Study On Transdermal Preparation Of Modified Total Alkaloid Fraction From Nux Vomica

In this paper, the extraction of the total alkaloid fraction （TAF） from nux vomica was established. Most strychnine was removed from TAF and the resulted modified total alkaloid fraction （MTAF） was obtained. Then a systematic determination of chemical composition and correlation studies were conducted:the determination of alkaloid composition of TAF and MTAF and fingerprint research. In contrast to TAF, the ratio of brucine to strychnine was adjusted from1:1.76to2.76:1in MTAF. Strychnine was removed by optimization to a great extent, but brucine and other alkaloids of Strychnos nux-vomica L. were substantially retained with little loss. Acute toxicity test was carried out to determine toxicity of MTAF and TAF by intravenous route. The results showed that LD⁵⁰ of MTAF and TAF were3.0870mg/kg and0.6330mg/kg, respectively. The toxicity was reduced near five times post optimization. Thus MTAF may possess more or equally potency with less toxicity compared with TAF, indicating the process was feasible.Ultrafiltration was employed to determine the rat plasma protein binding rate of alkaloids from the seed of Strychnou nux-vomica L.. The plasma concentrations were measured by RP-HPLC. Brucine and strychnine have medium capacity in binding to plasma protein. In comparison with the single components of the same concentration, the protein binding rate of brucine in TAF and MTAF shows little difference, while there seems to be an obvious decrease in strychnine. Competitive binding of plasma proteins arised from various components of effective fraction of Strychnou mix-vomica L., which provided a theoretical basis for clinical use.Considering that transdermal drug delivery formulations is an ideal dosage form, which can ensure therapy, significantly reduce toxicity, and quite suitable for clinical use at the same time, so hydrogel was used as a carrier to evaluate and compare the analgesic and anti-inflammatory activity of TAF, MTAF, brucine and strychnine dissolved in hydrogel after transdermal administration. Hot plate test、acetic acid-induced writhing response、tail pressure test were used to evaluate analgesic activity in all the chemical-, thermal-and physical-induced nociception models, xylene-induced ear edema was used to evaluate anti-inflammatory activity. It showed that TAF and strychnine demonstrated little activity in all those pharmacological tests, MTAF hydrogel exhibited the most potent analgesic and anti-inflammatory activity than the other three, which was far less toxic while possibly enhancing its therapeutic efficacy.Then the pharmacokinetic properties and skin permeation in vitro of brucine, TAF and MTAF after transdermal administration in mice were compared. It was obvious that brucine in MTAF was absorbed more completely than it alone at the same dosage of brucine after transdermal administration. The AUC^o-t of MTAF group was1.74and1.89times higher than the corresponding values of brucine and TAF group, respectively. The Cmax of MTAF group was2.09and2.42times higher than the corresponding values of brucine and TAF group, respectively. And the results indicated that TAF and MTAF exhibited an obvious promotion of skin permeation of brucine compared to brucine alone.On the basis of transdermal patch previously, MTAF patch of Strychnou nux-vomica L. was prepared. The whole content of MTAF were calculated by UV, the content of brucine and strychnine were determined by HPLC. Then in vitro skin permeation was conducted. After24h permeation, MTAF patch exhibited an constant and stable release of the drug, the cumulative amount of brucine were （62.95±0.44）%,and the accumulation in skin of brucine associated with brucine hydrogel reached （158.0±2.78） p.g/g, and the residue in patch was（75.2±3.11）μg/cm2.In accordance with the2010Pharmacopoeia of people’s republic of China, the MTAF patch was examined by quality criteria, including the content of mastic, limit of weight variation, recontour, adhesion and initiatory stability,the characterization was stable and controllable.In addition, complete Freund’s adjuvant （FCA） was used to induce adjuvant arthritis（AA） in rats. This study was to investigate the therapeutic effect of MTAF patch of three dosages on AA using the diclofenac diethylamine hydrogel as the positive drug. It showed that the high dose group has the best effect, which significantly attenuated the primary and secondary hind paw swelling, as well as improved pathology alteration. Meanwhile, it regulated the splenic T and B lymphocyte proliferation response induced by concanavalin A （ConA） or lipopolysaccharide （LPS）, remarkably down-regulated TNF-α productions from PMΦ, and may not work by the route of regulating IL-1β.These results indicated that the anti-inflammatory mechanism of MTAF patch on AA rats were mediated by modulating the immune function of abnormal cells and the balance of cytokines, which laid a sound foundations for AA in clinical therapy.