Molecular and Cellular Biochemistry 273: 193–208, 2005. c�Springer 2005

Antioxidant activity of fractionated extractsof rhizomes of high-altitude Podophyllumhexandrum: Role in radiation protection

Raman Chawla,1 Rajesh Arora,1 Raj Kumar,1 Ashok Sharma,1

Jagdish Prasad,1 Surendar Singh,1 Ravinder Sagar,1

Pankaj Chaudhary,1 Sandeep Shukla,1 Gurpreet Kaur,1

Rakesh Kumar Sharma,1 Satish Chander Puri,2 Kanaya Lal Dhar,2

Geeta Handa,2 Vinay Kumar Gupta2 and Ghulam Nabi Qazi21Division of Radiopharmaceuticals and Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, New Delhi,India; 2Natural Products Chemistry Division, Regional Research Laboratory (CSIR), Jammu, India

Received 12 October 2004; accepted 18 January 2005

Abstract

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radio-protection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar topolar) 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 ma-jor constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of theindividual 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 lipidperoxidation (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, fol-lowed 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 and92.249%, 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 activ-ity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations ascompared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are themost promising fractions, which can effectively tackle radiation-induced oxidative stress. (Mol Cell Biochem 273: 193–208,2005)

Key words: 2,2′-bipyridyl, free radical, lipid peroxidation, polyphenols, radioprotection, oxidative stress, Podophyllum hexan-drum

Address for offprints: R.K. Sharma, Division of Radiopharmaceuticals and Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Brig. SKMazumdar Road, New Delhi 110054, India (E-mail: rks@inmas.org)

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Introduction

Low linear energy transfer (LET) radiation causes damage tobiological systems by generating reactive oxygen/nitrogenspecies (ROS/RNS) like superoxide radicals (O•

2), hydro-gen peroxide (H2O2) singlet oxygen (O), hydroxyl radicals(OH•), nitric oxide (NO•) and peroxynitrite [1]. ROS/RNSinteract with various macromolecules like DNA, proteins andlipids leading to lipid peroxidation (LPO), which results inleaky membrane, DNA lesions, removal of sulfhydryl groupsfrom cellular proteins eventually leading to protein fragmen-tation and denaturation [2–4]. This can result in loss of cellu-lar homeostasis and even cell death [5]. Oxidative stress hasalso been implicated in the generation of several pathologicalconditions like arthritis [6], Alzheimer’s and Parkinson’s dis-ease, ageing, glycated oxidation in case of diabetes mellitus[7], low-density lipoprotein (LDL) oxidation in atheroscle-rosis [8], red blood cell hemolysis in glucose-6-phosphatedehydrogenase deficiency [9] and possibly play a causativerole in Acute Mountain Sickness [11].

The medicinal use of Podophyllum hexandrum Royale syn.P. emodi Wall (Himalayan Mayapple; family: Berberide-ceae), a high-altitude plant species native to the alpineand sub alpine areas of Himalayas, dates back to ancienttimes [11]. The plant has been described as ‘Aindri’ – adivine drug in the traditional Indian system of medicine– the Ayurveda [11], and has also been used in traditionalChinese System of Medicine [12] for treatment of a numberof ailments. In the modern allopathic system of medicine,the plant has been successfully used for the treatment ofvarious metabolic disorders [13], monocytoid leukemia,Hodgkin’s and non-Hodgkin’s lymphomas, bacterial andviral infections [14, 15], venereal warts [16], rheumatoidartharalgia associated with limb numbness and pycnogenicinfections of skin tissue [12], AIDS-associated Kaposissarcoma and different cancers of brain, lung and bladder[17]. The roots and rhizomes of P. hexandrum are known tosynthesize a plethora of secondary metabolites and bioactivecomponents like podophyllotoxin, epi-podophyllotoxin,podophyllotoxone, and other aryl tetrahydronaphthalenelignans, flavonoids like quercetin, quercetin-3-glycosides,4′-demethylpodophyllotoxin glycoside, podophyllotoxinglycoside, 4′-demethylpodophyllotoxone, deoxypodophyl-lotoxin, dehydropodophyllotoxin, kaempferol and astragalinor kaempferol-3-glucoside [11, 12] with a diverse arrayof biological activities. Etoposide and teniposide, semisynthetic derivatives of glycoside from podophyllotoxin,form an integral part of the therapeutic regimen used forchemotherapy [18], and have also triggered further studiesin the design and synthesis of other potentially usefulanticancer compounds [19–21]. The radioprotective effectsshown by this plant have been attributed to its ability toreduce the generation of reactive oxygen and nitrogen

species, stabilize the membrane potential and augment thelevels of glutathione [22], scavenge free radicals and protectmitochondria [23] against oxidative damage.

The radioprotective effect of P. hexandrum was first re-ported by our group [23, 25, 26]. Earlier studies in ourgroup have shown that the aqueous extract of P. hexandrumprovides over 80% radioprotection to Hep G2 cells [24],and also to Strain ‘A’ mice subjected to whole-body lethal(10 Gy) gamma radiation [23]. The crude (whole) extract ofP. hexandrum rhizomes has also been reported to modulateantioxidant enzyme levels, protect against ionizing radiation-induced DNA damage, protect gastrointestinal, reproductive,and central nervous system, against radiation-induced dam-age [23].

The role of antioxidant compounds in mitigating oxida-tive damage to biological systems has been reported by sev-eral workers [27–30]. However, the precise mode of actionof these compounds, e.g., whether they reduce free radicalgeneration by chelating transition metal ions or by scaveng-ing free radicals and/or modulating intrinsic antioxidants isrequired to be delineated to explain their role in renderingradioprotection. Therefore, in the present investigation, wefractionated the plant material of high-altitude P. hexandrum(HAPH) using solvents of differing polarity. The bioactivi-ties of five different fractions of P. hexandrum were evaluatedunder in vitro conditions by measuring reduction of radiation-induced LPO, chelation of metal (ferrous) ions, antioxidativeproperties and their ability to reduce DNA strand breaks.

Materials and methods

Materials

All chemicals and reagents used for the study were ofhigh purity. Ferric chloride, sodium sulphite, ferrous sul-phate, 2-2′-bipyridyl, potassium di-hydrogenorthophosphate,di-potassium hydrogen orthophosphate, potassium ferri-cyanide, trichloroacetic acid, di-sodium ethylene diaminetetra acetic acid, ascorbate, thiobarbituric acid (TBA), elec-trophoresis grade plasmid (pBR322) DNA, bromophenolblue, xylene cyanol FF, 15% Ficoll, glycerol, ethidium bro-mide, and molecular biology grade di-sodium tris-acetate-ethylene diamine tetra acetic acid (TAE), were purchasedfrom Sigma Chemicals (St. Louis, MO, USA). Metal-freemicro-centrifuge tubes, pipette tips and gamma rays steril-ized 35-mm petridishes were obtained from Tarsons (Kolkata,India). Dimethylsulfoxide and hydrochloric acid were ob-tained from BDH Chemical Co. (Toronto, Ontario, Canada).The rest of the chemicals utilized for this study were of an-alytical reagent (AR) grade and were obtained from reputedlocal suppliers in India.

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Plant material (high-altitude Podophyllum hexandrum(HAPH))

Collection, authentication and processingThe rhizome of P. hexandrum Royale (syn. Podophyllumemodi Wall) was collected from the high-altitude regions(>3000 m) of Leh and Ladakh, Jammu and Kashmir, India.The plant material was identified by Dr. Om Prakash Chaura-sia, Ethnobotanist, Field Research Laboratory (FRL), Leh(J&K), and further authenticated on the basis of botanicalcharacteristics by Dr. Rajesh Arora, INMAS, Delhi. Voucherspecimens have been deposited in the repositories, both atINMAS, Delhi and RRL, Jammu [voucher no.: INM/FRL-PH/Leh-2004]. Care was taken to free the plant material offoreign matter like soil, dust, insects and other extrinsic con-tamination. Organoleptic examination was conducted and therhizomes were found to be brittle and snapped upon apply-ing pressure. They possessed a phenolic odor with bitter taste.The powdered plant material was also subjected to analysisfor determination of microbial counts, total fungal counts,etc.

Standardization and quality control of HAPH

Standardization and quality control of herbal material of P.hexandrum was carried out as per FAO/UNDP/WHO norms(Table 1). The rhizome (3.5 kg) was air dried in shade aftercollection and powdered mechanically till a moderately finepowder was obtained, and then stored at room temperaturetill the material was subjected to extraction.

Table 1. Standardization and quality control of herbal material ofPodophyllum hexandrum

1. Loss on drying at 105 ◦C 5.94%

2. Total ash content 4.0679%

3. Acid insoluble ash 0.2938%

4. Microbial counts; Escherichiacoli (CFU/g)

<102 (within limits < 103)

Total fungal content (CFU/g) 7 × 103 (within limits < 104)

5. Pesticides residues (DDT,BHC, aldrin, dialdrin, melifoxand toxaphene)

Nil

6. Heavy metals (Pb, As, Zn) Within limits

7. Solvent extractive values

Hexane 0.56%

Chloroform 2.14%

Alcohol 18.28%

Ethyl alcohol:water (50:50) 14.29%

Water 18.00%

Phytochemical screening

The qualitative and phytochemical analysis of the powderedsample was carried out for the presence of various sec-ondary metabolites such as polyphenols, lignans and gly-cosides (Table 2) using the method of Harborne [31].

Fractionation of HAPHThe powdered plant material was transferred to a Soxhlet ap-paratus and consecutively extracted with solvents of increas-ing polarity viz., hexane, chloroform, alcohol, alcohol–waterand water for a minimum of three times using proportionateamount of solvent over the course of 24–72 h and the respec-tive filtrates were combined. The pooled filtrates were filteredthrough Whatman Paper No. 3, and concentrated by solventevaporation under reduced pressure in a rotary evaporator(Buchi, Switzerland) and dried. The yield was determinedon w/w basis separately for each fraction. The dried frac-tions were pulverized through a micropulverizer and passedthrough a number 40 sieve. The yield of extracts from n-hexane, chloroform, alcohol (ethanol), 50% alcohol, and wa-ter on w/w basis was 0.56, 2.14, 18.28, 14.29, 8.00%, respec-tively (Table 1). The extracts were designated as HE, CE, AE,HA, and WE, respectively.

Total phenolic contentThe phenolic content was estimated in different fractions ofHAPH using the method of Singleton and Rossi [32]. To analiquot (10 µl), taken from the stock solution (1 mg/ml) ofdifferent fractions, 10 ml of water and 1.5 ml of Folin Cio-calteu reagent were added. The mixture was kept for 5 min atroom temperature, and then 4 ml of 20% sodium carbonatesolution was added and the volume made upto 25 ml withdouble distilled water. The mixture was kept for 30 min andabsorbance of the color developed was recorded at 765 nm us-ing UV-visible spectrophotometer (Electronics Corporationof India Ltd., Hyderabad, India).

HPLC analysis

Isocratic analysisThe fractionated extracts were analyzed on Shimadzu LC-10AT VP HPLC machine isocratically utilizing E. Merck RP-18 e column (256 × 4.6 mm 5 µm) with diode array detec-tor SPD M-10 A VP/RF-10 AXL Fluorescent detector, andauto-injector SIL-10 AD VP. Elution was done with the mo-bile phase [MeOH:H2O; 65:35] for 30 min at a flow rateof 0.6 ml/min and a wavelength of 290 nm was used formeasurement.

Gradient analysisThe standard compounds and semi-purified fractions wereseparated on Shimadzu LC-10 AT VP, using diode detector

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Table 2. Lignans identified using LC/MS profiling and LC/MS/MS substructural analysis in Podophyllum hexandrum

Retention MolecularSample Compound time (RT) weight M+NH4 Other fragments peak

Standard 1 Podophyllotoxin 37.63 432.04 414.04, 396.93, 394.96, 352.97312.93

Standard 2 4′-Demethylpodophyllotoxin 28.91 418.04 400.04, 382.92, 298.92Hexane fraction (HE) 4′-Demethylpodophyllotoxin

glycoside28.17 580.16 399.98, 382.98, 299.96, 298.90, 245

Unidentified 28.97 636.17 594.11, 431.98, 412.00, 352.95,312.9, 245.84

4′-Demethylepipodophyllotoxin 31.83 418.01 382.96, 298.92, 245.894′-Demethylpodophyllotoxin 31.88 418.01 382.96, 298.92, 245.89Podophyllotoxin glycoside 32.87 594.15 411.98, 411.98, 352.97, 312.95Podophyllotoxin 36.46 432.04 396.93, 394.96, 352.97, 312.93

Chloroform fraction (CE) 4′-Demethylpodophyllotoxin 31.24 418.04 382.95, 298.92, 245.88Podophyllotoxin glycoside 32.39 594.18 432.05, 416.04, 397.01, 352.00,

312.86, 261.66epi-Podophyllotoxin 34.03 432.06 412.01, 52.94, 312.94, 397.00,

312.94Podophyllotoxin 36.01 432.05

Alcoholic fraction (AE) Unidentified 2.52 359.99 197.85, 179.84, 161.854′-Demethylpodophyllotoxin

glycoside27.95 580.15 448.91, 399.91, 382.96, 339.08,

299.74, 298.93, 286.89, 245.72,231.02

4′-Demethylpodophyllotoxin 31.73 418.02 382.94, 298.89, 245.67Podophyllotoxin glycoside 32.86 594.09 541.98, 411.98, 396.94, 352.95,

312.95, 245.86epi-podophyllotoxin 34.55 432.01 412.06, 352.96, 312.91, 118.83Podophyllotoxin 36.40 433.05 396.97, 394.94, 353.04, 312.95,

245.89Hydro-alcoholic fraction

(HA)Unidentified 2.45 522.08 475.06, 458.09, 44.08, 359.98,

304.96– 23.76 495.01 265.91, 197.83, 179.82, 132.82– 27.40 448.98 302.87, 302.85, 286.86Podophyllotoxin glycoside (iso) 29.14 594.18 411.95, 352.96, 312.93, 286.92epi-Podophyllotoxin 31.48 431.91 418.04, 382.95, 312.87, 245.75Podophyllotoxin glycoside 32.69 594.13 432.02, 411.97, 397.00, 352.94,

312.92Unidentified 34.29 312.95 245.87, 230.90, 213.86, 197.84,

136.89Podophyllotoxin 36.18 432.02 397.04, 263.89, 246.02

and Chromato-integrator. The mobile phase consisted ofmethanol (A) and water (B) 0–1 min (A:B; 65:35), 1–60 min(A:B; 35:65) and finally 60–70 min (A:B; 65:35). Separationwas carried out at 30 ◦C on RP-18 E. Merck column (5 µm,4.0×250 mm), with a flow rate of 0.6 ml/min. Measurementswere taken at 290 nm.

Analytical HPLC of the fractionated extracts

The fractions were analyzed by both isocratic HPLC for20 min at 0.6 ml/min, followed by 0.5 min. methanol washand re-equilibrium for 15 min [column RP-18 e (250×46 mm

5 µm) E. Merck] using a mobile phase of [MeOH:H2O;65:35] at an absorbance of 290 nm.

The compounds of the fraction were separated when a gra-dient of methanol (A) and water (B) mobile phase was usedin the following manner on RP-18 E. Merck column (5 µm,4.0 × 250 mm) at 30◦ and λ290 nm 0–1 min (A:B; 65:35)1–60 min (A:B; 35:65) and 60–70 min (A:B; 65:35) at a flowrate of 0.6 ml/min.

LC-APCI/MS/MSAll experiments were performed using a Thermo-Finnigan MAT8000 pneumatically assisted electrospray

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triple-quadruple mass spectrometer. The operating conditionsof LC-APCI/MS/MS were optimized for the MS/MS analy-sis of lignans in different fractionated extracts. The nebulizercurtain and collision gases were set at 1.25, 0.45 and 0.4 l/min,and the gas was supplied from a liquid nitrogen tank with ahead pressure of 50 psi. The ion spray voltage was 4800 V.The voltage at the orifice plates focusing ring deflector andchannel electron multiplier (CEM) was adjusted at 350–400and 2100 V, respectively. Positive ions were scanned in therange 350–700 Dalton (Da) using 10 ms dwell time and a 0.2Da step size during scans.

Experimental animalsAdult (6–8 weeks) Swiss albino strain ‘A’ mice (25 ± 2 g),bred locally in the animal house of the Institute of Nu-clear Medicine and Allied Sciences, Delhi (India), usedfor the study, were maintained under controlled tempera-ture (25 ± 2 ◦C; 12 h alternating dark and light cycle) inpolypropylene cages. Standard food pellets (M/s AmrutFeeds Pvt. Ltd., Kolkata, India) and drinking water were pro-vided ad libitum. Permission for use of animals was takenfrom the Institutional Animals Ethics Committee (IAEC) ofINMAS, and all experiments were carried out strictly in ac-cordance with the institutional guidelines laid down by theIndian National Science Academy (INSA) for the care anduse of laboratory animals for research purposes.

Irradiation

High dose radiation (250 Gy) was delivered to mice liverhomogenate from a 60Co-gamma chamber (Gamma Cell5000, Bhabha Radiation Isotope Technology, Mumbai) at adose rate of 3.54 kGy/h. Dosimetry was carried out usingBaldwin Farmer’s secondary dosimeter and Fricke’s Chemi-cal Dosimetry method.

Metal chelation activity of fractions of HAPH

Metal chelation assay was performed according to the methodof Harris and Livingstone [33]. Varied concentrations of frac-tionated extracts (2 ml) were mixed with 4 ml of ferric chlo-ride solution (5 µg/ml; 0.005N HCl) and then incubated atroom temperature for 10 min. Two milliliters of aliquots weretaken from each sample and mixed with sodium sulphite (fi-nal concentration: 0.05 M) and 2,2′-bipyridyl. The solutionwas then re-incubated in a hot water bath (Yorko, India 55 ◦C)for 5 min. The tubes were cooled to room temperature and theabsorbance recorded at 520 nm. The percentage inhibition offormation of metal 2,2′-bipyridyl complex (chromogen) wasevaluated as:

%inhibition = O.D.control − O.D.sample/O.D.control × 100

Antioxidant activity of fractions of HAPH

The antioxidant activity of different fractions was evaluated,in terms of reducing power using the method of Oyaizu [34].The different fractionated extracts (50 µl) were mixed with200 µl each of 0.2 M phosphate buffer (pH 6.5) and 0.1%potassium ferricyanide and incubated at 50 ◦C in a hot waterbath for 20 min. 250 microliters of 10% trichloroacetic acidwas added to the mixture and centrifuged at 3000 × g for10 min at room temperature in a Sorvall table-top centrifuge[Sorvall (Kendro) Instruments, USA). The resulting super-natant was taken and mixed with 500 µl of double distilleddemineralized water and 100 µl of 0.1% ferric chloride, andfurther incubated at 37 ◦C for 10 min. The absorbance wasrecorded at 700 nm using a spectrophotometer (ElectronicsCorporation of India Ltd., Hyderabad, India). Color devel-opment was measured against a blank containing phosphatebuffer, potassium ferricyanide and trichloroacetic acid. Thedifferent fractions were compared on the basis of their respec-tive concentrations (mg/ml) corresponding to unit absorbanceexpressed as mean ± S.D., using the formula:

Concentration (mg/ml)unit abs value

= C1/Abs.C1 + C2/Abs.C2 + C3/Abs.C3

where C1, C2 and C3 are three randomly selected concen-trations (mg/ml) from their linear response curve. Increasedabsorbance is indicative of increased reducing power.

Plasmid relaxation assayRadiation-induced damage to DNA was assessed elec-trophoretically using a modified method of Sambrook et al.[35]. To evaluate the radioprotective efficacy of the fraction-ated extracts, 2 µl of pBR322 plasmid DNA (25 µg/ml) wasmixed with different concentrations of the fractionate ex-tracts (10–50 µg/ml) along with 2 µl each of EDTA-Na2

(TE buffer, pH 8; 30 mM), and KH2PO4 buffer (50 mM, pH7.4) in the reaction mixture. The reaction mixture was ex-posed to 60Co-gamma radiation (250 Gy) in a Gamma Cell5000 (Board of Radiation Isotope Technology, India) at adose rate of 3.50 kGy/min. The final volume of the reactionmixture was then incubated for 1 h at 37 ◦C in an incuba-tor (Yorko, India). Three microliters of loading dye (15%bromophenol blue; 0.25% xylene cyanol FF, and 15% su-crose was added to the incubated mixture and 13 µl wasloaded onto 1% w/v agarose gel. Electrophoresis was car-ried out at 45 V (24 A) in a DNA submarine electrophore-sis unit (Bangalore Genie, Bangalore, India). The agarosegel was stained with ethidium bromide (0.5 µg/ml deion-ized distilled water) for 30 min. The ethidium bromide–stained DNA bands were visualized under ultraviolet lightusing a UV trans-illuminator (UVP, USA). The pictures wereanalyzed densitometrically using the Bio-Rad GEL-DOC

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system (Bio-Rad, USA). The percent retention of super-coiled form (%SC) represented protection, while percentageopen circular form (%OC) represented DNA damage and wasevaluated using integrated density values (IDV) of bands asfollows:

%SC form = (SCIDV/SCIDV + OCIDV) × 100

%OC form = (OCIDV/SCIDV + OCIDV) × 100

Estimation of lipid peroxidation in liver homogenate

The method of Srour et al. [36] was adopted for estimation ofLPO. Mice were randomly selected and sacrificed by cervicaldislocation, dissected and the abdominal cavity was perfusedwith 0.9% saline. Whole liver was taken out and visible bloodclots were carefully and maximally removed. Ten percent ho-mogenate was prepared in cold buffered saline (pH 7.4); usingPotter Elvejam homogenizer and filtered through a mesh toget a clear homogenate.

Estimation of iron/ascorbate- and radiation-inducedlipid peroxidation

Two milliliters of 10% liver homogenate was taken in a seriesof 35-mm petridishes to which desired amounts of differentfractions were added and mixed gently to form a homoge-nous solution. LPO was initiated by adding 20 µl of ferricchloride (0.5 mM) with 200 µl of ascorbate (1 mM) andalso by exposure to radiation (250 Gy) followed by incu-bation of petridishes at 37 ◦C for 30 min. One milliliter ofhomogenate was pippetted out for estimating LPO levels interms of thiobarbituric acid reactive substances (TBARS),measured by recording the absorbance at 535 nm. LPO val-ues are expressed as nanomoles of malonialdehyde formedper hour per gram of tissue.

Nanomoles of MDA formed/h/g of tissue

= O.D. (535 nm) × dilution factor/molar extinction

coefficientMDA:TBA complex (1.56 × 10−6)

Statistics

Each experiment was performed in triplicate and repeatedthree times. All results are expressed as mean ± S.D. or aspercentage. Statistical analysis of data was performed by us-ing Student’s t-test and a p value < 0.05 was consideredsignificant.

Results

Phytochemical analysis

The different fractions of HAPH were tested for the presenceof various secondary metabolites (Table 2). Total phenoliccontent was determined in different extracts and it was foundthat the percentage of total phenolic substances present indifferent fractions followed the order: AE (9.26 mg%) > HA(2.32 mg%) > WE (1.42 mg%) > CE (1.25 mg%) > HE(0.46 mg%).

In the liquid chromatographic separation, it was found thata gradient of methanol (A) and water (B) in the followingmanner: 1–60 min (A:B; 65:35), and finally 60–70 min (A:B;65:35) v/v at a flow rate of 0.6 ml/min was the most optimalmobile phase. The LC/MS pattern of reconstructed ion chro-matogram of three samples of P. hexandrum, collected at dif-ferent times, matched except for a very slight variation in therelative intensity of the peaks. HPLC profiles of different frac-tionated extracts is represented in Fig. 1. A number of arylte-trahydronapthalene and related lignans were identified in thedifferent fractions by analyzing the fragmentation patterns,as revealed in corresponding tandem mass spectra. The majorfragment ions observed in the respective tandem mass spec-tra are summarized in Table 2. The molecular weights of twoselected components (podophyllotoxin and podophyllotoxin-β-D-glucopyranoside) obtained online from the full scan ionspray mass spectrum at their corresponding retention time,are represented in Figs. 2(a) and 2(b).

Metal chelation activity of fractions of HAPH

In this test method, the metal ion chelating activity of dif-ferent fractions of HAPH was measured by 2,2′-bipyridylmethod. The metal chelation activity was found to increaseconcomitantly with increase in concentration (µg/ml) of thedifferent fractions in all the samples tested (Fig. 3). Maximummetal chelation ability was evaluated as percentage inhibi-tion of iron–2,2′-bipyridyl complex (chromogen) formation.The maximum percent inhibition observed in case of HE, CE,AE, HA, WE and quercetin (used as standard) was 26.6% (40µg/ml), 41.59% (10 µg/ml), 31.25% (2.5 µg/ml), 21.71% (5µg/ml), 20.48% (2.5 µg/ml) and 34.9% (50 µg/ml), respec-tively. All the values were found to be significant (p < 0.05)vis-a-vis control (0% inhibition).

Antioxidant activity of fractions of HAPH

The antioxidant activities of natural compounds are knownto have a direct correlation with their power to act as reduc-ing agents. The reducing power of the extract was, therefore,

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Fig. 1. LC profiles of (a) n-hexane, (b) chloroform, (c) alcohol and (d) hydro-alcoholic fractions from high-altitude Podophyllum hexandrum.

compared with butylated hydroxyl toluene (BHT), a standardantioxidant compound, which served as control. The concen-tration to attain unit absorbance at 700 nm was 0.230541 ±0.091607 for BHT and 0.331 ± 0.07092, 0.0750 ± 0.0021,0.0389 ± 0.00717, 0.044 ± 0.0063, 0.351 ± 0.0359 in caseof HE, CE, AE, HA and WE, respectively. The antioxidantactivity of AE and HA was found to be much higher than theother fractions of HAPH as well as BHT (Fig. 4).

Modulation of radiation-induced DNA damage assay

A comparative study was designed to evaluate the radiopro-tective effect of different fractions, viz., HE, CE, AE, HAand WE against 250-Gy gamma ray–induced DNA damagein pBR322 plasmid DNA. DNA-strand damage was semi-quantitatively measured by converting double stranded super-coiled DNA (fast migrating) into nicked open circular form(slow migrating) [37] (Figs. 5 and 6). Quercetin (a knownradioprotector) was used as a standard compound for com-parison. The untreated control (pBR322 DNA) comprised ofmore than 95% supercoiled form, while upon exposure to250-Gy gamma radiation, nearly 75% relaxed form (opencircular DNA) was observed. The different doses of fractions(in the range of 10–50 µg/ml) were evaluated for their pro-tective efficacy against radiation (data not shown), in terms

of percentage of supercoiled form of DNA retained. Thedoses that showed appreciable results were chosen for thepurpose of comparison (HE: 10 µg/ml, CE: 30 µg/ml, AE:40 µg/ml, HA: 10 µg/ml, WE: 10 µg/ml). A comparativeanalysis of pBR322 DNA pretreated (−1 h) with differentfractions and then irradiated (250 Gy) revealed that the bio-constituents present in CE and HE helped in, retention of68.09 and 68.02% SC form respectively, which was higherthan that of quercetin (30 µg/ml) treated group, which re-tained 63.64% of SC form. In comparison to non-polar frac-tions, the polar fractions viz., AE and WE showed 60.59 and51.13% retention of SC form, which was significantly pro-tective (p < 0.05) as compared to radiation control (25% SCform). Among the different fractions, the densitometric anal-ysis of gels showed that the protection (%SC form) againstradiation-induced damage follows the order:

CE (68.09%) > HE (68.02%) > quercetin (63.64%)

> AE (60.59%) > WE (51.13%) > HA (36.6%)

Modification of radiation (250 Gy)-inducedlipid peroxidation

The radiation-induced LPO activity in the liver homogenatewas found to decrease in a dose-dependant manner and

200

(a)

(b)

Fig. 2. MS/MS of (a) podophyllotoxin and (b) podophyllotoxin-β-D-glucopyranoside.

maximum inhibition i.e., 91.686, 90.135 and 92.249, 93.914,91.487% was observed at 2000 µg/ml in case of HE, CE, AE,HA and WE, respectively (Figs. 7a–7e). All the values werefound to be significant (p < 0.01) with respect to control(0% inhibition). The ratio of decrease in LPO activity (nmof MDA formed per hour per gram of tissue) in the range of50–2000 µg/ml was 8.4, 2.29, 1.33, 6.96, 7.57 for HE, CE,AE, HA and WE, respectively. Lower ratio is indicative ofeffectiveness of the fraction even at lower concentration.

Modification of iron/ascorbate-induced lipid peroxidation

The iron/ascorbate-induced LPO activity in the liver ho-mogenate was found to decrease in a dose-dependant man-ner and maximum inhibition, i.e. 99.35, 97.22, 99.76, 88.10,87.45% was observed at 2000 µg/ml for HE, CE, AE, HA andWE, respectively (Figs. 7a–7e). All the values were foundto be significant (p < 0.01) with respect to control (0%

inhibition). The ratio of decrease in LPO activity (nanomolesof MDA formed per hour per gram of tissue) at 50–2000µg/ml for HE, CE, AE, HA and WE was 149.8, 12.5, 312.6,6.9 and 6.54, respectively. Lower ratio is an indication ofeffectiveness of the fraction even at lower concentration.

Modification of iron/ascorbate- and radiation(250 Gy)-induced lipid peroxidation

The combined stress of iron/ascorbate with radiation(250 Gy) mimics the biological condition maximally. Theinitiators, radiation-induced (free radicals) and the ampli-fiers (iron) worked in a coordinated manner to generate alethal stress, hence maximum LPO activity was observed incontrol (0% inhibition). The different concentrations of thefractions decreased the activity in a dose-dependant man-ner and maximum inhibition i.e., 95.66, 93.92, 92.08, 93.05,91.4% was observed at 2000 µg/ml for HE, CE, AE, HA

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Fig. 3. Effect of different concentrations of various fractions of HAPH on chelation of metal ion. The percentage inhibition of M2+–2,2′-bipyridyl chromogencomplex containing all reagents, but without the drug was considered as 0% inhibition. ∗Maximum percentage inhibition of chromogen complex formationwas observed with respect to control (0% inhibition) (p < 0.05). Quercetin was used as a positive control.

Fig. 4. Evaluation of reducing power of different fractions of HAPH. The absorbance at 700 nm was recorded in triplicate and each experiment was repeatedthrice. The values are expressed as mean ± S.D. BHT, a standard synthetic antioxidant was used as control. ∗Fractions of Podophyllum hexandrum with respectto control (p < 0.05).

and WE, respectively (Figs. 7a–7e). All the values werefound to be significant (p < 0.01) with respect to control(0% inhibition). The ratio (activity at 50: 2000 µg/ml) ofdecrease in LPO activity (nanomoles of MDA formed per

hour per gram of tissue) for HE, CE, AE, HA and WE was18.31, 6.15, 13.70, 12.20, 9.14, respectively. Lower ratiois an indication of effectiveness of fraction even at lowerconcentration.

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Fig. 5. Densitometric analysis: 1, control (pBR322 plasmid DNA); 2, 250-Gy-treated pBR322 plasmid DNA; 3, pBR322 plasmid DNA + quercetin (30 µg/µl);4, pBR322 plasmid DNA + quercetin (30 µg/µl) + 250 Gy; 5, pBR322 plasmid DNA + HE (10 µg/µl); 6, pBR322 plasmid DNA + HE (10 µg/µl) + 250 Gy;7, pBR322 plasmid DNA + CE (30 µg/µl); 8, pBR322 plasmid DNA + CE (30 µg/µl) + 250 Gy; 9, pBR322 plasmid DNA + AE (40 µg/µl); 10, pBR322plasmid DNA + AE (40 µg/µl) + 250 Gy; 11, pBR322 plasmid DNA + HA (40 µg/µl); 12, pBR322 plasmid DNA + HA (40 µg/µl) + 250 Gy; 13, pBR322plasmid DNA + WE (30 µg/µl); 14, pBR322 plasmid DNA + WE (30 µg/µl) + 250 Gy.

Fig. 6. Effect of different fractions of HAPH against radiation (250 Gy)-induced DNA damage. %SC form (supercoiled form) of pBR322 DNA retainedrepresents percentage protection, while %OC form (open circular form) represents percentage damage. Each experiment was performed in triplicate and wasrepeated three times. ∗ p < 0.05. Drug + radiation (250 Gy) vs. radiation (250 Gy).

Discussion

Antioxidant compounds are known to influence peroxida-tion, DNA damage process mainly due to their free radicalscavenging, divalent ion chelation properties [38, 39].Homeostatis is maintained within the cell by the combinedaction of nutritionally occurring antioxidants, antioxidantenzymes like superoxide dismutase, catalase, glutathione-S-transferase (which catalyze the free radicals into unreactiveproducts), and certain high molecular weight proteins i.e.,ceruloplasmin, transferrin, albumin, which bind with transi-tion metals and clear them from the extracellular milieu, dueto their inherent capacity to react with hydrogen peroxideto form ferryl, perferryl species, which can initiate LPO[40].

In the present study, the iron chelation ability of differentfractionated extracts of P. hexandrum was tested using

2,2′-bipyridyl assay [33]. The activity of CE fraction wasfound to be substantially higher at even lower most concen-tration (1 µg/ml: 34.86%) and was almost equal to the metalchelation ability of quercetin (34.9%) and CE (35.4%) at50 µg/ml respectively. The inhibition of formation of chro-mogen complex by the different fractions followed the order:CE (41.59%) > quercetin > AE (31.25%) > HE (26.6%) >

HA (21.17%) > WE (20.48%). The aforementioned resultsdepict that CE fraction has highest metal chelation activityas compared to other fractions, which is likely due to theantagonistic and/or synergistic effect of the chloroform-soluble constituents, lignans including podophyllotoxin andits derivatives (Table 2). Exposure to ionizing radiation leadsto generation of free radicals, which increases LPO and alsoenhances the degradation of hemoglobin, ultimately leadingto increase in free cytosolic pool of iron [6], which actsas a secondary initiator. As secondary initiators, iron ions

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(a)

(b)

Fig. 7. Effect of different concentrations of (a) HE, (b) CE, (c) AE, (d) HA, and (e) WE against iron/ascorbate, radiation (250 Gy) and both [iron/ascorbate+ radiation (250 Gy)] as a combined stress-mediated lipid peroxidation evaluated in liver homogenate of strain ‘A’ mice. Each experiment was performed intriplicate and was repeated three times and the lipid peroxidation activity expressed as nanomoles of MDA (malonialdehyde) formed × 106. Lipid peroxidationin control represents 0% inhibition (maximum activity). (a) ∗ p < 0.05; HE + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; HE + iron/ascorbate vs.iron/ascorbate. ∗∗∗ p < 0.05; HE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (b) ∗ p < 0.05; CE + radiation (250 Gy) vs. radiation(250 Gy). ∗∗ p < 0.05; CE + iron/ascorbate vs. iron /ascorbate. ∗∗∗ p < 0.05; CE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (c)∗ p < 0.05; AE + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; AE + iron/ascorbate vs. iron/ascorbate. ∗∗∗ p < 0.05; AE + radiation (250 Gy)+ iron/ascorbate vs. radiation + iron/ascorbate. (d) ∗ p < 0.05; HA + radiation (250 Gy) vs. radiation (250 Gy). ∗∗ p < 0.05; HA + iron/ascorbate vs.iron/ascorbate. ∗∗∗ p < 0.05; HA + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate. (e) ∗ p < 0.05; WE + radiation (250 Gy) vs. radiation(250 Gy). ∗∗ p < 0.05; WE + iron/ascorbate vs. iron/ascorbate. ∗∗∗ p < 0.05; WE + radiation (250 Gy) + iron/ascorbate vs. radiation + iron/ascorbate.

(Continued on next page)

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catalyze OH• radical formation thereby accelerating LPO[41, 42]. The extent of initial damage caused by free radicalsis further amplified by Fenton reaction generated hydroxylradicals in the presence of superoxide and hydrogen peroxide[43]. Thus, the redox state and concentration of iron ionsin the cellular milieu plays a crucial role in amplificationof damage [44] as they interact with membranes to generatealkoxyl and peroxyl radicals, thereby inflicting furtherdamage to the cellular system [45]. Several workers havereported that chelating agents like diethylenetriamine

(c)

(d)

Fig. 7. (Continued)

pentaacetic acid exhibit antioxidant potential since they areable to occupy all the aqua-coordination sites of transitionmetals required for OH• generation [46]. The polypheno-lic components present in all fractions of P. hexandrumcontributed via their dual ability to donate hydrogen atoms(chain-breaking antioxidants) and chelate transition metalions (secondary antioxidants). The results clearly indicatedthat the fractionated extracts are able to modulate theconcentration of free iron in biological system and arethereby able to tackle the radiation-induced oxidative stress.

205

(e)

Fig. 7. (Continued)

Antioxidant activity of different fractions was assayed byusing the method of Oyaziu [34]. The antioxidant activities ofnatural compounds have a direct correlation with their reduc-ing ability (electron donation capacity). The antioxidant ac-tivity of different fractionated fractions extract, as comparedto BHT on the basis of their absorption unit values, was inthe following order: AE > HA > CE > BHT > HE > WE(Fig. 4). AE showed maximum antioxidant potential, whichcould be attributed partially to its polyphenolic content,which was maximally present in this fraction. On the otherhand, the fractions, viz., HE and WE, exhibited lesser antiox-idant potential (even lesser than that of BHT), which cor-related with the lower polyphenolic content of the fraction.CE showed intermediate antioxidant potential, overlappingwith that of BHT, which could be attributed to its polyphe-nolic content (>1%) and also due to the presence of lignanssuch as 4′-demethylpodophyllotoxin, epi- podophyllotoxin,podophyllotoxin, its glycoside and other compounds. Thisgives an indication that polyphenols and lignans play a vi-tal role in enhancing the overall electron donation capacityof extract acting in synergism and antagonism along withother constituents. The reducing power was found to in-crease with increasing fraction concentration. These resultscorrelate well with other published reports [30] through withdifferent extracts indicating that some of the compounds inthe extracts were electron donors and could react with freeradicals to terminate radical chain reactions and, therefore,were able to boost the natural antioxidant defence mechanism[47, 48].

Gamma radiation induces damage in biological systemsmainly via free radical generation, which interact withbiomolecules including DNA and lipid present in cell mem-branes [49, 50]. Plasmid relaxation assay was used for semi-quantitative assessment of the ionizing radiation-induced ox-idative damage to DNA. It is a more sensitive method to assessthe DNA damage as compared to chromatographic analysis of8-OHdG (8-oxo-7,8-dihydro-2′-deoxyguanosine) estimation[51]. Plasmid (pBR322) DNA in supercoiled form was nickedto generate open circular form, which was the result of single-stranded cleavage of supercoiled DNA. Wang and co-workershave suggested that single-strand damage in DNA is primarilydue to the generation of OH• radical in free solution, while theintrastrand cross-links might be formed following metal ionbinding to phosphate groups [52]. Radiation (250 Gy) causedextensive oxidative damage resulting in DNA fragmentationand degradation (Figs. 5 and 6). Exposure of pBR322 to 250Gy gamma-radiation caused complete conversion of super-coiled pBR322 DNA (fast mitigating), into open circular form(slow mitigating). In the present study, evaluation of the ef-fect of herbal fractions against 250-Gy gamma rays–inducedDNA damage in pBR322 plasmid DNA revealed their pro-tective efficacy against gamma radiation. Quercetin (a well-known standard flavonoid), tested along with different frac-tions, at a dose of 30 µg/µl, was found to render protectionagainst DNA damage. Non-polar fraction, viz., CE showedmaximum protection (68.09% SC form), which was evenhigher than that of quercetin (63.64% SC form) and polarfractions, viz., WE (51.13%). This can be attributed to the

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synergistic effect of polyphenolic compounds and lignanspresent in the different fractions. Quercetin and kaempferol,present in P. hexandrum are known to attenuate DNA damage[22, 24].

Anti-lipid peroxidation activity of HAPH was evaluatedon the basis of TBARS estimation [36]. The effect of dif-ferent fractions on induction of LPO in liver homogenate bynon-enzymatic (iron/ascorbate) method, radiation (250 Gy)and both iron and radiation in combination further unrav-eled the mechanism by which the fractions act at tissue level.The percentage inhibition of iron/ascorbate-induced LPO of-fered by AE, HE and CE was similar i.e., 99.76, 99.35 and97.22%, respectively, while that of HA and WE was compar-atively lower i.e, 88.10 and 87.45%, respectively. AE showedmaximal inhibition (99.76%), which is in conformity with itshigher metal chelation activity (31.25%), maximal polyphe-nolic content (9.26 mg%) and antioxidant potential. How-ever, on comparing the range of inhibition, polar fractionswere found to be effective at all the concentrations, tested inthe present study, while non-polar fractions and intermedi-ately polar fractions were mainly effective at higher concen-tration. AE showed maximum protection (92.25%) againstradiation-induced LPO. It can be concluded that AE providedprotection both via metal chelation and through electrondonation.

It is known that during irradiation, as a cascading effectdue to increase in high-energy electrons, followed by inter-action with water, hydroxyl, superoxide radicals and hydro-gen peroxide are formed [53, 54]. These reactive moleculesattack lipid molecules and again form new free radicals andthe chain continues. Iron ions further amplify the cascadeof reaction causing leaky membranes, which leads to ionexchanges, initiating the cell death pathway. In the state ofcombined stress (iron/ascorbate and radiation), HE inhibitedthe LPO maximally, but exhibited metal chelation activity toa moderate extent and had higher inhibitory ratio (149.83). Itwas, therefore, effective mainly at higher concentration. Onthe contrary, CE also inhibited the LPO (iron/ascorbate- andradiation-induced) in a similar way to HE, but showed lowerinhibitory ratio, maximum metal chelation activity (41.59%),high antioxidant activity and ability to provide DNA protec-tion (68.09% SC form retention). Similarly, the inhibitoryeffect of different fractions on iron/ascorbate-induced LPOcan be related to the presence of polyphenolic compoundslike quercetin, and kaempferol in the fractionated extracts,which exhibit antioxidant properties [55, 56] along with lig-nans (known for their potent free radical scavenging activity)and numerous glycoside derivatives. Polyphenols are com-posed of one (or more) aromatic rings bearing more than onehydroxyl groups and are capable of scavenging free radicalsby forming resonance stabilized phenoxy and quinone rad-icals [57]. Quercetin, which is known to have O-dihydroxyand 4,5-hydroxy ketone as substituent, exerts a strong scav-

enging activity effect, which is reported to be even better thanthat of trolox [58]. Polyphenolic compounds have been re-ported to exhibit the ability to donate their hydrogen atom inthe initial stage of LPO to compete with polyunsaturated fattyacids, thereby breaking the propagation chain. The plausiblemechanism by which these fractions inhibited LPO is indica-tive of free radical scavenging and to a certain extent metalion chelation capacity. Similar results have been reported byother workers [55, 56].

These results clearly indicated that fairly polar fraction(AE) has highest potential to mitigate the oxidative stressinduced by radiation. Further studies are needed at in vitro/invivo level to unravel the site-specific molecular mechanismsinvolved and other inherent activities that are relevant tocombat radiation stress.

Acknowledgements

We are thankful to the Directors of INMAS and RRL, Jammufor providing research facilities and to Col. (Dr.) B. Raut,Director, FRL, Leh and Dr. Om Prakash Chaurasia, Scien-tist, FRL, Leh for kindly providing the rhizomes of Podophyl-lum hexandrum. The work was supported by research fundsobtained from the DRDO’s CHARAK programme.

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