| As part of national economic initiative, manned spaceflight has become one ofmajor national projects in China. During the space flight, space radiation is verydangerous for human health. Thus, it’s important to keep healthy and improve theadaptability and tolerance of the astronauts when exposure to the outer spaceradiation.Crassulaceae Rhodiola L. plants are perennial herbage or subshrubs plants withsignificant anti-radiation, antioxidant and immune adjustment effects. Current studiesis only limited to extracts or Salidroside and few investigation has been conducted ontheir other ingredients and mechanism of action. Phenethyl and phenylpropanoidglycosides are two key active ingredients in Rhodiola L.. Due to their uniquebiological activity, Phenethyl and phenylpropanoid glycosides, including Salidroside,Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol have been drawn great attentions bymany researchers.In this paper, the antioxidant and immune protective effect of glycosides （aglycone）in Rhodiola were investigated for its application in radiation protection. In vitroantioxidant models and radiation-induced cell damage model were established to findits anti-radiation active ingredients. In addition, using Caco-2cell model, thepermeability and mechanism of transportation of the anti-radiation active ingredientswere investigated. Their potential as a radiation protective agent were evaluated.These studies have firstly investigated the oral absorption and bioavailability of itsanti-radiation active ingredients for the further development of these ingredients aspotential radio-protectors. The main studies were conducted as followed:1. Based on the anti-oxidative radiation protection mechanism using free radicalscavenging, six in vitro antioxidant models were built and optimized foranti-oxidative effect of glycosides （aglycone） in Rhodiola.Generally, the radiation can cause the ionization and excitation of the targetbiomolecule which lead to the production of reactive oxygen species （ROS）. TheseROS, including superoxide anion and hydroxyl radical et al., may contribute to radiation-induced injury of nucleic acid, protein and biofilm. The main mechanism ofradiation protection is clearing the genera ROS or restraining its production. In thisstudy, six in vitro antioxidant models （include DPPH radical scavenging assay,superoxide radical scavenging assay, hydroxyl radical scavenging assay, NO radicalscavenging assay, anti-lipid peroxidation assay, and total antioxidant activity） wereestablished and optimized. The antioxidative activities and the mechanisms action ofRutin, Salidroside, Isoquercitrin, Tyrosol, Rosarin, Arbutin, Rosavin and Cinnamylalcohol （six glycosides and two aglycones） were investigated. These studies haveshowed that eight glycosides （aglycone） showed anti-oxidative activity. Salidroside,Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol exhibited strong hydroxyl radicalscavenging activity (IC50value were18.97μg·mL-1,10.84μg·mL-1,61.84μg·mL-1，81.08μg·mL-1,26μg·mL-1). Rutin and Isoquercitrin showed strong DPPH radical,superoxide radical scavenging and anti-lipid peroxidation and total antioxidativeactivity. Arbutin showed DPPH scavenging activity and total anti oxidative activity.2. Based on the immune protection of radio-protectors, in vitro anti-radiation modelwas established using cellular and biomedical methods. Glycosides （aglycone） inRhodiola were evaluated for anti-radiation at the cellular level.Immune system is an important target organ of radiation, which are highly radiationsensitive. Radiation-induced lymphocytes damage will cause the suppression ofimmune function, so protection of lymphocytes from radiation damage is of important.In this study, in vitro anti-radiation model was established by comparing cellproliferation assays, investigating cell radio-sensitivity and investigating therelationship between proliferation and irradiation dose. Followed by toxicity studies,Lymphocytes （AHH-1） were exposed to10Gy60Co γ-ray to induce radiation injurymodel, and then MTS assay was used to detect the proliferation activity of AHH-1after treated by Rutin, Salidroside, Isoquercitrin, Tyrosol, Rosarin, Arbutin andRosavin. Comparing with the model groups, Salidroside (12.5μg.mL-1, P <0.05),Isoquercitrin (12.5μg.mL-1, P <0.05), Rosavin (12.5μg.mL-1, P <0.01), Rosarin (25μg.mL-1, P <0.05), Arbutin (12.5μg.mL-1, P <0.01) and Tyrosol (50μg.mL-1, P <0.05)could significantly improve the viability of radiation-induced AHH-1.3. Caco-2permeability and transportation assay was established to investigate thepermeability and mechanism of transportation of the anti-radiation active ingredients.The reliability of Caco-2cell monolayer was evaluated by cell morphology,transepithelial electrical resistance （TEER） and standard markers. Results showed that TEER steady raised in developing process of cell monolayer, and reached the standardvalue（>500Ω·cm2）.The Papp value of Atenolol (low permeability, Papp=2.20×10-7cm·s-1) and Propranolol (high permeability, Papp=1.01×10-5cm·s-1) were all withinthe reported range. So Caco-2cell model was validated for the study of the evaluationof the intestinal absorption. Anti-radiation active ingredients, including Salidroside,Tyrosol, Rosarin and Rosavin were selected for the studies of intestinal toxicity,absorption and transportation using Caco-2permeability and transportation model.The24h cytotoxicity of anti-radiation active ingredients was investigated. Intestinaltoxicity was evaluated and safe dose was calculated. In addition, bidirectionaltransport assay was also conducted using Caco-2monolayer model. Effects oftransport time （60min-180min） and drug concentration （50μM-200μM） wereinvestigated. The Papp values were determined by RP-HPLC methods. Salidroside(Papp=0.85±0.12×10-6cm·s-1), Rosarin (Papp=0.61±0.10×10-6cm·s-1) and Rosavin(Papp=0.74±0.12×10-6cm·s-1) were poor-absorbed ingredients (PappAPâ†'BL<1×10-6cm·s-1). While Tyrosol (Papp=1.52±0.16×10-6cm·s-1) showed higher permeabilitythan its glycosides Salidroside. The bidirectional transport of4glycosides （aglycone）at200μM was comparable, indicating a lack of directional preference; it was alsolinear with time and concentrations, suggesting a passive diffusion mechanism duringtheir transportation across the Caco-2cell monolayer. The poor-absorption ofSalidroside may be related to its efflux.In summary, glycosides （aglycone） in Rhodiola showed anti-oxidative activity.Salidroside, Tyrosol, Rosarin, Rosavin and Cinnamyl alcohol showed strong hydroxylradical scavenging. Comparing with the model groups, Salidroside, Isoquercitrin,Rosavin, Rosarin, Arbutin and Tyrosol can improve the viability of radiation-inducedAHH-1. The results suggested that salidroside, tyrosol, Rosavin and Rosarin wereanti-radiation active ingredients in Rhodiola. Salidroside, Rosarin and Rosavin werepoor-absorbed ingredients. There was higher permeability for Tyrosol than for itsglycosides Salidroside. |
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