| Objective:Observe whether FCC-1/HN schizont’s growth was inhibited by the serum containing ART or DHA of the healthy volunteer. The Plasmodium falciparum strains were selected by way of comparing dose-effect relationship and sensitivity of FCC-1/HN and 3D7 to ART To Compare titer of serum containing different doses of oral administration of ART or DHA, inhibiting Plasmodium falciparum schizont (PFS). Take the trial tests on in vitro drug efficacy of ART compound-prescriptioned serum; to evaluate effect of the formation of half-vitro inhibition of PFS. The minimum inhibitory concentration equivalent PFS were determined in vitro micro-method of ART or DHA. Explore a simple, sensitive, reproducible detection of in vivo determination of efficacy of anti-malarial drugs for malaria research and development of anti-malarial compound to provide reference.Methods:46 volunteers, with half-full-stomached, take the Artemisinin medicine first. Then they are regularly drawn 2-3ml of blood, which will be separated into serum or plasma and preserved in the low-temperature refrigerator afterwards. In vitro culture of Plasmodium falciparum FCC-1/HN or 3D7, relatively long time in the thick ring body synchronization Preparation of red blood cell parasite infection 0.2-1.0% or so with insects blood; containing blood serum and with insects in 37℃,5% CO2, under conditions of saturated humidity incubated 20-30h, before taking in the RBC schizont rupture of membranes coated with thick blood, dried 1-2 days, staining, microscopic examination. Results to determine:Schizonts were observed in control the number of well (3 or more cores)≥10% for the test valid; 200 no asexual schizonts within the body as negative.Results:(1) The effect of how the healthy volunteer’s serum containing ART/DHA to inhibiter FCC-1/HN schizont’s growth:after ART having been taken for 1-2 hours,2.5μl of serum interrupted schizont development. After 2-4 hours, the efficacy achieves to the peak.8 hours later,20μl of serum was still able to inhibit the development of schizont. After DHA having been taken for 1 hour,7.5μl of serum starts to suppress the schizont formation. After 6 hours,0.5μl of serum still can stop the schizont’s growth.8 hours later,0.5-7.5μl of serum still perform the suppression of schizont’s growth. It shows that DHA is with stronger effect.(2) Experiment ART sensitivities to FCC-l/HN and 3D7:in the group of high dosage level of FCC1/HN, the average formation rates of schizont are 0 for 2 hours,0.22% for 4 hours,5.14% for 6 hours,41.4% for 8 hours and 99.83% for 12 hours. In the group of middle dosage level of FCC1/HN, the average formation rates of schizont are 14.03% for 2 hours,10.33% for 4 hours,35.92% for 6 hours,59.75% for 8 hours and 99.50% for 12 hours. In the group of high dosage level of 3D7, the average formation rates of schizont are 0 for 2 hours,0.14% for 4 hours,0 for 6 hours,1.28% for 8 hours and 28.22% for 12 hours. In the group middle dosage level of 3D7, the average formation rates of schizont are 0.53% for 2 hours,0.81% for 4 hours,0.72% for 6 hours,8.53% for 8 hours and 52.81% for 12 hours. It is showed in statistics inspection that 3D7 is with higher sensitivity, meaning that with same dosage of serum, the average formation rates of schizont for 3D7 is much lower while for FCC1/HN is much higher.(3) Semi-in virto experiment how ART & DHA inhibite PSF formation:in ART 2 groups, there are 12 volunteers completing the experiment. After ART having been taken for 1 hour, only one volunteer (40μl of serum) performs the suppression to form PFS. After 2-4 hours, there are 11 volunteers are with the suppression to form PFS.8 hours later, most volunteers (9/12) can suppress PFS formation. However, in this experiment, no volunteer can still inhibite PFS formation after 12 hours. In DHA 2 groups, there are also 12 volunteers completing the experiment. After ART having been taken for 1 hour, there are 4 volunteers (60μl of 3 volunteers & 10μl of 1 volunteer) with the suppression to form PFS. After 2-4 hours, there are 8 volunteers are with the suppression to form PFS. Most volunteers (8/12) can still inhibite PFS formation for more than 12 hours. It can be concluded that DHA is with stronger efficacy.The experiment results for serum containing ART & DHA to inhibite PFS formation agree with normal distribution. A better way to examine efficacy of the medicated serum is to add the medicated serum into raise board’ wells individually in a double-diluted order.(4) Semi-in vitro experiment compound-prescriptioned ART:after serum/plasma containing compound-prescriptioned ART having been taken for 2 hours, it starts to inhibite PFS formation. The efficacy duration is to 15th day. The strength & weakness of drug efficacy and so on all demonstrate that the effects of serum/plasma containing compound-prescriptioned ART are basically consistent. This can prompt compound-prescriptioned ART study by using serum/plasma to proceed bioassay.Compound-prescriptioned ART is composed by ART and piperaquine phosphate. Having been taken for 2 hours, the quick-acting efficacy of ART starts to inhibite PFS growth. While the long-term efficacy of piperaquine phosphate, it can inhibite the Plasmodium growth in erythrocytic cycle for more than 20 days. ART is also with high efficacy. In the first 2 days, having been taken for 2-8 hours,1.25-2.5% of the medicated serum is capable to terminate PFS formation. Thus, there will be an obvious synergistic effect to take these two combinedly. With inexpensive price, ART should have more promotion and application in the malaria infected areas.(5) In vitro experiment ART’s & DHA’s the lowest density of inhibitation:ART can totally inhibite PFS formation-well 4 of Table 3. well 4 of Medicine Quality Density Table-31.25ng/ml. DHA can also totally inhibite PFS formation-well 7 of Table 3. Well 7 of Medicine Quality Density Table-3.47ng/ml. In this study, the efficacy of 3.47ng/ml DHA is about equal to the efficacy of 31.25ng/ml ART. Calculated by regression equation, the IC50 of ART and DHA are respectively 18.51nmol/L and 3.18nmol/L.ConclusionCombining serum pharmacology of traditional Chinese medicine and improved in vitro micromethod to continuously raise PF for examining efficacy is concluded to be feasible. Since there is no need to have large, high-end, precise or advanced equipment, it is very suitable to apply this method in the poorer malaria infected areas and the normal laboratories. There is a good way to examine efficacy of the medicated serum by adding the medicated serum into raise board’ holes individually in a double-diluted order. Using inexpensive medicines to develop compound-prescriptioned anti-malaria will be more advantageous for drugs promotion and application. If suitable animal models can be found to replace the human model, its prospective application is surely positive in the near future. |
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