The effect of Mullein capsule on uterine leiomyomas volume and the amount of menstrual bleeding: A randomized controlled trial

Journal Pre-proof The effect of Mullein capsule on uterine leiomyomas volume and the amount of menstrual bleeding: A randomized controlled trial Nafiseh Ghassab-Abdollahi, Behnaz Sadeghzade Oskouei, Parina Asgharian, Amirreza Jahanshahi, Azizeh Farshbaf-Khalili

PII:

S2210-8033(19)30064-8

DOI:

https://doi.org/10.1016/j.hermed.2019.100317

Reference:

HERMED 100317

To appear in:

Journal of Herbal Medicine

Received Date:

22 August 2018

Revised Date:

5 November 2019

Accepted Date:

5 November 2019

Please cite this article as: Ghassab-Abdollahi N, Sadeghzade Oskouei B, Asgharian P, Jahanshahi A, Farshbaf-Khalili A, The effect of Mullein capsule on uterine leiomyomas volume and the amount of menstrual bleeding: A randomized controlled trial, Journal of Herbal Medicine (2019), doi: https://doi.org/10.1016/j.hermed.2019.100317

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The effect of Mullein capsule on uterine leiomyomas volume and the amount of menstrual bleeding: A randomized controlled trial

Nafiseh Ghassab-Abdollahia, Behnaz Sadeghzade Oskoueib, Parina Asgharianc, Amirreza Jahanshahid, Azizeh Farshbaf‑ Khalili*e a

PhD student, Nutrition Research Centre, Department of Public Health and Management,

b

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School of Public Health, Tabriz University of Medical sciences, Tabriz, Iran Assistant Professor, Department of Midwifery Faculty of Nursing and Midwifery, Tabriz

University of Medical Sciences, Tabriz, Iran.

Assistant Professor of Pharmacognosy, Drug Applied Research Center, Faculty of Pharmacy,

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Tabriz University of Medical Sciences, Tabriz, Iran

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c

Assistant professor of radiology, Tabriz University of Medical Sciences, Tabriz, Iran.

e

Assistant Professor, Aging Research Institute, Physical medicine and rehabilitation Research

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d

*Corresponding

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Centre, Tabriz University of Medical Sciences, Tabriz, Iran. author:

Azizeh

Farshbaf‑ Khalili,

Tel.:

+ 984134796770,

Mobile:

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+ 989144023216, Fax: + 984134796969, E-mail: [email protected]

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Conflict of interest:

Authors state no conflict of interest.

This research has been supported financially by Tabriz University of Medical Sciences (grant number: 1395:1131)

Abstract Myomas are the most common benign tumors of the uterus. The role of inflammatory processes in myoma growth has led to the selection of Mullein (Verbascum Songaricum) for the treatment of myoma due to its potent anti-inflammatory properties. This study was conducted to evaluate the effect of oral Mullein capsule on asymptomatic uterine leiomyomas volume and the amount

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of menstrual bleeding. In this two-parallel arm triple-blind, randomized, controlled trial, 50 women were selected among women referred to the clinics affiliated to Tabriz University of Medical Sciences aged 18-50 years old, having at least one asymptomatic myoma greater than

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2.5-cm, in Iran over 2017-2018. The participants were randomized into two groups. The first group (n=25) received 400 mg of Mullein powder capsules and the second group (n=25)

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received placebo capsules twice a day for 8 weeks. The mean volume of the largest myoma indicated no statistically significant difference (adjusted mean difference (aMD) = -7.6 ml,

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95% CI: -22.0 to 6.6). Within-group analysis of the largest myoma volume showed a significant reduction in the Mullein group (MD = -6.8 ml, 95% CI: -13.0 to -6.3(. There was no significant

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difference in the first (aMD=1.3, 95% CI: -24.3 to 27.0) and second (aMD =7.3, 95% CI: -12.6 to 27 .2) cycles regarding the amount of menstrual bleeding. The results of this study showed

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that treatment with 800 mg of oral Mullein capsules per day for 2 months did not influence the myoma size, and amount of menstrual bleeding, although there was a significant reduction in

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the size of myoma in the Mullein group. Keywords: Mullein; Verbascum; leiomyoma; Myoma;Fibroid; Uterin bleeding Abbreviations: aMD

adjusted mean difference

BMI

Body mass index

COX-2

cyclooxygenase 2

IL-6

interleukin 6

IUD

intrauterine device

GnRH

Gonadotropin-releasing hormones

MD

mean difference

PBLAC

pictorial blood loss assessment chart

SD

standard deviation

TNF-α

tumor necrosis factor alpha

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Analysis of covariance

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ANCOVA

1. Introduction Uterine leiomyomas or myoma are masses originated from uterine smooth muscle. Myomas are the most common benign tumors of the uterus, that up to 80% of all women are affected by them in their reproductive age. (Aleksandrovych et al., 2015). Uterine myomas often cause no symptom, and many women discover them accidentally (Divakar, 2008). Nevertheless, they may create complications such as abnormal uterine

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bleeding, pain due to pressure on nerves or body organs, and reproductive disorders (Aleksandrovych et al., 2015). Evidence suggests that most procedures of hysterectomy are performed due to uterine myomas (Divakar, 2008).

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Although GnRH agonists are among the medical treatments for myomas, long-term use of them may produce menopause-like symptoms and bone mass loss (Moroni & Vieira, 2014). In

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addition, these agents may fail in permanent regression of myoma size, thereby lead to the

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increase in the chance of regrowth, which may even exceed the original size, following the withdrawal. Despite the beneficial effects of other hormonal agents on myomas, they result in

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fertility incompatibility through causing changes in estrogen and progesterone production or function (Sankaran & Manyonda, 2008). Although hysterectomy, myomectomy, and uterine artery embolization are common surgical treatments for myomas, they are invasive and have

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several complications (Evans & Brunsell, 2007). Following the increasing awareness regarding

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the uterine myomas biology, there is a reinforcing tendency towards discovering non-hormonal therapeutic methods. These methods should lead to myoma size regression, be cost-effective, cause least complications and not interfere with ovulation cycles (Sankaran & Manyonda, 2008). Herbal medicine, as a traditional medicine, is used by most people worldwide due to its accessibility, cost-effectiveness and its being highly accepted by culture (WHO, 2013).

Although several studies have been conducted on the effect of medicinal herbs on uterine myoma shrinkage, they lack quality. A Cochrane review study suggested that well-designed, double-blind, randomized controlled trials are necessary for evaluation of the effect of medicinal herbs on uterine myoma (Liu et al., 2013). Mullein or Verbascum Songaricum is an herbaceous plant of the Scrophulariacac family, known as Boosir in the Persian literature. The plant has long been used to treat respiratory diseases, asthma, diarrhea, tuberculosis, pharyngitis, and wounds (Mirhaidar, 2005). New

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studies have highlighted several therapeutic benefits for Mullein, including anthelmintic, antimicrobial, anti-inflammatory, anti-nociceptive, anti-cell proliferative and anti-cancer properties (Riaz et al., 2013). Some references have recommended the use of leaves and flowers

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of this plant for treatment of uterine myomas (Hobbs & Keville, 2007; Kathi & Robert, 2002).

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Verbascoside extracted from Mullein has shown anti-inflammatory effects in vitro. It is a substance with beneficial antioxidant and anti-cancer effects (Alipieva et al., 2014). The anti-

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inflammatory and analgesic activities of Mullein are attributed to the presence of substances such as aucubin and saponin (Kupeli et al., 2007). Saponins found in Mullein, are compounds

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with numerous biological properties, including anti-tumor properties (Riaz et al., 2013). Aucubin can reduce the cyclooxygenase activity by inhibiting the production of tumor necrosis

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factor alpha (TNF-α) and interleukin 6 (IL-6) (Jeong et al., 2002). Flavonoids in Mullein have antioxidant and anti-inflammatory activities (Riaz et al., 2013). The results of experimental

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studies on the myoma cell line have shown that flavonoids derived from medicinal herbs can induce apoptosis and inhibit cell proliferation (Sparic et al., 2016). Inflammation mechanism is a factor influencing the myoma size. Serum levels of preinflammatory cytokine IL-1α in women with uterine myoma were significantly higher than in healthy women (Sikorski et al., 2001). In addition, celecoxib, as a cyclooxygenase 2 (COX-2

(inhibitor, reduces the myoma cell proliferation in vitro by reducing the expression of IL-6 and TNF-α genes (Park et al., 2014). Despite the lack of clinical trials evaluating the effect of oral Mullein for any disease, several pharmaceutical companies are currently producing oral capsules ("PureControl Supplements", 2018) and Tablets ("Common Sense," 2018) of Mullein currently. Although the treatment of asymptomatic uterine myomas is rarely recommended, the possibility regarding the regression

who tend to have a pregnancy in the future (Divakar, 2008).

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of symptoms makes the use of pre-emptive measures reasonable, especially in young women

Considering the anti-inflammatory and anti-tumor effects of Mullein (Riaz et al., 2013), and

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the interference of inflammatory processes in the uterine myoma etiology (Protic et al., 2016), this study was carried out for the first time to evaluate the effect of oral Mullein capsule on the

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asymptomatic myoma volume and menstrual bleeding.

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2. Methods 2.1. Study design and participants

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This two-parallel arm, triple-blind, randomized, and controlled clinical trial, in which the participants, researcher, and outcome assessor (radiologist) were blinded, was conducted on

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women with asymptomatic myomas in Tabriz, Iran. Inclusion criteria were women of

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reproductive age (18-50 years old) with an asymptomatic myoma (without menorrhagia and pain), having at least one myoma greater than 2.5-cm confirmed by ultrasound, normal menstrual cycle (21-35 days), and literacy in terms of reading and writing skills, who were consented to participate in the study. Exclusion criteria were pregnancy and breastfeeding, the possibility of having a pregnancy in the next 3 months, uterine myoma greater than 12 cm, myomatous uterine size equal to or greater than 20 weeks of gestation, anemia requiring drug

therapy, history of chronic disease, concomitant use of drugs or other medicinal herbs, history of myomectomy, consumption of any hormonal agents over the past 3 months, hormonal IUD, severe allergy, and celiac disease. All participants were advised to use a barrier method of contraception during the study period and avoid the use of hormonal methods during the intervention. Since the effect of Mullein capsules on the uterine myoma was evaluated for the first time, women with asymptomatic myomas were only enrolled in the study in order to observe ethical considerations.

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The primary outcomes of this study were the myoma size at 8 weeks after treatment and the menstrual blood loss at 4 and 8 weeks after intervention. Secondary outcomes of this study included uterine size and hemoglobin and hematocrit levels at 8 weeks after treatment, as well

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as the complication frequency and patient satisfaction with the medicine.

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The total sample size estimation was equal to 50 patients using G*POWER (version 3.1.2). According to the study by Sayyah-Melli et al. it was found that the myoma size was variable

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and considering m1=376.53 m2=338.88 (by taking into account the post-intervention reduction in the myoma size by 10%), Sd1=Sd2=40.46, and power of 90%, n was obtained as 21.

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Regarding the possible loss of 20%, the estimated sample size for each group was equal to 25. Due to the lack of study regarding menstrual bleeding in women with asymptomatic myoma,

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the bleeding rates of 10 participants were piloted and the sample size was obtained accordingly. Considering m1=58.8 m2=47.04 (by taking into account the post-intervention bleeding

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reduction by 20%), Sd1=Sd2=11.6, and power of 90%, n was obtained as 18. Since the majority of studies on the effect of intervention on women with menorrhagia have considered a greater bleeding reduction (30-40%), the current study considered slighter reduction in menstruation bleeding (i.e. 20%), due to the effect of intervention on normal menstrual bleeding. 2.2. Sampling

Sampling was carried out over 2017-2018 after obtaining the approval of the Ethics Committee of Tabriz University of Medical Sciences under the code IR.TBZMED.REC.1395.1131 and its registration in the Clinical Trial Registration Center of Iran (available at: http://www.irct.ir) under the code IRCT2017012827311N2. Sampling sites included the clinics affiliated to Tabriz University of Medical Sciences. The Researcher attended the clinics, identified the eligible women based on ultrasound-confirmed myoma, explained the study and its objectives to them, and invited them to participate in the study. A written informed consent was signed by eligible

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participants who consented to participate. These women had been referred to the clinic for various reasons, including annual checkups (such as Pap smears), receiving contraceptive methods, or any cause (such as vaginitis or cervicitis) other than abnormal uterine bleeding,

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and were diagnosed with uterine myoma incidentally as confirmed in the ultrasound. Before the intervention, a control menstrual cycle was considered to measure menstrual bleeding.

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Participants with a Higham score of higher than 100 were excluded. Blood sampling was

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carried out after completion of the control menstrual cycle and Higham’s chart. This assessment is helpful in diagnosing patients with anemia to be excluded from the intervention. Then, an initial ultrasound was performed on participants by a radiologist, using only a single

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device.

2.3. Randomization and intervention

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After the completion of control menstrual cycle, blood tests, and ultrasound, the eligible

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patients were randomized into one of two groups. The first group received 400 mg of Mullein powder capsules and the placebo group received 400 mg of flour-containing capsules. The appearance of the placebo capsules resembled the Mullein powder capsules exactly. The random allocation sequence was determined using Random Allocation Software (RAS) based on a randomized block design with blocks of size 4 and 6 and a ratio of 1:1. Allocation concealment was carried out by concealing the capsules in sealed opaque envelopes numbered

sequentially from 1 to 50. The pockets were prepared and numbered by a person who was not involved in data collection and analysis. The intervention began after the completion of the control menstrual cycle, and the envelopes containing Mullein or placebo capsules were given to the participants according to enrollment from number 1 to 50, respectively. Two daily doses of 400 mg of Mullein or placebo capsules were given twice a day for 8 weeks. The participants were instructed on the medicine consumption. In order to ensure the use of medication, a sheet containing a table representing the week days was given to each participant and she was asked

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to fill out the table correspondingly after taking the capsule. A weekly SMS was sent to the participants to emphasize on the use of capsules. In this study, the participants, the researcher, and the outcome assessor (radiologist) were blinded to participant groups.

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2.4. Preparation of Mullein and placebo capsules

Verbascum Songaricum was collected and identified by an herbalist (Mr. Attaran) from the

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mountainous areas of the East Azarbaijan (Esparakhoon region) in September 2017 and was

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stored at the herbarium of the Faculty of Pharmacy with the herbarium number 1267. The aerial parts of the plants were rinsed and dried, and then were ground in to powder by an electric mill. In the next stage, the 400 mg of Mullein powder capsules were prepared by the Tabriz Faculty

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of Pharmacy. The participants were instructed to take one capsule every 12 hours along with a full glass of water or with food. Since this was the first clinical trial that administered oral

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Mullein and there was no similar clinical trial, a daily dose of 850 mg of capsules was estimated

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for humans based on a study by Tatli et al. (Tatli et al., 2008) study, which investigated the anti-inflammatory effects of 200 mg/kg of mullein, on mice. Regarding the total dose and treatment course of various commercial Mullein products (capsule and tablet), used for pulmonary diseases as a daily supplement ("Christopher's Mullein Leaves," 2018 ; "Common Sense," 2018), the treatment courses of 8 weeks with a daily dose of 800 mg of capsules seemed reasonable. Since Mullein was used orally, Mullein and placebo capsule samples were selected

randomly and assessed in terms of plausible microbial and fungal contamination caused by clostridium difficile, bacillus, fungus, and yeast tests. In order to standardize the samples, the total phenol test was performed and the results were obtained based on the phenol content in 1g of the Mullein powder and gallic acid. The placebo capsules were prepared in the Tabriz University of Pharmacy in the same shape, color, and weight of the Mullein capsules. There was no difference in the instructions of Mullein and placebo consumption.

2.5.1. Socio-economic and obstetric characteristics

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2.5. Data collection

In this study, a self-reporting questionnaire was used to collect socio-economic and obstetric

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characteristics of the participants. The validity of this questionnaire was confirmed by 10

the eligible participants.

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2.5.2. Hemoglobin and hematocrit levels

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faculty members. This questionnaire was filled out at the end of the control menstrual cycle by

Blood sampling was performed to compare the mean hemoglobin and hematocrit levels before and eight weeks after the intervention. All tests were carried out by a certain laboratory

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technician and device using flow cytometry. In order to ensure reliability, five samples with two different names were sent to the laboratory and their correlation was studied. The obtained

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correlation coefficient, 0.97, confirmed its reliability.

2.5.3. Assessment of myoma and uterine volume Abdominal ultrasound was performed to measure the myoma and uterus volume using a Samsung CA1-7A ultrasound probe. All ultrasound tests were taken using a single machine and results were analyzed by a radiologist. The machine was calibrated before the intervention at 6-month intervals. The measurement of the myoma dimensions (cm3) was carried out

through measuring three dimensions of myoma (length, width, height), using the ellipse volume measurement formula (0.532 × length × width × height) (Roshdy et al., 2013). In the case of multiple myomas, the size of the largest one was calculated. The uterus size was measured on three planes and its total volume was calculated. Ultrasound assessment was performed at baseline and eight weeks after intervention. 2.5.4. Assessment of menstrual blood loss The Higham’s Pictorial Blood Loss Assessment Chart (PBLAC) or the visual evaluation of

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menstrual bleeding was used to measure blood loss (Higham, O'Brien, & Shaw, 1990). PBLAC is a general visual scoring system that works by recording a count of sanitary pads or tampons used and its degree of soaking, along with the emergence of blood clots. Scores of 100 or more

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are equal to a menstrual blood loss of 80 ml or more (El-Nashar et al., 2015). In this study, menstrual bleeding was evaluated during three cycles: first, before the intervention in a control

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menstrual cycle; second, during the first menstrual cycles after the intervention; and third,

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during the second menstrual cycles after the intervention. Since this study investigated only asymptomatic myomas, women with Higham score of more than 100 were not included. The participants were instructed to complete the PBLAC. In order to homogenize research

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menstrual cycles.

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conditions, a specific type of pad was purchased and given to the participants for three

2.5.5. Side effects and satisfaction of the medicine In this study, the frequency of side effects was assessed using a [patient-rated] side effect questionnaire. Six possible side effects including headache, nausea, diarrhea, drowsiness, visual disturbances, and vomiting were addressed in this questionnaire. The questionnaire was given at the beginning of the intervention to the participants along with the medicines and they

were asked to rate the side effects, ranging from any, very low, low, moderate, high, and very high. Any other side effect was also written down by participants in the questionnaire. The satisfaction inventory was administered at the end of the intervention to measure participant satisfaction of the medicine on five-point scale: completely satisfied, satisfied, neutral, dissatisfied, and completely dissatisfied. 2.6. Statistical analysis The data obtained in the study was introduced into SPSS/Ver 23 and was analyzed statistically

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based on the objectives, at the significance level of 0.05. Qualitative data was summarized and reported using frequency and percentage, and quantitative data was summarized and reported using mean (standard deviation). The normality of quantitative data was investigated by groups

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using the Kolmogorov–Smirnov test. The chi-square test, chi-square test for trend, Fisher’s exact test, and independent t-test were applied to demographic, anthropometric, and obstetric

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variables to evaluate homogeneity of groups. Independent t-test was applied to compare

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continuous data with normal distribution at baseline. The analysis of covariance was used to compare the effect of the intervention by adjusting the baseline data and potential confounding data. The between-group comparisons were carried out regarding Higham scores using the

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independent t-test before the intervention, and repeated measure ANOVA was used after the intervention at different measurement times during the first and second menstrual cycles

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adjusted for baseline score. The between-group comparisons were conducted regarding

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hemoglobin level using the independent t-test before the intervention and the analysis of covariance was used after the intervention by adjusting the baseline values. The paired-sample t-test was used for within group comparison of myoma and uterus size. The confounding variables, such as age, parity, and obesity (BMI), were included in the demographic, anthropometric, midwifery questionnaire. Possible heterogeneities were adjusted using the statistical methods. The analysis was based on the intention-to-treat approach.

3. Results 3.1. Study population Participants were enrolled from May 2017 to May 2018 and followed-up. Among women referred to the clinic, 89 patients were evaluated for eligibility, out of which 50 patients were included in the study and randomized in one of two equal-sized groups, namely Mullein and placebo. However, three placebo patients discontinued consuming allocated capsules during the study; two of them did so because of unwillingness and one of them due to stomach pain.

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However, their outcomes were measured and considered in the final analysis (Figure 1). Due to unwillingness, blood samples were not taken from two of these patients after treatment.

There was no significant between-group difference in demographic and obstetric

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characteristics at the baseline (Table 1). The mean standard deviation (SD) age of participants was equal to 39.8±5.1 years old. Nearly half (46%) of the women had high school education.

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More than three quarters (80%) of women were housewives. Half (54%) of the participants

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reported that their expenses exceeded their incomes. Less than half (42%) of the participants were overweight (BMI= 25-29.9 kg/m2). Three quarters (76%) of the participants were married and approximately two-thirds (63%) of them used coitus interruptus as a birth control method.

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More than three quarters (82%) of myomas were intramural. All of the quantitative data in this study had normal distribution.

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3.2. The effect of Mullein on myoma and uterine volume

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Before the intervention, the mean standard deviation (SD) volume of the largest myoma was equal to 94.1 (107.7) ml in the Mullein group and 126.2 (129.1) ml in the placebo group, indicating no statistically significant difference (p=0.553). The mean (SD) volume of the uterine before the intervention was equal to 194.0 (107.5) ml in the Mullein group and 277.2 (179.5) ml in the placebo group, indicating no statistically significant difference (p=0.175).

After the intervention, the mean (SD) volume of the largest myoma adjusted for baseline reduced from 94.1 (107.7) ml to 87.2 (106.0) ml in the Mullein group and increased from 126.2 (129.1) ml to 128.2 (144.2) ml in the placebo group, indicating no statistically significant difference (adjusted mean difference (aMD) = -7.6 ml, 95% CI: -22.0 to 6.6). Within-group analysis of the largest myoma volume showed a significant reduction in the Mullein group (MD = -6.8 ml, 95% CI: -13.0 to -6.3(, but it showed no significant difference in the placebo group (MD= 2.0 ml, 95% CI: -11.4 to 15.6).

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The mean (SD) uterine volume increased from 194.0 (107.5) ml to 205.0 (105.1) ml after the intervention in the Mullein group and from 277.2 (179.5) ml to 280.8 (190.5) ml in the placebo group, which was not significantly different (aMD= -7.6 ml, 95% CI: -36.1 to 20.9). Within-

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group analysis showed no significant difference in the uterus volume in the Mullein (MD= 11.0 ml, 95% CI -5.8 to 27.8) and placebo (MD= 3.6 ml, 95% CI -17.8 to 25.1) groups (Table 2).

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The mean (SD) number of myomas before intervention was equal to 2.5 (1.3) in the Mullein

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group and 2.2 (1.3) in the placebo group, which was changed after the intervention to 2.4 (1.2) and 2.2 (1.2), respectively, indicating no statistically significant difference (Table 2). 3.3. The effect of Mullein on menstrual blood loss

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The mean (SD) higham score before the intervention was equal to 50.6 (20.1) in the Mullein group and 58.8 (26.5) in the placebo group, indicating no statistically significant difference

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(p=0.056). There was no significant between-group difference during the two-month

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intervention period in the first (aMD=1.3, 95% CI: -24.3 to 27.0) and second (aMD =7.3, 95% CI: -12.6 to 27.2) treatment cycles adjusted for baseline. However, the within-group analysis showed a significant increase in menstrual bleeding of the first cycle in the Mullein group after the intervention (MD =17.3, 95% CI 1.4 to 33.1) (Figure 2) (Table 3). The mean (SD) length of menstrual cycles before and after the intervention was equal to 27.6 (3.8) days in the Mullein group and 29.0 (3.1) days in the placebo group, indicating no

statistically significant difference (p=0.549). By controlling the values before intervention, the length of the menstrual cycle did not show a significant difference during the two-month treatment in the both groups (p=0.781) (Table 3). 3.4. The effect of Mullein on hemoglobin and hematocrit levels The mean (SD) hemoglobin levels before intervention was equal to 13.2 (0.9) g/dl in the Mullein group and 13.2 (1.0) g/dl in the placebo group, indicating no statistically significant difference (p=0.630). These values increased to 13.3 (1.1) g/dl in the Mullein group and

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remained at 13.2 (1.0) g/dl in the placebo group after the intervention, indicating no statistically significant difference (aMD=-0.04 g/dl, 95% CI: -0.5 to 0.4). There was no significant difference in hematocrit values between the two groups (aMD=-0.4 %, 95% CI: -1.7 to 0.7)

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(Table 4). 3.5. The satisfaction of the medicine

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Six persons (25%) in the Mullein group and 4 persons (16%) in the placebo group were totally

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satisfied with the drug. Eight participants (33%) in the Mullein group and 7 participants (29%) in the placebo group were satisfied with the drug. Seven (29%) participants in the Mullein group and 9 participants (37%) in the placebo group were neither satisfied nor dissatisfied with

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the drug. Three participants (12%) in the Mullein group and 2 participants (8%) in the placebo group were dissatisfied with the drug. None of the participants in the Mullein group and 2

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participants (8%) in the placebo group were totally dissatisfied with the drug. There was no

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significant between-group difference in terms of satisfaction with the drug (p=0.281). 3.6. Reported side effects There were no serious side effects during the treatment course. Moderate nausea was reported by one person, moderate and severe drowsiness reported by two persons in the Mullein group, and slight drowsiness reported by one person in the placebo group. Other side effects reported by the participants included fatigue experienced by one person in the placebo group,

dysmenorrhea experienced by two persons in the Mullein group and one person in the placebo group, spotting reported by one person in the Mullein group and one person in the placebo group, irregular bleeding observed by one person in the placebo group, stomach ache reported by two persons in the Mullein group and three persons in the placebo group, and constipation reported by two persons in the Mullein group and two persons in the placebo group. 4. Discussion The efficacy of Mullein on the uterine myoma volume and menstrual bleeding has not

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previously been studied by any clinical trial. This study was the first to examine the effect of oral Mullein capsules on myoma size and menstrual bleeding in women with asymptomatic uterine myoma. In this study, there was no significant difference in the myoma size after two

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months intervention in the two groups of Mullein and placebo adjusted for baseline values, but there was a significant reduction in the myoma size after the intervention in the Mullein group

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compared to the baseline. Menstrual bleeding, uterine size, and hemoglobin and hematocrit

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levels were not significantly different between the two groups, as adjusted for baseline. The role of inflammatory processes in myoma growth (Protic et al., 2016) has led to the selection of Mullein for the treatment of myoma due to its potent anti-inflammatory properties.

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The presence of inflammatory cells in the uterus is a reason for the involvement of inflammatory processes in the myoma pathology. For example, higher numbers of

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macrophages were found inside and in the vicinity of myoma compared to the more distant

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surrounding myometrium. In addition, considering that TNF-α is expressed by adipocytes and macrophages, evidence suggests that the intra- lipoleiomyomas macrophages are positive for TNF-α (Protic et al., 2016). The components extracted from Mullein have anti-inflammatory properties. A study conducted by Akkul et al. showed that saponins and iridoid glycosides extracted from methanolic extract of 100 and 200 mg/kg of Mullein had anti-inflammatory effect on mice. This study showed a reduction in cyclooxygenase activity by Mullein (Akkol

et al., 2007). Iridoid glycosides, such as aucubin and nigroside, reduce the production of nitrite oxide by macrophages and inhibit IL-6 and nitrite oxide (Dimitrova et al., 2012). Zhao et al. separated chemical elements of Mullein’s aerial parts and showed their promising anti proliferative activity with a clear effect on the induction of apoptosis in A549 lung cancer cells (Zhao et al., 2011). In addition to a significant increase in menstrual bleeding observed in the intervention group during the first cycle of treatment in the current study, a slighter increase was also observed in

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menstrual bleeding in the placebo group during the first cycle of treatment. Particularly, there was no significant between-group difference in the hemoglobin and hematocrit levels. A lack of any changes in hemoglobin and hematocrit levels may be due to the selection of women

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with asymptomatic uterine myoma. Since menstrual bleeding is normal in these subjects, perhaps no significant changes can be observed in hemoglobin and hematocrit levels.

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Regarding the lack of any previous clinical trials on the effect of Mullein on menstrual

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bleeding, the results of this study cannot be accurately interpreted. The effect of Mullein on menstrual bleeding has not been mentioned in the traditional medicine literature either. The dosage is a factor influencing the anti-inflammatory properties of Mullein, such that the

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administration of Mullein at 200 mg of dosage amplified the anti-inflammatory effects in animal models; whereas, no significant anti-inflammatory effect was observed following the

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administration of 20 mg of Mullein (Dimitrova et al., 2012). It seems that, non-significant

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effect of mullein on myoma size between two groups in the present study may be related to dose selection. Since this is the first clinical trial in which Mullein was administered orally and there is no prior data regarding its effect on human health, the dosage and duration of treatment were selected conservatively. The selected dose in this study was based on its effects on animal models and oral dose of commercial products. The Christopher Drug Company’s product contains 400 mg of Mullein in the form of 100 capsules to be used as daily supplements

("Christopher's Mullein Leaves," 2018). The Pure Control Supplements Company’s product contains 450 mg of Mullein in the form of 100 capsules ("PureControl Supplements ," 2018). In this study, no serious side effect was observed after the use of Mullein capsules. In addition, the traditional literature has not mentioned any side effect after consumption of Mullein (Duke, 2002; Mirhaidar, 2005). A study carried out by Escobar et al. on cytotoxic activity of methanolic extract, at doses of 100, 300, and 500 mg/kg, induced to the bone marrow of rats showed no toxicity, indicating the safety of this plant (Escobar et al., 2011). In the study by

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Tatli et al., the administration of 200 mg/kg of methanolic extract of Mullein orally did not result in any acute toxicity or gastric damage in mice (Tatli et al., 2008).

There was no statistically significant between-group difference in the degree of patient

tolerated for patients at the dose used in this study.

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satisfaction with the medicine. This indicates that the administration of Mullein capsule is well

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Among the limitations of the study were (i) the lack of investigation on the liver and kidney

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function due to financial constraints, (ii) using the plant powder instead of its extract because of costly extraction process and need for more plants, and (iii) short treatment course. Since this was the first oral administration of this plant to humans and due to the lack of sufficient

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evidence, we did not prolong the treatment course. In a Cochrane review study on herbal products for treatment of myoma, the estimated mean length of phytotherapy was reported to

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be 3 to 6 months (Liu et al., 2013). The small sample size was another limitation, which may

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inhibit the detection of some statistical differences. Evaluation of the effect of administrating oral Mullein capsule in humans for the first time is one of the strengths of this study. So far, Mullein has been used in clinical trials only as a topical cream for reducing the episiotomy pain in primiparous women (Taleb et al., 2016). The other strength of this study is evaluating the effect of pure plant on the treatment of myomas. Most phytotherapy trials on myomas using herbs have evaluated the effects of plant products,

which all consist of several herbs. Even though herbal products have a better effect on the treatment of myomas, the net effect of each plant and its potential side effects cannot be ascertained. The conduction of further studies with larger sample sizes, as well as administration of higher doses of Mullein is recommended. In addition, the use of plant extracts can result in higher efficacy. Laboratory tests, such as liver and kidney function examination, are also recommended for detection of probable hepatic and renal toxicity. This clinical trial can pave

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the way for further studies on the effects of oral Mullein on other diseases and conditions. 5. Conclusion

The results of the present study showed that administration of 800 mg of Mullein capsules

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orally per day for 2 months did not influence the myoma and uterus size, amount of menstrual bleeding and the levels of hemoglobin and hematocrit, although there was a significant

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decrease in the size of myoma in the Mullein group.

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. Acknowledgments

Authors appreciate the assistance of authorities and personnel of the clinic of Al-Zahra Hospital

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in East Azerbaijan Province, Iran and the cooperation of participants. Funding

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This article is based on a master's thesis in Midwifery Department of Nursing and Midwifery

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faculty of Tabriz University of Medical Sciences approved by the Regional Ethics Committee of

Tabriz

University

of

Medical

Sciences

under

the

ethical

code

(IR.TBZMED.REC.1395.1131). Authors would like to thank the assistance and financial support of the Research Deputy of Tabriz University of Medical Sciences. Conflict of interest Authors state no conflict of interest.

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Table 1: Baseline characteristics of participants in the placebo and Mullein group.

24.5 (4.1)

24.1 (5.2)

0.224a

27.1 (5.6) N (%)

27.8 (4.2) N (%)

0.594a

2 (8%) 4 (16%)

2 (8%) 4 (16%)

13 (52%) 6 (24%)

10 (40%) 9 (36%)

22 (88%) 3 (12%)

18 (72%) 7 (28%)

0 (0%) 10 (40%)

2 (8%) 11 (44%)

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15 (60%)

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P-value 0.543a 0.934a

0.634b

0.157c

0.503d

12 (48%)

19 (76.0%) 5 (20%) 1 (4%)

19 (76.0%) 4 (16%) 2 (8%)

1.0d

8 (42.1%) 4 (21.1%) 7 (36.8%)

6 (28.6%) 10 (47.6%) 5 (23.8%)

0.212c

11 (57.9%) 5 (26.3%) 3 (15.8%)

13 (61.9%) 7 (33.3%) 1 (4.8%)

0.484b

1 (5.3%) 8 (42.1%) 6 (31.6%) 4 (21.1%)

1 (4.8%) 8 (38.1%) 10 (47.6%) 2 (9.5%)

0.798b

4 (21.1%)

4 (21.1%)

13 (68.4%)

11 (57.9%)

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Education Primary school Secondary school High school University Job Housewife Employed Income Sufficient Relatively Adequate Inadequate Marital status Married Single divorced Gravid 1 2 ≥3 Number of abortion 0 1 ≥2 Number of children 0 1 2 3 Contraception condom Natural family planning

Mullein (n=25) Mean (SD) 40.9 (4.7) 13.04 (2.0)

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General characteristics Age (years) Menarche age (years) First pregnancy age (years) BMI (kg/m2)

Placebo (n = 25) Mean (SD) 38.8 (5.4) 12.9 (1.8)

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IUD or VS Condom+ Natural family planning or without contraception (Infertility) types of leiomyoma intramural Sub-serosal

0 (0%)

3 (15.8%)

2 (10.5%)

1 (5.3%)

19 (76%) 6 (24%)

22 (88%) 3 (12%)

0.463d

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Note. SD: standard deviation; BMI: Weight (kg)/ Height (m) 2. a=independent T test. b= chi2 for trend. c= Pearson Chi-Square. d= fisher exact test.

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Table 2:Effect of oral Mullein capsules on myoma and uterine volume in patients with uterine fibroids Mullein Placebo Adjusted MD P (n= 25) (n= 25) (95% CI) Mean (SD) Mean (SD) before 94.1 (107.7) 126.2 (129.1) 0.553a -7.6 after 87.2 (106.0) 128.2 (144.2) 0.286b Myoma (-22.0 to 6.6) volume MD (95% -6.8 2.0 (ml) CI) (-13.0 to -6.3) (-11.4 to 15.6) p 0.032c 0.753c before 194.0 (107.5) 277.2 (179.5) 0.175a -7.6 after 205.0 (105.1) 280.8 (190.5) 0.593b uterine (-36.1 to 20.9) volume MD (95% 11.0 3.63 (ml) CI) (-5.8 to 27.8) (-17.8 to 25.1) p 0.187c 0.729 c before 2.5 (1.3) 2.2 (1.3) 0.388d Number of after 2.4 (1.2) 2.2 (1.2) 0.414d myomas

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Note. SD: standard deviation, MD= Mean difference within groups before and after intervention. Adjusted MD= Mean difference between groups adjusted for baseline values, a= independent-samples t test for between groups’ comparison at baseline. b=Analysis of covariance for between groups’ comparison after intervention adjusted for baseline values. c= pairedsamples t test for within groups’ comparison d= Mann-Whitney Test

Cycle duration (day)

Baseline 1st cycle 2nd cycle

27.6 (3.8) 28.7 (4.4) 28.6 (6.7)

29.0 (3.1) 26.7 (4.3) 26.8 (2.8)

P 0.056a 0.110b 0.657b

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Table 3: Effect of oral Mullein capsules on blood loss in patients with uterine fibroids Mullein Placebo Adjusted MD (n= 25) (n= 25) (95% CI) Mean (SD) Mean (SD) PBAC Baseline 50.0 (20.1) 58.8 (26.5) score After 1st cycle 67.3 (28.2) 70.4 (54.0) 1.3 (-24.3 to 27.0) After 2nd cycle 60.6 ( 32.6) 61.9 (44.6) 7.3 (-12.6 to 27.2) After MD 17.3 11.6 1st (95% CI) (1.4 to 33.1) (-17.3 to 40.5) cycle P 0.029c 0.674c Baseline After MD 10.6 3.0 2nd (95% CI) (-6.3 to 27.6 ) (-14.9 to 21.0) cycle P 0.318c 0.963c After 1st After MD -6.7 -8.5 cycle 2nd (95% CI) (-20.1 to 6.7) (-37.2 to 20.0) cycle P 0.503c 0.832c

-0.006 (-1.9 to 1.9) 1.9 (-0.2 to 4.1)

0.549a 0.104b 0.781b

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Note. SD: standard deviation, MD= Mean difference within groups before and after intervention, PBAC= pictorial blood loss assessment chart. a= Independent-samples t test for between groups’ comparison at baseline. b= Repeated measure c= Analysis of covariance for within groups’ comparison

Table 4: : Effect of oral Mullein capsules on hemoglobin and Hematocrit levels in patients with uterine fibroids Mullein

Placebo

Adjusted

Mean (SD)

Mean (SD)

MD (95%

P

CI) Hb (g/dl)

Before (n= 50)

13.2 (0.9)

13.2 (1.0)

After (n= 48)

13.3 (1.1)

13.2 (1.0)

0.630a -0.04

0.856b

(-0.5 to 0.4) Before (n= 50)

39.6 (2.4)

39.4 (2.9)

(%)

After (n= 48)

39.2 (2.7)

39.4 (2.6)

0.501a -0.4

0.438b

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Hct

(-1.7 to 0.7)

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Note. MD= Mean difference, Hb= Hemoglobin, Hct= Hematocrit. a= independent samples test for between groups’ comparison at baseline. b= Analysis of covariance for between groups’ comparison after intervention adjusted for baseline values.

Excluded (n = 39) Declined to participate (n=10)

Assessed for eligibility (n = 89)

Severe anemia (n=6)

Enrollment

Higham score > 100 (n=7) Breastfeeding (n=2) Tendency to have a pregnancy (n=4) Severe allergy (n=1) Uterine myoma greater than 12 cm (n=3)

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myomatous uterine size greater than 20 weeks (n=1) history of myomectomy (n=3)

consumption of hormonal agents over the past 3 months (n=2)

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Randomized (n = 50)

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Allocation Allocated to placebo (n = 25)

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Received allocated intervention (n = 25)

Follow-up

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Lost to follow-up (n = 0)

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Discontinued intervention (n =3)  Stomach pain (n=1)  Unwillingness (n=2)

Analyzed (n = 25)

Allocated to placebo (n = 25)

Received allocated intervention (n = 25)

Lost to follow-up (n = 0) Discontinued intervention (n =0)

Analysis Analyzed (n = 25)

Figure 1. Flow diagram of the progress through the phases of the randomized controlled trial.

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Figure 2. The changes in the menstrual bleeding in the baseline, after 1st and 2st cycle of intervention.