| ObjectiveTo individually detect for the anti-HCMV activity including anti-adsorption, anti-penetration and anti-replication of jinye baidu granules medicated serum by MTT method and discuss the action mechanism of anti-HCMV activity of jinye baidu granules. Methods1. Human embryonic lung fibroblasts cells were cultured with the method of suspension culture.2. The AD169 strain of HCMV was passaged by cell culture and the TCID50 was quantitated by the method of Reed-Muench.3. To prepare Jinye baidu granules medicated serum :①Normal, adult, female Japanese white rabbits were randomized into low dose group , middle dose group , high dose group and blank control group , 3 in each group. The dosage of jinye baidu granules were 1.5 g/ Kg, 3 g/ Kg and 6 g/ Kg, the different dosage were 5, 10, 20 times that of an adult per KG each time, and the blank control group was given an equivalent amount of double distilled water.②The low, middle and high dose group was given corresponding dosage of jinye baidu granules by intragastric administration, and blank control group was given an equivalent amount of double distilled water, bid, keeping 3 days. The blood was extracted from the central artery of the rabbit ear at the time of 1h, 1.5h, 2h, 2.5h and 3h after the last time filling of drugs on the morning of the 4th day. The serum was separated by centrifugation under the condition of 3000 rpm for 20 minutes after standing for 2 hours, then the serum was inactivated at 56℃for 30 minutes and sterilized by filtration, at last was reserved in -80℃.③The serum was thawed at 37℃before using, then was dispensed in the cell maintenance solution to make the 5%, 10%, 20%medicated serum-DMEM culture medium. 4. To detect for the cytotoxicity of jinye baidu granules medicated serum: The human embryonic lung fibroblasts were inoculated into 96-well culture plates and the cell seeding density was 1×105 /ml, then were cultured for 24 hours under the condition of 37℃, 5% CO2. Pretreat cells with various medicated serum-DMEM culture medium, then observe the morphological character of cells by cytopathic effect (CPE) method at last, screen the noncytotoxic medicated serum-DMEM culture medium.5. To detect for the anti-virus activity of jinye baidu granules medicated serum: The human embryonic lung fibroblasts were inoculated into 96-well culture plates and the cell seeding density was 1×105 /ml, then were cultured for 24 hours under the condition of 37℃,5% CO2. Pretreat cells with HCMV AD169 100ul of 100TCID50/100ul and the noncytotoxic medicated serum-DMEM culture medium successively, then observe the morphological character of cells by CPE method and detect for OD value to calculate the rate of viral suppression, at last, screen the medicated serum-DMEM culture medium with the best anti-virus activity.6. Experimental research on antiviral mechanism of jinye baidu granules medicated serum : The human embryonic lung fibroblasts were inoculated into 96-well culture plates and the cell seeding density was 1×105 /ml, then were cultured for 24 hours under the condition of 37℃,5% CO2. Pretreat cells with HCMV AD169 100ul of 100TCID50/100ul and the medicated serum-DMEM culture medium with the best anti-virus activity successively by Synchronized infection assays, then observe the morphological character of cells by CPE method and detect for OD value to calculate the rate of viral suppression, at last, individually evaluate the anti-HCMV activity including anti-adsorption, anti-penetration and anti-replication of jinye baidu granules medicated serum.Results1. The TCID50 of the AD169 strain of HCMV was 10-3.4/100ul. 2. To detect for the cytotoxicity of jinye baidu granules medicated serum:①Low dose group medicated serum: The 5%, 10% medicated serum-DMEM culture medium, which was dispensed using the blood extracted at the time of 1h, 1.5h, 2h, 2.5h and 3h after the filling of drugs, had no cytotoxicity and showed no significant difference as compared with the blank control group(p>0.1). The 20% medicated serum-DMEM culture medium, which was dispensed using the blood extracted at the time of 1h and 1.5h after the filling of drugs, had no cytotoxicity and showed no significant difference as compared with the blank control group(p>0.1).②Middle dose group medicated serum: The 5%, 10% medicated serum-DMEM culture medium, which was dispensed using the blood extracted at the time of 1h, 1.5h, 2h, 2.5h and 3h after the filling of drugs, had no cytotoxicity and showed no significant difference as compared with the blank control group(p>0.1). The 20% medicated serum-DMEM culture medium, which was dispensed using the blood extracted at the time of 1h and 1.5h after the filling of drugs, had no cytotoxicity and showed no significant difference as compared with the blank control group(p>0.1).③High dose group medicated serum: The 5%, 10% medicated serum-DMEM culture medium, which was dispensed using the blood extracted at the time of 1h, 1.5h, 2h, 2.5h and 3h after the filling of drugs, had no cytotoxicity and showed no significant difference as compared with the blank control group(p>0.1). The 20% medicated serum-DMEM culture medium, which was dispensed using the blood extracted at the time of 1h after the filling of drugs, had no cytotoxicity and showed no significant difference as compared with the blank control group(p>0.1). 3. To detect for the anti-virus activity of jinye baidu granules medicated serum:①Middle and high dose group medicated serum had the high anti-HCMV activity showed significant difference as compared with the virus control group (p<0.01), the rate of viral suppression of middle, 1.5h and 5% medicated serum was 90.90%.②The anti-HCMV activity changed with time and the peak time was 1.5h and 2h, the effect of 1.5h group was equivalent to the 2h group and showed no significant difference as compared with each other (p>0.1).③When the time was 1.5h and 2h, the effect of middle dose medicated serum was equivalent to the high dose medicated serum and showed no significant difference as compared with each other (p>0.1).4. The rate of viral suppression of anti-adsorption, anti-penetration and anti-replication was 40.43%, 37.55% and 88.84% respectively and showed significant difference as compared with the virus control group(p<0.01), jinye baidu granules medicated serum had the strongest anti-virus effect on the replication of HCMV.Conclusion1. The best drug dosage was middle dose (3 g/ Kg) and the best time was 1.5h in the process of preparation fo medicated serum, the best didution of medicated serum in vitro was 5% in the reaction system. 2. Jinye baidu granules medicated serum had the anti-HCMV activity. 3. Jinye baidu granules medicated serum had the anti-HCMV activity including anti-adsorption, anti-penetration and anti-replication. 4. Jinye baidu granules medicated serum had the strongest anti-virus effect on the replication of HCMV. |
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