Cornuside ameliorated experimental autoimmune encephalomyelitis by limiting the recruitment of CD4+ T lymphocytes in the spinal cord

We conducted this study to determine whether cornuside could improve the neurological deficit symptoms of experimental autoimmune encephalomyelitis (EAE) rats, as well as determine the potential involvement of CD4+ T lymphocytes, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and tumor necrosis factor-α (TNF-α). Altogether, 32 Lewis rats were randomly divided into control, EAE, EAE/prednisolone, and EAE/cornuside, wherein their neurological function was assessed every day. CD4+ T lymphocyte recruitment into the spinal cord (SC) was evaluated using immunohistochemistry. The VCAM-1, ICAM-1 and TNF-α mRNA expressions in the SC were determined by real-time quantitative PCR, and the VCAM-1 and ICAM-1 proteins were determined by western blotting. Compared to the control group, the EAE group rats with neurological deficits had enhanced CD4+ T lymphocyte infiltration and higher expression levels of VCAM-1, ICAM-1, and TNF-α in the SC. Meanwhile, compared with the EAE group, the EAE/cornuside and EAE/prednisolone groups had lower neurological scores, less CD4+ T lymphocyte infiltrations, and lower expression levels of VCAM-1, ICAM-1, and TNF-α in the SC. Thus, cornuside ameliorated EAE, which could be owed to the inhibition of CD4+ T lymphocyte recruitment and VCAM-1, ICAM-1, and TNF-α expressions in the SC

Cornuside alleviates experimental autoimmune encephalomyelitis by inhibiting Th17 cell infiltration into the central nervous system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The present study was conducted to clarify the therapeutic effect of cornuside on experimental autoimmune encephalomyelitis (EAE) and its influence on T helper 17 (Th17) cell and regulatory T (Treg) cell infiltration into the central nervous system. Rats were randomly placed into four treatment groups: control, EAE, EAE+cornuside, and EAE+prednisolone. The neurological function scores of rats were assessed daily. On the second day after EAE rats began to show neurological deficit symptoms, the four groups were treated with normal saline, normal saline, cornuside (150 mg/kg), and prednisolone (5 mg/kg), respectively. The treatment was discontinued after two weeks, and the spinal cord was obtained for hematoxylin and eosin (H&E) and luxol fast blue staining, as well as retinoic acid receptor-related orphan receptor γ (RORγ) and forkhead box protein P3 (Foxp3) immunohistochemical staining. Blood was collected for Th17 and Treg cell flow cytometry testing, and the serum levels of interleukin (IL)-17A, IL-10, transforming growth factor-β (TGF-β), IL-6, IL-23, and IL-2 were measured via enzyme-linked immunosorbent assay (ELISA). Compared with rats in the EAE group, rats in the EAE+cornuside and EAE+prednisolone groups began to recover from neurological deficits earlier, and had a greater degree of improvement of symptoms. Focal inflammation, demyelination, and RORγ-positive cell infiltration were reduced by cornuside or prednisolone treatment, whereas the Foxp3-positive cell numbers were not significantly different. Meanwhile, the number of Th17 cells and the IL-17A, IL-6, and IL-23 levels were lower in the blood after cornuside or prednisolone treatment, whereas the number of Treg cells or the levels of IL-10, TGF-β, and IL-2 were not markedly different. Cornuside can alleviate symptoms of EAE neurological deficits through its anti-inflammatory and immunosuppressive effects, and Th17 cells may be one of its therapeutic targets.

Simultaneous determination of five constituents in Shanzhuyu Formulated Granules by QAMS / 中成药

AIM To establish a quantitative analysis of multi-components by single mark (QAMS) method for the simultaneous content determination of five constituents in Shanzhuyu Formulated Granules (Corni Fructus).METHODS The analysis of 80％ methanol extract of this drug was performed on a 30 ℃ thermostatic Wondasil C18column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.3％ phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 335 nm.With morroniside as an internal standard,the relative correction factors of gallic acid,5-hydroxymethylfurfural,loganin and cornuside were established,followed by the determination of their contents.RESULTS Gallic acid,5-hydroxymethylfurfural,morroniside,loganin and cornuside showed good linear relationships within the ranges of 0.0120-0.120,0.026 8-0.268,0.074 4-0.744,0.058 6-0.586 and 0.0086-0.086 μg (r≥ 0.9999),whose average recoveries (RSDs) were 103.43％ (1.45％),103.36％ (1.50％),104.47％ (0.30％),102.08％(1.74％) and 104.01％ (0.62％),respectively.The results obtained by QAMS approximated those obtained by external standard method.CONCLUSION This stable and reliable method can be used for the quality control of Shanzhuyu Formulated Granules.

Simultaneous determination of five constituents in Shanzhuyu Formulated Granules by QAMS / 中成药

AIM To establish a quantitative analysis of multi-components by single mark (QAMS) method for the simultaneous content determination of five constituents in Shanzhuyu Formulated Granules (Corni Fructus).METHODS The analysis of 80％ methanol extract of this drug was performed on a 30 ℃ thermostatic Wondasil C18column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.3％ phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 335 nm.With morroniside as an internal standard,the relative correction factors of gallic acid,5-hydroxymethylfurfural,loganin and cornuside were established,followed by the determination of their contents.RESULTS Gallic acid,5-hydroxymethylfurfural,morroniside,loganin and cornuside showed good linear relationships within the ranges of 0.0120-0.120,0.026 8-0.268,0.074 4-0.744,0.058 6-0.586 and 0.0086-0.086 μg (r≥ 0.9999),whose average recoveries (RSDs) were 103.43％ (1.45％),103.36％ (1.50％),104.47％ (0.30％),102.08％(1.74％) and 104.01％ (0.62％),respectively.The results obtained by QAMS approximated those obtained by external standard method.CONCLUSION This stable and reliable method can be used for the quality control of Shanzhuyu Formulated Granules.

Optimization of steaming time of Cornus officinalis by QAMS combined with electronic-eye and electronic-tongue techniques / 中草药

Objective: To establish a method of quantitative analysis of multi-components by single marker (QAMS) for medicinal materials and pieces of Cornus officinalis. This method was used in combination with electronic-eye and electronic-tongue technique, and the best steaming time of Cornus officinalis was selected. Methods: Medicinal materials and pieces of C. officinalis were used as the research objects. The contents of five components were determined by establishing the relative correction factor (RCF) of gallic acid, 5-hydroxymethyl furfural (5-HMF), morroniside, cornuside, and internal reference loganin in C. officinalis. Color and taste were measured by electronic eye and electronic tongue technique. The data were analyzed by principal component analysis (PCA), and the best steaming time was optimized by analyzing the results of three methods. Results: The five compounds were well separated. The RSD values of precision and reproducibility were all less than 2%. The stability was good in 24 h. The linear relationship among the concentration and peak areas of the five compounds was all linear (r ≥ 0.999 6). The average recoveries were between 98% and 100.1% and the RSD values were all less than 2%; The RCFs of loganin with the other four compounds were 0.560, 1.344, 1.255, and 0.972 in a linear range. In the principal component analysis (PCA), the sums of main components were 94.618% and 94.98% and the discrimination indexes (DI) were 98 and 93, which indicated that all the samples of C. officinalis could be distinguished well by the electronic-eye and the electronic-tongue. The results showed that the optimum steaming time of C. officinalis was 4 h. Conclusion: The best steaming time of C. officinalis can be optimized by the combination of QAMS with electronic-eye and electronic-tongue techniques.

Simultaneous determination of morroniside, loganin, cornuside, oleanolic acid, and ursolic in Corni Fructus terpenoids medicinal preparation base on RP-HPLC / 中草药

Objective: To establish RP-HPLC strategy to simultaneously determine the terpenoids constitute of Corni Fructus preparation which is useful for the intervention of acute immunological liver injury in mice induced by concanavalin A (ConA), including morroniside, loganin, cornuside, oleanolic acid and ursolic, thus providing a scientific basis on the quality controls of Corni Fructus terpenoids and related medicinal preparations. Methods: RP-HPLC method and Zorbax SB-C18 column (150 mm × 4.6 mm, 5 μm) chromatographic column were employed; 2 mmol/L γ-cyclodextrin was added into the mobile phase containing acetonitrile and 0.1% ortho-phosphoric acid for gradient elution; The volume flow was 1.0 mL/min, the column temperature was 30 ℃ and the detection wavelengths were 240, 360, and 210 nm; The injection volume was 3 μL. Results: The linear range in morroniside, loganin, cornuside, oleanolic acid, and ursolic acid was 10.42—333.33, 23.44—750.00, 9.11—291.67, 10.42—333.33, and 13.02—416.67 mg/L, respectively; The average recovery in the selected samples was 95.60%—98.02%, RSD was 1.47%—1.89%; The repeatability RSD was 1.46%—1.71%; The stability RSD was 1.29%—1.76%; Six batches of the Corni Fructus terpenoids medicinal preparation contained the average quality of morroniside, loganin, cornuside, oleanolic acid, and ursolic acid respectively was 669.6—680.2, 850.1—869.5, 94.1—96.4, 164.3—166.1, and 85.6—87.6 mg/L. Conclusion: The method established in this study is a credible way to determine the concents of morroniside, loganin, ornuside, oleanolic acid, and ursolic acid in the Corni Fructus terpenoids medicinal preparation with its simplicity, good repeatability, high sensibility and recovery rate.