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The drone pupae of honeybee (A. mellifera) were obtained from 3 regions in Korea namely Cheongyang, Gimje, and Changnyeong. The drone pupae were colonized by using the special drone hives and harvested on the 21th days (Fig. 1). All samples were immediately stored at -20°C after collection.

Fig. 1. 
The raw material of drone pupae of honeybee (A. mellifera).

The stored drone pupae were homogenized by grinding after freeze drying. The drone pupae powder (1g) were extracted with 10mL of 5% acetic acid using a stirrer in 4°C for 18h and then filtered using a 0.45μM filler (Whatman, Simga-Aldrich, MO, USA). Each extract was dispensed in a 700~800μL into 1.5mL tubes and then concentrated using a rotary vacuum evaporator (CVC-3100, Eyela, Japan) for 24 hours. The concentrated extract was diluted in 0.01% acetic acid as suitable concentration for experiment.

Anti-thrombosis activity was measured using Astrup and Mullertz fibrin plate method (Astrup and Mullertz 1952). 20U/mL Thrombin (Sigma-Aldrich, USA) and 0.5% (w/v) concentration of fibrinogen (Sigma-Aldrich, USA) were used in phosphate-buffered saline (PBS). 100μL of thrombin and 1mL of fibrinogen were mixed in a 6-well plate to prevent the formation of air bubbles. After, plates were completely sealed and put in a drying oven at 37°C for 1 hour to form a fibrin membrane. Fibrin plate method (Astrup and Mullertz, 1952) was used to measure the area where the fibrin hydrolyzed and become transparent. The relative area of fibrin dissolved by sample and plasmin were measured. The experiments were repeated with three times. Human plasmin (20U/mL, Simga-Aldrich) was used as a positive control while 5% acetic acid was used as a negative control.

In Fig. 2, the drone pupae extract (0.12mg/mL) was more effective in fibrinolytic activity than the human plasmin used as a positive control, thus its extract was excellent in thrombolytic activity. The results are shown in Fig. 2, and the numerical values are shown in Table 1.

Fig. 2. 
The fibrinolytic activity of the drone pupae extract compared with control groups. Negative control is 5% acetic acid. Positive control is human plasmin (20U/mL). Concentration of drone pupae extracts is 0.12mg/mL in 5% acetic acid.

Thrombolytic agents are very important because thrombus accumulation in blood vessels causes various vascular diseases. Currently, the kinds of available thrombolytic agents are tissue plasminogen activator (t-PA), urokinase, streptokinase, and staphylokinase. Of these, only urokinase is possible to be administered orally (Weitz and Crowther 2002; Choi et al., 2013). However, these thrombolytic agents are expensive, low thrombosis selectivity, and have side effects such as systemic hemorrhage and allergic reactions. For this reason, there is a growing need for economical and safe thrombolytic agents. Drone pupae extract had a sufficient value as a thrombolytic agent since it can be orally administered and it had excellent fibrinolytic activity. Moreover, it is possible to develop drone pupae extract not only as a medicine but also as a health food supplement.

The whole fibrinolytic activity test was carried out in two replicated experiments. In Table 1, the upper treatment was the first treatment and the lower treatment was the second treatment. The diameter of the ring dissolved in the positive control was 12.93mm and the diameter of the drone pupae extract was 20.83mm. Based on these results, it was confirmed that the extract of the pupae had better anti-thrombotic efficacy than human plasmin.

Commonly, fibrinolysis is directly related to antithrombosis. Thrombosis is formed when fibrinogen is converted to fibrin by thrombin. Blood clots accumulate in blood vessels and interfere with blood circulation by blocking nutrients and oxygen supply to each tissue resulting in an increase in blood pressure (Butenas and Mann, 2002). Therefore, not only inhibiting the production of fibrin but also dissolving the generated thrombus contributes to improvement of vascular diseases (Ryu et al., 2008).