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Gamma irradiation of medicinally important plants and the enhancement of

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 International Journal of Radiation Biology

ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20

Gamma irradiation of medicinally important

plants and the enhancement of secondary
metabolite production

P. Vivek Vardhan & Lata I. Shukla

To cite this article: P. Vivek Vardhan & Lata I. Shukla (2017) Gamma irradiation of medicinally
important plants and the enhancement of secondary metabolite production, International Journal of
Radiation Biology, 93:9, 967-979, DOI: 10.1080/09553002.2017.1344788

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INTERNATIONAL JOURNAL OF RADIATION BIOLOGY, 2017
VOL. 93, NO. 9, 967–979
https://doi.org/10.1080/09553002.2017.1344788

REVIEW

Gamma irradiation of medicinally important plants and the enhancement of

secondary metabolite production
P. Vivek Vardhan and Lata I. Shukla
Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry, India

ABSTRACT ARTICLE HISTORY

Purpose: The profitable production of some important plant-based secondary metabolites (ginseno- Received 23 March 2017
sides, saponins, camptothecin, shikonins etc.) in vitro by gamma irradiation is a current area of interest. Revised 14 June 2017
We reviewed different types of secondary metabolites, their mode of synthesis and effect of c-radiation Accepted 15 June 2017
on their yield for different plants, organs and in vitro cultures (callus, suspension, hairy root). Special
effort has been made to review the biochemical mechanisms underlying the increase in secondary KEYWORDS
metabolites. A comparison of yield improvement with biotic and abiotic stresses was made. Gamma irradiation;
Results: Phenolic compounds increase with c-irradiation in whole plants/plant parts; psoralen con- secondary metabolites;
tent in the common herb babchi (Psoralea corylifolia) was increased as high as 32-fold with c-irradi- phenolic compounds;
ation of seeds at 20 kGy. The capsaicinoids, a phenolic compound increased about 10% with 10 ginsenosides; camptothecin;
kGy in paprika (Capsicum annum L.). The in vitro studies show all the three types of secondary shikonins
metabolites are reported to increase with c-irradiation. Stevioside, total phenolic and flavonoids
content were slightly increased in 15 Gy-treated callus cultures of stevia (Stevia rebaudiana Bert.). In
terpenoids, total saponin and ginsenosides content were increased 1.4- and 1.8-fold, respectively,
with 100 Gy for wild ginseng (Panax ginseng Meyer) hairy root cultures. In alkaloids, camptothecin
yield increased as high as 20-fold with 20 Gy in callus cultures of ghanera (Nothapodytes foetida).
Shikonins increased up to 4-fold with 16 Gy in suspension cultures of purple gromwell
(Lithospermum erythrorhizon S.). The enzymes associated with secondary metabolite production were
increased with c-irradiation of 20 Gy; namely, phenylalanine ammonia-lyase (PAL) for phenolics, chal-
cone synthase (CHS) for flavonoids, squalene synthase (SS), squalene epoxidase (SE) and oxidosqua-
lene cyclases (OSC) for ginsenosides and PHB (p-hydroxylbenzoic acid) geranyl transferase for
shikonins.
Conclusions: An increase in secondary metabolites in response to various biotic and abiotic stresses is
compared with ionizing radiation. A 5- to 20-fold increase is noted with 20 Gy irradiation dose. It
increases the yield of secondary metabolites by enhancing the activity of certain key biosynthetic
enzymes. Identification of the optimum dose is the important step in the large-scale production of sec-
ondary metabolites at industrial level.

CONTACT Lata I. Shukla lishukla@gmail.com Asst. Professor, Department of Biotechnology, School of Life Sciences, Pondicherry University,
Pondicherry–605014, India
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
968 P. V. VARDHAN AND L. I. SHUKLA
Abbreviations: PAL: Phenylalanine ammonia-lyase; CHS: Chalcone synthase; CS: Capsaicinoid synthase;
SS: Squalene synthase; SE: Squalene epoxidase; OSC: Oxidosqualene cyclases; STR: Strictosidine syn-
thase; TIA: Terpenoid indole alkaloid; GBA: m-geranyl-p-hydroxylbenzoic acid; PHB: p-hydroxylbenzoic
acid; GPP: Gernaylpyrophosphate
Introduction
Secondary metabolites are the diverse group of low molecu-
lar weight, organic compounds which are distinguished from
primary metabolites. They assist plants to interact with the
environment both biotic and abiotic and to establish defense
mechanisms (Murthy et al. 2014). Their role is not crucial for
normal growth and development but being synthesized in
the particular needs, they are required for defense purposes
and other interspecies protection (Bennett & Wallsgrove
1994). There are some unique secondary metabolites which
are highly specific to certain plant species with peculiar qual-
ities which are useful for humanity (Dixon 2001). Various
types of secondary metabolites produced by plants have
been used by mankind since time immemorial for numerous
purposes (Bourgaud et al. 2001). Many of them have been
used as medicines, pharmaceuticals, therapeutics, flavorings,
narcotics etc. (Singh et al. 2003; Wallace 2004; Patra & Saxena
2010; Baenas et al. 2014). Secondary metabolites may often
be biosynthesized from primary metabolites or share sub-
strates of primary metabolite origin (Herbert 1989; Yamazaki
et al. 2003a). The regulation of carbon fluxes between pri-
mary and secondary metabolism is controlled by a trade-off
between the growth and defense mechanisms thus, acquir-
ing protective adaptation to environmental stresses (Cheynier
et al. 2013). Their quantity is very low (1% dry weight) and
depends greatly on the physiological and developmental
stages of the plant (Facchini 2001). Secondary metabolites
include phenolic compounds, terpenoids and nitrogen-con-
taining compounds.
Secondary metabolites may represent chemical adaptations
to a wide range of environmental stress conditions or they
may serve as defensive, protective or offensive chemical
agents against microorganisms, insects and higher herbivor-
ous predators (Namdeo 2007). The microbial invasion of plants
induces the synthesis of anti-microbial secondary metabolites
in the same way as abiotic stress factors like radiation, osmotic
shock, fatty acids, inorganic salts and heavy metal ions induce
the synthesis of protective secondary metabolites in plants.
Abiotic stresses such as ultra violet (UV) radiation, high light,
nutrient deficiencies, temperature and herbicide treatment
often increase the accumulation of phenylpropanoids.
The biotic and abiotic stresses often induce production of
secondary metabolites in plant tissue culture systems (Akula
& Ravishankar 2011). The effect of different biotic and abiotic
elicitors and their role in secondary metabolites production
have been reported and recently reviewed (Naik & Al-Khayri
2016). Enhanced secondary metabolite production can be
induced in cell cultures by using elicitors in the culture
medium (Ahlawat et al. 2014; Srivastava & Srivastava 2014;
Shakeran et al. 2015; Hashemi & Naghavi 2016). Some of
them are Asperigillus niger extract (Ahmed & Baig 2014),
yeast extract (Deepthi & Satheeshkumar 2016), jasmonate
and salicilate (Song & Byun 1998; Siva et al. 2014) and agaro-
pectin (Fukui et al. 1983). The comparison of enhancement
of different secondary metabolites by gamma radiation,
biotic and abiotic stresses is illustrated in Table 1. Gamma
radiation is sparsely ionizing radiation which typically causes
more indirect effect in respect to the direct effect of radi-
ation. The gamma irradiation typically has been given by
60
Co source with dose rates in a range of 0.01–0.27 Gy/s. The
increased production of plant secondary metabolites with
c-irradiation was reviewed and presented at the 2016 inter-
national conference on radiation biology (ICRB) (Vardhan &
Shukla 2016). They act by stimulating stress responses which
could lead to the accumulation of secondary metabolites.
The type of stress and its magnitude are the major factors
determining the effects on the production of secondary
metabolites (Eilert 1987).
This review addresses different secondary metabolites, the
effect of c-irradiation on their yield from different in vitro cul-
tures or plant parts and also the mechanisms associated with
the accumulation of secondary metabolites. The effect of
c-irradiation on different types of secondary metabolites is
discussed; namely, total phenolics and flavonoids (capsaici-
noids, stevioside, psoralen), terpenoids (saponins, ginseno-
sides) and alkaloids (camptothecin, shikonins). Effort has
been made to compare the elicitation effect of c-irradiation
with different elicitors.
Types of secondary metabolites
Based on their biosynthetic origin, secondary metabolites of
plants can be classified into three broad groups: phenolic
compounds, terpenoids and nitrogen-containing compounds.
Phenolic compounds
Phenolic compounds are one of the largest groups of sec-
ondary metabolites produced by plants which have a wide
range of applications (Kabera et al. 2014) (Figure 1).
Phenolics typically present as esters or glycosides conjugated
with other natural compounds such as flavonoids, alcohols
and sterols (Dai & Mumper 2010). Some of them have anti-
oxidant, anti-inflammatory, anti-bacterial, antiseptic, anthel-
mintic and other biological and pharmacological properties
(Cueva et al. 2010; Stankovic 2011). Phenolics are biosynthe-
sized via a complex pathway, the phenylpropanoid pathway
also known as shikimic acid pathway (Figure 2). This pathway
also synthesizes aromatic amino acids which constitutes the
primary metabolism. It is linked with many such metabolic
pathways leading to a complex systemic network (Pereira
et al. 2009). Each of them is under tight control regulating
 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 969

Figure 1. Schematic representation of classification of phenolic compounds.

Figure 2. Schematic representation of the phenylpropanoid pathway (Devic et al. 1999). The enzymes whose activity is enhanced with c-irradiation are PAL and
CHS (written in bold). PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase; CHS: chalcone synthase; CHI: chalcone isomerase.
970 P. V. VARDHAN AND L. I. SHUKLA
one another through influx and efflux of intermediates
(Treutter 2001).
Most of the phenolics are polymerized into larger mole-
cules such as tannins and lignans (polyphenols). Polyphenols
are very important among phenolic compounds due to their
higher abundance in nature, diversity and their probable role
in the prevention of different diseases such as cancer, cardio-
vascular and neurodegenerative diseases (Scalbert et al.
2005). They are a vast group of secondary metabolites which
comprises more than 8000 known compounds. Based on the
differences in chemical structure, polyphenols can be sub-
divided into two classes: flavonoids and non-flavonoids
(Galeotti et al. 2008).
Furthermore, flavonoids can also be divided into three
groups; anthocyanins, flavones and flavonols. Anthocyanins
are hydrophilic in nature with a typical red to blue color
which gives some plants the typical color. They occur mainly
as glycosides of anthocyanidins such as cyanidin, delphinidin,
peonidin, pelargonidin, petunidin and malvidin (He et al.
2010; Aza-Gonz
alez et al. 2012; Zhu et al. 2012). They pro-
mote cardiovascular health and also have anti-cancer activity
and anti-inflammatory properties (Hui et al. 2010; Kruger
et al. 2014; Wallace et al. 2016). Flavones are colorless crystal-
line water-insoluble aromatic ketones and their derivatives,
many of which occur as yellow plant pigments in the form of
glycosides and are used as dyes. Flavones include luteolin,
apigenin, tangeritin, chrysin, etc. They are associated with
overall anti-oxidant benefits and delaying the metabolism of
drugs (Cermak 2008; Roy et al. 2015). Flavonols are the
hydroxyl derivatives of flavones which are colorless crystalline
molecules that accumulate mainly in the outer and aerial tis-
sues. Flavonols are a widely distributed subgroup of flavo-
noids which includes quercetin and kaempferol. They are
associated with anti-histamine, anti-inflammatory and anti-
oxidant activities leading to chronic disease prevention
(Nassis et al. 1991; Guardia et al. 2001; H€am€al€ainen et al.
2007; Wang et al. 2016).
In non-flavonoids, tannins are the prominent and diverse
group which gives foods the specific astringency. They
include gallic acid esters and proanthocyanidins. Most poly-
phenols contain repetitive units of simple phenolic moieties
such as pyrocatechol, resorcinol, pyrogallol, and phlorogluci-
nol. In hydrolysable tannins, the monomeric units will be
connected by ester linkages and in condensed tannins they
will be connected by stable C–C bonds. They form complexes
with many organic compounds of plants such as proteins,
amino acids, starch and cellulose (White 1957; Okuda et al.
1993; Khanbabaee & van Ree 2001). Tannins play an import-
ant role in plants, protecting them from herbivory (Karowe
1989; Forkner et al. 2004). They are used in tanning leather,
anti-corrosive primers and wood adhesives (Bliss 1989;
Matamala et al. 2000; Rahim & Kassim 2008; Pinto et al.
2013).
Terpenoids
Terpenoids are the most important and diverse group of sec-
ondary metabolites which comprises over 25,000 known
active compounds (Gershenzon & Dudareva 2007). They
include steroids, carotenoids, and gibberellic acid which con-
stitute a little functional and structural relevance in common.
Terpenoids are polymeric isoprene derivatives biosynthesized
from acetate by the mevalonic acid pathway (Cheng et al.
2007). They are formed by polymerization of isoprene units
linked in head-to-tail fashion (Maffei 2010). Their classification
is mainly based on the number of isoprene units they con-
tain. Monoterpenes (C10) contain two units, sesquiterpenes
(C15) three, diterpenes (C20) four, sesterterpenes (C25) five, tri-
terpenes (C30) six and so on.
Many of them have active biological and pharmacological
properties and are used for treating diseases both in humans
and animals (Tolstikova et al. 2006; Nassar et al. 2010;
Thoppil & Bishayee 2011). Various saponins and ginsenosides
are known to have anti-cancer, anti-diabetic, anti-hepatotoxic
and antifungal properties (Lacaille-Dubois & Wagner 1996).
Most of the groups of terpenes (monoterpenes, sesquiter-
penes, diterpenes, sesterterpenes, triterpenes, quinones,
hydroquinones, dammarane, lupane and lactones) are found
to have anti-inflammatory properties (Souza et al. 2014).
Betulinic acid and other lupane derivatives possess promising
anti-tumor and antiviral activities (Tolstikova et al. 2006).
Nitrogen-containing compounds
A variety of plant secondary metabolites have nitrogen as
part of their structure such as alkaloids, cyanogenic glyco-
sides and non-protein amino acids. Alkaloids are the larg-
est class of nitrogen-containing secondary metabolites
found in approximately 20% of vascular plant species (Lu
et al. 2012; Ashok et al. 2015). They are water soluble,
positively-charged molecules synthesized from common
amino acids particularly lysine, tyrosine or tryptophan.
More than 3000 alkaloids have been isolated from plants
and are scattered almost in every group of plants.
Alkaloids are sub-divided into three sub groups; protoalka-
loids, true alkaloids and pseudo alkaloids. They are remark-
able for their diversity in structure and functions and
hence there is no uniform classification. Mostly their classi-
fication is based on the common source of availability of
these compounds.
Cyanogenic glycosides are a class of nitrogen-containing
secondary metabolites in plants which emit volatile poisons
or toxins when the plants are crushed (Vetter 2000). Thus,
they act as feeding deterrents to many insects and other her-
bivores (Cooper-Driver & Swain 1976; Gleadow & Woodrow
2002).
Non-protein amino acids are a group of over 200 different
amino acids which occur free in plant cells and are not incor-
porated into proteins. A good number of different kinds of
these amino acids are found in plants of the family
Leguminosae. Their main function appears to be protective
against herbivores (Huang et al. 2011; Mach 2015). Many of
them closely resemble protein amino acids in their structure
and they can block their uptake or they may wrongly be
incorporated into proteins which become non-functional
(Hohsaka & Sisido 2002).
 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 971

Table 1. Comparison of enhancement of secondary metabolites by different elicitors.

Magnitude of increase (fold)
Gamma Biotic Abiotic
Plant name Secondary metabolite Plant material irradiation stress stress References
Phenolic compounds
Psoralea corylifolia (babchi) Psoralen Seeds 32 – – Jan et al. 2011
Cells – 9 – Ahmed & Baig 2014
Root – – 6.6 Siva et al. 2014
Capsicum annuum (chili) Capsaicinoids Pods 0.1 – – Topuz & Ozdemir 2004
Calli – 2.9 – Brooks et al. 1986
Cells – – 2 Johnson et al. 1991
Rosmarinus officinalis (rosemary) Total phenols Calli 5 – – El-Beltagi et al. 2011
Brassica oleraceae (broccoli) “ Planlets – 2.2 – Lola-luz et al. 2014
Arachis hypogea (peanut) “ Seeds – – 0 Rudolf & Resurreccion 2005
Terpenoids
Panax ginseng (ginseng) Saponinis Hairy root 1.4 – – Zhang et al. 2011
Cells – 0.7 0.8 Hu et al. 2003, Wu & Lin 2002.
Alkaloids
Nothapodytes foetida (ghanera) Camptothecin Calli 20 – – Fulzele et al. 2015
Ophiorrhiza mungos “ Cells – 13 – Deepthi & Satheeshkumar 2016
Camptotheca acuminata “ Cells – – 11 Song & Byun 1998
Lithospermum erythrorhizon (purple gromwel) Shikonins Cells 4 – 2 Chung et al. 2006, Fukui et al. 1983.

Table 2. Increase of secondary metabolites (phenolic compounds, terpenoids and alkaloids) production by c-irradiation in different plant materials, seedlings,
fruits, callus cultures, hairy roots and suspension cultures.
Plant name Secondary metabolite Plant material Dose (Gy) Magnitude of increase (fold) Reference
Phenolic compounds
Eryngium foetidum (culantro) Total phenols & flavonoids Plantlet 40 1.7, 1.2 Mohamed 2009
Curcuma alismatifolia (summer tulip) Total phenols & flavonoids Roots 20 1.5, 1.8 Taheri et al. 2014
Centella asiatica (centella) Total flavonoids Roots 20, 30 minor Moghaddam et al. 2011
Anethum graveolens (dill) Total flavonoids Flower 640 4 Said-Al Ahl et al. 2015
Capsicum annuum (chili) Capsaicinoids Fruits 10000 0.1 Topuz & Ozdemir 2004
Psoralea corylifolia (babchi) Psoralen Seeds 20000 32 Jan et al. 2011
Phyllanthu odontadenius Total phenols & flavonoids Seedlings 100 – Nakweti et al. 2014
Terminalia arjuna (arjuna) Total phenols Seedlings 25, 150 2 Akshatha et al. 2013
Rosmarinus officinalis (rosemary) Total phenols & flavonoids Calli 20 5, 5.6 El-Beltagi et al. 2011
Stevia rebaudiana (stevia) Total phenols & flavonoids Calli 15 minor Khalil et al. 2015
Ferula gummosa Total phenols Calli 20 1.6 Ashouri et al. 2016
Terpenoids
Panax ginseng (ginseng) Saponins, Ginsenosides Hairy root 100 1.4, 1.8 Zhang et al. 2011
- Ginsenoside Rg3 Std. Rb1 30 25 Kim et al. 2012
Panax ginseng (ginseng) Ginsenosides Hairy root 50 1.7 Kim et al. 2013
Eryngium foetidum (culantro) Saponins Plantlet 40 1.4 Mohamed 2009
Phyllanthu odontadenius Saponins Seedlings 100 – Nakweti et al. 2014
Alkaloids
Nothapodytes foetida (ghanera) Camptothecin Calli 20 20 Fulzele et al. 2015
Lithospermum erythrorhizon (purple gromwel) Shikonins Cells 16 4 Chung et al. 2006

Effect of c-irradiation on in vitro cultures
Gamma irradiation has been widely applied in medicine and
biology in terms of biological effects induced by a counter
intuitive switch-over from low doses stimulation to high
doses inhibition (Charbaji & Nabulsi 1999). Relatively low
doses of c-irradiation exhibit accelerated cell proliferation,
germination rate, cell growth, enzyme activity, stress resist-
ance and crop yields (Davies 1970; Sparrow et al. 1971).
Previous studies have reported that it has shown almost all
the similar effects on cell cultures in vitro. It is proved in sev-
eral investigations that the treatment of cell cultures with
relative low doses of c-radiation significantly increased the
yield of secondary metabolites (Table 2).
Recent evidences suggest that reactive oxygen species
play an important role in the action of ionizing radiation.
c-irradiation enhances the production of ROS in a variety of
cells resulting in oxidative stress (Azzam et al. 2012; Kebeish
et al. 2015). ROS are the by-products of many degenerative
reactions, which will affect the regular metabolism by dam-
aging the cellular components (Noctor et al. 2002). Extensive
study on oxidative stress has demonstrated that exposure of
plants or cell cultures to adverse environmental conditions
induces the over production of ROS, such as superoxide
radical (O2 ), H2O2 and hydroxyl radical in plant cells. As a
consequence, plants evolved cellular adaptive responses like
up-regulation of oxidative stress protectors and accumulation
of protective solutes (Horling et al. 2003). Plants exposed to
c-radiation produce various defense and anti-oxidant
enzymes many of which produce secondary metabolites
which in turn alleviates the induced oxidative stress condi-
tions (Kim et al. 2004; Aly & El-Beltagi 2010; Zahran & Eliwa
2015). The reports on the c-irradiation of callus cultures show
interesting examples, where the irradiated cultures exhibited
an increase in secondary metabolites, total soluble proteins,
amino acids, soluble sugars and anti-oxidant enzymes.
972 P. V. VARDHAN AND L. I. SHUKLA
El-Beltagi et al. (2011) investigated the effect of different
doses of c-irradiation (0, 5, 10, 15 and 20 Gy with dose rate
0.23 Gy/s) on phenolic and flavonoid production and anti-oxi-
dant properties for 4-week-old callus cultures of rosemary
(Rosmarinus officinalis L.). The total phenolics and total flavo-
noids content were increased 5- and 5.6-fold, respectively, in
calli irradiated with 20 Gy. The highest total phenolic content
was 4.38 mg/g for calli irradiated with 20 Gy over the control
(0.89 mg/g) and the highest total flavonoids content was
3.35 mg/g for calli irradiated with 20 Gy over the control
(0.64 mg/g). The irradiated samples exhibited an 80% increase
in phenylalanine ammonia-lyase (PAL) activity. PAL, at the
first step of the phenylpropanoid pathway, is an important
enzyme for the synthesis of various phenolic compounds
(Figure 2). Moreover, the anti-oxidant defense enzymes like
catalase, ascorbate peroxidase, superoxide dismutase and
glutathione reductase showed a gradual increase in response
to doses.
Khalil et al. (2015) investigated the effect of different
doses of c-irradiation (5, 10, 15 and 20 Gy) on 30-day-old leaf
derived callus cultures of stevia (Stevia rebaudiana Bert.).
They evaluated the effect of irradiation on callus proliferation,
production of stevioside and total phenolics and flavonoids
content. They found that most of the irradiated doses signifi-
cantly reduced callus proliferation as compared to untreated
culture, except the 15 Gy which slightly enhanced callus bio-
mass. A total of 15 Gy slightly (92%) enhanced stevioside
content (0.251 mg/g) over the control (0.232 mg/g) along
with total phenolics and flavonoids contents.
Zhang et al. (2011) produced mutant cell lines of wild gin-
seng (Panax ginseng Meyer) hairy root cultures by irradiating
with different doses of c-radiation (5, 10, 25, 50, 75, 100, and
200 Gy). They evaluated the effect of irradiation on the
growth of hairy roots and the production of crude saponins
and ginsenosides content. The growth of the roots was inhib-
ited with the increasing doses of irradiation. However, the
crude saponins and total ginsenosides content were
enhanced 1.4- and 1.8-fold, respectively, in cell lines treated
with 100 Gy.
Fulzele et al. (2015) investigated the effect of different
doses of c-irradiation (5, 10, 15, 20, 25 and 30 Gy) on the pro-
duction of camptothecin for 3-week-old callus cultures of
ghanera (Nothapodytes foetida). The amount of camptothecin
showed a maximum increase with 15 and 20 Gy-treated calli.
For 9-methoxy-camptotheicin, 15 Gy had a mild effect and
20 Gy showed maximum increase. The total yields of campto-
thecin and 9-methoxy-camptotheicin was increased as high
as 20-fold by 20 Gy. The results revealed that there was a dir-
ect correlation between doses and camptothecin content in
irradiated calli. Nevertheless, the high c-strength (25 Gy)
adversely affected the growth and production levels. Hence,
the dose of 20 Gy was considered satisfactory for callus
attenuation.
Chung et al. (2006) investigated the effect of different
doses of c-irradiation (2, 16 and 32 Gy) on the production of
shikonins for 2-week-old suspension cultures of purple grom-
well (Lithospermum erythrorhizon) cells. c-irradiation signifi-
cantly increased the total shikonins yield by 4-fold at 16 Gy
over the control but, the doses higher than that of 32 Gy
could not affect the yield significantly. It was also found that
the PHB geranyltransferase activity was significantly increased
at 16 Gy. PHB geranyltransferase synthesizes m-geranyl-p-
hydroxylbenzoic acid (GBA), which provides the complete
carbon skeleton for shikonins (Figure 5). Hence, the data indi-
cates that the increase in PHB geranyltransferase activity
leads to accumulation of shikonins.
Effect of irradiation on phytochemical complexes in
whole plants/plant parts
Topuz and Ozdemir (2004) investigated the effect of different
doses of c-irradiation (2.5, 5.0, 7.5 and 10 kGy with dose rate
0.7 Gy/s) on the levels of different capsaicinoid components
in paprika (Capsicum annum L.). It was clearly shown that all
components of capsaicinoids were significantly increased
with the maximum applied dose of 10 kGy which is esti-
mated to be about 10%. However, the level of isodihydrocap-
saicin was not affected by irradiation doses. Doses up to 5
kGy of c-irradiation led to greater increases of capsaicin and
dihydrocapsaicin levels. With doses of more than 5 kGy, their
levels did not show significant differences, i.e. both 7.5 and
10 kGy doses had similar effects on capsaicin and dihydro-
capsaicin. The capsaicinoids increase with the effect of irradi-
ation treatments can be explained by changing the
conformation of the molecules and/or accompanying com-
pounds which affects the extraction yield.
Moghaddam et al. (2011) investigated the effect of differ-
ent doses of c-irradiation (20, 30 and 40 Gy) on total flavo-
noids content of two accessions of Centella asiatica (CA03
and CA23). It was shown that the total flavonoids content for
plants irradiated with 20 and 30 Gy was higher than the uni-
rradiated ones. The c-irradiation dose of 40 Gy was found to
be a median lethal dose (LD50) for CA23.
Gamma irradiation influencing the biosynthetic
pathways and other components to accumulate
secondary metabolites
The underlying mechanisms and possible reasons through
which irradiation influences the increased production of dif-
ferent types of secondary metabolites from different cell
cultures and plants/plant parts will be discussed in this
section.
Effect of c-irradiation on the yields of phenolic
compounds and flavonoids
Phenylalanine ammonia-lyase (PAL) is a key enzyme in the
phenylpropanoid pathway (Figure 2) responsible for the syn-
thesis of several plant phenolics. It lies at the first step of the
pathway, and catalyzes the conversion of phenylalanine to
cinnamic acid (Hahlbrock & Scheel 1989; Cheng & Breen
1991; Dixon & Paiva 1995). The involvement of PAL in biosyn-
thesis of various phenolics and flavonoids has been con-
firmed in several previous studies (Cahill & McComb 1992;
Lister et al. 1996; Jiang & Joyce 2003; Nadernejad et al.

2013). For example, the involvement of PAL in the biosyn-
thesis of rosmarinic acid (RA) has been confirmed by
up-regulation of its activity preceding the RA accumulation
in cell cultures of the Boraginaceae and Lamiaceae species
(Mizukami et al. 1992; Yan et al. 2006). It is also reported in
many instances that PAL activity may increase in response to
various biotic and abiotic stresses such as wounding,
drought, irradiation, nutrient deficiencies, herbicide treatment
and insect attacks (Wada et al. 2014; Wang et al. 2016).
Studies with several different species of plants have shown
PAL is activated by many environmental factors, which is
consistent with the increase of PAL activity in rice (Shehab
et al. 2010).
El-Beltagi et al. (2011) reported that c-irradiation treatment
significantly increased the PAL activity of rosemary (R. offici-
nalis L.) calli. There was a positive correlation between irradi-
ation doses and PAL activity. Benoit et al. (2000) reported
that c-irradiation significantly increased the PAL activity of
the Agaricus bisporus mushrooms. PAL activity was 4-fold
higher in samples treated with 2.5 kGy than the control.
Hussain et al. (2010) reported that c-irradiation significantly
increased the PAL activity of peach fruit over the control
samples. It was shown that the PAL activity exhibited almost
a linear increase with an increase in irradiation dose. Also, it
was observed that there was a positive correlation between
the PAL activity and total phenols, indicating that enhance-
ment in total phenols is accompanied by an increase in PAL
activity.
Also, the increase in phenolics might be due to the
release of phenolic compounds from glycosidic components
and the degradation of complex phenolics into simple ones
by c-irradiation (Harrison & Were 2007). Irradiation exerts its
Figure 3. Biosynthetic pathway of ginsenoids (Choi et al. 2005). The enzymes whose activity is enhanced with c-irradiation are SS, SE, DS, CAS and b-AS (written in
bold). MVA: mevalonate; FPP: farnesyl diphosphate; SS: squalene synthase; SE: squalene epoxidase; DS: dammarenediol synthase; CAS: cycloartenol synthase; b-AS:
b-amyrin synthase; LS: lupeol synthase.
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 973
effects by inducing oxidative stress in the cells. Radiolysis of
water results in the production of free radicals such as
hydroxyl radicals, hydroperoxide radicals and hydrated elec-
trons. These radicals may break the glycosidic bonds of pro-
cyanidin trimers, tetramers and hexamers that are present in
fruits, leading to the formation of procyanidin monomers,
which increase the total phenolic content in irradiated fruits
(Lee et al. 2009).
Capsaicinoids
Capsaicinoids are the active compounds associated with
the pungency of chili peppers which is unique to the
Capsicum species. Capsaicinoids belong to the group vanil-
lylamides which are synthesized via phenlypropanoid path-
way and branched-chain amino acid pathway. Topuz and
Ozdemir (2004) investigated the effect of different doses of
c-irradiation (2.5, 5.0, 7.5 and 10 kGy with dose rate
0.7 Gy/s) on the levels of different capsaicinoid components
in paprika. Pungent spice paprika is one of the oldest,
most important and widely used food flavorings and colo-
rants. It is also used as raw material for the pharmaceutical
industries. Since the substrate is the dead tissue, it is diffi-
cult to explain the influence of c-irradiation. However, they
suggested that it may be due to change in conformation
of the molecules and/or accompanying compounds which
affects the extraction yield. However, capsaicinoid synthase
(CS) is the last enzyme which is an acyltransferase, respon-
sible for the condensation of vanillylamine to a branched-
chain fatty acid moiety in the capsaicinoid biosynthetic
pathway. Further investigations are needed to evaluate the
role of CS in accumulation of capsaicinoids in response to
c-irradiation.
974 P. V. VARDHAN AND L. I. SHUKLA

Flavonoids
Since flavonoid biosynthesis is a prominent branch (Figure 2)
of the phenylpropanoid pathway, it is also affected by the
PAL activity. Oufedjikh et al. (2000) reported that c-irradiation
induces oxidative stress and de nova synthesis of flavonoids
by increased PAL activity. However, the key enzyme in the
flavonoid biosynthesis is the chalcone synthase (CHS) which
catalyzes the formation of chalcones from malonyl-coA and
coumaroyl-coA. Chalcones are the important intermediate in
the flavonoid pathway which gives rise to various classes of
flavonoids. El-Garhy et al. (2016) showed that there was a
correlation between up-regulation of CHS genes and
increased flavonoid content in response to c-irradiation, indi-
cating that the enhancement in flavonoids content is accom-
panied by an increase in CHS expression. The increase in
various phenolics and flavonoids may contribute to alleviate
the damage induced by the irradiation stress.

Effect of c-irradiation on the yields of terpenoids

(ginsenosides)
Ginsenosides are the most studied terpenoids which are
reported to increase in quantity with c-irradiation. They
are glycosylated triterpenes (saponins) and triterpenes of gin-
seng which can be classified into three groups based on
their aglycone structure; namely, the dammarane-type,
phytosterol-type and oleanane-type. The most common
ginsenosides are the dammarane-type, which can further be
sub-divided into Rb (Rb1, Rb2, Rc, and Rd) and Rg (Re, Rf,
and Rg1) groups derived from the 20(S)-protopanaxadiol and
20(S)-protopanaxatriol, respectively (Kim et al. 2006; Shi et al.
2007). The oleanane-type ginsenosides are only R0 in which
the fundamental skeletons are pentacyclic (Kim et al. 2013).
Ginsenosides are biosynthesized by the mevalonate path-
way in the cytosol in the roots of ginseng (Figure 3). The
P. ginseng squalene synthase (PgSS) and P. ginseng squalene
epoxidase (PgSE) genes encodes squalene synthase (SS) and
squalene epoxidase (SE) which catalyzes the initial steps of
the pathway which leads to various triterpenoids (Han et al.
2010). SS is involved in the biosynthesis of squalene from far-
nesyl diphosphate and SE converts squalene to oxidosqua-
lene (Choi et al. 2005). These enzymes are considered to
constitute potential regulatory points in the isoprenoid path-
way (Croteau et al. 2000). Kim et al. (2013) reported that the
transcription levels of PgSS and PgSE genes were significantly
increased with c-irradiation of ginseng hairy root cultures.
The first committed step in the ginsenosides biosynthetic
pathway involves the cyclization of oxidosqualene by oxidos-
qualene cyclases (OSC) (Choi et al. 2005). Three types of
OSCs, dammarenediol synthase, phytosterol synthase and
oleanane-type synthase, are encoded by the genes DDS
(dammarenediol synthase), PNX (phytosterol synthase) and
PNY (oleanane-type synthase), respectively. Dammarenediol
synthase yields dammarane-type ginsenosides, phytosterol
synthase yields phytosterols (cycloartenol) and oleanane-type
synthase yields oleanane-type (b-amyrin) ginsenosides. These
genes were also found to be significantly up-regulated in
response to c-irradiation along with enhanced ginsenoside
Figure 4. Biosynthetic pathway of camptothecin (Yamazaki et al. 2003b). The
activity of STR is enhanced with c-irradiation (written in bold). STR: strictosidine
synthase.
content (Kim et al. 2013). Thus, c-irradiation increases the
production of ginsenosides by enhancing the expression of
key enzymes of ginsenosides biosynthetic pathway.
Effect of c-irradiation on the yields of alkaloids
Camptothecin
Fulzele et al. (2015) investigated the effect of different
doses of c-irradiation (5, 10, 15, 20, 25 and 30 Gy) on the pro-
duction of camptothecin for 3-week-old callus cultures of
ghanera (N. foetida). The total yields of camptothecin and 9-
methoxy-camptotheicin was increased as high as 20-fold by
20 Gy. The amount of camptothecin showed a maximum
increase with 15 and 20 Gy-treated calli. For 9-methoxy-camp-
totheicin, 15 Gy had a mild effect and 20 Gy showed a max-
imum increase. Nevertheless, the high c-strength (25 Gy)
adversely affected the growth and production levels.
Camptothecin is a quinoline alkaloid, belonging to a class
of modified monoterpenoid indole alkaloids which shows
anti-tumor activity by inhibiting DNA topoisomerase-I (Hsiang
et al. 1985). Strictosidine synthase (STR) is an important
enzyme in the terpenoid indole alkaloid (TIA) biosynthetic
pathway (Figure 4) which catalyzes the formation of strictosi-
dine, a key metabolite which is a common precursor for all
TIAs including camptothecin (Lu et al. 2009). STR catalyzes
the stereospecific condensation of indole tryptamine and

Figure 5. Biosynthetic pathway of shikonin (Chung et al. 2006). The enzymes whose activity is enhanced with c-irradiation are PHB geranyl transfarase and PHB
glucosyl transferase (written in bold). GPP: geranylpyrophosphate; PHB: p-hydroxylbenzoic acid; GBA: m-geranyl-p-hydroxylbenzoic acid; GHQ: geranylhydroquinone.
monoterpenoid secologanin to form strictosidine (Yamazaki
et al. 2003b). Tryptamine is synthesized via the shikimate
pathway, and the secologanin part is synthesized via the
methylerythritol 4-phosphate (MEP) pathway (Yamazaki et al.
2004). Strictosidine is then converted to strictosamide, but
the remaining details and precise intermediates between
strictosamide and camptothecin are not completely defined
(Lorence & Nessler 2004). Although many studies report that
c-irradiation significantly influenced the yield of various
camptothecin derivatives, none of them have reported the
correlation between camptothecin accumulation and STR up-
regulation. Hence there will be scope in this line to evaluate,
through which mechanism c-irradiation is influencing the
camptothecin yields.
Shikonins
Shikonins and their derivatives are organic hydroxynaphtho-
quinones with a wide range of properties such as anti-inflam-
matory, antipyretic, analgesic, anti-oxidant activities which
act by inhibiting the biosynthesis of prostaglandins. The key
step in the biosynthesis of shikonins is the formation of
m-geranyl-p-hydroxylbenzoic acid (GBA) in a reaction cata-
lyzed by PHB geranyltransferase which is the first step of the
shikonin pathway (Figure 5). It catalyzes the formation of
GBA by linking PHB (p-hydroxylbenzoic acid) from the phe-
nylpropanoid pathway with GPP (gernaylpyrophosphate)
from isoprenoid pathway. Chung et al. (2006) reported that
the activity of PHB geranyltransferase was significantly
increased and the activity of PHB glucosyltransferase was sig-
nificantly decreased with c-irradiation of suspension cultures
of Lithospermum erythrorhizon cells as observed in the normal
enzymatic regulation of shikonin production. PHB
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 975
glucosyltransferase deviate the shikonin pathway by convert-
ing PHB into PHB-glucose. Hence, it is suggested that when
the activity of PHB geranyltrasferase is high with low activity
of PHB glucosyltransferase, the process of shikonin biosyn-
thesis would be boosted, resulting in the increase of shikonin
yields.
Conclusions
The plant secondary metabolites are in increasing demand
for commercial and industrial production of different medi-
cinal formulations. The production of secondary metabolites
in plants is in response to stress. These compounds could
facilitate mutualistic, antagonistic interactions of the plant
with its environment for coping with changing conditions.
Optimal gamma irradiation facilitates a higher amount of sec-
ondary metabolite assimilation. A reduction in production
with higher doses of c-irradiation is noted. Evidences are pro-
vided wherein the c-irradiation has led to synthesis in higher
concentrations of secondary metabolites namely; psoralen,
capsaicinoids, stevioside, saponins, ginsenosides, camptothe-
cin and shikonins. The c-irradiation showed many fold higher
accumulation when compared to those obtained by biotic
and abiotic elicitors. These results are indicative of the higher
gene plasticity of secondary metabolite production, which in
response to various elicitors like biotic, abiotic and c-irradi-
ation stress, shows differences in accumulative concentrations.
The enzymes associated with secondary metabolite produc-
tion – phenylalanine ammonia-lyase (PAL), chalcone synthase
(CHS), squalene synthase (SS), squalene epoxidase (SE), oxidos-
qualene cyclases (OSC) and PHB geranyl transferases – show
direct correlations with increases in radiation dose.
976 P. V. VARDHAN AND L. I. SHUKLA
The method of c-irradiation at different stages of second-
ary metabolite production could be used for yield improve-
ment of secondary metabolites in plants. Relative low doses
of c-irradiation (20–40 Gy) could be effective for the
enhanced production of secondary metabolites, as higher
doses (>50 Gy) affect the vigor and vitality of the tissue. A
detailed mechanistic understanding of the generation of sec-
ondary metabolites in response to c-irradiation elicitors could
facilitate a scale-up of operations for several fold increases in
secondary metabolite synthesis. This would be good for sus-
tainable agriculture relevance of medicinal plants and would
provide higher output and effective formulations. Further
work needs to be done to understand the underlying radio-
lytic mechanisms to develop standardized methods for profit-
able production of various secondary metabolites.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by DST-FIST [SR/FST/LSI-366/2008 Dt.
18.02.2009] and UGC-SAP [F.4-2/2015/DRS-III/(SAP-II) Dt. 31.03.2015].
Notes on contributors
Mr P. Vivek Vardhan is a PhD scholar at Department of Biotechnology,
Pondicherry University, India. He is a UGC-BSR fellow and holds an M.Sc.
degree in Biochemistry with distinction from GITAM University. He is
working in plant biotechnology and has made oral presentations at con-
ferences ICRB-2016, Indian Science Congress-2016 and IABMS-2016.
Dr Lata I. Shukla is a DST-BOYSCAST fellow, a plant biotechnologist,
working as an Assistant Professor at Department of Biotechnology,
Pondicherry University, India. Contributions in radiation biology include
the EPR of trapped electrons in irradiated seeds, first report of EPR and
sugar damage in DNA and conserved miRNA in plants.
ORCID
Lata I. Shukla http://orcid.org/0000-0003-4981-3224
References
Ahlawat S, Saxena P, Alam P, Wajid S, Abdin M. 2014. Modulation of arte-
misinin biosynthesis by elicitors, inhibitor and precursor in hairy root
cultures of Artemisia annua L. J Plant Interact. 9:811–824.
Ahmed SA, Baig MMV. 2014. Biotic elicitor enhanced production of psor-
alen in suspension cultures of Psoralea corylifolia L. Saudi J Biol Sci.
21:499–504.
Akshatha Chandrashekar KR, Somashekarappa HM, Souframanien J. 2013.
Effect of gamma irradiation on germination, growth, and biochemical
parameters of Terminalia arjuna Roxb. Radiat Prot Environ. 36:38–44.
Akula R, Ravishankar GA. 2011. Influence of abiotic stress signals on sec-
ondary metabolites in plants. Plant Signal Behav. 6:1720–1731.
Aly AA, El-Beltagi HS. 2010. Influence of ionizing irradiation on the anti-
oxidant enzymes of Vicia faba L. Grasas Y Aceites. 61:288–294.
Ashok P, Lathiya H, Murugesan S. 2015. Manzamine alkaloids as antileish-
manial agents: a review. Eur J Med Chem. 97:928–936.
Ashouri SA, Hassanpour H, Jonoubi P, Ghorbani NM, Nadimifar M. 2016.
The effect of gamma irradiation on in vitro total phenolic content and
antioxidant activity of Ferula gummosa Bioss. J Med Plants. 3:122–131.
Aza-Gonz
alez C, N
un~ez-Palenius HG, Ochoa-Alejo N. 2012. Molecular biol-
ogy of chili pepper anthocyanin biosynthesis. J Mex Chem Soc.
56:93–98.
Azzam EI, Jay-Gerin JP, Pain D. 2012. Ionizing radiation-induced meta-
bolic oxidative stress and prolonged cell injury. Cancer Lett.
327:48–60.
Baenas N, Garc
ıa-Viguera C, Moreno DA. 2014. Elicitation: a tool for
enriching the bioactive composition of foods. Molecules.
19:13541–13563.
Bennett RN, Wallsgrove RM. 1994. Secondary metabolites in plant
defense mechanisms. New Phytol. 127:617–633.
Benoit M, D’Aprano G, Lacroix M. 2000. Effect of gamma-irradiation on
phenylalanine ammonia-lyase activity, total phenolic content, and res-
piration of mushrooms (Agaricus bisporus). J Agric Food Chem.
48:6312–6316.
Bliss ED. 1989. Chemistry and significance of condensed tannins. In:
Hemingway RW, Karchesy JJ, Branham SJ, editors. Using tannins to
produce leather. New York: Springer; pp. 493–502.
Bourgaud F, Gravot A, Milesi S, Gontier E. 2001. Production of plant sec-
ondary metabolites: a historical perspective. Plant Sci. 161:839–851.
Brooks CJ, Watson DG, Freer IM. 1986. Elicitation of capsidiol accumula-
tion in suspended callus cultures of Capsicum annuum.
Phytochemistry. 25:1089–1092.
Cahill DM, McComb JA. 1992. A comparison of changes in phenylalanine
ammonia-lyase activity, lignin and phenolic synthesis in the roots of
Eucalyptus calophylla (field resistant) and E. marginata (susceptible)
when infected with Phytophthora cinnamomi. Physiol Mol Plant
Pathol. 40:315–332.
Cermak R. 2008. Effect of dietary flavonoids on pathways involved in
drug metabolism. Expert Opin Drug Metab Toxicol. 4:17–35.
Charbaji T, Nabulsi I. 1999. Effect of low doses of gamma irradiation on
in vitro growth of grapevine. Plant Cell Tissue Organ Cult. 57:129–132.
Cheng AX, Lou YG, Mao YB, Lu S, Wang LJ, Chen XY. 2007. Plant terpe-
noids: biosynthesis and ecological functions. J Integrat Plant Biol.
49:179–186.
Cheng GW, Breen PJ. 1991. Activity of phenylalanine ammonia-lyase
(PAL) and concentrations of anthocyanins and phenolics in develop-
ing strawberry fruit. J Am Soc Hortic Sci. 116:865–869.
Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S. 2013. Plant phe-
nolics: recent advances on their biosynthesis, genetics, and ecophysi-
ology. Plant Physiol Biochem. 72:1–20.
Choi DW, Jung J, Im Ha Y, Park HW, In DS, Chung HJ, Liu JR. 2005.
Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots
to identify genes involved in the biosynthesis of ginsenosides and
other secondary metabolites. Plant Cell Rep. 23:557–566.
Chung B, Lee YB, Baek MH, Kim JH, Wi S, Kim JS. 2006. Effects of low-
dose gamma-irradiation on production of shikonin derivatives in cal-
lus cultures of Lithospermum erythrorhizon S. Radiat Phys Chem.
75:1018–1023.
Cooper-Driver GA, Swain T. 1976. Cyanogenic polymorphism in bracken
in relation to herbivore predation. Nature. 260:604–604.
Croteau R, Kutchan TM, Lewis NG. 2000. Natural products (secondary
metabolites). In: Buchanan B, Gruissem W, Jones R, editors.
Biochemistry and molecular biology of plants. Rockville, MD: American
Society of Plant Physiologists; pp. 1250–1319.


Cueva C, Moreno-Arribas MV, Mart
ın-Alvarez PJ, Bills G, Vicente MF,
Basilio A, Rivas CL, Requena T, Rodr
ıguez JM, Bartolome
B. 2010.
Antimicrobial activity of phenolic acids against commensal, probiotic
and pathogenic bacteria. Res Microbiol. 161:372–382.
Dai J, Mumper RJ. 2010. Plant phenolics: extraction, analysis and their
antioxidant and anticancer properties. Molecules. 15:7313–7352.
Davies CR. 1970. Effects of gamma irradiation on growth and yield of
agricultural crops – II. Spring sown barley and other cereals. Radiat
Bot. 10:19–27.
Deepthi S, Satheeshkumar K. 2016. Enhanced camptothecin production
induced by elicitors in the cell suspension cultures of Ophiorrhiza
mungos Linn. Plant Cell Tiss Organ Cult. 124:483–493.

Devic M, Guilleminot J, Debeaujon I, Bechtold N, Bensaude E, Koornneef M,
Pelletier G, Delseny M. 1999. The BANYULS gene encodes a DFR-like
protein and is a marker of early seed coat development. Plant J.
19:387–398.
Dixon RA. 2001. Natural products and plant disease resistance. Nature.
411:843–847.
Dixon RA, Paiva NL. 1995. Stress-induced phenylpropanoid metabolism.
Plant Cell. 7:1085–1097.
Eilert U. 1987. Elicitation: methodology and aspects of application. In:
Vasil IK, Constabel F, editors. Cell culture and somatic cell genetics of
plants. Vol. 4. Amsterdam, The Netherlands: Elsevier Science;
pp. 153–196.
El-Beltagi HS, Ahmed OK, El-Desouky W. 2011. Effect of low doses
c-irradiation on oxidative stress and secondary metabolites production
of rosemary (Rosmarinus officinalis L.) callus culture. Radiat Phys
Chem. 80:968–976.
El-Garhy HA, Khattab S, Moustafa MM, Ali RA, Azeiz AZA, Elhalwagi A, El
Sherif F. 2016. Silybin content and overexpression of chalcone syn-
thase genes in Silybum marianum L. plants under abiotic elicitation.
Plant Physiol Biochem. 108:191–202.
Facchini PJ. 2001. Alkaloid biosynthesis in plants: biochemistry, cell biol-
ogy, molecular regulation, and metabolic engineering applications.
Annu Rev Plant Physiol Plant Mol Biol. 52:29–66.
Forkner RE, Marquis RJ, Lill JT. 2004. Feeny revisited: condensed tannins
as anti-herbivore defenses in leaf-chewing herbivore communities of
Quercus. Ecol Entomol. 29:174–187.
Fukui H, Yoshikawa N, Tabata M. 1983. Induction of shikonin formation
by agar in Lithospermum erythrorhizon cell suspension cultures.
Phytochemistry. 22:2451–2453.
Fulzele DP, Satdive R, Kamble S, Singh S, Singh S. 2015. Improvement of
anticancer drug camptothecin production by gamma irradiation on
callus cultures of Nothapodytes foetida. Int J Pharma Res Allied Sci.
4:19–27.
Galeotti F, Barile E, Curir P, Dolci M, Lanzotti V. 2008. Flavonoids from
carnation (Dianthus caryophyllus) and their antifungal activity.
Phytochem Lett. 1:44–48.
Gershenzon J, Dudareva N. 2007. The function of terpene natural prod-
ucts in the natural world. Nat Chem Biol. 3:408–414.
Gleadow RM, Woodrow IE. 2002. Mini-review: constraints on effectiveness
of cyanogenic glycosides in herbivore defense. J Chem Ecol.
28:1301–1313.
Guardia T, Rotelli AE, Juarez AO, Pelzer LE. 2001. Anti-inflammatory prop-
erties of plant flavonoids. Effects of rutin, quercetin and hesperidin on
adjuvant arthritis in rat. Farmaco. 56:683–687.
Hahlbrock K, Scheel D. 1989. Physiology and molecular biology of phe-
nylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol.
40:347–369.
H€am€al€ainen M, Nieminen R, Vuorela P, Heinonen M, Moilanen E. 2007.
Anti-inflammatory effects of flavonoids: genistein, kaempferol, quer-
cetin, and daidzein inhibit STAT-1 and NF-jB activations, whereas fla-
vone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-jB
activation along with their inhibitory effect on iNOS expression and NO
production in activated macrophages. Mediators Inflamm. 2007;45673.
Han JY, In JG, Kwon YS, Choi YE. 2010. Regulation of ginsenoside and
phytosterol biosynthesis by RNA interferences of squalene epoxidase
gene in Panax ginseng. Phytochemistry. 71:36–46.
Harrison K, Were L. 2007. Effect of gamma irradiation on total phenolic
content yield and antioxidant capacity of almond skin extracts. Food
Chem. 102:932–937.
Hashemi SM, Naghavi MR. 2016. Production and gene expression of mor-
phinan alkaloids in hairy root culture of Papaver orientale L. using abi-
otic elicitors. Plant Cell Tiss Organ Cult. 125:31–41.
He F, Mu L, Yan GL, Liang NN, Pan QH, Wang J, Reeves MJ, Duan CQ.
2010. Biosynthesis of anthocyanins and their regulation in colored
grapes. Molecules. 15:9057–9091.
Herbert RB. 1989. The biosynthesis of secondary metabolites.
Amsterdam, The Netherlands: Springer Science & Business Media.
Hohsaka T, Sisido M. 2002. Incorporation of non-natural amino acids into
proteins. Curr Opin Chem Biol. 6:809–815.
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 977
Horling F, Lamkemeyer P, Ko €nig J, Finkemeier I, Kandlbinder A, Baier M,
Dietz KJ. 2003. Divergent light-, ascorbate-, and oxidative stress-
dependent regulation of expression of the peroxiredoxin gene family
in Arabidopsis. Plant Physiol. 131:317–325.
Hsiang YH, Hertzberg R, Hecht S, Liu LF. 1985. Camptothecin induces
protein-linked DNA breaks via mammalian DNA topoisomerase I.
J Biol Chem. 260:14873–14878.
Hu X, Neill S, Cai W, Tang Z. 2003. Hydrogen peroxide and jasmonic
acid mediate oligogalacturonic acid-induced saponin accumulation in
suspension-cultured cells of Panax ginseng. Physiol Plant.
118:414–421.
Huang T, Jander G, de Vos M. 2011. Non-protein amino acids in plant
defense against insect herbivores: representative cases and opportuni-
ties for further functional analysis. Phytochemistry. 72:1531–1537.
Hui C, Bin Y, Xiaoping Y, Long Y, Chunye C, Mantian M, Wenhua L. 2010.
Anticancer activities of an anthocyanin-rich extract from black rice
against breast cancer cells in vitro and in vivo. Nutr Cancer.
62:1128–1136.
Hussain PR, Wani AM, Meena RS, Dar MA. 2010. Gamma irradiation
induced enhancement of phenylalanine ammonia-lyase (PAL) and
antioxidant activity in peach (Prunus persica Bausch, Cv. Elberta).
Radiat Phys Chem. 79:982–989.
Jan S, Parween T, Siddiqi TO. 2011. Gamma radiation effects on growth
and yield attributes of Psoralea corylifolia L. with reference to
enhanced production of psoralen. Plant Growth Regul. 64:163–171.
Jiang Y, Joyce DC. 2003. ABA effects on ethylene production, PAL activ-
ity, anthocyanin and phenolic contents of strawberry fruit. Plant
Growth Regul. 39:171–174.
Johnson TS, Ravishankar GA, Venkataraman LV. 1991. Elicitation of capsa-
icin production in freely suspended cells and immobilized cell cultures
of Capsicum frutescens Mill. Food Biotechnol. 5:197–205.
Kabera JN, Semana E, Mussa AR, He X. 2014. Plant secondary metabo-
lites: biosynthesis, classification, function and pharmacological proper-
ties. J Pharm Pharmacol. 2:377–392.
Karowe DN. 1989. Differential effect of tannic acid on two tree-feeding
Lepidoptera: implications for theories of plant anti-herbivore chemis-
try. Oecologia. 80:507–512.
Kebeish R, Hanan ED, El-Bialy N. 2015. Effects of gamma radiation on
growth, oxidative stress, antioxidant system and alliin producing gene
transcripts in Allium Sativum. Int J Res Stud Biosci. 3:161–174.
Khalil SA, Ahmad N, Zamir R. 2015. Gamma radiation induced variation
in growth characteristics and production of bioactive compounds dur-
ing callogenesis in Stevia rebaudiana (Bert.). New Negat Plant Sci.
1:1–5.
Khanbabaee K, van Ree T. 2001. Tannins: classification and definition. Nat
Prod Rep. 18:641–649.
Kim DS, Song M, Kim SH, Jang DS, Kim JB, Ha BK, Kim SH, Lee KJ, Kang
SY, Jeong IY. 2013. The improvement of ginsenoside accumulation in
Panax ginseng as a result of c-irradiation. J Ginseng Res. 37:332–340.
Kim JH, Baek MH, Chung BY, Wi SG, Kim JS. 2004. Alterations in the
photosynthetic pigments and antioxidant machineries of red pepper
(Capsicum annuum L.) seedlings from gamma-irradiated seeds. J Plant
Biol. 47:314–321.
Kim JH, Kwon SK, Sung NY, Jung PM, Choi J, Kim JK, Sharma AK, Lee JW.
2012. Effect of gamma irradiation on the conversion of ginsenoside
Rb1 to Rg3. Radiat Phys Chem. 81:1128–1131.
Kim MK, Lee BS, In JG, Sun H, Yoon JH, Yang DC. 2006. Comparative ana-
lysis of expressed sequence tags (ESTs) of ginseng leaf. Plant Cell Rep.
25:599–606.
Kruger MJ, Davies N, Myburgh KH, Lecour S. 2014. Proanthocyanidins,
anthocyanins and cardiovascular diseases. Food Res Int. 59:41–52.
Lacaille-Dubois MA, Wagner H. 1996. A review of the biological and
pharmacological activities of saponins. Phytomedicine. 2:363–386.
Lee JW, Kim JK, Srinivasan P, Choi J, Kim JH, Han SB, Kim DJ, Byun MW.
2009. Effect of gamma irradiation on microbial analysis, antioxidant
activity, sugar content and color of ready-to-use tamarind juice during
storage. LWT-Food Sci Tech. 42:101–105.
Lister CE, Lancaster JE, Walker JR. 1996. Phenylalanine ammonia-lyase
(PAL) activity and its relationship to anthocyanin and flavonoid levels
978 P. V. VARDHAN AND L. I. SHUKLA
in New Zealand-grown apple cultivars. J Am Soc Hortic Sci.
121:281–285.
Lola-Luz T, Hennequart F, Gaffney M. 2014. Effect on yield, total phenolic,
total flavonoid and total isothiocyanate content of two broccoli culti-
vars (Brassica oleraceae var. italica) following the application of a com-
mercial brown seaweed extract (Ascophyllum nodosum). Agric Food
Sci. 23:28–37.
Lorence A, Nessler CL. 2004. Camptothecin, over four decades of surpris-
ing findings. Phytochemistry. 65:2735–2749.
Lu JJ, Bao JL, Chen XP, Huang M, Wang YT. 2012. Alkaloids isolated from
natural herbs as the anticancer agents. Evid Based Complement
Alternat Med. 2012;485042.
Lu Y, Wang H, Wang W, Qian Z, Li L, Wang J, Zhou G, Kai G. 2009.
Molecular characterization and expression analysis of a new cDNA
encoding strictosidine synthase from Ophiorrhiza japonica. Mol Biol
Rep. 36:1845–1852.
Mach J. 2015. Production of the non-protein amino acid b-tyrosine in
rice. Plant Cell. 27:949.
Maffei M. 2010. Sites of synthesis, biochemistry and functional role of
plant volatiles. S Afr J Bot. 76:612–631.
Matamala G, Smeltzer W, Droguett G. 2000. Comparison of steel anticor-
rosive protection formulated with natural tannins extracted from aca-
cia and from pine bark. Corros Sci. 42:1351–1362.
Mizukami H, Ogawa T, Ohashi H, Ellis BE. 1992. Induction of rosmarinic
acid biosynthesis in Lithospermum erythrorhizon cell suspension cul-
tures by yeast extract. Plant Cell Rep. 11:480–483.
Moghaddam SS, Jaafar H, Ibrahim R, Rahmat A, Aziz MA, Philip E. 2011.
Effects of acute gamma irradiation on physiological traits and flavon-
oid accumulation of Centella asiatica. Molecules. 16:4994–5007.
Mohamed AA. 2009. Effect of low dose gamma irradiation on some phy-
tochemicals and scavenger ability of in vitro culantro (Eryngium foeti-
dum L.) plantlets. Med Aromat Plant Sci Biotechnol. 3:32–36.
Murthy HN, Lee EJ, Paek KY. 2014. Production of secondary metabolites
from cell and organ cultures: strategies and approaches for biomass
improvement and metabolite accumulation. Plant Cell Tiss Organ Cult.
118:1–16.
Nadernejad N, Ahmadimoghadam A, Hossyinifard J, Poorseyedi S. 2013.
Evaluation of PAL activity, phenolic and flavonoid contents in three
pistachio (Pistacia vera L.) cultivars grafted onto three different root-
stocks. J Stress Physiol Biochem. 9:84–97.
Naik PM, Al–Khayri JM. 2016. Abiotic and biotic elicitors-role in secondary
metabolites production through in vitro culture of medicinal plants.
In: Shanker AK, Shanker C, editors. Abiotic and biotic stress in plants –
recent advances and future perspectives. Rijeka: InTech; chapter 10.
Nakweti RK, Ndiku SL, Sinou V, Luyeye FL, Fundu TM, Hity DM, Kanianga
RC, Ndofunsu AD. 2014. Effects of gamma irradiation on seeds ger-
mination, plantlets growth and in vitro antimalarial activities of
Phyllanthus odontadenius M€ ull Arg. AJEA. 4:1435–1437.
Namdeo AG. 2007. Plant cell elicitation for production of secondary
metabolites: a review. Pharmacogn Rev. 1:69–79.
Nassar ZD, Aisha AA, Majid AMSA. 2010. The pharmacological properties
of terpenoids from Sandoricum koetjape. Webmedcentral Complement
Med. [Internet]. 2010;WMC001311. Available from: http://www.
webmedcentral.com/article_view/1311
Nassis CZ, Haebisch EM, Giesbrecht AM. 1991. Antihistamine activity of
Bryophyllum calycinum. Braz J Med Biol Res. 25:929–936.
Noctor G, Veljovic-Jovanovic S, Driscoll S, Novitskaya L, Foyer CH. 2002.
Drought and oxidative load in the leaves of C3 plants: a predominant
role for photorespiration? Ann Bot. 89:841–850.
Okuda T, Yoshida T, Hatano T. 1993. Classification of oligomeric hydrolys-
able tannins and specificity of their occurrence in plants.
Phytochemistry. 32:507–521.
Oufedjikh H, Mahrouz M, Amiot MJ, Lacroix M. 2000. Effect of c-irradi-
ation on phenolic compounds and phenylalanine ammonia-lyase
activity during storage in relation to peel injury from peel of Citrus
clementina Hort. Ex. Tanaka. J Agric Food Chem. 48:559–565.
Patra AK, Saxena J. 2010. A new perspective on the use of plant second-
ary metabolites to inhibit methanogenesis in the rumen.
Phytochemistry. 71:1198–1222.
Pereira DM, Valent~ao P, Pereira JA, Andrade PB. 2009. Phenolics: from
chemistry to biology. Molecules. 14:2202–2211.
Pinto PC, Sousa G, Crispim F, Silvestre AJ, Neto CP. 2013. Eucalyptus glob-
ulus bark as source of tannin extracts for application in leather indus-
try. ACS Sustain Chem Eng. 1:950–955.
Rahim AA, Kassim J. 2008. Recent development of vegetal tannins in cor-
rosion protection of iron and steel. MATS. 1:223–231.
Roy S, Mallick S, Chakraborty T, Ghosh N, Singh AK, Manna S, Majumdar S.
2015. Synthesis, characterisation and antioxidant activity of luteolin-
vanadium(II) complex. Food Chem. 173:1172–1178.
Rudolf JR, Resurreccion AV. 2005. Elicitation of resveratrol in peanut
kernels by application of abiotic stresses. J Agric Food Chem.
53:10186–10192.
Said-Al Ahl HA, Sarhan AM, Dahab ADMA, Abou-Zeid ESN, Ali MS,
Naguib NY. 2015. Bio-fertilizer and gamma radiation influencing flavo-
noids content at different parts of dill herb. Int J Life Sci Eng.
1:145–149.
Scalbert A, Johnson IT, Saltmarsh M. 2005. Polyphenols: antioxidants and
beyond. Am J Clin Nutr. 81:215S–217S.
Shakeran Z, Keyhanfar M, Asghari G, Ghanadian M. 2015. Improvement
of atropine production by different biotic and abiotic elicitors in hairy
root cultures of Datura metel. Turk J Biol. 39:111–118.
Shehab GG, Ahmed OK, El-Beltagi HS. 2010. Effects of various chemical
agents for alleviation of drought stress in rice plants (Oryza sativa L.).
Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 38:139–148.
Shi W, Wang Y, Li J, Zhang H, Ding L. 2007. Investigation of ginsenosides
in different parts and ages of Panax ginseng. Food Chem.
102:664–668.
Singh B, Bhat TK, Singh B. 2003. Potential therapeutic applications of
some antinutritional plant secondary metabolites. J Agric Food Chem.
51:5579–5597.
Siva G, Sivakumar S, Premkumar G, Kumar TS, Jayabalan N. 2014.
Enhanced production of psoralen through elicitors treatment in
adventitious root culture of Psoralea corylifolia L. Int J Pharm Pharm
Sci. 7:146–149.
Song SH, Byun SY. 1998. Characterization of cell growth and camptothe-
cin production in cell cultures of Camptotheca acuminata. J Microbiol
Biotechnol. 8:631–638.
Souza MT, Almeida JR, Araujo AA, Duarte MC, Gelain DP, Moreira JC,
Santos MR, Quintas-J
unior LJ. 2014. Structure-activity relationship of
terpenes with anti-inflammatory profile – a systematic review. Basic
Clin Pharmacol Toxicol. 115:244–256.
Sparrow AH, Schwemmer SS, Bottino P. 1971. The effects of external
gamma radiation from radioactive fallout on plants with special refer-
ence to crop production. Radiat Bot. 11:85–118.
Srivastava S, Srivastava A. 2014. Effect of elicitors and precursors on aza-
dirachtin production in hairy root culture of Azadirachta indica. Appl
Biochem Biotechnol. 172:2286–2297.
Stankovic MS. 2011. Total phenolic content, flavonoid concentration and
antioxidant activity of Marrubium peregrinum L. extracts. Kragujevac J
Sci. 33:63–72.
Taheri S, Abdullah TL, Karimi E, Oskoueian E, Ebrahimi M. 2014.
Antioxidant capacities and total phenolic contents enhancement with
acute gamma irradiation in Curcuma alismatifolia (Zingiberaceae)
leaves. Int J Mol Sci. 15:13077–13090.
Thoppil RJ, Bishayee A. 2011. Terpenoids as potential chemopreventive
and therapeutic agents in liver cancer. World J Hepatol. 3:228–249.
Tolstikova T, Sorokina I, Tolstikov G, Tolstikov A, Flekhter O. 2006.
Biological activity and pharmacological prospects of lupane terpe-
noids: I. Natural lupane derivatives. Russ J Bioorg Chem. 32:37–49.
Topuz A, Ozdemir F. 2004. Influences of gamma irradiation and storage
on the capsaicinoids of sun-dried and dehydrated paprika. Food
Chem. 86:509–515.
Treutter D. 2001. Biosynthesis of phenolic compounds and its regulation
in apple. Plant Growth Regul. 34:71–89.
Vardhan PV, Shukla LI. 2016. Gamma irradiation of commercially import-
ant plants enhances the secondary metabolite accumulation.
International conference on radiation biology: High LET Radiation
Biology and complex Natural Products in Biology & Medicine. Paper

presented at the 113th Biennial Meeting of the Indian Society for
Radiation Biology; Chennai, India.
Vetter J. 2000. Plant cyanogenic glycosides. Toxicon. 38:11–36.
Wada KC, Mizuuchi K, Koshio A, Kaneko K, Mitsui T, Takeno K. 2014.
Stress enhances the gene expression and enzyme activity of
phenylalanine ammonia-lyase and the endogenous content of sali-
cylic acid to induce flowering in pharbitis. J Plant Physiol.
171:895–902.
Wallace RJ. 2004. Antimicrobial properties of plant secondary metabo-
lites. Proc Nutr Soc. 63:621–629.
Wallace TC, Slavin M, Frankenfeld CL. 2016. Systematic review of antho-
cyanins and markers of cardiovascular disease. Nutrients. 8:13.
Wang G, Wu L, Zhang H, Wu W, Zhang M, Li X, Wu H. 2016. Regulation
of the phenylpropanoid pathway: a mechanism of selenium tolerance
in peanut (Arachis hypogaea L.) seedlings. J Agric Food Chem.
64:3626–3635.
White T. 1957. Tannins – their occurrence and significance. J Sci Food
Agric. 8:377–385.
Wu J, Lin L. 2002. Elicitor-like effects of low-energy ultrasound on
plant (Panax ginseng) cells: induction of plant defense responses
and secondary metabolite production. Appl Microbial Biotechnol.
59:51–57.
Yamazaki Y, Sudo H, Yamazaki M, Aimi N, Saito K. 2003a. Camptothecin
biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning,
View publication stats
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 979
characterization and differential expression in tissues and by stress
compounds. Plant Cell Physiol. 44:395–403.
Yamazaki Y, Urano A, Sudo H, Kitajima M, Takayama H, Yamazaki M, Aimi N,
Saito K. 2003b. Metabolite profiling of alkaloids and strictosidine synthase
activity in camptothecin producing plants. Phytochemistry. 62:461–470.
Yamazaki Y, Kitajima M, Arita M, Takayama H, Sudo H, Yamazaki M, Aimi
N, Saito K. 2004. Biosynthesis of camptothecin. In silico and in vivo
tracer study from [1-13C] glucose. Plant Physiol. 134:161–170.
Yan Q, Shi M, Ng J, Wu JY. 2006. Elicitor-induced rosmarinic acid accu-
mulation and secondary metabolism enzyme activities in Salvia mil-
tiorrhiza hairy roots. Plant Sci. 170:853–858.
Zahran AA, Eliwa NE. 2015. Influence of gamma radiation, glutathione
and ascorbic acid on some antioxidant enzymes in Egyptian clover
(Trifolium alexandrinum L.) under aluminum stress. Am Eurasian J
Agric Environ Sci. 15:1887–1894.
Zhang JY, Bae TW, Boo KH, Sun HJ, Song IJ, Pham CH, Ganesan M, Yang
DH, Kang HG, Ko SM, et al. 2011. Ginsenoside production and mor-
phological characterization of wild ginseng (Panax ginseng Meyer)
mutant lines induced by c-Irradiation (60Co) of adventitious roots.
J Ginseng Res. 35:283–293.
Zhu M, Zheng X, Shu Q, Li H, Zhong P, Zhang H, Xu Y, Wang L, Wang L.
2012. Relationship between the composition of flavonoids and flower
colors variation in tropical water lily (Nymphaea) cultivars. PLoS One.
7:e34335.