Ravinder Sagar

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.

Evaluation of radioprotective activities of Rhodiola imbricata Edgew – A high altitude plant

The present study reports the radioprotective properties of a hydro-alcoholic rhizome extract of Rhodiola imbricata (code named REC-7004), a plant native to the high-altitude Himalayas. The radioprotective effect, along with its relevant superoxide ion scavenging, metal chelation, antioxidant, anti-lipid peroxidation and anti-hemolytic activities was evaluated under both in vitro and in vivo conditions. Chemical analysis showed the presence of high content of polyphenolics (0.971 ± 0.01 mg% of quercetin). Absorption spectra analysis revealed constituents that absorb in the range of 220–290 nm, while high-performance liquid chromatography (HPLC) analysis confirmed the presence of four major peaks with retention times of 4.780, 5.767, 6.397 and 7.577 min. REC-7004 was found to lower lipid oxidation significantly (p < 0.05) at concentrations viz., 8 and 80 μg/ml respectively as compared to reduced glutathione, although the optimally protective dose was 80 μg/ml, which showed 59.5% inhibition of induction of linoleic acid degradation within first 24 h. The metal chelation activity of REC-7004 was found to increase concomitantly from 1 to 50 μg/ml. REC-7004 (10–50 μg/ml) exhibited significant metal chelation activity (p < 0.05), as compared to control, and maximum percentage inhibition (30%) of formation of iron-2,2′-bi-pyridyl complex was observed at 50 μg/ml, which correlated well with quercetin (34.9%), taken as standard. The reducing power of REC-7004 increased in a dose-dependent manner. The absorption unit value of REC-7004 was significantly lower (0.0183± 0.0033) as compared to butylated hydroxy toluene, a standard antioxidant (0.230± 0.091), confirming its high reducing ability. Superoxide ion scavenging ability of REC-7004 exhibited a dose-dependent increase (1–100 μg/ml) and was significantly higher (p < 0.05) than that of quercetin at lower concentrations (1–10 μg/ml), while at 100 μg/ml, both quercetin and REC-7004 scavenged over 90% superoxide anions. MTT assay in U87 cell line revealed an increase in percent survival of cells at doses between 25 and 125 μg/ml in case of drug + radiation group. In vivo evaluation of radio-protective efficacy in mice revealed that intraperitoneal administration of REC-7004 (maximally effective dose: 400 mg/kg b.w.) 30 min prior to lethal (10 Gy) total-body γ-irradiation rendered 83.3% survival. The ability of REC-7004 to inhibit lipid peroxidation induced by iron/ascorbate, radiation (250 Gy) and their combination [i.e., iron/ascorbate and radiation (250 Gy)], was also investigated and was found to decrease in a dose-dependent manner (0.05–2 mg/ml). The maximum percent inhibition of formation of MDA-TBA complex at 2 mg/ml in case of iron/ascorbate, radiation (250 Gy) and both i.e., iron/ascorbate with radiation (250 Gy) was 53.78, 63.07, and 51.76% respectively and were found to be comparable to that of quercetin. REC-7004 (1 μg/ml) also exhibited significant anti-hemolytic capacity by preventing radiation-induced membrane degeneration of human erythrocytes. In conclusion, Rhodiola renders in vitro and in vivo radioprotection via multifarious mechanisms that act in a synergistic manner.

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2′-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 μg/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 μg/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.

Podophyllum hexandrum Offers Radioprotection by Modulating Free Radical Flux: Role of Aryl-Tetralin Lignans

We have evaluated the effect of variation in aryl-tetralin lignans on the radioprotective properties of Podophyllum hexandrum. Two fractionated fractions of P. hexandrum [methanolic (S1) and chloroform fractions (S2)], with varying aryl-tetralin lignan content were utilized for the present study. The peroxyl ion scavenging potentials of S1 and S2 were found to be comparable [i.e. 45.88% (S1) and 41% (S2)] after a 48 h interval in a time-dependent study, whereas in a 2 h study, S2 exhibited significant (P < 0.05) antioxidant activity in different metal ion + flux states. In the aqueous phase, S2 exhibited non-site-specific reactive oxygen species scavenging activity, i.e. 73.12% inhibition at 500 μg ml−1. S1 exhibited 58.40 ± 0.8% inhibition (at 0.025 μg ml−1) of the formation of reactive nitrite radicals, comparable to S2 (52.45 ± 0.825%), and also showed 45.01% site-specific activity (1000 μg ml−1), along with significant (P < 0.05) electron donation potential (50–2000 μg ml−1) compared to S2. Such activities of S1 could be attributed to the significantly (P < 0.05) higher levels of podophyllotoxin β-d-glucopyranoside (16.5 times) and demethyl podophyllotoxin glucoside (2.9 times) compared with S2. Together, these findings clearly prove that aryl-tetralin lignan content influences the radiation protective potential of the Podophyllum fractions to a great extent.

Podophyllum hexandrum (Himalayan mayapple) extract provides radioprotection by modulating the expression of proteins associated with apoptosis.

Podophyllum hexandrum Royale (Himalayan mayapple), a high‐altitude Himalayan plant, has been shown to provide over 80% whole‐body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen‐activated protein kinase‐activated protein) kinase‐2 activation along with HSF‐1 (heat‐shock transcription factor‐1), leading to up‐regulation of HSP‐70 (heat‐shock protein‐70) as compared with sham‐irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis‐inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham‐irradiated group. Inhibition in the translocation of free NF‐κB (nuclear factor κB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation‐treated mice. P. hexandrum pre‐treatment (2 h before irradiation) resulted in inhibition of NF‐κB translocation, and the expression of tumour suppressor protein p53 was observed to be down‐regulated as compared with sham‐irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl‐2 (B‐cell chronic lymphocytic lymphoma 2), Ras‐GAP (Ras‐GTPase‐activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum‐pretreated irradiated mice as compared with sham‐irradiated controls. Caspase 3 activation resulted PARP [poly(ADP‐ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/− irradiation) as compared with the mice treated with radiation (+/− heat shock). The present study thus clearly demonstrated that P. hexandrum extract provides protection from γ‐radiation by the modulation of expression of proteins associated with cell death.

3-O-β-ᴅ-Galactopyranoside of Quercetin as an Active Principle from High Altitude Podophyllum hexandrum and Evaluation of its Radioprotective Properties

Abstract The aqueous-ethanolic extract (AEE) of high altitude Podophyllum hexandrum has earlier been reported to render a radioprotective effect against lethal gamma radiation in in vitro model. AEE has also been reported to possess metal chelating and DNA protecting properties. The present study was undertaken to isolate and characterize the bioactive principle present in AEE and investigate its role in radiation protection. A novel molecule was found to be present in AEE and was assigned as 3-O-β-ᴅ-galactoside of quercetin by acid hydrolysis, LC-MS, LC-APCI-MS/MS and 13C NMR spectra. Various biological activities were investigated at in vitro level. The antioxidant potential of AEE in lipid and aqueous phase was determined against numerous stresses. AEE was found to be significantly (p < 0.05) protective, i.e., against Fe2+ and Cu2+-induced linoleic acid degradation, respectively. Radiationinduced lipid oxidation studies revealed that AEE maximally works at a [lignan]/0.25 kGy ratio 400 (ratio of concentration of AEE divided by the radiation dose, i.e., 0.25 kGy) and no drug-induced lipid oxidation at all concentrations tested was found. In a time-dependent study, total antioxidant activity was maximally exhibited at 1 mg/ml. The site-specific and non-site-specific deoxyribose degradation assay exhibited a dose-dependant hydroxyl scavenging potential of AEE (0.05-500 μg/ml). The anti-lipid peroxidation ability of AEE against radiation (0.25 kGy)-induced lipid peroxidation was higher in case of neural tissue homogenate as compared to kidney homogenate [activity ratio: 0.039 (brain) < 0.24 (kidney)]. The protein protection study using bovine serum albumin was also done for two time intervals (2 h and 4 h) and significant (p < 0.05) protection was observed at 500 μg/ml (> 97%). This study implies that 3-O-β-ᴅ-galactoside present in AEE renders radioprotection by protecting lipids, proteins in renal and neural model system against supra-lethal (0.25 kGy) gamma radiation.

Radiomodulatory and Free-Radical Scavenging Activity of the Fractionated Aquo-Alcoholic Extract of the Adaptogenic Nutraceutical (Rhodiola imbricata)—A Comparative In Vitro Assessment with Ascorbate

Objective: Immense interest has been generated in recent years for the development of drugs of herbal origin for the mitigation of deleterious effects of environmental pollutants like ionizing radiation, mainly to protect against radiation leakages resulting from mishaps in nuclear reactors, deliberate use of dirty bombs, etc. Method: The radio modifying effects of a fractionated extract of the high-altitude Himalayan plant species Rhodiola imbricata, along with its electron-donation potential, super-oxide ion scavenging (IC50 ≤ 0.025 mg/ml), nitric oxide (NO) scavenging potential (IC50 = 0.5 mg/ml), and antihemolytic activity were evaluated in the present study. Reducing power, superoxide ion (O2•−), and nitric oxide scavenging ability of the fractionated extract increased in a dose-dependent manner. Rhodiola imbricata also exhibited antihemolytic potential preventing radiation-induced membrane degeneration of human erythrocytes. Conclusion: Thus, it can be stated that Rhodiola imbricata provides protection against gamma radiation via multifarious mechanisms that act in a synergistic manner. Rhodiola imbricata is widely used as a nutraceutical supplement in the trans-Himalayan region nations, and the current study shows that Rhodiola has immense potential for alleviation of biological damage in a radiation environment.