ABSTRACT
BACKGROUND: Hippophae rhamnoides, a high altitude habitat plant, has been extremely used in traditional medicinal practices for treating a variety of ailments. Recently, an extract (RH-3) prepared from berries of Hippophae rhamnoides has been reported to exhibit significant radioprotection against whole body lethal irradiation. OBJECTIVE: Present study was undertaken to elucidate the DNA binding ability of an extract (RH-3) prepared from berries of Hippophae rhamnoides and its role in modulating radiation induced frank and clustered DNA damage. METHOD: Agarose gel electrophoresis was employed as method to understand DNA binding potential and DNA protective ability of RH-3. RESULTS: RH-3 in a dose dependent fashion interacted with plasmid DNA (pUC18) reducing the mobility of supercoiled form and increasing the amount of the complex in the well indicating its ability to interact with plasmid DNA. RH-3 at higher concentrations (> 0.4 mg/ml) almost completely prevented the migration of supercoiled form without interfering with mobility of open circular form indicating its ability to selectively interact with supercoiled form. Studies done with supercoiled or open circular form also revealed the binding specificity of RH-3 for supercoiled form of plasmid. Both inhibited radiation induced strand breaks and DNA interaction by RH-3 were found to be dependent upon pH and the order of efficacy was found to be acidic pH> neutral pH > alkaline pH. RH-3 in a dose dependent manner inhibited radiation induced frank single, double strand breaks as well as endonuclease IV detectable abasic sites (clusters) and maximum reduction was observed at a concentration of 200 µg/ml. CONCLUSION: Results obtained in this study suggest that the ability of RH-3 to interact with DNA could be playing a significant role in preventing radiation induced DNA damage.
ABSTRACT
The aqueous extract of Podophyllum hexandrum (RP-1), which has been recently reported to manifest radioprotective and anti-tumour properties, has been investigated for its mode of action. RP-1, under in-vitro conditions dose-dependently chelated metal ions, inhibited radiation or metal ion-induced hydroxyl radicals and lipid peroxidation and scavenged superoxide anions. Intraperitoneal administration of RP-1 to mice pre-irradiation (10 Gy) induced more DNA fragmentation and lipid peroxidation in thymocytes maximally at 4 and 8 h, respectively, in comparison with RP-1 treatment or irradiation. Flow-cytometric quantification of sub-diploid peak, oligonucleosomal cleavage assay (ladder) and depletion of total thiols also corroborated the ability of RP-1 to enhance radiation-induced apoptosis. RP-1 in presence of 100 microM CuSO(4) induced strand breaks in plasmid DNA and addition of metal chelators (EDTA and deferoxamine) inhibited the strand scission. Treatment with a major constituent of RP-1, podophyllin, did not cause strand breaks, but isolated constituents of RP-1, quercetin or podophyllotoxin, induced strand breaks. Depending on its concentration in the milieu, RP-1 acted as a pro- or antioxidant modifying the radiation-induced apoptosis and therefore could be exploited for cancer management.
Subject(s)
Apoptosis/drug effects , Apoptosis/radiation effects , Plant Extracts/pharmacology , Podophyllum/chemistry , Radiation-Sensitizing Agents/pharmacology , Animals , Antioxidants/administration & dosage , Antioxidants/isolation & purification , Antioxidants/pharmacology , Chelating Agents/pharmacology , Chromatography, High Pressure Liquid/methods , DNA Damage/drug effects , DNA Damage/radiation effects , DNA Fragmentation/drug effects , DNA Fragmentation/radiation effects , Dose-Response Relationship, Drug , Gamma Rays , Hydroxyl Radical/metabolism , Injections, Intraperitoneal , Lipid Peroxidation/drug effects , Male , Mice , Plant Extracts/administration & dosage , Plant Extracts/isolation & purification , Radiation-Sensitizing Agents/administration & dosage , Radiation-Sensitizing Agents/isolation & purification , Rhizome/chemistry , Sulfhydryl Compounds/metabolism , Superoxides/metabolism , Thiobarbituric Acid Reactive Substances/metabolism , Thymus Gland/cytology , Thymus Gland/drug effects , Thymus Gland/metabolism , Time FactorsABSTRACT
Aqueous extract of T. cordifolia inhibited Fenton (FeSO4) reaction and radiation mediated 2-deoxyribose degradation in a dose dependent fashion with an IC50 value of 700 microg/ml for both Fenton and radiation mediated 2-DR degradation. Similarly, it showed a moderate but dose dependent inhibition of chemically generated superoxide anion at 500 microg/ml concentration and above with an IC50 value of 2000 microg/ml. Aqueous extract inhibited the formation of Fe2+-bipiridyl complex and formation of comet tail by chelating Fe2+ ions in a dose dependent manner with an IC50 value of 150 microg/ml for Fe2+-bipirydyl formation and maximally 200 microg/ml for comet tail formation, respectively. The extract inhibited ferrous sulphate mediated lipid peroxidation in a dose-dependent manner with an IC50 value of 1300 microg/ml and maximally (70%) at 2000 microg/ml. The results reveal that the direct and indirect antioxidant actions of T. cordifolia probably act in corroboration to manifest the overall radioprotective effects.
