Purification And Identification Of Syringin And Its Effects On Proliferation And Th1 Cytokines Of Con A-stimulated Mouse Lymphocyte

Objective: To separate and purify Syringin from Acanthopanax Senticosus by gel column chromatography, Sephadex LH20 chromatography and preparative high performance liquid chromatography(PHPLC). To research the effects of Syringin on the Con A-stimulated mouse lymphocytes proliferation and Th1 cytokines preliminary.Methods:(1) The dried Acanthopanax Senticosus were ground to a coarse powder and extracted with 70% ethanol. The 70% alcohol extract was subjected to HPD100 macroporous resin, the 10% alcohol fraction were obtained. Then the 10% alcohol fraction were subjected to Sephadex LH20 chromatography and the fraction A-1 and A-2 were obtained. Afterwards, through the optimization of PHPLC condition, fraction A-1 was concentrated with methanol-water gradient elution as the two mobile phase solvent and compound 1 was obtained. Then the purity of obtained chemical was determinated by UPLC, the structure of obtained component was identified by ESI-QTOF-MS, nuclear magnetic resonance hydrogen(1HNMR) and nuclear magnetic resonance carbon(13CNMR).(2) The survival rate of the normal mouse lymphocytes after treatment with Syringin within 2-256 μg/m L concertration range was detected by MTT. Then, the high, medium, low concertration groups were determined according to reference literature.(3) Experiment was divided into five groups, namely control, Con A and high, medium, low dose groups stimulated by Con A. The effects of Syringin on Con A-stimulated mouse lymphocytes proliferation were investigated by MTT. The effects of Syringin on expressions of cytokines IL-2, IFN-γ, TNF-α which secreted in Con A-stimulated mouse lymphocytes were determined by ELISA. The effects of Syringin on expressions of cytokines IL-2 m RNA, IFN-γ m RNA, TNF-α m RNA which secreted in Con A-stimulated mouse lymphocytes were determined by RT-q PCR.Results:(1) The dried Acanthopanax Senticosus was separated using HPD100 column chromatography and Sephadex LH20 chromatography, the fraction A-1 was obtained, compound 1 was obtained by PHPLC with methanol-water gradient elution as thetwo-phase solvent. The purity was determined by UPLC is 98.9%. The structure of the compound 1 was identified by ESI-QTOF-MS, 1HNMR and 13 CNMR to be Syringin.(2) The survival rate of mouse spleen lymphocytes were above 88% within 2-256 μg/m L concertration range of Syringin. Confirm high, medium, low concertration groups of Syringin as 256μg/m L、128μg/m L、64μg/m L.(3) MTT measurement results showed that compared with control group, Con A group can significantly promote the mouse lymphocytes proliferation, the proliferation rate is 91.77%, with significant difference(P<0.05). Compared with Con A group, different concertration groups significantly inhibite the mouse lymphocytes proliferation stimulated by Con A, the proliferation rate is 21.58%、26.09% 、 33.85% respectively, with significant difference(P<0.05).(4) ELISA measurement results showed that compared with control group: TNF-α(17.768±3.389 pg/m L)、IFN-γ(18.897±8.144 pg/m L)、IL-2(21.813±11.923 pg/m L), the expression of TNF-α(105.079±8.035 pg /m L)、IFN-γ(107.960±9.032 pg/m L)、IL-2(84.017±9.903 pg/m L) in Con A group were increased, with significant difference(P<0.05). Compared with Con A group, the expressions of different Syringin concertration groups on cytokines TNF-α(76.964±9.121pg/m L 、 32.539±7.044 pg/m L 、 27.96±8.513 pg/m L) 、 IFN-γ(72.823±10.317 pg/m L 、 46.951±11.504 pg/m L 、 40.123±12.884 pg/m L) 、 IL-2(41.113±9.981 pg/m L、31.795±8.762 pg/m L、 26.478±10.589 pg/m L) which secreted in Con A-stimulated mouse lymphocytes were decreased, with significant difference(P<0.05).(5) RT-q PCR measurement results showed that compared with control group: TNF-α m RNA(1.003±0.031)、IFN-γ m RNA(1.003±0.089)、IL-2 m RNA(1.002±0.075), the expression of TNF-α m RNA(2.547±0.184)、IFN-γ m RNA(2.378±0.218)、IL-2 m RNA(3.089±0.137) in Con A group were increased, with significant difference(P<0.05). Compared with Con A group, the expressions of different Syringin concertration groups on cytokines TNF-α m RNA(2.281±0.1911、1.781±0.122、1.391±0.075)、IFN-γ m RNA(1.995±0.0211、1.647±0.124、1.239±0.072)、IL-2 m RNA(1.819±0.085、1.648±0.090、1.182±0.038) which secreted in Con A-stimulated mouse lymphocytes were decreased, with significant difference(P<0.05).Conclusion:(1) The new technique of combination of gel column chromatography, Sadex LH20 chromatography Sephadex and PHPLC can isolate the Syringin from Acanthopanax Senticosus, and the purification is 98.9%, the method is efficient and reagent-saving.(2)The Syringin can inhibite the Con A-stimulated mouse lymphocytes proliferation, reduce the expressions of TNF-α, IFN-γ, IL-2 and TNF-α m RNA, IFN-γ m RNA, IL-2 m RNA stimulated by Con A. Syringin may play a role of immunosuppressive by reducing the expression level of IL-2, INF-γ, TNF-α.