Protective effects of acacetin isolated from Ziziphora clinopodioides Lam. (Xintahua) on neonatal rat cardiomyocytes

MTT was purchased from Amresco LLC (Pennsylvania, USA). Trypsin was from Amresco LLC (Pennsylvania, USA). Streptomycin was produced by North China Pharmaceutical Group Corporation (Shijiazhuang, China). DMEM (high glucose and low glucose) was from Invitrogen Corporation (Carlsbad, USA). Ampicillin sodium for injection was produced from China-promise Pharmaceutical Industry (Shijiazhuang, China). Malondialdehyde (MDA) was provided from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Acacetin was prepared in the Key Laboratory of Xinjiang Uighur Medicine (Urumqi, China). HPLC grade methanol and acetonitrile were purchased from Fisher Scientific (New Jersey, USA). Water (0.055 μS/cm) was purified by a Milli-Q system from Millipore (New Jersey, USA). Sephadex LH-20 silicagel was from Amersham Pharmacia Biotech (USA). Pillar layer chromatography silica gel (100-200 mesh) was from Qingdao Marine Chemical Plant (China). All other chemicals were of analytical grade.

Two batches of Z. clinopodioides were collected at the Astronomical Observatory (87°10′40″E, 43°28′14″N, with altitude of 2076 m) and the Chrysanthemum terrace (87°08′38″E, 43°27′14″N, with altitude of 2308 m), South Mountain of Tianshan Mountains in Urumqi, China, in September and August 2010. Another batch was from Xiao Dong Gou (88°07′57″E, 47°56′46″N, with altitude of 970 m) of Altai Mountains in Alaty, China, in August 2010. The plant materials were identified by associate researcher Jiang He (Xinjiang Institute of Materia Medica, Urumqi, China), according to Hudaberbi et al.[8], and voucher specimens (no. 100954, 100988, 100990, successively) were deposited in the plant herbarium, Institute of Metaria Medica.

NMR nuclear magnetic resonance was measured using JEOL ECP-500, INOVA400 and INOVA-600 (Varian, USA). Melting points were measured with a semi-automatic melting point apparatus (Yanagimoto MFG Co., uncorrected. HPLC analysis was performed using a Shimadzu-LC 2010C HPLC (Shimadzu, Japan) system. A BS124S Electronic Balance (Sartorius, Germany) was used for analysis. A DG-5031 ELISA Reader (Nanjing Huadong Electronics Group Medical Equipment Co., Ltd., China) and Shimadzu UV-2501 (Shimadzu, Japan) were used for cardiomyocyte experiments.

Neonatal Sprague-Dawley rats (1-3 days old) of either sex were maintained under standard environmental conditions. All animals were purchased from the Experimental Animal Centre of Xinjiang Medical University (Urumqi, China). Certificate Number: SCXK (Xin) 2003-2001. The study protocols were approved by the Ethics Committee on Animal Experiments, Xinjiang Material Medica, China (no.20110515).

The air-dried aerial portion of Z. clinopodioides (10 kg) was extracted with water and then the residue was extracted with methanol (MeOH) under reflux. The methanol extract was suspended in water and then successively extracted with CHCl₃ and EtOAc. Then, the solution was vacuum-distilled using a rotary vacuum evaporator (Rotavapor R-220; Buchi, Switzerland) to yield the CHCl₃ fraction (142.5 g) and EtOAc fraction (138.5 g). The EtOAc fraction was purified on silica gel eluted with a gradient of CHCl₃-MeOH. Eluates were combined according to thin layer chromatography (TLC) behavior using two solvent systems CHCl₃-MeOH (97:3) to offer compound 1 (320 mg).

All experiments were conducted with a Shimadzu-LC 2010C HPLC system. The mobile phase consisted of acetonitrile (A) and water with 1.0% glacial acetic acid (B), with the proportion of A:B held at 37:63. The chromatographic separation was performed using a YMC-Pack ODS-A (4.6 × 250 mm, 5 μm) column with a flow rate of 1.0 mL/min. The column temperature was maintained at 35°C. All analytes were monitored at 326 nm.

Sample was analyzed using the developed method to verify the stability of the sample. The stability test was carried out by analyzing the sample at 0, 2, 4, 8, and 24 h. The relative standard deviations (RSDs) of peak areas at different times were calculated.

Intra-day and inter-day variations were used to determine the precision of the developed method. The intra-day precision or inter-day precision was determined by analyzing replicated samples) on 1 day or over 3 consecutive days, respectively.

The accuracy of the developed method was evaluated by spike recovery. Acacetin was added into 0.5 g of sample. Then, the mixtures were extracted and analyzed. The spiked recovery was calculated as follows:

Primary cultures of neonatal rat cardiomyocytes were prepared from the ventricles of 1 to 3-day-old Sprague-Dawley rats as previously described [9],[10]. The cells were pre-plated three times for 30 min in a humidified incubator (95% air/5% CO₂ at 37°C) in DMEM supplemented with 2 mmol/L L-glutamine, 10% (v/v) foetal calf serum and penicillin/streptomycin (100 U/mL) to minimise fibroblast contamination. Cardiomyocyte-rich cultures (>90%) were plated onto fibronectin-coated 96-well plates, 100 μL per well, at a final density of 1.00 × 10⁵ cells per cm² in supplemented DMEM [11],[12].

The culture solution was discarded after 24 h co-cultivation of myocardial cells and different concentrations of acacetin, and then 180 μL DMEM and 20 μL MTT were added to each well for 4 h cultivation. The supernatant was discarded and 150 μL DMSO was added to each well, mixed evenly, and the absorbance (A) was measured at 570 nm within 10 min using a DG-5031 ELISA Reader [13].

After cultivation for 72 h, the medium was exchanged with one that was hypoxic (culture medium saturated with high concentrations of N₂ in advance), and the solution was placed in a hypoxic culture box (99.99% N₂) for 120 min. Then, the medium was replaced with one saturated with pure O₂, and the cells were exposed to a normoxic atmosphere containing 95% air and 5% CO₂ at 37°C (reoxygenation) for 30 min. A thiobarbituric acid (TBA) method was used to determine the content of MDA [6].

Experimental doses of acacetin were investigated using a cell viability test. The cells were divided into five experimental groups: group I served as a control (normal cell culture group, incubation for 3 h in the incubator), group II served as the myocardial cell injury control group (hypoxic 2 h, and reoxygenation 1 h), groups III, IV and V were treated with three different doses (25, 10, and 5 μg/mL,respectively) of acacetin (hypoxic 2 h, and reoxygenation 1 h). The inhibition rate was calculated from the absorbance of the medium containing added acacetin over the medium of the control (group I).

Compound 1 was yellow needle-like crystals. ¹H-NMR(DMSO-d ₆ , 600 M Hz) δ: 12.93 (1H, s, 5-OH), 10.87 (1H,br s, 7-OH), 8.05 (2H, d, J = 9.0 Hz, H-2′,6′), 7.11 (2H, d, J = 8.8 Hz, H-3′,5′), 6.89 (1H, s, H-3), 6.51 (1H, d, J = 1.8 Hz, H-8), 6.20 (1H, d, H-3, J = 2.4 Hz, H-6); ¹³CNMR(DMSO-d ₆ , 150 M Hz) δ: 181.8 (C-4), 164.2 (C-7), 163.3 (C-2), 162.3 (C-4′), 161.4(C-5), 157.3 (C-9), 128.3 (C-2′,6′), 122.8 (C-1′), 114.5 (C-3′,5′), 103.8 (C-10), 103.5 (C-3), 98.9 (C-6), 94.0 (C-8). Compound 1 was identified as acacetin by comparison of its physical and spectral data with the literature [14],[15].

Figure 1A shows an HPLC chromatogram for Acacetin, Figure 1B shows a chromatogram of extract of sample. The results of the quantitative analysis of three batches of Z. clinopodioides are shown in Table 2. No significant differences of acacetin content in Z. clinopodioides were found from one batch to another (ranging from 45.50 to 47.41 μg/g) (Table 2).

The cardiomyocyte viability was greater than 50% when subjected to an acacetin dose less than 12.5 μg/mL (Table 3).

The MDA content was significantly increased from 0.14 ± 21.34 to 30.72 ± 1.40 nmol/Lafter myocardial cell injury (Table 4). Compared with Group II (30.72 ± 1.40 nmol/L), the MDA content of Group III, Group VI, and Group V were reduced to 4.00 ± 2.91 nmol/L (P < 0.001), 10.64 ± 3.54 nmol/L (P < 0.001) and 15.45 ± 14.62 nmol/L (P = 0.033), respectively. These results confirmed that as the treatment concentration of acacetin increased, the MDA content in cardiomyocytes decreased. At 25 and 10 μg/mL acacetin, a significant reduction was observed (P < 0.001 and P < 0.001, respectively) (Table 4).