Radioprotective properties of apple polyphenols: an in vitro study.

Present study was undertaken to evaluate the radioprotective ability of total polyphenols extracted from edible portion (epicarp and mesocarp) of apple. Prior administration of apple polyphenols to murine thymocytes significantly countered radiation induced DNA damage (evaluated by alkaline halo assay) and cell death (trypan blue exclusion method) in a dose dependent manner maximally at a concentration of 2 and 0.2 mg/ml respectively. Apple polyphenols in a dose dependent fashion inhibited both radiation or Fenton reaction mediated 2-deoxyribose (2-DR) degradation indicating its ability to scavenge hydroxyl radicals and this activity was found to be unaltered in presence of simulated gastric juice. Similarly apple polyphenols in a dose dependent fashion scavenged DPPH radicals (maximum 69% at 1 mg/ml), superoxide anions (maximum 88% at 2 mg/ml), reduced Fe(3 +) to Fe(2 +) (maximum at 1 mg/ml) and inhibited Fenton reaction mediated lipid peroxidation (maximum 66% at 1.5 mg/ml) further establishing its antioxidative properties. Studies carried out with plasmid DNA revealed the ability of apple polyphenols to inhibit radiation induced single as well as double strand breaks. The results clearly indicate that apple polyphenols have significant potential to protect cellular system from radiation induced damage and ability to scavenge free radicals might be playing an important role in its radioprotective manifestation.

Induction of DNA-protein cross-links by Hippophae rhamnoides: implications in radioprotection and cytotoxicity.

Recently Hippophae rhamnoides has been reported to render chromatin compaction and significantly inhibit radiation induced DNA strand breaks. To investigate the mechanism of action of RH-3, a preparation of Hippophae rhamnoides, in this connection, present study was undertaken. Chromatin compaction induced by RH-3 (100 microg/ml or more) was maximum at alkaline pH but was completely negated by acidic pH (< 6) or presence of free radical scavengers like glycerol, DMSO etc. In a concentration dependent manner, RH-3 inhibited the intercalation of ethidium ions from Et Br into calf thymus DNA and also increased the precipitation of DNA-protein cross-links (DPC) in thymocytes. Chromatin compaction caused by RH-3 treatment did not permit the separation of proteins from DNA even after treatment with 2 M NaCl solution. SDS-PAGE profiles also revealed that RH-3 in a dose dependent manner compacted the chromatin organization, induced DPC and inhibited the extraction of both histone and non-histone matrix proteins from chromatin maximally at 80 microg/ml. More than 80 microg/ml of RH-3, though extracted low molecular weight histones but did not separate non-histone proteins. The RH-3 mediated DPCs were resistant even to 1% SDS, 4 M NaCl and 3.8 M hydroxyl amine hydrochloride but were prone to both urea (8 M) and guanidine hydrochloride (6 M) indicating covalent bonding between DNA and proteins (serine/threonine). RH-3 in a concentration dependent manner induced superoxide anions and the phenomenon was dependent upon nature of medium, presence of metal ions and pH. RH-3 at concentrations up to 100 microg/ml in presence of 50 microM copper sulfate inflicted significant damage to extraneously added 2-deoxyribose molecules and maximum TBARS were formed at a concentration of 100 microg/ml. Higher concentrations of RH-3 more than 100 microg/ml quenched free radicals and inhibited 2-deoxyribose degradation. RH-3 also induced strand breaks in plasmid DNA at concentrations lower than 100 microg/ml but completely inhibited at concentrations higher than 250 microg/ml, indicating bimodal function. Strand breaks induced by lower concentrations of RH-3 (up to 100 microg/ml) were inhibited by antioxidants like GSH, DFR etc. RH-3, in a concentration dependent mode also inhibited the relaxation of supercoiled plasmid DNA (PBR322) by topoisomerase I. Present study indicated that RH-3 caused compaction of reversible (< 100 micrpg/ml) and irreversible (> 100 microg/ml) nature which was related to the magnitude of DNA-protein cross-links formed. Maintenance of chromatin organization, induction of hypoxia, hydrogen atom donation, free radical scavenging and blocking of cell cycle at G2-M phase by interfering with topoisomerase I activity seem to contribute towards the radioprotective efficacy of RH-3.

Modulation of chromatin organization by RH-3, a preparation of Hippophae rhamnoides, a possible role in radioprotection.

The present study was aimed to understand the mode of action of alcoholic extract of whole berries of Hippophae rhamnoides (RH-3) which has already been reported to render more than 80 % protection against radiation induced mortality in mice. Direct and indirect antioxidant action (free radical scavenging and metal chelating potential) were assayed using 2-deoxy ribose degradation and 2,2'-bipiridyl assays. Effect of RH-3 on radiation and chemical oxidant mediated DNA damage was evaluated using single cell gel electrophoresis (Comet assay) and alkaline halo assay. Ability of RH-3 to bind with calf thymus DNA was assayed through change in melting temperature (Tm) while toxicity was assayed in thymocytes by trypan blue exclusion. RH-3 inhibited 2-deoxy ribose degradation in a dose dependent manner (IC 50 approximately 500 microg/ml). 2,2'-bipiridyl assay revealed the inability of RH-3 to chelate Fe2+ ions. RH-3 inhibited radiation and tertiary butyl hydroperoxide induced DNA strand breaks in a dose dependent manner and at concentrations of 100 and 120 microg/ml the length of comet tail was considerably reduced and became almost similar to that of untreated control. RH-3 at a concentration of 120 pg/ml or more induced a strong compaction of chromatin as was evident from lack of tail and appearance of intensely stained circular bodies. This made the nuclei resistant even to a radiation dose of 1,000 Gy. The compaction of chromatin was not reversed even by relaxation buffer indicating that salt concentration had no role in RH-3 induced chromatin compaction. Alkaline halo assay also corroborated the results of comet assay. Lower DNA-RH-3 concentrations (1:0.5 and 1:1) induced a shift of Tm towards left by 2 and 5 degrees C respectively; however higher concentrations (1:8 and 1:16) shifted the Tm towards right increasing it by 10 and 21 degrees C correspondingly. RH-3, evinced only a mild free radical scavenging activity at concentrations used in the present study, therefore its ability to protect DNA could mainly be attributed to direct modulation of chromatin organization. Further work to unravel these facts would be necessary.

Radioprotection by a herbal preparation of Hippophae rhamnoides, RH-3, against whole body lethal irradiation in mice.

PURPOSE: Hippophae rhamnoides L. has been well documented to have anti-oxidative, immunostimulative and regenerative properties and therefore a herbal preparation of H. rhamnoides coded as RH-3 was investigated for its radioprotective action. MATERIALS AND METHODS: RH-3 was administered intraperitonially (i.p.) to mice 30 minutes before whole body irradiation and whole body survival, spleen Colony forming units (CFU) and haematological parameters were studied. To investigate free radical scavenging and antioxidant potential, Fenton reaction, radiation mediated OH radical scavenging and chemically generated superoxide anions scavenging were studied in vitro while inhibition of lipid peroxidation was studied in liver homogenate of mice. RESULTS: A dose of 30 mg/kg body weight of RH-3 rendered 82% survival as compared to no survival in irradiated control. The endogenous CFU counts in mouse spleen on 10th post-irradiation day with and without RH-3 demonstrated radioprotective effect. Various hematological parameters also corroborated the radioprotective effect of RH-3. In a dose dependent manner, RH-3 inhibited Fenton reaction and radiation mediated generation of hydroxyl radicals in vitro, superoxide anion mediated Nitroblue tetrazolium (NBT) reduction and FeSO4 mediated lipid peroxidation in liver. CONCLUSION: Free radical scavenging, acceleration of stem cell proliferation and immunostimulation are the radioprotective attributes, which require further investigations.