Plant material
Leaves of Larrea divaricata Cav. used in this study were collected in the province of Cordoba, Argentina and identified using morphological, anatomical and histochemical analyses by Dr. Hernán Gerónimo Bach from the Museum of Pharmacobotany, School of Pharmacy and Biochemistry, University of Buenos Aires. One voucher specimen (BAFC no. 38) was deposited at the Museum of Pharmacobotany. The aqueous extract (AE) was prepared from air-dried leaves. Briefly, 750 mg were infused for 20 min with 10 ml of sterilized boiling distilled water and the supernatant was lyophilised [18].
NDGA quantification by HPLC
The high performance liquid chromatography (HPLC) analysis was performed in a Varian Pro Star instrument equipped with a Rheodyne injection valve (20 μl) and a photodiode array detector set at 260 nm. A reversed-phase Phenomenex-C18 (2) Luna column (250 mm × 4.6 mm and 5 μ pd) was used. Samples were eluted with a gradient of A: water:acetic acid (98:2) and B: methanol:acetic acid (98:2) from 15% B to 40% B in 30 min; 40% B to 75% B in 10 min; 75% B to 85% B in 5 min and 100% B in 5 min. Solution B (100%) was run for 10 min and back to initial conditions. The flow rate was 1.2 ml/min and the separation was done at room temperature (18–25 °C). The optical density was registered in a Varian Star 5.5 detector (USA). Lyophilised aqueous extracts (10 mg/ml) and the pure standard were dissolved in methanol:water (70:30). The water employed to prepare the working solution was of ultrapure quality (Milli-Q). Methanol (J.T. Baker) and acetic acid (Merck, Argentina) were HPLC grade. A pure standard of NDGA (Sigma, USA, lot 19C-0504) was employed [18].
Total polyphenol determination
The total polyphenols content was determined by spectrophotometry by the Folin-Ciocalteu’s method using gallic acid as standard. The lyophilised extract was weighed and dissolved in distilled water. Briefly, 1.0 ml of the extract was transferred to separate tubes containing 7.0 ml of distilled water, 0.5 ml of Folin–Ciocalteu’s reagent, and 1.5 ml of a 20% sodium carbonate anhydrous solution. Tubes were allowed to stand at room temperature for 60 min and the absorbance at 765 nm was measured in a UV-vis spectrophotometer. The concentration of polyphenols in the samples was derived from a standard curve of gallic acid ranging from 10 to 50 μg/ml (Pearson’s correlation coefficient: r2 = 0.9996) [19].
Animals
Female albino Wistar rats (n = 40) weighing between 150 and 200 g each were used. Animals were acclimatized to laboratory conditions for 7 days before the experiments and housed in groups of five in stainless steel cages and were kept at 22 ± 2 °C in an illumination controlled room (photoperiod: 14 h light and 10 h darkness); they were fed Purina Chow and allowed to have water ad libitum. Twenty four hours before the experiment, animals were fasted and separated in randomized groups: Twenty animals were treated i.p. with 1 X PBS (control group) and twenty animals were injected i.p. once with streptozotocin (STZ) (60 mg/kg). The number of animals was selected taking into account the statistical procedures.
Ten days after the treatment, blood samples were taken from the tail vain to determine the glucose levels. Accu-check Performa test strips (Roche) were employed. For the study, animals with glucose values ≥300 mg/ml were selected. Animals were sacrificed by cervical dislocation and the submandibular glands were dissected. Animals were handled according to Ethics Committee Guidelines from the Faculty of Pharmacy and Biochemistry, University of Buenos Aires that approved the experiments under Exp-FFyB 220,612–1 and the Guide to the Care and Use of Experimental Animals (DHEW Publication, NIH 80–23).
Submandibular gland preparations
All experiments were performed on submandibular glands removed from normal and STZ-treated female rats euthanised by cervical dislocation. Free connective tissue, fat and lymph nodes were removed under a magnifying glass and the dissected glands were weighed and incubated in Krebs-Henseleit buffer (pH: 7.4) containing 125NaCl; 4.0 mM KCl; 0.5 mM NaH2PO4; 0.1 mM MgCl2; 1.1 mM CaCl2 and 5.0 mM glucose; bubbled with 95% O2 and 5% CO2 at 37 °C.
Upon reaching equilibrium after 10 min, glands were incubated during 40 min in buffer to allow basal secretion. Glands were treated in vitro as follows: from 5 normal animals, five glands were treated with 500 μg/ml of AE using the opposite glands as normal controls. From the other five animals, five glands were treated with 1.5 μg/ml of NDGA, using the opposite glands as normal controls. The same procedure was applied to STZ-treated animals: from five STZ-treated animals, five glands were treated in vitro with 500 μg/ml of AE and the opposite glands were used as controls. From the remaining five animals, five glands were treated with 1.5 μg/ml of NDGA and the opposite glands used as control. The optimum concentrations of AE and NDGA had previously been determined [18].
The activity of peroxidase and superoxide dismutase were determined in the incubation medium and in glands to determine total activity. For determination of enzyme activity in glands, tissues were homogenised in a Sorvall Omni mixer (DuPont Instruments) in Krebs-Henseleit buffer containing 10− 4 M phenylmethylsulfonyl fluoride (PMSF, Sigma) and 10− 3 M ethylene diamine tetraacetic acid (EDTA). The homogenate was centrifuged at 5000 x g for15 min at 5 °C and the supernatant was used for the determination of the enzymatic activity. To determine the effect of either the AE or that of NDGA, glands were incubated for 40 min in presence of either the AE or NDGA (one gland of each animal was used to determine the basal values) [18]. Proteins were determined in submandibular gland homogenate by the Lowry’s method.
Determination of the antioxidant activity
Enzymatic antioxidant activities were determined in gland homogenates and in incubation medium from control glands (without any treatment) and from glands treated with either AE or NDGA from normal and STZ-treated animals.
The peroxidase activity was determined according to Herzog and Fahimi (1973) [20]. Briefly, samples were incubated with 775 μl of 5 × 10− 4 M 3,3 diaminobenzidine tetrahydrochloride (DAB, Sigma) and 25 μl of H2O2 (Parafarm R, 30% v/v diluted 1/86 in distilled water). The reaction was initiated by the addition of H2O2. DAB without H2O2 was used as blank. The change in absorbance readings was recorded at 30 s intervals for 5 min using a Shimadzu recording spectrophotometer UV-240 (graphic printer PR-1) set at 465 nm, and the ∆ absorbance/min was calculated. The activity of samples was derived from a standard curve displaying a linear relationship between the enzymatic activity and the ∆ absorbance/min. Measurements were performed in duplicate.
The activity of superoxide dismutase was determined by its ability to inhibit the spontaneous oxidation of adrenaline to adrenochrome, which was measured spectrophotometrically at 480 nm. The enzyme activity was calculated taking into account that 1 U of superoxide dismutase inhibits the auto-oxidation of adrenaline by a 50%. Results were expressed as total peroxidase or superoxide dismutase activity U/ml/g gland = activity of homogenised glands + activity determined in incubation medium (mean ± SEM of values from five measurements) [21].
Determination of hydrogen peroxide (H2O2) and superoxide anion levels
For the determination of H2O2, gland homogenates were incubated with 0.56 mM DAB in a buffer containing 140 mM NaCl, 10 mM potassium phosphate, 5.5 mM dextrose; and 0.01 mg/ml type II horseradish peroxidase (Sigma, St. Louis, MO, USA) as described before [22]. Results were expressed as H2O2 (M) /g gland. A standard curve of known molar concentrations of H2O2 in buffered DAB was run in each test.
The levels of superoxide anion were determined through the reduction of nitroblue tetrazolium (NBT) (Sigma, CA, USA) to formazan [23]. Briefly, gland homogenates were incubated with 300 μl of NBT during 30 min. The reaction was stopped with 1 N HCl (Tetrahedron, Buenos Aires, Argentina). The formazan generated was extracted with dioxane (Dorwill, Buenos Aires, Argentina) and the absorbance was measured in a microplate reader at 525 nm (Microplate Reader Benchmark. Bio-Rad, CA, USA). Results were expressed as mmol reduced NBT /g gland.
Total nitrite determination and immunoblot analysis of inducible nitric oxide synthase (iNOS)
Total nitrites were determined employing the Griess reagent [24]. Gland homogenates or serum samples were incubated with the Griess reagent for 20 min in the dark and the absorbance was read at 540 nm. The levels of total nitrites were calculated by interpolation in a standard curve constructed with known concentrations of nitrites.
For the determination of iNOS levels, glands homogenates from control and STZ-treated animals were dissolved in sample buffer (2% SDS, 10% (v/v) glycerol, 62.5 mM Tris· HCl, pH 6.8, 0.2% bromphenol blue and 10 mM 2-mercaptoethanol). Equal amounts of proteins were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Non-specific binding sites were blocked with blocking buffer (5% non-fat dry milk containing, 0.1% Tween 20 in 100 mM Tris·-HCl and 0.9% NaCl, pH 7.5,) for 2 h. The PVDF membrane was subsequently incubated with an anti-iNOS antibody (Sigma Chemical Co) for 18 h. An anti-actin antibody (Santa Cruz Biotechnology PAIS) was used as loading control. After washing with PBS-Tween, the membrane was incubated for 1 h with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (HRP) (Amersham Biotech PAIS) diluted 1:2000 in PBS-Tween. Immunoreactive bands were visualized using ECL technology (Amersham Pharmacia PAIS). A densitometric analysis was performed with the Image J software (version 5.1, Silk Scientific Corporation PAIS). Densitometry values of iNOS (arbitrary units) in each lane were normalized to the corresponding actin densitometry values [25].
Determination of lipid peroxidation
Lipid peroxidation levels were assayed by determining the rate of production of thiobarbituric acid-reactive components expressed as malondialdehyde (MDA), according to Ohkawa et al., (1979) with slight modifications [26]. Briefly, 10 μl of gland homogenate or 10 μl of serum were treated with a mixture of 100 μl of 20% TCA and 0.5% thiobarbituric acid (TBA) and 100 μl of butyl hydroxyl toluene (BHT) (4% in ethanol). The reactive mixture was heated at 90 °C for 30 min. The mixture was allowed to cool down and centrifuged for 10 min at 800 x g. The MDA content was determined spectrophotometrically at 532 nm and 600 nm (unspecific absorbance) and the concentration was determined by calculating the difference between the values measured at 532 nm and 600 nm using the molar extinction coefficient (155 mM− 1 cm− 1). Finally, the content of MDA was expressed in nmol/g gland.
Determination of glutathione levels
The levels of reduced glutathione (GSH) were determined according to Moron et al., (1979) [27]. Briefly, immediately after obtaining the homogenates, they were precipitated with 0.1 ml of 25% trichloroacetic acid (TCA). Samples were centrifuged and the precipitate was removed. Free sulfhydryl groups were determined in a total volume of 200 μl. One hundred and thirty four μl of 0.6 mM DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid, Sigma) and 56 μl 0.2 mM sodium phosphate buffer (pH 8.0) were added to 10 μl of each supernatant and the absorbance was read at 405 nm in a UV-VIS Systronics spectrophotometer. Glutathione (Sigma) was used as a standard to calculate μmol GSH/g tissue.
Protein carbonyl group assay
The assay for detection of protein oxidation was based on the generation of carbonyl groups. The generation of carbonyl groups was detected by derivatisation with 2,4-dinitrophenylhydrazine (DNPH), which leads to the formation of a stable 2,4-dinitrophenyl hydrazone product (DNP). This product was measured at 375 nm. The number of carbonyl groups was expressed as nmol/gland [28].
Detection of peroxidase and superoxide dismutase by western blot
Gland homogenates (40 μg of protein/lane) were size-fractionated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were incubated for 90 min in Tris-buffered saline (TBS, pH 7.5)-3% non-fat milk, washed and incubated overnight with a 1:200 dilution of a rabbit anti-superoxide dismutase antibody or an anti-peroxidase antibody (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA.). Membranes were washed with TBS-0.05%-Tween 20 and incubated with a 1:1000 dilution of a goat anti-rabbit conjugated horseradish peroxidase antiserum (Sigma, CA//California, USA). The immunodetection was performed with the Western Blot Chemiluminescence reagent kit (NEN Life Science, Boston, USA), according to the manufacturer’s directions. Immunoreactive protein bands were analysed with the Corel Photopaint 9.0 software [18].
Histological studies
For these studies, gland samples from a group of 10 normal control and 10 STZ-treated animals whose glands were treated in vitro with either the AE extract or with NDGA were fixed in a 10% formalin solution at room temperature. Tissues were embedded in paraffin, and 5 μm sections were cut and stained with Hematoxylin-Eosin, and mounted in glass slides for light microscopy and scanning. Specimens were analysed at 200 x in an Aperio CS Scancope.
Statistical analysis
The level of significance between two or more treatments in comparison with control groups was assessed by an ANOVA followed by the Dunnett’s test. To determine the level of significance between one treatment and a control group, the Student’s t test was used. Differences between means were considered significant if P < 0.05.