6-7, 9)
5. Reference(s) as to the efficacy of the formula, if any
(Page 6-7)
6. Pharmacologic study results of the formula, if any
(Supplemental
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languages: Chinese (Pinyin), Latin, or English. Names of the parts of the File 2-Table 1
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Interventions
4b
File 2 P.16-27) (Supplemental 7. Production method of the formula, if any
File 2 P.8) (Page 10,
8. Quality control of each ingredient and of the product of the formula, if Supplemental any. This would include any quantitative and/or qualitative testing method(s); when, where, how, and by whom these tests were conducted; whether the original data and samples were kept, and, if so, whether they are accessible.
File 2 P.9-15)
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
9. Safety assessment of the formula, including tests for heavy metals and
(Supplemental
toxic elements, pesticide residues, microbial limit, and acute/chronic
File 2 P.9-12,
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toxicity, if any. If yes, it should be stated when, where, how, and by whom 16) these tests were conducted; if the original data and samples were kept; and, if so, whether they are accessible.
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10. Dosage of the formula, and how the dosage was determined
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11. Administration route (e.g., oral, external)
5b. For individualized CHM formulas
(Page 9-10) (Page 9-10)
(N/A)
1. See recommendations 5a 1–11 2. Additional information: how, when, and by whom the formula was
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modified
5c. For patent proprietary CHM formulas 1. Reference to publicly available materials, such as pharmacopeia, for the details about the composition, dosage, efficacy, safety, and quality control of the formula 2. Illustration of the details of the formula, namely 1) the proprietary product name (i.e., brand name), 2) name of manufacturer, 3) lot number, 4) production date and expiry date, 5) name and percentage of added materials, and 6) whether any additional quality control measures were conducted 3. Statement of whether the patent proprietary formula used in the trial is for a condition that is identical to the publicly available reference
5d. Control groups
(N/A)
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Placebo control (Page 9-10)
2. Description of the similarity of placebo with the intervention (e.g.,
(Page 10,
color, smell, taste, appearance, packaging)
Supplemental
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1. Name and amount of each ingredient
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3. Quality control and safety assessment, if any
(Page 9-10)
5. Production information: where, when, how, and by whom the placebo
(Page 10,
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Completely defined prespecified primary and secondary outcome
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6a
(Page 10)
4. Administration route, regimen, and dosage
was produced
Outcomes
File 3)
Supplemental File 2 P.9)
Active control
(N/A)
1. If a CHM formula was used, see recommendations 5a–5c
(Page 9-10,
2. If a chemical drug was used, see item 5 of the CONSORT Statement
Additional File 2)
Illustration of outcome measures with Pattern in detail
Page 10-11
measures, including how and when they were assessed
Any changes to trial outcomes after the trial commenced, with reasons
N/A
7a
How sample size was determined
Page 12
7b
When applicable, explanation of any interim analyses and stopping
N/A
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Sample size
6b
guidelines Randomization
8a
Method used to generate the random allocation sequence
Page 11
8b
Type of randomization; details of any restriction (such as blocking and
Page 11
Sequence
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
9
Mechanism used to implement the random allocation sequence (such as
concealment
sequentially numbered containers), describing any steps taken to
mechanism
conceal the sequence until interventions were assigned 10
Blinding
participants, and who assigned participants to interventions 11a
If done, who was blinded after assignment to interventions (for example,
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Implementation
Who generated the random allocation sequence, who enrolled
Page 11
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Allocation
block size)
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generation
Page 11
Page 11
participants, care providers, those assessing outcomes) and how
12a
methods
Statistical methods used to compare groups for primary and secondary outcomes
12b
Methods for additional analyses, such as subgroup analyses and adjusted
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analyses Results Participant flow
13a
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Statistical
If relevant, description of the similarity of interventions
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11b
Page 10, Supplemental File 3 Page 12-13
N/A
For each group, the numbers of participants who were randomly
Page 13
(a diagram is
assigned, received intended treatment, and were analyzed for the
Figure 1
strongly
primary outcome
recommended) 13b
For each group, losses and exclusions after randomization, together with
Page 13
reasons
Figure 1
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Dates defining the periods of recruitment and follow-up
Page 13
14b
Why the trial ended or was stopped
N/A
15
A table showing baseline demographic and clinical characteristics for each group
16
analyzed Outcomes and
For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups
17a
estimation
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Numbers
For each primary and secondary outcome, results for each group, and the
estimated effect size and its precision (such as 95% confidence interval) 17b
For binary outcomes, presentation of both absolute and relative effect
18
Harms
Results of any other analyses performed, including subgroup analyses
Page 13 Figure 1 Page 13-16 Table 2
Page 13-16 Table 2
and adjusted analyses, distinguishing prespecified from exploratory 19
All important harms or unintended effects in each group (for specific (There is no extension for this item)
Discussion 20
Page 16 Supplemental
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guidance see CONSORT for harms)
Limitations
Table 1
N/A
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analyses
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sizes is recommended Ancillary
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Baseline data
14a
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Recruitment
File 6
Trial limitations; addressing sources of potential bias; imprecision; and,
Page 17
if relevant, multiplicity of analyses Generalizability
21
Generalizability (external validity, applicability) of the trial findings
Discussion of how the formula works on different TCM Patterns or diseases
Page 17
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Interpretation
22
Interpretation consistent with results, balancing benefits and harms, and Interpretation with TCM theory and Pattern is encouraged.
Page 17
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considering other relevant evidence Other
23
Registration number and name of trial registry
Protocol
24
Where the full trial protocol can be accessed, if available
Sources of funding and other support (such as supply of drugs), role of
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funders
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25
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Funding
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Registration
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information
Page 8 Supplemental File 1: Protocol Page 2
PII: DOI: Reference:
S1542-3565(18)30341-0 10.1016/j.cgh.2018.04.005 YJCGH 55782
To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 2 April 2018 Please cite this article as: Zhong LL, Cheng C-w, Kun W, Dai L, Hu D-d, Ning Z-w, Xiao H-t, Lin Cy, Zhao L, Huang T, Tian K, Chan K-h, Lam T-w, Chen X-r, Wong C-t, Li M, Lu A-p, Wu JC, Bian Zx, Efficacy of MaZiRenWan, a Chinese Herbal Medicine, in Patients With Functional Constipation in a Randomized Controlled Trial, Clinical Gastroenterology and Hepatology (2018), doi: 10.1016/ j.cgh.2018.04.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title page Title: Efficacy of MaZiRenWan, a Chinese Herbal Medicine, in Patients With Functional Constipation in a Randomized Controlled Trial
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Authors:
Dr. Linda LD Zhong1*, MD, PhD; Ms. Chung-wah Cheng1*, MPhil; Dr. Wai Kun1, MD; Mr. Liang Dai1, MPhil; Ms. Dong-dong Hu1, MPhil; Ms. Zi-wan Ning1, MPhil; Dr.
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Hai-tao Xiao1, PhD; Dr. Cheng-yuan Lin1, PhD; Ms. Ling Zhao1, MPhil; Dr. Tao Huang1, PhD; Dr. Ke Tian1, PhD; Dr. King-hong Chan2, MD; Dr. Ting-wa Lam3, MD; Dr.
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Xiao-rui Chen2, MD; Dr. Chi-tak Wong2, MD; Prof. Min Li1, MD; Prof. Ai-ping Lu1, MD, PhD; Prof. Justin CY Wu4, MD#; Prof. Zhao-xiang Bian1, MD, PhD#
1. Hong Kong Chinese Medicine Study Centre, Hong Kong Baptist University, Hong
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Kong, P. R. China
2. Department of Family Medicine & General Out-patient Clinics, Kowloon Central Cluster, Hospital Authority, Kowloon City, Hong Kong, P. R. China
R. China
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3. Department of Medicine, Queen Elizabeth Hospital, Hospital Authority, Hong Kong, P.
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4. Institute of Digestive Disease, Faculty of Medicine, The Chinese University of Hong Kong, P. R. China
*: Equal contribution.
Grant Support: This study was supported by the Health and Health Services Research Fund (HHSRF) from the Food and Health Bureau of Hong Kong (project no. 09101501).
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Abbreviations Used in This Paper: AE, adverse event; ANOVA, analysis of variance; CHM, Chinese herbal medicine; CSBM, complete spontaneous bowel movement; CI,
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confidence interval; FC, functional constipation; GMP, Good Manufactory Practice; HHSRF, Health and Health Services Research Fund; MZRW, MaZiRenWan; NHS, National Health Service; RCT, randomized controlled trial; SD, standard deviation; TCM,
Co-correspondence: Prof. Justin CY Wu, Institute of Digestive Disease, Faculty of
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#
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Traditional Chinese Medicine.
Medicine, The Chinese University of Hong Kong, P. R. China, Email address: [email protected], Telephone: 852 3505 3174, Fax: 852 2646 8915 and Prof. Zhao-xiang Bian, Hong Kong Chinese Medicine Study Centre, Hong Kong Baptist
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University. 3/F, Jockey Club School of Chinese Medicine Building, 7 Baptist University Road, Kowloon Tong, Hong Kong, P. R. China. E-mail address: [email protected],
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Telephone: 852 3411 2905, Fax 852 3411 2902.
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Disclosures: The authors declare that they have no competing interests.
Writing Assistance: We have asked Dr. Martha Dahlen for critical English editing.
Authors Contributions: All authors participated in the design of the study and performed the trial. Linda LD Zhong drafted and Chung-wah Cheng revised the manuscript. Justin CY Wu and Zhao-xiang Bian supervised and coordinated the clinical
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trial. Linda LD Zhong and Wai Kun participated in general trial coordination and recruited the participants. King-hong Chan, Ting-wa Lam and Xiao-rui Chen referred and recruited patients. Chung-wah Cheng, Liang Dai, Tao Huang and Ke Tian participated in
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randomization and statistical analysis. Dong-dong Hu, Zi-wan Ning, Cheng-yuan Lin, Ling Zhao and Hai-tao Xiao conducted all the quality control studies of MZRW. Min Li
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authors read and approved the final manuscript.
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and Ai-ping Lu gave critical advice on the design and implementation of the study. All
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Abstract Background & Aims: The Chinese herbal medicine, MaZiRenWan (MZRW), has been used for more than 2000 years to treat constipation, but it has not been tested in a
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randomized controlled trial. We performed a trial to evaluate the efficacy and safety of MZRW, compared with the stimulant laxative senna or placebo, for patients with
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functional constipation (FC).
Methods: We performed a double-blind, double-dummy, trial of 291 patients with FC
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based on Rome III criteria, seen at 8 clinics in Hong Kong from June 2013 through August 2015. Patients were observed for 2 weeks and then assigned randomly (1:1:1) to groups given MZRW (7.5 g, twice daily), senna (15 mg daily), or placebo for 8 weeks. Patients were then followed for 8 weeks and evaluated at baseline and weeks 4, 8 (end of
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treatment), and 16 (end of follow up). Participants recorded information on stool form and frequency, feeling of complete evacuation, and research medication taken. Data on individual bowel symptoms, global symptom improvement, and adverse events were
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collected. A complete response was defined as an increase ≥1 complete spontaneous bowel movement (CSBM)/week from baseline (the primary outcome). Secondary
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outcomes included response during the follow-up period, colonic transit, individual and global symptom assessments, quality of life measured with 36-item short form Chinese version, and adverse events.
Results: Although there was no statistically significant difference in proportions of patients with a complete response to MZRW (68%) vs. senna (57.7%) (P=.14) at week 8,
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there was a statistically significant difference vs. placebo (33.0%) (P<.005). At the 16-week timepoint (after the 8-week follow-up period), 47.4% of patients had a complete response to MZRW, 20.6% had a complete response to senna, and 17.5% had a complete
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response to placebo (P<.005 for MZRW vs. placebo). The group that received MZRW group also had significant increases in colonic transit and reduced severity of constipation, straining, incomplete evacuation, and global constipation symptoms compared with the
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groups that received placebo or senna in (P<.05 for all comparisons).
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Conclusion: In a randomized controlled trial of 291 patients with FC, we found MZRW to be well-tolerated and effective in increasing CSBM/week. MZRW did not appear to be more effective than senna, and might be considered as an alternative to this drug.
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ClincialTrials.gov no: NCT01695850.
KEY WORDS: alternative medicine, plant, Asia, functional bowel disorder
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(382 words-edited by CGH’s Science Editor)
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Background Constipation is a common chronic gastrointestinal disease with prevalence varying from 0.7% to 79% (median 16%) in the global population.1 Conventional drug therapies for
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constipation include fiber supplements, stool softeners or wetting agents, osmotic and stimulant laxatives, and, more recently, chloride channel activators, guanylate cyclase C activators, opioid receptor antagonists and serotonergic agonists.2 However, with the
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unsatisfactory response and immaturity and relatively high economic burden of new
herbal medicine (CHM).
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agents,3-5 many patients seek help from alternative medicine, mostly by taking Chinese
According to Traditional Chinese Medicine (TCM) theory, constipation can be divided into excessive and deficient Patterns based on the underlying etiology6,7. The former is
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characterized by the presence of heat or pathological accumulation of Qi (stagnation). The Chinese herbal compound formula, MaZiRenWan (MZRW), also called Hemp Seed Pills, was first recorded in the TCM classic, “Discussion of Cold-induced Disorders”
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(ShangHanLun). It comprises six herbs: Fructus Cannabis (HuoMaRen), Radix et Rhizoma Rhei (DaHuang), Radix Paeoniae Alba (BaiShao), Semen Armeniacae Amarum
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(KuXingRen), Fructus Aurantii Immaturus (ZhiShi) and Cortex Magnoliae Officinalis (HouPo). MZRW is marketed in the United States and many other countries as dietary supplement. Based on modern pharmaceutical studies, MZRW can stimulate intestinal mucosa, increase secretion, accelerate intestinal peristalsis and decrease water absorption6-9-12. From our previous studies of MZRW, the dose of 7.5g b.i.d. had better therapeutic effect than its lower dose (2.5g b.i.d) and recommendated dose (5.0g b.i.d)
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among 96 subjects. Furthermore, its efficacy was superior to placebo in a double-blinded, randomized, controlled trial (RCT) with 120 subjects13.
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Senna, due to its low cost, safety and ease of administration, is widely prescribed in clinical practice and recorded in Laxative Treatment Guideline 14-17. This study aimed to evaluate the efficacy and safety of a Chinese herbal medicine, MZRW, by comparing with
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stimulant laxative, senna, and placebo for patients with FC in excessive TCM syndrome.
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In reporting details of trial, we followed the recommendations of CONOSRT Extension for Chinese Herbal Medicine Formulas 2017 (CONSORT-CHM Formulas 2017).18
Methods
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Study Design
This trial was a multi-center, randomized, double-blinded, double-dummy, controlled trial conducted in the Lee Kee Memorial Dispensary (general outpatient clinic), the Li Ka Shing
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Specialist Clinic of Prince of Wales Hospital and six Chinese medicine clinics affiliated with the School of Chinese Medicine, Hong Kong Baptist University. After a 2-week
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run-in, eligible subjects were randomly assigned to the MZRW arm (MZRW plus placebo senna), the senna arm (placebo MZRW plus senna), or the placebo arm (placebo MZRW plus placebo senna). All subjects received eight weeks of treatment and eight weeks follow-up. Five visits, in total, were scheduled at week -2 (run-in), week 0 (baseline), week 4 (within treatment), week 8 (end of treatment) and week 16 (end of follow-up).
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The study protocol was approved by the Ethics Committee on the Use of Human Subjects for Teaching and Research (Approval no. HASC/10-11/16) in September 2011, registered at ClinicalTrials.gov (NCT01695850) in September 2012 and published in 2013
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(Supplemental file 1).19 The study design was based on the recommendations made in the 'Design of Treatment Trial for Functional Gastrointestinal Disorders’, as proposed by the Rome III Working Team20. The duration of treatment was based on our previous two
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clinical studies of MZRW13. Written informed consent was obtained from each subject
and approved the final manuscript.
Participants and Recruitment
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who agreed to participate. All the authors had access to the study data and had reviewed
The participants were recruited from the public through advertisements or press releases in
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local newspapers. Diagnosis of FC was based on Rome III criteria: 1) including two or more of the following in at least 25% of defecations: straining, lumpy or hard stools, sensation of incomplete evacuation, sensation of anorectal obstruction/blockage, manual
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maneuvers to facilitate, or less than 3 defecations per week; 2) rare presence of loose stools without the use of laxatives; 3) insufficient criteria for irritable bowel syndrome.20
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Excessive Pattern was defined as any three of the chief symptoms: 1) dry and hard stools; 2) difficult bowel movements; 3) abdominal distension, with or without tenderness; 4) belching; 5) dry mouth or halitosis; 6) red tongue with dry and/or yellow coating; 7) wiry pulse.6,7
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Subjects were recruited only if they fulfilled all of the followings: 1) the diagnostic criteria for FC; 2) the diagnostic criteria for Excessive Pattern; 3) aged 18 to 65 years; 4) complete spontaneous bowel movement (CSBM) ≤2times/week (CSBM was defined by feeling of
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complete passage of stool and without the use of laxatives or enemas within 24 hours);21 5) severity of constipation ≥3 points (on a 7-point scale); 6) total symptom score ≥8 points (on a 7-point scale for constipation-related symptoms); 7) normal colonic examination (barium
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enema or colonoscopy) within five years; 8) normal liver and renal function in blood test
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within three months.
In addition, patients were excluded if they had drug-induced or secondary causes of constipation, abdominal surgery within one year, taken other herbal medicine for constipation in the last three months, or severe diseases (e.g. cancer, acute asthma).
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Patients with a history of allergy to CHM, psychiatric or addictive disorders requiring medications with side effects of constipation, or women who were pregnant or
Interventions
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breast-feeding were also excluded from the study.
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Based on previous study result, a dose of 15g a day (7.5g, b.i.d) of MZRW granules was selected.13 The placebo granules were made from dextrin (76.03%), tea essence (23.61%), gardenin (0.02%) and caramel (0.34%).13 Patients were instructed to dissolve a sachet of granules in 150ml of hot water; and to take the solution orally twice daily, after breakfast and dinner. Both of them were prepared by PuraPharm Pharmaceuticals (Nanning, China). The entire manufacturing process was in strict compliance with the standards of Good
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Manufactory Practice (GMP) and Chinese Pharmacopoeia 2010.22 The composition of MZRW and placebo, and the authentication, production, pharmacology study and safety details are listed in Supplemental file 2. The senna tablets (trade name: Senokot)
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containing 7.5mg Sennoside B were manufactured by Reckitt Benckiser Company, UK.16,23 The placebo tablets were made of starch and pigment by Guangzhou Huahai Pharmaceuticals (Guangzhou, China). Patients were instructed to take two tablets at
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bedtime.
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Rescue medication/enema: Dulcolax tablets and Dulcolax suppositories were provided as rescue medicines to ensure bowel movements only in those patients without bowel movements for at least three consecutive days during the study.
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To ensure the success rate of blinding, the color, smell, taste, appearance and packaging of placebos were comparable to the interventions (see Supplemental file 3).
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Outcomes
Participants were requested to record their stool form and frequency, feeling of the
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completeness of evacuation, and research medication taken daily in the diary.
The primary outcome was the proportions of patients with a complete response during treatment. Participants with an increase ≥1CSBM/week from baseline were defined as complete response. The changes in colonic transit detected by using a commercially available radio-opaque Sitzmarks capsule (Konsyl Pharmaceuticals, US) was secondary
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outcome24-26. Slow transit constipation (STC) is defined as expulsion of less than 80% markers after 120 hours, i.e. more than 5 out of 24 markers remained. Other secondary outcomes included the complete response of CSBM during the follow-up period.
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Individual symptoms of severity of constipation, sensation of straining, incomplete evacuation, bloating, abdominal pain / cramping, nausea, and passing of gas) were self-assessed by subjects using a 7-point ordinal scale (0=not at all to 6=very severe) at
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every visit. Global symptom improvement was defined as the feeling of adequate relief of constipation in comparison with baseline with scores (0=markedly worse to 6=markedly
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better). The quality of life was measured by the 36-item Short Form (SF-36) Chinese version.27
Safety profiles of MZRW were assessed by determining important adverse events (AEs)
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reported or clinical laboratory evaluations. The success of blinding was evaluated for both investigators and patients, as which of MZRW, senna or placebo had been taken during the
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last visit.
Randomization and Blinding
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Block randomization was carried out in a 1:1:1 ratio according to the sequence generated with Random Allocation Software (Version 2.0), Isfahan, Iran. Research assistant assigned interventions according to the codes kept in opaque sealed envelopes with consecutive randomization numbers. Treatment assignments were not revealed and were blinded to patients and investigators (including statisticians) until the entire study was completed.
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Sample size calculation Based on the results of our previous placebo-control study, the proportions of a complete 13
Since there
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response of MZRW group and placebo groups were around 40% and 10%.
has been no RCT study of senna, we assumed the response rates for MZRW, senna and
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placebo were 40%, 20% and 10%, respectively.
Therefore, 82 patients per treatment group were deemed sufficient to achieve 80% power
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in detecting treatment differences, based on two-sided Chi-square test without continuity correction at a significance level of 0.025 (used to maintain the overall significance level at 5%). Further assuming a 15% dropout rate, a total of 291 patients (97 per arm) were recruited to ensure statistically significant results. The calculation was performed using
Statistical Analysis
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Study Size 2.0 software, London, UK.
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All efficacy and safety analyses were conducted according to the intention-to-treat (ITT) principle, as the ITT population included all patients who were randomized. Missing
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values were imputed by the last-observation-carried-forward method. The details of statistical analysis appear in Supplemental file 4.
Results
Patient Characteristics
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From June 2013 to August 2015, 843 potential subjects were screened; of these, 291 were randomized and assigned to MZRW, senna, or placebo groups (97 per arm). In total, 33 subjects withdrew from the study. Seven of them were lost to follow-up, 18 discontinued
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their interventions due to unsatisfactory effects, seven withdrew due to AEs and one was pregnant. For those who completed the study, 95.3% (246/258) were compliant with at least 80% of scheduled doses, which was determined by counting the medication returned.
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The flow diagram is shown in Figure 1. The baseline characteristics of the three groups
Primary Outcome Assessment
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were well balanced (P>.05) and are summarized in Table 1.
The proportion of complete response of MZRW group was comparable to senna group during treatment (68.0% vs. 57.7%, with P=.14), but both were significantly higher than
(P<.005).
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that of placebo group (33.0%, with P<.005), with significant inter-group difference
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Secondary Outcome Assessment Complete response and CSBMs
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The proportions of complete responses during the follow-up period were 47.4% in MZRW group, 20.6% in senna group, and 16.5% in placebo group (P<.005). The mean CSBM per week increased 2.2times (95%CI: 1.8, 2.6) in MZRW group, 2.0times (95%CI: 1.6, 2.3) in senna group and 1.2times (95%CI: 0.8, 1.6) in placebo group during treatment, with significant inter-group difference (P=.002). In contrast, the mean CSBM in the follow-up period increased 1.5 times (95% CI: 1.1, 1.9) in MZRW group, 0.6 times
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(95%CI: 0.3, 0.8) in senna group and 0.5times (95%CI: 0.2, 0.7) in placebo group, with significant inter-group difference (P<.0005) (Table 2). The weekly changes of complete response and CSBM are shown in Figure 2. Proportion of patients with weekly
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frequency CSBM ≥3 and an increase ≥1 from baseline among three groups is presented in Supplemental file 5.
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Colonic transit
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Before treatment, proportions of slow transit colonic (STC) patients (>5 out of 24 markers remained) were among 47.1% to 59.6%, without significant inter-group difference (P=.637) and post-hoc pairwise multiple comparison (P>.05). Significant change of STC proportion were found after 8-week treatment (P=0.006). Post-hoc pairwise multiple comparison of slow transit after 8-week treatment is as follows: MZRW
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vs. placebo: P=.002; MZRW vs. senna: P=.039; senna vs. placebo: P=.251. Proportion of STC subjects and no STC subjects were represented by percentage in Figure 3.
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Individual symptom assessment and rescue medicine
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In general, comparing outcomes with baselines for all three groups, scores for individual symptoms were lower after treatment and during follow-up period. MZRW was superior to senna and placebo in reducing the severity of constipation, sensation of straining, incomplete evacuation, and passing of gas (P<.05), while it was comparable to senna in managing abdominal bloating and abdominal pain/cramping (Table 2). The frequency on using rescue medicine per week was lower in the MZRW group than in both senna and
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placebo groups during treatment (P=.029 and .003, respectively) and the follow-up period (P=.007 and .001, respectively) (Table 2).
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Other secondary outcomes
The results of global symptom assessment and quality of life assessment are represented
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in Supplemental file 6.
Success of blinding
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With regard to participants, 34.5% (89/258; 35 in MZRW group, 26 in senna group and 28 in placebo group) correctly guessed their treatment. With regard to investigators, 55.4% (143/258; 57 in MZRW group, 49 in senna group and 37 in placebo group) correctly guessed. For subjects with correct answers, 68.5% (61/89) were based on the
intervention.
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efficacy of treatment, 22.5% (20/89) on the taste and 9.0% on the appearance of the
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Safety and adverse events
There were no significant differences in renal and liver function between and within the
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MZRW, senna and placebo groups at baseline and after treatments. Most subjects well tolerated the research medication, and no serious AEs reported (Supplemental file 7).
Discussion
The results from this study showed that both MZRW and senna had beneficial effects on increasing CSBM and higher proportion of complete response than placebo during
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treatment, and MZRW had even better than senna with regard to relieving most of constipation-related symptoms and improving the colonic transit during treatment and follow-up period. Why MZRW prolonged improvement in bowel movement even after
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treatment, i.e. during the follow-up period, might due to some specific metabolites, whose changes are significantly correlated with CSBM improvement. Our further research is
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focusing on MZRW’s mechanism28.
It has been reported in other studies of FC that placebo effects can range from 18.0% to
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37.5%29,30. In this study, we found the placebo effect was about 33.0% during treatment and 17.5% during follow-up, a rate consistent with those studies. But in our previous study with 120 patients reported placebo effect was 8.3%13. A number of factors could explain this variation, such as three-armed design (MZRW, senna and placebo), two types
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of medications (Chinese herbal medicine and conventional laxative) and larger sample sizes (291 vs. 120 subjects).31
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How to apply the randomized controlled clinical trial methodology in CHM research always challenges clinical investigators. Our comprehensive assessment of MZRW, from
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first identification of target intervention with a systematic review, determination of optimal dosage with a RCT, evaluation of its efficacy and safety with a placebo-controlled study, to further verification of therapeutic effects by comparison with standard conventional treatment, can be a reference study model for other CHM interventions. This evidenced-based approach with transparent and full disclosure of the composition, authentication, processing, production and quality control of MZRW
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following the recommendations of CONSORT-CHM Formula 2017 ensures the quality and reproducibility of intervention, as well as the study results.18
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The current study also investigated the efficacy and safety of senna by comparing with placebo. Results showed that senna was better than placebo in improving mean CSBM by 2.0/week (95%CI: 1.6, 2.3) vs. 1.2/week (95%CI: 0.8, 1.6) during treatment (P<.05). As
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being the first RCT comparing senna with placebo, the study results provide consolidated
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evidence of the efficacy of senna in the management of FC.
There are several limitations to this study. First, the majority of the study population was female. Second, although the Pattern concept was used to select eligible subjects, the changes in Pattern parameters were not quantitatively assessed nor calculated as an
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outcome. Thirdly, the sample size calculation was only based on MZRW and placebo; the effects of senna was assumed. If we want to investigate the superiority of MZRW as compared to senna, a larger sample size clinical trial would be required. Our results also
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suggest that combination therapy with both MZRW and senna is worthy of investigation. Fourthly, the population was all ethnic Chinese. There may have been a higher response
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rate to TCM due to preconceived cultural expectations or beliefs; the efficacy might be reduced, or different, in other ethnic populations.
In conclusion, MZRW is a well-tolerated and effective intervention for FC. It could be considered as an alternative remedy for FC. Further, the approach of assessing the efficacy and safety of MZRW by first having a systematic review, determining the
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optimal dose, followed by comparing with placebo and standard conventional treatment
Table Legends Table 1. Baseline characteristics of participants
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can be a reference for other CHM intervention studies.
Table 2. Comparison of treatment effect toward bowel movement, individual symptom
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Figure Legends
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assessment and rescue medicine used
Figure 1. FC = functional constipation; MZRW = MaZiRenWan; ITT analysis = intention-to-treat analysis, which consisted of all randomly assigned patients.
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Figure 2. a. Weekly CSBMs were recorded by patients in the stool and symptom diary. Error bars represented 95% confidence intervals. The MZRW group and senna group increased 2.2 and 2.0 mean CSBM/week from baseline during treatment, respectively
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(P>.05). The P values were calculated from two-way ANOVA analysis and t-test (*, P<.05, **, P<.01).
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b. Complete response was defined as an increase ≥1 CSBM/week from baseline. The P values were calculated from Fisher-exact test (*, P<.05; **, P<.01). CSBM = complete spontaneous bowel movement; MZRW = MaZiRenWan
Figure 3. The colonic transit was measured by using a radio-opaque capsule containing 24 markers. Slow transit constipation (STC) was defined as expulsion of less than 80%
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markers after 120 hours (>5 out of 24 markers remained). Proportion of STC subjects (cyan) and no STC subjects (purple) were represented by percentage. No significant differences among three groups were detected at baseline (pre-treatment, P=.637).
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Significant change of STC proportion were found after 8-week treatment (P=.006).
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MZRW = MaZiRenWan; ns=no significance.
References
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7. Maclean W, Lyttleton J. Clinical Handbook of Internal Medicine: the Treatment of Disease with Traditional Chinese Medicine. Sydney: University of Western Sydney, Macarthur, 2002.
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8. Bensky D. Chinese Herbal Medicine: Formulas & Strategies. Washington: Eastland Press, 1990.
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10. Zhu Y. Chinese Materia Medica: Chemistry, Pharmacology, and Applications. Amsterdam: Harwood Academic, 1998.
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13. Cheng CW, Bian ZX, Zhu LX, et al. Efficacy of a Chinese herbal proprietary medicine (Hemp Seed Pill) for functional constipation. Amercian Journal of
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Gastroenterology 2011;106:120-129. 14. Marciniak CM, Toledo S, Lee J, et al. Lubiprostone vs Senna in postoperative orthopedic surgery patients with opioid-induced constipation: a double-blind, active-comparator trial. World Journal of Gastroenterology 2014;20:16323-16333. 15. Santos-Jasso KA, Arredondo-García JL, Maza-Vallejos J, et al. Effectiveness of senna vs polyethylene glycol as laxative therapy in children with constipation related to
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17. National Institute for Health and Care Excellence. Clinical Knowledge Summary: CKS Constipation Guideline. https://cks.nice.org.uk/constipation. Accessed May 18, 2017.
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18. Cheng CW, Wu TX, Shang HC, et al. CONSORT extension for Chinese herbal
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medicine formulas 2017: recommendations, explanation, and elaboration. Annual of
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20. Drossman DA, Corazziari E, Delvaux M, et al. Rome III: The Functional Gastrointestinal Disorders. McLean, VA: Degnon Associates, 2006. 21. Tarumi Y, Wilson MP, Szafran O, et al. Randomized, double-blind, placebo-controlled
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trial of oral docusate in the management of constipation in hospice patients. Journal of Pain and Symptom Management 2013;45:2-13.
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22. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China, 2010th ed. Beijing: People’s Medical Publishing House, 2010. 23. Ford AC, Suares NC. Effect of laxatives and pharmacological therapies in chronic idiopathic constipation: systematic review and meta-analysis. Gut 2011;60:209-218. 24. Konsyl Pharmaceuticals. Sitzmarks: the preferred colonic transit diagnostic test. http://www.sitzmarks.com (For Professionals). Accessed May 19, 2017.
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25. Park JM, Choi MG, Choi H, et al. Measurement of colonic transit using a delayed-release capsule containing radio-opaque markers. Scandinavian Journal of Gastroenterology 2008;43:545-550.
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26. Drossman D, Chang L, Chey WD, et al. Rome IV Functional Gastrointestinal Disorder-Disorder of Gut Brain Interaction, 4th ed. Raleigh, NC: Rome Foundation, 2016.
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27. Lam CL. Reliability and construct validity of the Chinese (Hong Kong) SF-36 for patients in primary care. Hong Kong Practitioner 2003;25:468-475.
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28. Huang T, Ning ZW, Hu DD, et al. Uncovering the mechanisms of Chinese Herbal Medicine (MaZiRenWan) for functional constipation by focused network pharmacology approach.
29. Lacy BE, Schey R, Shiff SJ, et al. Linaclotide in chronic idiopathic constipation
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patients with moderate to severe abdominal bloating: a randomized, controlled trial. PLos One 2015;10:e0134349.
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Supplemental files Supplemental file 1: Published study protocol
safety details of MaZiRenWan Supplemental file 3: Packaging and appearance of interventions Supplemental file 4: Statistical analysis
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Supplemental file 2: Composition, authentication, preparation, pharmacokinetics and
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Supplemental file 5: The proportion of patients with weekly frequency ≥3times CSBM
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and an increase ≥1 CSBM from baseline
Supplemental file 6: Results of other secondary outcomes
Supplemental file 7: Adverse events occurred in different groups
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Supplemental file 8. CONSORT – Chinese Herbal Medicine Formulas Checklist
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ACCEPTED MANUSCRIPT Table 1 Baseline characteristics of participants Characteristic
All Patients
MZRW
Senna
Placebo
(N=291)
(n=97)
(n=97)
(n=97)
Sex, n (%)
P value
0·071 263 (90·4)
86 (88·7)
93 (95·9)
84 (86·6)
Male
28 (9·6)
11 (11·3)
4 (4·1)
13 (13·4)
Age, mean (SD), y
45·4 (12·0)
45·5 (12·0)
45·1 (11·7)
Duration of FC, mean (SD), y
15·6 (11·5)
15·9 (11·4)
16·2 (11·7)
SBM per week, mean (SD)
1·8 (0·9)
1·9 (1·0)
1·7 (0·6)
CSBM per week, mean (SD)
1·8 (0·9)
1·9 (1·1)
1·7 (0·8)
CSBM < 3 per week, n (%)
256 (88.0%)
83 (85.6%)
Severity of constipation
4·5 (1·1)
4·5 (1·1)
Sensation of straining
4·1 (1·5)
4·2 (1·6)
Incomplete evacuation
3·6 (1·5)
Bloating
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Female
0·934
14·6 (11·4)
0·579
2·0 (0·9)
0·187
1·9 (0·9)
0·340
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45·7 (12·4)
90 (92.8%)
83 (85.6%)
0.204
4·6 (1·0)
4·4 (1·2)
0·566
4·1 (1·4)
4·0 (1·4)
0·673
3·9 (1·3)
3·7 (1·5)
3·4 (1·6)
0·081
1·0 (1·4)
1·2 (1·5)
0·9 (1·4)
1·0 (1·4)
0·504
Abdominal pain/cramping
0·4 (1·1)
0·3 (1·0)
0·5 (1·2)
0·3 (1·0)
0·608
Passing of gas
3·1 (1·5)
3·2 (1·8)
3·3 (1·3)
2·9 (1·3)
0·198
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Individual symptoms, mean (SD)
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SBM = spontaneous bowel movement; CSBM = complete spontaneous bowel movement; FC = functional constipation; MZRW = MaZiRenWan
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Individual symptoms were assessed on a 7-point scale, from 0 = not at all to 6 = very severe.
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Table 2 Comparison of treatment effect toward bowel movement, individual symptom assessment and rescue medicine used MZRW
Senna
Placebo
MZRW vs. Senna Differences
MZRW vs. Placebo
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Outcome
P Value
Differences
(95%, CI) Bowel movement [Mean Changes From Baseline (95% CI)]
a) Spontaneous bowel movement 2.2 (1.8 to 2.6)
2.0 (1.6 to 2.4)
1.2 (0.8 to 1.6)
0.3 (-0.2 to 0.8)
Follow up
1.6 (1.2 to 2.0)
0.6 (0.4 to 0.9)
0.5 (0.3 to 0.7)
1.1 (0.7 to 1.5)
b) Complete spontaneous bowel movement Treatment
2.2 (1.8 to 2.6)
2.0 (1.6 to 2.3)
1.2 (0.8 to 1.6)
0.4 (-0.1 to 0.9)
Follow up
1.5 (1.1 to 1.9)
0.6 (0.3 to 0.8)
0.5 (0.2 to 0.7)
1.1 (0.6 to 1.6)
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Individual symptom assessment [Mean Changes From Baseline (95% CI)]
a) Severity of constipation
0.190.9 (0.4 to 1.4)
Differences
3 Groups P Value
P Value
(95%, CI)
0.001
0.9 (0.3 to 1.4)
0.004
0.001
<0.00051.0 (0.5 to 1.4)
<0.0005
0.2 (-0.3 to 0.6)
0.498
<0.0005
0.151.0 (0.5 to 1.5)
<0.0005
0.8 (0.2 to 1.4)
0.006
0.002
<0.00051.0 (0.6 to 1.5)
<0.0005
0.1 (-0.4 to 0.6)
0.691
<0.0005
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Treatment
2.
(95%, CI)
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1.
P Value
Senna vs. Placebo
-2.4 (-2.8 to -2.1)
-1.7 (-2.0 to -1.4)
-1.0 (-1.4 to -0.7)
-0.8 (-1.2 to -0.3)
0.001
-1.3 (-1.8 to -0.8)
<0.0005
-0.5 (-0.9 to -0.1)
0.026
<0.0005
Follow up
-1.8 (-2.1 to -1.5)
-0.8 (-1.1 to -0.5)
-0.4 (-0.6 to -0.1)
-1.1 (-1.5 to -0.7)
<0.0005
-1.4 (-1.8 to -1.0)
<0.0005
-0.3 (-0.6 to 0.1)
0.095
<0.0005
b) Sensation of straining
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Treatment
-2.3 (-2.6 to -2.0)
-1.7 (-2.0 to -1.4)
-0.9 (-1.2 to -0.7)
-0.7 (-1.1 to -0.2)
0.001
-1.2 (-1.6 to -0.8)
<0.0005
-0.5 (-0.9 to -0.1)
0.014
0.003
Follow up
-1.7 (-2.0 to -1.4)
-0.8 (-1.0 to -0.5)
-0.5 (-0.7 to -0.2)
-1.0 (-1.3 to -0.6)
<0.0005
-1.0 (-1.4 to -0.7)
<0.0005
-0.1 (-0.4 to 0.3)
0.661
<0.0005
c) Incomplete evacuation
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Treatment
Treatment
-1.9 (-2.2 to -1.5)
-1.2 (-1.5 to -0.8)
-0.5 (-0.8 to -0.1)
-0.5 (-1.0 to -0.1)
0.012
-0.9 (-1.3 to -0.5)
<0.0005
-0.4 (-0.8 to 0.1)
0.088
<0.0005
Follow up
-1.5 (-1.9 to -1.1)
-0.6 (-0.9 to -0.2)
-0.1 (-0.4 to 0.2)
-0.8 (-1.2 to -0.4)
<0.0005
-0.9 (-1.3 to -0.5)
<0.0005
-0.1 (-0.5 to 0.2)
0.464
<0.0005
d) Bloating
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-0.7 (-1.0 to -0.4)
0.1 (-0.3 to 0.4)
-0.5 (-0.8 to -0.2)
-0.5 (-0.9 to -0.2)
0.006
-0.03 (-0.3 to 0.2)
0.929
0.5 (0.2 to 0.9)
0.004
0.005
Follow up
-0.7 (-1.1 to -0.4)
0.01 (-0.3 to 0.3)
-0.4 (-0.7 to -0.1)
-0.5 (-0.8 to -0.2)
0.001
-0.1 (-0.4 to 0.1)
0.469
0.4 (0 to 0.7)
0.028
0.004
-0.2 (-0.4 to 0.04)
0.398
0.04 (-0.2 to 0.3)
0.778
0.36
-0.2 (-0.4 to 0.1)
0.697
0.1 (-0.2 to 0.3)
0.727
0.475
e) Abdominal pain/cramping
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Treatment
-0.2 (-0.4 to 0.001)
-0.1 (-0.3 to 0.04)
-0.02 (-0.05 to 0.01)
-0.2 (-0.5 to 0.01)
0.150
Follow up
-0.2 (-0.4 to 0.03)
-0.1 (-0.3 to 0.04)
-0.03 (-0.1 to 0.004)
-0.2 (-0.4 to 0.03)
0.24
Treatment
-1.1 (-1.5 to -0.8)
-0.7 (-1.0 to -0.4)
-0.3 (-0.6 to 0.01)
-0.6 (-1.0 to -0.2)
0.004
-0.6 (-1.0 to -0.3)
0.001
0.01 (-0.4 to 0.4)
0.955
<0.0005
Follow up
-1.1 (-1.4 to -0.7)
-0.4 (-0.7 to -0.1)
-0.2 (-0.4 to 0.1)
-0.8 (-1.2 to -0.5)
<0.0005
-0.6 (-1.0 to -0.3)
<0.0005
0.2 (-0.1 to 0.6)
0.246
<0.0005
2.
*Rescue medicine frequency per week [Mean (SD)]
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f) Passing of gas
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Treatment
1.2 (2.0)
1.3 (4.0)
0.9 (1.1)
-0.1 (-0.6 to -0.5)
0.151
-0.6 (-1.8 to -0.5)
0.419
-0.4 (-1.3 to 0.1)
0.504
0.077
Treatment
0.8 (2.2)
1.2 (4.5)
0.8 (2.7)
-0.3 (-0.6 to -0.03)
0.029
-0.4 (-0.6 to -0.1)
0.003
-0.1 (-0.3 to 0.2)
0.683
0.002
Follow up
0.8 (2.0)
1.2 (4.7)
0.8 (2.5)
-0.4 (-0.7 to -0.1)
0.007
-0.4 (-0.6 to -0.2)
0.001
-0.03 (-0.3 to 0.3)
0.859
<0.0005
CI = confidence interval; MZRW = MaZiRenWan; SD= standard derviation
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Baseline
Individual symptoms including severity of constipation, sensation of straining, incomplete evacuation, bloating, abdominal pain/cramping and passing of gas, were assessed on a 7-point scale, from 0 = not at all to 6 = very severe and presented in mean (95% CI).
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*Rescue medicine (Dulcolax tablets or Dulcolax suppositories) was provided only in those patients without bowel movements for at least three consecutive days during the whole study.
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P values were calculated from Post-hoc pairwise multiple comparison
ACCEPTED MANUSCRIPT Figure 1 Study Flow Diagram
Assessed for eligibility (n= 843)
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Enrolment
Excluded (n=552) Not FC (n=238) Declined to participate (n=89) Not excessive constipation (n=179) Exams with significant abnormality (n= 46)
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Allocated to MZRW (n=97) Received allocated intervention (n= 97) Did not receive allocated intervention (give reasons) (n=0)
Allocated to Senna (n=97) Received allocated intervention (n= 97) Did not receive allocated intervention (give reasons) (n=0)
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Lost to follow-up (n=2) Discontinued intervention (n=7) 2 due to bloating and passing of gas 5 due to unsatisfactory effects
Analysis
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Follow-up
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Allocation
Randomized (n=291)
ITT analysis (n=97)
Lost to follow-up (n=2) Discontinued intervention (n=8) 2 due to stomachache and headache 1 due to pregnant 5 due to unsatisfactory effects
ITT analysis (n=97)
Allocated to Placebo (n=97) Received allocated intervention (n= 97) Did not receive allocated intervention (give reasons) (n=0)
Lost to follow-up (n=3) Discontinued intervention (n=11) 3 due to insomnia 8 due to unsatisfactory effects
ITT analysis (n=97)
ACCEPTED MANUSCRIPT Figure 2 Weekly changes of CSBM and responder rate
Baseline period
a
Treatment period
Follow-up period
**
**
**
**
** **
2.0 ** **
1.5
*
*
1.0 0.5
-2
-1
0
1
2
3
4
**
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80 70 ** 60
* *
50
**
** *
40 30 20 10 0 -2
-1
0
1
2
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-3
5
**
3
6
7
*
4
5
8
**
9
10
11
12
** *
6
**
**
**
**
7
Week
8
9
10
11
13
**
14
**
**
15
16
Senna group Placebo group
**
**
**
MZRW group
**
**
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Responder rate (%)
**
*
*
0.0
b
**
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** **
2.5
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Change of CSBM from baseline
3.0
**
12
**
13
**
**
**
14
15
16
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ACCEPTED MANUSCRIPT
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Supplemental File 2: Composition, authentication, preparation, pharmacokinetics and safety details of MaZiRenWan (A) Composition and authentication of MZRW ingredients
MaZiRenWan (MZRW) is an herbal formula originally recorded in traditional Chinese medicine classic ‘Discussion of Cold-Induced Disorders’ (Shang Han Lun) (1). This
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formula has been commonly used to treat constipation in China for more than 2000 years. This formula comprised six herbs: Rhei Radix Et Rhizoma, Cannabis Frustus, Paeoniae Radix Alba, Magnoliae Officinalis Cortex, Aurantii Fructus Immaturus and Semen Armeniacae Amarum (2). To gain the quality of MZRW granules, the source and
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processing protocols of raw medicinal substance, as well as the operation of production should be highly standardized and critically controlled. Voucher numbers of individual specimens of MZRW ingredients were given and samples were kept at the Clinical Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China. Each herb was authenticated and tested by the Purapharm (Nanning) Pharmaceuticals Co. Ltd according to the methods recorded in
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corresponding monographs of Chinese Pharmacopoeia (2010) (3). Results of authentication were summarized in Supplement 2-Table 1.
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Chinese name (Pinyin) Huomaren
Medicinal
Source
Latin name Cannabis Frustus
Processing method
parts
Cannabis sativa L.
Dry seeds
Eliminating the foreign matter and pericarp
Radix
(DH)
Rhizoma
Et
Rheum tanguticum
Dry roots
Maxim,ex Balf.
Kuxingren
Armeniacae
Prunus
(KXR)
Semen Amarum
mandshurica
Dry seeds
(BS)
Alba
Radix
Paeonia
lactiflora
Pall.
chromatogram
with
reference herb
and cut into thick slices
total amount of aloe-emodin, rhein,
amount of aloe-emodin, rhein,
emodin, chrysophanic acid and
emodin, chrysophanic acid and
physcion
physcion
1. Thin layer chromatography
1. Identical
Breaking to pieces
Dry roots
Identical Contained
Eliminating the foreign matter
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Paeoniae
Thin layer chromatography Contained not less than 1.5% of the
(Maxim.) Koehne Baishao
Result
Eliminating the foreign matter
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Rhei
Methods / standards cited in the Chinese Pharmacopeia
(HMR) Dahuang
Authentication
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Ingredients
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Table 1. The name, source, processing method of each ingredient and its authentication report
and cutting into thin slices and
2. Contained not less than 3.0% of amygdalin Contained not less than 1.6% of the
2.2%
of
the
chromatogram
total
with
reference herb 2. Contained 6.1% of amygdalin Contained 3.0% of paeoniflorin
paeoniflorin
stir-bake
Magnoliae
Magnolia
(HP)
Officinalis Cortex
officinalis
Dry barks Rehd.et
Wils. Aurantii Fructus
(ZS)
Immaturus
Citrus aurantium L.
Dry fruits
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Zhishi
Scraping off the coarse bark and
Contained not less than 2.0% of the
Contained
cut into slivers
total amount of magnolol and
amount of magnolol and honokiol
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Houpo
3.2%
of
the
total
honokiol
Eliminating the foreign matter,
1. Thin layer chromatography
cutting
2. Contained not less than 0.3% of
into
thin
slices
stir-baking with bran
and
synephrine
1. Identical
chromatogram
with
reference herb 2. Contained 0.4% of synephrine
*Authentication methods and standards are referenced to the Pharmacopeia of the People’s Republic of China 2010, Vol. 1.
(B) Preparation of MZRW granules 250 kg raw medicinal materials in total were mixed by six herbs in proportion listed in Supplement 2-Table 2, and extracted with 8 times of boiling water (w/v) for 2.5 hours
ACCEPTED MANUSCRIPT
and filtered. The filtrate was concentrated to the density of 1.10~1.20 (60℃) under reduced pressure and then subjected to dry in spraying. Subsequently, the dried powders
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were thoroughly mixed with 70kg dextrin to gain 100 kg granules by dry method. Finally, the granules were packed into small quantities with sealed opaque aluminum sachets (7.5 g per sachet).
Table 2. The dosages of all ingredients for MZRW granule Composition (%)
Basic formulation (g)
Production formulation (kg)
Cannabis Frustus
35.7
892.9
89.29
Rhei Radix et Rhizoma
17.9
446.4
22.32
17.9
446.4
8.9
223.2
10.7
267.9
8.9
223.2
Paeoniae Radix Alba Magnoliae Officinalis Cortex Aurantii Fructus Immaturus
Each gram of MZRW granules is equivalent to 4.05 g of raw herbs.
(C) Preparation of placebo
21.7
5.4
10.9
22.32
5.4
10.9
44.64
2.7
5.4
26.79
3.3
6.5
2.7
5.4
44.64
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*
10.8
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Amarum
g/day
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Armeniacae Semen
g/sachet
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Ingredients
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Green tea was decocted with 10 times of water for 1.5 hours and filtered. The filtrate was then concentrated to sticky solution with a relative density of 1.10~1.20 at 60℃. Caramel (0.34kg) and gardenia yellow (0.02kg) dissolved in water were mixed with the green tea essence (23.61kg), followed by mixed with dextrin (76.03kg). The mixture was dried in vacuum and granulated. The granules were packed in sealed opaque aluminum sachets (7.5g per sachet).
(D) Quality report of MZRW granules
MZRW granules was manufactured in Purapharm (Nanning) Pharmaceuticals Co. Ltd with GMP standard. The granules used in this study were originated from one batch, and the qualities in appearance, taste, paeoniflorin identification using TLC, water content, particle size, dissolubility, loading difference, content of paeoniflorin, microbial
ACCEPTED MANUSCRIPT
limit, heavy metals limit and pesticide residue were determined according to the requirements of Chinese Pharmacopoeia (version 2010) for granules and quality standard of
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MZRW granules (as shown in Supplement 2-Table 3). In addition, under the accelerating storage condition of 40℃ and 75% relative humidity, the description, identification, determination of water, particle size, determination of dispersibility, assay and microbial limit test meet the product specification after storage for 3 months. Accelerated stability study was indicated that MZRW granules had good
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stability and the results were presented in Supplement 2-Table 4.
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Table 3. Quality report of MZRW granules
Description
Quality requirements
Results
Reference methods
Light yellow to brownish yellow in color, fragrant in smell,
Brownish yellow in color,
Chinese
acrid and slightly bitter in taste.
fragrant in smell, acrid and
2010, Vol. 1, Appendix I C)
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Test items
Pharmacopoeia
(Version
slightly bitter in taste. consistent distance travelled and color with spot of in the
The thin-layer chromatograms
chromatogram of paeoniflorin was observed.
were shown in S2-Figure 1.
Not more than 6.0%
Chinese
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Determination of
Pass
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Identification
Corresponding spot in chromatogram of test samples with
5.5%
water
Sum of weight of granules that cannot pass through sieve Particle size
No.1 and weight of powder that can pass through sieve NO.5 ≤ 15%
Determination of
Light turbidity is allowed and no foreign matters
Paeoniflorin no less than 0.70 mg/g
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Assay
The filling margin of error shall be plus or minus 5%
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Filling variation
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dispersibility
7%
Slight turbidity Comply
4.72mg/g The high performance liquid chromatograms were shown in
Pharmacopoeia
(Version
2010, Vol. 1, Appendix VI B) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix IX H) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix XI B) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix I C) Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix I C)
Chinese
Pharmacopoeia
(Version
2010, Vol. 1, Appendix VI D)
S2-Figure 2.
Microbial limit test
Chinese
-Total Aerobic Count
2010, Vol. 1, Appendix XIII C)
Not more than 500 colony/g
≤10 colony/g
Pharmacopoeia
(Version
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and
Yeast
Not more than 100 colony/g
≤10 colony/g
Count -Escherichia coli
Not detectable
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-Mould
Not detectable
Heavy metal limit
Chinese
Not more than 150 ppm
3.596ppm
-Arsenic (As)
Not more than 41.67 ppm
0.157ppm
-Cadmium (Cd)
Not more than 97.22 ppm
0.014ppm
-Mercury (Hg)
Not more than 4.97 ppm
0.264ppm
-Lead (Pb)
Not more than 1.00 ppm
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Not more than 0.05 ppm
-Chlordane
Not more than 0.05 ppm
-Total DDT’s
Not more than 1.0 ppm
-Total BHC’s
Not more than 0.3 ppm
-Eldrin
Not more than 0.05 ppm
-Heptachlor
Not more than 0.05 ppm
-Hexachlorobenzene
Not more than 0.1 ppm
-Lindane
Not more than 0.6 ppm
-Quintozene
Not more than 1.0 ppm
2010, Vol. 1, Appendix IX B)
＜0.001ppm
Pesticide residues -Aldrin & Dieldrin
(Version
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-Copper (Cu)
Pharmacopoeia
Not detectable
Kong (Application form: Registration
Not detectable
of proprietary Chinese Medicines)
Not detectable
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0.00504ppm
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Chinese Medicine Council of Hong
Not detectable Not detectable Not detectable Not detectable 0.00106ppm
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Table 4. The test report of accelerated stability study for MZRW granules
Identification
T=1(Jan. 19, 2013)
Light yellow to brownish yellow in
Brownish yellow in color,
color, fragrant in smell, acrid and slightly bitter in taste.
T=3 (Mar. 19, 2013)
Brownish yellow in color,
Brownish yellow in color,
Brownish yellow in color,
fragrant in smell, acrid and
fragrant in smell, acrid and
fragrant in smell, acrid and
fragrant in smell, acrid and
slightly bitter in taste.
slightly bitter in taste.
slightly bitter in taste.
slightly bitter in taste.
Same fluorescent spots observed in
Same
the
observed
corresponding
position
of
reference paeoniflorin
Determination of
T=2 (Feb. 19, 2013)
Not more than 6.0%
fluorescent
spots
in
weight of powder that can pass
foreign matters
Assay
Paeoniflorin no less than 0.70 mg/g
Microbial
limit
test -Total
Aerobic
Not more than 500 colony/g
Count -Mould Yeast Count
and
Not more than 100 colony/g
in
the
Same
fluorescent
observed
in
spots the
reference paeoniflorin
reference paeoniflorin
reference paeoniflorin
5.4%
5.3%
4.4%
8%
7%
7%
Slight turbidity
Slight turbidity
Slight turbidity
4.48mg/g
4.20mg/g
4.12mg/g
≤10 colony/g
≤10 colony/g
≤10 colony/g
≤10 colony/g
Negative
Negative
7%
Slight turbidity
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dispersibility
observed
spots
reference paeoniflorin
4.72mg/g
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Light turbidity is allowed and no
the
fluorescent
corresponding position of
through sieve NO.5 ≤ 15% Determination of
in
Same
corresponding position of
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cannot pass through sieve No.1 and
observed
spots
corresponding position of
Sum of weight of granules that Particle size
fluorescent
corresponding position of
5.5%
water
the
Same
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Description
T=0 (Dec. 19, 2012)
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Months
Quality requirements
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Test items
≤10 colony/g
≤10 colony/g
≤10 colony/g
≤10 colony/g
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Negative
Negative
Negative
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-Escherichia coli
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Figure 1. The thin-layer chromatograms (TLC) of MZRW granules
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1-3: the sample of MZRW granules; 4: negative control sample (without Paeoniae Radix Alba); 5: paeoniflorin reference standard
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Figure 2. Typical chromatograms of assay for MZRW granule
A. Paeoniflorin reference standard ; B. The sample solution of MZRW granule; C. Negative control sample (without Paeoniae Radix Alba)
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(E) Toxicity of MZRW granules
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We have done the acute toxicity test of MZRW granules in our previous study (2). 60 ICR mice weighed 18-22g (half male and female) were randomized into 5 groups. MZRW granules was dissolved with distilled water and administered at 20ml/kg via gavage. Single dose at 75g/kg, 37.5g/kg, 19.25g/kg, 9.625g/kg, 4.8125g/kg, which was equal to 300, 150, 75, 37.5, 19.75 times of clinical dose, was given. Mice were under daily inspection for 7 days. No death was recorded by the end of the study.
1.
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(F) Study on major active compounds of MZRW granules in rat plasma Major component identification
The aforementioned report is generally an acceptable quality control for a Chinese medicine intervention; however, it does not fully cover the phytochemical feature of
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MZRW granules. A comprehensive chemical profile of poly-chemical species is sought for establishing higher standard of quality control. LC/MS was chosen for its sensitivity, broad capability and spectral sensitivity (4).
Analysis was performed on UPLC-Q-TOF-MS/MS (Agilent G6520 (USA)). (Supplement 2-Figure 3 & Supplement 2-Figure 4). Around 300 compounds were found in MZRW granules with 80 compounds can be predicted (Supplement 2- Table 5). 41 out of 80 were contributed by (DH), 6 by (HMR), 13 by (BS), 9 by (HP), 12 by (ZS) and
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4 by (KXR).
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Figure 3. LC/MS chromatograms of extraction of MZRW granules
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LC-MS referred to the published article (5).
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This figure showed 3D total ion chromatograms (TIC) of MZRW granules. UPLC-Q-TOF-MS/MS analysis was performed for the compound components, nearly 300 compounds were found. Analytical conditions of
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Figure 4. Ion-current chromatograms obtained in negative-ion mode
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This figure showed ion-current chromatograms of the chemical component extract screened using negative-ion mode. 80 compounds were identified.
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Peak No. RT
Observed mass
Ion species
Molecular
Error
Plant
formula
(mDa)
source
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Table 5. Chemical profile of MZRW granules Identification
0.735
225.0614
[M + HCOO]-
C6 H12 O6
-1.12
KXR
Inositol
2
1.61
169.0133
[M - H]-
C7 H6 O5
-0.93
DH;BS
Gallic acid
3
1.61
331.0659
[M - H]-
C13 H16 O10
1.52
DH
Gallic acid 3-O-β-D-glucopyranoside
4
1.634
125.0253
[M - H]-
C6 H6 O3
0.89
BS
1,2,3-benzenetriol
5
1.984
331.066
[M - H]-
C13 H16 O10
1.09
DH
Gallic acid 4-O-β-D-glucopyranoside
6
2.588
493.1199
[M - H]-
C19 H26 O15
0.1
DH
6-O-galloylsucrose
7
3.871
451.1234
[M - H]-
C21 H24 O11
-1.41
ZS
Hesperetin-7-O-β-D-glucoside
8
4.509
451.1234
[M + HCOO]-
C20 H22 O9
-1.32
DH
Piceatannol 3-O-β-D-glucopyranoside
9
4.661
577.1335
[M - H]-
C30 H26 O12
-1.72
DH
Procyanidin B-1
10
4.852
451.1235
[M - H]-
C21 H24 O11
-2.82
DH
(+)-catechin-5-O-β-D-glucopyranoside
11
4.956
289.071
[M - H]-
C15 H14 O6
-0.87
DH;BS
(+)-catechin
12
4.956
483.0767
[M - H]-
C20 H20 O14
-0.54
DH
1,6-di-O-galloylycerol-β-D-glucopyranoside
13
5.268
417.1387
[M + HCOO]-
C17 H24 O9
-1.46
BS
Paeonin A
14
5.528
456.1511
[M - H]-
C20 H27 N O11 0.47
KXR
Amygdalin
15
5.828
289.0711
[M + HCOO]-
C14 H12 O4
-0.63
DH
Piceatannol
16
5.979
729.1466
[M - H]-
C37 H30 O16
0.61
DH
Procyanidin B-1-3-O-gallate
17
6.131
340.1037
[M + HCOO]-
C14 H17 N O6
-0.13
KXR
Prunasin
18
6.51
609.1829
[M - H]-
C28 H34 O15
7.36
ZS
Neohesperidin
19
6.51
623.2
[M - H]-
C29 H36 O15
1.71
HP
Acteoside
20
6.541
449.1467
[M + HCOO]-
C21 H24 O8
1.23
DH
Desoxyrhaponticin
21
6.541
479.1565
[M - H]-
C23 H28 O11
2.54
BS
Albiflorin
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6.541
525.1636
[M - H]-
C24 H30 O13
2.1
BS
Mudanpioside E
23
6.541
609.1843
[M + HCOO]-
C27 H32 O13
2.54
DH
10R-chrysaloin1-O-β-D-glucopyranoside
24
6.554
119.0503
[M - H]-
C8H8O
0.01
HP
Benzeneacetaldehyde
25
6.744
639.1921
[M + HCOO]-
C28 H34 O14
-0.15
ZS
Poncirin
26
6.962
567.2092
[M - H]-
C27H36O13
0.81
ZS
Citrusin B
27
7.02
269.0448
[M - H]-
C15 H10 O5
-0.73
HMR;ZS
Apigenin
28
7.02
863.2018
[2M - H]-
C21 H20 O10
-0.26
DH
Emodin-8-O-β-D-glucopyranoside
29
7.081
441.0817
[M - H]-
C22 H18 O10
-1.04
DH
(-)-Epicatechin-3-O-gallate
30
7.081
445.077
[M - H]-
C21 H18 O11
-0.48
DH
Rhein-8-O-β-D-glucopyranoside
31
7.142
417.1184
[M - H]-
C21 H22 O9
-5.11
DH
Cassialoin
32
7.172
477.1395
[M - H]-
C23 H26 O11
-0.97
DH
Lindleyin/isolindleyin
33
7.264
525.1606
[M + HCOO]-
C23 H28 O11
-3.17
BS
Paeoniflorin
34
7.35
623.1973
[M - H]-
C29 H36 O15
-1.03
DH
Physcion-8-O-β-D-gentiobioside
35
7.411
631.1659
[M - H]-
C30 H32 O15
1.6
BS
Albiflorin; 4-O-(3,4,5-Trihydroxybenzoyl)
36
7.597
609.1816
[M + HCOO]-
C27 H32 O13
-0.6
DH
Cascaroside C
37
7.668
861.1833
[M - H]-
C42H38O20
1.22
DH
Sennoside A or Sennoside B
38
7.696
579.1717
[M - H]-
C27 H32 O14
0
ZS
Naringin
39
7.935
298.1075
[M - H]-
C17 H17 N O4
-1.65
HMR
N-trans-caffeoyltyramine
40
8.026
355.1022
[M + HCOO]-
C15 H18 O7
-1.55
DH
2-O-cinnamoyl-β-D-glucose
41
8.13
609.1823
[M + HCOO]-
C27 H32 O13
-0.22
DH
Cascaroside D
42
8.13
609.1824
[M - H]-
C28 H34 O15
-0.49
ZS
Hesperidin
43
8.398
189.0546
[M - H]-
C11 H10 O3
-1.06
DH
2,5-dimethyl-7-hydroxychromone
44
8.398
393.1182
[M - H]-
C19 H22 O9
-1.43
DH
Aloesone-7-O-β-D-glucopyranoside
45
8.428
431.0975
[M - H]-
C21 H20 O10
-0.76
DH
Kaempferol-3-O-rhamnoside
46
8.428
525.1595
[M + HCOO]-
C23 H28 O11
-2.09
BS
Mudanpioside I
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ACCEPTED MANUSCRIPT
8.652
593.1917
[M - H]-
C34H30N2O8
-0.62
HMR
Cannabisin A
48
8.667
461.1086
[M + HCOO]-
C21 H20 O9
-0.23
DH
Chrysophaein
49
8.778
595.2073
[M - H]-
C34 H32 N2 O8 -1.36
HMR
Cannabisin B
50
8.877
315.1232
[M - H]-
C18 H20 O5
-0.61
HP
Honokitriol; (7R*,8R*)-form
51
9.615
461.1082
[M - H]-
C22 H22 O11
-0.47
DH
1-O-galloyl-6-O-cinnamoyl-β-D-glucose
52
9.645
639.1922
[M + HCOO]-
C28 H34 O14
-0.94
ZS
Neoponcirin
53
9.798
415.1019
[M - H]-
C21 H20 O9
0.47
DH
Chrysophanol-8-O-β-D-glucopyranoside
54
9.872
847.2097
[M - H]-
C42 H40 O19
4.51
DH
Sennoside C
55
9.949
313.0344
[M - H]-
C16 H10 O7
-1
DH
laccaic acid D
56
10.065 253.0496
[M - H]-
C15 H10 O4
-1.01
DH
Chrysophanol
57
10.065 831.2135
[2M - H]-
C21 H20 O9
-0.43
DH
Chrysophanol-1-O-β-D-glucopyranoside
58
10.385 517.0975
[M - H]-
C24 H22 O13
-1.16
DH
Emodin-8-O-(6'-O-malonyl)-glucoside/7-O-glucosyl-6''-malonyl genistein
59
10.39
[M - H]-
C18 H20 O4
-0.86
HP
Magnolignan A
60
10.449 629.1871
[M + HCOO]-
C30 H32 O12
-0.29
BS
6'-O-galloylalbiflorin
61
10.449 629.1871
[M - H]-
C31 H34 O14
-0.37
BS
Mudanpioside B
62
10.613 629.187
[M + HCOO]-
C30 H32 O12
-1.03
BS
Benzoylpaeoniflorin
63
10.707 723.2134
[M - H]-
C33 H40 O18
-0.51
ZS
Melitidin
64
10.745 285.0396
[M + HCOO]-
C14 H8 O4
-0.78
DH
1,8-Dihydroxyanthraquinone
65
10.745 285.0395
[M - H]-
C15 H10 O6
-1.05
DH
Citreorosein
66
10.916 283.0607
[M - H]-
C16 H12 O5
-0.47
DH
Physcion
67
10.916 445.113
[M - H]-
C22 H22 O10
-1.18
DH
Physcion-1-O-β-D-glucopyranoside
68
11.05
241.0858
[M - H]-
C15 H14 O3
-1.28
HP
Magnatriol B
69
11.7
297.0396
[M - H]-
C16 H10 O6
-0.9
DH
6-methyl-rhein
70
12.133 253.0865
[M - H]-
C16 H14 O3
-0.65
HP
Magnaldehyde D
71
12.193 515.1925
[M + HCOO]-
C26H30O8
0.11
ZS
Limonin
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299.1281
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12.245 283.0242
[M - H]-
C15 H8 O6
-0.49
DH
Rhein
73
12.558 269.0813
[M - H]-
C16 H14 O4
-0.66
ZS
Imperatorin
74
12.59
[2M + HCOO]-
C15 H12 O5
-0.68
ZS
Naringenin
75
13.379 269.045
[M - H]-
C15 H10 O5
-0.64
DH
Emodine
76
13.809 265.1243
[M - H]-
C18H18O2
0.81
HP
Honokiol
77
13.991 281.1182
[M - H]-
C18 H18 O3
-0.19
HP
Obovatol
78
14.055 265.1247
[M - H]-
C18 H18 O2
1.25
HP
Magnolol
79
16.084 327.2535
[M + HCOO]-
C18H34O2
-0.64
HMR;KXR Oleic acid
80
22.057 279.223
[M - H]-
C18H32O2
0.08
HMR
2. Quantification of ten active compounds in MZRW granules
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Linoeic acid
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589.1344
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Remission of constipation by ten major components in MZRW granules has been highlighted by recent literature and our pilot study. Quantification of these components, including rhein, emodin, aloe emodin, hesperidin, naringin, paeoniflorin, albiflorin, magnolol, honokiol and amygdalin (Supplement 2-Figure 5), was performed with
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appropriate reference compounds (Supplement 2-Figure 6 and Supplement 2- Table 6).
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Supplement 2-Figure 5. Chemical structures of biologically active components on constipation of MZRW granules
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Amygdalin Aloe emodin O
OH
O
OH
Rhein OH
O
OH
NC HO
CH 2OH CH3
COOH OH
O
O
OH
C 15H 10O5 exact mass: 270.0528
O
OH
OH
HO
OH
O
O
OH
H 3C
O
O H OH
O
O
C23H 28O11 exact mass: 480.1632
C 27H 32O14 Exact mass: 580.1792 OH OH O
O
HO
CH3 OH
O
C28H34O 15 Exact Mass: 610.1898
O
OCH3
honokiol OH
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OH
O
O
O CH3 O
C23H 28 O11 exact mass: 480.1632
magnolol OH HO
OH
O
HO
O
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Hesperidin HO
OH
O
O
HO
Alibiflorin
OH
OH
O
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HO
HO
O
O O
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OH
HO
Peaoniflorin
OH
O
HO
OH
OH
Naringin
O
HO
O
OH
C 20H 27 NO 11 Exact mass: 457.1584
C 15 H 18 O6 exact mass: 284.0321
CH 3 HO
OH
HO
O
C 15 H10O5 exact mass: 270.0528
OH
O
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HO
O
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Emodin
C 18H 18O2 Exact Mass: 266.1307
C 18H18 O2 Exact Mass: 266.1307
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Figure 6. Total ion chromatography of 10 mixed standard solution, MZRW solution and SRM chromatography for each compound and I.S.
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MZRW granules
Plant source
Amygdalin
11.03±0.15
KXR
Albiflorin
0.56±0.01
BS
Paeoniflorin
1.3±0.03
BS
Naringin
0.67±0.01
ZS
Hesperidin
12.25±0.13
ZS
Aloe emodin
1.53±0.02
DH
Rhein
18.23±0.10
DH
Emodin
6.15±0.03
DH
Honokiol
6.87±0.05
HP
Magnolol
1.24±0.02
HP
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Active compounds
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Table 6. The content of each compound in MZRW granules (mg/g, Mean± SD, n = 5)
3. The pharmacokinetics assessment of major compounds
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We elaborated the pharmacokinetic features in rats of these ten compounds when they were orally administrated in the form of MZRW granules. The concentration-time (C-T) profile of rhein was appeared with dual peaks, which may be probably due to the replenishment of rehein by metabolites of some similar anthraquinones and anthraquinone derivatives in this formula, and another possibility is because of enterohepatic circulation of these chemicals. The t1/2 of rhein, emodin and aloe emodin were more than 10 h,
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though they were rapidly absorbable (Tmax was less than half an hour). This well interpreted the both quick and long-lasting effects of MZRW. The C-T curve of amygdalin showed that it was detectable in rat plasma within 5min post-dosing; and a shorter Tmax (0.75 h) indicated that amygdalin was rapidly absorbed after oral administration. Elimination of amygdalin from plasma was also relatively fast, with the t1/2 values at approximately 3 h. Probably due to the similarity in chemical structure, albiflorin and paeoniflorin exhibited consistent tendencies in plasma C-T profiles and similar Tmax (both at 0.75 h) after oral administration of MZRW. The t1/2 values of these two compounds were (2.81±1.09) h and (5.90±1.36) h, respectively, suggesting a rapid elimination from plasma of the two compounds. Naringin was absorbed into the body with Tmax at 0.75 h and eliminated with t1/2 near 4 h after oral administration of MZRW. The rapid elimination may be due to the fact that orally administered naringin can be
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quickly metabolized into naringenin and naringenin glucuronide. The plasma concentration and AUC of magnolol were both higher than that of honokiol, while in MZRW the
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amount of magnolol (1.24 mg/g) was much lower than honokiol (6.87 mg/g). This data suggest that magnolol was better absorbed by the gastrointestinal tract than honokiol. Illustrating the pharmacokinetic profile of each compound can foster better understanding of the efficacy of MZRW, and can facilitate clinical study and quality evaluation for development of this formula into even more effective pharmaceuticals (5). Further clinical investigation about its pharmacokinetics in human is necessary in the future.
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References
1. Zhang B, Dong J, Zhou Z. Traditional Chinese Internal Medicine [in Chinese]. Shang Hai Science and Technology Press: Shanghai, 1985.
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2. Cheng CW, Bian ZX, Zhu LX et al. Efficacy of a Chinese herbal proprietary medicine (Hemp Seed Pill) for functional constipation. Am J Gastroenterol 2011;106(1):120-9.
3. Chinese Pharmacopeia Commission. Hemp Seed Pills. In: Pharmacopeia of the People’s Republic of China, Vol. 1, 2010 edn. China Medical Science Publisher: Beijing, 2010, pp 1093.
4. Wang G, Fu H, Ye W et al. Comprehensive characterization of the in vitro and in vivo metabolites of ziyuglycoside I in rat microsome, intestinal flora, excretion specimen and fresh tissues based on LC-Q-TOF/MS. J Pharm Biomed Anal 2016; (128):191-200.
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5. Hu DD, Han QB, Zhong LL et al. Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of
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Ma-Zi-Ren-Wan. J Chromatogr B Analyt Technol Biomed Life Sci 2015; 1000:136-46.
ACCEPTED MANUSCRIPT Supplemental File 3: Packaging and appearance of interventions
a
MZRW
Placebo
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c
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b
(I) Packaging and appearance of MaZiRenWan (MZRW) granules and its placebo a The one-week course package of MZRW granules (a zip lock bag contained 14 sealed aluminum sachets. is placebo granules).
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b The appearance of MZRW granules and placebo granules (Left side is MZRW granules and right side
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c The clear appearance of MZRW granules.
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a
c
Placebo
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Senna
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b
(II) Packaging and appearance of Senna and its placebo
a The 4-week course package of Senna and placebo (a plastic bottle contained 56 tablets). b The appearance of Senna and placebo (Left side is Senna tablets and right side is placebo tablets).
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c The appearance of Senna.
ACCEPTED MANUSCRIPT Supplemental file 4: Statistical analysis Data are presented by mean and standard deviation (SD), 95% confidence interval
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(95% CI) or percentages. Differences among three groups were assessed using one-way Analysis of variance (ANOVA) or Kruskal-Wallis non-parametric test for continuous variables, or using Chi-square test or Fisher’s exact test for categorical variables.
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Analysis of covariance (ANCOVA) was also applied with baseline as covariate if
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needed. Post-hoc pairwise multiple comparisons were made with LSD or Dunnett’s procedure if significant differences were found among three groups. Changes in the eight domains of SF-36 before and after treatment in three groups were calculated and the within-group comparisons were performed using paired t-test or Wilcoxon
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22.0.
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matched-pairs signed rank sum test. Analyses were carried out using SPSS version
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MZRW
Senna
MZRW vs. Senna$
Placebo
χ2
P Value
csbm>=1 than baseline and csbm>=3/wk (%)
MZRW vs. Placebo$
Senna vs. Placebo$
3 Groups
χ2
P Value
χ2
P Value
χ2
P Value
7.774
0.005
5.446
0.020
9.183
0.01
23.918
<0.0001
2.212
0.137
26.356
<0.0001
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Outcome
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Supplement File 8: The proportion of patients with weekly frequency ≥3times CSBM and an increase ≥1 CSBM from baseline
68 (70.1%)
65 (67.0%)
49 (50.5%)
0.215
0.643
Follow up
53 (54.6%)
29 (29.9%)
20 (20.6%)
12.167
<0.0001
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Treatment
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$: Post-hoc pairwise comparison of chi-squared test with applying the Bonferroni corrected alpha level (corrected α = 0.05/3 for three compared pairs)
*: P<0.05/3, vs. Placebo; #: P<0.05/3, vs. Senna.
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Supplemental file 6: Results of other secondary outcomes (A) Comparison of the treatment effect on changes of global symptom assessment among three groups
Global Symptom Assessment
MZRW
Senna
Placebo
MZRW vs. Senna
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MZRW vs.
3 Groups
Senna vs. placebo
placebo
(%)
(n=89)
(n=88)
(n=90)
P-value
P-value P-value
72.7%
45.6%
Treatment
Same
9.0%
26.1%
51.1%
Wk6
Worse
1.1%
1.1%
3.3%
Improved
91.0%
76.1%
51.1%
Treatment
Same
9.0%
21.6%
45.6%
Wk10
Worse
0.0%
2.3%
Improved
70.8%
34.1%
28.9%
Same
27.0%
64.8%
67.8%
Worse
2.2%
1.1%
3.3%
Follow up
0.004
<0.0005
0.001
<0.0005
0.013
<0.0005
0.001
<0.0005
<0.0005
<0.0005
0.505
<0.0005
3.3%
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End of
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89.9%
Within
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Improved
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P-value
Wk18
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MZRW = MaZiRenWan Global symptom improvement was assessed the participant’s subjective feeling of adequate relief of symptoms as “improved”, “same” and “worse”.
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P values were calculated from Fisher’s Exact test.
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Higher proportion of subjects in the MZRW group improved during the treatment period, with significant differences among three groups (P<.0005). Post-hoc
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pairwise multiple comparison results is as follows: MZRW vs. placebo: P<.0005 (Wk6 & 10); MZRW vs. senna: P=.004 (Wk6) and P=.013 (Wk10); senna vs. placebo: P=.001 (Wk 6 & 10). In the follow-up period, over 70% subjects of the MZRW group sustained this improvement, with significant inter-group
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difference (P<.0005). Post-hoc pairwise multiple comparison: MZRW vs. placebo: P<.0005; MZRW vs. senna: P<.0005; senna vs. placebo: P=.505.
(B) Comparison of different domains of SF-36 questionnaires within different groups
ACCEPTED MANUSCRIPT Variables
MZRW (n=97)
Senna (n=97)
Pre-Treatment
Post-Treatment
Mean
Mean
SD
P-value*
SD
Placebo (n=97)
Pre-Treatment
Post-Treatment
Mean
Mean
SD
P-value*
SD
Pre-Treatment
Post-Treatment
Mean
Mean
SD
P-value*
SD
SF-36 18.2
88.2
10.5
0.588
85.6
15.3
86.3
11.1
Role-Physical
81.1
19.1
78.2
18.8
0.118
81.1
18.2
77.8
19.8
Bodily Pain
63.6
27.7
65.3
22.5
0.425
62.3
26.3
60.3
23.1
General Health
50.5
22.7
53.5
18.3
0.021
50.6
21.5
52.0
19.7
Vitality
50.6
14.4
52.9
18.0
0.035
49.2
15.1
51.4
17.6
Social Functioning
75.4
24.2
79.7
19.2
0.024
75.9
23.4
73.3
Role-Emotional
79.6
22.1
75.0
21.6
0.031
78.6
22.2
74.8
Mental Health
59.9
13.9
62.5
14.4
0.162
59.0
14.2
18.5
87.3
10.3
0.681
81.5
18.9
79.5
17.6
0.288
0.310
61.9
26.0
62.4
23.9
0.809
0.350
51.4
21.8
53.9
18.1
0.159
0.012
50.9
13.0
52.9
14.9
0.060
22.4
0.171
74.9
23.8
75.6
20.6
0.747
21.3
0.044
79.4
22.8
75.1
21.5
0.033
59.3
0.824
59.3
12.8
60.0
12.6
0.540
14.9
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#P value was from an analysis of covariance (ANCOVA) with baseline as covariate
86.6
0.039
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*P value was from an analysis of Paired-sample T-test
0.694
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87.1
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Physical Functioning
The eight domains of SF-36 questionnaires (physical functioning, role–physical, bodily pain, general health perceptions, vitality, social functioning,
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role-emotional, and mental health) were compared before and after treatments in three groups. We found that three groups had significantly decreased on the
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scores of role-emotional after treatment (P<.05). The MZRW group showed significant improvement on general health, vitality and social functioning (P<.05). The senna group also showed significant improvement on vitality, and worse on role-physical (P<.05). As life satisfaction is associated with number of doctor visits, it may be the reason the scores of role-emotional and role-physical after treatment.
ACCEPTED MANUSCRIPT
MZRW (n = 97)
Senna (n = 97)
Placebo (n=97)
Bloating and passing of gas Mild diarrhea Headache Dizziness
2 2 1 1
0 0 1 0
0 0 1 0
Abdominal pain Vomiting
3 0
3 1
0
1
Worsening of straining Worsening of sleeping quality
0
4
0
0
Discomfort of stomach
2
a
2
0 0 1
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Stomach ache
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Event
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Supplement file 7 Adverse events occurred in three group a
1 3
5
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There were no significant differences in any adverse event among three groups, as examined with Fisher Exact test.
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Item Section / Tropic
Reported Standard CONSORT Checklist Item
abstract 1a
Page Number
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No. Title,
Identification as a randomized trial in the title
Statement of whether the trial targets a TCM Pattern, a Western Page 1
and keywords
medicine–defined disease, or a Western medicine–defined disease with a
Structured summary of trial design, methods, results, and conclusions
Illustration the name and form of TCM formula used, and the TCM
1c
Page 4
Pattern applied, if applicable.
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(for specific guidance, see CONSORT for abstracts)
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specific TCM Pattern, if applicable 1b
Determination of appropriate keywords, including “Chinese herbal Page 5 medicine formula” and “randomized control trial”
2a
Scientific background and explanation of rationale
2b
Specific objectives or hypotheses
Methods 3a
3b
Pattern or western medicine disease with a specific TCM Pattern.
Description of trial design (such as parallel, factorial), including allocation ratio
Page 6-7
Whether the TCM formula targeting on western medicine disease, TCM Page 7
Page 7, 11
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Trial design
Statement with biomedical science approaches and/or TCM approaches
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objectives
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Introduction Background and
Important changes to methods after trial commencement (such as
N/A
eligibility criteria), with reasons Participants
4a
on
Extension for TCM Formulas
Eligibility criteria for participants
Statement of whether the formula targets a Western medicine–defined Page 8 disease, a TCM Pattern, or a Western medicine–defined disease with a specific TCM Pattern
ACCEPTED MANUSCRIPT
Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Page 7-8
5
The interventions for each group with sufficient details to allow replication, including how and when they were actually administered
Description(s) for different types of formulas should include the following:
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Settings and locations where the data were collected
5a. For fixed CHM formulas
(Page 6-7, 9)
2. Name, source, processing method, and dosage of each medical
(Page 9-10,
substance. Names of substances should be presented in at least 2
Supplemental
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1. Name, source, and dosage form (e.g., decoctions, granules, powders)
substances used should be specified.
& 2)
3. Authentication method of each ingredient and how, when, where, and
(Supplemental
by whom it was conducted; statement of whether any voucher specimen
File 2 P.6,
was retained, and if so, where they were kept and whether they are
Supplemental
accessible
File 2-Table 1)
4. Principles, rationale, and interpretation of forming the formula
(Page 6-7, 9)
5. Reference(s) as to the efficacy of the formula, if any
(Page 6-7)
6. Pharmacologic study results of the formula, if any
(Supplemental
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languages: Chinese (Pinyin), Latin, or English. Names of the parts of the File 2-Table 1
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Interventions
4b
File 2 P.16-27) (Supplemental 7. Production method of the formula, if any
File 2 P.8) (Page 10,
8. Quality control of each ingredient and of the product of the formula, if Supplemental any. This would include any quantitative and/or qualitative testing method(s); when, where, how, and by whom these tests were conducted; whether the original data and samples were kept, and, if so, whether they are accessible.
File 2 P.9-15)
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
9. Safety assessment of the formula, including tests for heavy metals and
(Supplemental
toxic elements, pesticide residues, microbial limit, and acute/chronic
File 2 P.9-12,
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toxicity, if any. If yes, it should be stated when, where, how, and by whom 16) these tests were conducted; if the original data and samples were kept; and, if so, whether they are accessible.
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10. Dosage of the formula, and how the dosage was determined
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11. Administration route (e.g., oral, external)
5b. For individualized CHM formulas
(Page 9-10) (Page 9-10)
(N/A)
1. See recommendations 5a 1–11 2. Additional information: how, when, and by whom the formula was
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modified
5c. For patent proprietary CHM formulas 1. Reference to publicly available materials, such as pharmacopeia, for the details about the composition, dosage, efficacy, safety, and quality control of the formula 2. Illustration of the details of the formula, namely 1) the proprietary product name (i.e., brand name), 2) name of manufacturer, 3) lot number, 4) production date and expiry date, 5) name and percentage of added materials, and 6) whether any additional quality control measures were conducted 3. Statement of whether the patent proprietary formula used in the trial is for a condition that is identical to the publicly available reference
5d. Control groups
(N/A)
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Placebo control (Page 9-10)
2. Description of the similarity of placebo with the intervention (e.g.,
(Page 10,
color, smell, taste, appearance, packaging)
Supplemental
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1. Name and amount of each ingredient
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3. Quality control and safety assessment, if any
(Page 9-10)
5. Production information: where, when, how, and by whom the placebo
(Page 10,
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Completely defined prespecified primary and secondary outcome
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6a
(Page 10)
4. Administration route, regimen, and dosage
was produced
Outcomes
File 3)
Supplemental File 2 P.9)
Active control
(N/A)
1. If a CHM formula was used, see recommendations 5a–5c
(Page 9-10,
2. If a chemical drug was used, see item 5 of the CONSORT Statement
Additional File 2)
Illustration of outcome measures with Pattern in detail
Page 10-11
measures, including how and when they were assessed
Any changes to trial outcomes after the trial commenced, with reasons
N/A
7a
How sample size was determined
Page 12
7b
When applicable, explanation of any interim analyses and stopping
N/A
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Sample size
6b
guidelines Randomization
8a
Method used to generate the random allocation sequence
Page 11
8b
Type of randomization; details of any restriction (such as blocking and
Page 11
Sequence
ACCEPTED MANUSCRIPT
Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
9
Mechanism used to implement the random allocation sequence (such as
concealment
sequentially numbered containers), describing any steps taken to
mechanism
conceal the sequence until interventions were assigned 10
Blinding
participants, and who assigned participants to interventions 11a
If done, who was blinded after assignment to interventions (for example,
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Implementation
Who generated the random allocation sequence, who enrolled
Page 11
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Allocation
block size)
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generation
Page 11
Page 11
participants, care providers, those assessing outcomes) and how
12a
methods
Statistical methods used to compare groups for primary and secondary outcomes
12b
Methods for additional analyses, such as subgroup analyses and adjusted
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analyses Results Participant flow
13a
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Statistical
If relevant, description of the similarity of interventions
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11b
Page 10, Supplemental File 3 Page 12-13
N/A
For each group, the numbers of participants who were randomly
Page 13
(a diagram is
assigned, received intended treatment, and were analyzed for the
Figure 1
strongly
primary outcome
recommended) 13b
For each group, losses and exclusions after randomization, together with
Page 13
reasons
Figure 1
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Dates defining the periods of recruitment and follow-up
Page 13
14b
Why the trial ended or was stopped
N/A
15
A table showing baseline demographic and clinical characteristics for each group
16
analyzed Outcomes and
For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups
17a
estimation
M AN U
Numbers
For each primary and secondary outcome, results for each group, and the
estimated effect size and its precision (such as 95% confidence interval) 17b
For binary outcomes, presentation of both absolute and relative effect
18
Harms
Results of any other analyses performed, including subgroup analyses
Page 13 Figure 1 Page 13-16 Table 2
Page 13-16 Table 2
and adjusted analyses, distinguishing prespecified from exploratory 19
All important harms or unintended effects in each group (for specific (There is no extension for this item)
Discussion 20
Page 16 Supplemental
AC C
guidance see CONSORT for harms)
Limitations
Table 1
N/A
EP
analyses
TE D
sizes is recommended Ancillary
RI PT
Baseline data
14a
SC
Recruitment
File 6
Trial limitations; addressing sources of potential bias; imprecision; and,
Page 17
if relevant, multiplicity of analyses Generalizability
21
Generalizability (external validity, applicability) of the trial findings
Discussion of how the formula works on different TCM Patterns or diseases
Page 17
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Supplemental File 6: CONSORT – Chinese Herbal Medicine Formulas Checklist
Interpretation
22
Interpretation consistent with results, balancing benefits and harms, and Interpretation with TCM theory and Pattern is encouraged.
Page 17
RI PT
considering other relevant evidence Other
23
Registration number and name of trial registry
Protocol
24
Where the full trial protocol can be accessed, if available
Sources of funding and other support (such as supply of drugs), role of
TE D
funders
EP
25
AC C
Funding
M AN U
Registration
SC
information
Page 8 Supplemental File 1: Protocol Page 2