Diabetes mellitus is a serious public health problem characterized by deficient plasma glucose regulation due to tissue insulin resistance and/or β
-cell failure which causes high morbidity and mortality rates. Type 2 diabetes (T2DM) accounts for the majority cases of diabetes (about 90%) and is becoming more prevalent due to the increasing rates of obesity in youth and adulthood and sedentary lifestyles [1
Dyslipidaemia is also common among diabetic patients and plays a critical role in the development of cardiovascular complications. Metabolic dyslipidaemia is characterised by high levels of triglycerides, associated with low levels of high-density lipoprotein cholesterol–HDL-C with or without a raise in low-density lipoprotein cholesterol–LDL-C [2
]. These imbalances in the internal metabolic environment, combined with the characteristic low antioxidant defences of diabetics can lead to oxidative stress and cellular damage. Oxidative stress has been demonstrated to be a contributor to the progression of the disease, accelerating both β
-cell failure and cardiovascular complications. Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) play crucial roles in the cellular protection against oxidative damage eliminating reactive oxygen species (ROS) [6
The increased expression of heat shock proteins (Hsp) is regarded as one of the most powerful means of cytoprotection against loss of cellular homeostasis and Hsp levels have been shown to be involved in tissue insulin responsiveness [8
]. The study of levels of cell protection in the most relevant insulin sensitive tissues is highly invasive and the easily accessible lymphocyte may provide valuable biomarkers of health status [9
]. The Hsp70 levels of lymphocytes may therefore provide information on effects on insulin response.
T2DM is preventable through lifestyles changes (including diet changes, physical exercise and weight loss) and pharmacological interventions with drugs such as metformin and acarbose [11
]. Herbal teas with glucose-lowering properties may offer low-cost alternatives to pharmacological interventions to limit the progression of the disease while having good acceptance. In particular Momordica charantia
has been shown to improve insulin secretion in β
-cells, increase peripheral glucose uptake, significantly reduce serum cholesterol and triglycerides levels at the same time as increasing HDL-C levels [14
]; Coccinia indica
improves antioxidant status by increasing antioxidant defences such as SOD, CAT and reduced glutathione levels and shows a significant hypoglycaemic action by decreasing blood glucose levels and increasing hepatic glycogen synthesis in animal models [15
] and Camellia sinensis
, has been associated with weight reduction, decrease in blood pressure and blood glucose levels, protection against lipid peroxidation and improvement of blood lipid profile which suggest beneficial effects against obesity, cardiovascular diseases (CVD) and T2DM [17
Common sage (Salvia officinalis
) is among the plants to which antidiabetic properties have been attributed by popular medicine and its extracts showed to possess hypoglycaemic effects in normal and diabetic animals [20
]. In a previous study we have shown that treatment with sage tea for 14 days lowered fasting plasma glucose levels but had no effects on glucose clearance in response to an intraperitoneal glucose tolerance test (ipGTT) in rats [22
]. Using hepatocyte primary cultures a decreased gluconeogenesic response to glucagon and a higher responsiveness to insulin were found after in vivo
treatment with sage tea [22
]. In vivo
treatments with Salvia fruticosa
tea also reduced plasma glucose in STZ rats (unpublished observations).
With the purpose of studying the effects of sage tea consumption on glucose regulation in humans, a pilot trial with human volunteers was carried out where a number of parameters relevant to diabetes were analysed such as fasting and postprandial blood glucose, response to an oral glucose tolerance test–OGTT, lipid profile, liver toxicity and antioxidant defences. Demonstration that there is no toxicity or adverse effects associated with sage consumption paves the way for future studies involving diabetic patients where the true antidiabetic potential of sage will have to be tested.
3. Experimental Section
3.1. Subjects and Study Design
Six healthy female volunteers (aged 40–50) participated in this trial after signing an informed consent form. The whole study was carried out in accordance with the principles of the Declaration of Helsinki. Smokers and subjects on regular medication were excluded from the study. Effects of sage tea drinking on body weight, blood pressure and heart rate at rest were recorded at first week of baseline and the end of each of the eight weeks of the trial. Weekly records of perceived negative events and concomitant medication were also kept. All the volunteers completed the study and reported no side effects. A non-randomized crossover study, where individuals serve as their own controls, was carried out in three phases: two weeks of baseline, four weeks of sage tea treatment and two weeks of wash-out (Figure 4
). The two-week baseline phase was included in order to obtain control values for all the volunteers. During this phase, all the parameters were measured and values are presented in figures and tables as basal levels. A treatment phase with sage tea followed, where 300 mL of tea were taken twice daily for four weeks. Sampling was carried out at the end of second and fourth week of sage treatment. A two-week wash-out phase was included after treatment with the aim to assess the duration of sage tea effects beyond the treatment period.
3.2. Plant Material and Preparation of S. officinalis Tea
Salvia officinalis L.
plants were grown in an experimental farm located in Arouca, Portugal, and were collected in April, 2001. The aerial parts of plants were lyophilized and kept at −20 °C. The sage tea was routinely prepared by pouring 300 mL of boiling water onto 4 g dried plant material and allowing to steep for 5 min [32
]. This infusion yielded about 3.5 ± 0.1 mg lyophilized extract dry weight per mL, where rosmarinic acid (362 mg/mL infusion) and luteolin-7-glucoside (115.3 mg/mL infusion) were the major phenolic compounds, and 1,8-cineole, cis
-thujone, camphor and borneol the major volatile components (4.8 mg/mL infusion). For full extract characterization see [32
3.3. Blood Samples, Erythrocytes’ Hemolysates and Lymphocytes Lysates
At the different sampling points (baseline–B, second week of treatment–T2, fourth week of treatment–T4 and at the end of wash-out–W), venous blood samples were collected postprandially in EDTA vacutainers (Vacuett®, Greiner Bio-one GmbH, Austria). An aliquot of blood was used for measuring glucose levels. Immediately after sampling, about 3 mL of blood were centrifuged at 200× g (KUBOTA 2100, Tokyo, Japan) for 10 min to separate the plasma. Plasma aliquots were stored at −80 °C for later measurements of transaminases, total cholesterol, HDL-C and LDL-C levels. The remaining erythrocyte enriched fraction was haemolysed to analyse SOD and CAT activity. About 10 mL of blood were used to separate peripheral blood lymphocytes (PBLs) by a Ficoll density gradient centrifugation following the procedure provided by the Ficoll manufacturer (Ficoll Paque-Plus, GE Healthcare, Piscataway, NJ, USA). The resultant PBL fraction was collected, washed with PBS and the cell pellet was homogenised with lysis buffer (25 mM KH2PO4, pH 7.5, 2 mM MgCl2, 5 mM KCl, 1 mM EDTA, 1 mM EGTA, with 0.1 mM PMSF and 2 mM DTT added fresh). Protein concentration from lymphocyte lysates was measured with the Bradford reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA) and aliquots kept at −80 °C for later quantification of Hsp70.
3.4. Measurement of Blood and Plasma Parameters
3.4.1. Quantification of Glucose Levels and Oral Glucose Tolerance Test (OGTT)
Fasting and postprandial glycaemia were measured with the Accutrend® GCT device (Roche Diagnostics GmbH, Mannheim, Germany) using Accutrend® test strips for glucose (Roche Diagnostics GmbH). Two OGTTs were performed after an overnight fast one at baseline and the other at week four of sage tea treatment. For that, 1 g of glucose per Kg body weight of each volunteer was given in up to 300 mL of warm water, which was consumed within 5 min of start. The OGTT started when the subjects began drinking with blood sampling taken before as well as at 45 min and 165 min after the oral glucose load. Blood glucose concentration was measured as above.
3.4.2. Characterization of Lipid Profile
Total plasma cholesterol, LDL-C and HDL-C levels were measured in plasma using spectrophotometric commercial kits from Spinreact (Girona, Spain), according to the manufacturer’s specifications.
3.4.3. Quantification of Plasma Aminotransferases
The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured spectrophotometrically in plasma following the NADH oxidation method at 340 nm on a plate reader (Spectra Max 340 pc, Molecular Devices, Sunnyvale, CA, USA), as previously described [32
3.4.4. Quantification of Erythrocytes’ Antioxidant Defences
The haemolysate fraction was used to determine SOD activity using the Ransod kit (Randox, Crumlin, UK) following the manufacturer’s specifications. The SOD activity in haemolysates was expressed as U/mL, with 1U corresponding to 50% of inhibition of 2-(4-iodophenyl)-3-(4-nitro-phenol)-5-phenyltetrazolium chloride (INT) reduction under assay conditions. The same haemolysates were used to measure CAT activity as described elsewhere [46
]. In brief, the decomposition of H2
was followed at 240 nm in a spectrophotometer (Cary IE, UV-Visible Spectrophotometer Varian, Australia) and the activity expressed as U/mL (U being μmol of H2
decomposed per minute) using the molar extinction coefficient of 0.0394 mL μmol–1
3.4.5. Western Blot Analyses
The quantification of Hsp70 protein in lymphocyte lysates was assessed by Western Blot in which proteins (20 μg per sample) were separated by SDS-PAGE using the mini-PROTEAN 3 electrophoresis cell (BioRad Laboratories, Inc., Hercules, CA, USA). Proteins were then transferred onto Hybond-P polyvinylidene difluoride membrane (GE Healthcare, UK) and membranes blocked in 5% (w/v) non-fat dry milk in TPBS (0.05% (v/v) Tween 20 in PBS, pH 7.4). Blotted membranes were probed with mouse monoclonal antibodies against Hsp72 (StressGen, Assay Designs, Inc., Ann Arbor, MI, USA) and β-actin (Sigma; used as loading control). Bound antibodies were then detected by chemiluminescence using appropriate secondary antibodies and the reactive bands acquired with a ChemiDoc XRS (BioRad) imaging densitometer. Band intensity was quantified using the Quantity One image analysis software (BioRad).
3.5. Statistical Analysis
Data are expressed as means ± SEM (n = 6). For statistical analysis the different parameters were analysed by repeated one-way ANOVA measurements followed by the Student-Newman-Keuls post-test (GraphPad Prism, version 4.03; GraphPad Software Inc., San Diego, CA, USA) to identify differences between studied time points. P values ≤ 0.05 were considered statistically significant (with a confidence interval of 95%).