Study Of Relationship Between Purity Of Evodia Rutaecarpa Extracts And Transdermal Behavior Of EVO And RUT And Its Topical Analgesic Effect

OBJECTIVESThe main bioactive agents from Evodia rutaecarpa extract (EE) are evodiamine and rutaecarpine, which are probably a kind of multi-pathway analgesic by depleting neuropeptides from peripheral nerve terminals and down-regulating the expression and activity of COX-2. However, calcitonin gene-related peptide (CGRP) induced by activating the vanilloid receptor 1 may bring broad side effect. Delivery of pharmacologically active compounds across skin is an attractive alternative. The main aim of this paper was to investigate effect of purities on solubility, apparent partition coefficient and permeation of EVO and RUT from EE and enhancement of chemical enhancers; to prepare and evaluate its topical preparation. And to study the analgesic effect and the In Vivo mechanism of EE topical formulation in hot plate and formalin test and provide experimental basis for a new indication and administration of EE.METHODS AND RESULTS1. Effect of purities on solubility and apprant partition coefficientTo determine solubility and apparent partition coefficient of EVO and RUT from EE with different purities (high, medium and low) by equilibrium method and shaking flask method.EVO and RUT which were contained 71.0%,49.2% and 25.4% in high, medium and low purity extract were dissolved in PEG-400, about 8～14 mg/mL and 13～19 mg/mL respectively, while less than 7 mg/mL in isopropanol. Solvents in sequence of dissolvability of evodiamine and rutaecarpine is PEG-400>Tween-80> RH-40>MCT> ethanol≈1,2-propyleneglycol≈1,3-butyleneglycol≈isopropanol. The range of log of apparent partition coefficient is between 0.6～1.2. Purity is in contrast to the EVO and RUT's dissolvability in hydrophile and lipophilic solvents (p<0.01 or p<0.05) but is irrelevant to that in surfactants and their apparent partition coefficient (p>0.05); pH has no effect on the dissolvability and partition.2. Effect of purities and chemical enhancers on permeationTo establish a HPLC method for the determination of EVO and RUT; to investigate effect of purity, chemical enhancers and stratum corneum on the penetration of EVO and RUT and to elucidate the possible mechanism of penetration enhancement by DSC.The steady state flux of EVO and RUT from EE with different purities is between 0.15～0.18μg/h·cm2 and 0.17～0.21μg/h·cm2, respectively, and the lag time is at 5.46～6.63 h and 7.68～8.83 h, the cumulative penetration amount for 24 h is between 1.88～3.35μg/cm2. Their characteristic of penetration is low transdermal rate, long lag time and little cumulative amount. No significance of EVO or RUT's cumulative amount was observed between different purities extract (p>0.05). The steady state flux and cumulative amount through tape-stripped epidermis were significantly increased (p<0.01 or p<0.05), and the lag time show no difference (p>0.05), indicating that the main transdermal obstacle is the stratum corneum, while diffusion through stripped epidermis is the rate-limiting step. Chemical enhancers with high logP and hydrophilic surfactant can help to promote flux rate and shorten lag time. Penetration enhancerment of chemical enhancers is attributed to the increase of lipid fluidity of stratum corneum.3. The preparation and evaluation of topical formulationSingle factor study was carried out to evaluate the type of emulsion, solvent, HLB value and emulsifier combinations by cream appearance, partical shape and cumulative penetration amount. The optimal prescription was as follows:high-purity EE (0.1%-0.4%), PEG-400 (25%), glyceryl monostearate (1%), octadecanol (8%), nerolidol (5%), Span 60 (4.1%), RH-40(3.9%) and propylene glycol (5%).EE cream has a yellow milky semi-solid and uniform appearance. pH was 6.81±0.04 and the cone penetration was between 102.4-119.7 mm. Drug loading of cream was great improved by adding PEG-400 to the oil phase. PEG-400 in dispersed phase did not transfer to continuous phase. The appearance of cream remain unchanged at the temperature of 40℃and -20℃in vials for ten days. The cumulative penetration amount of EVO and RUT from 0.1%,0.2% and 0.4% cream is drug-loading dependent (p>0.05).4. Analgesic activity and mechanism of EE creamTo investigate the analgesic activity of the topical formulation to heat and chemical stimulus in Kuming mice. Elisa and histological section were taken to evaluate the expresstion of CGRP and PGE2 and the level of inflammatory cell infiltration in aching part skin so as to elucidate the possible analgesic mechamism in vivo.EE cream produced significant drug loading-dependent inhibition of pain response elicited by formalin and hot plate (p<0.01 or p<0.05). Results from Elisa showed that EE cream can markly decrease the expression of CGRP at 3 min after a subcutaneous injection of formalin (p<0.05), and the change of CGRP's expression (p<0.05), while the cream has no effect to the contents of PGE2 (p>0.05). Histological section results indicated that the level of inflammatory cell infiltration can be inhibited by EE cream (p<0.05).The analgesical mechanism was likely to exhaust and interdict conduction of CGRP and cooperate with the inflammatory activity, while the exact inflammatory mechanism need further study.CONCLUSIONSThe solubility of EVO and RUT can be mediated by extract putiry, which has no effect on apparent partition coefficient and penetration. The main transdermal obstacle of EVO and RUT is the stratum corneum, while diffusion through stripped epidermis is the rate-limiting step with the penetration characteristic of low transdermal rate, long lag time and little cumulative amount, which may provide guidance to the design of formulation. Penetration enhancerment of chemical enhancers is attributed to the increase of lipid fluidity of stratum corneum. EE cream possesses analgesic activity, whose mechanism is multiple, namely to exhaust and interdict conduction of CGRP and cooperate with the inflammatory effect.