In this double-blind, randomized clinical trial, 36 healthy male volunteers were randomly distributed into three groups (
Interest in phytotherapeutic agents is growing in North America and Europe [
The aerial parts of
Preclinical studies have revealed various pharmacological actions of
The health regulatory agencies of the Federal Republic of Germany established the German Commission E in 1974. This commission approved the use of
The present study assessed the diuretic action and the short-term safety of a standardized dry extract of the aerial parts of
Three compounds were prepared: a standardized dry extract of the aerial parts of
The tested substances were placed in capsules, which were placed in vials and labeled with the letters A, B, and C. One of the investigators established this code, which remained secret until the statistical analyses were complete.
The clinical trial complied with the guidelines of Resolution 196/96 of the National Health Council and the Declaration of Helsinki for research on human beings. The research ethics committee of UFG, which is accredited by the National Commission of Research Ethics of the National Health Council/Health Ministry, approved the protocol (no. 312/10).
The volunteers completed an informed consent form that granted the researchers the right to use the collected materials to perform the required tests to establish the correct medication use, as described in the experimental protocol.
The Recruitment Department of the Institute of Pharmaceutical Science (Instituto de Ciências Farmacêuticas—ICF) recruited and selected all of the volunteers using an institutional database of volunteers who were duly oriented to the importance of appropriate and reliable data collection. Furthermore, the records of these volunteers indicated that the results of biochemical screening tests three months before the onset of the study had been normal.
A total of 36 healthy male volunteers who were 20 to 55 years of age, 1.50 to 1.85 m tall, and 50 to 90 kg in weight were selected on the basis of their medical histories, past medication use, physical examinations, vital signs, and laboratory results.
Smokers and individuals with congestive heart failure, arterial hypertension, or a history of kidney disease, cardiovascular diseases, endocrine disorders, or other underlying diseases were excluded.
The other exclusion criteria included a lack of proper compliance with the protocol at any stage of the study and the appearance of unexpected or exacerbated reactions.
The 36 volunteers (numbered 1 to 36) were randomly allocated to six different drug administration sequences (
Drug administration sequences of study according to the proposed design by Williams.
Stage | |||||
---|---|---|---|---|---|
First | Second | Third | |||
Treatment: 4 days | 10 days | Treatment: 4 days | 10 days | Treatment: 4 days | |
Seq. 1 ABC | A |
Washout | B |
Washout | C |
Seq. 2 CAB | C |
Washout | A |
Washout | B |
Seq. 3 BCA | B |
Washout | C |
Washout | A |
Seq. 4 CBA | C |
Washout | B |
Washout | A |
Seq. 5 BAC | B |
Washout | A |
Washout | C |
Seq. 6 ACB | A |
Washout | C |
Washout | B |
Day 0 corresponded to the day before treatment onset. Days 1, 2, 3, and 4 were the treatment days, which were followed by the 10-day washout period. Therefore, day 10 of any washout period was also day 0 of the following stage.
The volunteers recorded their fluid input and urinary output over 24 h (24 h fluid balance) on day 0 and during the four days of the treatment to assess the effects of the treatments. On day 0 and the day immediately following the end of each treatment stage, the volunteers underwent the medical assessments and laboratory tests that were defined in the protocol.
The capsules used in all the treatments exhibited an identical appearance (size 0 gel capsules). The volunteers received two vials, labeled Vial 1 and Vial 2, at each treatment stage. Vial 1 contained four blue capsules to be taken once per day in the morning for four days, and Vial 2 contained eight capsules to be taken twice per day for four days. The capsules were delivered randomly to the volunteers at the Clinical Research Unit of ICF.
All the volunteers underwent blood and urine tests. The blood tests included a complete blood count and assessments of the concentrations of fasting blood glucose, albumin, aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transpeptidase (GGT), total bilirubin, direct and indirect bilirubins, urea, creatinine, sodium, potassium, chlorine, phosphorus, and magnesium. The urine tests included a urinalysis and analyses of the sodium and potassium levels.
The blood and urine samples were collected at the Clinical Research Unit of ICF by pharmacists who were affiliated with the Group of Toxicopharmacological Studies and Research of UFG (NEPET-UFG). All the samples were appropriately packaged and were transported to the Rômulo Rocha Laboratory of the School of Pharmacy of UFG for analysis. The samples were collected exclusively on the day before treatment onset and the day after the end of treatment for each stage of the study.
All the volunteers underwent two ECG tests: the first test before the onset of the study and the second immediately following the end of the study.
The medical assessments were performed and the treatments were prescribed in the Clinical Research Unit of ICF. The complete clinical history of each volunteer was obtained at the beginning of the study, and a clinical assessment was conducted at each stage of the study prior to and after the treatments to record clinical manifestations, possible adverse reactions, and physical examination results.
The volunteers were provided with guidelines for their lifestyle, diet, and physical activity before the onset of the experimental phase. The volunteers were requested not to change their diet during the study and to avoid drug use, alcoholic drinks, and beverages with a diuretic effect (e.g., coffee and tea). The volunteers were also asked to perform their usual physical and daily routine activities and avoid more intense or long-lasting exercises than usual.
The volunteers were provided with backpacks to carry continually throughout the study. Each backpack included two separate compartments to store (in a hygienic manner) a graduated measuring cup to measure fluid intake, a vessel with a cap to measure urine, a notebook, and a pen to record the volumes of ingested fluids and urination. All of the volunteers signed a form indicating the receipt of the materials and their commitment to comply with the provided instructions. From day 0 to day 4, the volunteers recorded the volume of urine after each urination and, immediately after any intake of fluid, the volume of fluid ingested. These data served as the basis for the fluid balance (FB) calculations.
Therefore, the FB represented the difference between the volumes of fluid intake and excreted urine over 24 h, from the first urination in the morning after awakening to the final urination before arising from bed the following morning [
The diuretic effects were assessed using the following criteria. Assessment of the intragroup (A, B, and C) final FB: the final FB corresponded to the difference between the posttreatment FB and the initial FB (without treatment; FB0). Therefore, final FB = posttreatment FB − FB0. Assessment of the intergroup (A, B, and C) final FB: the final FBs were compared between the groups. Assessment of the effects of the treatments on urine electrolyte concentrations: the results of the measurements on day 4 were compared to the results on day 0 at each stage of the experiment.
The volunteers were asked to report any symptoms and the use of any emergency medications. A researcher recorded all the reported adverse events during the clinical trial on case report forms (CRFs) for clinical followup. The reported symptoms were classified on the basis of their intensity: mild, moderate, or severe [
Possible acute toxic effects related to liver, kidney, and heart functions were investigated during the clinical and laboratory examinations that were performed before and after the treatments at each stage of the study. ECGs were performed at the onset and conclusion of the study.
The following resources were used to reduce and monitor noncontrolled parameters, such as fluid and sodium intake, physical activity, and the effects of other drugs. The study included volunteers who were aware of the importance of clinical research and who were recruited, selected, and oriented by an independent institute of clinical research with experience in clinical trials (ICF, Goiânia). The volunteers were asked to maintain their usual diet and exercise routines and to avoid the use of products and physical activities that might interfere with the study results. The volunteers were contacted daily by telephone during the three stages of medication use, and they underwent weekly clinical assessments throughout the 42-day study period. The volunteers received orientation at recruitment meetings and in a written format. The calculation of the actual fluid balance, instead of the controlled volume of fluid intake minus the urine volume, represents an organism’s global response to real-life factors that controlled studies do not consider. However, commercially available drugs do consider this global response.
The following measures were applied to determine the variability of the noncontrolled real-life variables between the individuals and groups, which might have interfered with the study results. Statistical tests were used to assess the groups’ homogeneity relative to the FB0 at each stage and to assess the reliability of this parameter for the assessment of diuretic activity. A crossover design and the Williams design for randomization were used to reduce the impact of the noncontrolled variables and to increase the data reliability.
The data were stored in Microsoft Excel for Windows for later analysis with the
The Kolmogorov-Smirnov test was used to establish the normality of the data distribution.
A paired Student’s
An analysis of variance was used to evaluate the significance of differences between the variables for treatments A, B, and C.
Wilcoxon’s test and Pearson’s correlation test were used to analyze the volunteers’ urinary sodium and potassium concentrations prior to and after the treatments.
The confidence level was established as 95% (i.e.,
Laboratory tests confirmed that the dry extract used in the study corresponded to
The individuals in the various treatment groups did not exhibit significant differences in age (
The FB values of all the volunteers on day 0 (FB0) were compared at each stage of the study. The FB0s were not different between the individual volunteers or between the groups that were established using the Williams design. A comparison of the FB0s of groups A, B, and C revealed no significant intergroup differences (
The fluid intake volumes of the volunteers were similar in all three stages of the experiment. The volume of urine, as a whole, also did not differ between the three data collection stages (
These data support the homogeneity of the volunteers and confirm the reliability of the FB0 value as a reference parameter.
The FB0 values exhibited a normal distribution according to the Kolmogorov-Smirnov test, despite demonstrating a wide variation. This result indicated that the FB would be highly variable even among individuals of homogeneous age, weight, height, and BMI.
The diuretic effects of the treatments were assessed using the following criteria, according to the parameters in Section
Intragroup comparisons of fluid balance (
Group | Fluid Balance | |||
---|---|---|---|---|
FB0 (mL) | FB (mL) | FFB (mL) |
|
|
A |
|
|
|
<0.001* |
B |
|
|
|
0.067 |
C |
|
|
|
0.164 |
Groups: A:
Comparisons of the fluid balance between groups (
The negative final FB of hydrochlorothiazide was similar to that of
The dose of
Our experimental data confirm the ethnopharmacology and traditional use of
The results of the additional tests (Section
The results of the laboratory tests remained within the normal ranges for all three stages of the study prior to and after treatment. No signs of liver, kidney, hematological, or electrolyte toxicity were identified.
The volunteers in Group A exhibited consistent reductions in GGT during the second and third stages (
All the treatments were well tolerated overall. The few symptoms that occurred exhibited a homogeneous distribution across all three groups. One volunteer complained of a strong headache during the first stage of
The systolic pressure was significantly reduced after treatment compared to pretreatment levels only at the first stage in the group that underwent the BCA sequence (
Future studies are required to elucidate the mechanism of the diuretic action of
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank the Goiás State Research Foundation (Fundação de Apoio à Pesquisa do Estado de Goiás, Brasil—FAPEG) for financial support; the Institute of Pharmaceutical Science, Brazil (ICF) for its collaboration during the entire experimental phase of the study; Farmácia Artesanal (Artesanal Pharmacy), which generously supplied the drugs for the present study; and the Laboratory of Natural Products Research (LPPN) of the School of Pharmacy of UFG for the quality control analysis of the