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

Background The total flavonoids from ethanol extract of the aerial part of Ziziphora clinopodioides Lam. (Lamlaceae) (Xintahua) showed protective activities against rat acute myocardial ischemia in rats. This study aims to isolate acacetin, a flavonoid, from the aerial part of Z. clinopodioides, to develop an HPLC method for its detection, and to evaluate its protective effects on neonatal rat cardiomyocytes. Methods Sephadex LH-20 silicagel and pillar layer chromatography silica gel were applied for the isolation and purification of acacetin and its structure was elucidated on the basis of 1H and 13C NMR spectroscopy. The content of acacetin in Z. clinopodioides collected from three different origins was determined by HPLC. The neonatal rat cardiomyocytes were isolated and cultured in vitro to establish a hypoxia/reoxygenation injury model. The viability of cardiomyocytes was measured by the MTT method. Changes of malondialdehyde (MDA) content in the medium were also determined. Results The acacetin content in various batches of Z. clinopodioides ranged from 45.50 to 47.41 μg/g. Acacetin of 25, 10, 5 μg/mL significantly decreased the MDA content in a model of hypoxia/reoxygenation injury (P < 0.001, P < 0.001 and P = 0.033, respectively). Conclusions Acacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity.

The trichloromethane (CHCl 3 ) and ethyl acetate (EtOAc) portions from the ethanol extract of the aerial part of Z. clinopodioides showed protective effects on rat acute myocardial ischemia and neonatal rat cardiomyocytes [6]. Detailed analysis indicated that the total flavonoids were the primary contributor to the observed activities [7].
This study aims to isolate acacetin, a flavonoid, from the aerial part of Z. clinopodioides, to develop a HPLC method for its detection, and to evaluate its protective effects on neonatal rat cardiomyocytes.

Standards and reagents
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.

Extraction and isolation
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 CHCl 3 and EtOAc. Then, the solution was vacuumdistilled using a rotary vacuum evaporator (Rotavapor R-220; Buchi, Switzerland) to yield the CHCl 3 fraction (142.5 g) and EtOAc fraction (138.5 g). The EtOAc fraction was purified on silica gel eluted with a gradient of CHCl 3 -MeOH. Eluates were combined according to thin layer chromatography (TLC) behavior using two solvent systems CHCl 3 -MeOH (97:3) to offer compound 1 (320 mg).

Sample preparation for determination of acacetin
Dried powder (1.0 g) of the aerial portion of Z. clinopodioides was refluxed in 30 mL of methanol for 1 h after soaking for 20 min. After extraction, solvent was added to the extraction vessel until the final weight was equal to the starting weight to counter solvent loss. The extract was thoroughly mixed on a vortex mixer, and filtered through a 0.45 μm syringe filter prior to HPLC injection.

HPLC analysis of acacetin
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.

Calibration curves
Stock standard solutions of acacetin were prepared in MeOH and diluted to different concentrations to build calibration curves, e.g., plotting the peak areas versus the concentrations of each analyte.

Stability test
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.

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

Accuracy test
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:

Cell viability
The culture solution was discarded after 24 h cocultivation 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].

Model of hypoxia/reoxygenation injury
After cultivation for 72 h, the medium was exchanged with one that was hypoxic (culture medium saturated with high concentrations of N 2 in advance), and the solution was placed in a hypoxic culture box (99.99% N 2 ) for 120 min. Then, the medium was replaced with one saturated with pure O 2 , and the cells were exposed to a normoxic atmosphere containing 95% air and 5% CO 2 at 37°C (reoxygenation) for 30 min. A thiobarbituric acid (TBA) method was used to determine the content of MDA [6].

Cell experimental protocol
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).

Statistical analysis
The results were reported as the mean ± standard derivation (SD) of at least three measurements. The analysis of MDA data was performed with the SPSS 10.0 statistical package (IBM, USA), while simple linear regression was performed in Excel (Microsoft, Redmond, WA, USA). Results with P values less than 0.05 were considered significant.  [14,15].

Stability test
The RSD of peak areas at different times were less than 1.13%, indicating that the sample was stable for at least 24 h.

Precision test
The RSD value of intra-day and inter-day precision was 0.11% and 1.64%, respectively, which suggested that the developed method was precise enough for determining acacetin in Z. clinopodioides.

Accuracy test
The recoveries of acacetin were 97.33-103.92%, which indicated the developed method was suitable for determination of acacetin from Z. clinopodioides (Table 1). Figure 1A shows an HPLC chromatogram for Acacetin, Figure 1B shows a chromatogram of extract of sample.

Quantitative analysis
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).

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

Discussion
Flavonoids are polyphenol compounds, which are widely distributed in a variety of plants and have many pharmacological activities associated with cardiovascular protection such as antioxidation [16], anti-inflammatory, blood vessel expansion, arrhythmia inhibition, and antiplatelet aggregation [17]. Some flavonoids also have antitumor activities [18,19]. Acacetin exists in plants of asteraceae [20][21][22], and violaceae [23], but was rarely identified in lamiaceae. Acacetin is an atrium-selective agent that prolongs the atrial refractory period without prolonging the corrected QT interval and effectively prevents atrial fibrillation in anesthetized dogs after intraduodenal administration. These results indicate that oral acacetin might be a promising atriumselective agent for the treatment of AF [24]. However, the antioxidant activity of acacetin has not been thoroughly investigated.
In cardiomyocyte injury induced by hypoxia/reoxygenation, which is similar to heart ischemia-reperfusion injury in vitro, free radical injury was involved [25]. After myocardial ischemia-reperfusion, the body produces oxygen free radicals (OFR), and OFR-mediated cell membranes and subcellular membrane lipid peroxidation (LPO), while MDA is the LPO reaction product induced by OFR attacking the biomembrane. The amount of MDA reflects the degree of LPO, and is usually used to evaluate the degree of exposure to OFR [26]. In this study, after subjecting the cardiomyocytes to hypoxia/reoxygenation, the content of MDA in the medium increased significantly. Treatment with acacetin prevented the increase in MDA content, hence improving the antioxidant capacity of the myocardial cells.

Conclusions
Acacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity.   Results are expressed as the mean ± S.D (n = 6). * P < 0.05, significant difference vs. group II. ** P < 0.01 significant difference vs. group II.