| Centella asiatica (L.) Urb., the entire plants of Centella asiatica of Umbelliferae, hasthe effects of clearing away heat evil and promoting diuresis and detumescence bydetoxification. Its major components are triterpenes, flavones, polyacetylene alkenes andessential oils, and it has been used for the remedy for damp jaundice, heatstroke anddiarrhea, urinary sandy calculi and bloody stranguria, carbuncle and bruises. The recentreports indicate that the total saponins whose major active constituents are madecassosideand asiaticoside and essential oils whose major active constituents is β-caryophyllene haveantidepressive effect in Centella asiatica. In the present paper, the antidepressivecomponents of Centella asiatica (L.) Urb. were chosen as the subject to be researched fromthe following aspects:1. Near-infrared spectroscopy technology for online monitoring of the columnseparation and purification process of madecassoside and asiaticoside of Centella asiatica(L.) Urb.At first, determination method of madecassoside and asiaticoside was developedusing HPLC method. Aqueous extract of Centella asiatica (L.) Urb. was obtained usingdecocting method and was separated by D101macroporous resin, then was eluted tocolorless in order by water,0.1%NaOH-10%ethanol solution and10%ethanol solution.10mL50%-ethanol eluant of Centella asiatica (L.) Urb. extract was as one samplecollected every five minutes, then the NIR spectra of samples were detected and thecontents of madecassoside and asiaticoside in samples were determined by HPLC.Combination with PLS, the quantitative analysis models of the above two compounds wereestablished. The results showed that R2and RMSECV of madecassoside were96.44and0.08480, respectively, and96.07and0.00099were respectively for asiaticoside. Theaccuracy and predicted results of the model was satisfactory. This method was proved to befast, convenient and precise. It can be used in online monitoring and quality control of themanufacturing of madecassoside and asiaticoside.2. Study on chemical components by GC-IT-MS and in vitro anti-oxidant activity ofessential oils from Centella asiatica (L.) Urb.A GC-IT-MS technique was developed to analyze chemical components of essentialoils of Centella asiatica (L.) Urb. extracted by steam distillation method.45compoundswere characterized, which were mainly terpenes including monoterpene, sesquiterpenes and their oxygenated derivatives. With VitC as the positive control drug, the scavengingability of essential oils on·OH, DPPH and ABTS+radical was investigated. As a result,essential oil showed significant anti-oxidant activities on·OH, DPPH and ABTS+radicaland the IC50were0.5629,2.6643and0.6683mg/mL, respectively.3. Preparationand physicochemical characterization ofβ-caryophyllene/β-cyclodextrin inclusion complexThe β-caryophyllene/β-cyclodextrin inclusion complex was prepared by thecoprecipitation method and characterized by different analytical techniques includingUV-vis, DTA and FT-IR. The inclusion ratio of β-caryophyllene was62.04%, which wasdetermined by steam distillation method. According to the Ch.P (2010Edition, Part2,Appendix XC. No.2method), in vitro dissolution study of the inclusion complex wasrespectively performed in the mediums of hydrochloric acid (0.1mol/L) and phosphatebuffer (pH6.8). After90min, approximately90%and60%of β-caryophyllene werereleased from inclusion complex into the above two mediums, respectively, denoting thatbioavailability of β-caryophyllene in inclusion complex would be improved than that offree β-caryophyllene.4. Study on pharmacokinetics of β-caryophyllene in SD RatA fast and sensitive GC-MS/SIM quantitative method was developed for thedetermination of β-caryophyllene in SD rat plasma and tissues. The pretreatment methodof biological samples was LLE. According to pre-clinical research technical guidelines forchemicals, we carried out overall methodological evaluation. As a result, no endogenoussubstances in biological samples interfered with the assay of β-caryophyllene. Thecalibration curve showed good linearity within the range of0.053-5.30μg/mL. And theaccuracy, precision, absolute recovery, matrix effect and stability were satisfied for therequirements of the guidelines. The method could be used for the pharmacokinetic study ofβ-caryophyllene on SD rat.The validated GC-MS/SIM method was used to determine the concentration ofβ-caryophyllene in rat plasma after a single oral dose of free β-caryophyllene (50mg/kg)or different doses of β-caryophyllene/β-cyclodextrin inclusion complex (25mg/kg,50mg/kg,100mg/kg). The pharmacokinetic parameters of both formulations were calculatedby non-compartmental model and pharmacokinetic feature of β-caryophyllene wasidentified. With free β-caryophyllene (50mg/kg) as reference, mean relative bioavailability(AUC0-12h) of β-caryophyllene after administration of β-caryophyllene/β-cyclodextrin inclusion complex (50mg/kg) was264.63±126.54%, suggesting that bioavailability ofβ-caryophyllene in inclusion complex increased about2.6times than that of freeβ-caryophyllene. The AUC0-12hof β-caryophyllene presented the positive correlationrelationship with the doses after administration of inclusion complex (25mg/kg,50mg/kg,100mg/kg) and the r was0.9989, which indicated that pharmacokinetic process ofβ-caryophyllene in SD rat was belong to linear pharmacokinetics.In order to understand its feature of tissue distribution and effect target organ, theconcentration of β-caryophyllene in different tissues was determined by GC-MS/SIM aftera single oral dose of inclusion complex50mg/kg on SD rats. As a result, the concentrationof β-caryophyllene in all tissues reached to the maximum value at3h. Among them, itsconcentration in liver was the highest, less in heart, kidney and brain, and least in spleenand lung. The content of β-caryophyllene in liver was the most which was related toabundant blood flow and the relative more in brain prompted that brain might be its effecttarget organ, which was corresponding to its biological activity. |
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