| Objective: Separation and purification of iridoid glycosides and crocetin derivatives from Gardenia jasminoides Ellis and five alkaloids from Aconitum duclouxii Levl by HSCCC. UPLC determination of geniposide and crocin-1 in six Chinese patent drugs. A study on the Neuroprotective effect and mechanism of Gardenia jasminoides Ellis to MPTP induced Parkinson’s disease mouse model.Methods:(1) Separation and purification of iridoid glycosides and crocetin derivatives from Gardenia jasminoides Ellis and five alkaloids from Aconitum duclouxii Levl by HSCCC. Four factors, which inculd the two-phase solvent system, the flow rate of mobile phase, revolution speed and temperature were optimized. The purity of obtained compounds were determined by UPLC and identified by 1H-NMR,13C-NMR and UPLC-QTOF-MS.(2) Pure geniposide and crocin-1 were used as standards for UPLC determination of samples, geniposide and crocin-1 concentration in six Chinese patent drugs(Qinghuozhimai Pian, Zhizijinhua Wan, Fukefenqing Wan, Qiongjushangqing Pian, Jieyuanshen Keli and Lingyangqingfei Keli) were measured.(3) Mice PD model was established by intraperitoneal injection of MPTP. C57 Mice were randomly divided into control, MPTP and treatment groups(25, 50, 100 mg/Kg). Behavior effects of GJE were tested by Rotarod, Pole, Pulling Force. IHC test for TH positive neurons were performed to study the neuroprotective effects of GJE. The levels of DA and its metabolites in striatum were determined by UPLC-QTOF-MS. Peripheral immunomodulatory responses were also investigated.Results:(1) n-butanol:ethanol:water(10:1:10, v/v), ethyl acetate:n-butanol:water(2:1.5:3, v/v), hexane:ethylacetate:n-butanol:water(1:2:1:5, v/v), hexane:ethylacetate:methanol:water(0.3:1:0.3:1, v/v) was selected to purify gardenoside, 6β-hydroxy geniposide, geniposidic acid, geniposide, crocin-1, crocin-2, crocin-3, crocin-4 and gardecin from Gardenia jasminoides Ellis. Their purities were determined to be 91.7%, 93.4%, 92.5%, 98.2%, 94.1%, 96.3%, 94.1%, 98.9% and 97.4% by UPLC.(2) The two-phase solvent system composed of n-hexane: ethyl acetate: methanol:Water:NH3·H2O(25%)(1:1:1:1:0.1, v/v) was selected and benzoylaconine, N-deethylaconitine, aconitine, deoxyaconitine and ducloudine A were obtained from Aconitum alkaloids extract. Their purities were determined to be 98.4%, 97.2%, 98.2%, 96.8%, and 96.6% by UPLC.(3) The concentration of geniposide and crocin-1 were 3.58, 5.32, 2.17, 4.57, 1.23, 0.38 mg/g and 1.52, 1.06, 0.35, 0.55, 0.068, 0.099 mg/g in six Chinese patent drugs. The two compounds were well separated. The accuracy,precision and stability of method were high.(3) Remarkable behavioral changes showed on MPTP group, the landing time of pole test and pulling force of MPTP group were significantly lower than control group(P<0.05), The landing time of Rotarod were significantly longer than control group(P<0.001). For group of MPTP, the number of TH positive neuron in SNpc area underwent a significantly dropping down compare with the control(P<0.01). MPTP injection led to a significant reduction of DA and its metabolites levels in striatal, compared to the saline-treated control mice(P<0.001). MPTP treatment caused marked elevations of inflammatory cytokine level in serum(P<0.001), a significant reduction of DAO level in serum(P<0.001), compared to the control group. The results of this study indicated that GJE reduced bradykinesia(P<0.05), inhibited the loss of striatal dopamine and its metabolites(P<0.05), and led to an increase in tyrosine hydroxylase in PD mice(P<0.01). It also significantly suppressed the inflammatory cytokine levels in serum(P<0.01). Furthermore, the gut immune biomarkers such as serum diamine oxidase were significantly enhanced by GJE treatment(P<0.01).Conclusion:(1) The present results demonstrate that the main iridoid glycosides and crocetin derivatives in Gardenia can be obtained efficiently from extracts using HSCCC. This work makes a foundation for further studies on the pharmacological research and quality control of Gardenia jasminoides Ellis fruits.(2) The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloidsare suitable for biochemical and toxicological investigation in Aconitum duclouxii Levl.(3) This study establishes a UPLC method for the simultaneous determination of geniposide and crocin-1 in 20 minutes. This method is precise,time-saving,stable and suitable for the quality control of geniposide and crocin-1 in the Chinese patent drugs.(4) The GJE could significantly improve the behavior impairment induced by MPTP, plus it could inhibit decreasing of the TH positive neuron and the loss of striatal dopamine and its metabolites. Probably because of blocking activation of microglia and the expression of inflammatory cytokine, GJE against impaired DA neurons and protect DA neurons. |
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