4.1. Pilot Clinical Trial
An experimental prospective, pilot, parallel design, single blind, randomized, placebo-controlled study was conducted; the study was based on a screening evaluation, three follow-up visits and one final visit. Patients with stage 1–2 SAH, according to Official Mexican Standard, NOM-030-SSA2-1999 “For prevention, treatment, and control of arterial hypertension”, under stable treatment with ACE inhibitors (captopril, enalapril, lisinopril) were included; medications were given by their physician at therapeutic and recommended doses. This clinical trial was carried out in accordance with the declaration of Helsinki and was approved by Centro Especializado en Diabetes, Obesidad y Prevención de Enfermedades Cardiovasculares, S.C. (CEDOPEC) Ethics Committee No. 7-08-2014, and by the Research Committee of the Faculty of Medicine, UNAM, Mexico.
Inclusion criteria: patients who decided their voluntary participation, who understood the nature of the study and the procedures and the restrictions that involved the participation of the same, and patients older than 18 years.
Exclusion criteria: uncontrolled, stage 3 or systolic isolated hypertension, patients without pharmacological treatment, smoking history, secondary hypertension, women under pregnancy or in lactation period or who planned to become pregnant during treatment period; presence of coronary or peripheral vascular disease, diagnosed through medical history and physical examination; transaminase levels 2–3 times higher than upper normal limit, creatinine levels up to 1.5 mg/dL in screening visit.
The procedures were performed in screening, follow-up and final visits. The screening visit was carried out two weeks prior to patients’ randomization, informed consent was obtained, also demographical data, medical history, physical examination, blood pressure measurement, vital signs, electrocardiogram, and safety labs (hematology, serum chemistry, and urine analysis). If patients were eligible, they were randomized in visit 1.
The following procedures were performed in visit 1: review for inclusion and exclusion criteria, concomitant drugs review, questionnaire for quality of life in hypertension, physical examination, vital signs, blood pressure measurement and blood sampling for baseline determination of endothelial damage indicators and oxidative stress status. Additionally, in this visit SM or placebo were dispensed according with the randomization list, in sufficient quantity for patients’ consumption at the established dose until the following visit. The follow up visits 2 and 3 were performed in weeks 4 and 8 after randomization; in these visits a review of concomitant medication, adverse events and physical examination, vital signs and blood pressure measurement was performed. The final visit was performed in week 12, and the following procedures were performed: physical examination, review for adverse events, questionnaire for quality of life in hypertension, safety labs (hematology, serum chemistry, urine analysis), blood sampling for measuring endothelial damage indicators and antioxidant status, as well as an electrocardiogram. Once these procedures were completed, the clinical study was terminated for each patient who had complied with all the mentioned activities.
4.1.1. Outcome Measures
The outcome measures were blood pressure levels, changes in endothelial damage indicators, antioxidant status and quality of life. The quality of life of patients with hypertension was assessed through the application of CHAL questionnaire, developed and modified in Spain by Roca-Cusachs [59
]. The questionnaire assesses different aspects of the disease as well as patients’ daily life, which is affected by the disease and constant medication; it has a Cronbach’s alpha reliability index of 0.89 to 0.96. This instrument contains two dimensions: mood and somatic manifestations, which explore dimensions of hypertensive patients’ quality of life. In this sense, the CHAL questionnaire has been applied to the Mexican population [60
], indicating its usefulness in this population. Blood pressure measurement was performed by a physician, following the recommendations of NOM-030-SSA2-2009, using a calibrated digital blood pressure monitor (Citizen, model CH-308B).
Safety and tolerability of SM or placebo was carried out through the presence and severity of adverse events, in accordance with the provisions of the Official Mexican Standard, NOM-220-SSA1-2016 “Installation and operation of Pharmacovigilance. Safety labs (hematology, serum chemistry and urine analysis) and electrocardiogram performed in the screening visit and final visit by certified laboratory and interpreted by specialist physicians, according to their standard operating procedures.
4.3. Antioxidant Status Measures
After blood sampling in weeks 0 and 12, the samples were centrifuged at 4500 rpm to obtain the blood plasma, which was transferred to two polypropylene cryotubes duly labeled and stored at −78 °C under controlled conditions in ultrafreezer (Thermo Fisher Scientific, Whaltham, MA, USA) until the moment of their analyses. The sample analyses were performed in a period of 30 days after blood sampling. All the reactive were purchased from Sigma-Aldrich Química, S. de RL. de CV (Mexico City, Mexico). All spectrophotometric measurements were performed using a Genesys-10 uv spectrophotometer (Thermo Electron Corporation, Whaltham, MA, USA). Biochemical measurements were performed in duplicate with a coefficient of variation <0.05.
The activity of CAT was performed by the method proposed by Aebi [61
], which is based on the reduction of hydrogen peroxide, through the decrease in absorbance at 240 nm. The result of the enzyme activity is expressed as catalytic units on milliliter of plasma (kat/mL).
4.3.2. Superoxide Dismutase
Total activity of the SOD enzyme was based on the experiments carried out by Kono [62
]. The method is based on the ability of superoxide dismutase to inhibit the reduction of nitroblue tetrazolium (NBT). The results were expressed in units/mL of plasma, where one unit is the amount of enzyme that causes the maximum inhibition of NBT by photooxidation of hydroxylamine hydrochloride. Spectrophotometric reading was performed at 560 nm.
4.3.3. Glutathione Reductase
The activity of GR was based on the consumption of NADPH. The results were expressed as moles of NADPH/mL plasma per minute. The disappearance of NADPH was recorded spectrophotometrically at a wavelength of 340 nm [63
4.3.4. Glutathione Peroxidase
Glutathione peroxidase activity was based on the method developed by Paglia and Valentine where the moles of oxidized NADPH per minute/mL of plasma are determined. This technique is based on the ability of peroxidase to reduce organic peroxides through the oxidation of two GSH molecules. The oxidation of NADPH was monitored at 340 nm [64
4.3.5. Reduced and Oxidized Glutathione
Measurement of GSH was based on the Ellman reaction, a plasma sample reacted with dinitrobenzene in the presence of phosphate buffer. A standard curve was made with known concentrations of glutathione, the results being expressed as mg of GSH/mL of plasma. The spectrophotometric reading was performed at 412 nm. For the case of GSSG, the samples were treated with 4-vinylpyridine, to precipitate the reduced glutathione, leaving only the oxidized glutathione as substrate for the assay.
4.3.6. Thiobarbituric Acid Reactive Substances
The final products of the peroxidation were evaluated as TBARS as described by Torres-Durán et al. [65
]. A standard curve was made with tetraethoxypropane and the results were obtained extrapolating from the standard curve, expressing them as ng/mL plasma.