| Objective:Heating reflux and aqueous two-phase extraction were used to extract、separate and purify pueraria flavonids. Orthogonal experiment design was used to optimize the process and get pueraria flavonids liniment with high concentration and purity. Stability and transdermal permeation of the liniment were investigated. Cervical spondylotic radiculopathy rats model were established, paint the pueraria flavonids liniment to the side which had the operation. The effects of pueraria flavonoids liniment on behavior and pathological morphology changes were observed.Methods:1. Determine the content of pueraria flavonidsHPLC and UV were established to determin the content of pueraria flavonids.2. The separation and purification of pueraria flavonidsATPE was used to separate and purify pueraria flavonids. The effects of the content of drug、p H value、mass fraction of PEG and phosphate to the distribution coefficient(K) and yield(Y) of pueraria flavonids were investigated by single factor experiment. According to the results, optimize the process with orthogonal experiment disign.3. The stability of pueraria flavonids linimentTook a certain amount of pueraria flavonids liniment, the stability was investigated by three aspects, including single factor experiment、constant temperature acceleration test、long-term experiment. To observe wheather the appearance character、content and related substance were altered or not.4. The transdermal permeation of pueraria flavonids linimentFranz diffusion cell and separated rat skin were used to measure the permeation of pueraria. The content of penetration in different time were determined by HPLC and the transdermal absorption rate and cumulation amount were calculated, choose puerarin content as the major quantitative index. The effect of different dosage、high polymer material and azone on the transdermal permeation of pueraria flavonids were observed, respectively.5. The pharmacodynamics study of pueraria flavonoids linimentCervical spondylotic radiculopathy rat model was established by inserting nylon thread to spinal canal. The rats were randomized into 5 groups, including high-dose group, middle-dose group, low-dose group, positive control group and model group. The effects of pueraria flavonoids liniment on behavior and pathological morphology changes were observed.Results:1. The methods of HPLC and UV to determine the content of pueraria flavonids were accurate, sensitive and repeatable.2. The results of single factor experiment indicated that the mass fraction of PEG and phosphate、p H value、the dosage of pueraria flavonids extraction had an effect on the results of ATPE. The optimum aqueous two-phase system is: the total volume was 10 m L, added 3 g pueraria flavonids extraction, the mass fraction of PEG and phosphate were 22% and 18%, the p H value was 7.5. On this condition, the distribution coefficient of pueraria flavonids was 40.19, the yield was 98.05%. The results of variance analysis indicated that the mass fraction of PEG and phosphate had a significant effect on ATPE.3. The stability of pueraria flavonids liniment was good during the experiment time, it was resistant to moisture and light. In order to avoid forming layer, the liniment should keep in cool place.4. The relationship between cumulation amount of pueraria and time was linear, the transdermal absorption rate was 40.69 μg/(cm2·h), cumulation amount of 24 hours was(907.1±22.9) μg/cm2; the absorption rate of liniment had a significant improvement compared with pueraria flavonoids extraction(P<0.05); the promotion effect of azone on percutaneous absorption of pueraria flavonoids was not significant under 3% concentration(P>0.05).5. After modeling, the foreleg of operation side was bent, five fingers huddled together, lifted the feet as soon as touched the ground. After 10 days with medication, the symptoms were not improved in model group. The spontaneous pain value and gait score of pueraria flavonoids liniment in high-dose、middle-dose group and positive control group were decreased, swimming time were increased, the differences were significant compared with model group(P < 0.05), the number of neuron vacuolar degeneration 、 cellular necrosis、axon distortion have been decreased.Conclusion:The content and purity of pueraria flavonids liniment based on ATPE was high, it was resistant to moisture and light, the property of stability and transdermal permeation were good under trial conditions. The animal experiment showed that pueraria flavonids liniment could relieve the radicular pain of cervical spondylotic radiculopathy and improve the damage of nerve,it was expected to be a new dosage form for clinical use. |
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