2. Review of literature 2.1. Interaction of radiation with matter

Review of literature

6 | P a g e

2. Review of literature

2.1. Interaction of radiation with matter - Physical and chemical aspects

Ionizing radiations are either electromagnetic or particulate in nature. X-rays and gamma ()

rays, the two forms of electromagnetic radiation, are similar in their properties, but differ in

their modes of production. X-rays are produced when accelerating electrons collide with a

metal target and suddenly decelerate, whereas, -rays are emitted by radioactive decay [22].

Particulate radiation is a stream of high energy sub-atomic particles, such as alpha particles,

beta particles (electrons), protons, neutrons and charged ions [23].

Ionizing radiation, while passing through matter, transfers its energy to molecules, producing

ion pairs. Whenever the energy imparted to a molecule exceeds its ionization potential, the

molecule gets ionized [22]. The effectiveness of radiation in producing biological damage is

often related to linear energy transfer (LET) [24]. The rate at which an electron transfers

energy to a material is known as LET, defined as the amount of energy transferred per unit

distance traveled. The actual relationship between the destructive efficiency of radiation and

LET values depends on the biological effect considered. For some effects, the efficiency

increases with an increase in LET, for some it decreases, and for others it increases up to a

point and then decreases. For a given biological effect, there is an LET value that produces an

optimum energy concentration within the tissue. Radiations with lower LET values do not

produce an adequate concentration of energy, whereas radiations with higher LET values

tend to deposit more energy than is needed to produce the effect; leading to wastage of

energy and decreased efficiency [24].

Interaction of ionizing radiations with biological systems may be direct or indirect [23].

Radiation interacts directly with critical intracellular targets by energy transfer, leading to

ionization or excitation of the target atoms, thus triggering a series of biological changes.

Indirect interaction occurs when radiation interacts with other atoms or molecules


7 | P a g e

(particularly water) in the cell to produce free radicals, also known as reactive species. A free

radical may be defined as any species capable of independent existence, containing one or

more unpaired electrons [25, 26]. It is the presence of unpaired electron(s) that renders free

radicals highly unstable, reactive and capable of damaging critical targets such as proteins,

lipids and even DNA by diffusing deep into the nucleus [27]. Reactive species are mainly

classified into reactive oxygen species (ROS) and reactive nitrogen species (RNS). Reactive

oxygen species (ROS) such as hydroxyl radicals (●OH), hydrogen radical (H

●), hydrated

electron (eaq–

), superoxide radical (HO2●), hydronium ion (H3O

+), etc. are generated in cells

not only under the influence of xenobiotics and radiation (UV, X-rays, -rays), but also

endogenously, as by-products of the oxygen metabolism (mitochondrial respiration) [28] as

shown in Figure 2.1.

Figure 2.1. Sources of ROS generation in cells

Endogenous ROS play a major role in oxidative DNA lesions [29]. The genotoxic effect of

endogenous ROS is mainly mediated via ●OH generated from hydrogen peroxide. This


8 | P a g e

occurs by a redox reaction with traces of reduced transitional metal ions, mainly ferrous, via

Fenton chemistry [30]. The major difference between endogenous ROS and those generated

by radiation (exogenous ROS) is that, while the spatial distribution of ●OH produced from

endogenous ROS is diffused, radiation generates clusters of ●OH within a nanometer scale,

resulting in multiple radical attacks on DNA [31-33]. Consequently, the relative frequency of

DNA lesions is much higher for exogenous sources than the endogenous ones. As ROS

implies potential risk for cells, an endogenous antioxidant defense system exists to maintain a

steady state level of ROS.

2.2. Bio-molecular targets of radiation

Ionizing radiations damage living tissues through a series of molecular events, triggered by

the very first step of free radical generation. Majority of the radicals, generated as a result of

indirect interaction of radiation, react with the cellular macromolecules such as DNA, RNA,

proteins, membrane lipids, etc. and cause cell dysfunction and mortality [34-36]. Radiation

damage is manifested mainly in the form of lipid peroxidation, protein oxidation and DNA

lesions (Figure 2.2). A broad spectrum of DNA lesions, including damage to nucleotide

bases, cross-linkages, and DNA single/double-strand breaks are induced. This is followed by

altered cell division, cell death, depletion of stem cell pools, organ system dysfunction and, if

the radiation dose is sufficiently high, even death. Reports also state that inappropriately

repaired DNA breaks can lead to induction of chromosomal abnormalities, mutations and

cancer [37]. Radiation-induced lipid peroxidation alters cell membrane fluidity leading to

degradation and impaired biological defense [38].


9 | P a g e

Figure 2.2. Bio-molecular targets of radiation

2.2.1. DNA lesions

Considerable evidence suggests that ionizing radiation primarily target DNA, leading to

mutations, carcinogenesis and cytotoxicity [39, 40]. Two well recognized distinct

mechanisms are involved in radiation-induced DNA damage. Firstly by direct ionization of

atoms in the DNA molecule (direct effect) and secondly by free radicals, generated from

radiolysis of surrounding water molecules (indirect effect) [41]. The major contributor to the

indirect effect is ●OH, as evident from studies involving

●OH scavengers [42]. Direct DNA

damage is mediated mainly by ●OH, generated in water molecules intimately associated with

DNA, since these radicals cannot be scavenged effectively [43]. Indirect damage, affecting

almost two-third of the DNA, is mediated by scavengeable radicals [42].

DNA lesions include changes in the deoxyribose ring and base structures [44], intra- and

inter-strand DNA-DNA cross-links, DNA single strand breaks (SSBs) and double strand


10 | P a g e

breaks (DSBs), and DNA–protein cross-links [45, 46]. The most susceptible sites of free

radical attack on DNA are depicted in Figure 2.3.A.

Figure 2.3.A. Susceptible sites for free radical attack on DNA

2.2.1.1. Damages to DNA bases and sugars

●OH radicals either abstract a hydrogen atom or add a double bond to nucleotide bases

producing reactive intermediates [47, 48], which destabilize the bridge between the base and

the sugar. Disruption of the sugar-base bond results in loss of nitrogenous bases and

formation of basic deoxyribose residues. Abstraction of hydrogen atoms from the sugar–

phosphate backbone of DNA generates 2-deoxyribose radicals that attack molecular oxygen

or endogenous thiols, leading to strand damage [47]. Sugar radicals might either release an

unchanged base from DNA [49] or may react with base residues on the same nucleotide to

yield an inter-strand nucleotide cross-link [47].


11 | P a g e

8-hydroxydeoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications resulting

from direct addition of ●OH to nucleotide base [50]. Evidences indicate that singlet oxygen

(1O2) may also be involved in oxidative damage to DNA following UV radiation [51].

Extracellular 8-oxo-dG content has been used as a sensitive marker for oxidative stress.

2.2.1.2. DNA strand and DNA-protein cross-links

Radiation-induced coupling of pyrimidine radicals to a neighboring purine base produces an

intra-strand cross-link lesion [52]. Nucleotide radicals can covalently bond to the deoxyribose

of the same nucleotide or to its neighboring base, yielding cyclonucleosides and nucleobase–

nucleobase inter- or intra-strand cross-links, respectively as shown in Figure 2.3.B. [53].

These alkyl radicals may react with other bases, resulting in inter-strand cross-linked DNA

[54]. Biochemical studies have demonstrated that these lesions, if not repaired, can block

DNA replication and transcription.

Figure 2.3.B. DNA strand cross links

Furthermore, there are evidences suggesting that the repair of inter-strand cross-links

involves the generation of a DSB. DSBs are extremely cytotoxic and mutagenic, more


12 | P a g e

specifically, a DSB with only one strand repaired. Repair of strand breaks is very crucial for

cell survival. In case of imperfect repair, a DSB can potentially result in chromosome

aberration, leading to genetic instability, mutation and chromosome rearrangements [55, 56].

DNA-protein cross links can block normal DNA transcription and replication in cells and are

therefore considered detrimental [53].

2.2.2. Lipid peroxidation

A continuous supply of free radicals initiate membrane lipid peroxidation leading to altered

membrane permeability and function. The entire process of lipid peroxidation is illustrated in

Figure 2.4. In brief, lipid peroxidation is initiated by radiolytic products of water such as

●OH, HO2

●, etc. that attack the unsaturated fatty acids forming carbon-centered radicals.

These radicals undergo molecular rearrangement to form stable diene conjugates. The radical

conjugates rapidly incorporate oxygen to form peroxyl radicals, which trigger a chain of

reactions by abstracting hydrogen from nearby unsaturated fatty acid, resulting in the

formation of lipid hydroperoxide and carbon-centered radical. In the presence of metal

catalysts, lipid hydroperoxides are cleaved to form lipid alkoxy and peroxyl radicals. Lipid

alkoxy radicals are prone to form cytotoxic aldehydes such as malondialdehyde (MDA) and

4-hydroxynonenal (4HNE), through beta cleavage [57]. MDA and 4HNE are quite stable and

employed as biomarkers of lipid peroxidation. Intensive studies on these aldehydes have

revealed their apoptotic potential in cells [58-61].


13 | P a g e

Figure 2.4. Sequence of events involved in lipid peroxidation

2.2.3. Protein oxidation

Protein oxidation is defined as the covalent modification of a protein, induced either directly

by ROS or indirectly by reaction with secondary by-products of oxidative stress [62].

Hydrogen atom abstraction at the alpha carbon or alkyl carbon of protein backbone initiates

the fragmentation process (Figure 2.5). The amino acid residues of protein are also prone to

free radical attack. Oxidation of aliphatic side-chains, by hydrogen atom abstraction, results

in the formation of hydroxylated derivatives such as peroxides, alcohols and carbonyls [63-

65]. Reaction of aromatic side-chains is mediated primarily by hydrogen addition. Some

specific aromatic side-chain oxidation products, such as di-tyrosine, ortho-tyrosine and meta-

tyrosine have been used as biomarkers of protein oxidation [66].


14 | P a g e

O

HN

R

O

NH

R

H

O

HN

R

O

NH

R

.

O2

O

HN

R

O

NH

R

OO

O

N

OR

R

NH

-HO2

H2O

O

NH2

R

+

R

O

O

NH

O

HN

R

O

NH

R

O

RH

-R

O

HN

R

O

NH

R

OOH

O

HN

R

R

O

+ NH

O

. .

.

.

.RH

-R.

O

HN

R OH

O

NH

R

X.

O

HN

R

O

NH

R

H

X

.

.

O

HN

R

O

NH

H

ROO.

O2

O

HN

R

O

NH

H

RO.

Protein backbone

Side chain radical alpha-Carbon radical

alpha-Carbon peroxyl radicalSide chain peroxyl radicalSide chain alkoxyl radical

alpha-Hydroxyalkyl radical

O2

alpha-Hydroxyalkyl peroxyl radical

Further reactions

Alkoxy radical

alpha-Carbon alcohol

beta-Scisson

Backbone imine

Further reactions

Figure 2.5. Radical-induced protein backbone fragmentation


15 | P a g e

2.3. Radiation injuries – a result of imbalance in endogenous defenses

Two of the biologically important endogenous ROS are superoxide anion radical (O2●-

) and

hydrogen peroxide (H2O2). Endogenous superoxide, a result of mitochondrial respiration

[30], is converted to hydrogen peroxide efficiently by cellular SOD. Although O2●-

and H2O2

are relatively long lived species and can diffuse over considerable distance, these are unable

to damage DNA directly [29]. As mentioned earlier in section 2.1, cells and tissues are

equipped with endogenous enzymes e.g. superoxide dismutase (SOD), glutathione peroxidase

(GPx) and catalase (Figure 2.6). These antioxidant enzymes facilitate the neutralization of

endogenously generated free-radicals, thus limiting ROS-induced cytotoxicity [67].

Figure 2.6. Endogenous antioxidant enzymes- defense against oxidative stress

Reduced glutathione (GSH), a very important endogenous antioxidant, attenuates radiation

toxicity by scavenging free radicals and by maintaining thiol-disulphide balance and cellular

ATP levels [68]. Radiation can lead to increase in ROS levels, creating oxidative stress, thus

leading to a depletion in cellular antioxidant stores [69]. Once the level of ROS increases

above tolerable limits, the endogenous system fails to protect the cells from the hazardous

effects of free radicals. Therefore, despite the existence of efficient defense mechanisms


16 | P a g e

(endogenous antioxidants and DNA repair genes), cellular generation of ROS imposes a

serious threat to the cellular integrity [70].

Toxicity of high-dose ionizing radiation manifests as acute radiation syndrome, affecting

highly proliferative systems such as the hematopoietic system (HP) and the gastrointestinal

tract (GI) [71, 72]. Radiation doses of 7-8 Gy induce hematopoietic damage, as hematopoietic

system is the most radiosensitive, due to a high cell turnover [68, 73]. The primary cause of

mortality during the early phases of radiation-induced hematopoietic syndrome is sepsis. This

results from opportunistic infections due to reduced blood neutrophil count. The situation is

further complicated by thrombocytopenia and defective immune system [74]. Radiation doses

above 12 Gy damage gastrointestinal barrier thereby facilitating the entry of bacteria in

interstitial space, leading to bacteremia. Also, there is water and electrolytes loss resulting in

dehydration, collectively termed as gastrointestinal syndrome [75].

2.4. Global efforts in the search for radioprotectors

Radioprotection is an area of great significance due to wide application in planned

radiotherapy as well as unplanned radiation exposure [76]. Research in the development of

radioprotectors worldwide has focused on screening a variety of chemical and biological

compounds. The initial development of radioprotectors led to the discovery of effective,

synthetic thiol compounds (Figure 2.7) [77-90], however, the side effect of these agents

necessitated the search for second-generation drugs that are more effective, less toxic and

easier to administer [75].

The potential of certain substances in protecting against ionizing radiation-induced damages

was first published in 1949 [91]. Patt et al., first reported the in vivo radioprotection by a

naturally occurring amino acid, cysteine [83]. Studies on similar lines showed that aminothiol


17 | P a g e

compounds like cysteine and cysteamine fulfill the structural requirements for radioprotection

and the presence of functional SH moiety in these compounds renders radioprotective

property [92]. However, these compounds are limited in their use due to serious side effects

and toxicity at all radioprotective doses. Preclinical research on radioprotectors has shown

several compounds quite effective in lab, but transitional extrapolation to humans have turned

unsuccessful due to toxicity and high risk of side effects [3, 27].

Treatments, which either reduce the risk/severity of damage to normal tissue or facilitate the

healing of radiation injury, are being developed. Toll-like receptor 5 (TLR5) activation by the

flagellin derivative, CBLB502, as radioprotector was one such approach [72, 93, 94]. A

single injection of CBLB502 (0.2 mg/kg) before lethal (> 13 Gy) total-body irradiation

protected mice from both gastrointestinal and hematopoietic acute radiation syndromes and

improved survival. CBLB502 also enhanced survival post irradiation, but at lower radiation

doses (9 Gy). The suggested mechanism of CBLB502 action was enhanced expression of NF-

B-induced antioxidant superoxide dismutase 2 (SOD2). CBLB502 is currently at an

advanced stage of development for biodefense applications as a medical radiation

countermeasure.


18 | P a g e

Figure 2.7. Milestones in the development of radioprotectors


19 | P a g e

Various natural and synthetic compounds including antioxidants, cytoprotective agents,

immunomodulators, vitamins and DNA binding molecules, have been evaluated extensively

for radioprotective potential in both in vitro and in vivo models [70, 95-98]. In recent years,

an array of immunomodulatory agents, haemopoietic growth and stimulating factors,

synthetic chelating agents and natural antioxidants have been examined for their ability to

ameliorate radiation-induced damage [75]. Emphasis has been on natural and synthetic

compounds based research, including organometallic compounds and biological response

modifiers (Table 2.1) [40, 99-139]. Immunomodulators are non-cytokine drugs proposed as

alternative stimulator of haematopoietic stem cells. These stimulate growth, differentiation

and proliferation of haematopoietic progenitor and stem cells, thus protecting and repairing

radiation-induced abnormalities [119]. Reduction of oxidative stress-induced damage by such

natural antioxidants provides a degree of protection against radiation injury. Natural

compounds in the diet provide functional antioxidants such as vitamins, minerals and

enzymes. Randomized clinical trials on antioxidant vitamins, suggest that use of high doses

of antioxidants as adjuvant therapy might limit toxic effects of radiotherapy, without

compromising efficacy [16]. A group of synthetic SOD mimetic compounds, with a metal ion

(Cu, Fe, Mn and Zn) at their active centers, have also been developed [75]. All such agents

have shown varying extent of protection when administered to cancer patients undergoing

radiotherapy, and have shown encouraging results. However, the strategy becomes

jeopardized when it comes to using synthetic molecules during radiotherapy to reduce the

unwanted radiation side effects. WR-2721 is the best radioprotector studied so far, but it has

failed to find acceptability in routine radiotherapy due to undesirable side effects and

exorbitant cost [140, 141]. Therefore, the basic property of free radical scavenging has been

the central theme in the development of radioprotectors.


20 | P a g e

Table 2.1. List of biological modifiers used in radioprotection

Biological response modifiers DRF/DMF/Percentage

Survival

Reference (s)

Cytokines

IL-1 1.2 -1.25 [123]

TNF-α 1.15 [102]

PGEs 1.5 [106-110, 134]

Leucotrienes 1.65-2.07 [135]

Polysaccharides

Bacterial lipopolysaccharide 1.22 [125]

Glucan 1.08 [125]

Bacillus Calmette-Guerin * [99]

Carboxymethylglucans 1.21-1.4 [119, 120, 124-

126, 128, 131]

Mannane mannozyne 2.16 [132]

Immunomodulators

Ammonium trichloroethylene-o-o’-tellurate * [113-117, 132]

Ribomunyl * [133]

Metals and metal complexes

Zinc aspartate * [104, 105]

Selenium with thiols-cysteamine * [136]

Selenomethionine 80% at 9 Gy [138]

Selenomethionine + WR-2721 2.6 [137]

3,3'-Diselenodipropionic acid (DSePA), a

diselenide and a derivative of selenocystine 35.3% at 10 Gy

[142]

Simple salts of Cu and Zn * [129, 130]

Copper complexes such as copper glycinate,

copper(II) 2(3,5-diisopropylsaicylate) and

copper(II)(chloride)

67 %

[40, 111]

Nitroxide tempol 1.25-2.5 [121]

Diltiazem 100% [103]

Cimetidine > 1.5 [122]

Captopril I and II 1.1-1.3 [139]

Naturally occurring substances

Vitamins E, A and C, superoxide dismutase * [8]

Algal mutant Chlorella vulgaris 1.11-1.15 [127]

Melatonin * [100, 101]

Methylxanthines (1.2-2.0)(1.3-2.3) [112]

Caffeine 1.1-1.2 [118]

*Data not available on dose reduction/modifying factors; DMF= Dose modifying factor, DRF= Dose reduction

factor

2.5. Herbal radioprotectors – approach towards safer alternatives

A number of plants have been utilized successfully for the treatment of free radical-mediated

diseases [16, 17]. It is, therefore, reasonable to expect that plants may contain certain


21 | P a g e

compounds that confer protection against ROS-mediated damage. A recent review

emphasizes the potential of natural products in radioprotector drug discovery [5].

A number of medicinal plants have shown protective effects against the damaging effects of

ionizing radiations [143-154]. Plant extracts, eliciting radioprotective efficacy, contain a

plethora of compounds, including antioxidants, immunostimulants, cell proliferation

stimulators, anti-inflammatory and antimicrobial agents, some of which may act in isolation

or in combination with other constituents from the same plant. Ayurveda and several other

traditional systems (Chinese, Japanese, Korean, Siddha, European, Tibetan, Unani, etc.) of

medicine recommend the use plant extracts/constituents in treating radiation-injuries. Herbal

sources with proven radioprotective efficacies include cruciferous vegetables (e.g. cabbage

and broccoli), green tea (polyphenols), Spirulina platensis, Mentha arvensis Linn. (mint),

Podophyllum hexandrum Linn., Syzygium cumini Linn. (Jamun, black plum), Panax ginseng

Linn., Aspalathus linearis (N.L.Burm.) (rooibos tea), soy products, venoruton (rutoside),

bixin (carotenoid), Gingko biloba Linn. extract (flavone glycosides and terpene lactones),

milk thistle (silymarin), grape seed extract, triphala extracts, Eleutherococcus senticosus

(Rupr. & Maxim.) or Shigoka extract, curcumin, chlorogenic acid, quercetin, garlic (allicin),

lycopene, methylxanthines, melatonin, ellagic acid, etc. Most studies using plants have

focused on the evaluation of radioprotective efficacy of whole extracts or poly-herbal

formulations, and in some cases, fractionated extracts and isolated constituents [143, 144,

146], [155-170]. A few plants, their extracts and fractions are listed in Table 2.2.


22 | P a g e

Table 2.2. Traditional herbal plants showing therapeutic activities relevant to radioprotection

Plant Family Use in radioprotection

Optimum

radioprotec

tive dose

Reference(s)

Aegle marmelos Corr. ex

Roxb.[171, 172]

Rutaceae

Provided protection against radiation-induced sickness and

mortality in mice

15 mg/kg

b.wt. [171, 172]

Acanthopanax senticosus

(Rupr. and Maxim.)[173, 174]

Araliaceae

Shigoka extract increased leukocyte counts and diminished cerebral

haemorrhage

24 mg/kg

b.wt. ; 24 h

i.p. pre

irradiation

[173, 174]

Aphanamixis polystachya

(Wall.) R.N. Parker[175]

Meliaceae

Ethyl acetate fraction significantly reduced frequency chromosomal

aberrations like acentric fragments, chromatid and chromosome

breaks, centric rings, dicentrics, exchanges

7.5 mg/kg b.

wt. pre 1-5 Gy

of whole body

-radiation

[175]

Ageratum conyzoides

Linn.[176]

Asteraceae

Alcoholic extract effectively protected mice against 10 Gy-induced

gastro intestinal and bone marrow injury

75 mg/kg

b.wt. ; 1 h pre

irradiation

[176]

Allium cepa Linn.[70]

Alliaceae Dried bulb effective against X-rays 20 mg/kg

b.wt. [70]


23 | P a g e

Allium sativum L. Gaertn.

[177, 178]

Alliaceae

Aged garlic extract (S-allylcysteine, S-allylmercaptocysteine,

allixin and selenium) possess significant antioxidant and anti-

carcinogenic properties

* [177, 178]

Aloe arborescens Mill.[179]

Liliaceae Protected mouse skin against soft X-rays by scavenging hydroxyl

radicals and reducing alterations in enzyme activity * [179]

Archangelica officinalis

Hoffm.[163]

Apiaceae

Combination of Archangelica officinalis and Ledum palustre

extracts increased survival by 70% (DMF: 1.48) in mice

5-15 min

before 7.5 Gy

irradiation

[163]

Angelica sinensis (Oliver)

Diels[180]

Apiaceae

The polysaccharide fraction, containing a ferulic acid, increased

survival in irradiated mice (> 30 days) by promoting haemopoietic

stem cell proliferation

i.v. route

(post-

irradiation)

[180]

Amaranthus paniculatus

Linn.[181]

Amaranthaceae

Leaf extract protected mice against 5 Gy by reducing lipid

peroxidation, glycogen and cholesterol levels in brain

600 mg/kg

b.wt. for 2

weeks

[181]

Biophytum sensitivum

(Linn.) DC.[182]

Oxalidaceae

Biophytum sensitivum attenuated the enhanced serum ALP, GPT,

LPO and liver GSH in irradiated animals. Protective effect on

radiation-induced haemopoietic damage is mediated through

immunomodulation as well as sequential induction of IL-1ß, GM-

CSF and IFN-γ

50 mg/kg

b.wt. [182]

Centella asiatica (Linn.) Mackinlayaceae Aqueous extract protects Sprague Dawley rats against the adverse 100 mg/kg [167]


24 | P a g e

Urban [167]

effects of low-dose ionizing radiation (2 Gy). Administered orally,

provides total body protection in mice against sublethal (8 Gy) 60

Co

gamma radiation

b.wt; i.p.;

single dose; -1

h

Coronopus didymus

Linn.[21, 183, 184]

Brassicaceae

Optimum radioprotection was observed upon i.p. administration,30

min prior to 10 Gy irradiation; DRF 1.07

400 mg/kg

b.wt.

[21, 183,

184]

Curcuma longa Linn.[185-

187]

Zingiberaceae

Curcumin and its analogs are effective radiomodifiers as they

selectively protected normal lymphocytes and isolated rat

hepatocytes against -radiation, with no protection to tumor cells.

Also, these compounds enhanced radiation effect on cancer cells by

increasing tumor response

* [185-187]

Emblica officinalis

Linn.[188]

Euphorbiaceae

Prevented -radiation-induced lipid peroxidation, protected

mitochondrial SOD and radiation-induced DNA strand breaks in a

concentration-dependent manner

* [188]

Ginkgo biloba Linn.[189-

191]

Cycadaceae

Ethanolic extract (30%) effective against hydroxyl radical-induced

apoptosis in human cell culture and on rat cerebellar neuronal cell

culture

100 µg/ml [189-191]

Glycyrrhiza glabra

Linn.[192]

Fabaceae

70% methanolic extract protected rat microsomal membranes from

-radiation-induced lipid peroxidation 100 μg/ml [192]

Hypericum perforatum

Linn.[193]

Hypericaceae

Aqueous extract protected mice bone marrow and intestinal mucosa

against X-ray * [193]


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Hippophae rhamnoides

Linn.[194]

Elaegnaceae

An aqueous-alcohol extract of the berries increased life span and

survival (at 30 days) by 80% in irradiated Strain ‘A’ mice

30 mg/kg b.

wt. [194]

Lycium chinense Mill.[195]

Solanaceae

Root extract significantly improved the recovery of leucocyte,

erythrocyte and thrombocyte counts and hematocrit in ICR strain

mice irradiated with X-ray

* [195]

Mentha arvensis Linn.[147]

Lamiaceae Chloroform extract protected mice against gastrointestinal and bone

marrow death (DMF: 1.2) * [147]

Moringa oleifera Lam.[196]

Moringaceae

Leaf extract significantly reduced the proportion of aberrant

chromosomes, in the metaphase stage of cell division by day 7

post-irradiation in mice

150 mg/kg

single dose,

pretreatment

i.p.

[196]

Ocimum sanctum

Linn.[153]

Lamiaceae

Flavonoids orientin and vicenin significantly increased mouse

survival against γ-irradiation. DMF: 1.37; Vicenin > DMF: 1.30;

orientin in murine models.

50 µg/kg/i.p. [153]

Panax ginseng Linn.[197,

198]

Araliaceae Water-soluble whole plant extract of ginseng provided the best

protection against 60

Co gamma radiation in C3H mice * [197, 198]

Podophyllum hexandrum

Royle[199]

Berberidaceae

Provided protection to haematopoietic, gastrointestinal,

reproductive and central nervous system (CNS) * [199]

Piper longum Linn.[200]

Piperaceae Ethanolic extract of fruits protected mice against radiation-induced

decline in WBC, bone marrow cells and GSH * [200]


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Pilea microphylla (Linn.)

Liebm. [201]

Urticaceae

Ethanolic extract conferred 80% protection in Swiss albino mice

and a DMF of about 1.12

900 mg/kg

b.wt. [201]

Syzygium cuminii (Linn.)

Skeels [76, 148]

Myrtaceae

Leaf extract reduced radiation-induced sickness, gastrointestinal

syndrome, bone marrow damage and micronuclei formation in

human peripheral blood lymphocytes

80 mg/kg

b.wt.;100

μg/ml

[76, 148]

Tephrosia purpurea

(Linn.) Pers.[202]

Fabaceae

Extract protected Swiss albino mice against 5 Gy-induced

hemopoetic injury * [202]

Tinospora cordifolia

(Thunb.) Miers.[194]

Menispermaceae

A pure arabinogalactan polysaccharide, genistein, provides some

protection against radiation-induced intestinal damage in mice

(DMF: 1.16)

* [194]


27 | P a g e

Many plants exhibit a diverse array of biological activities including mitigation of ionizing

radiation-induced damage in mammalian systems. However, so far, only a fraction of these

plants have been investigated systematically. Isolation of the bioactive constituents from plants

with radioprotection potential needs attention, considering the current trend of radioprotector

developmental process [203].

In an article published on Jan 6th

, 2012 in The Times of India (TOI) – Bhubaneswar edition,

scientists at the Defense Research and Development Organization (DRDO) announced the

success of the phase I clinical trial on radioprotective potential of Ocimum sanctum (Tulsi).

Tulsi-based herbal medicines are being screened through second phase of clinical trials as was

reported in a consecutive article published in TOI – Bhubaneswar edition on Jan 8th

, 2012.

Besides Tulsi, sea-buckthorn and Podophyllum hexandrum (Himalayan May Apple) were other

herbs chosen to develop medicines, not just to treat those affected by nuclear radiations but also

as a prophylactic measure for rescue work in radiation-affected areas. It is worth mentioning that

a significant part of the preliminary radioprotection studies on Tulsi, have been carried out in our

University.

2.6. Research on synthetic and herbal radioprotectors in our lab

Our team has also worked on a few synthetic molecules in this direction and the findings are

summarized below.

1. Pre-treatment with 5-amino salicylic acid (5ASA) significantly reduced the micronuclei

counts to 40-50% compared to radiation control, with a dose modification factor (DMF) 2.02

-2.53 [204].


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2. Sulfasalazine (SAZ) treatment at 120 mg/kg optimally protected mice, without any toxicity

per se. At this dose, SAZ produced >60% reduction in the radiation-induced percent aberrant

metaphases and micronucleated erythrocytes. SAZ protected mice against radiation-induced

chromosomal damage and cell cycle progression delay. SAZ also protected plasmid DNA

(pGEM-7Zf) against Fenton reaction-induced breaks, suggesting free radical scavenging as

one of the possible mechanism for radioprotection [205].

3. 3,3'-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was

evaluated for in vivo radioprotective effects in Swiss albino mice, at 2 mg/kg, i.p., for 5 days

before whole-body exposure to -radiation. DSePA improved the 30-day survival in

irradiated mice by 35.3%. The mRNA expression analysis of genes revealed that DSePA

augmented GADD45 and inhibited p21 in both spleen and liver tissues. In response to

radiation-induced DNA damage, cells activate p21 and GADD45 genes that regulate the

cell cycle arrest at G1/S and G2/M respectively. GADD45α is specifically associated with

DNA repair and prevent apoptosis in normal cells [206, 207]. DSePA also inhibited

radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the

expression of the pro-apoptotic BAX and the anti-apoptotic Bcl-2 genes [142].

In our laboratory, the free radical scavenging, antioxidant and radioprotective potential of plants

such as Coronopus didymus (Linn.) Sm., Pilea microphylla (Linn.) Liebm., Ocimum sanctum

Linn., Ficus racemosa Roxb. (to name a few important ones) have also been carried out. A part

of the collaborative project on the flavonoids (orientin and vicenin) derived from Ocimum

sanctum (Tulsi) showed a significant in vitro free radical scavenging activity and protection

against radiation-induced lipid peroxidation in mouse liver [153]. A few plants screened for

radioprotection potential are given below.


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1. Systematic chemical investigation of Coronopus didymus (Linn.) Sm. revealed the presence

of flavonoids such as chrysoeriol and its glucoside, which are potent antioxidants and free

radical scavengers [208]. CDF1, the most active fraction of Coronopus didymus, conferred

maximum in vivo radioprotection of 70% at a dose of 400 mg/kg prior to 10 Gy -radiation in

Swiss albino mice [21]. Dose reduction factor (DRF) at 400 mg/kg was found to be 1.07.

CDF1 pre-treatment improved the levels of endogenous antioxidant enzymes to normal

levels. Isolation of active principles and screening them for in vitro and in vivo

radioprotection is the aim of the present study.

2. Active fraction of Pilea microphylla (Linn.) Liebm. (PM1), showed 80% protection at a dose

of 900 mg/kg, with a DRF of about 1.12 in Swiss albino mice. PM1 protected livers of

irradiated mice from depletion of endogenous antioxidants like glutathione, GST, SOD,

catalase and thiols. PM1 conferred protection to the gastrointestinal and hematopoietic

system contributing to overall radioprotection [201]. Active constituents responsible for

radioprotection have been isolated from this plant [209, 210].

3. Ethanolic extract of another plant, Ficus racemosa Roxb. (FRE), exhibited significant

antioxidant activity (DPPH, ABTS, hydroxyl radical and superoxide radical scavenging and

inhibition of lipid peroxidation), higher than standard compounds. In vitro radioprotective

potential of FRE, studied using micronucleus assay in irradiated Chinese hamster lung

fibroblasts (V79), revealed maximum radioprotection at 20 µg/ml of FRE, when

administered 1 h prior to 0.5, 1, 2, 3 and 4 Gy -radiation [191].

From the above studies it is clear that the presence of polyphenolic constituents, majority of

which are flavonoids in nature, imparts radioprotective potential to the plants.


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2.7. Flavonoids as exogenous radioprotective agents

Flavonoids are polyphenolic compounds possessing diverse chemical structure and

characteristics. Present ubiquitously in plants, flavonoids are mainly found in fruits, vegetables,

nuts, seeds, flowers, and bark and are generally present in the form of glycosides [211, 212].

Till date, more than 9000 different flavonoids, exhibiting important biological roles have been

identified in plants [213]. In general, all flavonoids are derivatives of the 2-phenylchromone

parent compound composed of three phenolic rings referred to as A, B and C rings (Figure 2.8).

Figure 2.8. Basic structure of flavonoid nucleus

Flavonoids are classified according to their chemical structure. The major flavonoid classes

include flavones, flavonols, flavanones, catechins (or flavanols), anthocyanidins, isoflavones,

dihydroflavonols and chalcones (Figure 2.9).


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Figure 2.9. Classification of flavonoids with basic structure and examples


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Flavonoids confer radioprotection by three mechanisms (Figure 2.10), which are mainly

ROS scavenging

Improving cellular antioxidant levels

Protecting genomic integrity

Figure 2.10. Mechanisms of radioprotection by flavonoids [53].

2. Review of literature 2.1. Interaction of radiation with matter

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