Collagen and adenosine 5′-diphosphate (ADP) were purchased from Chrono-Log Co. (Havertown, PA, USA). Aspirin, fibrinogen, thrombin, plasmin and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thromboplastin and calcium chloride were purchased from Instrumentation Laboratory Co. (Milan, Italy). Other chemicals were of analytical grade. The standard marker compounds, (+)-catechin and (−)-epicatechin were purchased from Sigma-Aldrich (purity ≥98%). HPLC-grade reagents, methanol and water were obtained from J.T. Baker (Phillipsburg, NJ, USA).

Male Sprague-Dawely rats weighing between 240–250 g were obtained from Orient Co. (Seoul, Korea) and maintained in a standard laboratory animal facility with free access to feed and water and were allowed to acclimatize for at least two weeks prior to the start of the study. The animal studies were carried out in accordance with the Korea Institute of Oriental Medicine Care Committee Guidelines.

Ulmus macrocarpa was obtained as a dried herb from JungDo Co. (Seoul, Korea) and was authenticated, based on its microscopic and macroscopic characteristics, by the Classification and Identification Committee of the Korea Institute of Oriental Medicine. The 70% ethanol extract from Ulmus macrocarpa was made according to the following procedure. The roots of the plant were boiled twice at 70–80°C each time for 2 h. The filtrates were collected, concentrated and evaporated to dryness at 40–50°C under vacuum. The dosage of the extract was indicated in the powdered form. The powder was dissolved in 20 or 50% DMSO (v/v, in PBS) for the experiments.

HPLC analysis was performed on a Waters 2695 system (Waters Co. Milford, MA, USA), consisting of a solvent delivery unit, an online degasser, an autosampler and a photodiode array detector. A Luna C18 column (250 ×4.6 mm; particle size 5 μm, Phenomenex, Torrance, CA, USA) was used for analysis and the mobile phase was water (A) and MeOH (B). The gradient flow was as follows: (A)/(B) = 10/90 (0 min) → (A)/(B) = 40/60 (30 min) → (A)/(B) = 0/100 (32 min; hold for 5 min) → (A)/(B) = 10/90 (38 min; hold for 12 min). The flow rate was 1.0 ml/min, the injection volume was 10 μl and the detection wavelength was 280 nm.

A standard stock solution mixture of (+)-catechin and (−)-epicatechin were prepared in 70% EtOH at a concentration of 1,000 μg/ml, respectively, and stored at 4°C. UME powder (500 mg) was transferred to a 100 ml volumetric flask and dissolved up to the 100 ml mark with methanol. The mixture was subsequently sonicated for 30 min at room temperature and filtered through a 0.2 μm membrane filter. Calibration of the standard mixture was prepared by serial dilution of the standard stock solution mixture to yield concentrations of 10–1,000 μg/ml for (+)-catechin and (−)-epicatechin. These standard component solutions at five different concentrations were injected in triplicate. Calibration curves were generated by plotting the peak areas vs. the concentrations of the two components.

Arterial thrombus formation in vivo was investigated as previously described (14). UME was orally administered on a daily basis at doses of 300 and 600 mg/kg for 3 days. The rats were fasted overnight, and then orally administered the extract or the 1% carboxymethylcellulose (CMC) solution as a vehicle. The rats were i.p. anaesthetized with 60 mg/kg pentobarbital sodium salt and placed on a heat source. A segment of the right carotid artery was isolated and dissected free of the vagus nerve and surrounding tissues. Aortic blood flow was measured with a Doppler velocimeter (ADInstruments, Colorado Springs, CO, USA). Arterial thrombus formation was induced by wrapping a 2 mm² Whatmanno no. 1 filter paper saturated with 50% FeCl₃ around the carotid artery near the probe for 10 min. The time required for occlusion was measured for up to 60 min. An occlusion time of 60 min was assigned for vessels that did not occlude within 60 min.

Ex vivo platelet aggregation was investigated as previously described (14). The rats (n=7) were orally administered UME at a dose of 300 mg/kg for 3 days. Platelet-rich plasma (PRP) was obtained by centrifuging the blood sample at 180 × g for 10 min, and platelet poor plasma (PPP) was obtained by centrifuging the PRP at 2,100 × g for 10 min continuously. PRP was adjusted to 4×10⁸ platelets/ml with PPP. Platelet aggregation was measured with an aggregometer (Chrono-Log Co.), and collagen (5 μg/ml) and ADP (5 μM) were used as aggregation stimulators. The plasma-activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured with an Automated Coagulation Laboratory 7000 Instrument (Instrumentation Laboratory) as previously described (14). PPP was incubated at 37°C for 7 min, and subsequently 100 μl of incubated plasma was mixed with 50 μl of cephalin in the process plate. Coagulation was initiated by the addition of CaCl₂ plus 100 μl thromboplastin or 100 μl polybrene for the APTT and PT assays, respectively.

PRP was obtained by centrifuging the blood samples at 180 × g for 10 min, and PPP was obtained by centrifuging the PRP at 2,100 × g for 10 min. PRP was adjusted to 4×10⁸ platelets/ml with PPP. The number of platelets was counted using a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL, USA).

Evaluating platelet aggregation was undertaken by optical platelet aggregometry in an aggregometer (Chrono-Log Co.). Aggregation was recorded as the percentage change in light transmission; the baseline value was set using PRP and the maximal transmission using PPP. They were incubated at 37°C for 4 min in the aggregometer. Following incubation, platelet aggregation was induced by the addition of collagen (5 μg/ml) or ADP (5 μM). The inhibition of platelet aggregation for each sample was expressed as the percentage decrease in the maximal transmittance compared with the stimulated control.

Fibrinolytic activity was evaluated according to the method of Astrup and Mullertz with minor modifications (15). The fibrin plate was prepared by mixing 0.6% human fibrinogen solution (Sigma-Aldrich) in 1X PBS (pH 7.4) with 50 NIH units of human thrombin. In order to speed up the clotting process, the plates were incubated at 37°C for 30 min. Plates were prepared fresh each time. UME (250, 500 and 1,000 mg/ml), standard solutions (plasmin) and control solutions were placed separately onto membrane discs in the fibrin plate. We used 20% DMSO in PBS as a control. The plates were incubated at 37°C for 15 h. The diameters of the transparent rings were measured with calipers (Mitutoyo Co., Tokyo, Japan), and subsequently the fibrinolysis area (mm²) was calculated. The mean diameter of the hydrolyzed clear zone was measured and volume of lysis caused by each sample was calculated.

Experimental results were expressed as the means ± SD. Differences between the groups were used for multiple comparisons followed by an unpaired Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

We employed HPLC to confirm and quantify the standard contents of UME, such as catechin and epicatechin, for standardization and quality control. The representative chromatogram at 280 nm reveals that UME contained two marker compounds, catechin and epicatechin whose peaks were retained at 18.55 and at 24.83 min, respectively (Fig. 1). Correlation coefficients were better than 0.999 for all standard components. The limits of detection (LOD) and quantification (LOQ) for both components were 1.5 and 5.0 μg/ml, respectively. The catechin and epicatechin levels in UME were quantified as 1.397 and 0.069%, respectively.

The effect of UME on arterial thrombus formation in vivo was evaluated by using the FeCl₃-induced rat carotid artery injury model. Following the application of 50% FeCl₃, injured vessels of the control group occluded within 21.0 ± 5.7 min (n=7). Oral UME treatment for 3 days significantly extended the occlusion times to 13.4 ± 8.0 (n =7) and 13.9 ± 13.6 min (n =7) for doses of 300 and 600 mg/kg, respectively (Fig. 2).

UME significantly inhibited ADP- and collagen-induced platelet aggregations ex vivo (Fig. 3A). Collagen- and ADP-stimulated aggregations were inhibited by 18.0±10.0 and 8.0±9.4% at 300mg/kg, and 24.7±7.3 and 9.3±3.3% at 600mg/kg, respectively (n=7). The APTT and PT of the control group were 17.5±1.7 and 14.7±10.0 sec, respectively (Fig. 3B). UME treatment did not significantly alter APTT and PT, which were 17.8±0.8 and 15.2±0.9 sec at 300mg/kg, and 17.3±0.9 and 15.1±0.7 sec at 600 mg/kg, respectively. These data indicate that UME significantly inhibits platelet aggregation but does not affect the coagulation system. Aspirin (20 mg/kg), was used as a positive control and completely blocked collagen-induced aggregation.

UME significantly inhibited collagen-(5 μg/ml), and ADP (5 μM)-induced platelet aggregations in vitro in a concentration-dependent manner, with IC₅₀ values of 915.7±4.6 and 833.3±2.5 μg/ml, respectively (Fig. 4). This result supports the anti-platelet effect on platelet aggregation ex vivo.

In the fibrin plate, fibrinolysis was increased by treatment with UME in a dose-dependent manner (Fig. 5). UME induced fibrinolysis at 1,000 μg/ml, compared with the control group (2.2±0.4 vs. 0.0±0.0 mm²), respectively.