Evidence-based antioxidant activity of the essential oil from Fructus A. zerumbet on cultured human umbilical vein endothelial cells’ injury induced by ox-LDL

The essential oil was extracted from the fruit of A. zerumbet, which was collected in Zhenfeng county, Guizhou province, China, in October 2009. The fruit was identified by Professor Chen Zu-yun, and a voucher specimen (No.20091026) was deposited at the Research Division of Pharmacology, Guiyang Medical University. The isolation of the essential oil was carried out at the school of Pharmacy of the Guiyang Medical University, according to a method described elsewhere [13]. Briefly, chopped fruit was placed in a glass flask connected at one end to a glass vessel with water and at the other end to a water-cooled condenser. The water was heated to boiling point, and the steam percolated through the chopped fruit and collected in the condenser. After condensation, the watery phase with its solutes, termed the ‘hydrolate’, was separated from the oily phase; the essential oil when re-diluted in water is termed the ‘pseudo-hydrolate’. Sixty-two compounds were separated and 58 were identified from the essential oil. The composition of EOFAZ was determined by gas chromatography and mass spectrometry: β-phellandrene (16.388%), β-pinene (15.056%), 1,8-cineole (10.956%), camphene (10.120%), α-pinene (9.275%), linalool L (4.026%), camphor (3.657%), O-cymene (3.384%), β-myrcene (3.189%), borneol L (2.446%), caryophyllene oxide (1.778%) and terpinen-4-ol (1.756%); over 12 volatile compounds accounted for 82.03% of the total [12]. Pravastatin sodium standard substance (PRA) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China, Beijing.

Cell cultures were carried out essentially as described previously [14, 15], with some modifications. Endothelial cells were isolated and pooled from human umbilical cords obtained less than 3 h after delivery (cords were obtained from three healthy puerperae donors, who gave informed consent, and the acquisition of tissues was carried out in line with the principles of the Declaration of Helsinki and all procedures with patients were approved by the ethical review board of Guiyang Medical University according to local and national guidelines). After rinsing, the veins were cannulated and incubated with 50 U/ml collagenase (Sigma-Aldrich, St. Louis, MO, USA) in serum-free M199 medium for 10 min. Primary cultures were seeded at a concentration of approximately 4×10⁴ cells/cm² onto 75-cm² flasks precoated with 0.01% (w/v) polygeline in phosphate-buffered saline (PBS). The culture medium was used M199 with Earle’s salts (Sigma-Aldrich), containing 20% (v/v) human serum (Shanghai Hengyuan, Shanghai, China), 5 U/ml penicillin G, 5 pg/ml streptomycin sulfate and 150 pg/ml endothelial cell growth supplement (Sigma-Aldrich). Cells reached confluency within approximately 4 d, and were cultured at 37°C in a humidified atmosphere with 5% CO₂. The medium was changed a day after seeding and every 2 d thereafter.

For all experiments, cells were used at passages 3 to 6 and seeded at a concentration of 1×10⁵ cells/ml in 96-well plates, 24-well plates, or six-well plates. Treatments were carried out on a confluent monolayer of cells. EOFAZ was freshly prepared as a stock solution in DMSO and diluted with culture medium. DMSO was present at an equal concentration (0.02% final concentration) in all groups except for the PRA group. ox-LDL was used for no more than 2 wk from the date of production (Yiyuan Biotechnology, Guangzhou, China). Cells were preincubated for 30 min with EOFAZ or PRA before being exposed to 100 mg/L ox-LDL for 24 h except for the control group. After treatment, the cells cultured in the 96-well plates were used for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay, those in the six-well plates were used for trypan blue (Beyotime, Haimen, China) exclusion staining (TBES), and those in the 24-well plates were assayed for the contents of malondialdehyde (MDA), glutathione (GSH) and LDH, and for the anti-oxidative enzymatic activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). All experiments were performed in triplicate (four or five cultures were used for the experiments), however, the TBES assay was performed only once, but on four cultures.

Cell viability was measured by the MTT reduction assay as previously described [16]. Cells from at least six wells were used for each group. Briefly, after a 24 h exposure to ox-LDL (100 mg/L), the supernatant was removed and the cells were gently washed with PBS followed by 80 μl of fresh culture medium and addition of 20 μl MTT (5 mg/ml). After 4 h of incubation, 200 μl of DMSO, the solubilization/stop solution, was added to dissolve the formazan crystals, and the absorbance was read by a microplate reader (Sunrise RC, Tecan, Switzerland) at a wavelength of 570 nm. Inhibition of cell damage (%) was calculated with the following formula:

The TBES method is another classic cell viability method [17]. HUVECs in six-well plates were pretreated with EOFAZ or PRA for 30 min and then stimulated with 100 mg/L ox-LDL for 24 h. Then the cells were harvested and centrifuged to remove the medium. Cells were washed three times with PBS and resuspended to make a suspension of 1×10⁶ cells/ml. The suspension was mixed with 0.4% trypan blue dye for 5 min at 25°C, and the unstained (viable) and stained (nonviable) cells were counted on a hemacytometer within 5 min in five 40× microscope fields per well.

To evaluate cell injury, the LDH released from the cytosol into the culture medium was measured as previously described [18]. After treatment with EOFAZ or PRA, followed by incubation with 100 mg/L ox-LDL for 24 h, the medium was collected from each well. Supernatants were obtained by centrifugation at 12,000×g at 4°C for 10 min. LDH release was determined using an LDH assay kit according to the manufacturer’s instructions (Nanjing Jiancheng Co., Nan Jing, China). In brief, 100 μl supernatant, 250 μl buffer and 50 μl coenzyme were mixed and incubated for 15 min at 37°C, followed by addition of 250 μl 2,4-dinitrophenylhydrazine and another 15 min incubation at 37°C in the dark. Finally, 2.5 ml NaOH (0.4 mol/L) were added to the reaction mixture. Three minutes later, 200 μl of each reaction mixture were transferred into a new 96-well plate. The absorbance was read at 440 nm with an ELISA reader.

HUVECs were pretreated with EOFAZ or PRA for 30 min and then incubated with 100 mg/L ox-LDL for 24 h in 24-well plates. The cells were lysed with extraction buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin and 1 mM PMSF). Cell lysates from each well were collected and used for determination of MDA and GSH contents and anti-oxidative enzymatic activity. Protein concentrations of cell extracts were determined by the BCA assay (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

The total levels of MDA, the lipid peroxidation product in the lysates, were measured by the thiobarbituric acid-reactive substance (TBARS) assay as described previously [19]. The lysates (100 μl) were mixed in glass test tubes with 3 ml of 1% phosphoric acid, 1 ml of 0.67% thiobarbituric acid and 0.04% butylated hydroxytoluene, and the mixtures were incubated in a boiling water bath for 60 min. Marbles were placed on the tops of the tubes during the incubation period to avoid excessive loss of reaction mixture. After cooling the tubes in ice, 1.5 ml of n-butanol were added and the reaction mixture was centrifuged at 1000×g for 10 min. The absorbance of the supernatant was read at 535 nm. The concentrations of TBARS were calculated using tetraethoxypropane as a reference standard. Results were expressed in nmol/mg protein.

Intracellular GSH was measured using the enzymatic recycling assay [20]. Aliquots (20 μl) were mixed with 100 mM TEA, 600 mM DTNB, and 210 mM NADPH in 6.3 mM EDTA-0.125 mM NaH₂PO₄, pH 7.5 buffer. The final volume was 200 μl and the reaction was initiated by adding 0.1 units GSH reductase. Absorbance was read at 405 nm in a 96-well plate format using an ELX800 microplate reader (Bio Tek, Winooski, VT, USA), and concentrations were determined using purified GSH as standard and expressed as nmoles of GSH per mg of protein (nmol/mg protein).

SOD activity was measured by the xanthine/xanthine oxidase mediated ferricytochrome c reduction assay [21]. One unit of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%. Fifty microliters of lysates were added to 2.9 ml of reaction buffer (0.5 μmol xanthine, 0.1 mM NaOH and 2 μmol cytochrome c in 50 mM K₂HPO₄-Na₂HPO₄/0.1 mM EDTA, pH 7.8). The reaction was initiated by adding 50 μl of xanthine oxidase solution (0.2 U/ml in 0.1 mM EDTA). The absorbance change was monitored for 3 min at 25°C. Activities were calculated using a concurrently run standard curve and expressed per mg of protein. The SOD activity results were expressed as U/mg protein.

The GSH-Px activity assay was conducted by quantifying the rate of oxidation of the reduced glutathione (GSH) to the oxidized glutathione by H₂O₂, which is catalyzed by GSH-Px. One unit of GSH-Px was defined as the amount that reduced the level of GSH by 1 μmol/L in 1 min per mg protein. GSH-Px activity was measured in a 1.0 ml cuvette containing 400 μl of 0.25 M potassium phosphate buffer (pH 7.0), 200 μl of sample, 100 μl of 10mM GSH, 100 μl of 2.5 mM NADPH and 100 μl of glutathione reductase (6 U/ml). Hydrogen peroxide (100 μl of 12 mM) was then added, and the change in absorbance was measured at 1min intervals for 5 min at 366 nm [22]. GSH-Px activity was expressed as U/mg protein compared to the standard.

CAT activity was detected by methods of our lab [23]. Briefly, 2.8 ml of 10 mmol/L H₂O₂ were added to a 5 ml cuvette that contained 0.2 ml of 1 mol/L Tris–HCl (pH 8.0) in an ice-water bath, and absorbance was measured immediately at 240 nm, which was OD₀. Then 10 μl of lysate were added to the cuvette. The mixture was incubated at 25°C for 4 min, and then absorbance was measured, which was OD₄. For the nonenzymatic reaction cuvette, the procedure was the same as for the enzymatic cuvette, but the cell lysate supernatant was substituted by distilled water, which was OD_n. The results were expressed as catalytic activity following the equation: U/mg protein = (OD₀ − OD₄ − OD_n) × CH₂O₂/C_protein × 4.

The MTT assay utilizes the yellow MTT, which is metabolized by the mitochondrial succinate dehydrogenase to yield a purple formazan reaction product. Exposure of endothelial cells to 100 mg/L ox-LDL for 24 h significantly reduced cell viability (Table 1; 1.85 ± 0.11 in control vs. 0.98 ± 0.12 in 100 mg/L ox-LDL alone, p < 0.01). Preincubation with 0.1 mg/L EOFAZ restored viability significantly, but 0.01 mg/L EOFAZ had no effect (1.25±0.20 and 1.09±0.25 vs. 0.98±0.12, respectively, p<0.01 and p>0.05; Table 1). A similar protective effect was observed in the PRA treated group. The potential toxicity of EOFAZ to HUVECs was examined by addition of EOFAZ at 0.01, 0.1, 1 and 10 mg/L to the vehicle control medium. These concentrations had no effect on the survival of HUVECs, except for the 10 mg/L that led to a significant reduction in viability data were not shown. These results suggest that EOFAZ at concentrations of up to 1 mg/L is effective and safe to use with HUVECs. The trypan blue staining exclusion test is widely used to determine the number of viable cells present in a cell suspension. After incubation with 100 mg/L ox-LDL for 24 h, the number of stained cells significantly increased compared with the control group, and EOFAZ-pretreatment significantly decreased the cell blue staining ratio (Figure 1).

A 24 h exposure to 100 mg/L ox-LDL resulted in a marked and significant facilitation of LDH release from the cells, compared with the vehicle control group (555.15±59.67 U/L vs. 269.12 ± 97.80 U/L, p < 0.01). In contrast, the LDH activity in the supernatant was significantly decreased in the samples pretreated with 0.1 mg/L EOFAZ compared with the samples treated with ox-LDL alone (328.68 ± 61.29 U/L vs. 555.15±59.67 U/L, p < 0.01; Figure 2).

In the present study, incubation of HUVECs with 100 mg/L ox-LDL for 24 h resulted in a significant increase in the MDA contents, which was significantly attenuated by EOFAZ or PRA pretreatment (Figure 3). However, the exposure of HUVECs to ox-LDL 100 mg/L for 24 h resulted in a significant decrease in GSH contents, which was ameliorated by preincubation with EOFAZ or PRA (Figure 4).

The main scavengers responsible for inactivation and termination of free oxygen radicals are SOD, CAT, and the glutathione system. Our results indicated that the activities of the anti-oxidative enzymes were significantly decreased after HUVECs were exposed to 100 mg/L ox-LDL, and that EOFAZ and PRA improved these activities (Figures 5, 6 and 7). Preincubation with 0.1 mg/L EOFAZ significantly ameliorated the activities of SOD, CAT and GSH-Px in the cell lysates. Preincubation with 0.01 mg/L EOFAZ enhanced the activities of these enzymes, however, these results were not statistically significant. PRA (10 μmol/L) alleviated the decreased activities of SOD and CAT (**p < 0.01, compared with the ox-LDL treatment), but did not improve the GSH-Px activity.

This study was approved by the ethical review board of Guiyang Medical University according to local and national guidelines. Care of the patients in this study was in accordance with the Declaration of Helsinki and all relevant laws.