Preclinical Studies On The Earthworm Injection

Earthworm had been used for thousands of years as an animal medicinal substance. This injection used aqua pro injectione to extract the Earthworm active components and alcohol to remove useless elements, adding appreciable proportion adjuvant by its pharmacological properties. The way we used is simple, convenience in clinically. We also studied its stability, safety and effectiveness.Stable life prediction for earthworm injections was carried out by permanent stability test and the content of hypoxanthine was detected by HPLC-UV. The HPLC equipped with a constant-flow pump was used, together with a variable-wave-length UV detector operated at 254 nm. The separation was performed on C18(5μm, 4.6mm×250mm I.D, Agilent)with water-methanol-tetrahydrofuran(100:0.12:0.04,v/v/v)as the mobile phase at a flow-rate of 0.5mL·min-1 at room temperature, and a 10μL sample was injected into the HPLC system .According to"the technological specification of stability test for veterinary drug (trial performance)", the thermal stress tests (6 months, according to Jees-Davis method), light accelerater tests and long-term stability tests of earthworm injections were carried out. After the samples had been kept under ambient condition, illumination (4500±500Lx) and temperature (25±2℃) for 10 days, relative humidity (75±5%) and temperature (40±2℃) for 6 months, relative humidity (60±5%) for 12 months, appearance character, hypoxanthine content and pH were investigated. At the end of the test, except for color, all programs such as pellucidity, pH and decomposed product had no significant change; the hypoxanthine content of the preparation is consistented with the quality standard. The experimental results indicated that the preparation had good stability. We suggest that the period of validity about the injection could be temporarily determined at 2 years at room temperature. Drench by mouth and inject by abdominal cavity to mice and local stimulus to rabbits tests are used to evaluate earthworm injection's safety. The dosage and concentration we used are the maximum for each administration way. The experimental results indicated that the earthworm injection hardly have toxic action and side effect.We viewed its therapeutic action with three tests, I.e.relieve asthma and fever test and immunity enhancement test. The asthma model are caused by histamine phosphate- acetylcholine chloride (1:1); the high fever model are finished by injection Staphylococcus aureus 29213(1×109/mL) into rats. Earthworm injection was gived to guinea pigs which had asthma and rats which had a high fever. E-rosette test andα-HAc naphtalin carboxylesterase test are used to detect the earthworm injection whether raise nonspecific immunity. The experimental results indicated that earthworm injection has a good relief of asthma and fever and highly enhances nonspecific immunity.Plate chromatography and HPLC are used to control earthworm injection's quality. The quality controls are finished by plate chromatography to Ala and Val and by HPLC to hypoxanthine. The experimental results indicated that it is totally feasible.In conclusion, the earthworm injections have good stability, high bioavaibility. The therapeutic effect of relieve asthma and fever and enhancement of immunity are evident. Its successful development can provide a new formulation for veterinary clinical use of Earthworm.